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

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

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cot A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Cot A的最适反应温度为60℃,最适p H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±5μmol/L,kcat为39.1±1/s,最大反应速率为0.005μmol/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Cot A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Cot A的比活力。

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 cot A gene from 168 strains of Bacillus subtilis was cloned by PCR amplification. The size of the laccase gene cot A is 1 542 bp,consisting of one open reading frame,which encodes a polypeptide of 513 amino acids and a termination codon. The cot A gene was cloned and expressed in E. coli Transetta（ DE3） under microaerobic conditions. The recombinant protein Cot A was purified by Ni-NTA column and characterized. The optimal temperature and p H of Cot A 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,kcatand Vmaxof purified recombinant protein Cot A with ABTS as the substrate were approximate 63. 9 ± 5 μmol / L,39. 1 ± 1 / s and0. 005 μmol / L·min·mg, respectively. Moreover, the specific activity of recombinant protein Cot A 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 Cot A.