Golyshin PN

References (32)

Title : Thermophilic Carboxylesterases from Hydrothermal Vents of the Volcanic Island of Ischia Active on Synthetic and Biobased Polymers and Mycotoxins - Distaso_2023_Appl.Environ.Microbiol__e0170422
Author(s) : Distaso MA , Chernikova TN , Bargiela R , Coscolin C , Stogios P , Gonzalez-Alfonso JL , Lemak S , Khusnutdinova AN , Plou FJ , Evdokimova E , Savchenko A , Lunev EA , Yakimov MM , Golyshina OV , Ferrer M , Yakunin AF , Golyshin PN
Ref : Applied Environmental Microbiology , :e0170422 , 2023
Abstract : Hydrothermal vents are geographically widespread and host microorganisms with robust enzymes useful in various industrial applications. We examined microbial communities and carboxylesterases of two terrestrial hydrothermal vents of the volcanic island of Ischia (Italy) predominantly composed of Firmicutes, Proteobacteria, and Bacteroidota. High-temperature enrichment cultures with the polyester plastics polyhydroxybutyrate and polylactic acid (PLA) resulted in an increase of Thermus and Geobacillus species and to some extent Fontimonas and Schleiferia species. The screening at 37 to 70 degreesC of metagenomic fosmid libraries from above enrichment cultures identified three hydrolases (IS10, IS11, and IS12), all derived from yet-uncultured Chloroflexota and showing low sequence identity (33 to 56%) to characterized enzymes. Enzymes expressed in Escherichia coli exhibited maximal esterase activity at 70 to 90 degreesC, with IS11 showing the highest thermostability (90% activity after 20-min incubation at 80 degreesC). IS10 and IS12 were highly substrate promiscuous and hydrolyzed all 51 monoester substrates tested. Enzymes were active with PLA, polyethylene terephthalate model substrate, and mycotoxin T-2 (IS12). IS10 and IS12 had a classical alpha/beta-hydrolase core domain with a serine hydrolase catalytic triad (Ser155, His280, and Asp250) in their hydrophobic active sites. The crystal structure of IS11 resolved at 2.92 A revealed the presence of a N-terminal beta-lactamase-like domain and C-terminal lipocalin domain. The catalytic cleft of IS11 included catalytic Ser68, Lys71, Tyr160, and Asn162, whereas the lipocalin domain enclosed the catalytic cleft like a lid and contributed to substrate binding. Our study identified novel thermotolerant carboxylesterases with a broad substrate range, including polyesters and mycotoxins, for potential applications in biotechnology. IMPORTANCE High-temperature-active microbial enzymes are important biocatalysts for many industrial applications, including recycling of synthetic and biobased polyesters increasingly used in textiles, fibers, coatings and adhesives. Here, we identified three novel thermotolerant carboxylesterases (IS10, IS11, and IS12) from high-temperature enrichment cultures from Ischia hydrothermal vents and incubated with biobased polymers. The identified metagenomic enzymes originated from uncultured Chloroflexota and showed low sequence similarity to known carboxylesterases. Active sites of IS10 and IS12 had the largest effective volumes among the characterized prokaryotic carboxylesterases and exhibited high substrate promiscuity, including hydrolysis of polyesters and mycotoxin T-2 (IS12). Though less promiscuous than IS10 and IS12, IS11 had a higher thermostability with a high temperature optimum (80 to 90 degreesC) for activity and hydrolyzed polyesters, and its crystal structure revealed an unusual lipocalin domain likely involved in substrate binding. The polyesterase activity of these enzymes makes them attractive candidates for further optimization and potential application in plastics recycling.
ESTHER : Distaso_2023_Appl.Environ.Microbiol__e0170422
PubMedSearch : Distaso_2023_Appl.Environ.Microbiol__e0170422
PubMedID: 36719236
Gene_locus related to this paper: 9bact-estC55.8n1 , 9bact-IS10

Title : The Mobility of the Cap Domain Is Essential for the Substrate Promiscuity of a Family IV Esterase from Sorghum Rhizosphere Microbiome - Distaso_2023_Appl.Environ.Microbiol__e0180722
Author(s) : Distaso M , Cea-Rama I , Coscolin C , Chernikova TN , Tran H , Ferrer M , Sanz-Aparicio J , Golyshin PN
Ref : Applied Environmental Microbiology , :e0180722 , 2023
Abstract : Metagenomics offers the possibility to screen for versatile biocatalysts. In this study, the microbial community of the Sorghum bicolor rhizosphere was spiked with technical cashew nut shell liquid, and after incubation, the environmental DNA (eDNA) was extracted and subsequently used to build a metagenomic library. We report the biochemical features and crystal structure of a novel esterase from the family IV, EH(0), retrieved from an uncultured sphingomonad after a functional screen in tributyrin agar plates. EH(0) (optimum temperature [T(opt)], 50 degreesC; melting temperature [T(m)], 55.7 degreesC; optimum pH [pH(opt)], 9.5) was stable in the presence of 10 to 20% (vol/vol) organic solvents and exhibited hydrolytic activity against p-nitrophenyl esters from acetate to palmitate, preferably butyrate (496 U mg(-1)), and a large battery of 69 structurally different esters (up to 30.2 U mg(-1)), including bis(2-hydroxyethyl)-terephthalate (0.16 +/- 0.06 U mg(-1)). This broad substrate specificity contrasts with the fact that EH(0) showed a long and narrow catalytic tunnel, whose access appears to be hindered by a tight folding of its cap domain. We propose that this cap domain is a highly flexible structure whose opening is mediated by unique structural elements, one of which is the presence of two contiguous proline residues likely acting as possible hinges, which together allow for the entrance of the substrates. Therefore, this work provides a new role for the cap domain, which until now was thought to be an immobile element that contained hydrophobic patches involved in substrate prerecognition and in turn substrate specificity within family IV esterases. IMPORTANCE A better understanding of structure-function relationships of enzymes allows revelation of key structural motifs or elements. Here, we studied the structural basis of the substrate promiscuity of EH(0), a family IV esterase, isolated from a sample of the Sorghum bicolor rhizosphere microbiome exposed to technical cashew nut shell liquid. The analysis of EH(0) revealed the potential of the sorghum rhizosphere microbiome as a source of enzymes with interesting properties, such as pH and solvent tolerance and remarkably broad substrate promiscuity. Its structure resembled those of homologous proteins from mesophilic Parvibaculum and Erythrobacter spp. and hyperthermophilic Pyrobaculum and Sulfolobus spp. and had a very narrow, single-entry access tunnel to the active site, with access controlled by a capping domain that includes a number of nonconserved proline residues. These structural markers, distinct from those of other substrate-promiscuous esterases, can help in tuning substrate profiles beyond tunnel and active site engineering.
ESTHER : Distaso_2023_Appl.Environ.Microbiol__e0180722
PubMedSearch : Distaso_2023_Appl.Environ.Microbiol__e0180722
PubMedID: 36602332
Gene_locus related to this paper: 9bact-EH0

