Jaeger KE

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Full name : Jaeger Karl-Erich

First name : Jaeger Karl-Erich

Mail : Institut fur Molekulare Enzymtechnologie, Heinrich Heine Universitat Dusseldorf and Forschungszentrum Julich, Wilhelm-Johnen-Strasse, 52426 Julich

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Country : Germany

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References (101)

Title : Biodegradation of poly(ester-urethane) coatings by Halopseudomonas formosensis - de Witt_2024_Microb.Biotechnol_17_e14362
Author(s) : de Witt J , Molitor R , Gatgens J , Ortmann de Percin Northumberland C , Kruse L , Polen T , Wynands B , van Goethem K , Thies S , Jaeger KE , Wierckx N
Ref : Microb Biotechnol , 17 :e14362 , 2024
Abstract : Impranil((a)) DLN-SD is a poly(ester-urethane) (PEU) that is widely used as coating material for textiles to fine-tune and improve their properties. Since coatings increase the complexity of such plastic materials, they can pose a hindrance for sustainable end-of-life solutions of plastics using enzymes or microorganisms. In this study, we isolated Halopseudomonas formosensis FZJ due to its ability to grow on Impranil DLN-SD and other PEUs as sole carbon sources. The isolated strain was exceptionally thermotolerant as it could degrade Impranil DLN-SD at up to 50 degreesC. We identified several putative extracellular hydrolases of which the polyester hydrolase Hfor_PE-H showed substrate degradation of Impranil DLN-SD and thus was purified and characterized in detail. Hfor_PE-H showed moderate temperature stability (T(m) = 53.9 degreesC) and exhibited activity towards Impranil DLN-SD as well as polyethylene terephthalate. Moreover, we revealed the enzymatic release of monomers from Impranil DLN-SD by Hfor_PE-H using GC-ToF-MS and could decipher the associated metabolic pathways in H.formosensis FZJ. Overall, this study provides detailed insights into the microbial and enzymatic degradation of PEU coatings, thereby deepening our understanding of microbial coating degradation in both contained and natural environments. Moreover, the study highlights the relevance of the genus Halopseudomonas and especially the novel isolate and its enzymes for future bio-upcycling processes of coated plastic materials.
ESTHER : de Witt_2024_Microb.Biotechnol_17_e14362
PubMedSearch : de Witt_2024_Microb.Biotechnol_17_e14362
PubMedID: 37991424
Gene_locus related to this paper: 9gamm-a0a1i6bky1

Title : An archaeal lid-containing feruloyl esterase degrades polyethylene terephthalate - Perez-Garcia_2023_Commun.Chem_6_193
Author(s) : Perez-Garcia P , Chow J , Costanzi E , Gurschke M , Dittrich J , Dierkes RF , Molitor R , Applegate V , Feuerriegel G , Tete P , Danso D , Thies S , Schumacher J , Pfleger C , Jaeger KE , Gohlke H , Smits SHJ , Schmitz RA , Streit WR
Ref : Commun Chem , 6 :193 , 2023
Abstract : Polyethylene terephthalate (PET) is a commodity polymer known to globally contaminate marine and terrestrial environments. Today, around 80 bacterial and fungal PET-active enzymes (PETases) are known, originating from four bacterial and two fungal phyla. In contrast, no archaeal enzyme had been identified to degrade PET. Here we report on the structural and biochemical characterization of PET46 (RLI42440.1), an archaeal promiscuous feruloyl esterase exhibiting degradation activity on semi-crystalline PET powder comparable to IsPETase and LCC (wildtypes), and higher activity on bis-, and mono-(2-hydroxyethyl) terephthalate (BHET and MHET). The enzyme, found by a sequence-based metagenome search, is derived from a non-cultivated, deep-sea Candidatus Bathyarchaeota archaeon. Biochemical characterization demonstrated that PET46 is a promiscuous, heat-adapted hydrolase. Its crystal structure was solved at a resolution of 1.71 A. It shares the core alpha/beta-hydrolase fold with bacterial PETases, but contains a unique lid common in feruloyl esterases, which is involved in substrate binding. Thus, our study widens the currently known diversity of PET-hydrolyzing enzymes, by demonstrating PET depolymerization by a plant cell wall-degrading esterase.
ESTHER : Perez-Garcia_2023_Commun.Chem_6_193
PubMedSearch : Perez-Garcia_2023_Commun.Chem_6_193
PubMedID: 37697032
Gene_locus related to this paper: 9arch-PETcan211 , 9cren-PETcan204 , 9arch-PET46

Title : Resolution of Maximum Entropy Method-Derived Posterior Conformational Ensembles of a Flexible System Probed by FRET and Molecular Dynamics Simulations - Dittrich_2023_J.Chem.Theory.Comput__
Author(s) : Dittrich J , Popara M , Kubiak J , Dimura M , Schepers B , Verma N , Schmitz B , Dollinger P , Kovacic F , Jaeger KE , Seidel CAM , Peulen TO , Gohlke H
Ref : J Chem Theory Comput , : , 2023
Abstract : Maximum entropy methods (MEMs) determine posterior distributions by combining experimental data with prior information. MEMs are frequently used to reconstruct conformational ensembles of molecular systems for experimental information and initial molecular ensembles. We performed time-resolved Forster resonance energy transfer (FRET) experiments to probe the interdye distance distributions of the lipase-specific foldase Lif in the apo state, which likely has highly flexible, disordered, and/or ordered structural elements. Distance distributions estimated from ensembles of molecular dynamics (MD) simulations serve as prior information, and FRET experiments, analyzed within a Bayesian framework to recover distance distributions, are used for optimization. We tested priors obtained by MD with different force fields (FFs) tailored to ordered (FF99SB, FF14SB, and FF19SB) and disordered proteins (IDPSFF and FF99SBdisp). We obtained five substantially different posterior ensembles. As in our FRET experiments the noise is characterized by photon counting statistics, for a validated dye model, MEM can quantify consistencies between experiment and prior or posterior ensembles. However, posterior populations of conformations are uncorrelated to structural similarities for individual structures selected from different prior ensembles. Therefore, we assessed MEM simulating varying priors in synthetic experiments with known target ensembles. We found that (i) the prior and experimental information must be carefully balanced for optimal posterior ensembles to minimize perturbations of populations by overfitting and (ii) only ensemble-integrated quantities like inter-residue distance distributions or density maps can be reliably obtained but not ensembles of atomistic structures. This is because MEM optimizes ensembles but not individual structures. This result for a highly flexible system suggests that structurally varying priors calculated from varying prior ensembles, e.g., generated with different FFs, may serve as an ad hoc estimate for MEM reconstruction robustness.
ESTHER : Dittrich_2023_J.Chem.Theory.Comput__
PubMedSearch : Dittrich_2023_J.Chem.Theory.Comput__
PubMedID: 37023001

Title : Analysis of protein secretion in Bacillus subtilis by combining a secretion stress biosensor strain with an in vivo split GFP assay - Lenz_2023_Microb.Cell.Fact_22_203
Author(s) : Lenz P , Bakkes PJ , Muller C , Malek M , Freudl R , Oldiges M , Drepper T , Jaeger KE , Knapp A
Ref : Microb Cell Fact , 22 :203 , 2023
Abstract : BACKGROUND: Bacillus subtilis is one of the workhorses in industrial biotechnology and well known for its secretion potential. Efficient secretion of recombinant proteins still requires extensive optimization campaigns and screening with activity-based methods. However, not every protein can be detected by activity-based screening. We therefore developed a combined online monitoring system, consisting of an in vivo split GFP assay for activity-independent target detection and an mCherry-based secretion stress biosensor. The split GFP assay is based on the fusion of a target protein to the eleventh beta-sheet of sfGFP, which can complement a truncated sfGFP that lacks this beta-sheet named GFP1-10. The secretion stress biosensor makes use of the CssRS two component quality control system, which upregulates expression of mCherry in the htrA locus thereby allowing a fluorescence readout of secretion stress. RESULTS: The biosensor strain B. subtilis PAL5 was successfully constructed by exchanging the protease encoding gene htrA with mCherry via CRISPR/Cas9. The Fusarium solani pisi cutinase Cut fused to the GFP11 tag (Cut11) was used as a model enzyme to determine the stress response upon secretion mediated by signal peptides SP(Pel), SP(Epr) and SP(Bsn) obtained from naturally secreted proteins of B. subtilis. An in vivo split GFP assay was developed, where purified GFP1-10 is added to the culture broth. By combining both methods, an activity-independent high-throughput method was created, that allowed optimization of Cut11 secretion. Using the split GFP-based detection assay, we demonstrated a good correlation between the amount of secreted cutinase and the enzymatic activity. Additionally, we screened a signal peptide library and identified new signal peptide variants that led to improved secretion while maintaining low stress levels. CONCLUSION: Our results demonstrate that the combination of a split GFP-based detection assay for secreted proteins with a secretion stress biosensor strain enables both, online detection of extracellular target proteins and identification of bottlenecks during protein secretion in B. subtilis. In general, the system described here will also enable to monitor the secretion stress response provoked by using inducible promoters governing the expression of different enzymes.
ESTHER : Lenz_2023_Microb.Cell.Fact_22_203
PubMedSearch : Lenz_2023_Microb.Cell.Fact_22_203
PubMedID: 37805580
Gene_locus related to this paper: fusso-cutas

Title : Accelerated strain construction and characterization of C. glutamicum protein secretion by laboratory automation - Muller_2022_Appl.Microbiol.Biotechnol_106_4481
Author(s) : Muller C , Bakkes PJ , Lenz P , Waffenschmidt V , Helleckes LM , Jaeger KE , Wiechert W , Knapp A , Freudl R , Oldiges M
Ref : Applied Microbiology & Biotechnology , 106 :4481 , 2022
Abstract : Secretion of bacterial proteins into the culture medium simplifies downstream processing by avoiding cell disruption for target protein purification. However, a suitable signal peptide for efficient secretion needs to be identified, and currently, there are no tools available to predict optimal combinations of signal peptides and target proteins. The selection of such a combination is influenced by several factors, including protein biosynthesis efficiency and cultivation conditions, which both can have a significant impact on secretion performance. As a result, a large number of combinations must be tested. Therefore, we have developed automated workflows allowing for targeted strain construction and secretion screening using two platforms. Key advantages of this experimental setup include lowered hands-on time and increased throughput. In this study, the automated workflows were established for the heterologous production of Fusarium solani f. sp. pisi cutinase in Corynebacterium glutamicum. The target protein was monitored in culture supernatants via enzymatic activity and split GFP assay. Varying spacer lengths between the Shine-Dalgarno sequence and the start codon of Bacillus subtilis signal peptides were tested. Consistent with previous work on the secretory cutinase production in B. subtilis, a ribosome binding site with extended spacer length to up to 12 nt, which likely slows down translation initiation, does not necessarily lead to poorer cutinase secretion by C. glutamicum. The best performing signal peptides for cutinase secretion with a standard spacer length were identified in a signal peptide screening. Additional insights into the secretion process were gained by monitoring secretion stress using the C. glutamicum K9 biosensor strain. KEY POINTS: Automated workflows for strain construction and screening of protein secretion Comparison of spacer, signal peptide, and host combinations for cutinase secretion Signal peptide screening for secretion by C. glutamicum using the split GFP assay.
ESTHER : Muller_2022_Appl.Microbiol.Biotechnol_106_4481
PubMedSearch : Muller_2022_Appl.Microbiol.Biotechnol_106_4481
PubMedID: 35759036

Title : Critical assessment of structure-based approaches to improve protein resistance in aqueous ionic liquids by enzyme-wide saturation mutagenesis - El Harrar_2022_Comput.Struct.Biotechnol.J_20_399
Author(s) : El Harrar T , Davari MD , Jaeger KE , Schwaneberg U , Gohlke H
Ref : Comput Struct Biotechnol J , 20 :399 , 2022
Abstract : Ionic liquids (IL) and aqueous ionic liquids (aIL) are attractive (co-)solvents for green industrial processes involving biocatalysts, but often reduce enzyme activity. Experimental and computational methods are applied to predict favorable substitution sites and, most often, subsequent site-directed surface charge modifications are introduced to enhance enzyme resistance towards aIL. However, almost no studies evaluate the prediction precision with random mutagenesis or the application of simple data-driven filtering processes. Here, we systematically and rigorously evaluated the performance of 22 previously described structure-based approaches to increase enzyme resistance to aIL based on an experimental complete site-saturation mutagenesis library of Bacillus subtilis Lipase A (BsLipA) screened against four aIL. We show that, surprisingly, most of the approaches yield low gain-in-precision (GiP) values, particularly for predicting relevant positions: 14 approaches perform worse than random mutagenesis. Encouragingly, exploiting experimental information on the thermostability of BsLipA or structural weak spots of BsLipA predicted by rigidity theory yields GiP = 3.03 and 2.39 for relevant variants and GiP = 1.61 and 1.41 for relevant positions. Combining five simple-to-compute physicochemical and evolutionary properties substantially increases the precision of predicting relevant variants and positions, yielding GiP = 3.35 and 1.29. Finally, combining these properties with predictions of structural weak spots identified by rigidity theory additionally improves GiP for relevant variants up to 4-fold to -10 and sustains or increases GiP for relevant positions, resulting in a prediction precision of -90% compared to -9% in random mutagenesis. This combination should be applicable to other enzyme systems for guiding protein engineering approaches towards improved aIL resistance.
ESTHER : El Harrar_2022_Comput.Struct.Biotechnol.J_20_399
PubMedSearch : El Harrar_2022_Comput.Struct.Biotechnol.J_20_399
PubMedID: 35070165

Title : The periplasmic chaperone Skp prevents misfolding of the secretory lipase A from Pseudomonas aeruginosa - Papadopoulos_2022_Front.Mol.Biosci_9_1026724
Author(s) : Papadopoulos A , Busch M , Reiners J , Hachani E , Baeumers M , Berger J , Schmitt L , Jaeger KE , Kovacic F , Smits SHJ , Kedrov A
Ref : Front Mol Biosci , 9 :1026724 , 2022
Abstract : Pseudomonas aeruginosa is a wide-spread opportunistic human pathogen and a high-risk factor for immunodeficient people and patients with cystic fibrosis. The extracellular lipase A belongs to the virulence factors of P. aeruginosa. Prior to the secretion, the lipase undergoes folding and activation by the periplasmic foldase LipH. At this stage, the enzyme is highly prone to aggregation in mild and high salt concentrations typical for the sputum of cystic fibrosis patients. Here, we demonstrate that the periplasmic chaperone Skp of P. aeruginosa efficiently prevents misfolding of the lipase A in vitro. In vivo experiments in P. aeruginosa show that the lipase secretion is nearly abolished in absence of the endogenous Skp. Small-angle X-ray scattering elucidates the trimeric architecture of P. aeruginosa Skp and identifies two primary conformations of the chaperone, a compact and a widely open. We describe two binding modes of Skp to the lipase, with affinities of 20 nM and 2 microM, which correspond to 1:1 and 1:2 stoichiometry of the lipase:Skp complex. Two Skp trimers are required to stabilize the lipase via the apolar interactions, which are not affected by elevated salt concentrations. We propose that Skp is a crucial chaperone along the lipase maturation and secretion pathway that ensures stabilization and carry-over of the client to LipH.
ESTHER : Papadopoulos_2022_Front.Mol.Biosci_9_1026724
PubMedSearch : Papadopoulos_2022_Front.Mol.Biosci_9_1026724
PubMedID: 36353734

Title : Polar substitutions on the surface of a lipase substantially improve tolerance in organic solvents - Davari_2022_ChemSusChem__
Author(s) : Davari MD , Cui H , Vedder M , Zhang L , Jaeger KE , Schwaneberg U
Ref : ChemSusChem , : , 2022
Abstract : Biocatalysis in organic solvents (OSs) enables more efficient routes to the synthesis of various valuable chemicals. However, OSs often reduce enzymatic activity which limits the use enzymes in OSs. Herein, we report a comprehensive understanding of interactions between surface polar substitutions and DMSO by integrating the molecular dynamics (MD) simulations of 45 variants from Bacillus subtilis lipase A (BSLA) and substitution landscape in “BSLA-SSM library. By systematically analyzing 39 structural-, solvation-, and interaction energy-based observables, we discovered hydration shell maintenance, DMSO reduction, and decreased local flexibility simultaneously govern the stability of polar variants in OS. Moreover, the fingerprints of 1644 polar-related variants in three OSs demonstrated substituting aromatic to polar residue(s) hold great potential to highly improve OSs resistance. Hence, surface polar engineering enable to be a powerful and general strategy for generating OS-tolerant lipases and other enzymes, thereby adapting the catalyst to the desired reaction and process with OSs.
ESTHER : Davari_2022_ChemSusChem__
PubMedSearch : Davari_2022_ChemSusChem__
PubMedID: 35007408
Gene_locus related to this paper: bacsu-lip

Title : Evidence for a bacterial Lands cycle phospholipase A: Structural and mechanistic insights into membrane phospholipid remodeling - Bleffert_2021_Biorxiv__
Author(s) : Bleffert F , Granzin J , Caliskan M , Schott-Verdugo SN , Siebers M , Thiele B , Rahme L , Felgner S , Dormann P , Gohlke H , Batra-Safferling R , Jaeger KE , Kovacic F
Ref : Biorxiv , : , 2021
Abstract : Cells steadily adapt their membrane glycerophospholipid (GPL) composition to changing environmental and developmental conditions. While the regulation of membrane homeostasis via GPL synthesis in bacteria has been studied in detail, the mechanisms underlying the controlled degradation of endogenous GPLs remain unknown. Thus far, the function of intracellular phospholipases A (PLAs) in GPL remodeling (Lands cycle) in bacteria is not clearly established. Here, we identified the first cytoplasmic membrane-bound phospholipase A 1 (PlaF) from Pseudomonas aeruginosa involved in the Lands cycle. PlaF is an important virulence factor, as the P. aeruginosa delta plaF mutant showed strongly attenuated virulence in Galleria mellonella and macrophages. We present a 2.0-A-resolution crystal structure of PlaF, the first structure that reveals homodimerization of a single-pass transmembrane (TM) full-length protein. PlaF dimerization, mediated solely through the intermolecular interactions of TM and juxtamembrane regions, inhibits its activity. A dimerization site and the catalytic sites are linked by an intricate ligand-mediated interaction network which likely explains the product (fatty acid) feedback inhibition observed with the purified PlaF protein. We used molecular dynamics simulations and configurational free energy computations to suggest a model of PlaF activation through a coupled monomerization and tilting of the monomer in the membrane, which constrains the active site cavity into contact with the GPL substrates. Thus, these data show the importance of the GPL remodeling pathway for virulence and pave the way for the development of a novel therapeutic class of antibiotics targeting PlaF-mediated membrane GPL remodeling. Synopsis Membrane homeostasis can be regulated by phospholipase-controlled deacylation of endogenous glycerophospholipids (GPLs) followed by reacylation of products, known as the Lands cycle in eukaryotes. Here we show that the human pathogen Pseudomonas aeruginosa uses intracellular phospholipase A 1 (PlaF) to modulate membrane GPL composition, which is the first example in bacteria. This newly identified PLA 1 indirectly regulates the bacterial virulence properties by hydrolyzing a specific set of membrane GPLs. The crystal structure of full-length PlaF dimers bound to natural ligands, MD simulations, and biochemical approaches provide insights into the molecular mechanism of dimerization-mediated inactivation of this single-pass transmembrane PLA 1 . Our findings shed light on a mechanism by which bacterial intracellular PLAs might regulate membrane homeostasis what showcases these enzymes as a promising target for a new class of antibiotics.
ESTHER : Bleffert_2021_Biorxiv__
PubMedSearch : Bleffert_2021_Biorxiv__
PubMedID:
Gene_locus related to this paper: pseae-PA2949

Title : Substrate access mechanism in a novel membrane-bound 1 phospholipase A of Pseudomonas aeruginosa concordant with specificity and regioselectivity - Ahmad_2021_Biorxiv__
Author(s) : Ahmad S , Strunk CH , Schott-Verdugo SN , Jaeger KE , Kovacic F , Gohlke H
Ref : Biorxiv , : , 2021
Abstract : PlaF is a cytoplasmic membrane-bound phospholipase A1 from Pseudomonas aeruginosa that alters the membrane glycerophospholipid (GPL) composition and fosters the virulence of this human pathogen. PlaF activity is regulated by a dimer-to-monomer transition followed by tilting of the monomer in the membrane. However, how substrates reach the active site and how the characteristics of the active site tunnels determine the activity, specificity, and regioselectivity of PlaF for natural GPL substrates has remained elusive. Here, we combined unbiased and biased all-atom molecular dynamics (MD) simulations and configurational free energy computations to identify access pathways of GPL substrates to the catalytic center of PlaF. Our results map out a distinct tunnel through which substrates access the catalytic center. PlaF variants with bulky tryptophan residues in this tunnel revealed decreased catalysis rates due to tunnel blockage. The MD simulations suggest that GPLs preferably enter the active site with the sn-1 acyl chain first, which agrees with the experimentally demonstrated PLA1 activity of PlaF. We propose that the acyl chain-length specificity of PlaF is determined by the structural features of the access tunnel, which results in favorable free energy of binding of medium-chain GPLs. The suggested egress route conveys fatty acid products to the dimerization interface and, thus, contributes to understanding the product feedback regulation of PlaF by fatty acid-triggered dimerization. These findings open up opportunities for developing potential PlaF inhibitors, which may act as antibiotics against P. aeruginosa.
ESTHER : Ahmad_2021_Biorxiv__
PubMedSearch : Ahmad_2021_Biorxiv__
PubMedID:
Gene_locus related to this paper: pseae-PA2949

