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Inverting enantioselectivity of Burkholderia gladioli esterase EstB by directed and designed evolution

Paper ID Volume ID Publish Year Pages File Format Full-Text
25258 43563 2007 14 PDF Available
Title
Inverting enantioselectivity of Burkholderia gladioli esterase EstB by directed and designed evolution
Abstract

Esterase EstB from Burkholderia gladioli, showing moderate S-enantioselectivity (ES = 6.1) in the hydrolytic kinetic resolution of methyl-β-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 (ERapp = 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 ERapp = 1.5 for the parent mutant to ERapp = 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.

Keywords
Directed evolution; Enantioselectivity; Esterase; High-throughput screening
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Inverting enantioselectivity of Burkholderia gladioli esterase EstB by directed and designed evolution
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 129, Issue 1, 30 March 2007, Pages 109–122
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