Title : Assigning Functions of Unknown Enzymes by High-Throughput Enzyme Characterization - Molina-Espeja_2023_Methods.Mol.Biol_2555_181
Author(s) : Molina-Espeja P , Fernandez-Lopez L , Golyshin PN , Ferrer M
Ref : Methods Mol Biol , 2555 :181 , 2023
Abstract : The discovery of new enzymes is strongly enabled by the implementation of high-throughput screening methods to detect enzymatic activity in single organisms or clone expression libraries, or to benchmark their performances against known prototypes. In this chapter, a number of methods, applicable at high-throughput scale, are described that allow the screening and characterization of enzymes relevant to biotechnology, particularly, ester-hydrolases (esterases, lipases, phospholipases, and polyester hydrolases).
ESTHER : Molina-Espeja_2023_Methods.Mol.Biol_2555_181
PubMedSearch : Molina-Espeja_2023_Methods.Mol.Biol_2555_181
PubMedID: 36306087

Title : Harnessing extremophilic carboxylesterases for applications in polyester depolymerisation and plastic waste recycling - Williams_2023_Essays.Biochem__
Author(s) : Williams GB , Ma H , Khusnutdinova AN , Yakunin AF , Golyshin PN
Ref : Essays Biochem , : , 2023
Abstract : The steady growth in industrial production of synthetic plastics and their limited recycling have resulted in severe environmental pollution and contribute to global warming and oil depletion. Currently, there is an urgent need to develop efficient plastic recycling technologies to prevent further environmental pollution and recover chemical feedstocks for polymer re-synthesis and upcycling in a circular economy. Enzymatic depolymerization of synthetic polyesters by microbial carboxylesterases provides an attractive addition to existing mechanical and chemical recycling technologies due to enzyme specificity, low energy consumption, and mild reaction conditions. Carboxylesterases constitute a diverse group of serine-dependent hydrolases catalysing the cleavage and formation of ester bonds. However, the stability and hydrolytic activity of identified natural esterases towards synthetic polyesters are usually insufficient for applications in industrial polyester recycling. This necessitates further efforts on the discovery of robust enzymes, as well as protein engineering of natural enzymes for enhanced activity and stability. In this essay, we discuss the current knowledge of microbial carboxylesterases that degrade polyesters (polyesterases) with focus on polyethylene terephthalate (PET), which is one of the five major synthetic polymers. Then, we briefly review the recent progress in the discovery and protein engineering of microbial polyesterases, as well as developing enzyme cocktails and secreted protein expression for applications in the depolymerisation of polyester blends and mixed plastics. Future research aimed at the discovery of novel polyesterases from extreme environments and protein engineering for improved performance will aid developing efficient polyester recycling technologies for the circular plastics economy.
ESTHER : Williams_2023_Essays.Biochem__
PubMedSearch : Williams_2023_Essays.Biochem__
PubMedID: 37334661

Title : Structure and evolutionary trace-assisted screening of a residue swapping the substrate ambiguity and chiral specificity in an esterase - Cea-Rama_2021_Comput.Struct.Biotechnol.J_19_2307
Author(s) : Cea-Rama I , Coscolin C , Katsonis P , Bargiela R , Golyshin PN , Lichtarge O , Ferrer M , Sanz-Aparicio J
Ref : Comput Struct Biotechnol J , 19 :2307 , 2021
Abstract : Our understanding of enzymes with high substrate ambiguity remains limited because their large active sites allow substrate docking freedom to an extent that seems incompatible with stereospecificity. One possibility is that some of these enzymes evolved a set of evolutionarily fitted sequence positions that stringently allow switching substrate ambiguity and chiral specificity. To explore this hypothesis, we targeted for mutation a serine ester hydrolase (EH(3)) that exhibits an impressive 71-substrate repertoire but is not stereospecific (e.e. 50%). We used structural actions and the computational evolutionary trace method to explore specificity-swapping sequence positions and hypothesized that position I244 was critical. Driven by evolutionary action analysis, this position was substituted to leucine, which together with isoleucine appears to be the amino acid most commonly present in the closest homologous sequences (max. identity, ca. 67.1%), and to phenylalanine, which appears in distant homologues. While the I244L mutation did not have any functional consequences, the I244F mutation allowed the esterase to maintain a remarkable 53-substrate range while gaining stereospecificity properties (e.e. 99.99%). These data support the possibility that some enzymes evolve sequence positions that control the substrate scope and stereospecificity. Such residues, which can be evolutionarily screened, may serve as starting points for further designing substrate-ambiguous, yet chiral-specific, enzymes that are greatly appreciated in biotechnology and synthetic chemistry.
ESTHER : Cea-Rama_2021_Comput.Struct.Biotechnol.J_19_2307
PubMedSearch : Cea-Rama_2021_Comput.Struct.Biotechnol.J_19_2307
PubMedID: 33995922
Gene_locus related to this paper: 9zzzz-a0a2k8jn75

Title : Genetically engineered proteins with two active sites for enhanced biocatalysis and synergistic chemo- and biocatalysis - Alonso_2020_Nat.Catal_3_319
Author(s) : Alonso S , Santiago G , Cea-Rama I , Fernandez-Lopez L , Coscolin C , Modregger J , Ressmann A , Martinez-Martinez M , Marrero H , Bargiela R , Pita M , Gonzalez-Alfonso JL , Briand M , Rojo D , Barbas C , Plou FJ , Golyshin PN , Shahgaldian P , Sanz-Aparicio J , Guallar V , Ferrer M
Ref : Nature Catalysis , 3 :319 , 2019
Abstract : Enzyme engineering has allowed not only the de novo creation of active sites catalysing known biological reactions with rates close to diffusion limits, but also the generation of abiological sites performing new-to-nature reactions. However, the catalytic advantages of engineering multiple active sites into a single protein scaffold are yet to be established. Here, we report on pro-teins with two active sites of biological and/or abiological origin, for improved natural and non-natural catalysis. The approach increased the catalytic properties, such as enzyme efficiency, substrate scope, stereoselectivity and optimal temperature window, of an esterase containing two biological sites. Then, one of the active sites was metamorphosed into a metal-complex chemocatalytic site for oxidation and Friedel-Crafts alkylation reactions, facilitating synergistic chemo- and biocatalysis in a single protein. The transformations of 1-naphthyl acetate into 1,4-naphthoquinone (conversion approx. 100%) and vinyl crotonate and benzene into 3-phenylbutyric acid (>=83%; e.e. >99.9%) were achieved in one pot with this artificial multifunc-tional metalloenzyme.
ESTHER : Alonso_2020_Nat.Catal_3_319
PubMedSearch : Alonso_2020_Nat.Catal_3_319
PubMedID:
Gene_locus related to this paper: 9bact-LAE6

