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Karl-erich Jaeger - One of the best experts on this subject based on the ideXlab platform.
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identification of organic solvent tolerant Carboxylic Ester hydrolases for organic synthesis
Applied and Environmental Microbiology, 2020Co-Authors: Alexander Bollinger, Rebecka Molitor, Cristina Coscolin, Rainhard Koch, Karl-erich Jaeger, Stephan Thies, Manuel FerrerAbstract:Biocatalysis has emerged as an important tool in synthetic organic chemistry enabling the chemical industry to execute reactions with high regio- or enantioselectivity and under usually mild reaction conditions while avoiding toxic waste. Target substrates and products of reactions catalyzed by Carboxylic Ester hydrolases are often poorly water soluble and require organic solvents, whereas enzymes are evolved by nature to be active in cells, i.e., in aqueous rather than organic solvents. Therefore, biocatalysts that withstand organic solvents are urgently needed. Current strategies to identify such enzymes rely on laborious tests carried out by incubation in different organic solvents and determination of residual activity. Here, we describe a simple assay useful for screening large libraries of Carboxylic Ester hydrolases for resistance and activity in water-miscible organic solvents. We have screened a set of 26 enzymes, most of them identified in this study, with four different water-miscible organic solvents. The triglyceride tributyrin was used as a substrate, and fatty acids released by enzymatic hydrolysis were detected by a pH shift indicated by the indicator dye nitrazine yellow. With this strategy, we succeeded in identifying a novel highly organic-solvent-tolerant Esterase from Pseudomonas aestusnigri In addition, the newly identified enzymes were tested with sterically demanding substrates, which are common in pharmaceutical intermediates, and two enzymes from Alcanivorax borkumensis were identified which outcompeted the gold standard Ester hydrolase CalB from Candida antarctica IMPORTANCE Major challenges hampering biotechnological applications of Esterases include the requirement to accept nonnatural and chemically demanding substrates and the tolerance of the enzymes toward organic solvents which are often required to solubilize such substrates. We describe here a high-throughput screening strategy to identify novel organic-solvent-tolerant Carboxylic Ester hydrolases (CEs). Among these enzymes, CEs active against water-insoluble bulky substrates were identified. Our results thus contribute to fostering the identification and biotechnological application of CEs.
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organic solvent tolerant Carboxylic Ester hydrolases for organic synthesis
Applied and Environmental Microbiology, 2020Co-Authors: Alexander Bollinger, Rebecka Molitor, Cristina Coscolin, Rainhard Koch, Karl-erich Jaeger, Stephan Thies, Manuel FerrerAbstract:ABSTRACT Biocatalysis has emerged as an important tool in synthetic organic chemistry enabling the chemical industry to execute reactions with high regio- or enantioselectivity and under usually mild reaction conditions while avoiding toxic waste. Target substrates and products of reactions catalyzed by Carboxylic Ester hydrolases are often poorly water soluble and require organic solvents, whereas enzymes are evolved by nature to be active in cells, i.e., in aqueous rather than organic solvents. Therefore, biocatalysts that withstand organic solvents are urgently needed. Current strategies to identify such enzymes rely on laborious tests carried out by incubation in different organic solvents and determination of residual activity. Here, we describe a simple assay useful for screening large libraries of Carboxylic Ester hydrolases for resistance and activity in water-miscible organic solvents. We have screened a set of 26 enzymes, most of them identified in this study, with four different water-miscible organic solvents. The triglyceride tributyrin was used as a substrate, and fatty acids released by enzymatic hydrolysis were detected by a pH shift indicated by the indicator dye nitrazine yellow. With this strategy, we succeeded in identifying a novel highly organic-solvent-tolerant Esterase from Pseudomonas aestusnigri. In addition, the newly identified enzymes were tested with sterically demanding substrates, which are common in pharmaceutical intermediates, and two enzymes from Alcanivorax borkumensis were identified which outcompeted the gold standard Ester hydrolase CalB from Candida antarctica. IMPORTANCE Major challenges hampering biotechnological applications of Esterases include the requirement to accept nonnatural and chemically demanding substrates and the tolerance of the enzymes toward organic solvents which are often required to solubilize such substrates. We describe here a high-throughput screening strategy to identify novel organic-solvent-tolerant Carboxylic Ester hydrolases (CEs). Among these enzymes, CEs active against water-insoluble bulky substrates were identified. Our results thus contribute to fostering the identification and biotechnological application of CEs.
