Schwab H

References (27)

Title : Simple plug-in synthetic step for the synthesis of (-)-camphor from renewable starting materials - Calderini_2021_Chembiochem__
Author(s) : Calderini E , Drienovska I , Myrtollari K , Pressnig M , Sieber V , Schwab H , Kourist R , Hofer M
Ref : Chembiochem , : , 2021
Abstract : Racemic camphor and isoborneol are readily available as industrial side products, whereas (1R)-camphor is available from natural sources. Optically pure (1S)-camphor, however, is much more difficult to obtain. The synthesis of racemic camphor from a-pinene proceeds via an intermediary racemic isobornyl ester, which is then hydrolysed and oxidized to give camphor. We reasoned that enantioselective hydrolysis of isobornyl esters would give facile access to optically pure isoborneol and camphor isomers, respectively. While screening of a set of commercial lipases and esterases in the kinetic resolution of racemic monoterpenols did not lead to the identification of any enantioselective enzymes, the cephalosporin Esterase B from Burkholderia gladioli (EstB) and Esterase C (EstC) from Rhodococcus rhodochrous showed outstanding enantioselectivity (E>100) towards the butyryl esters of isoborneol borneol and fenchol. The enantioselectivity was higher with increasing chain length of the acyl moiety of the substrate. The kinetic resolution of isobornyl butyrate can be easily integrated into the production of camphor from a-pinene and thus allows the facile synthesis of optically pure monoterpenols from a renewable side-product.
ESTHER : Calderini_2021_Chembiochem__
PubMedSearch : Calderini_2021_Chembiochem__
PubMedID: 34033201

Title : Surface engineering of a cutinase from Thermobifida cellulosilytica for improved polyester hydrolysis - Herrero_2013_Biotechnol.Bioeng_110_2581
Author(s) : Herrero Acero E , Ribitsch D , Dellacher A , Zitzenbacher S , Marold A , Steinkellner G , Gruber K , Schwab H , Guebitz GM
Ref : Biotechnol Bioeng , 110 :2581 , 2013
Abstract : Modeling and comparison of the structures of the two closely related cutinases Thc_Cut1 and Thc_Cut2 from Thermobifida cellulosilytica DSM44535 revealed that dissimilarities in their electrostatic and hydrophobic surface properties in the vicinity to the active site could be responsible for pronounced differences in hydrolysis efficiencies of polyester (i.e., PET, polyethyleneterephthalate). To investigate this hypothesis in more detail, selected amino acids of surface regions outside the active site of Thc_Cut2, which hydrolyzes PET much less efficiently than Thc_Cut1 were exchanged by site-directed mutagenesis. The mutants were expressed in E. coli BL21-Gold(DE3), purified and characterized regarding their specific activities and kinetic parameters on soluble substrates and their ability to hydrolyze PET and the PET model substrate bis(benzoyloxyethyl) terephthalate (3PET). Compared to Thc_Cut2, mutants carrying Arg29Asn and/or Ala30Val exchanges showed considerable higher specific activity and higher kcat /KM values on soluble substrates. Exchange of the positively charged arginine (Arg19 and Arg29) located on the enzyme surface to the non-charged amino acids serine and asparagine strongly increased the hydrolysis activity for 3PET and PET. In contrast, exchange of the uncharged glutamine (Glu65) by the negatively charged glutamic acid lead to a complete loss of hydrolysis activity on PET films. These findings clearly demonstrate that surface properties (i.e., amino acids located outside the active site on the protein surface) play an important role in PET hydrolysis. Biotechnol. Bioeng. 2013;110: 2581-2590. (c) 2013 Wiley Periodicals, Inc.
ESTHER : Herrero_2013_Biotechnol.Bioeng_110_2581
PubMedSearch : Herrero_2013_Biotechnol.Bioeng_110_2581
PubMedID: 23592055

Title : Fusion of binding domains to Thermobifida cellulosilytica cutinase to tune sorption characteristics and enhancing PET hydrolysis - Ribitsch_2013_Biomacromolecules_14_1769
Author(s) : Ribitsch D , Yebra AO , Zitzenbacher S , Wu J , Nowitsch S , Steinkellner G , Greimel K , Doliska A , Oberdorfer G , Gruber CC , Gruber K , Schwab H , Stana-Kleinschek K , Herrero Acero E , Guebitz GM
Ref : Biomacromolecules , 14 :1769 , 2013
Abstract : A cutinase from Thermomyces cellullosylitica (Thc_Cut1), hydrolyzing the synthetic polymer polyethylene terephthalate (PET), was fused with two different binding modules to improve sorption and thereby hydrolysis. The binding modules were from cellobiohydrolase I from Hypocrea jecorina (CBM) and from a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (PBM). Although both binding modules have a hydrophobic nature, it was possible to express the proteins in E. coli . Both fusion enzymes and the native one had comparable kcat values in the range of 311 to 342 s(-1) on pNP-butyrate, while the catalytic efficiencies kcat/Km decreased from 0.41 s(-1)/ muM (native enzyme) to 0.21 and 0.33 s(-1)/muM for Thc_Cut1+PBM and Thc_Cut1+CBM, respectively. The fusion enzymes were active both on the insoluble PET model substrate bis(benzoyloxyethyl) terephthalate (3PET) and on PET although the hydrolysis pattern was differed when compared to Thc_Cut1. Enhanced adsorption of the fusion enzymes was visible by chemiluminescence after incubation with a 6xHisTag specific horseradish peroxidase (HRP) labeled probe. Increased adsorption to PET by the fusion enzymes was confirmed with Quarz Crystal Microbalance (QCM-D) analysis and indeed resulted in enhanced hydrolysis activity (3.8x for Thc_Cut1+CBM) on PET, as quantified, based on released mono/oligomers.
ESTHER : Ribitsch_2013_Biomacromolecules_14_1769
PubMedSearch : Ribitsch_2013_Biomacromolecules_14_1769
PubMedID: 23718548
Gene_locus related to this paper: thefu-q6a0i4