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 : Rational Engineering of Multiple Active Sites in an Ester Hydrolase - Santiago_2018_Biochemistry_57_2245
Author(s) : Santiago G , Martinez-Martinez M , Alonso S , Bargiela R , Coscolin C , Golyshin PN , Guallar V , Ferrer M
Ref : Biochemistry , 57 :2245 , 2018
Abstract : Effects of altering the properties of an active site in an enzymatic homogeneous catalyst have been extensively reported. However, the possibility of increasing the number of such sites, as commonly done in heterogeneous catalytic materials, remains unexplored, particularly because those have to accommodate appropriate residues in specific configurations. This possibility was investigated by using a serine ester hydrolase as the target enzyme. By using the Protein Energy Landscape Exploration software, which maps ligand diffusion and binding, we found a potential binding pocket capable of holding an extra catalytic triad and oxyanion hole contacts. By introducing two mutations, this binding pocket became a catalytic site. Its substrate specificity, substrate preference, and catalytic activity were different from those of the native site of the wild type ester hydrolase and other hydrolases, due to the differences in the active site architecture. Converting the binding pocket into an extra catalytic active site was proven to be a successful approach to create a serine ester hydrolase with two functional reactive groups. Our results illustrate the accuracy and predictive nature of modern modeling techniques, opening novel catalytic opportunities coming from the presence of different catalytic environments in single enzymes.
ESTHER : Santiago_2018_Biochemistry_57_2245
PubMedSearch : Santiago_2018_Biochemistry_57_2245
PubMedID: 29600855
Gene_locus related to this paper: 9bact-LAE6

Title : Screening and Characterization of Novel Polyesterases from Environmental Metagenomes with High Hydrolytic Activity against Synthetic Polyesters - Hajighasemi_2018_Environ.Sci.Technol_52_12388
Author(s) : Hajighasemi M , Tchigvintsev A , Nocek B , Flick R , Popovic A , Hai T , Khusnutdinova AN , Brown G , Xu X , Cui H , Anstett J , Chernikova TN , Bruls T , Le Paslier D , Yakimov MM , Joachimiak A , Golyshina OV , Savchenko A , Golyshin PN , Edwards EA , Yakunin AF
Ref : Environ Sci Technol , 52 :12388 , 2018
Abstract : The continuous growth of global plastics production, including polyesters, has resulted in increasing plastic pollution and subsequent negative environmental impacts. Therefore, enzyme-catalyzed depolymerization of synthetic polyesters as a plastics recycling approach has become a focus of research. In this study, we screened over 200 purified uncharacterized hydrolases from environmental metagenomes and sequenced microbial genomes and identified at least 10 proteins with high hydrolytic activity against synthetic polyesters. These include the metagenomic esterases MGS0156 and GEN0105, which hydrolyzed polylactic acid (PLA), polycaprolactone, as well as bis(benzoyloxyethyl)-terephthalate. With solid PLA as a substrate, both enzymes produced a mixture of lactic acid monomers, dimers, and higher oligomers as products. The crystal structure of MGS0156 was determined at 1.95 A resolution and revealed a modified alpha/beta hydrolase fold, with a lid domain and highly hydrophobic active site. Mutational studies of MGS0156 identified the residues critical for hydrolytic activity against both polyester and monoester substrates, with two-times higher polyesterase activity in the MGS0156 L169A mutant protein. Thus, our work identified novel, highly active polyesterases in environmental metagenomes and provided molecular insights into their activity, thereby augmenting our understanding of enzymatic polyester hydrolysis.
ESTHER : Hajighasemi_2018_Environ.Sci.Technol_52_12388
PubMedSearch : Hajighasemi_2018_Environ.Sci.Technol_52_12388
PubMedID: 30284819
Gene_locus related to this paper: 9zzzz-a0a0g3fj39 , 9zzzz-a0a0g3fj48 , 9zzzz-A0A0G3FEJ8 , 9bact-a4uz10

Title : Activity screening of environmental metagenomic libraries reveals novel carboxylesterase families - Popovic_2017_Sci.Rep_7_44103
Author(s) : Popovic A , Hai T , Tchigvintsev A , Hajighasemi M , Nocek B , Khusnutdinova AN , Brown G , Glinos J , Flick R , Skarina T , Chernikova TN , Yim V , Bruls T , Paslier DL , Yakimov MM , Joachimiak A , Ferrer M , Golyshina OV , Savchenko A , Golyshin PN , Yakunin AF
Ref : Sci Rep , 7 :44103 , 2017
Abstract : Metagenomics has made accessible an enormous reserve of global biochemical diversity. To tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. We have validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalytic residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools.
ESTHER : Popovic_2017_Sci.Rep_7_44103
PubMedSearch : Popovic_2017_Sci.Rep_7_44103
PubMedID: 28272521
Gene_locus related to this paper: 9zzzz-a0a0g3fj39 , 9zzzz-a0a0g3fj48 , 9zzzz-A0A0G3FEJ8

Title : Biochemical and Structural Insights into Enzymatic Depolymerization of Polylactic Acid and Other Polyesters by Microbial Carboxylesterases - Hajighasemi_2016_Biomacromolecules_17_2027
Author(s) : Hajighasemi M , Nocek BP , Tchigvintsev A , Brown G , Flick R , Xu X , Cui H , Hai T , Joachimiak A , Golyshin PN , Savchenko A , Edwards EA , Yakunin AF
Ref : Biomacromolecules , 17 :2027 , 2016
Abstract : Polylactic acid (PLA) is a biodegradable polyester derived from renewable resources, which is a leading candidate for the replacement of traditional petroleum-based polymers. Since the global production of PLA is quickly growing, there is an urgent need for the development of efficient recycling technologies, which will produce lactic acid instead of CO2 as the final product. After screening 90 purified microbial alpha/beta-hydrolases, we identified hydrolytic activity against emulsified PLA in two uncharacterized proteins, ABO2449 from Alcanivorax borkumensis and RPA1511 from Rhodopseudomonas palustris. Both enzymes were also active against emulsified polycaprolactone and other polyesters as well as against soluble alpha-naphthyl and p-nitrophenyl monoesters. In addition, both ABO2449 and RPA1511 catalyzed complete or extensive hydrolysis of solid PLA with the production of lactic acid monomers, dimers, and larger oligomers as products. The crystal structure of RPA1511 was determined at 2.2 A resolution and revealed a classical alpha/beta-hydrolase fold with a wide-open active site containing a molecule of polyethylene glycol bound near the catalytic triad Ser114-His270-Asp242. Site-directed mutagenesis of both proteins demonstrated that the catalytic triad residues are important for the hydrolysis of both monoester and polyester substrates. We also identified several residues in RPA1511 (Gln172, Leu212, Met215, Trp218, and Leu220) and ABO2449 (Phe38 and Leu152), which were not essential for activity against soluble monoesters but were found to be critical for the hydrolysis of PLA. Our results indicate that microbial carboxyl esterases can efficiently hydrolyze various polyesters making them attractive biocatalysts for plastics depolymerization and recycling.
ESTHER : Hajighasemi_2016_Biomacromolecules_17_2027
PubMedSearch : Hajighasemi_2016_Biomacromolecules_17_2027
PubMedID: 27087107
Gene_locus related to this paper: marav-a1u5n0 , rhopa-q6n9m9 , alcbs-q0vlq1

