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.
Highlights
Laccases (EC 1.10.3.2) are a family of multicopper oxidases which catalyze the oxidation of a wide range of inorganic and aromatic substrates by coupling four-electron reduction of dioxygen to water [1,2]
The 1602 bp nucleotide sequence of intact multicopper oxidase (MCO) open reading frame cloned from Ochrobactrum sp. 531 is shown in Figure 2. 533 amino acids were deduced for Ochrobactrum sp. 531 MCO based on its DNA sequence
Analysis of the primary structure suggested that Ochrobactrum sp. 531 MCO could be a multicopper oxidase that might act as a laccase
Summary
Laccases (EC 1.10.3.2) are a family of multicopper oxidases which catalyze the oxidation of a wide range of inorganic and aromatic substrates by coupling four-electron reduction of dioxygen to water [1,2]. Like fungal and plant laccases, bacterial laccases contain four copper atoms: type I or blue copper, type II or normal copper, and type III or coupled binuclear copper center [15]. Bacterial laccases show little overall amino acid sequence similarity, low oxidation rate, and supplementation of Cu2+ [16]. Because of these differences, bacterial enzyme is often defined as “multicopper oxidase (MCO)”, and its activity as “laccase-like enzyme”
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