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identification of organic solvent tolerant Carboxylic Ester hydrolases for organic synthesis
Applied and Environmental Microbiology, 2020Co-Authors: Alexander Bollinger, Rebecka Molitor, Cristina Coscolin, Rainhard Koch, Karl-erich Jaeger, Stephan Thies, Manuel FerrerAbstract:Biocatalysis has emerged as an important tool in synthetic organic chemistry enabling the chemical industry to execute reactions with high regio- or enantioselectivity and under usually mild reaction conditions while avoiding toxic waste. Target substrates and products of reactions catalyzed by Carboxylic Ester hydrolases are often poorly water soluble and require organic solvents, whereas enzymes are evolved by nature to be active in cells, i.e., in aqueous rather than organic solvents. Therefore, biocatalysts that withstand organic solvents are urgently needed. Current strategies to identify such enzymes rely on laborious tests carried out by incubation in different organic solvents and determination of residual activity. Here, we describe a simple assay useful for screening large libraries of Carboxylic Ester hydrolases for resistance and activity in water-miscible organic solvents. We have screened a set of 26 enzymes, most of them identified in this study, with four different water-miscible organic solvents. The triglyceride tributyrin was used as a substrate, and fatty acids released by enzymatic hydrolysis were detected by a pH shift indicated by the indicator dye nitrazine yellow. With this strategy, we succeeded in identifying a novel highly organic-solvent-tolerant Esterase from Pseudomonas aestusnigri In addition, the newly identified enzymes were tested with sterically demanding substrates, which are common in pharmaceutical intermediates, and two enzymes from Alcanivorax borkumensis were identified which outcompeted the gold standard Ester hydrolase CalB from Candida antarctica IMPORTANCE Major challenges hampering biotechnological applications of Esterases include the requirement to accept nonnatural and chemically demanding substrates and the tolerance of the enzymes toward organic solvents which are often required to solubilize such substrates. We describe here a high-throughput screening strategy to identify novel organic-solvent-tolerant Carboxylic Ester hydrolases (CEs). Among these enzymes, CEs active against water-insoluble bulky substrates were identified. Our results thus contribute to fostering the identification and biotechnological application of CEs.
Alexander Bollinger - One of the best experts on this subject based on the ideXlab platform.
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identification of organic solvent tolerant Carboxylic Ester hydrolases for organic synthesis
Applied and Environmental Microbiology, 2020Co-Authors: Alexander Bollinger, Rebecka Molitor, Cristina Coscolin, Rainhard Koch, Karl-erich Jaeger, Stephan Thies, Manuel FerrerAbstract:Biocatalysis has emerged as an important tool in synthetic organic chemistry enabling the chemical industry to execute reactions with high regio- or enantioselectivity and under usually mild reaction conditions while avoiding toxic waste. Target substrates and products of reactions catalyzed by Carboxylic Ester hydrolases are often poorly water soluble and require organic solvents, whereas enzymes are evolved by nature to be active in cells, i.e., in aqueous rather than organic solvents. Therefore, biocatalysts that withstand organic solvents are urgently needed. Current strategies to identify such enzymes rely on laborious tests carried out by incubation in different organic solvents and determination of residual activity. Here, we describe a simple assay useful for screening large libraries of Carboxylic Ester hydrolases for resistance and activity in water-miscible organic solvents. We have screened a set of 26 enzymes, most of them identified in this study, with four different water-miscible organic solvents. The triglyceride tributyrin was used as a substrate, and fatty acids released by enzymatic hydrolysis were detected by a pH shift indicated by the indicator dye nitrazine yellow. With this strategy, we succeeded in identifying a novel highly organic-solvent-tolerant Esterase from Pseudomonas aestusnigri In addition, the newly identified enzymes were tested with sterically demanding substrates, which are common in pharmaceutical intermediates, and two enzymes from Alcanivorax borkumensis were identified which outcompeted the gold standard Ester hydrolase CalB from Candida antarctica IMPORTANCE Major challenges hampering biotechnological applications of Esterases include the requirement to accept nonnatural and chemically demanding substrates and the tolerance of the enzymes toward organic solvents which are often required to solubilize such substrates. We describe here a high-throughput screening strategy to identify novel organic-solvent-tolerant Carboxylic Ester hydrolases (CEs). Among these enzymes, CEs active against water-insoluble bulky substrates were identified. Our results thus contribute to fostering the identification and biotechnological application of CEs.