Title : Isolation and characterization of novel lipases\/esterases from a bovine rumen metagenome - Prive_2015_Appl.Microbiol.Biotechnol_99_5475
Author(s) : Prive F , Newbold CJ , Kaderbhai NN , Girdwood SG , Golyshina OV , Golyshin PN , Scollan ND , Huws SA
Ref : Applied Microbiology & Biotechnology , 99 :5475 , 2015
Abstract : Improving the health beneficial fatty acid content of meat and milk is a major challenge requiring an increased understanding of rumen lipid metabolism. In this study, we isolated and characterized rumen bacterial lipases/esterases using functional metagenomics. Metagenomic libraries were constructed from DNA extracted from strained rumen fluid (SRF), solid-attached bacteria (SAB) and liquid-associated rumen bacteria (LAB), ligated into a fosmid vector and subsequently transformed into an Escherichia coli host. Fosmid libraries consisted of 7,744; 8,448; and 7,680 clones with an average insert size of 30 to 35 kbp for SRF, SAB and LAB, respectively. Transformants were screened on spirit blue agar plates containing tributyrin for lipase/esterase activity. Five SAB and four LAB clones exhibited lipolytic activity, and no positive clones were found in the SRF library. Fosmids from positive clones were pyrosequenced and twelve putative lipase/esterase genes and two phospholipase genes retrieved. Although the derived proteins clustered into diverse esterase and lipase families, a degree of novelty was seen, with homology ranging from 40 to 78 % following BlastP searches. Isolated lipases/esterases exhibited activity against mostly short- to medium-chain substrates across a range of temperatures and pH. The function of these novel enzymes recovered in ruminal metabolism needs further investigation, alongside their potential industrial uses.
ESTHER : Prive_2015_Appl.Microbiol.Biotechnol_99_5475
PubMedSearch : Prive_2015_Appl.Microbiol.Biotechnol_99_5475
PubMedID: 25575887

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 : Diversity of hydrolases from hydrothermal vent sediments of the Levante Bay, Vulcano Island (Aeolian archipelago) identified by activity-based metagenomics and biochemical characterization of new esterases and an arabinopyranosidase - Placido_2015_Appl.Microbiol.Biotechnol_99_10031
Author(s) : Placido A , Hai T , Ferrer M , Chernikova TN , Distaso M , Armstrong D , Yakunin AF , Toshchakov SV , Yakimov MM , Kublanov IV , Golyshina OV , Pesole G , Ceci LR , Golyshin PN
Ref : Applied Microbiology & Biotechnology , 99 :10031 , 2015
Abstract : A metagenomic fosmid expression library established from environmental DNA (eDNA) from the shallow hot vent sediment sample collected from the Levante Bay, Vulcano Island (Aeolian archipelago) was established in Escherichia coli. Using activity-based screening assays, we have assessed 9600 fosmid clones corresponding to approximately 350 Mbp of the cloned eDNA, for the lipases/esterases/lactamases, haloalkane and haloacid dehalogenases, and glycoside hydrolases. Thirty-four positive fosmid clones were selected from the total of 120 positive hits and sequenced to yield ca. 1360 kbp of high-quality assemblies. Fosmid inserts were attributed to the members of ten bacterial phyla, including Proteobacteria, Bacteroidetes, Acidobateria, Firmicutes, Verrucomicrobia, Chloroflexi, Spirochaetes, Thermotogae, Armatimonadetes, and Planctomycetes. Of ca. 200 proteins with high biotechnological potential identified therein, we have characterized in detail three distinct alpha/beta-hydrolases (LIPESV12_9, LIPESV12_24, LIPESV12_26) and one new alpha-arabinopyranosidase (GLV12_5). All LIPESV12 enzymes revealed distinct substrate specificities tested against 43 structurally diverse esters and 4 p-nitrophenol carboxyl esters. Of 16 different glycosides tested, the GLV12_5 hydrolysed only p-nitrophenol-alpha-(L)-arabinopyranose with a high specific activity of about 2.7 kU/mg protein. Most of the alpha/beta-hydrolases were thermophilic and revealed a high tolerance to, and high activities in the presence of, numerous heavy metal ions. Among them, the LIPESV12_24 was the best temperature-adapted, retaining its activity after 40 min of incubation at 90 degrees C. Furthermore, enzymes were active in organic solvents (e.g., >30 % methanol). Both LIPESV12_24 and LIPESV12_26 had the GXSXG pentapeptides and the catalytic triads Ser-Asp-His typical to the representatives of carboxylesterases of EC 3.1.1.1.
ESTHER : Placido_2015_Appl.Microbiol.Biotechnol_99_10031
PubMedSearch : Placido_2015_Appl.Microbiol.Biotechnol_99_10031
PubMedID: 26266751
Gene_locus related to this paper: 9bact-a0a0h4tgu6 , 9bact-a0a0k1z4z5

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 : Single residues dictate the co-evolution of dual esterases: MCP hydrolases from the alpha\/beta hydrolase family - Alcaide_2013_Biochem.J_454_157
Author(s) : Alcaide M , Tornes J , Stogios PJ , Xu X , Gertler C , Di Leo R , Bargiela R , Lafraya A , Guazzaroni ME , Lopez-Cortes N , Chernikova TN , Golyshina OV , Nechitaylo TY , Plumeier I , Pieper DH , Yakimov MM , Savchenko A , Golyshin PN , Ferrer M
Ref : Biochemical Journal , 454 :157 , 2013
Abstract : Several members of the C-C MCP (meta-cleavage product) hydrolase family demonstrate an unusual ability to hydrolyse esters as well as the MCPs (including those from mono- and bi-cyclic aromatics). Although the molecular mechanisms responsible for such substrate promiscuity are starting to emerge, the full understanding of these complex enzymes is far from complete. In the present paper, we describe six distinct alpha/beta hydrolases identified through genomic approaches, four of which demonstrate the unprecedented characteristic of activity towards a broad spectrum of substrates, including p-nitrophenyl, halogenated, fatty acyl, aryl, glycerol, cinnamoyl and carbohydrate esters, lactones, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate and 2-hydroxy-6-oxohepta-2,4-dienoate. Using structural analysis and site-directed mutagenesis we have identified the three residues (Ser32, Val130 and Trp144) that determine the unusual substrate specificity of one of these proteins, CCSP0084. The results may open up new research avenues into comparative catalytic models, structural and mechanistic studies, and biotechnological applications of MCP hydrolases.
ESTHER : Alcaide_2013_Biochem.J_454_157
PubMedSearch : Alcaide_2013_Biochem.J_454_157
PubMedID: 23750508
Gene_locus related to this paper: 9bact-r9qzf7 , 9gamm-k0c6t6

