D-泛解酸内酯水解酶的定向进化  

Directed Evolution of D-lactonohydrolase by Error Prone PCR and DNA Shuffling

作　　者：柳志强[1] 孙志浩[1] 郑璞[1] 冷泳[1] 钱嘉南[1] 

机构地区：[1]江南大学生物工程学院生物催化研究室,无锡214036

出　　处：《生物工程学报》2005年第5期773-781,共9页Chinese Journal of Biotechnology

基　　金：国家重点基础研究发展规划(973)项目(No.2003CB716008);国家自然科学基金项目(No.20476039)资助。~~

摘　　要：易错PCR结合DNA改组方法向D泛解酸内酯水解酶基因中引入突变,并构建突变体库。利用酶的催化特点和产物特性建立了基于平板初筛和高效液相复筛的两步法D泛解酸内酯水解酶活性筛选系统。用该筛选系统以酶活力和pH稳定性为指标对突变体库进行筛选,最终获得一株酶活力高且在低pH条件下稳定性好的突变体MutE861。该突变体的酶活力是野生型酶的5.5倍。对突变体和野生型酶在pH6.0和pH5.0条件下的残余酶活进行对比,在这两种pH条件下,突变体酶的酶活残留分别为75%和50%,而野生型酶只能保持原来的40%和20%。通过软件对突变体MutE861酶基因和野生型酶基因进行分析对比,发现突变体MutE861酶基因发生了三处点突变,其中突变使两处氨基酸取代,另一处为沉默突变,未引起氨基酸的变化。D-lactonohydrolase is useful in the procedure of resolution of racemie pantolactone to produce D-pantolactone, but the activity and stability under low pH of the wild type enzyme is not satisfactory enough to be applied to industrial production. The expected properties of wild type enzyme were enhanced by directed evolution. According to the formation of products and pH indicators, a screening system was designed. After three sequential en＇or prone PCR and one round DNA shuffling followed by screening, Mut E-861, the best mutant with improved activity and stability under low pH situation was obtained. Gene analysis of the Mut E-861 mutant indicated that the mutant enzyme had A352C, G721A mutations and a silent mutation of position 1038. Moreover, the activity and stability of Mut E-861 were determined. The results showed that the activity of this mutant was 5.5-fold higher than that of wild type, and the stability under low pH was improved at no expense of D-lactonohydrolase activity. After incubated at pH 6.0 and pH 5.0 the activity of D-lactonohydrolase could be retained 75 % to 50% , however, compared with 40% to 20% for wild type.

关 键 词：串珠镰孢霉菌 D-泛解酸内酯水解酶 易错PCR DNA改组 筛选 定向进化 

分 类 号：Q55[生物学—生物化学]