漆酶Lac1338的酶学特性测定及定向突变对其酶解染料影响

Determination of Enzymatic Properties of a Laccase Lac1338,and Effects of Directed Mutants on the Degradations of Different Dyes

为了获得表达量高、热稳定性好的漆酶,通过密码子优化合成漆酶基因lac1338、连接到pET-32a(+)载体上并在Escherichia coli BL21(DE3)中表达,获得HIS-Lac1338蛋白。酶学性质测定结果显示,以ABTS为底物时,HIS-Lac1338的比活力高达22.8 U/mg,Km和Vmax分别为567μmol/L和2.8 mmol/(L·min·g);HIS-Lac1338的最适反应温度为55℃,最适pH为6.0;在55℃以下保温2 h能保留50%的酶活性,在pH 4-8范围内孵育4 h仍保留50%以上的活性;HIS-Lac1338对Cu^(2+)抗性强,Ca^(2+)、Na^+、K^+对HIS-Lac1338有促进作用,而Co^(2+)、Fe^(2+)、Hg^(2+)、Ag^+等重金属离子对HIS-Lac1338有抑制作用。易错PCR方法得到的Lac1338的突变酶Lac16与HIS-Lac1338相比,对酸性紫7、溴酚蓝、考马斯亮蓝的降解率分别由10.9%、20%和25%提高到90.5%、67.8%和85%。结果表明,HIS-Lac1338具有较好的温度及pH稳定性,而通过易错PCR技术定向突变获得的突变酶Lac16的染料降解率大大提高。

In order to obtain the laccase with high expression and high thermal stability,a laccase gene lac1338 synthesized was clonedby codon optimization,ligated to the vector p ET-32a（＋）,then expressed in Escherichia coli BL21（DE3）,and the recombinant proteinHIS-Lac1338 was obtained.Its enzymatic properties showed that while using ABTS as a substrate,the specific activity of HIS-Lac1338 wasup 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 temperatureand 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 Cu^（2＋）,while its activity was promoted by Ca^（2＋）,Na~＋,K~＋ and inhibited by heavy metal ions such as Co^（2＋）,Fe^（2＋）,Hg^（2＋）,Ag~＋,etc.Comparing with HIS-Lac1338,mutant enzyme Lac16 of Lac1338 by sequential error-prone PCR improved the degradation rates ofAcidviolet 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.