来源于枯草芽孢杆菌的漆酶cotA基因克隆与表达及其酶学性质研究

漆酶作为一种含铜离子的多酚氧化酶，在环境保护、生物能源、食品工业和纸浆漂白等工业中有重要的应用价值。从枯草芽孢杆菌168菌株中克隆漆酶cotA基因，全长1 542 bp，编码513个氨基酸，外加一个终止密码子。将该基因在大肠杆菌Transetta（DE3）菌株中通过微好氧发酵法进行异源表达和纯化，获得的重组酶蛋白CotA的最适反应温度为60℃，最适pH为4.5。该酶在60℃具有较好的热稳定性，保温90 min后仍有71.70%的剩余酶活。最适反应条件下，重组酶对ABTS的Km为63.9±5 μmol/L，kcat为39.1±1/s，最大反应速率为0.005 μmol/L·min·mg，且在最适反应条件下，微好氧发酵法获得的重组酶蛋白CotA的比活（557.8 U/mg）是低温诱导法（0.2 U/mg）的2 655.4倍。因此，通过微好氧发酵法可以显著提高重组漆酶CotA的比活力。

Gene Cloning,Expression and Characterization of cotA,a Laccase from Bacillus subtilis

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.