Title : Insights into the completely annotated genome of Lactobacillus buchneri CD034, a strain isolated from stable grass silage - Heinl_2012_J.Biotechnol_161_153
Author(s) : Heinl S , Wibberg D , Eikmeyer F , Szczepanowski R , Blom J , Linke B , Goesmann A , Grabherr R , Schwab H , Puhler A , Schluter A
Ref : J Biotechnol , 161 :153 , 2012
Abstract : Lactobacillus buchneri belongs to the group of heterofermentative lactic acid bacteria and is a common member of the silage microbiome. Here we report the completely annotated genomic sequence of L. buchneri CD034, a strain isolated from stable grass silage. The whole genome of L. buchneri CD034 was sequenced on the Roche Genome Sequencer FLX platform. It was found to consist of four replicons, a circular chromosome, and three plasmids. The circular chromosome was predicted to encode 2319 proteins and contains a genomic island and two prophages which significantly differ in G+C-content from the remaining chromosome. It possesses all genes for enzymes of a complete phosphoketolase pathway, whereas two enzymes necessary for glycolysis are lacking. This confirms the classification of L. buchneri CD034 as an obligate heterofermentative lactic acid bacterium. A set of genes considered to be involved in the lactate degradation pathway and genes putatively involved in the breakdown of plant cell wall polymers were identified. Moreover, several genes encoding putative S-layer proteins and two CRISPR systems, belonging to the subclasses I-E and II-A, are located on the chromosome. The largest plasmid pCD034-3 was predicted to encode 57 genes, including a putative polysaccharide synthesis gene cluster, whereas the functions of the two smaller plasmids, pCD034-1 and pCD034-2, remain cryptic. Phylogenetic analysis based on sequence comparison of the conserved marker gene rpoA reveals that L. buchneri CD034 is more closely related to Lactobacillus hilgardii strains than to Lactobacillus brevis and Lactobacillus plantarum strains. Comparison of the L. buchneri CD034 core genome to other fully sequenced and closely related members of the genus Lactobacillus disclosed a high degree of conservation between L. buchneri CD034 and the recently sequenced L. buchneri strain NRRL B-30929 and a more distant relationship to L. buchneri ATCC 11577 and L. brevis ssp. gravesensis ATCC 27305, which cluster together with L. hilgardii type strain ATCC 8290. L. buchneri CD034 genome information will certainly provide the basis for further postgenome studies with the objective to optimize application of the strain in silage production.
ESTHER : Heinl_2012_J.Biotechnol_161_153
PubMedSearch : Heinl_2012_J.Biotechnol_161_153
PubMedID: 22465289
Gene_locus related to this paper: lacbn-f4fwx4

Title : Extracellular serine proteases from Stenotrophomonas maltophilia: Screening, isolation and heterologous expression in E. coli - Ribitsch_2012_J.Biotechnol_157_140
Author(s) : Ribitsch D , Heumann S , Karl W , Gerlach J , Leber R , Birner-Gruenberger R , Gruber K , Eiteljoerg I , Remler P , Siegert P , Lange J , Maurer KH , Berg G , Guebitz GM , Schwab H
Ref : J Biotechnol , 157 :140 , 2012
Abstract : A large strain collection comprising antagonistic bacteria was screened for novel detergent proteases. Several strains displayed protease activity on agar plates containing skim milk but were inactive in liquid media. Encapsulation of cells in alginate beads induced protease production. Stenotrophomonas maltophilia emerged as best performer under washing conditions. For identification of wash-active proteases, four extracellular serine proteases called StmPr1, StmPr2, StmPr3 and StmPr4 were cloned. StmPr2 and StmPr4 were sufficiently overexpressed in E. coli. Expression of StmPr1 and StmPr3 resulted in unprocessed, insoluble protein. Truncation of most of the C-terminal domain which has been identified by enzyme modeling succeeded in expression of soluble, active StmPr1 but failed in case of StmPr3. From laundry application tests StmPr2 turned out to be a highly wash-active protease at 45 degrees C. Specific activity of StmPr2 determined with suc-L-Ala-L-Ala-L-Pro-l-Phe-p-nitroanilide as the substrate was 17+/-2U/mg. In addition we determined the kinetic parameters and cleavage preferences of protease StmPr2.
ESTHER : Ribitsch_2012_J.Biotechnol_157_140
PubMedSearch : Ribitsch_2012_J.Biotechnol_157_140
PubMedID: 21983234

Title : A New Esterase from Thermobifida halotolerans Hydrolyses Polyethylene Terephthalate (PET) and Polylactic Acid (PLA) - Ribitsch_2012_Polymers_4_617
Author(s) : Ribitsch D , Herrero Acero E , Greimel K , Dellacher A , Zitzenbacher S , Marold A , Rodriguez RD , Steinkellner G , Gruber K , Schwab H , Guebitz GM
Ref : Polymers , 4 :617 , 2012
Abstract : A new esterase from Thermobifida halotolerans (Thh_Est) was cloned and expressed in E. coli and investigated for surface hydrolysis of polylactic acid (PLA) and polyethylene terephthalate (PET). Thh_Est is a member of the serine hydrolases superfamily containing the -GxSxG- motif with 8587% homology to an esterase from T. alba, to an acetylxylan esterase from T. fusca and to various Thermobifida cutinases. Thh_Est hydrolyzed the PET model substrate bis(benzoyloxyethyl)terephthalate and PET releasing terephthalic acid and mono-(2-hydroxyethyl) terephthalate in comparable amounts (19.8 and 21.5 mmol/mol of enzyme) while no higher oligomers like bis-(2-hydroxyethyl) terephthalate were detected. Similarly, PLA was hydrolyzed as indicated by the release of lactic acid. Enzymatic surface hydrolysis of PET and PLA led to a strong hydrophilicity increase, as quantified with a WCA decrease from 90.8 and 75.5 to 50.4 and to a complete spread of the water drop on the surface, respectively
ESTHER : Ribitsch_2012_Polymers_4_617
PubMedSearch : Ribitsch_2012_Polymers_4_617
PubMedID:
Gene_locus related to this paper: 9actn-h6wx58