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organic solvent tolerant Carboxylic Ester hydrolases for organic synthesis
Applied and Environmental Microbiology, 2020Co-Authors: Alexander Bollinger, Rebecka Molitor, Cristina Coscolin, Rainhard Koch, Karl-erich Jaeger, Stephan Thies, Manuel FerrerAbstract:ABSTRACT Biocatalysis has emerged as an important tool in synthetic organic chemistry enabling the chemical industry to execute reactions with high regio- or enantioselectivity and under usually mild reaction conditions while avoiding toxic waste. Target substrates and products of reactions catalyzed by Carboxylic Ester hydrolases are often poorly water soluble and require organic solvents, whereas enzymes are evolved by nature to be active in cells, i.e., in aqueous rather than organic solvents. Therefore, biocatalysts that withstand organic solvents are urgently needed. Current strategies to identify such enzymes rely on laborious tests carried out by incubation in different organic solvents and determination of residual activity. Here, we describe a simple assay useful for screening large libraries of Carboxylic Ester hydrolases for resistance and activity in water-miscible organic solvents. We have screened a set of 26 enzymes, most of them identified in this study, with four different water-miscible organic solvents. The triglyceride tributyrin was used as a substrate, and fatty acids released by enzymatic hydrolysis were detected by a pH shift indicated by the indicator dye nitrazine yellow. With this strategy, we succeeded in identifying a novel highly organic-solvent-tolerant Esterase from Pseudomonas aestusnigri. In addition, the newly identified enzymes were tested with sterically demanding substrates, which are common in pharmaceutical intermediates, and two enzymes from Alcanivorax borkumensis were identified which outcompeted the gold standard Ester hydrolase CalB from Candida antarctica. IMPORTANCE Major challenges hampering biotechnological applications of Esterases include the requirement to accept nonnatural and chemically demanding substrates and the tolerance of the enzymes toward organic solvents which are often required to solubilize such substrates. We describe here a high-throughput screening strategy to identify novel organic-solvent-tolerant Carboxylic Ester hydrolases (CEs). Among these enzymes, CEs active against water-insoluble bulky substrates were identified. Our results thus contribute to fostering the identification and biotechnological application of CEs.
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identification of organic solvent tolerant Carboxylic Ester hydrolases for organic synthesis
Applied and Environmental Microbiology, 2020Co-Authors: Alexander Bollinger, Rebecka Molitor, Cristina Coscolin, Rainhard Koch, Karl-erich Jaeger, Stephan Thies, Manuel FerrerAbstract:Biocatalysis has emerged as an important tool in synthetic organic chemistry enabling the chemical industry to execute reactions with high regio- or enantioselectivity and under usually mild reaction conditions while avoiding toxic waste. Target substrates and products of reactions catalyzed by Carboxylic Ester hydrolases are often poorly water soluble and require organic solvents, whereas enzymes are evolved by nature to be active in cells, i.e., in aqueous rather than organic solvents. Therefore, biocatalysts that withstand organic solvents are urgently needed. Current strategies to identify such enzymes rely on laborious tests carried out by incubation in different organic solvents and determination of residual activity. Here, we describe a simple assay useful for screening large libraries of Carboxylic Ester hydrolases for resistance and activity in water-miscible organic solvents. We have screened a set of 26 enzymes, most of them identified in this study, with four different water-miscible organic solvents. The triglyceride tributyrin was used as a substrate, and fatty acids released by enzymatic hydrolysis were detected by a pH shift indicated by the indicator dye nitrazine yellow. With this strategy, we succeeded in identifying a novel highly organic-solvent-tolerant Esterase from Pseudomonas aestusnigri In addition, the newly identified enzymes were tested with sterically demanding substrates, which are common in pharmaceutical intermediates, and two enzymes from Alcanivorax borkumensis were identified which outcompeted the gold standard Ester hydrolase CalB from Candida antarctica IMPORTANCE Major challenges hampering biotechnological applications of Esterases include the requirement to accept nonnatural and chemically demanding substrates and the tolerance of the enzymes toward organic solvents which are often required to solubilize such substrates. We describe here a high-throughput screening strategy to identify novel organic-solvent-tolerant Carboxylic Ester hydrolases (CEs). Among these enzymes, CEs active against water-insoluble bulky substrates were identified. Our results thus contribute to fostering the identification and biotechnological application of CEs.
