Cloning of multicopper oxidase gene from Ochrobactrum sp. 531 and characterization of its alkaline laccase activity towards phenolic substrates

Yang Li, Wenfeng Zuo, Yadong Li, Xingguo Wang

Faculty of Life Science, Hubei University, Wuhan, China.

DOI: 10.4236/abc.2012.23031                

Abstract

A 1602 bp fragment was cloned from a soil bacterium Ochrobactrum sp. 531. It contained an open reading frame (ORF) of 1092 bp which was identified as a multicopper oxidase (MCO) with potential laccase activity. After inserting the cloned gene into the expression vector pET23a, it was expressed in E. coli BL21(DE3)pLysS, and its product was purified to homogeneity through chromatography. The Ochrobactrum sp. 531 MCO, consisting of 533 amino acids with a molecular mass of 57.8 kDa, was quite stable in neutral pH and showed laccase-like activity oxidizing 2,6-dimethoxyphenol (DMP), 2,2’-azino-bis(3-ethylbe- nzthiazolinesulfonic acid) (ABTS), and syringaldazine (SGZ). The enzyme showed optimum activity towards DMP, ABTS, and SGZ at the pH 8.0, 3.6, and 7.5 respectively. Kinetic studies gave this enzyme Km, kcat and kcat//Km values of: 0.09 mM, 7.94 s–1, and 88.22 s–1?mM–1 for DMP; 0.072 mM, 2.95 s^–1, and 40.97 s^–1.mM^–1 for ABTS; and 0.015 mM, 2.4 s^–1, and 160 s^–1.mM^–1 for SGZ. Our results demonstrate that Ochrobactrum sp. 531 MCO is a bacterial laccase which oxidized phenolic substrates DMP and SGZ effectively under alkaline conditions. These unusual properties make the enzyme an interesting biocatalyst in applications for which classical laccases are unsuitable.

Keywords

Ochrobactrum sp. 531; Multicopper Oxidase; Bacterial Laccase; Enzyme Kinetics

Conflicts of Interest

The authors declare no conflicts of interest.

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