Title : Crystal structures of a novel family IV esterase in free and substrate-bound form - Hoppner_2021_FEBS.J_288_3570
Author(s) : Hoppner A , Bollinger A , Kobus S , Thies S , Coscolin C , Ferrer M , Jaeger KE , Smits SHJ
Ref : Febs J , 288 :3570 , 2021
Abstract : Bacterial lipolytic enzymes of family IV are homologs of the mammalian hormone-sensitive lipases (HSL) and have been successfully used for various biotechnological applications. The broad substrate specificity and ability for enantio-, regio-, and stereoselective hydrolysis are remarkable features of enzymes from this class. Many crystal structures are available for esterases and lipases, but structures of enzyme-substrate or enzyme-inhibitor complexes are less frequent although important to understand the molecular basis of enzyme substrate interaction and to rationalize biochemical enzyme characteristics. Here, we report on the structures of a novel family IV esterase isolated from a metagenomic screen which shows a broad substrate specificity. We solved the crystal structures in the apo form and with a bound substrate analogue at 1.35 and 1.81 resolution, respectively. This enzyme named PtEst1 hydrolyzed more than 60 out 96 structurally different ester substrates thus being substrate promiscuous. Its broad substrate specificity is in accord with a large active site cavity, which is covered by an alpha-helical cap domain. The substrate analogue methyl 4-methylumbelliferyl hexylphosphonate was rapidly hydrolyzed by the enzyme leading to a complete inactivation caused by covalent binding of phosphinic acid to the catalytic serine. Interestingly, the alcohol leaving group 4-methylumbelliferone was found remaining in the active site cavity and additionally, a complete inhibitor molecule was found at the cap domain next to the entrance of the substrate tunnel. This unique situation allowed gaining valuable insights into the role of the cap domain for enzyme-substrate interaction of esterases belonging to family IV.
ESTHER : Hoppner_2021_FEBS.J_288_3570
PubMedSearch : Hoppner_2021_FEBS.J_288_3570
PubMedID: 33342083
Gene_locus related to this paper: pseth-a0a1m6y2k1

Title : Less unfavorable salt-bridges on the enzyme surface results in more organic cosolvent resistance - Cui_2021_Angew.Chem.Int.Ed.Engl__
Author(s) : Cui H , Eltoukhy L , Zhang L , Markel U , Jaeger KE , Davari MD , Schwaneberg U
Ref : Angew Chem Int Ed Engl , : , 2021
Abstract : Biocatalysis for the synthesis of fine chemicals is highly attractive but usually requires organic (co-)solvents (OSs). However, native enzymes often have low activity and resistance in OSs and at elevated temperatures. Herein, we report a smart salt bridge design strategy for simultaneously improving OS resistance and thermostability of the model enzyme, Bacillus subtilits Lipase A (BSLA). We combined comprehensive experimental studies of 3450 BSLA variants and molecular dynamics simulations of 36 systems. Iterative recombination of four beneficial substitutions yielded superior resistant variants with up to 7.6-fold (D64K/D144K) improved resistance toward three OSs while exhibiting significant thermostability (thermal resistance up to 137-fold, and half-life up to 3.3-fold). Molecular dynamics simulations revealed that locally refined flexibility and strengthened hydration jointly govern the highly increased resistance in OSs and at 50-100 degreesC. The salt bridge redesign provides protein engineers with a powerful and likely general approach to design OSs- and/or thermal-resistant lipases and other alpha/beta-hydrolases.
ESTHER : Cui_2021_Angew.Chem.Int.Ed.Engl__
PubMedSearch : Cui_2021_Angew.Chem.Int.Ed.Engl__
PubMedID: 33687787
Gene_locus related to this paper: bacsu-LIPB

Title : Biosensor-Based Optimization of Cutinase Secretion by Corynebacterium glutamicum - Bakkes_2021_Front.Microbiol_12_750150
Author(s) : Bakkes PJ , Lenz P , Muller C , Bida A , Dohmen-Olma D , Knapp A , Oldiges M , Jaeger KE , Freudl R
Ref : Front Microbiol , 12 :750150 , 2021
Abstract : The industrial microbe Corynebacterium glutamicum is gaining substantial importance as a platform host for recombinant protein secretion. We recently developed a fluorescence-based (eYFP) C. glutamicum reporter strain for the quantification of Sec-dependent protein secretion by monitoring the secretion-related stress response and now demonstrate its applicability in optimizing the secretion of the heterologous enzyme cutinase from Fusarium solani pisi. To drive secretion, either the poor-performing Pel(SP) or the potent NprE(SP) Sec signal peptide from Bacillus subtilis was used. To enable easy detection and quantification of the secreted cutinase we implemented the split green fluorescent protein (GFP) assay, which relies on the GFP11-tag fused to the C-terminus of the cutinase, which can complement a truncated GFP thereby reconstituting its fluorescence. The reporter strain was transformed with different mutant libraries created by error-prone PCR, which covered the region of the signal peptide and the N-terminus of the cutinase. Fluorescence-activated cell sorting (FACS) was performed to isolate cells that show increased fluorescence in response to increased protein secretion stress. Five Pel(SP) variants were identified that showed a 4- to 6-fold increase in the amount and activity of the secreted cutinase (up to 4,100 U/L), whereas two improved NprE(SP) variants were identified that showed a -35% increase in secretion, achieving -5,500 U/L. Most of the isolated variants carried mutations in the h-region of the signal peptide that increased its overall hydrophobicity. Using site-directed mutagenesis it was shown that the combined mutations F11I and P16S within the hydrophobic core of the Pel(SP) are sufficient to boost cutinase secretion in batch cultivations to the same level as achieved by the NprE(SP). Screening of a Pel(SP) mutant library in addition resulted in the identification of a cutinase variant with an increased specific activity, which was attributed to the mutation A85V located within the substrate-binding region. Taken together the biosensor-based optimization approach resulted in a substantial improvement of cutinase secretion by C. glutamicum, and therefore represents a valuable tool that can be applied to any secretory protein of interest.
ESTHER : Bakkes_2021_Front.Microbiol_12_750150
PubMedSearch : Bakkes_2021_Front.Microbiol_12_750150
PubMedID: 34777299

Title : Promiscuous Esterases Counterintuitively Are Less Flexible than Specific Ones - Nutschel_2021_J.Chem.Inf.Model__
Author(s) : Nutschel C , Coscolin C , David B , Mulnaes D , Ferrer M , Jaeger KE , Gohlke H
Ref : J Chem Inf Model , : , 2021
Abstract : Understanding mechanisms of promiscuity is increasingly important from a fundamental and application point of view. As to enzyme structural dynamics, more promiscuous enzymes generally have been recognized to also be more flexible. However, examples for the opposite received much less attention. Here, we exploit comprehensive experimental information on the substrate promiscuity of 147 esterases tested against 96 esters together with computationally efficient rigidity analyses to understand the molecular origin of the observed promiscuity range. Unexpectedly, our data reveal that promiscuous esterases are significantly less flexible than specific ones, are significantly more thermostable, and have a significantly increased specific activity. These results may be reconciled with a model according to which structural flexibility in the case of specific esterases serves for conformational proofreading. Our results signify that an esterase sequence space can be screened by rigidity analyses for promiscuous esterases as starting points for further exploration in biotechnology and synthetic chemistry.
ESTHER : Nutschel_2021_J.Chem.Inf.Model__
PubMedSearch : Nutschel_2021_J.Chem.Inf.Model__
PubMedID: 33949194

Title : CompassR Yields Highly Organic-Solvent-Tolerant Enzymes through Recombination of Compatible Substitutions - Cui_2021_Chemistry_27_2789
Author(s) : Cui H , Jaeger KE , Davari MD , Schwaneberg U
Ref : Chemistry , 27 :2789 , 2021
Abstract : The CompassR (computer-assisted recombination) rule enables, among beneficial substitutions, the identification of those that can be recombined in directed evolution. Herein, a recombination strategy is systematically investigated to minimize experimental efforts and maximize possible improvements. In total, 15 beneficial substitutions from Bacillus subtilis lipaseA (BSLA), which improves resistance to the organic cosolvent 1,4-dioxane (DOX), were studied to compare two recombination strategies, the two-gene recombination process (2GenReP) and the in silico guided recombination process (InSiReP), employing CompassR. Remarkably, both strategies yielded a highly DOX-resistant variant, M4 (I12R/Y49R/E65H/N98R/K122E/L124K), with up to 14.6-fold improvement after screening of about 270 clones. M4 has a remarkably enhanced resistance in 60% (v/v) acetone (6.0-fold), 30% (v/v) ethanol (2.1-fold), and 60% (v/v) methanol (2.4-fold) compared with wild-type BSLA. Molecular dynamics simulations revealed that attracting water molecules by charged surface substitutions is the main driver for increasing the DOX resistance of BSLA M4. Both strategies and obtained molecular knowledge can likely be used to improve the properties of other enzymes with a similar alpha/beta-hydrolase fold.
ESTHER : Cui_2021_Chemistry_27_2789
PubMedSearch : Cui_2021_Chemistry_27_2789
PubMedID: 33186477

Title : The length of ribosomal binding site spacer sequence controls the production yield for intracellular and secreted proteins by Bacillus subtilis - Volkenborn_2020_Microb.Cell.Fact_19_154
Author(s) : Volkenborn K , Kuschmierz L , Benz N , Lenz P , Knapp A , Jaeger KE
Ref : Microb Cell Fact , 19 :154 , 2020
Abstract : BACKGROUND: Bacillus subtilis is widely used for the industrial production of recombinant proteins, mainly due to its high secretion capacity, but higher production yields can be achieved only if bottlenecks are removed. To this end, a crucial process is translation initiation which takes place at the ribosome binding site enclosing the Shine Dalgarno sequence, the start codon of the target gene and a short spacer sequence in between. Here, we have studied the effects of varying spacer sequence lengths in vivo on the production yield of different intra- and extracellular proteins. RESULTS: The shuttle vector pBSMul1 containing the strong constitutive promoter P(HpaII) and the optimal Shine Dalgarno sequence TAAGGAGG was used as a template to construct a series of vectors with spacer lengths varying from 4 to 12 adenosines. For the intracellular proteins GFPmut3 and beta-glucuronidase, an increase of spacer lengths from 4 to 7-9 nucleotides resulted in a gradual increase of product yields up to 27-fold reaching a plateau for even longer spacers. The production of secreted proteins was tested with cutinase Cut and swollenin EXLX1 which were N-terminally fused to one of the Sec-dependent signal peptides SPPel, SPEpr or SPBsn. Again, longer spacer sequences resulted in up to tenfold increased yields of extracellular proteins. Fusions with signal peptides SPPel or SPBsn revealed the highest production yields with spacers of 7-10nt length. Remarkably, fusions with SPEpr resulted in a twofold lower production yield with 6 or 7nt spacers reaching a maximum with 10-12nt spacers. This pattern was observed for both secreted proteins fused to SPEpr indicating a dominant role also of the nucleotide sequence encoding the respective signal peptide for translation initiation. This conclusion was corroborated by RT qPCR revealing only slightly different amounts of transcript. Also, the effect of a putative alternative translation initiation site could be ruled out. CONCLUSION: Our results confirm the importance of the 5' end sequence of a target gene for translation initiation. Optimizing production yields thus may require screenings for optimal spacer sequence lengths. In case of secreted proteins, the 5' sequence encoding the signal peptide for Sec-depended secretion should also be considered.
ESTHER : Volkenborn_2020_Microb.Cell.Fact_19_154
PubMedSearch : Volkenborn_2020_Microb.Cell.Fact_19_154
PubMedID: 32727460

Title : Agar plate-based screening methods for the identification of polyester hydrolysis by Pseudomonas species - Molitor_2020_Microb.Biotechnol_13_274
Author(s) : Molitor R , Bollinger A , Kubicki S , Loeschcke A , Jaeger KE , Thies S
Ref : Microb Biotechnol , 13 :274 , 2020
Abstract : Hydrolases acting on polyesters like cutin, polycaprolactone or polyethylene terephthalate (PET) are of interest for several biotechnological applications like waste treatment, biocatalysis and sustainable polymer modifications. Recent studies suggest that a large variety of such enzymes are still to be identified and explored in a variety of microorganisms, including bacteria of the genus Pseudomonas. For activity-based screening, methods have been established using agar plates which contain nanoparticles of polycaprolactone or PET prepared by solvent precipitation and evaporation. In this protocol article, we describe a straightforward agar plate-based method using emulsifiable artificial polyesters as substrates, namely Impranil((R)) DLN and liquid polycaprolactone diol (PLD). Thereby, the currently quite narrow set of screening substrates is expanded. We also suggest optional pre-screening with short-chain and middle-chain-length triglycerides as substrates to identify enzymes with lipolytic activity to be further tested for polyesterase activity. We applied these assays to experimentally demonstrate polyesterase activity in bacteria from the P. pertucinogena lineage originating from contaminated soils and diverse marine habitats.
ESTHER : Molitor_2020_Microb.Biotechnol_13_274
PubMedSearch : Molitor_2020_Microb.Biotechnol_13_274
PubMedID: 31016871

Title : A Novel Polyester Hydrolase From the Marine Bacterium Pseudomonas aestusnigri - Structural and Functional Insights - Bollinger_2020_Front.Microbiol_11_114
Author(s) : Bollinger A , Thies S , Knieps-Grunhagen E , Gertzen C , Kobus S , Hoppner A , Ferrer M , Gohlke H , Smits SHJ , Jaeger KE
Ref : Front Microbiol , 11 :114 , 2020
Abstract : Biodegradation of synthetic polymers, in particular polyethylene terephthalate (PET), is of great importance, since environmental pollution with PET and other plastics has become a severe global problem. Here, we report on the polyester degrading ability of a novel carboxylic ester hydrolase identified in the genome of the marine hydrocarbonoclastic bacterium Pseudomonas aestusnigri VGXO14T. The enzyme, designated PE-H, belongs to the type IIa family of PET hydrolytic enzymes as indicated by amino acid sequence homology. It was produced in Escherichia coli, purified and its crystal structure was solved at 1.09 A resolution representing the first structure of a type IIa PET hydrolytic enzyme. The structure shows a typical alpha/beta-hydrolase fold and high structural homology to known polyester hydrolases. PET hydrolysis was detected at 30C with amorphous PET film (PETa), but not with PET film from a commercial PET bottle (PETb). A rational mutagenesis study to improve the PET degrading potential of PE-H yielded variant PE-H (Y250S) which showed improved activity, ultimately also allowing the hydrolysis of PETb. The crystal structure of this variant solved at 1.35 A resolution allowed to rationalize the improvement of enzymatic activity. A PET oligomer binding model was proposed by molecular docking computations. Our results indicate a significant potential of the marine bacterium P. aestusnigri for PET degradation.
ESTHER : Bollinger_2020_Front.Microbiol_11_114
PubMedSearch : Bollinger_2020_Front.Microbiol_11_114
PubMedID: 32117139
Gene_locus related to this paper: 9psed-peh

Title : Organic-Solvent-Tolerant Carboxylic Ester Hydrolases for Organic Synthesis - Bollinger_2020_Appl.Environ.Microbiol_86_e00106
Author(s) : Bollinger A , Molitor R , Thies S , Koch R , Coscolin C , Ferrer M , Jaeger KE
Ref : Applied Environmental Microbiology , 86 :e00106 , 2020
Abstract : Biocatalysis has emerged as an important tool in synthetic organic chemistry enabling the chemical industry to execute reactions with high regio- or enantioselectivity and under usually mild reaction conditions while avoiding toxic waste. Target substrates and products of reactions catalyzed by carboxylic ester hydrolases are often poorly water soluble and require organic solvents, whereas enzymes are evolved by nature to be active in cells, i.e., in aqueous rather than organic solvents. Therefore, biocatalysts that withstand organic solvents are urgently needed. Current strategies to identify such enzymes rely on laborious tests carried out by incubation in different organic solvents and determination of residual activity. Here, we describe a simple assay useful for screening large libraries of carboxylic ester hydrolases for resistance and activity in water-miscible organic solvents. We have screened a set of 26 enzymes, most of them identified in this study, with four different water-miscible organic solvents. The triglyceride tributyrin was used as a substrate, and fatty acids released by enzymatic hydrolysis were detected by a pH shift indicated by the indicator dye nitrazine yellow. With this strategy, we succeeded in identifying a novel highly organic-solvent-tolerant esterase from Pseudomonas aestusnigri In addition, the newly identified enzymes were tested with sterically demanding substrates, which are common in pharmaceutical intermediates, and two enzymes from Alcanivorax borkumensis were identified which outcompeted the gold standard ester hydrolase CalB from Candida antarctica IMPORTANCE Major challenges hampering biotechnological applications of esterases include the requirement to accept nonnatural and chemically demanding substrates and the tolerance of the enzymes toward organic solvents which are often required to solubilize such substrates. We describe here a high-throughput screening strategy to identify novel organic-solvent-tolerant carboxylic ester hydrolases (CEs). Among these enzymes, CEs active against water-insoluble bulky substrates were identified. Our results thus contribute to fostering the identification and biotechnological application of CEs.
ESTHER : Bollinger_2020_Appl.Environ.Microbiol_86_e00106
PubMedSearch : Bollinger_2020_Appl.Environ.Microbiol_86_e00106
PubMedID: 32111588
Gene_locus related to this paper: 9psed-peh , alcbs-q0vt77 , alcbs-q0vtl7 , aneth-d3xb96 , alcbs-q0vl36 , alcbs-q0vq49 , 9psed-CE24 , 9psed-CE23 , 9psed-CE22 , 9psed-CE20 , 9psed-CE18 , 9psed-CE15 , 9psed-CE13 , alcbs-q0vmp2 , alcbs-q0vlp6 , marav-a1u5n0 , alcbs-q0vlk5 , 9psed-a0a1h5udv9

Title : The Membrane-Integrated Steric Chaperone Lif Facilitates Active Site Opening of Pseudomonas aeruginosa Lipase A - Verma_2020_J.Comput.Chem_41_500
Author(s) : Verma N , Dollinger P , Kovacic F , Jaeger KE , Gohlke H
Ref : J Comput Chem , 41 :500 , 2020
Abstract : Lipases are essential and widely used biocatalysts. Hence, the production of lipases requires a detailed understanding of the molecular mechanism of its folding and secretion. Lipase A from Pseudomonas aeruginosa, PaLipA, constitutes a prominent example that has additional relevance because of its role as a virulence factor in many diseases. PaLipA requires the assistance of a membrane-integrated steric chaperone, the lipase-specific foldase Lif, to achieve its enzymatically active state. However, the molecular mechanism of how Lif activates its cognate lipase has remained elusive. Here, we show by molecular dynamics simulations at the atomistic level and potential of mean force computations that Lif catalyzes the activation process of PaLipA by structurally stabilizing an intermediate PaLipA conformation, particularly a beta-sheet in the region of residues 17-30, such that the opening of PaLipA's lid domain is facilitated. This opening allows substrate access to PaLipA's catalytic site. A surprising and so far not fully understood aspect of our study is that the open state of PaLipA is unstable compared to the closed one according to our computational and in vitro biochemical results. We thus speculate that further interactions of PaLipA with the Xcp secretion machinery and/or components of the extracellular matrix contribute to the remaining activity of secreted PaLipA. (c) 2019 Wiley Periodicals, Inc.
ESTHER : Verma_2020_J.Comput.Chem_41_500
PubMedSearch : Verma_2020_J.Comput.Chem_41_500
PubMedID: 31618459
Gene_locus related to this paper: pseae-llipa

Title : Structural and dynamic insights revealing how lipase binding domain MD1 of Pseudomonas aeruginosa foldase affects lipase activation - Viegas_2020_Sci.Rep_10_3578
Author(s) : Viegas A , Dollinger P , Verma N , Kubiak J , Viennet T , Seidel CAM , Gohlke H , Etzkorn M , Kovacic F , Jaeger KE
Ref : Sci Rep , 10 :3578 , 2020
Abstract : Folding and cellular localization of many proteins of Gram-negative bacteria rely on a network of chaperones and secretion systems. Among them is the lipase-specific foldase Lif, a membrane-bound steric chaperone that tightly binds (K(D) = 29 nM) and mediates folding of the lipase LipA, a virulence factor of the pathogenic bacterium P. aeruginosa. Lif consists of five-domains, including a mini domain MD1 essential for LipA folding. However, the molecular mechanism of Lif-assisted LipA folding remains elusive. Here, we show in in vitro experiments using a soluble form of Lif (sLif) that isolated MD1 inhibits sLif-assisted LipA activation. Furthermore, the ability to activate LipA is lost in the variant sLif(Y99A), in which the evolutionary conserved amino acid Y99 from helix alpha1 of MD1 is mutated to alanine. This coincides with an approximately three-fold reduced affinity of the variant to LipA together with increased flexibility of sLif(Y99A) in the complex as determined by polarization-resolved fluorescence spectroscopy. We have solved the NMR solution structures of P. aeruginosa MD1 and variant MD1(Y99A) revealing a similar fold indicating that a structural modification is likely not the reason for the impaired activity of variant sLif(Y99A). Molecular dynamics simulations of the sLif:LipA complex in connection with rigidity analyses suggest a long-range network of interactions spanning from Y99 of sLif to the active site of LipA, which might be essential for LipA activation. These findings provide important details about the putative mechanism for LipA activation and point to a general mechanism of protein folding by multi-domain steric chaperones.
ESTHER : Viegas_2020_Sci.Rep_10_3578
PubMedSearch : Viegas_2020_Sci.Rep_10_3578
PubMedID: 32107397
Gene_locus related to this paper: pseae-llipa

