Gene cloning, identification, and characterization of the multicopper oxidase CumA from Pseudomonas sp. 593.
The cumA, 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. Cu2+remarkably enhanced the activity of the CumA. By contrast, the activity of the CumA was inhibited by the addition of Fe2+. Kinetic studies gave the Km, kcat, and kcat/Kmvalues 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 cumA from Pseudomonas and characterization of the CumA.
The Faculty of Life Sciences, Hubei University, Wuhan, People's Republic of China.|Hubei Collaborative innovation Center for Green Transformation of Bio-Resources, Hubei University, Wuhan, People's Republic of China.
Sheng
S
Yang
College of Life Sciences, Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), State Key Laboratory of Virology, Wuhan University, Wuhan, People's Republic of China.
Yan
Y
Long
The Faculty of Life Sciences, Hubei University, Wuhan, People's Republic of China.|Hubei Collaborative innovation Center for Green Transformation of Bio-Resources, Hubei University, Wuhan, People's Republic of China.
Hong
H
Yan
The Faculty of Life Sciences, Hubei University, Wuhan, People's Republic of China.|Hubei Collaborative innovation Center for Green Transformation of Bio-Resources, Hubei University, Wuhan, People's Republic of China.
Huawan
H
Cai
The Faculty of Life Sciences, Hubei University, Wuhan, People's Republic of China.|Hubei Collaborative innovation Center for Green Transformation of Bio-Resources, Hubei University, Wuhan, People's Republic of China.
Yadong
Y
Li
The Faculty of Life Sciences, Hubei University, Wuhan, People's Republic of China.|Hubei Collaborative innovation Center for Green Transformation of Bio-Resources, Hubei University, Wuhan, People's Republic of China.
Xingguo
X
Wang
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D010088:Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D010088:Oxidoreductases
Pseudomonas sp. 593; bacterial laccase; enzyme kinetics; multicopper oxidase; protein structure