Title : Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispira antarctica - Kube_2013_Nat.Commun_4_2156
Author(s) : Kube M , Chernikova TN , Al-Ramahi Y , Beloqui A , Lopez-Cortez N , Guazzaroni ME , Heipieper HJ , Klages S , Kotsyurbenko OR , Langer I , Nechitaylo TY , Lunsdorf H , Fernandez M , Juarez S , Ciordia S , Singer A , Kagan O , Egorova O , Petit PA , Stogios P , Kim Y , Tchigvintsev A , Flick R , Denaro R , Genovese M , Albar JP , Reva ON , Martinez-Gomariz M , Tran H , Ferrer M , Savchenko A , Yakunin AF , Yakimov MM , Golyshina OV , Reinhardt R , Golyshin PN
Ref : Nat Commun , 4 :2156 , 2013
Abstract : Ubiquitous bacteria from the genus Oleispira drive oil degradation in the largest environment on Earth, the cold and deep sea. Here we report the genome sequence of Oleispira antarctica and show that compared with Alcanivorax borkumensis--the paradigm of mesophilic hydrocarbonoclastic bacteria--O. antarctica has a larger genome that has witnessed massive gene-transfer events. We identify an array of alkane monooxygenases, osmoprotectants, siderophores and micronutrient-scavenging pathways. We also show that at low temperatures, the main protein-folding machine Cpn60 functions as a single heptameric barrel that uses larger proteins as substrates compared with the classical double-barrel structure observed at higher temperatures. With 11 protein crystal structures, we further report the largest set of structures from one psychrotolerant organism. The most common structural feature is an increased content of surface-exposed negatively charged residues compared to their mesophilic counterparts. Our findings are relevant in the context of microbial cold-adaptation mechanisms and the development of strategies for oil-spill mitigation in cold environments.
ESTHER : Kube_2013_Nat.Commun_4_2156
PubMedSearch : Kube_2013_Nat.Commun_4_2156
PubMedID: 23877221
Gene_locus related to this paper: olean-olei00960 , olean-r4ym14 , olean-r4yv64 , olean-r4ys13

Title : Genome Sequence of Thalassolituus oleivorans MIL-1 (DSM 14913T) - Golyshin_2013_Genome.Announc_1_e0014113
Author(s) : Golyshin PN , Werner J , Chernikova TN , Tran H , Ferrer M , Yakimov MM , Teeling H , Golyshina OV
Ref : Genome Announc , 1 :e0014113 , 2013
Abstract : Thalassolituus oleivorans is one of the most prevalent marine gammaproteobacteria in microbial communities, emerging after oil spills in coastal, estuarine, and surface seawaters. Here, we present the assembled genome of strain T. oleivorans MIL-1 (DSM 14913(T)), which is 3,920,328 bp with a G+C content of 46.6%.
ESTHER : Golyshin_2013_Genome.Announc_1_e0014113
PubMedSearch : Golyshin_2013_Genome.Announc_1_e0014113
PubMedID: 23599290
Gene_locus related to this paper: 9gamm-m5dq85 , 9gamm-m5dt68 , 9gamm-m5dm97

Title : Gene sets for utilization of primary and secondary nutrition supplies in the distal gut of endangered Iberian lynx - Alcaide_2012_PLoS.One_7_e51521
Author(s) : Alcaide M , Messina E , Richter M , Bargiela R , Peplies J , Huws SA , Newbold CJ , Golyshin PN , Simon MA , Lopez G , Yakimov MM , Ferrer M
Ref : PLoS ONE , 7 :e51521 , 2012
Abstract : Recent studies have indicated the existence of an extensive trans-genomic trans-mural co-metabolism between gut microbes and animal hosts that is diet-, host phylogeny- and provenance-influenced. Here, we analyzed the biodiversity at the level of small subunit rRNA gene sequence and the metabolic composition of 18 Mbp of consensus metagenome sequences and activity characteristics of bacterial intra-cellular extracts, in wild Iberian lynx (Lynx pardinus) fecal samples. Bacterial signatures (14.43% of all of the Firmicutes reads and 6.36% of total reads) related to the uncultured anaerobic commensals Anaeroplasma spp., which are typically found in ovine and bovine rumen, were first identified. The lynx gut was further characterized by an over-representation of 'presumptive' aquaporin aqpZ genes and genes encoding 'active' lysosomal-like digestive enzymes that are possibly needed to acquire glycerol, sugars and amino acids from glycoproteins, glyco(amino)lipids, glyco(amino)glycans and nucleoside diphosphate sugars. Lynx gut was highly enriched (28% of the total glycosidases) in genes encoding alpha-amylase and related enzymes, although it exhibited low rate of enzymatic activity indicative of starch degradation. The preponderance of beta-xylosidase activity in protein extracts further suggests lynx gut microbes being most active for the metabolism of beta-xylose containing plant N-glycans, although beta-xylosidases sequences constituted only 1.5% of total glycosidases. These collective and unique bacterial, genetic and enzymatic activity signatures suggest that the wild lynx gut microbiota not only harbors gene sets underpinning sugar uptake from primary animal tissues (with the monotypic dietary profile of the wild lynx consisting of 80-100% wild rabbits) but also for the hydrolysis of prey-derived plant biomass. Although, the present investigation corresponds to a single sample and some of the statements should be considered qualitative, the data most likely suggests a tighter, more coordinated and complex evolutionary and nutritional ecology scenario of carnivore gut microbial communities than has been previously assumed.
ESTHER : Alcaide_2012_PLoS.One_7_e51521
PubMedSearch : Alcaide_2012_PLoS.One_7_e51521
PubMedID: 23251564
Gene_locus related to this paper: 9zzzz-j9g462 , 9zzzz-j9bqr9 , 9zzzz-j9gjr1 , 9zzzz-j9gm47 , 9zzzz-j9ddn5 , 9zzzz-j9g9e5 , 9zzzz-j9fnv6

