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Enhancement of a bacterial laccase thermostability through directed mutagenesis of a surface loop

Paper ID Volume ID Publish Year Pages File Format Full-Text
17387 42665 2011 7 PDF Available
Title
Enhancement of a bacterial laccase thermostability through directed mutagenesis of a surface loop
Abstract

Laccases (benzenediol oxygen oxidoreductases, EC 1.10.3.2) are used in many biotechnological processes, including removal of polyphenols in beverages, decolorizing and detoxifying effluents, drug analysis and bioremediation. In the present work, we have tried to increase thermal stability of laccase from Bacillus HR03 using site directed point mutations. Glu188 was substituted with 2 positive (Lys and Arg) and one hydrophobic (Ala) residues. All mutations showed improved thermal stability. Thermal activation of laccase was also increased after introducing the mutations. Remarkably, the Glu188Lys variant showed 3-fold higher thermal activation and higher T50 (5 °C) with respect to the native enzyme. Furthermore steady-state kcat and Km values were influenced despite the distance between the mutated position and the catalytic site. In Glu188Arg mutation, the kcat was improved 3-fold and Km reduced by 25%. Interestingly, all three variants showed higher stability against urea as a chemical denaturant. Structural analyses of the native and mutated variants were carried out using fluorescence and far-UV circular dichroism.

► Results proved improve of laccase thermostability by directed mutagenesis of a surface loop. ► Substitution of Glu188 with Lys, Arg and Ala showed the heat-resistance in high temperatures. ► The Glu188Lys mutation of laccase at a surface loop has gained highest thermal activation. ► Glu188Arg variant has raised catalytic efficiency through increased kcat and decreased Km. ► Results confirmed improve of chemical tolerance in the all mutated laccase variants.

Keywords
Laccase; Site-directed mutagenesis; Thermostability; Thermal activation
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Enhancement of a bacterial laccase thermostability through directed mutagenesis of a surface loop
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 49, Issue 5, 10 October 2011, Pages 446–452
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