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Improved thermostability of esterase from Aspergillus fumigatus by site-directed mutagenesis

Paper ID Volume ID Publish Year Pages File Format Full-Text
16999 42630 2014 6 PDF Available
Title
Improved thermostability of esterase from Aspergillus fumigatus by site-directed mutagenesis
Abstract

•Site-directed mutagenesis was applied to obtain more thermostable esterases.•The mutants prolong the T1/2 by 2–24 times than that of the WT.•The improved thermostability may be due to the newly formed favorable interaction.•The additional hydrogen bonds may be of value to improve the thermostability.•The mutation sites were regarded as new clues for structure–function relationship.

A 1.020-bp esterase gene, estQ, encoding for a protein of 339 amino acids, was cloned from Aspergillus fumigatus and expressed in E. coli. EstQ exhibited the optimal activity around 40 °C and pH 9.0. In order to obtain more thermostable esterases, three mutants (A134T, V160T, A134T–V160T) were constructed by site-directed mutagenesis and also characterized for further research. Compared to A134T and V160T displaying their optimum activity at 40 °C, A134T–V160T exhibited a 5 °C higher optimal temperature and a longer half-life more than 24 times than that of WT at 50 °C. All the mutants displayed favorable effects on thermostability and retained 53–76% activity after pre-incubation for 30 min at 45 °C, about 20–40% higher than that of the WT. With an increase in Km of the three mutants, a decrease in catalytic efficiency in kcat/Km was observed in mutant V160T and A134T–V160T against p-nitrophenyl butyrate. Homology models of WT and A134T–V160T were built to understand the structure–function relationship. The analysis results showed that the improved thermostability may be due to the favorable interaction and additional hydrogen bonds formed in the mutants by substitution of hydrophobic residues with hydrophilic residues. This study provide useful theoretical reference for enzyme evolution in vitro.

Keywords
Esterase; Site-directed mutagenesis; Thermostability; Half-life
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Improved thermostability of esterase from Aspergillus fumigatus by site-directed mutagenesis
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volumes 64–65, October 2014, Pages 11–16
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
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Don't Miss Today's Special Offer
Price was $35.95
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Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
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Any Questions? feel free to contact us