Title : The biotechnological potential of marine bacteria in the novel lineage of Pseudomonas pertucinogena - Bollinger_2020_Microb.Biotechnol_13_19
Author(s) : Bollinger A , Thies S , Katzke N , Jaeger KE
Ref : Microb Biotechnol , 13 :19 , 2020
Abstract : Marine habitats represent a prolific source for molecules of biotechnological interest. In particular, marine bacteria have attracted attention and were successfully exploited for industrial applications. Recently, a group of Pseudomonas species isolated from extreme habitats or living in association with algae or sponges were clustered in the newly established Pseudomonas pertucinogena lineage. Remarkably for the predominantly terrestrial genus Pseudomonas, more than half (9) of currently 16 species within this lineage were isolated from marine or saline habitats. Unlike other Pseudomonas species, they seem to have in common a highly specialized metabolism. Furthermore, the marine members apparently possess the capacity to produce biomolecules of biotechnological interest (e.g. dehalogenases, polyester hydrolases, transaminases). Here, we summarize the knowledge regarding the enzymatic endowment of the marine Pseudomonas pertucinogena bacteria and report on a genomic analysis focusing on the presence of genes encoding esterases, dehalogenases, transaminases and secondary metabolites including carbon storage compounds.
ESTHER : Bollinger_2020_Microb.Biotechnol_13_19
PubMedSearch : Bollinger_2020_Microb.Biotechnol_13_19
PubMedID: 29943398

Title : Classification of Lipolytic Enzymes from Bacteria - Kovacic_2019_Handb.Hydrocarbon.Lipid.Microbiol__255
Author(s) : Kovacic F , Babic N , Krauss U , Jaeger KE
Ref : Handbook of Hydrocarbon and Lipid Microbiology , Aerobic Utilization of Hydrocarbons, Oils, and Lipids :255 , 2019
Abstract : Lipolytic enzymes including lipases and esterases comprise a versatile group of enzymes with diverse amino acid sequences but related three-dimensional structures. Despite the large number of bacterial lipolytic enzymes so far identified (~5000), only a small portion (<10%) was cloned, expressed and experimentally studied. Twenty years ago, Arpigny and Jaeger published a seminal study which systematically grouped bacterial lipolytic enzymes into eight families according to similarity of their amino acid sequences and physiological properties (Arpigny and Jaeger 1999). Here, we present a comprehensive overview as an extension of the original Arpigny and Jaeger classification covering all nineteen presently known families of lipolytic enzymes. The conserved features of sequences and structures are described for all families in order to simplify the assignment of newly discovered lipolytic enzymes to the respective family. Furthermore, we have correlated the biochemical properties of some enzymes with the nature of the often extremophilic microorganism from which the respective enzyme was isolated. This may help to identify lipases families with potential as biocatalysts in industrial applications. As an example, family XV enzymes are stable and active at elevated temperatures, thus, enzymes of this family represent a potential source for novel biocatalysts
ESTHER : Kovacic_2019_Handb.Hydrocarbon.Lipid.Microbiol__255
PubMedSearch : Kovacic_2019_Handb.Hydrocarbon.Lipid.Microbiol__255
PubMedID:
Gene_locus related to this paper: psywf-a5wgv1

Title : Pseudomonas aeruginosa esterase PA2949, a bacterial homolog of the human membrane esterase ABHD6: expression, purification and crystallization - Bleffert_2019_Acta.Crystallogr.F.Struct.Biol.Commun_75_270
Author(s) : Bleffert F , Granzin J , Gohlke H , Batra-Safferling R , Jaeger KE , Kovacic F
Ref : Acta Crystallographica F Struct Biol Commun , 75 :270 , 2019
Abstract : The human membrane-bound alpha/beta-hydrolase domain 6 (ABHD6) protein modulates endocannabinoid signaling, which controls appetite, pain and learning, as well as being linked to Alzheimer's and Parkinson's diseases, through the degradation of the key lipid messenger 2-arachidonylglycerol (2-AG). This makes ABHD6 an attractive therapeutic target that lacks structural information. In order to better understand the molecular mechanism of 2-AG-hydrolyzing enzymes, the PA2949 protein from Pseudomonas aeruginosa, which has 49% sequence similarity to the ABHD6 protein, was cloned, overexpressed, purified and crystallized. Overexpression of PA2949 in the homologous host yielded the membrane-bound enzyme, which was purified in milligram amounts. Besides their sequence similarity, the enzymes both show specificity for the hydrolysis of 2-AG and esters of medium-length fatty acids. PA2949 in the presence of n-octyl beta-D-glucoside showed a higher activity and stability at room temperature than those previously reported for PA2949 overexpressed and purified from Escherichia coli. A suitable expression host and stabilizing detergent were crucial for obtaining crystals, which belonged to the tetragonal space group I4122 and diffracted to a resolution of 2.54 A. This study provides hints on the functional similarity of ABHD6-like proteins in prokaryotes and eukaryotes, and might guide the structural study of these difficult-to-crystallize proteins.
ESTHER : Bleffert_2019_Acta.Crystallogr.F.Struct.Biol.Commun_75_270
PubMedSearch : Bleffert_2019_Acta.Crystallogr.F.Struct.Biol.Commun_75_270
PubMedID: 30950828
Gene_locus related to this paper: pseae-PA2949

Title : Interaction of carbohydrate-binding modules with poly(ethylene terephthalate) - Weber_2019_Appl.Microbiol.Biotechnol_103_4801
Author(s) : Weber J , Petrovic D , Strodel B , Smits SHJ , Kolkenbrock S , Leggewie C , Jaeger KE
Ref : Applied Microbiology & Biotechnology , 103 :4801 , 2019
Abstract : Poly(ethylene terephthalate) (PET) is one of the most widely applied synthetic polymers, but its hydrophobicity is challenging for many industrial applications. Biotechnological modification of PET surface can be achieved by PET hydrolyzing cutinases. In order to increase the adsorption towards their unnatural substrate, the enzymes are fused to carbohydrate-binding modules (CBMs) leading to enhanced activity. In this study, we identified novel PET binding CBMs and characterized the CBM-PET interplay. We developed a semi-quantitative method to detect CBMs bound to PET films. Screening of eight CBMs from diverse families for PET binding revealed one CBM that possesses a high affinity towards PET. Molecular dynamics (MD) simulations of the CBM-PET interface revealed tryptophan residues forming an aromatic triad on the peptide surface. Their interaction with phenyl rings of PET is stabilized by additional hydrogen bonds formed between amino acids close to the aromatic triad. Furthermore, the ratio of hydrophobic to polar contacts at the interface was identified as an important feature determining the strength of PET binding of CBMs. The interaction of CBM tryptophan residues with PET was confirmed experimentally by tryptophan quenching measurements after addition of PET nanoparticles to CBM. Our findings are useful for engineering PET hydrolyzing enzymes and may also find applications in functionalization of PET.
ESTHER : Weber_2019_Appl.Microbiol.Biotechnol_103_4801
PubMedSearch : Weber_2019_Appl.Microbiol.Biotechnol_103_4801
PubMedID: 30993383

Title : Marine Biosurfactants: Biosynthesis, Structural Diversity and Biotechnological Applications - Kubicki_2019_Mar.Drugs_17_
Author(s) : Kubicki S , Bollinger A , Katzke N , Jaeger KE , Loeschcke A , Thies S
Ref : Mar Drugs , 17 : , 2019
Abstract : Biosurfactants are amphiphilic secondary metabolites produced by microorganisms. Marine bacteria have recently emerged as a rich source for these natural products which exhibit surface-active properties, making them useful for diverse applications such as detergents, wetting and foaming agents, solubilisers, emulsifiers and dispersants. Although precise structural data are often lacking, the already available information deduced from biochemical analyses and genome sequences of marine microbes indicates a high structural diversity including a broad spectrum of fatty acid derivatives, lipoamino acids, lipopeptides and glycolipids. This review aims to summarise biosyntheses and structures with an emphasis on low molecular weight biosurfactants produced by marine microorganisms and describes various biotechnological applications with special emphasis on their role in the bioremediation of oil-contaminated environments. Furthermore, novel exploitation strategies are suggested in an attempt to extend the existing biosurfactant portfolio.
ESTHER : Kubicki_2019_Mar.Drugs_17_
PubMedSearch : Kubicki_2019_Mar.Drugs_17_
PubMedID: 31323998

Title : Determinants and prediction of esterase substrate promiscuity patterns - Martinez-Martinez_2018_ACS.Chem.Biol_13_225
Author(s) : Martinez-Martinez M , Coscolin C , Santiago G , Chow J , Stogios PJ , Bargiela R , Gertler C , Navarro-Fernandez J , Bollinger A , Thies S , Mendez-Garcia C , Popovic A , Brown G , Chernikova TN , Garcia-Moyano A , Bjergah GE , Perez-Garcia P , Hai T , Del Pozo MV , Stokke R , Steen IH , Cui H , Xu X , Nocek BP , Alcaide M , Distaso M , Mesa V , Pelaez AI , Sanchez J , Buchholz PCF , Pleiss J , Fernandez-Guerra A , Glockner FO , Golyshina OV , Yakimov MM , Savchenko A , Jaeger KE , Yakunin AF , Streit WR , Golyshin PN , Guallar V , Ferrer M
Ref : ACS Chemical Biology , 13 :225 , 2018
Abstract : Esterases receive special attention because their wide distribution in biological systems and environments and their importance for physiology and chemical synthesis. The prediction of esterases substrate promiscuity level from sequence data and the molecular reasons why certain such enzymes are more promiscuous than others, remain to be elucidated. This limits the surveillance of the sequence space for esterases potentially leading to new versatile biocatalysts and new insights into their role in cellular function. Here we performed an extensive analysis of the substrate spectra of 145 phylogenetically and environmentally diverse microbial esterases, when tested with 96 diverse esters. We determined the primary factors shaping their substrate range by analyzing substrate range patterns in combination with structural analysis and protein-ligand simulations. We found a structural parameter that helps ranking (classifying) promiscuity level of esterases from sequence data at 94% accuracy. This parameter, the active site effective volume, exemplifies the topology of the catalytic environment by measuring the active site cavity volume corrected by the relative solvent accessible surface area (SASA) of the catalytic triad. Sequences encoding esterases with active site effective volumes (cavity volume/SASA) above a threshold show greater substrate spectra, which can be further extended in combination with phylogenetic data. This measure provides also a valuable tool for interrogating substrates capable of being converted. This measure, found to be transferred to phosphatases of the haloalkanoic acid dehalogenase superfamily and possibly other enzymatic systems, represents a powerful tool for low-cost bioprospecting for esterases with broad substrate ranges, in large scale sequence datasets.
ESTHER : Martinez-Martinez_2018_ACS.Chem.Biol_13_225
PubMedSearch : Martinez-Martinez_2018_ACS.Chem.Biol_13_225
PubMedID: 29182315
Gene_locus related to this paper: 9zzzz-a0a2k8jn75 , 9zzzz-a0a2k8jt94 , 9zzzz-a0a0g3fj44 , 9zzzz-a0a0g3fh10 , 9zzzz-a0a0g3fh03 , 9bact-a0a1s5qkj8 , 9zzzz-a0a0g3feh5 , 9zzzz-a0a0g3fkz4 , 9zzzz-a0a0g3fh07 , 9zzzz-a0a0g3fh34 , 9zzzz-a0a0g3fh31 , 9bact-KY458167 , alcbs-q0vqa3 , 9bact-a0a1s5qki8 , 9zzzz-a0a0g3feq8 , 9zzzz-a0a0g3feh8 , 9zzzz-a0a0g3fh19 , 9bact-KY203037 , 9bact-a0a1s5ql22 , 9bact-a0a1s5qm34 , 9bact-KY203034 , 9bact-r9qzg0 , 9bact-a0a1s5qly8 , 9zzzz-a0a0g3fkz8 , 9zzzz-a0a0g3feg9 , 9zzzz-KY203033 , 9zzzz-a0a0g3fes4 , 9zzzz-a0a0g3fh42 , 9bact-a0a1s5qlx2 , 9zzzz-KY483651 , 9bact-a0a1s5qmh4 , 9zzzz-KY203032 , 9zzzz-EH87 , 9zzzz-a0a0g3fei1 , 9zzzz-a0a0g3fet2 , 9zzzz-KY483647 , 9zzzz-EH82 , 9zzzz-a0a0g3fe15 , 9bact-KY203031 , 9bact-t1w006 , 9zzzz-a0a0g3fet6 , 9bact-KY458164 , geoth-g8myf3 , 9bact-a0a1s5ql04 , 9gamm-a0a1y0ihk7 , 9bact-a0a1s5qly6 , 9bact-a0a1s5qkg4 , 9bact-a0a1s5qkm4 , 9gamm-s5tv80 , 9gamm-a0a0c4zhg2 , 9zzzz-t1b379 , 9gamm-KY483646 , 9bact-KY458160 , 9zzzz-a0a0g3fj57 , 9gamm-s5t8349 , 9arch-KY203036 , 9bact-KY458168 , 9zzzz-a0a0g3fes0 , 9zzzz-t1be47 , 9bact-KY458159 , 9zzzz-a0a0g3fh39 , 9bact-t1vzd5 , 9prot-EH41 , 9bact-Lip114 , alcbs-q0vt77 , 9bact-a0a1s5qke6 , 9bact-a0a1s5qkf3 , 9prot-SRP030024 , 9gamm-s5t532 , 9bact-a0a1s5qkl2 , 9bact-a0a1s5qkk8 , 9zzzz-KY203030 , 9zzzz-t1d4I7 , 9prot-KY019260 , 9bact-a0a1s5qm38 , 9arch-KY458161 , 9prot-KY010302 , 9zzzz-a0a0g3fl25 , 9actn-KY010298 , 9gamm-s5u059 , 9bact-a0a1s5qmi7 , 9bact-KY010297 , 9bact-KY483642 , 9bact-a0a1s5qkj1 , 9bact-KY010299 , 9bact-KY483648 , alcbs-q0vtl7 , 9bact-a0a1s5qf1 , 9bact-a0a1s5qkg0 , 9bact-a0a0h4tgu6 , 9bact-MilE3 , 9bact-LAE6 , 9alte-MGS-MT1 , 9bact-r9qzf7 , 9gamm-k0c6t6 , alcbs-q0vl36 , alcbs-q0vlq1 , alcbs-q0vq49 , bacsu-pnbae , canar-LipB , canan-lipasA , geost-lipas , marav-a1u5n0 , pseps-i7k8x5 , staep-GEHD , symth-q67mr3 , altma-s5cfn7 , cycsp-k0c2b8 , alcbs-q0vlk5 , 9bact-k7qe48 , 9bact-MGS-M1 , 9bact-MGS-M2 , 9bact-a0a0b5kns5 , 9zzzz-a0a0g3fej4 , 9zzzz-a0a0g3fj60 , 9zzzz-a0a0g3fej0 , 9zzzz-a0a0g3fj64 , 9bact-a0a0b5kc16 , 9zzzz-a0a0g3feg6 , 9zzzz-a0a0g3feu6

Title : First Insights into the Genome Sequence of Pseudomonas oleovorans DSM 1045 - Poehlein_2017_Genome.Announc_5_
Author(s) : Poehlein A , Daniel R , Thurmer A , Bollinger A , Thies S , Katzke N , Jaeger KE
Ref : Genome Announc , 5 : , 2017
Abstract : The Gram-negative proteobacterium Pseudomonas oleovorans DSM 1045 is considered a promising source for enzymes of biotechnological interest, e.g., hydrolases and transaminases. Here, we present a draft sequence of its 4.86-Mb genome, enabling the identification of novel biocatalysts.
ESTHER : Poehlein_2017_Genome.Announc_5_
PubMedSearch : Poehlein_2017_Genome.Announc_5_
PubMedID: 28798180

Title : Activity-independent screening of secreted proteins using split GFP - Knapp_2017_J.Biotechnol_258_110
Author(s) : Knapp A , Ripphahn M , Volkenborn K , Skoczinski P , Jaeger KE
Ref : J Biotechnol , 258 :110 , 2017
Abstract : The large-scale industrial production of proteins requires efficient secretion, as provided, for instance, by the Sec system of Gram-positive bacteria. Protein engineering approaches to optimize secretion often involve the screening of large libraries, e.g. comprising a target protein fused to many different signal peptides. Respective high-throughput screening methods are usually based on photometric or fluorimetric assays enabling fast and simple determination of enzymatic activities. Here, we report on an alternative method for quantification of secreted proteins based on the split GFP assay. We analyzed the secretion by Bacillus subtilis of a homologous lipase and a heterologous cutinase by determination of GFP fluorescence and enzyme activity assays. Furthermore, we identified from a signal peptide library a variant of the biotechnologically relevant B. subtilis protein swollenin EXLX1 with up to 5-fold increased secretion. Our results demonstrate that the split GFP assay can be used to monitor secretion of enzymatic and non-enzymatic proteins in B. subtilis in a high-throughput manner.
ESTHER : Knapp_2017_J.Biotechnol_258_110
PubMedSearch : Knapp_2017_J.Biotechnol_258_110
PubMedID: 28619616

Title : A combination of mutational and computational scanning guides the design of an artificial ligand-binding controlled lipase - Kaschner_2017_Sci.Rep_7_42592
Author(s) : Kaschner M , Schillinger O , Fettweiss T , Nutschel C , Krause F , Fulton A , Strodel B , Stadler A , Jaeger KE , Krauss U
Ref : Sci Rep , 7 :42592 , 2017
Abstract : Allostery, i.e. the control of enzyme activity by a small molecule at a location distant from the enzyme's active site, represents a mechanism essential for sustaining life. The rational design of allostery is a non-trivial task but can be achieved by fusion of a sensory domain, which responds to environmental stimuli with a change in its structure. Hereby, the site of domain fusion is difficult to predict. We here explore the possibility to rationally engineer allostery into the naturally not allosterically regulated Bacillus subtilis lipase A, by fusion of the citrate-binding sensor-domain of the CitA sensory-kinase of Klebsiella pneumoniae. The site of domain fusion was rationally determined based on whole-protein site-saturation mutagenesis data, complemented by computational evolutionary-coupling analyses. Functional assays, combined with biochemical and biophysical studies suggest a mechanism for control, similar but distinct to the one of the parent CitA protein, with citrate acting as an indirect modulator of Triton-X100 inhibition of the fusion protein. Our study demonstrates that the introduction of ligand-dependent regulatory control by domain fusion is surprisingly facile, suggesting that the catalytic mechanism of some enzymes may be evolutionary optimized in a way that it can easily be perturbed by small conformational changes.
ESTHER : Kaschner_2017_Sci.Rep_7_42592
PubMedSearch : Kaschner_2017_Sci.Rep_7_42592
PubMedID: 28218303

Title : A membrane-bound esterase PA2949 from Pseudomonas aeruginosa is expressed and purified from Escherichia coli - Kovacic_2016_FEBS.Open.Bio_6_484
Author(s) : Kovacic F , Bleffert F , Caliskan M , Wilhelm S , Granzin J , Batra-Safferling R , Jaeger KE
Ref : FEBS Open Bio , 6 :484 , 2016
Abstract : Pseudomonas aeruginosa strain 1001 produces an esterase (EstA) that can hydrolyse the racemic methyl ester of beta-acetylthioisobutyrate to produce the (D)-enantiomer, which serves as a precursor of captopril, a drug used for treatment of hypertension. We show here that PA2949 from P. aeruginosa PA01, a homologue of EstA, can efficiently be expressed in an enzymatically active form in E. coli. The enzyme is membrane-associated as demonstrated by cell fractionation studies. PA2949 was purified to homogeneity after solubilisation with the nonionic detergent, Triton X-100, and was shown to possess a conserved esterase catalytic triad consisting of Ser137-His258-Asp286. Our results should allow the development of an expression and purification strategy to produce this biotechnologically relevant esterase in a pure form with a high yield.
ESTHER : Kovacic_2016_FEBS.Open.Bio_6_484
PubMedSearch : Kovacic_2016_FEBS.Open.Bio_6_484
PubMedID: 27419054
Gene_locus related to this paper: pseae-PA2949

Title : Structural features determining thermal adaptation of esterases - Kovacic_2016_Protein.Eng.Des.Sel_29_65
Author(s) : Kovacic F , Mandrysch A , Poojari C , Strodel B , Jaeger KE
Ref : Protein Engineering Des Sel , 29 :65 , 2016
Abstract : The adaptation of microorganisms to extreme living temperatures requires the evolution of enzymes with a high catalytic efficiency under these conditions. Such extremophilic enzymes represent valuable tools to study the relationship between protein stability, dynamics and function. Nevertheless, the multiple effects of temperature on the structure and function of enzymes are still poorly understood at the molecular level. Our analysis of four homologous esterases isolated from bacteria living at temperatures ranging from 10 degrees C to 70 degrees C suggested an adaptation route for the modulation of protein thermal properties through the optimization of local flexibility at the protein surface. While the biochemical properties of the recombinant esterases are conserved, their thermal properties have evolved to resemble those of the respective bacterial habitats. Molecular dynamics simulations at temperatures around the optimal temperatures for enzyme catalysis revealed temperature-dependent flexibility of four surface-exposed loops. While the flexibility of some loops increased with raising the temperature and decreased with lowering the temperature, as expected for those loops contributing to the protein stability, other loops showed an increment of flexibility upon lowering and raising the temperature. Preserved flexibility in these regions seems to be important for proper enzyme function. The structural differences of these four loops, distant from the active site, are substantially larger than for the overall protein structure, indicating that amino acid exchanges within these loops occurred more frequently thereby allowing the bacteria to tune atomic interactions for different temperature requirements without interfering with the overall enzyme function.
ESTHER : Kovacic_2016_Protein.Eng.Des.Sel_29_65
PubMedSearch : Kovacic_2016_Protein.Eng.Des.Sel_29_65
PubMedID: 26647400

