白腐菌漆酶LAC1基因重要功能区段的随机突变掺入及定向筛选  

Random mutation incorporation and directional screening of important functional segments of laccase LAC1 gene in white-rot fungus

作　　者：李晓彤 肖君华 李凯 周宇荀 LI Xiaotong;XIAO Junhua;LI Kai;ZHOU Yuxun(Key Laboratory of Science&Technology of Eco-Textile Ministry of Education College of Chemistry Chemical Engineering and Biotechnology Donghua University,Shanghai 201620 China)

机构地区：[1]东华大学化学化工与生物工程学院生态纺织教育部重点实验室,上海201620

出　　处：《生物学杂志》2023年第1期27-33,共7页Journal of Biology

基　　金：中央高校基本科研业务费专项资金资助项目(2232020G-04);中央高校基本科研业务费学科交叉重点计划项目(2232020A-07)。

摘　　要：为了提高蛋白产量,通过密码子优化漆酶基因LAC1,构建pET-32a(+)-LAC1重组载体,并在Escherichiacoli BL21(DE3)中进行表达。其酶学性质表明,目的漆酶蛋白分子质量约为65 ku,其活性在55℃以下及pH 3.5~5.0时较为稳定,5 mmol/L的Ca2+、Cu2+、Mg2+对重组漆酶活性具有促进作用,镍柱纯化后漆酶比活力为32.3 U/mg。通过分析重组LAC1漆酶的结构及活性中心,并与其他真菌漆酶进行序列比对,找出其差异性区段,在化学合成该区段DNA片段时,在特定位点掺入任意碱基,从而引入随机突变,构建随机突变文库并对库容量进行测定,通过批量筛选得到1株酶活提升的突变体,同源建模预测其三维结构。结果表明,通过定向突变获得的突变酶共有4处氨基酸发生变化,其酶活性提高约2.3倍。In order to improve protein yield,laccase gene LAC1 was optimized by codon,and pET-32a(+)-LAC1 recombinant vector was constructed and expressed in Escherichia coli BL21(DE3).The enzyme properties showed that the molecular weight of the target laccase protein was about 65 ku,and its activity was stable below 55℃and pH 3.5-5.0.5 mmol/L Ca2+,Cu2+,Mg2+promoted the activity of the recombinant laccase,and the specific activity of the recombinant laccase was 32.3 U/mg after purification by nickel column.By analyzing the structure and active center of recombinant LAC1 laccase,the gene sequence was compared with that of other fungal laccase sequences,and the difference section was found.For this section,random mutation was introduced during the chemical synthesis of DNA fragments,and the random mutation library was constructed and the library capacity was measured.A mutant with enzyme activity enhancement was obtained by batch screening,and its three-dimensional structure was predicted by homologous modeling.The results showed that four amino acids of the mutated enzyme were changed and the enzyme activity was greatly increased by 2.3 times.

关 键 词：漆酶 酶学性质 随机突变 批量筛选 同源建模 

分 类 号：Q55[生物学—生物化学] Q78