Title : High-quality genome sequence of Pichia pastoris CBS7435 - Kuberl_2011_J.Biotechnol_154_312
Author(s) : Kuberl A , Schneider J , Thallinger GG , Anderl I , Wibberg D , Hajek T , Jaenicke S , Brinkrolf K , Goesmann A , Szczepanowski R , Puhler A , Schwab H , Glieder A , Pichler H
Ref : J Biotechnol , 154 :312 , 2011
Abstract : The methylotrophic yeast Pichia pastoris (Komagataella phaffii) CBS7435 is the parental strain of commonly used P. pastoris recombinant protein production hosts making it well suited for improving the understanding of associated genomic features. Here, we present a 9.35 Mbp high-quality genome sequence of P. pastoris CBS7435 established by a combination of 454 and Illumina sequencing. An automatic annotation of the genome sequence yielded 5007 protein-coding genes, 124 tRNAs and 29 rRNAs. Moreover, we report the complete DNA sequence of the first mitochondrial genome of a methylotrophic yeast. Fifteen genes encoding proteins, 2 rRNA and 25 tRNA loci were identified on the 35.7 kbp circular, mitochondrial DNA. Furthermore, the architecture of the putative alpha mating factor protein of P. pastoris CBS7435 turned out to be more complex than the corresponding protein of Saccharomyces cerevisiae.
ESTHER : Kuberl_2011_J.Biotechnol_154_312
PubMedSearch : Kuberl_2011_J.Biotechnol_154_312
PubMedID: 21575661
Gene_locus related to this paper: picp7-f2qwe3 , picpa-cbpy , picpa-KEX1 , picpg-c4qw07 , picpg-c4r0k6 , picpg-c4r5w7 , picpg-c4r6t8 , picpg-c4r272 , picpg-c4r403 , picpg-c4r663 , picpg-c4r788 , picpa-a0a1b2jh35

Title : Enzymatic Surface Hydrolysis of PET: Effect of Structural Diversity on Kinetic Properties of Cutinases from Thermobifida. - Herrero-Acero_2011_Macromolecules_44_4632
Author(s) : Herrero Acero E , Ribitsch D , Steinkellner G , Gruber K , Greimel K , Eiteljoerg I , Trotscha E , Wei R , Zimmermann W , Zinn M , Cavaco-Paulo A , Freddi G , Schwab H , Guebitz G
Ref : Macromolecules , 44 :4632 , 2011
Abstract : In this study cutinases from Thermobifida cellulosilytica DSM44535 (Thc_Cut1 and Thc_Cut2) and Thermobifida fusca DSM44342 (Thf42_Cut1) hydrolyzing poly(ethylene terephthalate) (PET) were successfully cloned and expressed in E.coli BL21-Gold(DE3). Their ability to hydrolyze PET was compared with other enzymes hydrolyzing natural polyesters, including the PHA depolymerase (ePhaZmcl) from Pseudomonas fluorescens and two cutinases from T. fusca KW3. The three isolated Thermobifida cutinases are very similar (only a maximum of 18 amino acid differences) but yet had different kinetic parameters on soluble substrates. Their kcat and Km values on pNP-acetate were in the ranges 2.4-211.9 s-1 and 127-200 micoM while on pNP-butyrate they showed kcat and Km values between 5.3 and 195.1 s-1 and between 1483 and 2133 microM. Thc_Cut1 released highest amounts of MHET and terephthalic acid from PET and bis(benzoyloxyethyl) terephthalate (3PET) with the highest concomitant increase in PET hydrophilicity as indicated by water contact angle (WCA) decreases. FTIR-ATR analysis revealed an increase in the crystallinity index A1340/A1410 upon enzyme treatment and an increase of the amount of carboxylic and hydroxylic was measured using derivatization with 2-(bromomethyl)naphthalene. Modeling the covalently bound tetrahedral intermediate consisting of cutinase and 3PET indicated that the active site His-209 is in the proximity of the O of the substrate thus allowing hydrolysis. On the other hand, the models indicated that regions of Thc_Cut1 and Thc_Cut2 which differed in electrostatic and in hydrophobic surface properties were able to reach/interact with PET which may explain their different hydrolysis efficiencies.
ESTHER : Herrero-Acero_2011_Macromolecules_44_4632
PubMedSearch : Herrero-Acero_2011_Macromolecules_44_4632
PubMedID:
Gene_locus related to this paper: bacsu-pnbae , thefu-q6a0i4 , thefu-q6a0i3

Title : Hydrolysis of polyethyleneterephthalate by p-nitrobenzylesterase from Bacillus subtilis - Ribitsch_2011_Biotechnol.Prog_27_951
Author(s) : Ribitsch D , Heumann S , Trotscha E , Herrero Acero E , Greimel K , Leber R , Birner-Gruenberger R , Deller S , Eiteljoerg I , Remler P , Weber T , Siegert P , Maurer KH , Donelli I , Freddi G , Schwab H , Guebitz GM
Ref : Biotechnol Prog , 27 :951 , 2011
Abstract : From a screening on agar plates with bis(benzoyloxyethyl) terephthalate (3PET), a Bacillus subtilis p-nitrobenzylesterase (BsEstB) was isolated and demonstrated to hydrolyze polyethyleneterephthalate (PET). PET-hydrolase active strains produced clearing zones and led to the release of the 3PET hydrolysis products terephthalic acid (TA), benzoic acid (BA), 2-hydroxyethyl benzoate (HEB), and mono-(2-hydroxyethyl) terephthalate (MHET) in 3PET supplemented liquid cultures. The 3PET-hydrolase was isolated from non-denaturating polyacrylamide gels using fluorescein diacetate (FDA) and identified as BsEstB by LC-MS/MS analysis. BsEstB was expressed in Escherichia coli with C-terminally fused StrepTag II for purification. The tagged enzyme had a molecular mass of 55.2 kDa and a specific activity of 77 U/mg on p-nitrophenyl acetate and 108 U/mg on p-nitrophenyl butyrate. BsEstB was most active at 40 degrees C and pH 7.0 and stable for several days at pH 7.0 and 37 degrees C while the half-life times decreased to 3 days at 40 degrees C and only 6 h at 45 degrees C. From 3PET, BsEstB released TA, MHET, and BA, but neither bis(2-hydroxyethyl) terephthalate (BHET) nor hydroxyethylbenzoate (HEB). The kcat values decreased with increasing complexity of the substrate from 6 and 8 (s-1) for p-nitrophenyl-acetate (4NPA) and p-nitrophenyl-butyrate (4NPB), respectively, to 0.14 (s-1) for bis(2-hydroxyethyl) terephthalate (BHET). The enzyme hydrolyzed PET films releasing TA and MHET with a concomitant decrease of the water-contact angle (WCA) from 68.2 degrees +/-1.7 degrees to 62.6 degrees +/-1.1 degrees due to formation of novel hydroxyl and carboxyl groups. These data correlated with a fluorescence emission intensity increase seen for the enzyme treated sample after derivatization with 2-(bromomethyl)naphthalene.
ESTHER : Ribitsch_2011_Biotechnol.Prog_27_951
PubMedSearch : Ribitsch_2011_Biotechnol.Prog_27_951
PubMedID: 21574267
Gene_locus related to this paper: bacsu-pnbae

