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Enhancing activity and thermostability of lipase A from Serratia marcescens by site-directed mutagenesis

Paper ID Volume ID Publish Year Pages File Format Full-Text
16738 42608 2016 11 PDF Available
Title
Enhancing activity and thermostability of lipase A from Serratia marcescens by site-directed mutagenesis
Abstract

•We examine the effect of site-direct mutagenesis on the thermal stability and activity of SML.•Four mutant lipases of SML were constructed by site-direct mutagenesis.•The thermal stability and activity of SML (wild type and mutants) was compared.•Using protein modeling program and creating mutation, can enhance lipase activity and/or thermostability of SML.

Lipases as significant biocatalysts had been widely employed to catalyze various chemical reactions such as ester hydrolysis, ester synthesis, and transesterification. Improving the activity and thermostability of enzymes is desirable for industrial applications. The lipase of Serratia marcescens belonging to family I.3 lipase has a very important pharmaceutical application in production of chiral precursors. In the present study, to achieve improved lipase activity and thermostability, using computational predictions of protein, four mutant lipases of SML (MutG2P, MutG59P, Mut H279K and MutL613WA614P) were constructed by site-directed mutagenesis. The recombinant mutant proteins were over-expressed in E. coli and purified by affinity chromatography on the Ni-NTA system. Circular dichroism spectroscopy, differential scanning calorimetry and kinetic parameters (Km and kcat) were determined. Our results have shown that the secondary structure of all lipases was approximately similar to one another. The MutG2P and MutG59P were more stable than wild type by approximately 2.3 and 2.9 in T1/2, respectively. The catalytic efficiency (kcat/Km) of MutH279K was enhanced by 2-fold as compared with the wild type (p < 0.05). These results indicate that using protein modeling program and creating mutation, can enhance lipase activity and/or thermostability of SML and it also could be used for improving other properties of enzyme to the desired requirements as well as further mutations.

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Keywords
Family I.3 lipase; Site-directed mutagenesis; Serratia marcescens; Differential scanning calorimetry; CD spectra
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Enhancing activity and thermostability of lipase A from Serratia marcescens by site-directed mutagenesis
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volumes 93–94, November 2016, Pages 18–28
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