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Molecular evolution of Fome lignosus laccase by ethyl methane sulfonate-based random mutagenesis in vitro

Paper ID Volume ID Publish Year Pages File Format Full-Text
14083 1144 2007 6 PDF Available
Title
Molecular evolution of Fome lignosus laccase by ethyl methane sulfonate-based random mutagenesis in vitro
Abstract

In order to improve the laccase activity, mutant libraries are constructed through ethyl methane sulfonate-based (EMS) random mutagenesis. Mutagenesis improved expression 3.7-fold to 144 mg l−1 laccase in yeast, together with a 1.4-fold increase in Kcat. Thus, the total activity is enhanced 5-fold for 2,2′-azino-bis 3-ethylbenzothiaoline-6-sulfonic acid (ABTS). In the presence of 0.6 mM copper, the highest activity value reached 30 U ml−1 after a 3-day cultivation at a temperature of 30 °C. In comparison with the wild type, the best mutant enzymatic properties (Km for ABTS and guaiacol, thermo- and pH stability, optimal pH) are not changed. Moreover, amino acid sequence analysis indicates that there are four substitutions in the best mutant laccase (Gly160Asp, Ala167Thr, Gly174Asp, and Glu234Gly). The best mutant laccase model showed that the Gly160 and Ala167 are to be found near the water channel; especially the distance of Ala167 to the Cu3a is 14.46 Å. This implies that it is likely involved in the formation of water channel and that it helps facilitate the easy incoming and outgoing of water.

Keywords
Ethyl methane sulfonate; Laccase; Pichia pastoris
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Molecular evolution of Fome lignosus laccase by ethyl methane sulfonate-based random mutagenesis in vitro
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biomolecular Engineering - Volume 24, Issue 6, December 2007, Pages 619–624
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