Title : Microbial beta-glucosidases from cow rumen metagenome enhance the saccharification of lignocellulose in combination with commercial cellulase cocktail - Del Pozo_2012_Biotechnol.Biofuels_5_73
Author(s) : Del Pozo MV , Fernandez-Arrojo L , Gil-Martinez J , Montesinos A , Chernikova TN , Nechitaylo TY , Waliszek A , Tortajada M , Rojas A , Huws SA , Golyshina OV , Newbold CJ , Polaina J , Ferrer M , Golyshin PN
Ref : Biotechnol Biofuels , 5 :73 , 2012
Abstract : BACKGROUND: A complete saccharification of plant polymers is the critical step in the efficient production of bio-alcohols. Beta-glucosidases acting in the degradation of intermediate gluco-oligosaccharides produced by cellulases limit the yield of the final product.
RESULTS: In the present work, we have identified and then successfully cloned, expressed, purified and characterised 4 highly active beta-glucosidases from fibre-adherent microbial community from the cow rumen. The enzymes were most active at temperatures 45-55 degrees C and pH 4.0-7.0 and exhibited high affinity and activity towards synthetic substrates such as p-nitrophenyl-beta-D-glucopyranoside (pNPbetaG) and pNP-beta-cellobiose, as well as to natural cello-oligosaccharides ranging from cellobiose to cellopentaose. The apparent capability of the most active beta-glucosidase, herein named LAB25g2, was tested for its ability to improve, at low dosage (31.25 units g-1 dry biomass, using pNPbetaG as substrate), the hydrolysis of pre-treated corn stover (dry matter content of 20%; 350 g glucan kg-1 dry biomass) in combination with a beta-glucosidase-deficient commercial Trichoderma reseei cellulase cocktail (5 units g-1 dry biomass in the basis of pNPbetaG). LAB25g2 increased the final hydrolysis yield by a factor of 20% (44.5 +/- 1.7% vs. 34.5 +/- 1.5% in control conditions) after 96-120 h as compared to control reactions in its absence or in the presence of other commercial beta-glucosidase preparations. The high stability (half-life higher than 5 days at 50 degrees C and pH 5.2) and 2-38000 fold higher (as compared with reported beta-glucosidases) activity towards cello-oligosaccharides may account for its performance in supplementation assays.
CONCLUSIONS: The results suggest that beta-glucosidases from yet uncultured bacteria from animal digestomes may be of a potential interest for biotechnological processes related to the effective bio-ethanol production in combination with low dosage of commercial cellulases.
ESTHER : Del Pozo_2012_Biotechnol.Biofuels_5_73
PubMedSearch : Del Pozo_2012_Biotechnol.Biofuels_5_73
PubMedID: 22998985
Gene_locus related to this paper: 9bact-m4pza5

Title : Structure and activity of the cold-active and anion-activated carboxyl esterase OLEI01171 from the oil-degrading marine bacterium Oleispira antarctica - Lemak_2012_Biochem.J_445_193
Author(s) : Lemak S , Tchigvintsev A , Petit P , Flick R , Singer AU , Brown G , Evdokimova E , Egorova O , Gonzalez CF , Chernikova TN , Yakimov MM , Kube M , Reinhardt R , Golyshin PN , Savchenko A , Yakunin AF
Ref : Biochemical Journal , 445 :193 , 2012
Abstract : The uncharacterized alpha/beta-hydrolase protein OLEI01171 from the psychrophilic marine bacterium Oleispira antarctica belongs to the PF00756 family of putative esterases, which also includes human esterase D. In the present paper we show that purified recombinant OLEI01171 exhibits high esterase activity against the model esterase substrate alpha-naphthyl acetate at 5-30 degrees C with maximal activity at 15-20 degrees C. The esterase activity of OLEI01171 was stimulated 3-8-fold by the addition of chloride or several other anions (0.1-1.0 M). Compared with mesophilic PF00756 esterases, OLEI01171 exhibited a lower overall protein thermostability. Two crystal structures of OLEI01171 were solved at 1.75 and 2.1 A resolution and revealed a classical serine hydrolase catalytic triad and the presence of a chloride or bromide ion bound in the active site close to the catalytic Ser148. Both anions were found to co-ordinate a potential catalytic water molecule located in the vicinity of the catalytic triad His257. The results of the present study suggest that the bound anion perhaps contributes to the polarization of the catalytic water molecule and increases the rate of the hydrolysis of an acyl-enzyme intermediate. Alanine replacement mutagenesis of OLEI01171 identified ten amino acid residues important for esterase activity. The replacement of Asn225 by lysine had no significant effect on the activity or thermostability of OLEI01171, but resulted in a detectable increase of activity at 35-45 degrees C. The present study has provided insight into the molecular mechanisms of activity of a cold-active and anion-activated carboxyl esterase.
ESTHER : Lemak_2012_Biochem.J_445_193
PubMedSearch : Lemak_2012_Biochem.J_445_193
PubMedID: 22519667
Gene_locus related to this paper: olean-d0vwz4

Title : Diversity of glycosyl hydrolases from cellulose-depleting communities enriched from casts of two earthworm species - Beloqui_2010_Appl.Environ.Microbiol_76_5934
Author(s) : Beloqui A , Nechitaylo TY , Lopez-Cortes N , Ghazi A , Guazzaroni ME , Polaina J , Strittmatter AW , Reva O , Waliczek A , Yakimov MM , Golyshina OV , Ferrer M , Golyshin PN
Ref : Applied Environmental Microbiology , 76 :5934 , 2010
Abstract : The guts and casts of earthworms contain microbial assemblages that process large amounts of organic polymeric substrates from plant litter and soil; however, the enzymatic potential of these microbial communities remains largely unexplored. In the present work, we retrieved carbohydrate-modifying enzymes through the activity screening of metagenomic fosmid libraries from cellulose-depleting microbial communities established with the fresh casts of two earthworm species, Aporrectodea caliginosa and Lumbricus terrestris, as inocula. Eight glycosyl hydrolases (GHs) from the A. caliginosa-derived community were multidomain endo-beta-glucanases, beta-glucosidases, beta-cellobiohydrolases, beta-galactosidase, and beta-xylosidases of known GH families. In contrast, two GHs derived from the L. terrestris microbiome had no similarity to any known GHs and represented two novel families of beta-galactosidases/alpha-arabinopyranosidases. Members of these families were annotated in public databases as conserved hypothetical proteins, with one being structurally related to isomerases/dehydratases. This study provides insight into their biochemistry, domain structures, and active-site architecture. The two communities were similar in bacterial composition but significantly different with regard to their eukaryotic inhabitants. Further sequence analysis of fosmids and plasmids bearing the GH-encoding genes, along with oligonucleotide usage pattern analysis, suggested that those apparently originated from Gammaproteobacteria (pseudomonads and Cellvibrio-like organisms), Betaproteobacteria (Comamonadaceae), and Alphaproteobacteria (Rhizobiales).
ESTHER : Beloqui_2010_Appl.Environ.Microbiol_76_5934
PubMedSearch : Beloqui_2010_Appl.Environ.Microbiol_76_5934
PubMedID: 20622123
Gene_locus related to this paper: 9zzzz-d8vn29

