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Gene cloning, identification, and characterization of the multicopper oxidase CumA from Pseudomonas sp. 593
Author(s) -
Yang Sheng,
Long Yan,
Yan Hong,
Cai Huawan,
Li Yadong,
Wang Xingguo
Publication year - 2016
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1002/bab.1501
Subject(s) - thermostability , multicopper oxidase , escherichia coli , pseudomonas , oxidase test , abts , biology , molecular cloning , chemistry , enzyme , biochemistry , bacteria , complementary dna , laccase , gene , antioxidant , genetics , dpph
The cum A, a gene encoding a multicopper oxidase (MCO), was cloned from the soil‐dwelling bacterium Pseudomonas sp. 593. Its corresponding product was overexpressed in Escherichia coli BL21 (DE3) pLysS and purified to homogeneity through Ni‐affinity chromatography. The amino acid sequence of the CumA of Pseudomonas sp. 593 was strongly homologous to that of CumA as previously reported. The CumA was quite stable in neutral pH and had poor thermostability. Meanwhile, its optimum pH and temperature toward laccase substrates 2,6‐dimethoxyphenol (DMP), syringaldazine (SGZ), and 2,2′‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulfonic acid) (ABTS) were 5.0 and 55 °C, 7.5 and 60 °C, and 5.0 and 60 °C, respectively. Cu 2+ remarkably enhanced the activity of the CumA. By contrast, the activity of the CumA was inhibited by the addition of Fe 2+ . Kinetic studies gave the K m , k cat , and k cat / K m values of 0.438 mmol·L −1 , 0.056 Sec −1 , and 0.128 Sec −1 ·mmol −1 ·L for DMP; 0.017 mmol·L −1 , 0.031 Sec −1 , and 1.824 Sec −1 ·mmol −1 ·L for SGZ; and 0.101 mmol·L −1 , 0.393 Sec −1 , and 3.891 Sec −1 ·mmol −1 ·L for ABTS. To our knowledge, this is the first report of cloning, expressing in E. coli of the cum A from Pseudomonas and characterization of the CumA.