Carsten Carlberg - One of the best experts on this subject based on the ideXlab platform.
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critical role of helix 12 of the vitamin d3 receptor for the partial agonism of Carboxylic Ester antagonists
Journal of Molecular Biology, 2002Co-Authors: Sami Vaisanen, Andreas Steinmeyer, Mikael Perakyla, Jouni I Karkkainen, Carsten CarlbergAbstract:Abstract The carboxy-terminal α-helix of a nuclear receptor ligand-binding domain (LBD), helix 12, contains a critical, ligand-modulated interface for the interaction with coactivator proteins. In this study, using the example of the vitamin D receptor (VDR) and the partial antagonist ZK159222, the role of helix 12 (residues 417-427) for both antagonistic and agonistic receptor actions was investigated. Amino acid residue G423 was demonstrated to be critical for partial agonism of ZK159222, but not for the activity of the natural VDR agonist, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). The amount of partial agonism of ZK159222 increased when helix 12 was truncated by the last four amino acid residues (Δ424-27) and augmented even more, when in addition helix 12 of VDR’s dimerization partner, retinoid X receptor (RXR), was truncated. In contrast, the low agonism of a structural derivative of ZK159222, ZK168281, was not affected comparably, whereas other close structural relatives of ZK159222 even demonstrated the same agonistic activity as that of 1α,25(OH)2D3. The amount of agonism of ZK159222 and ZK168281 at different variations of helix 12 correlated well with VDR’s ability to complex with coactivator proteins and inversely correlated with the strength of the compound’s antagonistic action on 1α,25(OH)2D3 signalling. Molecular dynamics simulations of the LBD complexed with the two antagonists could explain their different action by demonstrating a more drastic displacement of helix 12 through ZK168281 than through ZK159222. Moreover, the modelling could indicate a kink of helix 12 at amino acid residue G423, which provides the last four amino acid residues of helix 12 with a modulatory role for the partial agonism of some VDR antagonists, such as ZK159222. In conclusion, partial agonism of a VDR antagonist is lower the more it disturbs helix 12 in taking the optimal position for coactivator interaction.
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different molecular mechanisms of vitamin d3 receptor antagonists
Molecular Pharmacology, 2001Co-Authors: Andrea Toell, Seiichi Ishizuka, Manuel Macias Gonzalez, Dagmar Ruf, Andreas Steinmeyer, Carsten CarlbergAbstract:Two structurally different antagonists of the nuclear hormone 1α,25-dihydroxyvitamin D3[1α,25(OH)2D3], the 25-Carboxylic Ester ZK159222 and the 26,23-lactone TEI-9647, have recently been described. In this study, the molecular mechanisms and the efficacy of both antagonists were compared. ZK159222 showed similar potency and sensitivity to 1α,25(OH)2D3 in ligand-dependent gel shift assays using the vitamin D receptor (VDR), the retinoid X receptor, and specific DNA binding sites, whereas TEI-9647 displayed reduced potency and >10-fold lower sensitivity in this assay system. Limited protease digestion and gel shift clipping assays showed that the two antagonists stabilized individual patterns of VDR conformations. Both antagonists prevented the interaction of the VDR with coactivator proteins, as demonstrated by GST-pull-down and supershift assays; like the natural hormone, however, they were able to induce a dissociation of corepressor proteins. Interestingly, ZK159222 demonstrated functional antagonism in reporter gene assays both in HeLa and MCF-7 cells, whereas TEI-9647 functioned as a less sensitive antagonist only in MCF-7 cells. In conclusion, the two 1α,25(OH)2D3 analogs act in part via different molecular mechanisms, which allows us to speculate that ZK159222 is a more complete antagonist and TEI-9647 a more selective antagonist.
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structure activity relationship of Carboxylic Ester antagonists of the vitamin d3 receptor
Molecular Pharmacology, 2000Co-Authors: Yvonne Bury, Andreas Steinmeyer, Carsten CarlbergAbstract:A 25-Carboxylic Ester analog of 1α,25-dihydroxyvitamin D3[1α,25(OH)2D3], ZK159222 (compound 1), was recently described as a novel type of antagonist of 1α,25(OH)2D3 signaling. In this study five derivatives of compound 1 (compounds 2–6) were selected because of their sensitivity in facilitating complex formation between the 1α,25(OH)2D3 receptor (VDR) and the retinoid X receptor on a 1α,25(OH)2D3 response element that was comparable to that of the natural hormone (0.2–0.9 nM). Most derivatives of compound 1 reacted as typical agonists, because they were able to promote ligand-dependent interaction of the VDR with the coactivator TIF2, stabilized the VDR preferentially in its agonistic conformation c1LPD, and stimulated VDR-dependent gene activity with a potency similar to 1α,25(OH)2D3. In contrast, only compound 2 showed the antagonistic profile of compound 1, which includes the incompetence to induce a VDR-TIF2 contact, the stabilization of the antagonistic conformation c2LPD, and only a very weak and insensitive functional activity. Accordingly, only compounds 1 and 2, but not compounds 3 to 6, showed prominent antagonistic effects in cellular systems. The comparison of the structures of the compounds indicates that the essential requirements for an antagonistic function are a cyclopropyl ring at carbon 25, a hydroxy group at carbon 24, and at least a butylEster. Interestingly, compound 2 was an approximately 3 times more sensitive antagonist than compound 1 and even displayed a lower residual agonistic activity. In conclusion, only a very limited number of structural variations of compound 1 are possible to keep its antagonistic profile, but the tools presented here for their in vitro evaluation allow an accurate prediction of the effects and are suited to screening for even more potent 1α,25(OH)2D3 antagonists.
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Carboxylic Ester antagonists of 1α 25 dihydroxyvitamin d3 show cell specific actions
Chemistry & Biology, 2000Co-Authors: Michaela Herdick, Andreas Steinmeyer, Carsten CarlbergAbstract:Abstract Background: The nuclear hormone 1α,25-dihydroxyvitamin D 3 (1α,25(OH) 2 D 3 ) acts through the transcription factor vitamin D receptor (1α,25(OH) 2 D 3 receptor, VDR) via combined contact with the retinoid X receptor (RXR), coactivator proteins and specific DNA binding sites (1α,25(OH) 2 D 3 response elements, VDREs). Ligand-mediated conformational changes of the VDR are the basis of the molecular mechanisms of nuclear 1α,25(OH) 2 D 3 signaling. Cell-specific VDR antagonists would allow to dissect and fine regulate the pleiotropic 1α,25(OH) 2 D 3 endocrine system affecting the regulation of calcium homeostasis, bone mineralization and other cellular functions. Results: Two Carboxylic Ester analogues of 1α,25(OH) 2 D 3 , ZK159222 and ZK168281, which have additional cyclopropyl rings and allylic alcohol substructures in their side chain, were characterized in different 1α,25(OH) 2 D 3 target tissues as functional antagonists of 1α,25(OH) 2 D 3 signaling. In all tested systems, ZK168281 showed lower residual agonistic activity and higher antagonistic effects than ZK159222, but the strength of these effects was cell-specific. Both antagonists were shown to act via the same mechanisms: they selectively stabilize an antagonistic conformation of the ligand-binding domain of the VDR within VDR–RXR–VDRE complexes, which then inhibits the interaction of the VDR with coactivator proteins and an induction of transactivation. Interestingly, cells that have been treated with antagonists were found to contain VDR–RXR heterodimers in a different conformation than cells that were stimulated with an agonist. Moreover, the strength of the functional antagonism of ZK159222 and ZK168281 appears to depend on the VDR/RXR expression ratio and high RXR levels were found to reduce the antagonistic effect of both compounds. In support of this observation, the overexpression of an transactivation function 2 (AF-2) deletion mutant of RXR resulted for both ZK159222 and ZK168281 in a reduced agonistic activity and an increased antagonistic effect. Conclusions: A novel, more potent VDR antagonist, ZK168281, was identified, which stabilizes VDR–RXR heterodimers in living cells in a different conformation than agonists. In addition, the VDR/RXR ratio was found as the major discriminating factor for understanding cell-specific effects of VDR antagonists.
Rebecka Molitor - One of the best experts on this subject based on the ideXlab platform.
