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Enhanced acylation activity of esterase BioH from Escherichia coli by directed evolution towards improved hydrolysis activity

Paper ID Volume ID Publish Year Pages File Format Full-Text
3282 162 2013 5 PDF Available
Title
Enhanced acylation activity of esterase BioH from Escherichia coli by directed evolution towards improved hydrolysis activity
Abstract

•Directed evolution was used to improve the hydrolysis activity of BioH.•p-NPB was used as the substrate in the high-throughput screening for improved hydrolysis activity.•Some of the mutants with improved hydrolysis activity also showed enhanced acylation activity.•Combination of site mutations at hot spots further improved the acylation activity.

Esterase BioH is a critical enzyme for Biotin synthesis in Escherichia coli, which has been previously found to be active in the acylation of secondary alcohols and amines. Directed evolution towards improved acylation activity requires a high-throughput screening method. The aim of this study is to explore whether the acylation activity of BioH can be improved by directed evolution of its hydrolysis activity. A colorimetric method based on p-nitrophenyl butyrate hydrolysis was adopted in the high-throughput determination of hydrolysis activity. The wild-type BioH showed a hydrolysis activity of 18 U/mg, and the specific activities for α-phenylethanol and α-phenylethylamine acylation were 12.8 U/mg and 3.5 U/mg, respectively. After two rounds of directed evolution, seven mutants with improved hydrolysis activity were obtained, among which, K213E, Q70L/M170T and M197L/K213E also showed improvement in acylation activity. To further improve the acylation activity, site mutations were generated in different combinations at the four hot spots Q70L, M170T, M197L and K213E. Among the resulting mutants, Q70L/M197L/K213E showed the highest activity in α-phenylethylamine acylation with a 120% improvement, while Q70L/K213E had the highest α-phenylethanol acylation activity, which was improved by 70%. The results demonstrated that directed evolution of the hydrolysis activity might be a possible approach to improve the acylation activity of the esterase BioH.

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Keywords
Enzyme technology; Biocatalysis; Enantioseparation; Enzyme activity; Escherichia coli BioH; Directed evolution
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Enhanced acylation activity of esterase BioH from Escherichia coli by directed evolution towards improved hydrolysis activity
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biochemical Engineering Journal - Volume 79, 15 October 2013, Pages 182–186
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us