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Engineering of Candida antarctica lipase B for hydrolysis of bulky carboxylic acid esters

Paper ID Volume ID Publish Year Pages File Format Full-Text
24133 43500 2010 7 PDF Available
Title
Engineering of Candida antarctica lipase B for hydrolysis of bulky carboxylic acid esters
Abstract

Candida antarctica lipase B (CALB) is a widely used biocatalyst with high activity and specificity for a wide range of primary and secondary alcohols. However, the range of converted carboxylic acids is more narrow and mainly limited to unbranched fatty acids. To further broaden the biotechnological applications of CALB it is of interest to expand the range of converted carboxylic acid and extend it to carboxylic acids that are branched or substituted in close proximity of the carboxyl group.An in silico library of 2400 CALB variants was built and screened in silico by substrate-imprinted docking, a four step docking procedure. First, reaction intermediates of putative substrates are covalently docked into enzyme active sites. Second, the geometry of the resulting enzyme–substrate complex is optimized. Third, the substrate is removed from the complex and then docked again into the optimized structure. Fourth, the resulting substrate poses are rated by geometric filter criteria as productive or non-productive poses.Eleven enzyme variants resulting from the in silico screening were expressed in Escherichia coli BL21 and measured in the hydrolysis of two branched fatty acid esters, isononanoic acid ethyl ester and 2-ethyl hexanoic acid ethyl esters. Five variants showed an initial increase in activity. The variant with the highest wet mass activity (T138S) was purified and further characterized. It showed a 5-fold increase in hydrolysis of isononanoic acid ethyl ester, but not toward sterically more demanding 2-ethyl hexanoic acid ethyl ester.

Keywords
CALB, Candida antarctica lipase B; INA, isononanoic acid; INA-EE, isononanoic acid ethyl ester; EHA, 2-ethyl hexanoic acid; EHA-EE, 2-ethyl hexanoic acid ethyl esterIsononanoic acid; Rational design; Mutant library; Protein engineering; Virtual screening;
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Engineering of Candida antarctica lipase B for hydrolysis of bulky carboxylic acid esters
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 150, Issue 4, December 2010, Pages 474–480
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