Title : Inter-conversion of catalytic abilities in a bifunctional carboxyl\/feruloyl-esterase from earthworm gut metagenome - Vieites_2010_Microb.Biotechnol_3_48
Author(s) : Vieites JM , Ghazi A , Beloqui A , Polaina J , Andreu JM , Golyshina OV , Nechitaylo TY , Waliczek A , Yakimov MM , Golyshin PN , Ferrer M
Ref : Microb Biotechnol , 3 :48 , 2010
Abstract : Carboxyl esterases (CE) exhibit various reaction specificities despite of their overall structural similarity. In present study we have exploited functional metagenomics, saturation mutagenesis and experimental protein evolution to explore residues that have a significant role in substrate discrimination. We used an enzyme, designated 3A6, derived from the earthworm gut metagenome that exhibits CE and feruloyl esterase (FAE) activities with p-nitrophenyl and cinnamate esters, respectively, with a [(k(cat)/K(m))](CE)/[(k(cat)/K(m))](FAE) factor of 17. Modelling-guided saturation mutagenesis at specific hotspots (Lys(281), Asp(282), Asn(316) and Lys(317)) situated close to the catalytic core (Ser(143)/Asp(273)/His(305)) and a deletion of a 34-AA-long peptide fragment yielded mutants with the highest CE activity, while cinnamate ester bond hydrolysis was effectively abolished. Although, single to triple mutants with both improved activities (up to 180-fold in k(cat)/K(m) values) and enzymes with inverted specificity ((k(cat)/K(m))(CE)/(k(cat)/K(m))(FAE) ratio of approximately 0.4) were identified, no CE inactive variant was found. Screening of a large error-prone PCR-generated library yielded by far less mutants for substrate discrimination. We also found that no significant changes in CE activation energy occurs after any mutation (7.3 to -5.6 J mol(-1)), whereas a direct correlation between loss/gain of FAE function and activation energies (from 33.05 to -13.7 J mol(-1)) was found. Results suggest that the FAE activity in 3A6 may have evolved via introduction of a limited number of 'hot spot' mutations in a common CE ancestor, which may retain the original hydrolytic activity due to lower restrictive energy barriers but conveys a dynamic energetically favourable switch of a second hydrolytic reaction.
ESTHER : Vieites_2010_Microb.Biotechnol_3_48
PubMedSearch : Vieites_2010_Microb.Biotechnol_3_48
PubMedID: 21255305

Title : Novel hybrid esterase-haloacid dehalogenase enzyme -
Author(s) : Beloqui A , Polaina J , Vieites JM , Reyes-Duarte D , Torres R , Golyshina OV , Chernikova TN , Waliczek A , Aharoni A , Yakimov MM , Timmis KN , Golyshin PN , Ferrer M
Ref : Chembiochem , 11 :1975 , 2010
PubMedID: 20715265

Title : The 'pH optimum anomaly' of intracellular enzymes of Ferroplasma acidiphilum - Golyshina_2006_Environ.Microbiol_8_416
Author(s) : Golyshina OV , Golyshin PN , Timmis KN , Ferrer M
Ref : Environ Microbiol , 8 :416 , 2006
Abstract : A wide range of microorganisms, the so-called acidophiles, inhabit acidic environments and grow optimally at pH values between 0 and 3. The intracellular pH of these organisms is, however, close to neutrality or slightly acidic. It is to be expected that enzymatic activities dedicated to extracellular functions would be adapted to the prevailing low pH of the environment (0-3), whereas intracellular enzymes would be optimally active at the near-neutral pH of the cytoplasm (4.6-7.0). The genes of several intracellular or cell-bound enzymes, a carboxylesterase and three alpha-glucosidases, from Ferroplasma acidiphilum, a cell wall-lacking acidophilic archaeon with a growth optimum at pH 1.7, were cloned and expressed in Escherichia coli, and their products purified and characterized. The Ferroplasmaalpha-glucosidases exhibited no sequence similarity to known glycosyl hydrolases. All enzymes functioned and were stable in vitro in the pH range 1.7-4.0, and had pH optima much lower than the mean intracellular pH of 5.6. This 'pH optimum anomaly' suggests the existence of yet-undetected cellular compartmentalization providing cytoplasmic pH patchiness and low pH environments for the enzymes we have analysed.
ESTHER : Golyshina_2006_Environ.Microbiol_8_416
PubMedSearch : Golyshina_2006_Environ.Microbiol_8_416
PubMedID: 16478448
Gene_locus related to this paper: 9eury-q2pce5

Title : Genome sequence of the ubiquitous hydrocarbon-degrading marine bacterium Alcanivorax borkumensis - Schneiker_2006_Nat.Biotechnol_24_997
Author(s) : Schneiker S , Martins dos Santos VA , Bartels D , Bekel T , Brecht M , Buhrmester J , Chernikova TN , Denaro R , Ferrer M , Gertler C , Goesmann A , Golyshina OV , Kaminski F , Khachane AN , Lang S , Linke B , McHardy AC , Meyer F , Nechitaylo T , Puhler A , Regenhardt D , Rupp O , Sabirova JS , Selbitschka W , Yakimov MM , Timmis KN , Vorholter FJ , Weidner S , Kaiser O , Golyshin PN
Ref : Nat Biotechnol , 24 :997 , 2006
Abstract : Alcanivorax borkumensis is a cosmopolitan marine bacterium that uses oil hydrocarbons as its exclusive source of carbon and energy. Although barely detectable in unpolluted environments, A. borkumensis becomes the dominant microbe in oil-polluted waters. A. borkumensis SK2 has a streamlined genome with a paucity of mobile genetic elements and energy generation-related genes, but with a plethora of genes accounting for its wide hydrocarbon substrate range and efficient oil-degradation capabilities. The genome further specifies systems for scavenging of nutrients, particularly organic and inorganic nitrogen and oligo-elements, biofilm formation at the oil-water interface, biosurfactant production and niche-specific stress responses. The unique combination of these features provides A. borkumensis SK2 with a competitive edge in oil-polluted environments. This genome sequence provides the basis for the future design of strategies to mitigate the ecological damage caused by oil spills.
ESTHER : Schneiker_2006_Nat.Biotechnol_24_997
PubMedSearch : Schneiker_2006_Nat.Biotechnol_24_997
PubMedID: 16878126
Gene_locus related to this paper: alcbo-Q9F9H0 , alcbs-q0vl04 , alcbs-q0vl36 , alcbs-q0vl92 , alcbs-q0vlp5 , alcbs-q0vlq1 , alcbs-q0vlt9 , alcbs-q0vm92 , alcbs-q0vmd2 , alcbs-q0vmd6 , alcbs-q0vmn9 , alcbs-q0vnu3 , alcbs-q0vp43 , alcbs-q0vpa9 , alcbs-q0vpc7 , alcbs-q0vpg7 , alcbs-q0vpn2 , alcbs-q0vps0 , alcbs-q0vq49 , alcbs-q0vsg4 , alcbs-q0vth9 , marav-a1u5n0 , alcbs-q0vp59 , alcbs-q0vlk5