Title : Structural Rigidity and Protein Thermostability in Variants of Lipase A from Bacillus subtilis - Rathi_2015_PLoS.One_10_e0130289
Author(s) : Rathi PC , Jaeger KE , Gohlke H
Ref : PLoS ONE , 10 :e0130289 , 2015
Abstract : Understanding the origin of thermostability is of fundamental importance in protein biochemistry. Opposing views on increased or decreased structural rigidity of the folded state have been put forward in this context. They have been related to differences in the temporal resolution of experiments and computations that probe atomic mobility. Here, we find a significant (p = 0.004) and fair (R2 = 0.46) correlation between the structural rigidity of a well-characterized set of 16 mutants of lipase A from Bacillus subtilis (BsLipA) and their thermodynamic thermostability. We apply the rigidity theory-based Constraint Network Analysis (CNA) approach, analyzing directly and in a time-independent manner the statics of the BsLipA mutants. We carefully validate the CNA results on macroscopic and microscopic experimental observables and probe for their sensitivity with respect to input structures. Furthermore, we introduce a robust, local stability measure for predicting thermodynamic thermostability. Our results complement work that showed for pairs of homologous proteins that raising the structural stability is the most common way to obtain a higher thermostability. Furthermore, they demonstrate that related series of mutants with only a small number of mutations can be successfully analyzed by CNA, which suggests that CNA can be applied prospectively in rational protein design aimed at higher thermodynamic thermostability.
ESTHER : Rathi_2015_PLoS.One_10_e0130289
PubMedSearch : Rathi_2015_PLoS.One_10_e0130289
PubMedID: 26147762
Gene_locus related to this paper: bacsu-lip

Title : Exchange of single amino acids at different positions of a recombinant protein affects metabolic burden in Escherichia coli - Rahmen_2015_Microb.Cell.Fact_14_10
Author(s) : Rahmen N , Fulton A , Ihling N , Magni M , Jaeger KE , Buchs J
Ref : Microb Cell Fact , 14 :10 , 2015
Abstract : Background Escherichia coli is commonly used in academia and industry for expressing recombinant proteins because of its well-characterized molecular genetics and the availability of numerous expression vectors and strains. One important issue during recombinant protein production is the so-called `metabolic burden inverted question mark: the material and energy normally reserved for microbial metabolism which is sapped from the bacterium to produce the recombinant protein. This material and energy drain harms biomass formation and modifies respiration. To the best of our knowledge, no research has investigated so far whether a single amino acid exchange in a recombinant protein affects the metabolic burden phenomenon. Thus, in this study, 15 E. coli BL21(DE3) clones expressing either the fusion tags, a recombinant wild type lipase, or 13 different lipase variants are investigated to quantitatively analyze the respective effects of single amino acid exchanges at different positions on respiration, biomass and protein production of each clone. Therefore, two small-scale online monitoring systems, namely a Respiration Activity MOnitoring System (RAMOS) and a microtiter plate based cultivation system (BioLector) are applied.ResultsUpon expression of all enzyme variants, strong variations were found in the Oxygen Transfer Rate (OTR), biomass and protein (lipase) production of the respective E. coli clones. Two distinct patterns of respiration behavior were observed and, so, the clones could be classified into two groups (Type A and B). Potential factors to explain these patterns were evaluated (e.g. plasmid copy number, inclusion body formation). However, no decisive factor could yet be identified. Five distinct cultivation phases could be determined from OTR curves which give real-time information about carbon source consumption, biomass and protein production. In general, it was found that the quantity of product increased with the duration of active respiration.ConclusionsThis work demonstrates that single amino acid exchanges in a recombinant protein influence the metabolic burden during protein production. The small-scale online monitoring devices RAMOS and BioLector enable the real-time detection of even smallest differences in respiration behavior, biomass and protein production in the E. coli clones investigated. Hence, this study underscores the importance of parallel online monitoring systems to unveil the relevance of single amino acid exchanges for the recombinant protein production.
ESTHER : Rahmen_2015_Microb.Cell.Fact_14_10
PubMedSearch : Rahmen_2015_Microb.Cell.Fact_14_10
PubMedID: 25612616

Title : Pressure adaptation is linked to thermal adaptation in salt-saturated marine habitats - Alcaide_2015_Environ.Microbiol_17_332
Author(s) : Alcaide M , Stogios PJ , Lafraya A , Tchigvintsev A , Flick R , Bargiela R , Chernikova TN , Reva ON , Hai T , Leggewie CC , Katzke N , La Cono V , Matesanz R , Jebbar M , Jaeger KE , Yakimov MM , Yakunin AF , Golyshin PN , Golyshina OV , Savchenko A , Ferrer M
Ref : Environ Microbiol , 17 :332 , 2015
Abstract : The present study provides a deeper view of protein functionality as a function of temperature, salt and pressure in deep-sea habitats. A set of eight different enzymes from five distinct deep-sea (3040-4908 m depth), moderately warm (14.0-16.5 degrees C) biotopes, characterized by a wide range of salinities (39-348 practical salinity units), were investigated for this purpose. An enzyme from a 'superficial' marine hydrothermal habitat (65 degrees C) was isolated and characterized for comparative purposes. We report here the first experimental evidence suggesting that in salt-saturated deep-sea habitats, the adaptation to high pressure is linked to high thermal resistance (P value = 0.0036). Salinity might therefore increase the temperature window for enzyme activity, and possibly microbial growth, in deep-sea habitats. As an example, Lake Medee, the largest hypersaline deep-sea anoxic lake of the Eastern Mediterranean Sea, where the water temperature is never higher than 16 degrees C, was shown to contain halopiezophilic-like enzymes that are most active at 70 degrees C and with denaturing temperatures of 71.4 degrees C. The determination of the crystal structures of five proteins revealed unknown molecular mechanisms involved in protein adaptation to poly-extremes as well as distinct active site architectures and substrate preferences relative to other structurally characterized enzymes.
ESTHER : Alcaide_2015_Environ.Microbiol_17_332
PubMedSearch : Alcaide_2015_Environ.Microbiol_17_332
PubMedID: 25330254
Gene_locus related to this paper: 9alte-MGS-MT1 , 9bact-MGS-M1 , 9bact-MGS-M2 , 9bact-a0a0b5kns5

Title : Exploring the Protein Stability Landscape: Bacillus subtilis Lipase A as a Model for Detergent Tolerance - Fulton_2015_Chembiochem_16_930
Author(s) : Fulton A , Frauenkron-Machedjou VJ , Skoczinski P , Wilhelm S , Zhu L , Schwaneberg U , Jaeger KE
Ref : Chembiochem , 16 :930 , 2015
Abstract : A systematic study was conducted with Bacillus subtilis lipase A (BSLA) to determine the effect of every single amino acid substitution on detergent tolerance. BSLA is a minimal alpha/beta-hydrolase of 181 amino acids with a known crystal structure. It can be expressed in Escherichia coli and is biochemically well characterized. Site saturation mutagenesis resulted in a library of 3439 variants, each with a single amino acid exchange as confirmed by DNA sequencing. The library was tested against four detergents, namely SDS, CTAB, Tween 80, and sulfobetaine. Surface remodeling emerged as an effective engineering strategy to increase tolerance towards detergents. Amino acid residues that significantly affect the tolerance for each of the four detergents were identified. In summary, this systematic analysis provides an experimental dataset to help derive novel protein engineering strategies as well as to direct modeling efforts.
ESTHER : Fulton_2015_Chembiochem_16_930
PubMedSearch : Fulton_2015_Chembiochem_16_930
PubMedID: 25773356

Title : Purification and simultaneous immobilization of Arabidopsis thaliana hydroxynitrile lyase using a family 2 carbohydrate-binding module - Kopka_2015_Biotechnol.J_10_811
Author(s) : Kopka B , Diener M , Wirtz A , Pohl M , Jaeger KE , Krauss U
Ref : Biotechnol J , 10 :811 , 2015
Abstract : Tedious, time- and labor-intensive protein purification and immobilization procedures still represent a major bottleneck limiting the widespread application of enzymes in synthetic chemistry and industry. We here exemplify a simple strategy for the direct site-specific immobilization of proteins from crude cell extracts by fusion of a family 2 carbohydrate-binding module (CBM) derived from the exoglucanase/xylanase Cex from Cellulomonas fimi to a target enzyme. By employing a tripartite fusion protein consisting of the CBM, a flavin-based fluorescent protein (FbFP), and the Arabidopsis thaliana hydroxynitrile lyase (AtHNL), binding to cellulosic carrier materials can easily be monitored via FbFP fluorescence. Adsorption properties (kinetics and quantities) were studied for commercially available Avicel PH-101 and regenerated amorphous cellulose (RAC) derived from Avicel. The resulting immobilizates showed similar activities as the wild-type enzyme but displayed increased stability in the weakly acidic pH range. Finally, Avicel, RAC and cellulose acetate (CA) preparations were used for the synthesis of (R)-mandelonitrile in micro-aqueous methyl tert-butyl ether (MTBE) demonstrating the applicability and stability of the immobilizates for biotransformations in both aqueous and organic reaction systems.
ESTHER : Kopka_2015_Biotechnol.J_10_811
PubMedSearch : Kopka_2015_Biotechnol.J_10_811
PubMedID: 25755120
Gene_locus related to this paper: arath-HNL

Title : The environment shapes microbial enzymes: five cold-active and salt-resistant carboxylesterases from marine metagenomes - Tchigvintsev_2015_Appl.Microbiol.Biotechnol_99_2165
Author(s) : Tchigvintsev A , Tran H , Popovic A , Kovacic F , Brown G , Flick R , Hajighasemi M , Egorova O , Somody JC , Tchigvintsev D , Khusnutdinova A , Chernikova TN , Golyshina OV , Yakimov MM , Savchenko A , Golyshin PN , Jaeger KE , Yakunin AF
Ref : Applied Microbiology & Biotechnology , 99 :2165 , 2015
Abstract : Most of the Earth's biosphere is cold and is populated by cold-adapted microorganisms. To explore the natural enzyme diversity of these environments and identify new carboxylesterases, we have screened three marine metagenome gene libraries for esterase activity. The screens identified 23 unique active clones, from which five highly active esterases were selected for biochemical characterization. The purified metagenomic esterases exhibited high activity against alpha-naphthyl and p-nitrophenyl esters with different chain lengths. All five esterases retained high activity at 5 degrees C indicating that they are cold-adapted enzymes. The activity of MGS0010 increased more than two times in the presence of up to 3.5 M NaCl or KCl, whereas the other four metagenomic esterases were inhibited to various degrees by these salts. The purified enzymes showed different sensitivities to inhibition by solvents and detergents, and the activities of MGS0010, MGS0105 and MGS0109 were stimulated three to five times by the addition of glycerol. Screening of purified esterases against 89 monoester substrates revealed broad substrate profiles with a preference for different esters. The metagenomic esterases also hydrolyzed several polyester substrates including polylactic acid suggesting that they can be used for polyester depolymerization. Thus, esterases from marine metagenomes are cold-adapted enzymes exhibiting broad biochemical diversity reflecting the environmental conditions where they evolved.
ESTHER : Tchigvintsev_2015_Appl.Microbiol.Biotechnol_99_2165
PubMedSearch : Tchigvintsev_2015_Appl.Microbiol.Biotechnol_99_2165
PubMedID: 25194841

Title : Uml2 is a novel CalB-type lipase of Ustilago maydis with phospholipase A activity - Buerth_2014_Appl.Microbiol.Biotechnol_98_4963
Author(s) : Buerth C , Kovacic F , Stock J , Terfruchte M , Wilhelm S , Jaeger KE , Feldbrugge M , Schipper K , Ernst JF , Tielker D
Ref : Applied Microbiology & Biotechnology , 98 :4963 , 2014
Abstract : CalB of Pseudozyma aphidis (formerly named Candida antarctica) is one of the most widely applied enzymes in industrial biocatalysis. Here, we describe a protein with 66 % sequence identity to CalB, designated Ustilago maydis lipase 2 (Uml2), which was identified as the product of gene um01422 of the corn smut fungus U. maydis. Sequence analysis of Uml2 revealed the presence of a typical lipase catalytic triad, Ser-His-Asp with Ser125 located in a Thr-Xaa-Ser-Xaa-Gly pentapeptide. Deletion of the uml2 gene in U. maydis diminished the ability of cells to hydrolyse fatty acids from tributyrin or Tween 20/80 substrates, thus demonstrating that Uml2 functions as a lipase that may contribute to nutrition of this fungal pathogen. Uml2 was heterologously produced in Pichia pastoris and recombinant N-glycosylated Uml2 protein was purified from the culture medium. Purified Uml2 released short- and long-chain fatty acids from p-nitrophenyl esters and Tween 20/80 substrates. Furthermore, phosphatidylcholine substrates containing long-chain saturated or unsaturated fatty acids were effectively hydrolysed. Both esterase and phospholipase A activity of Uml2 depended on the Ser125 catalytic residue. These results indicate that Uml2, in contrast to CalB, exhibits not only esterase and lipase activity but also phospholipase A activity. Thus, by genome mining, we identified a novel CalB-like lipase with different substrate specificities.
ESTHER : Buerth_2014_Appl.Microbiol.Biotechnol_98_4963
PubMedSearch : Buerth_2014_Appl.Microbiol.Biotechnol_98_4963
PubMedID: 24469105
Gene_locus related to this paper: ustma-q4pep1

Title : Fusion of a flavin-based fluorescent protein to hydroxynitrile lyase from Arabidopsis thaliana improves enzyme stability - Scholz_2013_Appl.Environ.Microbiol_79_4727
Author(s) : Scholz KE , Kopka B , Wirtz A , Pohl M , Jaeger KE , Krauss U
Ref : Applied Environmental Microbiology , 79 :4727 , 2013
Abstract : Hydroxynitrile lyase from Arabidopsis thaliana (AtHNL) was fused to different fluorescent reporter proteins. Whereas all fusion constructs retained enzymatic activity and fluorescence in vivo and in vitro, significant differences in activity and pH stability were observed. In particular, flavin-based fluorescent reporter (FbFP) fusions showed almost 2 orders of magnitude-increased half-lives in the weakly acidic pH range compared to findings for the wild-type enzyme. Analysis of the quaternary structure of the respective FbFP-AtHNL fusion proteins suggested that this increased stability is apparently caused by oligomerization mediated via the FbFP tag. Moreover, the increased stability of the fusion proteins enabled the efficient synthesis of (R)-mandelonitrile in an aqueous-organic two-phase system at a pH of <5. Remarkably, (R)-mandelonitrile synthesis is not possible using wild-type AtHNL under the same conditions due to the inherent instability of this enzyme below pH 5. The fusion strategy presented here reveals a surprising means for the stabilization of enzymes and stresses the importance of a thorough in vitro characterization of in vivo-employed fluorescent fusion proteins.
ESTHER : Scholz_2013_Appl.Environ.Microbiol_79_4727
PubMedSearch : Scholz_2013_Appl.Environ.Microbiol_79_4727
PubMedID: 23728815
Gene_locus related to this paper: arath-HNL

Title : Interaction between extracellular lipase LipA and the polysaccharide alginate of Pseudomonas aeruginosa - Tielen_2013_BMC.Microbiol_13_159
Author(s) : Tielen P , Kuhn H , Rosenau F , Jaeger KE , Flemming HC , Wingender J
Ref : BMC Microbiol , 13 :159 , 2013
Abstract : BACKGROUND: As an opportunistic human pathogen Pseudomonas aeruginosa is able to cause acute and chronic infections. The biofilm mode of life significantly contributes to the growth and persistence of P. aeruginosa during an infection process and mediates the pathogenicity of the bacterium. Within a biofilm mucoid strains of P. aeruginosa simultaneously produce and secrete several hydrolytic enzymes and the extracellular polysaccharide alginate. The focus of the current study was the interaction between extracellular lipase LipA and alginate, which may be physiologically relevant in biofilms of mucoid P. aeruginosa.
RESULTS: Fluorescence microscopy of mucoid P. aeruginosa biofilms were performed using fluorogenic lipase substrates. It showed a localization of the extracellular enzyme near the cells. A microtiter plate-based binding assay revealed that the polyanion alginate is able to bind LipA. A molecular modeling approach showed that this binding is structurally based on electrostatic interactions between negatively charged residues of alginate and positively charged amino acids of the protein localized opposite of the catalytic centre. Moreover, we showed that the presence of alginate protected the lipase activity by protection from heat inactivation and from degradation by the endogenous, extracellular protease elastase LasB. This effect was influenced by the chemical properties of the alginate molecules and was enhanced by the presence of O-acetyl groups in the alginate chain. CONCLUSION: We demonstrate that the extracellular lipase LipA from P. aeruginosa interacts with the polysaccharide alginate in the self-produced extracellular biofilm matrix of P. aeruginosa via electrostatic interactions suggesting a role of this interaction for enzyme immobilization and accumulation within biofilms. This represents a physiological advantage for the cells. Especially in the biofilm lifestyle, the enzyme is retained near the cell surface, with the catalytic centre exposed towards the substrate and is protected from denaturation and proteolytic degradation.
ESTHER : Tielen_2013_BMC.Microbiol_13_159
PubMedSearch : Tielen_2013_BMC.Microbiol_13_159
PubMedID: 23848942

Title : Synthesis of chiral cyanohydrins by recombinant Escherichia coli cells in a micro-aqueous reaction system - Scholz_2012_Appl.Environ.Microbiol_78_5025
Author(s) : Scholz KE , Okrob D , Kopka B , Grunberger A , Pohl M , Jaeger KE , Krauss U
Ref : Applied Environmental Microbiology , 78 :5025 , 2012
Abstract : Synthesis of chiral cyanohydrins is performed in a monophasic micro-aqueous reaction system using whole recombinant Escherichia coli cells expressing the Arabidopsis thaliana hydroxynitrile lyase (AtHNL). Microscopy studies employing a fusion of AtHNL with a flavin-based fluorescent protein (FbFP) reveal that the cells remain intact in the reaction system.
ESTHER : Scholz_2012_Appl.Environ.Microbiol_78_5025
PubMedSearch : Scholz_2012_Appl.Environ.Microbiol_78_5025
PubMedID: 22544258
Gene_locus related to this paper: arath-HNL

Title : The metagenome-derived enzymes LipS and LipT increase the diversity of known lipases - Chow_2012_PLoS.One_7_e47665
Author(s) : Chow J , Kovacic F , Dall Antonia Y , Krauss U , Fersini F , Schmeisser C , Lauinger B , Bongen P , Pietruszka J , Schmidt M , Menyes I , Bornscheuer UT , Eckstein M , Thum O , Liese A , Mueller-Dieckmann J , Jaeger KE , Streit WR
Ref : PLoS ONE , 7 :e47665 , 2012
Abstract : Triacylglycerol lipases (EC 3.1.1.3) catalyze both hydrolysis and synthesis reactions with a broad spectrum of substrates rendering them especially suitable for many biotechnological applications. Most lipases used today originate from mesophilic organisms and are susceptible to thermal denaturation whereas only few possess high thermotolerance. Here, we report on the identification and characterization of two novel thermostable bacterial lipases identified by functional metagenomic screenings. Metagenomic libraries were constructed from enrichment cultures maintained at 65 to 75 degrees C and screened resulting in the identification of initially 10 clones with lipolytic activities. Subsequently, two ORFs were identified encoding lipases, LipS and LipT. Comparative sequence analyses suggested that both enzymes are members of novel lipase families. LipS is a 30.2 kDa protein and revealed a half-life of 48 h at 70 degrees C. The lipT gene encoded for a multimeric enzyme with a half-life of 3 h at 70 degrees C. LipS had an optimum temperature at 70 degrees C and LipT at 75 degrees C. Both enzymes catalyzed hydrolysis of long-chain (C(12) and C(14)) fatty acid esters and additionally hydrolyzed a number of industry-relevant substrates. LipS was highly specific for (R)-ibuprofen-phenyl ester with an enantiomeric excess (ee) of 99%. Furthermore, LipS was able to synthesize 1-propyl laurate and 1-tetradecyl myristate at 70 degrees C with rates similar to those of the lipase CalB from Candida antarctica. LipS represents the first example of a thermostable metagenome-derived lipase with significant synthesis activities. Its X-ray structure was solved with a resolution of 1.99 A revealing an unusually compact lid structure.
ESTHER : Chow_2012_PLoS.One_7_e47665
PubMedSearch : Chow_2012_PLoS.One_7_e47665
PubMedID: 23112831
Gene_locus related to this paper: symth-q67mr3 , 9bact-k7qe48