Title : The SGNH-hydrolase of Streptomyces coelicolor has (aryl)esterase and a true lipase activity - Bielen_2009_Biochimie_91_390
Author(s) : Bielen A , Cetkovic H , Long PF , Schwab H , Abramic M , Vujaklija D
Ref : Biochimie , 91 :390 , 2009
Abstract : The Streptomyces coelicolor A3(2) gene SCI11.14c was overexpressed and purified as a His-tagged protein from heterologous host, Streptomyces lividans. The purification procedure resulted in 34.1-fold increase in specific activity with an overall yield of 21.4%. Biochemical and physical properties of the purified enzyme were investigated and it was shown that it possesses (aryl)esterase and a true lipase activity. The enzyme was able to hydrolyze p-nitrophenyl-, alpha- and beta-naphthyl esters and poly(oxyethylene) sorbitan monoesters (Tween 20-80). It showed pronounced activity towards p-nitrophenyl and alpha- and beta-naphthyl esters of C(12)-C(16). Higher activity was observed with alpha-naphthyl esters. The enzyme hydrolyzed triolein (specific activity: 91.9 U/mg) and a wide range of oils with a preference for those having higher content of linoleic or oleic acid (C18:2; C18:1, cis). The active-site serine specific inhibitor 3,4-dichloroisocoumarin (DCI) strongly inhibited the enzyme, while tetrahydrofurane and 1,4-dioxane significantly increased (2- and 4- fold, respectively) hydrolytic activity of lipase towards p-nitrophenyl caprylate. The enzyme exhibited relatively high temperature optimum (55 degrees C) and thermal stability. CD analysis revealed predominance of alpha-helical structure (54% alpha-helix, 21% beta-sheet) and a T(m) value at 66 degrees C. Systematic bioinformatic analysis of deduced amino acid sequence of S. coelicolor enzyme placed it to the SGNH-hydrolase family. Phylogenetic analysis of the predicted protein homologous to the S. coelicolor SGNH-hydrolase generated three distinct groups consisting of proteins from Actinomycetales, Ascomycota and Nematoda. At present it seems that these enzymes are most conserved among soil inhabiting organisms.
ESTHER : Bielen_2009_Biochimie_91_390
PubMedSearch : Bielen_2009_Biochimie_91_390
PubMedID: 19041687

Title : Alternative pig liver esterase (APLE) - cloning, identification and functional expression in Pichia pastoris of a versatile new biocatalyst - Hermann_2008_J.Biotechnol_133_301
Author(s) : Hermann M , Kietzmann MU , Ivancic M , Zenzmaier C , Luiten RG , Skranc W , Wubbolts M , Winkler M , Birner-Gruenberger R , Pichler H , Schwab H
Ref : J Biotechnol , 133 :301 , 2008
Abstract : Isolated from pig liver as a crude, inhomogeneous enzyme fraction, pig liver esterase (PLE) was found to metabolize a wide range of substrates; often in a highly stereoselective manner. This crude esterase preparation, however, contains several iso-enzymes at proportions varying from batch to batch. Racemic methyl-(4E)-5-chloro-2-isopropyl-4-pentenoate is cleaved enantioselectively by crude PLE, but not by recombinantly expressed gamma-isoform of PLE. Concluding that another PLE iso-enzyme must carry the relevant activity, we cloned and sequenced cDNAs of several PLE isoforms and functionally expressed them in Pichia pastoris. One novel isoform termed alternative pig liver esterase (APLE) was found to hydrolyze methyl-(2R,4E)-5-chloro-2-isopropyl-4-pentenoate in a highly stereoselective manner (E>200). When heterologously expressed and directed for secretion in P. pastoris, APLE was found to be localized in the periplasm. The presence or absence of a putative C-terminal ER retention signal did neither influence functional expression nor cellular localization. The recombinant enzyme, purified by ion exchange chromatography, had a specific activity of 36U (mg protein)(-1) towards racemic methyl-(4E)-5-chloro-2-isopropyl-4-pentenoate.
ESTHER : Hermann_2008_J.Biotechnol_133_301
PubMedSearch : Hermann_2008_J.Biotechnol_133_301
PubMedID: 18078679
Gene_locus related to this paper: pig-a9gyw6

Title : A novel screening assay for hydroxynitrile lyases suitable for high-throughput screening - Krammer_2007_J.Biotechnol_129_151
Author(s) : Krammer B , Rumbold K , Tschemmernegg M , Pochlauer P , Schwab H
Ref : J Biotechnol , 129 :151 , 2007
Abstract : Hydroxynitrile lyases (Hnls) are important biocatalysts for the synthesis of optically pure cyanohydrins, which are used as precursors and building blocks for a wide range of high price fine chemicals. Although two Hnl enzymes, from the tropical rubber tree Hevea brasiliensis and from the almond tree Prunus amygdalus, are already used for large scale industrial applications, the enzymes still need to be improved and adapted to the special demands of industrial processes. In many cases directed evolution has been the method of choice to improve enzymes, which are applied as industrial biocatalysts. The screening procedure is the most crucial point in every directed evolution experiment. Herein, we describe the successful development of a novel screening assay for Hnls and its application in high-throughput screening of Escherichia coli mutant libraries. The new assay allows rapid screening of mutant libraries and facilitates the discovery of improved enzyme variants. Hnls catalyze the cleavage of cyanohydrins to hydrocyanic acid and the corresponding aldehyde or ketone. The enzyme assay is based on the detection of hydrocyanic acid produced, making it an all-purpose screening assay, without restriction to any kind of substrate. The gaseous HCN liberated within the Hnl reaction is detected by a visible colorimetric reaction. The facile, highly sensitive and reproducible screening method was validated by identifying new enzyme variants with novel substrate specificities.
ESTHER : Krammer_2007_J.Biotechnol_129_151
PubMedSearch : Krammer_2007_J.Biotechnol_129_151
PubMedID: 17157404

