Laccase is a multi-copper oxidase, which has important applications in food, textile, environmental remediation, and papermaking. To improve the fermentation level of laccase, Bacillus subtilis laccase (CotA) was expressed in Escherichia coli BL21 (DE3), and the effects of ribosome binding site, induction expression condition, and co-expression of lysis protein on its expression efficiency were investigated. The results showed that the recombinant strain with the optimal ribosome binding site (CotA-RBS5) achieved 1 236 U/L of intracellular CotA, 1.75 times higher than that of before optimization. Based on the orthogonal experimental, the optimal condition of induction expression was as follows: the final concentration of 2 mmol/L Cu2+ and 0.1 mmol/L IPTG were added when the biomass OD600 reached 0.8; then, induction condition was performed at 30℃. Under the optimal condition, the intracellular enzyme activity of the recombinant strain increased to 7 926 U/L, 5.40 times higher than that of before optimization; meanwhile, the extracellular enzyme activity reached 4 984 U/L. Co-expression of lysis protein E promoted cell lysis. With the expression of cleavage protein E was induced at 3.5 of OD600, the extracellular CotA activity reached 10 283 U/L as it fermented to 30 h. The results will provide important basic data for the industrial production of laccase.
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