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identification of organic solvent tolerant Carboxylic Ester hydrolases for organic synthesis
Applied and Environmental Microbiology, 2020Co-Authors: Alexander Bollinger, Rebecka Molitor, Cristina Coscolin, Rainhard Koch, Karl-erich Jaeger, Stephan Thies, Manuel FerrerAbstract:Biocatalysis has emerged as an important tool in synthetic organic chemistry enabling the chemical industry to execute reactions with high regio- or enantioselectivity and under usually mild reaction conditions while avoiding toxic waste. Target substrates and products of reactions catalyzed by Carboxylic Ester hydrolases are often poorly water soluble and require organic solvents, whereas enzymes are evolved by nature to be active in cells, i.e., in aqueous rather than organic solvents. Therefore, biocatalysts that withstand organic solvents are urgently needed. Current strategies to identify such enzymes rely on laborious tests carried out by incubation in different organic solvents and determination of residual activity. Here, we describe a simple assay useful for screening large libraries of Carboxylic Ester hydrolases for resistance and activity in water-miscible organic solvents. We have screened a set of 26 enzymes, most of them identified in this study, with four different water-miscible organic solvents. The triglyceride tributyrin was used as a substrate, and fatty acids released by enzymatic hydrolysis were detected by a pH shift indicated by the indicator dye nitrazine yellow. With this strategy, we succeeded in identifying a novel highly organic-solvent-tolerant Esterase from Pseudomonas aestusnigri In addition, the newly identified enzymes were tested with sterically demanding substrates, which are common in pharmaceutical intermediates, and two enzymes from Alcanivorax borkumensis were identified which outcompeted the gold standard Ester hydrolase CalB from Candida antarctica IMPORTANCE Major challenges hampering biotechnological applications of Esterases include the requirement to accept nonnatural and chemically demanding substrates and the tolerance of the enzymes toward organic solvents which are often required to solubilize such substrates. We describe here a high-throughput screening strategy to identify novel organic-solvent-tolerant Carboxylic Ester hydrolases (CEs). Among these enzymes, CEs active against water-insoluble bulky substrates were identified. Our results thus contribute to fostering the identification and biotechnological application of CEs.
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organic solvent tolerant Carboxylic Ester hydrolases for organic synthesis
Applied and Environmental Microbiology, 2020Co-Authors: Alexander Bollinger, Rebecka Molitor, Cristina Coscolin, Rainhard Koch, Karl-erich Jaeger, Stephan Thies, Manuel FerrerAbstract:ABSTRACT Biocatalysis has emerged as an important tool in synthetic organic chemistry enabling the chemical industry to execute reactions with high regio- or enantioselectivity and under usually mild reaction conditions while avoiding toxic waste. Target substrates and products of reactions catalyzed by Carboxylic Ester hydrolases are often poorly water soluble and require organic solvents, whereas enzymes are evolved by nature to be active in cells, i.e., in aqueous rather than organic solvents. Therefore, biocatalysts that withstand organic solvents are urgently needed. Current strategies to identify such enzymes rely on laborious tests carried out by incubation in different organic solvents and determination of residual activity. Here, we describe a simple assay useful for screening large libraries of Carboxylic Ester hydrolases for resistance and activity in water-miscible organic solvents. We have screened a set of 26 enzymes, most of them identified in this study, with four different water-miscible organic solvents. The triglyceride tributyrin was used as a substrate, and fatty acids released by enzymatic hydrolysis were detected by a pH shift indicated by the indicator dye nitrazine yellow. With this strategy, we succeeded in identifying a novel highly organic-solvent-tolerant Esterase from Pseudomonas aestusnigri. In addition, the newly identified enzymes were tested with sterically demanding substrates, which are common in pharmaceutical intermediates, and two enzymes from Alcanivorax borkumensis were identified which outcompeted the gold standard Ester hydrolase CalB from Candida antarctica. IMPORTANCE Major challenges hampering biotechnological applications of Esterases include the requirement to accept nonnatural and chemically demanding substrates and the tolerance of the enzymes toward organic solvents which are often required to solubilize such substrates. We describe here a high-throughput screening strategy to identify novel organic-solvent-tolerant Carboxylic Ester hydrolases (CEs). Among these enzymes, CEs active against water-insoluble bulky substrates were identified. Our results thus contribute to fostering the identification and biotechnological application of CEs.
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identification of organic solvent tolerant Carboxylic Ester hydrolases for organic synthesis
Applied and Environmental Microbiology, 2020Co-Authors: Alexander Bollinger, Rebecka Molitor, Cristina Coscolin, Rainhard Koch, Karl-erich Jaeger, Stephan Thies, Manuel FerrerAbstract:Biocatalysis has emerged as an important tool in synthetic organic chemistry enabling the chemical industry to execute reactions with high regio- or enantioselectivity and under usually mild reaction conditions while avoiding toxic waste. Target substrates and products of reactions catalyzed by Carboxylic Ester hydrolases are often poorly water soluble and require organic solvents, whereas enzymes are evolved by nature to be active in cells, i.e., in aqueous rather than organic solvents. Therefore, biocatalysts that withstand organic solvents are urgently needed. Current strategies to identify such enzymes rely on laborious tests carried out by incubation in different organic solvents and determination of residual activity. Here, we describe a simple assay useful for screening large libraries of Carboxylic Ester hydrolases for resistance and activity in water-miscible organic solvents. We have screened a set of 26 enzymes, most of them identified in this study, with four different water-miscible organic solvents. The triglyceride tributyrin was used as a substrate, and fatty acids released by enzymatic hydrolysis were detected by a pH shift indicated by the indicator dye nitrazine yellow. With this strategy, we succeeded in identifying a novel highly organic-solvent-tolerant Esterase from Pseudomonas aestusnigri In addition, the newly identified enzymes were tested with sterically demanding substrates, which are common in pharmaceutical intermediates, and two enzymes from Alcanivorax borkumensis were identified which outcompeted the gold standard Ester hydrolase CalB from Candida antarctica IMPORTANCE Major challenges hampering biotechnological applications of Esterases include the requirement to accept nonnatural and chemically demanding substrates and the tolerance of the enzymes toward organic solvents which are often required to solubilize such substrates. We describe here a high-throughput screening strategy to identify novel organic-solvent-tolerant Carboxylic Ester hydrolases (CEs). Among these enzymes, CEs active against water-insoluble bulky substrates were identified. Our results thus contribute to fostering the identification and biotechnological application of CEs.
Cristina Coscolin - One of the best experts on this subject based on the ideXlab platform.
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identification of organic solvent tolerant Carboxylic Ester hydrolases for organic synthesis
Applied and Environmental Microbiology, 2020Co-Authors: Alexander Bollinger, Rebecka Molitor, Cristina Coscolin, Rainhard Koch, Karl-erich Jaeger, Stephan Thies, Manuel FerrerAbstract:Biocatalysis has emerged as an important tool in synthetic organic chemistry enabling the chemical industry to execute reactions with high regio- or enantioselectivity and under usually mild reaction conditions while avoiding toxic waste. Target substrates and products of reactions catalyzed by Carboxylic Ester hydrolases are often poorly water soluble and require organic solvents, whereas enzymes are evolved by nature to be active in cells, i.e., in aqueous rather than organic solvents. Therefore, biocatalysts that withstand organic solvents are urgently needed. Current strategies to identify such enzymes rely on laborious tests carried out by incubation in different organic solvents and determination of residual activity. Here, we describe a simple assay useful for screening large libraries of Carboxylic Ester hydrolases for resistance and activity in water-miscible organic solvents. We have screened a set of 26 enzymes, most of them identified in this study, with four different water-miscible organic solvents. The triglyceride tributyrin was used as a substrate, and fatty acids released by enzymatic hydrolysis were detected by a pH shift indicated by the indicator dye nitrazine yellow. With this strategy, we succeeded in identifying a novel highly organic-solvent-tolerant Esterase from Pseudomonas aestusnigri In addition, the newly identified enzymes were tested with sterically demanding substrates, which are common in pharmaceutical intermediates, and two enzymes from Alcanivorax borkumensis were identified which outcompeted the gold standard Ester hydrolase CalB from Candida antarctica IMPORTANCE Major challenges hampering biotechnological applications of Esterases include the requirement to accept nonnatural and chemically demanding substrates and the tolerance of the enzymes toward organic solvents which are often required to solubilize such substrates. We describe here a high-throughput screening strategy to identify novel organic-solvent-tolerant Carboxylic Ester hydrolases (CEs). Among these enzymes, CEs active against water-insoluble bulky substrates were identified. Our results thus contribute to fostering the identification and biotechnological application of CEs.