Title : Inverting enantioselectivity of Burkholderia gladioli esterase EstB by directed and designed evolution - Ivancic_2007_J.Biotechnol_129_109
Author(s) : Ivancic M , Valinger G , Gruber K , Schwab H
Ref : J Biotechnol , 129 :109 , 2007
Abstract : Esterase EstB from Burkholderia gladioli, showing moderate S-enantioselectivity (E(S)=6.1) in the hydrolytic kinetic resolution of methyl-beta-hydroxyisobutyrate, was subjected to directed evolution in order to reverse its enantioselectivity. After one round of ep-PCR, saturation mutagenesis and high-throughput screening, it was found that different mutations at position 152 (in the vicinity of the active site) increase, decrease and even reverse the natural enantioselectivity of this enzyme. The newly created R-enantioselectivity of the esterase mutein (E(Rapp)=1.5) has been further enhanced by a designed evolution strategy involving random mutations close to the active site. Based on the three-dimensional structure nineteen amino acid residues have been selected as mutation sites for saturation mutagenesis. Mutations at three sites (135, 253 and 351) were found to increase R-enantioselectivity. Successive rounds of saturation mutagenesis at these "hot spots" resulted in an increase in R-enantioselectivity from E(Rapp)=1.5 for the parent mutant to E(Rapp)=28.9 for the best variant which carried four amino acid substitutions. Our results prove designed evolution followed by high-throughput screening to be an efficient strategy for engineering enzyme enantioselectivity.
ESTHER : Ivancic_2007_J.Biotechnol_129_109
PubMedSearch : Ivancic_2007_J.Biotechnol_129_109
PubMedID: 17147964

Title : Cell surface expression of bacterial esterase A by Saccharomyces cerevisiae and its enhancement by constitutive activation of the cellular unfolded protein response - Breinig_2006_Appl.Environ.Microbiol_72_7140
Author(s) : Breinig F , Diehl B , Rau S , Zimmer C , Schwab H , Schmitt MJ
Ref : Applied Environmental Microbiology , 72 :7140 , 2006
Abstract : Yeast cell surface display is a powerful tool for expression and immobilization of biocatalytically active proteins on a unicellular eukaryote. Here bacterial carboxylesterase EstA from Burkholderia gladioli was covalently anchored into the cell wall of Saccharomyces cerevisiae by in-frame fusion to the endogenous yeast proteins Kre1p, Cwp2p, and Flo1p. When p-nitrophenyl acetate was used as a substrate, the esterase specific activities of yeast expressing the protein fusions were 103 mU mg(-1) protein for Kre1/EstA/Cwp2p and 72 mU mg(-1) protein for Kre1/EstA/Flo1p. In vivo cell wall targeting was confirmed by esterase solubilization after laminarinase treatment and immunofluorescence microscopy. EstA expression resulted in cell wall-associated esterase activities of 2.72 U mg(-1) protein for Kre1/EstA/Cwp2p and 1.27 U mg(-1) protein for Kre1/EstA/Flo1p. Furthermore, esterase display on the yeast cell surface enabled the cells to effectively grow on the esterase-dependent carbon source glycerol triacetate (Triacetin). In the case of Kre1/EstA/Flo1p, in vivo maturation within the yeast secretory pathway and final incorporation into the wall were further enhanced when there was constitutive activation of the unfolded protein response pathway. Our results demonstrate that esterase cell surface display in yeast, which, as shown here, is remarkably more effective than EstA surface display in Escherichia coli, can be further optimized by activating the protein folding machinery in the eukaryotic secretion pathway.
ESTHER : Breinig_2006_Appl.Environ.Microbiol_72_7140
PubMedSearch : Breinig_2006_Appl.Environ.Microbiol_72_7140
PubMedID: 16980424

Title : Cloning and sequence analysis of the glyceraldehyde-3-phosphate dehydrogenase gene from the zygomycetes fungus Rhizomucor miehei - Vastag_2004_Antonie.Van.Leeuwenhoek_86_111
Author(s) : Vastag M , Kasza Z , Acs K , Papp T , Schwab H , Vagvolgyi C
Ref : Antonie Van Leeuwenhoek , 86 :111 , 2004
Abstract : Rhizomucor miehei is important from a biotechnological aspect in consequence of its content of aspartic proteinase, which has high milk-clotting activity. A genomic library of R. miehei NRRL 5901 has been constructed in a phage (Lambda Fix II) vector. The glyceraldehyde-3-phosphate dehydrogenase (gpd) gene was isolated from this library by hybridization of the recombinant phage clones with a gpd-specific gene probe generated by polymerase chain reaction. The complete nucleotide sequence encodes a putative polypeptide chain of 336 amino acids interrupted by 5 introns. The predicted amino acid sequence of this gene shows a high degree of sequence similarity to the glyceraldehyde-3-phosphate dehydrogenase proteins from yeast and filamentous fungi. The promoter region, containing a consensus TATA box, and 246-bp downstream from the putative stop codon were also determined. The possibility of using the gpd promoter in the construction of new transformation vectors is discussed.
ESTHER : Vastag_2004_Antonie.Van.Leeuwenhoek_86_111
PubMedSearch : Vastag_2004_Antonie.Van.Leeuwenhoek_86_111
PubMedID: 15280645