Title : Natural microbial diversity in superficial sediments of Milazzo Harbor (Sicily) and community successions during microcosm enrichment with various hydrocarbons - Yakimov_2005_Environ.Microbiol_7_1426
Author(s) : Yakimov MM , Denaro R , Genovese M , Cappello S , D'Auria G , Chernikova TN , Timmis KN , Golyshin PN , Giluliano L
Ref : Environ Microbiol , 7 :1426 , 2005
Abstract : Hydrocarbon-contaminated superficial sediments collected from the Harbor of Milazzo (Tirrenean Sea, northern Sicily), a zone strongly affected by anthropogenic activities, were examined for in situ biodegradative capacities. A culture-independent molecular phylogenetic approach was used to study the influence of hydrocarbon and nutrient addition on the activity and diversity of the indigenous microbiota during a microcosm evaluation. The autochthonous microbial community in non-polluted sediments was represented by eubacterial phylotypes grouped within Proteobacteria, CFB and Firmicutes. The archaeal domain was represented by members of Marine Group I of Crenarchaeota. The majority of recovered sequences was affiliated with heterotrophic genera Clostridium and Vibrio, typical members of eutrophic coastal environments. Amendments of hydrocarbons and mineral nutrients to microcosms dramatically changed the initial diversity of the microbial community. Only bacterial phylotypes affiliated with Proteobacteria and CFB division were detected. The decrease in diversity observed in several microcosms could be explained by the strong selection for microorganisms belonging to group of marine hydrocarbonoclastic gamma-Proteobacteria, namely Alcanivorax, Cycloclasticus, Marinobacter, Marinobacterium/Neptunomonas and Thalassolituus. This study demonstrated that nutrient amendment to hydrocarbon-contaminated superficial sediments enhanced the indigenous microbial biodegradation activity and that highly specialized marine hydrocarbonoclastic bacteria, representing a minor fraction in the natural microbial community, play an important role in the biodegradation of petroleum hydrocarbons accidentally entering the coastal environment.
ESTHER : Yakimov_2005_Environ.Microbiol_7_1426
PubMedSearch : Yakimov_2005_Environ.Microbiol_7_1426
PubMedID: 16104865
Gene_locus related to this paper: 9bact-MilE3

Title : Novel hydrolase diversity retrieved from a metagenome library of bovine rumen microflora - Ferrer_2005_Environ.Microbiol_7_1996
Author(s) : Ferrer M , Golyshina OV , Chernikova TN , Khachane AN , Reyes-Duarte D , Santos VA , Strompl C , Elborough K , Jarvis G , Neef A , Yakimov MM , Timmis KN , Golyshin PN
Ref : Environ Microbiol , 7 :1996 , 2005
Abstract : A metagenome expression library of bulk DNA extracted from the rumen content of a dairy cow was established in a phage lambda vector and activity-based screening employed to explore the functional diversity of the microbial flora. Twenty-two clones specifying distinct hydrolytic activities (12 esterases, nine endo-beta-1,4-glucanases and one cyclodextrinase) were identified in the library and characterized. Sequence analysis of the retrieved enzymes revealed that eight (36%) were entirely new and formed deep-branched phylogenetic lineages with no close relatives among known ester- and glycosyl-hydrolases. Bioinformatic analyses of the hydrolase gene sequences, and the sequences and contexts of neighbouring genes, suggested tentative phylogenetic assignments of the rumen organisms producing the retrieved enzymes. The phylogenetic novelty of the hydrolases suggests that some of them may have potential for new applications in biocatalysis.
ESTHER : Ferrer_2005_Environ.Microbiol_7_1996
PubMedSearch : Ferrer_2005_Environ.Microbiol_7_1996
PubMedID: 16309396
Gene_locus related to this paper: 9zzzz-q2yi75 , 9zzzz-q2yi81 , 9zzzz-q2yi93

Title : Microbial enzymes mined from the Urania deep-sea hypersaline anoxic basin - Ferrer_2005_Chem.Biol_12_895
Author(s) : Ferrer M , Golyshina OV , Chernikova TN , Khachane AN , Martins dos Santos VA , Yakimov MM , Timmis KN , Golyshin PN
Ref : Chemical Biology , 12 :895 , 2005
Abstract : We created a metagenome expression library from the brine:seawater interface of the Urania hypersaline basin, screened it for esterases, and characterized five of these. Two had no significant sequence homology to known esterases, hydrolyzed both carboxylesters and thioesters, and exhibited unusual, habitat-specific characteristics (preference for high hydrostatic pressure and salinity). One has an unusual structural signature incorporating three catalytic active centers mediating distinct hydrolytic activities and an adaptive tertiary-quaternary structure that alters between three molecular states, according to the prevailing physicochemical conditions. Some of the esterases have high activities, specificities, enantioselectivities, and exceptional stability in polar solvents, and they are therefore potentially useful for industrial biotransformations. One possesses the highest enantioselectivity toward an ester of the important chiral synthon solketal (E: 126[S]; 98%ee).
ESTHER : Ferrer_2005_Chem.Biol_12_895
PubMedSearch : Ferrer_2005_Chem.Biol_12_895
PubMedID: 16125101
Gene_locus related to this paper: 9zzzz-q335p2 , 9zzzz-q335p5

Title : Conversion of a carboxylesterase into a triacylglycerol lipase by a random mutation -
Author(s) : Reyes-Duarte D , Polaina J , Lopez-Cortes N , Alcalde M , Plou FJ , Elborough K , Ballesteros A , Timmis KN , Golyshin PN , Ferrer M
Ref : Angew Chem Int Ed Engl , 44 :7553 , 2005
PubMedID: 16254934

Title : Expression of a temperature-sensitive esterase in a novel chaperone-based Escherichia coli strain - Ferrer_2004_Appl.Environ.Microbiol_70_4499
Author(s) : Ferrer M , Chernikova TN , Timmis KN , Golyshin PN
Ref : Applied Environmental Microbiology , 70 :4499 , 2004
Abstract : A new principle for expression of heat-sensitive recombinant proteins in Escherichia coli at temperatures close to 4 degrees C was experimentally evaluated. This principle was based on simultaneous expression of the target protein with chaperones (Cpn60 and Cpn10) from a psychrophilic bacterium, Oleispira antarctica RB8(T), that allow E. coli to grow at high rates at 4 degrees C (maximum growth rate, 0.28 h(-1)). The expression of a temperature-sensitive esterase in this host at 4 to 10 degrees C yielded enzyme specific activity that was 180-fold higher than the activity purified from the non-chaperonin-producing E. coli strain grown at 37 degrees C (32,380 versus 190 micromol min(-1) g(-1)). We present evidence that the increased specific activity was not due to the low growth temperature per se but was due to the fact that low temperature was beneficial to folding, with or without chaperones. This is the first report of successful use of a chaperone-based E. coli strain to express heat-labile recombinant proteins at temperatures below the theoretical minimum growth temperature of a common E. coli strain (7.5 degrees C).
ESTHER : Ferrer_2004_Appl.Environ.Microbiol_70_4499
PubMedSearch : Ferrer_2004_Appl.Environ.Microbiol_70_4499
PubMedID: 15294778
Gene_locus related to this paper: olean-q6a2s8

Title : A putative lichenysin A synthetase operon in B.licheniformis: initial charachterization. -
Author(s) : Yakimov MM , Kroeger A , Slepak TN , Giuliano L , Timmis KN , Golyshin PN
Ref : Biochimica & Biophysica Acta , 1399 :141 , 1998
PubMedID: 9765590
Gene_locus related to this paper: bacli-LICC , bacli-LICTE