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

• Published in: Enzyme and microbial technology Vol. 44; no. 5; pp. 274 - 280
• Main Authors: Shao, Xiaohu, Gao, Yinghui, Jiang, Mengtian, Li, Lin
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Inc 06.05.2009; Elsevier
• Subjects: Biological and medical sciences; Biotechnology; Characterization; Enhancement; Enzyme active; Escherichia coli; Fundamental and applied biological sciences. Psychology; Laccase; Mutagenesis; Shigella dysenteriae; Characterization; Shigella dysenteriae; Mutagenesis; Enzyme active; Enhancement; Laccase; Enzyme; Site directed mutagenesis; Thermal stability; Enzymatic activity; Deletion; Bacteria; Oxidoreductases; Enterobacteriaceae
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/j.enzmictec.2008.12.013

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 ( K m = 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 NaN 3 and 60 mM thioglycolic acid. Activity was stimulated by Cu 2+; 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.