Organic-Solvent-Tolerant Carboxylic Ester Hydrolases for Organic Synthesis
| Autor: | Stephan Thies, Rebecka Molitor, Alexander Bollinger, Cristina Coscolín, Manuel Ferrer, Rainhard Koch, Karl-Erich Jaeger |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Tributyrin
Chemistry Techniques Synthetic Alcanivoraceae high-throughput screening Applied Microbiology and Biotechnology Esterase carboxylic ester hydrolases 03 medical and health sciences chemistry.chemical_compound Pseudomonas Enzymatic hydrolysis ddc:570 Alcanivorax borkumensis Organic chemistry 030304 developmental biology 2. Zero hunger chemistry.chemical_classification 0303 health sciences Ecology biology 030306 microbiology Substrate (chemistry) biology.organism_classification High-Throughput Screening Assays polar organic solvent Enzyme chemistry Biocatalysis Solvents Candida antarctica Organic synthesis Pseudomonas aestusnigri Food Science Biotechnology |
| Zdroj: | Applied and environmental microbiology 86(9), e00106-20 (2020). doi:10.1128/AEM.00106-20 Applied and Environmental Microbiology |
| Popis: | 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. 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. |
| Databáze: | OpenAIRE |
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