牦牛溶菌酶的定向进化研究

Investigation on directed evolution of yak lysozyme

为了通过定向进化的方法提高牦牛溶菌酶的活性,试验选用3个牦牛基因YSL1a、YSL3c和YML,通过DNA洗牌技术进行序列重组以及酶活筛选,获得了一个重组基因HYL,检测其酶活,构建重组子基因HYL的真核表达载体,转化毕赤酵母X33,并对重组HYL溶菌酶的活性进行检测。结果表明:HYL与YSL1a、YSL3c和YML的序列相似性分别为85.36%、96.04%和83.11%,突变核苷酸分别为65,17,75个;重组序列与3个亲本基因相比均有序列缺失。筛选到一个酶活达667.72 U/mg的阳性克隆,其活性高于亲本基因中YSL3c真核表达产物。说明本试验中牦牛HYL的序列发生了序列重组,而且其真核表达产物具有活性。

To explore the increasing of yak lysozyme activity by directed evolution, a recombinant gene, named HYL, was obtained by DNA shuffling and enzyme activity screening method according to 3 lysozyme genes （ YSL1 a,YSL3c and YML）. To confirm whether the enzyme activity was increased for the new HYL enzyme, the HYL gene was cloned into the eukaryotic expression vector （ pPICZαA）, then transformed into the yeast expression strain（ X33 ）. The heterotogous expressed HYL was collected and then the enzyme activity was detected by using turbidimetric method. The results showed that the sequence similarity between the new recombinant HYL gene and YSLla,YSL3c and YML was 85.36% ,96.04% and 83.11% , respectively. The number of mutant nucleotides was 65, 17 and 75, respectively. Compared with the 3 parental genes, the recombinant HYL gene has deletions in its nucleotide sequence. The enzyme activity of recombinant HYL enzyme was 667.72 U/rag which was slightly higher than the heterologous YSL3c that was expressed in the same eukaryotic expression system. These data indicate that the sequence of HYL is recombinanted, and the lysozyme activity can be detected in the heterologous HYL by eukaryotic expression system.