Title : Observation of a short, strong hydrogen bond in the active site of hydroxynitrile lyase from Hevea brasiliensis explains a large pKa shift of the catalytic base induced by the reaction intermediate - Stranzl_2004_J.Biol.Chem_279_3699
Author(s) : Stranzl GR , Gruber K , Steinkellner G , Zangger K , Schwab H , Kratky C
Ref : Journal of Biological Chemistry , 279 :3699 , 2004
Abstract : The hydroxynitrile lyase from Hevea brasiliensis (HbHNL) uses a catalytic triad consisting of Ser(80)-His(235)-Asp(207) to enhance the basicity of Ser(80)-O gamma for abstracting a proton from the OH group of the substrate cyanohydrin. Following the observation of a relatively short distance between a carboxyl oxygen of Asp(207) and the N delta(1)(His(235)) in a 1.1 A crystal structure of HbHNL, we here show by (1)H and (15)N-NMR spectroscopy that a short, strong hydrogen bond (SSHB) is formed between the two residues upon binding of the competitive inhibitor thiocyanate to HbHNL: the proton resonance of H-N delta 1(His(235)) moves from 15.41 ppm in the free enzyme to 19.35 ppm in the complex, the largest downfield shift observed so far upon inhibitor binding. Simultaneously, the D/H fractionation factor decreases from 0.98 to 0.35. In the observable pH range, i.e. between pH 4 and 10, no significant changes in chemical shifts (and therefore hydrogen bond strength) were observed for free HbHNL. For the complex with thiocyanate, the 19.35 ppm signal returned to 15.41 ppm at approximately pH 8, which indicates a pK(a) near this value for the H-N epsilon(2)(His(235)). These NMR results were analyzed on the basis of finite difference Poisson-Boltzmann calculations, which yielded the relative free energies of four protonation states of the His(235)-Asp(207) pair in solution as well as in the protein environment with and without bound inhibitor. The calculations explain all the NMR features, i.e. they suggest why a short, strong hydrogen bond is formed upon inhibitor binding and why this short, strong hydrogen bond reverts back to a normal one at approximately pH 8. Importantly, the computations also yield a shift of the free energy of the anionic state relative to the zwitterionic reference state by about 10.6 kcal/mol, equivalent to a shift in the apparent pK(a) of His(235) from 2.5 to 10. This huge inhibitor-induced increase in basicity is a prerequisite for His(235) to act as general base in the HbHNL-catalyzed cyanohydrin reaction.
ESTHER : Stranzl_2004_J.Biol.Chem_279_3699
PubMedSearch : Stranzl_2004_J.Biol.Chem_279_3699
PubMedID: 14597632
Gene_locus related to this paper: manes-hnl

Title : Reaction mechanism of hydroxynitrile lyases of the alpha\/beta-hydrolase superfamily: the three-dimensional structure of the transient enzyme-substrate complex certifies the crucial role of LYS236 - Gruber_2004_J.Biol.Chem_279_20501
Author(s) : Gruber K , Gartler G , Krammer B , Schwab H , Kratky C
Ref : Journal of Biological Chemistry , 279 :20501 , 2004
Abstract : The hydroxynitrile lyases (HNLs) from Hevea brasiliensis (HbHNL) and from Manihot esculenta (MeHNL) are both members of the alpha/beta-hydrolase superfamily. Mechanistic proposals have been put forward in the past for both enzymes; they differed with respect to the role of the active-site lysine residue for which a catalytic function was claimed for the Hevea enzyme but denied for the Manihot enzyme. We applied a freeze-quench method to prepare crystals of the complex of HbHNL with the biological substrate acetone cyanohydrin and determined its three-dimensional structure. Site-directed mutagenesis was used to prepare the mutant K236L, which is inactive although its three-dimensional structure is similar to the wild-type enzyme. However, the structure of the K236L-acetone cyanohydrin complex shows the substrate in a different orientation from the wild-type complex. Finite difference Poisson-Boltzmann calculations show that in the absence of Lys(236) the catalytic base His(235) would be protonated at neutral pH. All of this suggests that Lys(236) is instrumental for catalysis in several ways, i.e. by correctly positioning the substrate, by stabilizing the negatively charged reaction product CN(-), and by modulating the basicity of the catalytic base. These data complete the elucidation of the reaction mechanism of alpha/beta-hydrolase HNLs, in which the catalytic triad acts as a general base rather than as a nucleophile; proton abstraction from the substrate is performed by the serine, and reprotonation of the product cyanide is performed by the histidine residues. Together with a threonine side chain, the active-site serine and lysine are also involved in substrate binding.
ESTHER : Gruber_2004_J.Biol.Chem_279_20501
PubMedSearch : Gruber_2004_J.Biol.Chem_279_20501
PubMedID: 14998991
Gene_locus related to this paper: hevbr-hnl

Title : Esterase EstE from Xanthomonas vesicatoria ( Xv_EstE) is an outer membrane protein capable of hydrolyzing long-chain polar esters - Talker-Huiber_2003_Appl.Microbiol.Biotechnol_61_479
Author(s) : Talker-Huiber D , Jose J , Glieder A , Pressnig M , Stubenrauch G , Schwab H
Ref : Applied Microbiology & Biotechnology , 61 :479 , 2003
Abstract : A new esterase gene from Xanthomonas vesicatoria (formerly X. campestris) DSM 50861 was identified, cloned from a chromosomal gene library and overexpressed in Escherichia coli. The corresponding DNA fragment contains an ORF of 1,818 bp, encoding a hydrolase of the GDSL esterase family. A protein of about 67 kDa, named Xv_EstE, was expressed from this fragment. A N-terminal signal peptide was processed under low-expression conditions, yielding a 63-kDa mature protein. The predicted amino acid sequence showed distinct homology to esterases of the GDSL family. Based on homology, a catalytic triad Gly-Asp-Ser could be defined. Amino acid sequence alignments and computer-assisted structure prediction indicated the presence of a carboxyl-terminal beta-barrel membrane domain which might facilitate binding of Xv_EstE to the outer membrane. This could be verified by differential cell fractionation experiments, in which Xv_EstE was exclusively found in the outer membrane fraction. Xv_EstE showed preferential hydrolytic activity on short chain (up to C(8)) and para-substituted nitrophenylesters as substrates. However, only long-chain 1-hydroxy-pyrene-3,6,8-trisulfonic acid (HPTS)-fatty acid esters were hydrolyzed. Xv_EstE was also found to be active on a series of substrates of industrial interest, such as 1-methylprop-2-ynyl acetate, for which an enantioselectivity up to 93% ee could be recognized.
ESTHER : Talker-Huiber_2003_Appl.Microbiol.Biotechnol_61_479
PubMedSearch : Talker-Huiber_2003_Appl.Microbiol.Biotechnol_61_479
PubMedID: 12764562

