关键词: 漆酶；微好氧发酵；基因克隆；基因表达

Abstract:

As a multicopper oxidase, laccase is important for its applications in industries such as environmental protection, biological energy, food industry, and paper biobleaching. In this paper, full-length sequence of cotA gene from 168 strains of Bacillus subtilis was cloned by PCR amplification. The size of the laccase gene cotA is 1 542 bp, consisting of one open reading frame, which encodes a polypeptide of 513 amino acids and a termination codon. The cotA gene was cloned and expressed in E. coli Transetta (DE3) under microaerobic conditions. The recombinant protein CotA was purified by Ni-NTA column and characterized. The optimal temperature and pH of CotA were 60℃ and 4.5, respectively. This enzyme showed better thermostability at 60℃. There was still 71.70% residual enzyme activity after 90 min heat preservation. Under the optimum reaction condition, the Km, kcat and Vmax of purified recombinant protein CotA with ABTS as the substrate were approximate 63.9±5 μmol/L, 39.1±1 /s and 0.005 μmol/L·min·mg, respectively. Moreover, the specific activity of recombinant protein CotA under microaerobic conditions was 2 655.4-fold higher than that of aerobically grown cells. Accordingly, the microaerobic fermentation method can significantly improve the specific activity of recombinant protein CotA.

Key words: laccase, microaerobic fermentation, gene cloning, gene expression