关键词: 漆酶, 克隆表达, 酶学性质, 定向进化, 染料降解

Abstract: In order to obtain the laccase with high expression and high thermal stability,a laccase gene lac1338 synthesized was cloned by codon optimization,ligated to the vector pET-32a（+）,then expressed in Escherichia coli BL21（DE3）,and the recombinant protein HIS-Lac1338 was obtained. Its enzymatic properties showed that while using ABTS as a substrate,the specific activity of HIS-Lac1338 was up to 22.8 U/mg,Km and Vmax of HIS-Lac1338 was 567 μmol/L and 2.8 mmol/（min·g protein）,respectively. The optimal temperature and pH of HIS-Lac1338 were 55℃ and 6.0 respectively. Its activity remained 50% at 55℃ for 2 h and in the pH range of 4-8. HIS-Lac1338 was strongly resistant to Cu²⁺,while its activity was promoted by Ca²⁺,Na⁺,K⁺ and inhibited by heavy metal ions such as Co²⁺,Fe²⁺,Hg²⁺,Ag⁺,etc. Comparing with HIS-Lac1338,mutant enzyme Lac16 of Lac1338 by sequential error-prone PCR improved the degradation rates of Acidviolet 7,Bromophenol blue,and Coomssie brilliant blue from 10.9%,20%,and 25% to 90.5%,67.8%,and 85%,respectively. Above results reveal that HIS-Lac1338 is stable to temperature and pH,the degradation rate of dye increases greatly with the mutant enzyme Lac16 via directed evolution of sequential error-prone PCR.

Key words: laccase, cloning and expression, enzymatic properties, directed evolution, degradation to dyes