Title : Comprehensive step-by-step engineering of an (R)-hydroxynitrile lyase for large-scale asymmetric synthesis -
Author(s) : Glieder A , Weis R , Skranc W , Poechlauer P , Dreveny I , Majer S , Wubbolts M , Schwab H , Gruber K
Ref : Angew Chem Int Ed Engl , 42 :4815 , 2003
PubMedID: 14562357

Title : Cloning and characterization of EstC from Burkholderia gladioli, a novel-type esterase related to plant enzymes - Reiter_2000_Appl.Microbiol.Biotechnol_54_778
Author(s) : Reiter B , Glieder A , Talker D , Schwab H
Ref : Applied Microbiology & Biotechnology , 54 :778 , 2000
Abstract : By screening a genomic library of Burkholderia gladioli (formerly Pseudomonas marginata) for clones exhibiting esterolytic activity, the gene for a novel-type esterase (EstC) showing significant homology to plant enzymes could be isolated. High homology was found to two hydroxynitrile lyases originating from Hevea brasiliensis (tropical rubber tree) and Manihot esculenta (cassava), and to two proteins from Oryza sativa (rice) that are specifically induced upon infection by Pseudomonas syringae pv. syringae. The sequenced ORF encodes for a protein of 298 amino acids. The enzyme was efficiently overexpressed in Escherichia coli, purified and characterized with respect to enzymatic capabilities. The enzyme was able to hydrolyze a variety of esterase substrates of low to medium carbonic acid chain length, but no triglycerides were hydrolyzed. Despite the high sequence homology, no hydroxynitrile lyase activity could be recognized.
ESTHER : Reiter_2000_Appl.Microbiol.Biotechnol_54_778
PubMedSearch : Reiter_2000_Appl.Microbiol.Biotechnol_54_778
PubMedID: 11152069
Gene_locus related to this paper: burgl-EstC

Title : The defense-related rice gene Pir7b encodes an alpha\/beta hydrolase fold protein exhibiting esterase activity towards naphthol AS-esters - Waspi_1998_Eur.J.Biochem_254_32
Author(s) : Waspi U , Misteli B , Hasslacher M , Jandrositz A , Kohlwein SD , Schwab H , Dudler R
Ref : European Journal of Biochemistry , 254 :32 , 1998
Abstract : Acquired resistance of rice to Pyricularia oryzae, the causing agent of rice blast, can be induced by inoculation with the non-host pathogen Pseudomonas syringae pv. syringae. We have previously cloned a cDNA and a corresponding gene (Pir7b) whose transcripts accumulate upon infiltration with the resistance-inducing bacteria. The putative encoded product Pir7b exhibits significant sequence similarity to two recently cloned hydroxynitrile lyases from Manihot esculenta (cassava) and Hevea brasisliensis, enzymes involved in the release of hydrogen cyanide from cyanogenic glycosides. As rice does not contain cyanogenic glycosides, a similar function of Pir7b appears unplausible. In order to functionally characterize the protein, recombinant Pir7b was produced in Escherichia coli and Saccharomyces cerevisiae. We show that recombinant Pir7b does not have hydroxynitrile lyase activity, but exhibits esterase activity towards naphthol AS-acetate. Using Pir7b-specific antibodies, we show that the protein accumulates in rice leaves inoculated with P. syringae pv. syringae. Both the recombinant and the authentic proteins have an apparent molecular mass of 32 kDa (28.8 kDa calculated) and seem to be active as monomers. Pir7b esterase also exhibits sequence similarity to several expressed sequence tags of Arabidopsis thaliana, indicating that it belongs to a family of proteins widely occuring in plants.
ESTHER : Waspi_1998_Eur.J.Biochem_254_32
PubMedSearch : Waspi_1998_Eur.J.Biochem_254_32
PubMedID: 9652390
Gene_locus related to this paper: orysa-pir7b

Title : High-level intracellular expression of hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts - Hasslacher_1997_Protein.Expr.Purif_11_61
Author(s) : Hasslacher M , Schall M , Hayn M , Bona R , Rumbold K , Luckl J , Griengl H , Kohlwein SD , Schwab H
Ref : Protein Expr Purif , 11 :61 , 1997
Abstract : (S)-Hydroxynitrile lyase (Hnl) from the tropical rubber tree Hevea brasiliensis catalyzes the formation of (S)-cyanohydrins from hydrocyanic acid and aldehydes or ketones. This enzyme accepts aliphatic, aromatic, and heterocyclic carbonyl compounds as substrates and is therefore considered a potent biocatalyst for the industrial production of optically active chemicals. Limitations in enzyme supply from natural resources were overcome by production of the enzyme in the microbial host systems Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris. Expression of Hnl in the prokaryotic system led to the formation of inclusion bodies whereas in both yeast hosts high levels of soluble protein were obtained. Highest yields were obtained in a high cell density batch fermentation of a P. pastoris transformant that expressed heterologous Hnl to about 50% of the soluble cytosolic protein. At a cell density of 100 g/liter cell dry weight, a volume yield of 22 g/liter of heterologous product was obtained. Attempts to produce the Hnl protein extracellularly with the yeast hosts by applying different leader peptide strategies were not successful. Immunofluorescence microscopy studies indicated that the secretion-directed heterologous Hnl protein accumulated in the plasma membrane forming aggregated clusters of inactive protein.
ESTHER : Hasslacher_1997_Protein.Expr.Purif_11_61
PubMedSearch : Hasslacher_1997_Protein.Expr.Purif_11_61
PubMedID: 9325140