Title : Identification of amino acids involved in the hydrolytic activity of lipase LipBL from Marinobacter lipolyticus - Perez_2012_Microbiology_158_2192
Author(s) : Perez D , Kovacic F , Wilhelm S , Jaeger KE , Garcia MT , Ventosa A , Mellado E
Ref : Microbiology , 158 :2192 , 2012
Abstract : The lipolytic enzyme family VIII currently includes only seven members but represents a group of lipolytic enzymes with interesting properties. Recently, we identified a gene encoding the family VIII lipase LipBL from the halophilic bacterium Marinobacter lipolyticus. This enzyme, like most lipolytic enzymes from family VIII, possesses two possible nucleophilic serines located in an S-X-X-K beta-lactamase motif and a G-X-S-X-G lipase motif. The serine in the S-X-X-K motif is a catalytic residue, but the role of serine within the common lipase consensus sequence G-X-S-X-G has not yet been systematically studied. Here, the previously reported time-intensive procedure for purification of recombinant LipBL was replaced by one-step metal-affinity chromatography purification in the presence of ATP. Heterologous co-expression of His(6)-tagged LipBL with the cytoplasmic molecular chaperones GroEL/GroES was necessary to obtain catalytically active LipBL. Site-directed mutagenesis performed to map the active site of LipBL revealed that mutation of serine and lysine in the beta-lactamase motif (S(72)-M-T-K(75)) to alanine abolished the enzyme activity of LipBL, in contrast to mutation of the serine in the lipase consensus motif (S321A). Furthermore, mutagenesis was performed to understand the role of the G-X-S-X-G motif and other amino acids that are conserved among family VIII esterases. We describe how mutations in the conserved G-X-S-X-G motif altered the biochemical properties and substrate specificity of LipBL. Molecular modelling results indicate the location of the G-X-S(321)-X-G motif in a loop close to the catalytic centre of LipBL, presumably representing a substrate-binding site of LipBL.
ESTHER : Perez_2012_Microbiology_158_2192
PubMedSearch : Perez_2012_Microbiology_158_2192
PubMedID: 22609754

Title : Novel broad host range shuttle vectors for expression in Escherichia coli, Bacillus subtilis and Pseudomonas putida - Troeschel_2012_J.Biotechnol_161_71
Author(s) : Troeschel SC , Thies S , Link O , Real CI , Knops K , Wilhelm S , Rosenau F , Jaeger KE
Ref : J Biotechnol , 161 :71 , 2012
Abstract : Novel shuttle vectors named pEBP were constructed to allow the gene expression in different bacterial hosts including Escherichia coli, Bacillus subtilis and Pseudomonas putida. These vectors share the inducible promoters P(T7) and P(Xyl) and a cos site to enable packaging of plasmid DNA into phage, and carry different multiple cloning sites and antibiotic resistance genes. Vector pEBP41 generally replicates episomally while pEBP18 replicates episomally in Gram-negative bacteria only, but integrates into the chromosome of B. subtilis. Plasmid copy numbers determined for E. coli and P. putida were in the range of 5-50 per cell. The functionality of pEBP18 and pEBP41 was confirmed by expression of two lipolytic enzymes, namely lipase A from B. subtilis and cutinase from the eukaryotic fungus Fusarium solani pisi in three different host strains. Additionally, we report here the construction of a T7 RNA polymerase-based expression strain of P. putida.
ESTHER : Troeschel_2012_J.Biotechnol_161_71
PubMedSearch : Troeschel_2012_J.Biotechnol_161_71
PubMedID: 22440389

Title : Mechanism of acetaldehyde-induced deactivation of microbial lipases - Franken_2011_BMC.Biochem_12_10
Author(s) : Franken B , Eggert T , Jaeger KE , Pohl M
Ref : BMC Biochem , 12 :10 , 2011
Abstract : BACKGROUND: Microbial lipases represent the most important class of biocatalysts used for a wealth of applications in organic synthesis. An often applied reaction is the lipase-catalyzed transesterification of vinyl esters and alcohols resulting in the formation of acetaldehyde which is known to deactivate microbial lipases, presumably by structural changes caused by initial Schiff-base formation at solvent accessible lysine residues. Previous studies showed that several lipases were sensitive toward acetaldehyde deactivation whereas others were insensitive; however, a general explanation of the acetaldehyde-induced inactivation mechanism is missing. RESULTS: Based on five microbial lipases from Candida rugosa, Rhizopus oryzae, Pseudomonas fluorescens and Bacillus subtilis we demonstrate that the protonation state of lysine epsilon-amino groups is decisive for their sensitivity toward acetaldehyde. Analysis of the diverse modification products of Bacillus subtilis lipases in the presence of acetaldehyde revealed several stable products such as alpha,beta-unsaturated polyenals, which result from base and/or amino acid catalyzed aldol condensation of acetaldehyde. Our studies indicate that these products induce the formation of stable Michael-adducts at solvent-accessible amino acids and thus lead to enzyme deactivation. Further, our results indicate Schiff-base formation with acetaldehyde to be involved in crosslinking of lipase molecules. CONCLUSIONS: Differences in stability observed with various commercially available microbial lipases most probably result from different purification procedures carried out by the respective manufacturers. We observed that the pH of the buffer used prior to lyophilization of the enzyme sample is of utmost importance. The mechanism of acetaldehyde-induced deactivation of microbial lipases involves the generation of alpha,beta-unsaturated polyenals from acetaldehyde which subsequently form stable Michael-adducts with the enzymes. Lyophilization of the enzymes from buffer at pH 6.0 can provide an easy and effective way to stabilize lipases toward inactivation by acetaldehyde.
ESTHER : Franken_2011_BMC.Biochem_12_10
PubMedSearch : Franken_2011_BMC.Biochem_12_10
PubMedID: 21342514

Title : The lipase LipA (PA2862) but not LipC (PA4813) from Pseudomonas aeruginosa influences regulation of pyoverdine production and expression of the sigma factor PvdS - Funken_2011_J.Bacteriol_193_5858
Author(s) : Funken H , Knapp A , Vasil ML , Wilhelm S , Jaeger KE , Rosenau F
Ref : Journal of Bacteriology , 193 :5858 , 2011
Abstract : A key element in iron-dependent regulation of iron metabolism and virulence-related functions for Pseudomonas aeruginosa is the sigma factor PvdS. PvdS expression itself is also influenced by iron-independent stimuli. We show that pyoverdine production and pvdS expression depend on one of the two lipases of P. aeruginosa.
ESTHER : Funken_2011_J.Bacteriol_193_5858
PubMedSearch : Funken_2011_J.Bacteriol_193_5858
PubMedID: 21840975

Title : Lipase LipC affects motility, biofilm formation and rhamnolipid production in Pseudomonas aeruginosa - Rosenau_2010_FEMS.Microbiol.Lett_309_25
Author(s) : Rosenau F , Isenhardt S , Gdynia A , Tielker D , Schmidt E , Tielen P , Schobert M , Jahn D , Wilhelm S , Jaeger KE
Ref : FEMS Microbiology Letters , 309 :25 , 2010
Abstract : Pseudomonas aeruginosa produces and secretes several lipolytic enzymes, among them the lipases LipA and LipC. LipA is encoded within the lipA/lipH operon, together with its cognate foldase LipH, which was also found to be required for the functional expression of LipC. At present, the physiological function of LipC is unknown. We have cloned a synthetic operon consisting of the lipC structural gene and the foldase gene lipH obtained from the lipA/lipH operon and have constructed, in parallel, a lipC-deficient P. aeruginosa mutant. Inactivation of the lipC gene significantly impaired type IV pilus-dependent twitching and swarming motility, but also the flagella-mediated swimming motility of P. aeruginosa. Moreover, for the lipC mutant, we observed a significant decrease in the amount of extracellular rhamnolipids. Also, the P. aeruginosa lipC mutant showed a significantly altered biofilm architecture. Proteome analysis revealed the accumulation of the response regulator protein PhoP in the lipC mutant.
ESTHER : Rosenau_2010_FEMS.Microbiol.Lett_309_25
PubMedSearch : Rosenau_2010_FEMS.Microbiol.Lett_309_25
PubMedID: 20546309
Gene_locus related to this paper: pseae-clipa

Title : Lipolytic Enzymes from Bacteria - Hausmann_2010_Handb.Hydrocarbon.Lipid.Microbiol__1099
Author(s) : Hausmann S , Jaeger KE
Ref : Handbook of Hydrocarbon and Lipid Microbiology , Aerobic Utilization of Hydrocarbons, Oils, and Lipids :1099 , 2010
Abstract : Lipolytic enzymes comprising carboxylesterases and lipases represent a highly diverse group of hydrolases. Presently, about 900 of these enzymes are identified to originate from bacteria. In a landmark publication which appeared 10 years ago (Arpigny and Jaeger, 1999), lipolytic enzymes were grouped into eight families based on amino acid sequence homology and physiological properties. Here, we present the current status of this classification framework into which we have included numerous novel bacterial lipolytic enzymes. We further describe their biochemical properties and characteristic structural features. Additionally, diverse physiological functions of selected lipolytic enzymes are discussed.
ESTHER : Hausmann_2010_Handb.Hydrocarbon.Lipid.Microbiol__1099
PubMedSearch : Hausmann_2010_Handb.Hydrocarbon.Lipid.Microbiol__1099
PubMedID:

Title : Probing enzyme promiscuity of SGNH hydrolases - Lescic_2010_Chembiochem_11_2158
Author(s) : Lescic Asler I , Ivic N , Kovacic F , Schell S , Knorr J , Krauss U , Wilhelm S , Kojic-Prodic B , Jaeger KE
Ref : Chembiochem , 11 :2158 , 2010
Abstract : Several hydrolases of the SGNH superfamily, including the lipase SrLip from Streptomyces rimosus (Q93MW7), the acyl-CoA thioesterase I TesA from Pseudomonas aeruginosa (Q9HZY8) and the two lipolytic enzymes EstA (from P. aeruginosa, O33407) and EstP (from Pseudomonas putida, Q88QS0), were examined for promiscuity. These enzymes were tested against four chemically different classes of a total of 34 substrates known to be hydrolysed by esterases, thioesterases, lipases, phospholipases, Tweenases and proteases. Furthermore, they were also analysed with respect to their amino acid sequences and structural homology, and their phylogenetic relationship was determined. The Pseudomonas esterases EstA and EstP each have an N-terminal domain with catalytic activity together with a C-terminal autotransporter domain, and so the hybrid enzymes EstA(N)-EstP(C) and EstP(N)-EstA(C) were constructed by swapping the corresponding N- and C-terminal domains, and their hydrolytic activities were compared. Interestingly, substrate specificity and kinetic measurements indicated a significant influence of the autotransporter domains on the catalytic activities of these enzymes in solution. TesA, EstA and EstP were shown to function as esterases with different affinities and catalytic efficacies towards p-nitrophenyl butyrate. Of all the enzymes tested, only SrLip revealed lipase, phospholipase, esterase, thioesterase and Tweenase activities.
ESTHER : Lescic_2010_Chembiochem_11_2158
PubMedSearch : Lescic_2010_Chembiochem_11_2158
PubMedID: 20931591

Title : Enantioselective kinetic resolution of phenylalkyl carboxylic acids using metagenome-derived esterases - Fernandez-Alvaro_2010_Microb.Biotechnol_3_59
Author(s) : Fernandez-Alvaro E , Kourist R , Winter J , Bottcher D , Liebeton K , Naumer C , Eck J , Leggewie C , Jaeger KE , Streit WR , Bornscheuer UT
Ref : Microb Biotechnol , 3 :59 , 2010
Abstract : Enantiomerically pure beta-arylalkyl carboxylic acids are important synthetic intermediates for the preparation of a wide range of compounds with biological and pharmacological activities. A library of 83 enzymes isolated from the metagenome was searched for activity in the hydrolysis of ethyl esters of three racemic phenylalkyl carboxylic acids by a microtiter plate-based screening using a pH-indicator assay. Out of these, 20 enzymes were found to be active and were subjected to analytical scale biocatalysis in order to determine their enantioselectivity. The most enantioselective and also enantiocomplementary biocatalysts were then used for preparative scale reactions. Thus, both enantiomers of each of the three phenylalkyl carboxylic acids studied could be obtained in excellent optical purity and high yields.
ESTHER : Fernandez-Alvaro_2010_Microb.Biotechnol_3_59
PubMedSearch : Fernandez-Alvaro_2010_Microb.Biotechnol_3_59
PubMedID: 21255306
Gene_locus related to this paper: 9bact-Est8.6Y9K

Title : LOVely enzymes - towards engineering light-controllable biocatalysts - Krauss_2010_Microb.Biotechnol_3_15
Author(s) : Krauss U , Lee J , Benkovic SJ , Jaeger KE
Ref : Microb Biotechnol , 3 :15 , 2010
Abstract : Light control over enzyme function represents a novel and exciting field of biocatalysis research. Blue-light photoreceptors of the Light, Oxygen, Voltage (LOV) family have recently been investigated for their applicability as photoactive switches. We discuss here the primary photochemical events leading to light activation of LOV domains as well as the proposed signal propagation mechanism to the respective effector domain. Furthermore, we describe the construction of LOV fusions to different effector domains, namely a dihydrofolate reductase from Escherichia coli and a lipase from Bacillus subtilis. Both fusion partners retained functionality, and alteration of enzyme activity by light was also demonstrated. Hence, it appears that fusion of LOV photoreceptors to functional enzyme target sites via appropriate linker structures may represent a straightforward strategy to design light controllable biocatalysts.
ESTHER : Krauss_2010_Microb.Biotechnol_3_15
PubMedSearch : Krauss_2010_Microb.Biotechnol_3_15
PubMedID: 21255302

Title : Mutations towards enantioselectivity adversely affect secretion of Pseudomonas aeruginosa lipase - Hausmann_2008_FEMS.Microbiol.Lett_282_65
Author(s) : Hausmann S , Wilhelm S , Jaeger KE , Rosenau F
Ref : FEMS Microbiology Letters , 282 :65 , 2008
Abstract : Lipases are important biocatalysts used as detergent additives to manufacture biodiesel, and in particular, for the production of enantiopure compounds such as alcohols, amines and carboxylic acids. Extensive efforts were conducted trying to optimize lipase properties and lipase LipA of Pseudomonas aeruginosa comprises the best-studied example in terms of optimizing enantioselectivity by application of numerous directed evolution methods. Its enantioselectivity in the asymmetric hydrolysis of the model substrate 2-methyldecanoic acid p-nitrophenyl ester was increased from E=1.1 for the wild-type enzyme to E=51 for the best (S)-enantioselective variant which carried six amino acid exchanges. We have observed that overexpression of this variant in the homologous host resulted in only marginal yields of enzyme in the bacterial culture supernatant, suggesting that the enantioselective LipA variant was secreted with only low efficiency. Hence, we have analysed the secretion of this lipase variant and compared it to variants carrying either the respective single mutations or some combinations. We report here the identification of two amino acid substitutions located on the protein surface, which significantly impair lipase secretion.
ESTHER : Hausmann_2008_FEMS.Microbiol.Lett_282_65
PubMedSearch : Hausmann_2008_FEMS.Microbiol.Lett_282_65
PubMedID: 18355276
Gene_locus related to this paper: pseae-llipa

Title : Ultrahigh-throughput screening to identify E. coli cells expressing functionally active enzymes on their surface - Becker_2007_Chembiochem_8_943
Author(s) : Becker S , Michalczyk A , Wilhelm S , Jaeger KE , Kolmar H
Ref : Chembiochem , 8 :943 , 2007
Abstract : We show here that E. coli bacteria that display esterases or lipases on their cell surface together with horseradish peroxidase (HRP) are capable of hydrolysing carboxylic acid esters of biotin tyramide. The tyramide radicals generated by the coupled lipase-peroxidase reaction were short-lived and therefore became covalently attached to reactive tyrosine residues that were located in close vicinity on the surface of a bacterial cell that displayed lipase activity. Up to 120 000 biotinylated tyramide derivatives could be covalently coupled through HRP activation to the surface of a single living E. coli cell. Differences in cellular esterase activity were found to correlate with the amount of biotin tyramide deposited on the cell surface. Selective biotin tyramide labelling of cells that had lipase activity allowed their isolation by magnetic cell sorting from a 1:10(6) mixture of control cells. This strategy of covalently attaching a biotin label to esterase-proficient bacteria might open new avenues to ultrahigh-throughput screening of enzyme libraries for hydrolytic enzymes with enhanced activities or enantioselectivities.
ESTHER : Becker_2007_Chembiochem_8_943
PubMedSearch : Becker_2007_Chembiochem_8_943
PubMedID: 17458914

Title : A calcium-gated lid and a large beta-roll sandwich are revealed by the crystal structure of extracellular lipase from Serratia marcescens - Meier_2007_J.Biol.Chem_282_31477
Author(s) : Meier R , Drepper T , Svensson V , Jaeger KE , Baumann U
Ref : Journal of Biological Chemistry , 282 :31477 , 2007
Abstract : Lipase LipA from Serratia marcescens is a 613-amino acid enzyme belonging to family I.3 of lipolytic enzymes that has an important biotechnological application in the production of a chiral precursor for the coronary vasodilator diltiazem. Like other family I.3 lipases, LipA is secreted by Gram-negative bacteria via a type I secretion system and possesses 13 copies of a calcium binding tandem repeat motif, GGXGXDXUX (U, hydrophobic amino acids), in the C-terminal part of the polypeptide chain. The 1.8-A crystal structure of LipA reveals a close relation to eukaryotic lipases, whereas family I.1 and I.2 enzymes appear to be more distantly related. Interestingly, the structure shows for the N-terminal lipase domain a variation on the canonical alpha/beta hydrolase fold in an open conformation, where the putative lid helix is anchored by a Ca(2+) ion essential for activity. Another novel feature observed in this lipase structure is the presence of a helical hairpin additional to the putative lid helix that exposes a hydrophobic surface to the aqueous medium and might function as an additional lid. The tandem repeats form two separated parallel beta-roll domains that pack tightly against each other. Variations of the consensus sequence of the tandem repeats within the second beta-roll result in an asymmetric Ca(2+) binding on only one side of the roll. The analysis of the properties of the beta-roll domains suggests an intramolecular chaperone function.
ESTHER : Meier_2007_J.Biol.Chem_282_31477
PubMedSearch : Meier_2007_J.Biol.Chem_282_31477
PubMedID: 17728256
Gene_locus related to this paper: serma-lipasA

Title : Learning from directed evolution: Further lessons from theoretical investigations into cooperative mutations in lipase enantioselectivity - Reetz_2007_Chembiochem_8_106
Author(s) : Reetz MT , Puls M , Carballeira JD , Vogel A , Jaeger KE , Eggert T , Thiel W , Bocola M , Otte N
Ref : Chembiochem , 8 :106 , 2007
Abstract : An earlier experimental study, which involved the directed evolution of enantioselective lipase variants from Pseudomonas aeruginosa as catalysts in the hydrolytic kinetic resolution of 2-methyl-decanoic acid p-nitrophenyl ester, provided a mutant with six mutations. Consequently, the selectivity factor was found to increase from E = 1.1 for the wild-type to E = 51 for the best mutant. Only one of the amino acid exchanges in this mutant was found to occur next to the binding pocket, the other mutations being remote. Our previous theoretical analysis with molecular-dynamics simulations helped to unveil the source of enhanced enantioselectivity: a relay mechanism that involves two of the six mutations was shown to induce strong cooperativity. In this investigation, single, double, and triple mutants were constructed and tested as enantioselective catalysts. This study supports our original postulate regarding the relay mechanism, offers further mechanistic insight into the role of individual mutations, and provides mutants that display even higher enantioselectivity (E of up to 64).
ESTHER : Reetz_2007_Chembiochem_8_106
PubMedSearch : Reetz_2007_Chembiochem_8_106
PubMedID: 17133645

Title : Functional cell-surface display of a lipase-specific chaperone - Wilhelm_2007_Chembiochem_8_55
Author(s) : Wilhelm S , Rosenau F , Becker S , Buest S , Hausmann S , Kolmar H , Jaeger KE
Ref : Chembiochem , 8 :55 , 2007
Abstract : Lipases are important enzymes in biotechnology. Extracellular bacterial lipases from Pseudomonads and related species require the assistance of specific chaperones, designated "Lif" proteins (lipase specific foldases). Lifs, a unique family of steric chaperones, are anchored to the periplasmic side of the inner membrane where they convert lipases into their active conformation. We have previously shown that the autotransporter protein EstA from P. aeruginosa can be used to direct a variety of proteins to the cell surface of Escherichia coli. Here we demonstrate for the first time the functional cell-surface display of the Lif chaperone and FACS (fluorescence-activated cell sorting)-based analysis of bacterial cells that carried foldase-lipase complexes. The model Lif protein, LipH from P. aeruginosa, was displayed at the surface of E. coli cells. Surface exposed LipH was functional and efficiently refolded chemically denatured lipase. The foldase autodisplay system reported here can be used for a variety of applications including the ultrahigh-throughput screening of large libraries of foldase variants generated by directed evolution.
ESTHER : Wilhelm_2007_Chembiochem_8_55
PubMedSearch : Wilhelm_2007_Chembiochem_8_55
PubMedID: 17173269

