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Deletion and site-directed mutagenesis of laccase from Shigella dysenteriae results in enhanced enzymatic activity and thermostability

Paper ID Volume ID Publish Year Pages File Format Full-Text
17915 42705 2009 7 PDF Available
Title
Deletion and site-directed mutagenesis of laccase from Shigella dysenteriae results in enhanced enzymatic activity and thermostability
Abstract

A putative laccase gene was cloned from Shigella dysenteriae W202 and expressed in Escherichia coli as a soluble fusion protein with high yield. The purified product (Wlac) was characterized as the CueO-like laccase from E. coli, a monomer of molecular mass 55 kDa, with a maximum activity of 24.4 U/mg (Km = 0.086) and a pH optimum of 2.5, in a standard assay using ABTS (2,2′-azino-di(3-ethyl-benzthiazoline-6-sulfonate) as the substrate. Activity was stable at 0–25 °C but inhibited above 40 °C. Purified Wlac was completely inhibited by 200 mM EDTA and partially by 32 mM SDS, 50 mM NaN3 and 60 mM thioglycolic acid. Activity was stimulated by Cu2+; other metal ions had only slight or negative effects. Two mutated variants, WlacS and WlacD, were obtained by substituting Glu 106 with Phe 106, and adding a deletion of an α-helix domain (from Leu 351 to Gly 378). WlacS had a 2.2-fold (52.9 U/mg) and WlacD a 3.5-fold (85.1 U/mg) higher enzyme activity than the wild-type laccase and WlacD showed greater thermostability at higher temperatures. Sce VMA intein-associated fusion proteins maintained ∼80% of total enzyme activity. Thus, deletion and site-directed mutagenesis of laccases are capable of promoting both enzymatic activity and thermostability.

Keywords
Laccase; Shigella dysenteriae; Characterization; Mutagenesis; Enzyme active; Enhancement
First Page Preview
Deletion and site-directed mutagenesis of laccase from Shigella dysenteriae results in enhanced enzymatic activity and thermostability
Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 44, Issue 5, 6 May 2009, Pages 274–280
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering