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Expression, purification, and characterization of a novel laccase from Setosphaeria turcica in Eschericha coli
Author(s) -
Ma Shuangxin,
Liu Ning,
Jia Hui,
Dai Dongqing,
Zang Jinping,
Cao Zhiyan,
Dong Jingao
Publication year - 2018
Publication title -
journal of basic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.58
H-Index - 54
eISSN - 1521-4028
pISSN - 0233-111X
DOI - 10.1002/jobm.201700212
Subject(s) - laccase , abts , western blot , enzyme , recombinant dna , escherichia coli , specific activity , chemistry , molecular mass , biochemistry , nuclear chemistry , biology , chromatography , dpph , antioxidant , gene
Laccases are multicopper oxidases (E.C. 1.10.3.2) that catalyze the oxidation of many phenolic compounds. In this study, a novel laccase, Stlac4, from Setosphaeria turcica was cloned and expressed in Escherichia coli by insertion into the pET‐30a expression plasmid. The recombinant laccase was purified and visualized on SDS–PAGE as a single band with an apparent molecular weight of 71.5 KDa, and confirmed by Western blot. The maximum activity of the purified laccase was 127.78 U · mg −1 , the optimum temperature and pH value were 60 °C and 4.0 respectively, measured by oxidation of 2,2′‐Azinobis‐(3‐ethylbenzthiazoline‐6‐sulphonate) (ABTS). Purified laccase activity under different metal ions and an inhibitor were tested, revealing that laccase activity increased by approximately 434.8% with Fe 3+ , and 217.4% with Cu 2+ at 10 mmol · L −1 concentrations, Mn 2+ increased the laccase activity only at 5 mmol · L −1 , while Na + increased activity at 1 mmol · L −1 but inhibited activity at 5 and 10 mmol · L −1 . SDS increased laccase activity at 1 mmol · L −1 , and inhibited activity at 5 and 10 mmol · L −1 .