Title : Isolation and biochemical characterization of two novel metagenome-derived esterases - Elend_2006_Appl.Environ.Microbiol_72_3637
Author(s) : Elend C , Schmeisser C , Leggewie C , Babiak P , Carballeira JD , Steele HL , Reymond JL , Jaeger KE , Streit WR
Ref : Applied Environmental Microbiology , 72 :3637 , 2006
Abstract : The metagenomes of uncultured microbial communities are rich sources for novel biocatalysts. In this study, esterase EstA3 was derived from a drinking water metagenome, and esterase EstCE1 was derived from a soil metagenome. Both esterases are approximately 380 amino acids in size and show similarity to beta-lactamases, indicating that they belong to family VIII of the lipases/esterases. EstA3 had a temperature optimum at 50 degrees C and a pH optimum at pH 9.0. It was remarkably active and very stable in the presence of solvents and over a wide temperature and pH range. It is active in a multimeric form and displayed a high level of activity against a wide range of substrates including one secondary ester, 7-[3-octylcarboxy-(3-hydroxy-3-methyl-butyloxy)]-coumarin, which is normally unreactive. EstCE1 was active in the monomeric form and had a temperature optimum at 47 degrees C and a pH optimum at pH 10. It exhibited the same level of stability as EstA3 over wide temperature and pH ranges and in the presence of dimethyl sulfoxide, isopropanol, and methanol. EstCE1 was highly enantioselective for (+)-menthylacetate. These enzymes display remarkable characteristics that cannot be related to the original environment from which they were derived. The high level of stability of these enzymes together with their unique substrate specificities make them highly useful for biotechnological applications.
ESTHER : Elend_2006_Appl.Environ.Microbiol_72_3637
PubMedSearch : Elend_2006_Appl.Environ.Microbiol_72_3637
PubMedID: 16672512

Title : Identification of novel benzoylformate decarboxylases by growth selection - Henning_2006_Appl.Environ.Microbiol_72_7510
Author(s) : Henning H , Leggewie C , Pohl M , Muller M , Eggert T , Jaeger KE
Ref : Applied Environmental Microbiology , 72 :7510 , 2006
Abstract : A growth selection system was established using Pseudomonas putida, which can grow on benzaldehyde as the sole carbon source. These bacteria presumably metabolize benzaldehyde via the beta-ketoadipate pathway and were unable to grow in benzoylformate-containing selective medium, but the growth deficiency could be restored by expression in trans of genes encoding benzoylformate decarboxylases. The selection system was used to identify three novel benzoylformate decarboxylases, two of them originating from a chromosomal library of P. putida ATCC 12633 and the third from an environmental-DNA library. The novel P. putida enzymes BfdB and BfdC exhibited 83% homology to the benzoylformate decarboxylase from P. aeruginosa and 63% to the enzyme MdlC from P. putida ATCC 12633, whereas the metagenomic BfdM exhibited 72% homology to a putative benzoylformate decarboxylase from Polaromonas naphthalenivorans. BfdC was overexpressed in Escherichia coli, and the enzymatic activity was determined to be 22 U/ml using benzoylformate as the substrate. Our results clearly demonstrate that P. putida KT2440 is an appropriate selection host strain suitable to identify novel benzoylformate decarboxylase-encoding genes. In principle, this system is also applicable to identify a broad range of different industrially important enzymes, such as benzaldehyde lyases, benzoylformate decarboxylases, and hydroxynitrile lyases, which all catalyze the formation of benzaldehyde.
ESTHER : Henning_2006_Appl.Environ.Microbiol_72_7510
PubMedSearch : Henning_2006_Appl.Environ.Microbiol_72_7510
PubMedID: 17012586

Title : Biocatalytic production of enantiopure cyclohexane-trans-1,2-diol using extracellular lipases from Bacillus subtilis - Detry_2006_Appl.Microbiol.Biotechnol_72_1107
Author(s) : Detry J , Rosenbaum T , Lutz S , Hahn D , Jaeger KE , Muller M , Eggert T
Ref : Applied Microbiology & Biotechnology , 72 :1107 , 2006
Abstract : Two extracellular lipases from Bacillus subtilis, B. subtilis lipase A and lipase B, have been expressed in the heterologous host Escherichia coli, biochemically characterized and used for the kinetic resolution of (rac)-trans-1,2-diacetoxycyclohexane. Both enzymes were selectively acting on the (R,R)-enantiomer of the racemic substrate, highly specifically hydrolyzing only one of the two ester groups present, thus allowing the preparation of enantiopure (R,R)- and (S,S)-cyclohexane-trans-1,2-diol. The reaction conditions for the use of purified enzyme and crude cell lyophilizate were optimized and reactions in batch and repetitive batch modes were carried out on a preparative scale to yield enantiopure product (>99% enantiomeric excess).
ESTHER : Detry_2006_Appl.Microbiol.Biotechnol_72_1107
PubMedSearch : Detry_2006_Appl.Microbiol.Biotechnol_72_1107
PubMedID: 16586103

Title : Combination of computational prescreening and experimental library construction can accelerate enzyme optimization by directed evolution - Funke_2005_Protein.Eng.Des.Sel_18_509
Author(s) : Funke SA , Otte N , Eggert T , Bocola M , Jaeger KE , Thiel W
Ref : Protein Engineering Des Sel , 18 :509 , 2005
Abstract : Chiral compounds can be produced efficiently by using biocatalysts. However, wild-type enzymes often do not meet the requirements of a production process, making optimization by rational design or directed evolution necessary. Here, we studied the lipase-catalyzed hydrolysis of the model substrate 1-(2-naphthyl)ethyl acetate both theoretically and experimentally. We found that a computational equivalent of alanine scanning mutagenesis based on QM/MM methodology can be applied to identify amino acid positions important for the activity of the enzyme. The theoretical results are consistent with concomitant experimental work using complete saturation mutagenesis and high-throughput screening of the target biocatalyst, a lipase from Bacillus subtilis. Both QM/MM-based calculations and molecular biology experiments identify histidine 76 as a residue that strongly affects the catalytic activity. The experiments demonstrate its important influence on enantioselectivity.
ESTHER : Funke_2005_Protein.Eng.Des.Sel_18_509
PubMedSearch : Funke_2005_Protein.Eng.Des.Sel_18_509
PubMedID: 16203748

Title : A generic system for the Escherichia coli cell-surface display of lipolytic enzymes - Becker_2005_FEBS.Lett_579_1177
Author(s) : Becker S , Theile S , Heppeler N , Michalczyk A , Wentzel A , Wilhelm S , Jaeger KE , Kolmar H
Ref : FEBS Letters , 579 :1177 , 2005
Abstract : EstA is an outer membrane-anchored esterase from Pseudomonas aeruginosa. An inactive EstA variant was used as an anchoring motif for the Escherichia coli cell-surface display of lipolytic enzymes. Flow cytometry analysis and measurement of lipase activity revealed that Bacillus subtilis lipase LipA, Fusarium solani pisi cutinase and one of the largest lipases presently known, namely Serratia marcescens lipase were all efficiently exported by the EstA autotransporter and also retained their lipolytic activities upon cell surface exposition. EstA provides a useful tool for surface display of lipases including variant libraries generated by directed evolution thereby enabling the identification of novel enzymes with interesting biological and biotechnological ramifications.
ESTHER : Becker_2005_FEBS.Lett_579_1177
PubMedSearch : Becker_2005_FEBS.Lett_579_1177
PubMedID: 15710409

Title : Multiplex-PCR-based recombination as a novel high-fidelity method for directed evolution - Eggert_2005_Chembiochem_6_1062
Author(s) : Eggert T , Funke SA , Rao NM , Acharya P , Krumm H , Reetz MT , Jaeger KE
Ref : Chembiochem , 6 :1062 , 2005
Abstract : A new and convenient method for the in vitro recombination of single point mutations is presented. This method efficiently reduces the introduction of novel point mutations, which usually occur during recombination processes. A multiplex polymerase chain reaction (multiplex-PCR) generates gene fragments that contain preformed point mutations. These fragments are subsequently assembled into full-length genes by a recombination-PCR step. The process of multiplex-PCR-based recombination (MUPREC) does not require DNase I digestion for gene-fragmentation and is therefore easy to perform, even with small amounts of target DNA. The protocol yields high frequencies of recombination without creating a wild-type background. Furthermore, the low error rate results in high-quality variant libraries of true recombinants, thereby minimizing the screening efforts and saving time and money. The MUPREC method was used in the directed evolution of a Bacillus subtilis lipase that can catalyse the enantioselective hydrolysis of a model meso-compound. Thereby, the method was proved to be useful in producing a reliable second-generation library of true recombinants from which better performing variants were identified by using a high-throughput electrospray ionization mass spectrometry (ESI-MS) screening system.
ESTHER : Eggert_2005_Chembiochem_6_1062
PubMedSearch : Eggert_2005_Chembiochem_6_1062
PubMedID: 15880674

Title : Prospecting for biocatalysts and drugs in the genomes of non-cultured microorganisms - Streit_2004_Curr.Opin.Biotechnol_15_285
Author(s) : Streit WR , Daniel R , Jaeger KE
Ref : Curr Opin Biotechnol , 15 :285 , 2004
Abstract : Modern biotechnology has a steadily increasing demand for vitamins, antibiotics and, in particular, novel biocatalysts for use in the production of flavors, agrochemicals, pharmaceuticals and high-value fine chemicals. Novel experimental approaches are being developed in attempts to identify such molecules. However, it is known that up to 99.8% of the microbes present in many environments are not readily culturable; hence, they cannot be exploited for biotechnology. The 'metagenome technology' offers a solution to this problem by developing culture-independent methods to isolate, clone and express environmental DNA. So far, metagenome-based approaches have led to the isolation of many novel biocatalysts and a variety of other molecules with a high potential for downstream applications.
ESTHER : Streit_2004_Curr.Opin.Biotechnol_15_285
PubMedSearch : Streit_2004_Curr.Opin.Biotechnol_15_285
PubMedID: 15296926

Title : Lipase-specific foldases - Rosenau_2004_Chembiochem_5_152
Author(s) : Rosenau F , Tommassen J , Jaeger KE
Ref : Chembiochem , 5 :152 , 2004
Abstract : Lipases represent the most important class of enzymes used in biotechnology. Many bacteria produce and secrete lipases but the enzymes originating from Pseudomonas and Burkholderia species seem to be particularly useful for a wide variety of different biocatalytic applications. These enzymes are usually encoded in an operon together with a second gene which codes for a lipase-specific foldase, Lif, which is necessary to obtain enzymatically active lipase. A detailed analysis based on amino acid homology has suggested the classification of Lif proteins into four different families and also revealed the presence of a conserved motif, Rx1x2FDY(F/C)L(S/T)A. Recent experimental evidence suggests that Lifs are so-called steric chaperones, which exert their physiological function by lowering energetic barriers during the folding of their cognate lipases, thereby providing essential steric information needed to fold lipases into their enzymatically active conformation.
ESTHER : Rosenau_2004_Chembiochem_5_152
PubMedSearch : Rosenau_2004_Chembiochem_5_152
PubMedID: 14760735

Title : Enantioselective biocatalysis optimized by directed evolution - Jaeger_2004_Curr.Opin.Biotechnol_15_305
Author(s) : Jaeger KE , Eggert T
Ref : Curr Opin Biotechnol , 15 :305 , 2004
Abstract : Directed evolution methods are now widely used for the optimization of diverse enzyme properties, which include biotechnologically relevant characteristics like stability, regioselectivity and, in particular, enantioselectivity. In principle, three different approaches are followed to optimize enantioselective reactions: the development of whole-cell biocatalysts through the creation of designer organisms; the optimization of enzymes with existing enantioselectivity for process conditions; and the evolution of novel enantioselective biocatalysts starting from non-selective wild-type enzymes.
ESTHER : Jaeger_2004_Curr.Opin.Biotechnol_15_305
PubMedSearch : Jaeger_2004_Curr.Opin.Biotechnol_15_305
PubMedID: 15358000

Title : Learning from directed evolution: theoretical investigations into cooperative mutations in lipase enantioselectivity - Bocola_2004_Chembiochem_5_214
Author(s) : Bocola M , Otte N , Jaeger KE , Reetz MT , Thiel W
Ref : Chembiochem , 5 :214 , 2004
Abstract : Molecular modeling with classical force-fields has been used to study the reactant complex and the tetrahedral intermediate in lipase-catalyzed ester hydrolysis in 20 enzyme/substrate combinations. The R and S enantiomers of alpha-methyldecanoic acid ester served as substrates for the wild-type lipase from Pseudomonas aeruginosa and nine selected mutants. After suitable preparation of initial structures from an available wild-type crystal structure, each system was subjected to 1 ns CHARMM force-field molecular dynamics simulations. The resulting geometric and energetic changes allow interpretation of some experimentally observed effects of mutations, particularly with regard to the "hot spots" at residues 155 and 162. The replacement S155F enhances S enantiopreference through a steric relay involving Leu162. The double mutation S53P + L162G improves S enantioselectivity by creating a new binding pocket for the S enantiomer with an additional stabilizing hydrogen bond to His83. The simulations provide insight into remote and cooperative effects of mutations.
ESTHER : Bocola_2004_Chembiochem_5_214
PubMedSearch : Bocola_2004_Chembiochem_5_214
PubMedID: 14760743
Gene_locus related to this paper: pseae-llipa

Title : Metagenome survey of biofilms in drinking-water networks - Schmeisser_2003_Appl.Environ.Microbiol_69_7298
Author(s) : Schmeisser C , Stockigt C , Raasch C , Wingender J , Timmis KN , Wenderoth DF , Flemming HC , Liesegang H , Schmitz RA , Jaeger KE , Streit WR
Ref : Applied Environmental Microbiology , 69 :7298 , 2003
Abstract : Most naturally occurring biofilms contain a vast majority of microorganisms which have not yet been cultured, and therefore we have little information on the genetic information content of these communities. Therefore, we initiated work to characterize the complex metagenome of model drinking water biofilms grown on rubber-coated valves by employing three different strategies. First, a sequence analysis of 650 16S rRNA clones indicated a high diversity within the biofilm communities, with the majority of the microbes being closely related to the Proteobacteria: Only a small fraction of the 16S rRNA sequences were highly similar to rRNA sequences from Actinobacteria, low-G+C gram-positives and the Cytophaga-Flavobacterium-Bacteroides group. Our second strategy included a snapshot genome sequencing approach. Homology searches in public databases with 5,000 random sequence clones from a small insert library resulted in the identification of 2,200 putative protein-coding sequences, of which 1,026 could be classified into functional groups. Similarity analyses indicated that significant fractions of the genes and proteins identified were highly similar to known proteins observed in the genera Rhizobium, Pseudomonas, and Escherichia: Finally, we report 144 kb of DNA sequence information from four selected cosmid clones, of which two formed a 75-kb overlapping contig. The majority of the proteins identified by whole-cosmid sequencing probably originated from microbes closely related to the alpha-, beta-, and gamma-Proteobacteria: The sequence information was used to set up a database containing the phylogenetic and genomic information on this model microbial community. Concerning the potential health risk of the microbial community studied, no DNA or protein sequences directly linked to pathogenic traits were identified.
ESTHER : Schmeisser_2003_Appl.Environ.Microbiol_69_7298
PubMedSearch : Schmeisser_2003_Appl.Environ.Microbiol_69_7298
PubMedID: 14660379
Gene_locus related to this paper: 9bact-q6wlc7

Title : Prospecting for novel biocatalysts in a soil metagenome - Voget_2003_Appl.Environ.Microbiol_69_6235
Author(s) : Voget S , Leggewie C , Uesbeck A , Raasch C , Jaeger KE , Streit WR
Ref : Applied Environmental Microbiology , 69 :6235 , 2003
Abstract : The metagenomes of complex microbial communities are rich sources of novel biocatalysts. We exploited the metagenome of a mixed microbial population for isolation of more than 15 different genes encoding novel biocatalysts by using a combined cultivation and direct cloning strategy. A 16S rRNA sequence analysis revealed the presence of hitherto uncultured microbes closely related to the genera Pseudomonas, Agrobacterium, Xanthomonas, Microbulbifer, and Janthinobacterium. Total genomic DNA from this bacterial community was used to construct cosmid DNA libraries, which were functionally searched for novel enzymes of biotechnological value. Our searches in combination with cosmid sequencing resulted in identification of four clones encoding 12 putative agarase genes, most of which were organized in clusters consisting of two or three genes. Interestingly, nine of these agarase genes probably originated from gene duplications. Furthermore, we identified by DNA sequencing several other biocatalyst-encoding genes, including genes encoding a putative stereoselective amidase (amiA), two cellulases (gnuB and uvs080), an alpha-amylase (amyA), a 1,4-alpha-glucan branching enzyme (amyB), and two pectate lyases (pelA and uvs119). Also, a conserved cluster of two lipase genes was identified, which was linked to genes encoding a type I secretion system. The novel gene aguB was overexpressed in Escherichia coli, and the enzyme activities were determined. Finally, we describe more than 162 kb of DNA sequence that provides a strong platform for further characterization of this microbial consortium.
ESTHER : Voget_2003_Appl.Environ.Microbiol_69_6235
PubMedSearch : Voget_2003_Appl.Environ.Microbiol_69_6235
PubMedID: 14532085
Gene_locus related to this paper: 9bact-q6xci1 , 9bact-q6xci2

Title : Extracellular lipases from Bacillus subtilis: regulation of gene expression and enzyme activity by amino acid supply and external pH - Eggert_2003_FEMS.Microbiol.Lett_225_319
Author(s) : Eggert T , Brockmeier U , Droge MJ , Quax WJ , Jaeger KE
Ref : FEMS Microbiology Letters , 225 :319 , 2003
Abstract : Bacillus subtilis secretes two lipases LipA and LipB into the culture medium. Both enzyme genes were differentially expressed depending on the growth conditions as determined by activity assays and Western blotting in B. subtilis mutant strains lipA, lipB, and the corresponding lipA/lipB double mutant. In minimal medium, LipA was produced at wild-type level in a lipB mutant, however, no LipB protein was detected in a lipA mutant. Interestingly, LipA was produced and secreted at wild-type level in rich medium, but the enzyme remained enzymatically inactive, presumably being caused by a shift of the growth medium to acid pH. Furthermore, expression of the lipase genes was studied using transcriptional fusions with the lacZ reporter gene. The expression of lipA was repressed by high amino acid concentrations, whereas the lipB gene expression remained unaffected.
ESTHER : Eggert_2003_FEMS.Microbiol.Lett_225_319
PubMedSearch : Eggert_2003_FEMS.Microbiol.Lett_225_319
PubMedID: 12951259

Title : Lipases for biotechnology - Jaeger_2002_Curr.Opin.Biotechnol_13_390
Author(s) : Jaeger KE , Eggert T
Ref : Curr Opin Biotechnol , 13 :390 , 2002
Abstract : Lipases constitute the most important group of biocatalysts for biotechnological applications. The high-level production of microbial lipases requires not only the efficient overexpression of the corresponding genes but also a detailed understanding of the molecular mechanisms governing their folding and secretion. The optimisation of industrially relevant lipase properties can be achieved by directed evolution. Furthermore, novel biotechnological applications have been successfully established using lipases for the synthesis of biopolymers and biodiesel, the production of enantiopure pharmaceuticals, agrochemicals, and flavour compounds.
ESTHER : Jaeger_2002_Curr.Opin.Biotechnol_13_390
PubMedSearch : Jaeger_2002_Curr.Opin.Biotechnol_13_390
PubMedID: 12323363

Title : Directed Evolution of an Enantioselective Enzyme through Combinatorial Multiple-Cassette Mutagenesis -
Author(s) : Reetz MT , Wilensek S , Zha D , Jaeger KE
Ref : Angew Chem Int Ed Engl , 40 :3589 , 2001
PubMedID: 11592190

Title : Disulfide bond in Pseudomonas aeruginosa lipase stabilizes the structure but is not required for interaction with its foldase - Liebeton_2001_J.Bacteriol_183_597
Author(s) : Liebeton K , Zacharias A , Jaeger KE
Ref : Journal of Bacteriology , 183 :597 , 2001
Abstract : Pseudomonas aeruginosa secretes a 29-kDa lipase which is dependent for folding on the presence of the lipase-specific foldase Lif. The lipase contains two cysteine residues which form an intramolecular disulfide bond. Variant lipases with either one or both cysteines replaced by serines showed severely reduced levels of extracellular lipase activity, indicating the importance of the disulfide bond for secretion of lipase through the outer membrane. Wild-type and variant lipase genes fused to the signal sequence of pectate lyase from Erwinia carotovora were expressed in Escherichia coli, denatured by treatment with urea, and subsequently refolded in vitro. Enzymatically active lipase was obtained irrespective of the presence or absence of the disulfide bond, suggesting that the disulfide bond is required neither for correct folding nor for the interaction with the lipase-specific foldase. However, cysteine-to-serine variants were more readily denatured by treatment at elevated temperatures and more susceptible to proteolytic degradation by cell lysates of P. aeruginosa. These results indicate a stabilizing function of the disulfide bond for the active conformation of lipase. This conclusion was supported by the finding that the disulfide bond function could partly be substituted by a salt bridge constructed by changing the two cysteine residues to arginine and aspartate, respectively.
ESTHER : Liebeton_2001_J.Bacteriol_183_597
PubMedSearch : Liebeton_2001_J.Bacteriol_183_597
PubMedID: 11133953
Gene_locus related to this paper: pseae-llipa