Title : Hydroxynitrile lyase from Hevea brasiliensis: molecular characterization and mechanism of enzyme catalysis - Hasslacher_1997_Proteins_27_438
Author(s) : Hasslacher M , Kratky C , Griengl H , Schwab H , Kohlwein SD
Ref : Proteins , 27 :438 , 1997
Abstract : (S)-Hydroxynitrile lyase (Hnl) from the tropical rubber tree Hevea brasiliensis is a 29 kDa single chain protein that catalyses the breakdown or formation of a C--C bond by reversible addition of hydrocyanic acid to aldehydes or ketones. The primary sequence of Hnl has no significant homology to known proteins. Detailed homology investigations employing PROFILESEARCH and secondary structure prediction algorithms suggest that Hnl is a member of the alpha/beta hydrolase fold protein family and contains a catalytic triad as functional residues for catalysis. The significance of predicted catalytic residues was tested and confirmed by site-directed mutagenesis and expression of mutant and wild-type proteins in the yeast, Saccharomyces cerevisiae. Based on these data we suggest a mechanistic model for the (S)-cyanohydrin synthesis catalyzed by hydroxynitrile lyase from Hevea brasiliensis.
ESTHER : Hasslacher_1997_Proteins_27_438
PubMedSearch : Hasslacher_1997_Proteins_27_438
PubMedID: 9094745
Gene_locus related to this paper: hevbr-hnl

Title : Crystallization and preliminary X-ray diffraction studies of a hydroxynitrile lyase from Hevea brasiliensis - Wagner_1996_Acta.Crystallogr.D.Biol.Crystallogr_52_591
Author(s) : Wagner UG , Schall M , Hasslacher M , Hayn M , Griengl H , Schwab H , Kratky C
Ref : Acta Crystallographica D Biol Crystallogr , 52 :591 , 1996
Abstract : Crystals of the hydroxynitrile lyase from Hevea brasiliensis overexpressed in Pichia pastoris have been obtained by the hanging-drop technique at 294 K with ammonium sulfate and PEG 400 as precipitants. The crystals belong to the orthorhombic space group C222(1) with cell dimensions of a = 47.6, b = 106.8 and c = 128.2 A. The crystals diffract to about 2.5 A resolution on a rotating-anode X-ray source.
ESTHER : Wagner_1996_Acta.Crystallogr.D.Biol.Crystallogr_52_591
PubMedSearch : Wagner_1996_Acta.Crystallogr.D.Biol.Crystallogr_52_591
PubMedID: 15299689

Title : Molecular cloning of the full-length cDNA of (S)-hydroxynitrile lyase from Hevea brasiliensis. Functional expression in Escherichia coli and Saccharomyces cerevisiae and identification of an active site residue - Hasslacher_1996_J.Biol.Chem_271_5884
Author(s) : Hasslacher M , Schall M , Hayn M , Griengl H , Kohlwein SD , Schwab H
Ref : Journal of Biological Chemistry , 271 :5884 , 1996
Abstract : The full-length cDNA of (S)-hydroxynitrile lyase (Hnl) from leaves of Hevea brasiliensis (tropical rubber tree) was cloned by an immunoscreening and sequenced. Hnl from H. brasiliensis is involved in the biodegradation of cyanogenic glycosides and also catalyzes the stereospecific synthesis of aliphatic, aromatic, and heterocyclic cyanohydrins, which are important as precursors for pharmaceutical compounds. The open reading frame identified in a 1. 1-kilobase cDNA fragment codes for a protein of 257 amino acids with a predicted molecular mass of 29.2 kDa. The derived protein sequence is closely related to the (S)-hydroxynitrile lyase from Manihot esculenta (Cassava) and also shows significant homology to two proteins of Oryza sativa with as yet unknown enzymatic function. The H. brasiliensis protein was expressed in Escherichia coli and Saccharomyces cerevisiae and isolated in an active form from the respective soluble fractions. Replacement of cysteine 81 by serine drastically reduced activity of the heterologous enzyme, suggesting a role for this amino acid residue in the catalytic action of Hnl.
ESTHER : Hasslacher_1996_J.Biol.Chem_271_5884
PubMedSearch : Hasslacher_1996_J.Biol.Chem_271_5884
PubMedID: 8621461
Gene_locus related to this paper: hevbr-hnl

Title : (S)-hydroxynitrile lyase from Hevea brasiliensis -
Author(s) : Hasslacher M , Schall M , Hayn M , Griengl H , Kohlwein SD , Schwab H
Ref : Annals of the New York Academy of Sciences , 799 :707 , 1996
PubMedID: 8958122
Gene_locus related to this paper: hevbr-hnl

Title : Mechanism of cyanogenesis: the crystal structure of hydroxynitrile lyase from Hevea brasiliensis - Wagner_1996_Structure_4_811
Author(s) : Wagner UG , Hasslacher M , Griengl H , Schwab H , Kratky C
Ref : Structure , 4 :811 , 1996
Abstract : BACKGROUND: Over three thousand species of plants, including important food crops such as cassava, use cyanogenesis, the liberation of HCN upon tissue damage, as a defense against predation. Detoxification of cyanogenic food crops requires disruption of the cyanogenic pathway. Hydroxynitrile lyase is one of the key enzymes in cyanogenesis, catalyzing the decomposition of an alpha-cyanohydrin to form HCN plus the corresponding aldehyde or ketone. These enzymes are also of potential utility for industrial syntheses of optically pure chiral cyanohydrins, being used to catalyze the reverse reaction. We set out to gain insight into the catalytic mechanism of this important class of enzymes by determining the three-dimensional structure of hydroxynitrile lyase from the rubber tree, Hevea brasiliensis.
RESULTS: The crystal structure of the enzyme has been determined to 1.9 A resolution. It belongs to the alpha/beta hydrolase superfamily, with an active site that is deeply buried within the protein and connected to the outside by a narrow tunnel. The catalytic triad is made up of Ser80, His235 and Asp207. By analogy with known mechanisms of other members of this superfamily, catalysis should involve an oxyanion hole formed by the main chain NH of Cys81 and the side chains of Cys81 and Thr11. Density attributed to a histidine molecule or ion is found in the active site.
CONCLUSIONS: By analogy with other alpha/beta hydrolases, the reaction catalyzed by hydroxynitrile lyase involves a tetrahedral hemiketal or hemiacetal intermediate formed by nucleophilic attack of Ser80 on the substrate, stabilized by the oxyanion hole. The SH group of Cys81 is probably involved in proton transfer between the HCN and the hydroxynitrile OH. This mechanism is significantly different from the corresponding uncatalyzed solution reaction.
ESTHER : Wagner_1996_Structure_4_811
PubMedSearch : Wagner_1996_Structure_4_811
PubMedID: 8805565
Gene_locus related to this paper: hevbr-hnl