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organic solvent tolerant Carboxylic Ester hydrolases for organic synthesis
Applied and Environmental Microbiology, 2020Co-Authors: Alexander Bollinger, Rebecka Molitor, Cristina Coscolin, Rainhard Koch, Karl-erich Jaeger, Stephan Thies, Manuel FerrerAbstract:ABSTRACT Biocatalysis has emerged as an important tool in synthetic organic chemistry enabling the chemical industry to execute reactions with high regio- or enantioselectivity and under usually mild reaction conditions while avoiding toxic waste. Target substrates and products of reactions catalyzed by Carboxylic Ester hydrolases are often poorly water soluble and require organic solvents, whereas enzymes are evolved by nature to be active in cells, i.e., in aqueous rather than organic solvents. Therefore, biocatalysts that withstand organic solvents are urgently needed. Current strategies to identify such enzymes rely on laborious tests carried out by incubation in different organic solvents and determination of residual activity. Here, we describe a simple assay useful for screening large libraries of Carboxylic Ester hydrolases for resistance and activity in water-miscible organic solvents. We have screened a set of 26 enzymes, most of them identified in this study, with four different water-miscible organic solvents. The triglyceride tributyrin was used as a substrate, and fatty acids released by enzymatic hydrolysis were detected by a pH shift indicated by the indicator dye nitrazine yellow. With this strategy, we succeeded in identifying a novel highly organic-solvent-tolerant Esterase from Pseudomonas aestusnigri. In addition, the newly identified enzymes were tested with sterically demanding substrates, which are common in pharmaceutical intermediates, and two enzymes from Alcanivorax borkumensis were identified which outcompeted the gold standard Ester hydrolase CalB from Candida antarctica. IMPORTANCE Major challenges hampering biotechnological applications of Esterases include the requirement to accept nonnatural and chemically demanding substrates and the tolerance of the enzymes toward organic solvents which are often required to solubilize such substrates. We describe here a high-throughput screening strategy to identify novel organic-solvent-tolerant Carboxylic Ester hydrolases (CEs). Among these enzymes, CEs active against water-insoluble bulky substrates were identified. Our results thus contribute to fostering the identification and biotechnological application of CEs.
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identification of organic solvent tolerant Carboxylic Ester hydrolases for organic synthesis
Applied and Environmental Microbiology, 2020Co-Authors: Alexander Bollinger, Rebecka Molitor, Cristina Coscolin, Rainhard Koch, Karl-erich Jaeger, Stephan Thies, Manuel FerrerAbstract:Biocatalysis has emerged as an important tool in synthetic organic chemistry enabling the chemical industry to execute reactions with high regio- or enantioselectivity and under usually mild reaction conditions while avoiding toxic waste. Target substrates and products of reactions catalyzed by Carboxylic Ester hydrolases are often poorly water soluble and require organic solvents, whereas enzymes are evolved by nature to be active in cells, i.e., in aqueous rather than organic solvents. Therefore, biocatalysts that withstand organic solvents are urgently needed. Current strategies to identify such enzymes rely on laborious tests carried out by incubation in different organic solvents and determination of residual activity. Here, we describe a simple assay useful for screening large libraries of Carboxylic Ester hydrolases for resistance and activity in water-miscible organic solvents. We have screened a set of 26 enzymes, most of them identified in this study, with four different water-miscible organic solvents. The triglyceride tributyrin was used as a substrate, and fatty acids released by enzymatic hydrolysis were detected by a pH shift indicated by the indicator dye nitrazine yellow. With this strategy, we succeeded in identifying a novel highly organic-solvent-tolerant Esterase from Pseudomonas aestusnigri In addition, the newly identified enzymes were tested with sterically demanding substrates, which are common in pharmaceutical intermediates, and two enzymes from Alcanivorax borkumensis were identified which outcompeted the gold standard Ester hydrolase CalB from Candida antarctica IMPORTANCE Major challenges hampering biotechnological applications of Esterases include the requirement to accept nonnatural and chemically demanding substrates and the tolerance of the enzymes toward organic solvents which are often required to solubilize such substrates. We describe here a high-throughput screening strategy to identify novel organic-solvent-tolerant Carboxylic Ester hydrolases (CEs). Among these enzymes, CEs active against water-insoluble bulky substrates were identified. Our results thus contribute to fostering the identification and biotechnological application of CEs.