Title : The crystal structure of Bacillus subtilis lipase: a minimal alpha\/beta hydrolase fold enzyme - van Pouderoyen_2001_J.Mol.Biol_309_215
Author(s) : van Pouderoyen G , Eggert T , Jaeger KE , Dijkstra BW
Ref : Journal of Molecular Biology , 309 :215 , 2001
Abstract : The X-ray structure of the lipase LipA from Bacillus subtilis has been determined at 1.5 A resolution. It is the first structure of a member of homology family 1.4 of bacterial lipases. The lipase shows a compact minimal alpha/beta hydrolase fold with a six-stranded parallel beta-sheet flanked by five alpha-helices, two on one side of the sheet and three on the other side. The catalytic triad residues, Ser77, Asp133 and His156, and the residues forming the oxyanion hole (backbone amide groups of Ile12 and Met78) are in positions very similar to those of other lipases of known structure. However, no lid domain is present and the active-site nucleophile Ser77 is solvent-exposed. A model of substrate binding is proposed on the basis of a comparison with other lipases with a covalently bound tetrahedral intermediate mimic. It explains the preference of the enzyme for substrates with C8 fatty acid chains.
ESTHER : van Pouderoyen_2001_J.Mol.Biol_309_215
PubMedSearch : van Pouderoyen_2001_J.Mol.Biol_309_215
PubMedID: 11491291
Gene_locus related to this paper: bacsu-lip

Title : Directed evolution and the creation of enantioselective biocatalysts - Jaeger_2001_Appl.Microbiol.Biotechnol_55_519
Author(s) : Jaeger KE , Eggert T , Eipper A , Reetz MT
Ref : Applied Microbiology & Biotechnology , 55 :519 , 2001
Abstract : Directed evolution has emerged as a key technology to generate enzymes with new or improved properties that are of major importance to the biotechnology industry. A directed evolution approach starts with the identification of a target enzyme to be optimized and the cloning of the corresponding gene. An efficient expression system is needed before the target gene is subjected to random mutagenesis and/or in vitro recombination, thereby creating molecular diversity. Subsequently, improved enzyme variants are identified, preferably after being secreted into culture medium, by screening or selection for the desired property. The genes encoding the improved enzymes are then used to parent the next round of directed evolution. Enantioselectivity is a biocatalyst property of major biotechnological importance that is, however, difficult to deal with. We discuss recent examples of creating enantioselective biocatalysts by directed evolution.
ESTHER : Jaeger_2001_Appl.Microbiol.Biotechnol_55_519
PubMedSearch : Jaeger_2001_Appl.Microbiol.Biotechnol_55_519
PubMedID: 11414315

Title : Lipolytic enzymes LipA and LipB from Bacillus subtilis differ in regulation of gene expression, biochemical properties, and three-dimensional structure - Eggert_2001_FEBS.Lett_502_89
Author(s) : Eggert T , van Pouderoyen G , Dijkstra BW , Jaeger KE
Ref : FEBS Letters , 502 :89 , 2001
Abstract : Bacillus subtilis secretes the lipolytic enzymes LipA and LipB. We show here that they are differentially expressed depending on the composition of the growth medium: LipA is produced in rich and in minimal medium, whereas LipB is present only in rich medium. A comparison of biochemical characteristics revealed that LipB is thermostable at pH 11 but becomes thermolabile at pH 5. However, construction of a variant carrying the substitution A76G in the conserved lipase pentapeptide reversed these effects. The atomic coordinates from the LipA crystal structure were used to build a three-dimensional structural model of LipB, which revealed that 43 out of 45 residues different from LipA are surface-located allowing to rationalize the differences observed in the substrate preferences of the two enzymes.
ESTHER : Eggert_2001_FEBS.Lett_502_89
PubMedSearch : Eggert_2001_FEBS.Lett_502_89
PubMedID: 11583117
Gene_locus related to this paper: bacsu-lip , bacsu-LIPB

Title : A novel extracellular esterase from Bacillus subtilis and its conversion to a monoacylglycerol hydrolase - Eggert_2000_Eur.J.Biochem_267_6459
Author(s) : Eggert T , Pencreac'h G , Douchet I , Verger R , Jaeger KE
Ref : European Journal of Biochemistry , 267 :6459 , 2000
Abstract : A novel gene lipB, which encodes an extracellular lipolytic enzyme, was identified in the Bacillus subtilis genomic DNA sequence. We have cloned and overexpressed lipB in B. subtilis and Escherichia coli and have also purified the enzyme from a B. subtilis culture supernatant to electrophoretic homogeneity. Four different lipase assays were used to determine its catalytic activity: pH-stat, spectrophotometry, fluorimetry and the monomolecular film technique. LipB preferentially hydrolysed triacylglycerol-esters and p-nitrophenyl-esters of fatty acids with short chain lengths of <= 10 carbon atoms. Triolein, which is a typical substrate for true lipases, was not hydrolysed at all. These results led us to classify LipB as an esterase rather than a lipase. The catalytic triad of LipB consists of residues Ser78, Asp134, and His157 as demonstrated by amino-acid sequence alignments and site-directed mutagenesis. The nucleophile Ser78 is located in a lipase-specific consensus sequence, which is Ala-X-Ser-X-Gly for most Bacillus lipases. All other bacterial lipases contain a glycine residue instead of the alanine at position-2 with respect to the catalytic serine. We have investigated the role of this alanine residue by constructing LipB variant A76G, thereby restoring the lipase-specific consensus motif. When compared with LipB this variant showed a markedly reduced thermostability but an increased stability at pH 5-7. Determination of the specific activities of wild-type LipB and variant A76G using a monomolecular film of the substrate monoolein revealed an interesting result: the A76G substitution had converted the esterase LipB into a monoacylglycerol hydrolase.
ESTHER : Eggert_2000_Eur.J.Biochem_267_6459
PubMedSearch : Eggert_2000_Eur.J.Biochem_267_6459
PubMedID: 11029590
Gene_locus related to this paper: bacsu-LIPB

Title : Directed evolution of an enantioselective lipase - Liebeton_2000_Chem.Biol_7_709
Author(s) : Liebeton K , Zonta A , Schimossek K , Nardini M , Lang D , Dijkstra BW , Reetz MT , Jaeger KE
Ref : Chemical Biology , 7 :709 , 2000
Abstract : BACKGROUND: The biocatalytic production of enantiopure compounds is of steadily increasing importance to the chemical and biotechnological industry. In most cases, however, it is impossible to identify an enzyme that possesses the desired enantioselectivity. Therefore, there is a strong need to create by molecular biological methods novel enzymes which display high enantioselectivity.
RESULTS: A bacterial lipase from Pseudomonas aeruginosa (PAL) was evolved to catalyze with high enantioselectivity the hydrolysis of the chiral model substrate 2-methyldecanoic acid p-nitrophenyl ester. Successive rounds of random mutagenesis by ep-PCR and saturation mutagenesis resulted in an increase in enantioselectivity from E=1.1 for the wild-type enzyme to E=25.8 for the best variant which carried five amino acid substitutions. The recently solved three-dimensional structure of PAL allowed us to analyze the structural consequences of these substitutions.
CONCLUSIONS: A highly enantioselective lipase was created by increasing the flexibility of distinct loops of the enzyme. Our results demonstrate that enantioselective enzymes can be created by directed evolution, thereby opening up a large area of novel applications in biotechnology.
ESTHER : Liebeton_2000_Chem.Biol_7_709
PubMedSearch : Liebeton_2000_Chem.Biol_7_709
PubMedID: 10980451

Title : Crystal structure of pseudomonas aeruginosa lipase in the open conformation. The prototype for family I.1 of bacterial lipases - Nardini_2000_J.Biol.Chem_275_31219
Author(s) : Nardini M , Lang DA , Liebeton K , Jaeger KE , Dijkstra BW
Ref : Journal of Biological Chemistry , 275 :31219 , 2000
Abstract : The x-ray structure of the lipase from Pseudomonas aeruginosa PAO1 has been determined at 2.54 A resolution. It is the first structure of a member of homology family I.1 of bacterial lipases. The structure shows a variant of the alpha/beta hydrolase fold, with Ser(82), Asp(229), and His(251) as the catalytic triad residues. Compared with the "canonical" alpha/beta hydrolase fold, the first two beta-strands and one alpha-helix (alphaE) are not present. The absence of helix alphaE allows the formation of a stabilizing intramolecular disulfide bridge. The loop containing His(251) is stabilized by an octahedrally coordinated calcium ion. On top of the active site a lid subdomain is in an open conformation, making the catalytic cleft accessible from the solvent region. A triacylglycerol analogue is covalently bound to Ser(82) in the active site, demonstrating the position of the oxyanion hole and of the three pockets that accommodate the sn-1, sn-2, and sn-3 fatty acid chains. The inhibited enzyme can be thought to mimic the structure of the tetrahedral intermediate that occurs during the acylation step of the reaction. Analysis of the binding mode of the inhibitor suggests that the size of the acyl pocket and the size and interactions of the sn-2 binding pocket are the predominant determinants of the regio- and enantio-preference of the enzyme.
ESTHER : Nardini_2000_J.Biol.Chem_275_31219
PubMedSearch : Nardini_2000_J.Biol.Chem_275_31219
PubMedID: 10893416
Gene_locus related to this paper: pseae-llipa

Title : Directed evolution of enantioselective enzymes for organic chemistry - Jaeger_2000_Curr.Opin.Chem.Biol_4_68
Author(s) : Jaeger KE , Reetz MT
Ref : Curr Opin Chemical Biology , 4 :68 , 2000
Abstract : The production of enantiopure compounds is of steadily increasing importance to the chemical and biotechnological industries. In principal, the application of directed evolution in combination with newly developed screening methods enables the generation of enzymes with improved enantioselectivity. The first and most advanced example relates to a bacterial lipase from Pseudomonas aeruginosa. This enzyme was evolved towards a model substrate to yield in a lipase mutant showing > 90% enantiomeric excess as compared to 2% for the wild-type lipase. The creation of enantioselective enzymes by directed evolution will become an important technology in the near future.
ESTHER : Jaeger_2000_Curr.Opin.Chem.Biol_4_68
PubMedSearch : Jaeger_2000_Curr.Opin.Chem.Biol_4_68
PubMedID: 10679373

Title : Enantioselective enzymes for organic synthesis created by directed evolution - Reetz_2000_Chemistry_6_407
Author(s) : Reetz MT , Jaeger KE
Ref : Chemistry , 6 :407 , 2000
Abstract : A new concept for the creation of enzymes displaying improved enantioselectivity in a given reaction is described; it is based on "evolution in the test tube". Accordingly, proper molecular biological methods for random mutagenesis, gene expression, and high-throughput screening systems for the rapid assay of enantioselectivity are combined. Several rounds of mutagenesis and screening are generally necessary in order to create mutant enzymes that show high degrees of enantioselectivity, as in the case of the lipase-catalyzed hydrolytic kinetic resolution of a chiral ester in which the original enantioselectivity of 2 % ee (E = 1) increases to > 90% ee (E = 25).
ESTHER : Reetz_2000_Chemistry_6_407
PubMedSearch : Reetz_2000_Chemistry_6_407
PubMedID: 10747404

Title : Rapid gene inactivation in Pseudomonas aeruginosa - Windgassen_2000_FEMS.Microbiol.Lett_193_201
Author(s) : Windgassen M , Urban A , Jaeger KE
Ref : FEMS Microbiology Letters , 193 :201 , 2000
Abstract : A rapid and efficient method to inactivate genes in Pseudomonas aeruginosa has been developed. It is based on pKnockout vectors which carry either a gentamicin or a streptomycin/spectinomycin resistance cassette allowing for selection in P. aeruginosa where these vectors do not replicate. A PCR fragment of the gene of interest carrying 5'- and 3'-truncations is cloned into a pKnockout vector, mobilized into P. aeruginosa, and subsequently integrated into the chromosomal copy of the target gene. The orientation of the fragment determines whether (i) the target gene is disrupted without blocking the transcription of downstream genes or (ii) the insertion exerts a polar effect thereby leading to inactivation of a whole operon.
ESTHER : Windgassen_2000_FEMS.Microbiol.Lett_193_201
PubMedSearch : Windgassen_2000_FEMS.Microbiol.Lett_193_201
PubMedID: 11111024

Title : Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases - Jaeger_1999_Annu.Rev.Microbiol_53_315
Author(s) : Jaeger KE , Dijkstra BW , Reetz MT
Ref : Annu Rev Microbiol , 53 :315 , 1999
Abstract : Bacteria produce and secrete lipases, which can catalyze both the hydrolysis and the synthesis of long-chain acylglycerols. These reactions usually proceed with high regioselectivity and enantioselectivity, and, therefore, lipases have become very important stereoselective biocatalysts used in organic chemistry. High-level production of these biocatalysts requires the understanding of the mechanisms underlying gene expression, folding, and secretion. Transcription of lipase genes may be regulated by quorum sensing and two-component systems; secretion can proceed either via the Sec-dependent general secretory pathway or via ABC transporters. In addition, some lipases need folding catalysts such as the lipase-specific foldases and disulfide-bond-forming proteins to achieve a secretion-competent conformation. Three-dimensional structures of bacterial lipases were solved to understand the catalytic mechanism of lipase reactions. Structural characteristics include an alpha/beta hydrolase fold, a catalytic triad consisting of a nucleophilic serine located in a highly conserved Gly-X-Ser-X-Gly pentapeptide, and an aspartate or glutamate residue that is hydrogen bonded to a histidine. Four substrate binding pockets were identified for triglycerides: an oxyanion hole and three pockets accommodating the fatty acids bound at position sn-1, sn-2, and sn-3. The differences in size and the hydrophilicity/hydrophobicity of these pockets determine the enantiopreference of a lipase. The understanding of structure-function relationships will enable researchers to tailor new lipases for biotechnological applications. At the same time, directed evolution in combination with appropriate screening systems will be used extensively as a novel approach to develop lipases with high stability and enantioselectivity.
ESTHER : Jaeger_1999_Annu.Rev.Microbiol_53_315
PubMedSearch : Jaeger_1999_Annu.Rev.Microbiol_53_315
PubMedID: 10547694

Title : Specificity of the lipase-specific foldases of gram-negative bacteria and the role of the membrane anchor - El Khattabi_1999_Mol.Gen.Genet_261_770
Author(s) : El Khattabi M , Ockhuijsen C , Bitter W , Jaeger KE , Tommassen J
Ref : Molecular & General Genetics , 261 :770 , 1999
Abstract : Folding of lipases that are secreted by Pseudomonads and other gram-negative bacteria via the type II secretion pathway is facilitated by dedicated chaperones, called lipase-specific foldases (Lifs). Lifs are membrane-anchored proteins with a large periplasmic domain. The functional interaction between the Lif and its cognate lipase is specific, since the Pseudomonas aeruginosa Lif was found not to substitute for Lifs from Burkholderia glumae or Acinetobacter calcoaceticus. However, the P. aeruginosa Lif was able to activate the lipase from the closely related species P. alcaligenes. Hybrid proteins constructed from parts of the P. aeruginosa and B. glumae Lifs revealed that the C-terminal 138 amino acids of the B. glumae Lif determine the specificity of the interaction with the cognate lipase. Furthermore, the periplasmic domain of the B. glumae Lif was functional when cloned in frame with a cleavable signal sequence, which demonstrates that the membrane anchor is not essential for Lif function in vivo. However, the recombinant Lif was released into the medium, indicating that the function of the membrane anchor is to prevent secretion of the Lif together with the lipase.
ESTHER : El Khattabi_1999_Mol.Gen.Genet_261_770
PubMedSearch : El Khattabi_1999_Mol.Gen.Genet_261_770
PubMedID: 10394914

Title : Bacterial lipolytic enzymes: classification and properties - Arpigny_1999_Biochem.J_343_177
Author(s) : Arpigny JL , Jaeger KE
Ref : Biochemical Journal , 343 :177 , 1999
Abstract : Knowledge of bacterial lipolytic enzymes is increasing at a rapid and exciting rate. To obtain an overview of this industrially very important class of enzymes and their characteristics, we have collected and classified the information available from protein and nucleotide databases. Here we propose an updated and extensive classification of bacterial esterases and lipases based mainly on a comparison of their amino acid sequences and some fundamental biological properties. These new insights result in the identification of eight different families with the largest being further divided into six subfamilies. Moreover, the classification enables us to predict (1) important structural features such as residues forming the catalytic site or the presence of disulphide bonds, (2) types of secretion mechanism and requirement for lipase-specific foldases, and (3) the potential relationship to other enzyme families. This work will therefore contribute to a faster identification and to an easier characterization of novel bacterial lipolytic enzymes.
ESTHER : Arpigny_1999_Biochem.J_343_177
PubMedSearch : Arpigny_1999_Biochem.J_343_177
PubMedID: 10493927

Title : A novel lipolytic enzyme located in the outer membrane of Pseudomonas aeruginosa - Wilhelm_1999_J.Bacteriol_181_6977
Author(s) : Wilhelm S , Tommassen J , Jaeger KE
Ref : Journal of Bacteriology , 181 :6977 , 1999
Abstract : A lipase-negative deletion mutant of Pseudomonas aeruginosa PAO1 still showed extracellular lipolytic activity toward short-chain p-nitrophenylesters. By screening a genomic DNA library of P. aeruginosa PAO1, an esterase gene, estA, was identified, cloned, and sequenced, revealing an open reading frame of 1,941 bp. The product of estA is a 69.5-kDa protein, which is probably processed by removal of an N-terminal signal peptide to yield a 67-kDa mature protein. A molecular mass of 66 kDa was determined for (35)S-labeled EstA by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The amino acid sequence of EstA indicated that the esterase is a member of a novel GDSL family of lipolytic enzymes. The estA gene showed high similarity to an open reading frame of unknown function located in the trpE-trpG region of P. putida and to a gene encoding an outer membrane esterase of Salmonella typhimurium. Amino acid sequence alignments led us to predict that this esterase is an autotransporter protein which possesses a carboxy-terminal beta-barrel domain, allowing the secretion of the amino-terminal passenger domain harboring the catalytic activity. Expression of estA in P. aeruginosa and Escherichia coli and subsequent cell fractionation revealed that the enzyme was associated with the cellular membranes. Trypsin treatment of whole cells released a significant amount of esterase, indicating that the enzyme was located in the outer membrane with the catalytic domain exposed to the surface. To our knowledge, this esterase is unique in that it exemplifies in P. aeruginosa (i) the first enzyme identified in the outer membrane and (ii) the first example of a type IV secretion mechanism.
ESTHER : Wilhelm_1999_J.Bacteriol_181_6977
PubMedSearch : Wilhelm_1999_J.Bacteriol_181_6977
PubMedID: 10559163

Title : A novel heat-stable lipolytic enzyme from Sulfolobus acidocaldarius DSM 639 displaying similarity to polyhydroxyalkanoate depolymerases - Arpigny_1998_FEMS.Microbiol.Lett_167_69
Author(s) : Arpigny JL , Jendrossek D , Jaeger KE
Ref : FEMS Microbiology Letters , 167 :69 , 1998
Abstract : A fragment of genomic DNA from Sulfolobus acidocaldarius DSM 639 encoding a lipolytic enzyme was cloned and sequenced. The 314-amino acid polypeptide displays a maximum sequence similarity (43%) to a putative polyhydroxyalkanoate depolymerase from Pseudomonas oleovorans and contains the pentapeptide G-X1-S-X2-G which is typical of serine hydrolases. The protein is highly thermostable and is able to hydrolyse a variety of lipid substrates thus providing a promising tool for potential biotechnological applications.
ESTHER : Arpigny_1998_FEMS.Microbiol.Lett_167_69
PubMedSearch : Arpigny_1998_FEMS.Microbiol.Lett_167_69
PubMedID: 9785454
Gene_locus related to this paper: sulac-sal

Title : Microbial lipases form versatile tools for biotechnology - Jaeger_1998_Trends.Biotechnol_16_396
Author(s) : Jaeger KE , Reetz MT
Ref : Trends Biotechnol , 16 :396 , 1998
Abstract : Lipases are secreted into the culture medium by many bacteria and fungi. They catalyse not only the hydrolysis but also the synthesis of long-chain acylglycerols. Important uses in biotechnology include their addition to detergents, the manufacture of food ingredients, pitch control in the pulp and paper industry, and biocatalysis of stereoselective transformations. This makes them the most widely used class of enzymes in organic chemistry. Immobilization in hydrophobic sol-gel matrices and in vitro evolution are promising novel approaches to increasing the stability or enantioselectivity, respectively, of lipases.
ESTHER : Jaeger_1998_Trends.Biotechnol_16_396
PubMedSearch : Jaeger_1998_Trends.Biotechnol_16_396
PubMedID: 9744114

Title : Overexpression, immobilization and biotechnological application of Pseudomonas lipases - Reetz_1998_Chem.Phys.Lipids_93_3
Author(s) : Reetz MT , Jaeger KE
Ref : Chemistry & Physic of Lipids , 93 :3 , 1998
Abstract : Pseudomonas lipases play an important role in biotechnology both as hydrolases for detergent additives and as synthases catalyzing the kinetic resolution of racemic compounds. Large-scale production of Pseudomonas lipases requires correct folding and secretion through the bacterial membranes. Controllable expression of the gene lipH encoding a lipase-specific foldase proves to be important for overexpression in the homologous host Escherichia coli. Construction of appropriate His-tagged fusion proteins permitted overexpression, secretion and one-step purification of lipase from culture supernatants of the homologous host Pseudomonas aeruginosa. The immobilization of lipases in hydrophobic sol-gel materials derived from alkylsilane precursors of the type RSi(OCH3)3 or mixtures of RSi(OCH3)3 and Si(OCH3)4 provides highly active chemically and thermally stable heterogeneous biocatalysts. The entrapped lipases are excellent catalysts in a variety of synthetic organic transformations. Using directed evolution based on error prone PCR, the enantioselectivity of the hydrolysis of a chiral ester, catalyzed by the lipase from P. aeruginosa, can be increased from ee 2 to ee 81% in just four mutagenesis cycles.
ESTHER : Reetz_1998_Chem.Phys.Lipids_93_3
PubMedSearch : Reetz_1998_Chem.Phys.Lipids_93_3
PubMedID: 9720245

Title : Anaerobically controlled expression system derived from the arcDABC operon of Pseudomonas aeruginosa: application to lipase production - Winteler_1996_Appl.Environ.Microbiol_62_3391
Author(s) : Winteler HV , Schneidinger B , Jaeger KE , Haas D
Ref : Applied Environmental Microbiology , 62 :3391 , 1996
Abstract : The anaerobically inducible arcDABC operon encodes the enzymes of the arginine deiminase pathway in Pseudomonas aeruginosa. Upon induction, the arcAB mRNAs and proteins reach high intracellular levels, because of a strong anaerobically controlled promoter and mRNA processing in arcD, leading to stable downstream transcripts. We explored the usefulness of this system for the construction of expression vectors. The lacZ gene of Escherichia coli was expressed to the highest levels when fused close to the arc promoter. Insertion of lacZ further downstream into arcA or arcB did not stabilize the intrinsically unstable lacZ mRNA. On the contrary, lacZ mRNA appeared to be a vulnerable endonuclease target destabilizing arcAB mRNAs in the 5'-to-3' direction in P. aeruginosa. The native arc promoter was modified for optional expression in the -10 sequence and in the -40 region, which is a binding site for the anaerobic regulator ANR. In P. aeruginosa grown either anaerobically or with oxygen limitation in unshaken cultures, this promoter was stronger than the induced tac promoter. The P. aeruginosa lipAH genes, which encode extracellular lipase and lipase foldase, respectively, were fused directly to the modified arc promoter in an IncQ vector plasmid. Semianaerobic static cultures of P. aeruginosa PAO1 carrying this recombinant plasmid overproduced extracellular lipase 30-fold during stationary phase compared with the production by strain PAO1 without the plasmid. Severe oxygen limitation, in contrast, resulted in poor lipase productivity despite effective induction of the ANR-dependent promoter, suggesting that secretion of active lipase is blocked by the absence of oxygen. In conclusion, the modified arc promoter is useful for driving the expression of cloned genes in P. aeruginosa during oxygen-limited growth and stationary phase.
ESTHER : Winteler_1996_Appl.Environ.Microbiol_62_3391
PubMedSearch : Winteler_1996_Appl.Environ.Microbiol_62_3391
PubMedID: 8795231

Title : Role of Pseudomonas aeruginosa lipase in inflammatory mediator release from human inflammatory effector cells (platelets, granulocytes, and monocytes - Konig_1996_Infect.Immun_64_3252
Author(s) : Konig B , Jaeger KE , Sage AE , Vasil ML , Konig W
Ref : Infect Immun , 64 :3252 , 1996
Abstract : Previously, we have shown that Pseudomonas aeruginosa lipase and phospholipase C (PLC), two extracellular lipolytic enzymes, interact with each other during 12-hydroxyeicosatetraenoic acid (HETE) generation from human platelets. In this regard. the addition of purified P. aeruginosa lipase to PLC-containing crude P. aeruginosa culture supernatants enhances the generation of the chemotactically active 12-HETE from human platelets. Therefore, we analyzed the interaction of purified P. aeruginosa lipase and purified hemolytic P. aeruginosa PLC with regard to inflammatory mediator release from human platelets, neutrophilic and basophilic granulocytes, and monocytes. Purified P. aeruginosa PLC, but not purified lipase by itself, induced 12-HETE generation from human platelets, the generation of leukotriene B4 (LTB4) and oxygen metabolites, enzyme release from human neutrophils, and histamine release from basophils but diminished interleukin-8 (IL-8) release from human monocytes in a dose-dependent manner. The addition of purified lipase enhanced PLC-induced 12-HETE and LTB4 generation, did not influence enzyme, histamine, or IL-8 release, but diminished the PLC-induced chemiluminescent response. Similar results were obtained when the hemolytic PLC from Clostridium perfringens was used instead of P. aeruginosa PLC. For further comparison, we used the well-defined calcium ionophore A23187 and phorbol-12-myristate-13-acetate (PMA) as stimuli. Lipase enhanced calcium ionophore-induced LTB4 generation and beta-glucuronidase release but reduced calcium ionophore-induced and PMA-induced chemiluminescence. In parallel, we analyzed the role of lipase in a crude P. aeruginosa culture supernatant containing PLC and lipase. Lipase activity in the P. aeruginosa culture supernatant was inhibited by treatment with the lipase-specific inhibitor hexadecylsulfonyl fluoride, leaving the activity of PLC unaffected. The capacity of "lipase-inactivated culture supernatant" to induce 12-HETE and LTB4 generation was diminished by 50 to 100%. Our results suggest that the simultaneous secretion of lipase and PLC by P. aeruginosa residing in an infected host may result in severe pathological effects which cannot be explained by the sole action of the individual virulence factor on inflammatory effector cells.
ESTHER : Konig_1996_Infect.Immun_64_3252
PubMedSearch : Konig_1996_Infect.Immun_64_3252
PubMedID: 8757861

Title : Substrate specificities of bacterial polyhydroxyalkanoate depolymerases and lipases: bacterial lipases hydrolyze poly(omega-hydroxyalkanoates) - Jaeger_1995_Appl.Environ.Microbiol_61_3113
Author(s) : Jaeger KE , Steinbuchel A , Jendrossek D
Ref : Applied Environmental Microbiology , 61 :3113 , 1995
Abstract : The substrate specificities of extracellular lipases purified from Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas alcaligenes, Pseudomonas fluorescens, and Burkholderia cepacia (former Pseudomonas cepacia) and of extracellular polyhydroxyalkanoate (PHA) depolymerases purified from Comamonas sp., Pseudomonas lemoignei, and P. fluorescens GK13, as well as that of an esterase purified from P. fluorescens GK 13, to various polyesters and to lipase substrates were analyzed. All lipases and the esterase of P. fluorescens GK13 but none of the PHA depolymerases tested hydrolyzed triolein, thereby confirming a functional difference between lipases and PHA depolymerases. However, most lipases were able to hydrolyze polyesters consisting of an omega-hydroxyalkanoic acid such as poly(6-hydroxyhedxanoate) or poly(4-hydroxybutyrate). The dimeric ester of hydroxyhexanoate was the main product of enzymatic hydrolysis of polycaprolactone by P. aeruginosa lipase. Polyesters containing side chains in the polymer backbone such as poly (3-hydroxybutyrate) and other poly(3-hydroxyalkanoates) were not or were only slightly hydrolyzed by the lipases tested.
ESTHER : Jaeger_1995_Appl.Environ.Microbiol_61_3113
PubMedSearch : Jaeger_1995_Appl.Environ.Microbiol_61_3113
PubMedID: 7487042

Title : Gene cloning, sequence analysis, purification, and secretion by Escherichia coli of an extracellular lipase from Serratia marcescens - Li_1995_Appl.Environ.Microbiol_61_2674
Author(s) : Li X , Tetling S , Winkler UK , Jaeger KE , Benedik MJ
Ref : Applied Environmental Microbiology , 61 :2674 , 1995
Abstract : The gene encoding extracellular lipase of Serratia marcescens has been identified from a phage lambda genomic library. Formation of orange-red fluorescent plaques on rhodamine B-triolein plates was used to identify phages carrying the lipase gene. A 2.8-kb SalI fragment was subcloned into a plasmid, and lipase was expressed in Escherichia coli. Extracellular lipase was detected in the presence of the secretion plasmid pGSD6 carrying the genes prtD, -E, and -F, which guide the secretion of protease from Erwinia chrysanthemi. Determination of the nucleotide sequence of the entire cloned fragment revealed an open reading frame coding for a 613-amino-acid protein with a predicted M(r) of 64,800. Analysis of the amino acid sequence revealed significant homology (around 70%) to lipases of Pseudomonas fluorescens strains. The lipase-specific consensus sequence G-X1-S-X2-G resided in the amino-terminal part of the protein, and carboxyl-terminal consensus sequences were an L-X-G-G-B-G-B-B-X repeat motif and a so-called aspartate box, respectively, which are both found in proteins secreted by the class I secretion pathway. Lipase was purified from the supernatant of a culture carrying a lipase expression vector, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an M(r) of 64,000 for the purified protein. Our results suggest that the lipase of S. marcescens belongs to the group of extracellular enzyme proteins secreted by the class I secretion pathway.
ESTHER : Li_1995_Appl.Environ.Microbiol_61_2674
PubMedSearch : Li_1995_Appl.Environ.Microbiol_61_2674
PubMedID: 7618881
Gene_locus related to this paper: serma-lipasA

Title : Lipase from Chromobacterium viscosum: biochemical characterization indicating homology to the lipase from Pseudomonas glumae - Taipa_1995_Biochim.Biophys.Acta_1256_396
Author(s) : Taipa MA , Liebeton K , Costa JV , Cabral JM , Jaeger KE
Ref : Biochimica & Biophysica Acta , 1256 :396 , 1995
Abstract : Previous purification of a commercial lipolytic preparation from Chromobacterium viscosum using gel filtration chromatography yielded two enzymatically active fractions, named lipases A and B. Characterization of these fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that lipase A consisted of a high molecular weight aggregate of lipase protein with lipopolysaccharides. This complex could be dissociated by treatment with EDTA-Tris buffer containing the non-ionic detergent n-octyl-beta-D-glucopyranoside and subsequent isoelectric focusing in an agarose gel containing the same detergent. Both lipases A and B revealed a major peak corresponding to an isoelectric point of 7.1. SDS-PAGE analysis of lipases A and B after purification by gel filtration or by IEF revealed one major protein band of M(r) of 33 K. Determination of N-terminal amino acid sequences confirmed that both fractions A and B contained the same lipase protein. Furthermore, the N-terminal amino acid sequence of the C. viscosum lipase was identical to the one of Pseudomonas glumae lipase.
ESTHER : Taipa_1995_Biochim.Biophys.Acta_1256_396
PubMedSearch : Taipa_1995_Biochim.Biophys.Acta_1256_396
PubMedID: 7786905
Gene_locus related to this paper: burgl-lipas

Title : The structure-function relationship of the lipases from Pseudomonas aeruginosa and Bacillus subtilis - Misset_1994_Protein.Eng_7_523
Author(s) : Misset O , Gerritse G , Jaeger KE , Winkler U , Colson C , Schanck K , Lesuisse E , Dartois V , Blaauw M , Ransac S , et al.
Ref : Protein Engineering , 7 :523 , 1994
Abstract : Within the BRIDGE T-project on lipases we investigate the structure-function relationships of the lipases from Bacillus subtilis and Pseudomonas aeruginosa. Construction of an overproducing Bacillus strain allowed the purification of > 100 mg lipase from 30 l culture supernatant. After testing a large variety of crystallization conditions, the Bacillus lipase gave crystals of reasonable quality in PEG-4000 (38-45%), Na2SO4 and octyl-beta-glucoside at 22 degrees C, pH 9.0. A 2.5 A dataset has been obtained which is complete from 15 to 2.5 A resolution. P.aeruginosa wild-type strain PAC1R was fermented using conditions of maximum lipase production. More than 90% of the lipase was cell bound and could be solubilized by treatment of the cells with Triton X-100. This permitted the purification of approximately 50 mg lipase. So far, no crystals of sufficient quality were obtained. Comparison of the model we built for the Pseudomonas lipase, on the basis of sequences and structures of various hydrolases which were found to possess a common folding pattern (alpha/beta hydrolase fold), with the X-ray structure of the P.glumae lipase revealed that it is possible to correctly build the structure of the core of a protein even in the absence of obvious sequence homology with a protein of known 3-D structure.
ESTHER : Misset_1994_Protein.Eng_7_523
PubMedSearch : Misset_1994_Protein.Eng_7_523
PubMedID: 8029207

Title : Bacterial lipases - Jaeger_1994_FEMS.Microbiol.Rev_15_29
Author(s) : Jaeger KE , Ransac S , Dijkstra BW , Colson C , van Heuvel M , Misset O
Ref : FEMS Microbiology Reviews , 15 :29 , 1994
Abstract : Many different bacterial species produce lipases which hydrolyze esters of glycerol with preferably long-chain fatty acids. They act at the interface generated by a hydrophobic lipid substrate in a hydrophilic aqueous medium. A characteristic property of lipases is called interfacial activation, meaning a sharp increase in lipase activity observed when the substrate starts to form an emulsion, thereby presenting to the enzyme an interfacial area. As a consequence, the kinetics of a lipase reaction do not follow the classical Michaelis-Menten model. With only a few exceptions, bacterial lipases are able to completely hydrolyze a triacylglycerol substrate although a certain preference for primary ester bonds has been observed. Numerous lipase assay methods are available using coloured or fluorescent substrates which allow spectroscopic and fluorimetric detection of lipase activity. Another important assay is based on titration of fatty acids released from the substrate. Newly developed methods allow to exactly determine lipase activity via controlled surface pressure or by means of a computer-controlled oil drop tensiometer. The synthesis and secretion of lipases by bacteria is influenced by a variety of environmental factors like ions, carbon sources, or presence of non-metabolizable polysaccharides. The secretion pathway is known for Pseudomonas lipases with P. aeruginosa lipase using a two-step mechanism and P. fluorescens lipase using a one-step mechanism. Additionally, some Pseudomonas lipases need specific chaperone-like proteins assisting their correct folding in the periplasm. These lipase-specific foldases (Lif-proteins) which show a high degree of amino acid sequence homology among different Pseudomonas species are coded for by genes located immediately downstream the lipase structural genes. A comparison of different bacterial lipases on the basis of primary structure revealed only very limited sequence homology. However, determination of the three-dimensional structure of the P. glumae lipase indicated that at least some of the bacterial lipases will presumably reveal a conserved folding pattern called the alpha/beta-hydrolase fold, which has been described for other microbial and human lipases. The catalytic site of lipases is buried inside the protein and contains a serine-protease-like catalytic triad consisting of the amino acids serine, histidine, and aspartate (or glutamate). The Ser-residue is located in a strictly conserved beta-epsilon Ser-alpha motif. The active site is covered by a lid-like alpha-helical structure which moves away upon contact of the lipase with its substrate, thereby exposing hydrophobic residues at the protein's surface mediating the contact between protein and substrate.
ESTHER : Jaeger_1994_FEMS.Microbiol.Rev_15_29
PubMedSearch : Jaeger_1994_FEMS.Microbiol.Rev_15_29
PubMedID: 7946464

Title : Topological characterization and modeling of the 3D structure of lipase from Pseudomonas aeruginosa - Jaeger_1993_FEBS.Lett_332_143
Author(s) : Jaeger KE , Ransac S , Koch HB , Ferrato F , Dijkstra BW
Ref : FEBS Letters , 332 :143 , 1993
Abstract : Lipase from Pseudomonas aeruginosa is a M(r) 29 kDa protein with a single functional disulfide bond as shown by a shift in electrophoretic mobility after treatment with dithiothreitol and iodoacetamide. Limited proteolysis of lipase with Staphylococcus aureus protease V8 resulted in cleavage after amino acid residues Asp38 and Glu46. Comparison of the lipase amino acid sequence with those of other hydrolases with known 3D structures indicated that the folding pattern might be compatible with the alpha/beta hydrolase fold, thereby allowing us to construct a 3D model which fitted the biochemical properties. The model predicts a catalytic triad consisting of Ser82, Asp229 and His251, and contains a disulfide bond connecting residues Cys183 and Cys235. Residues Asp38 and Glu46 are located at the surface of the enzyme, whereas the disulfide bond is rather inaccessible, which is in agreement with the finding that the protein needed to be partly unfolded before a reduction of the disulfide bond could take place. A striking prediction from the model was the lack of a lid-like alpha-helical loop structure covering the active site which confers to other well-characterized lipases a unique property known as interfacial activation. Experimental determination of lipase activity under conditions where the substrate existed either as monomeric solutions or aggregates confirmed the absence of interfacial activation.
ESTHER : Jaeger_1993_FEBS.Lett_332_143
PubMedSearch : Jaeger_1993_FEBS.Lett_332_143
PubMedID: 8405431

Title : Extracellular lipase from Pseudomonas aeruginosa is an amphiphilic protein - Jaeger_1992_Biochim.Biophys.Acta_1120_315
Author(s) : Jaeger KE , Adrian FJ , Meyer HE , Hancock RE , Winkler UK
Ref : Biochimica & Biophysica Acta , 1120 :315 , 1992
Abstract : Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) secreted by Pseudomonas aeruginosa PAC1R was purified from cell-free growth medium by preparative isoelectric focusing. After blotting the N-terminal amino acid sequence and the amino acid composition were determined and compared to P. fragi and P. cepacia lipases yielding significant homology between all three species. Additionally, a consensus sequence K-Y-P-i-v-l-V-H-G was identified residing at the N-terminus of Pseudomonas lipases and in the central part of Staphylococcus lipases. Treatment of lipase with the serine-specific inhibitor diethyl p-nitrophenyl phosphate caused a rapid and complete inhibition of enzyme activity indicating the presence of a serine at the catalytic site as expected from lipase consensus sequences. Upon charge-shift electrophoresis the electrophoretic mobility of purified lipase was shifted either anodally or cathodally in the presence of sodium deoxycholate and cetyltrimethylammoniumbromide, respectively. This result demonstrates that extracellular lipase of P. aeruginosa exhibits an amphiphilic character like intrinsic membrane proteins.
ESTHER : Jaeger_1992_Biochim.Biophys.Acta_1120_315
PubMedSearch : Jaeger_1992_Biochim.Biophys.Acta_1120_315
PubMedID: 1576157
Gene_locus related to this paper: pseae-llipa

Title : Extracellular lipase of Pseudomonas aeruginosa: biochemical characterization and effect on human neutrophil and monocyte function in vitro - Jaeger_1991_Microb.Pathog_10_173
Author(s) : Jaeger KE , Kharazmi A , Hoiby N
Ref : Microb Pathog , 10 :173 , 1991
Abstract : Lipase was isolated from P. aeruginosa by ultrafiltration of sterile-filtered culture supernatant. Gel filtration on Sepharose 4B yielded a broad peak corresponding to a molecular mass range of 100 to 1000 kDa. Electron microscopy of a negatively stained lipase preparation after Sepharose 4B chromatography revealed spherical particles with diameters ranging from 5 to 20 nm. Biochemical characterization and SDS polyacrylamide gel electrophoresis suggested that these particles consisted of protein and carbohydrate including lipopolysaccharide with the major enzyme activity being lipase. Various concentrations of this lipase preparation were preincubated with human peripheral blood neutrophils and monocytes. The chemotaxis and chemiluminescence of these cells were then determined. It was shown that lipase inhibited the monocyte chemotaxis and chemiluminescence, whereas it had no or very little effect on neutrophils. The inhibitory effect was concentration dependent and was abolished by heat treatment of the enzyme at 100 degrees C. Since monocytes are one of the important cells of the host defence system the inhibition of the function of these cells may contribute to the pathogenesis of infections caused by P. aeruginosa.
ESTHER : Jaeger_1991_Microb.Pathog_10_173
PubMedSearch : Jaeger_1991_Microb.Pathog_10_173
PubMedID: 1910141
Gene_locus related to this paper: pseae-llipa

Title : Specific and sensitive plate assay for bacterial lipases - Kouker_1987_Appl.Environ.Microbiol_53_211
Author(s) : Kouker G , Jaeger KE
Ref : Applied Environmental Microbiology , 53 :211 , 1987
Abstract : A plate assay to detect bacterial lipase (EC 3.1.1.3) in a medium containing trioleoylglycerol and the fluorescent dye rhodamine B is presented. Substrate hydrolysis causes the formation of orange fluorescent halos around bacterial colonies visible upon UV irradiation. The logarithm of lipase activity from cell-free culture supernatants is linearly correlated with the diameter of halos, thereby allowing quantitation of lipase activities ranging from 1 to 30 nkat.
ESTHER : Kouker_1987_Appl.Environ.Microbiol_53_211
PubMedSearch : Kouker_1987_Appl.Environ.Microbiol_53_211
PubMedID: 3103532

Title : Purification of extracellular lipase from Pseudomonas aeruginosa - Stuer_1986_J.Bacteriol_168_1070
Author(s) : Stuer W , Jaeger KE , Winkler UK
Ref : Journal of Bacteriology , 168 :1070 , 1986
Abstract : Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) was excreted by Pseudomonas aeruginosa PAC1R during the late logarithmic growth phase. Characterization of cell-free culture supernatants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of significant amounts of lipopolysaccharide, part of which seemed to be tightly bound to lipase. After concentration of culture supernatants by ultrafiltration, lipase-lipopolysaccharide complexes were dissociated by treatment with EDTA-Tris buffer and subsequent sonication in the presence of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized lipase was purified by isoelectric focusing in an agarose gel containing the same detergent; the lipase activity appeared in a single peak corresponding to a distinct band in the silver-stained gel. The isoelectric point was 5.8. Analysis of purified lipase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and scanning revealed an apparent molecular weight of 29,000 and a specific activity of 760 mu kat/mg of protein. Estimations based on these data showed that a single P. aeruginosa cell excreted about 200 molecules of lipase, each having a molecular activity of 2.2 X 10(4) per s.
ESTHER : Stuer_1986_J.Bacteriol_168_1070
PubMedSearch : Stuer_1986_J.Bacteriol_168_1070
PubMedID: 3096967