易错PCR技术提高耐碱木聚糖酶基因xylBYG耐热性的研究  

Study on improving the thermostability of xylanase gene xylBYG by error-prone PCR

作　　者：张雯[1] 石丽娜[1] 付宁[1] 杨明明[1] 龚月生[1] 

机构地区：[1]西北农林科技大学动物科技学院,陕西杨凌712100

出　　处：《黑龙江畜牧兽医》2013年第2期1-4,166,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(30871813)

摘　　要：为了研究耐碱木聚糖酶基因xylBYG耐热性,试验采用易错PCR技术对耐碱木聚糖酶基因xylBYG进行定向进化,经过2轮易错PCR产生突变基因产物,重组于表达载体pET-32a(+)中,并导入大肠杆菌BL21构建突变体文库。结果表明:经筛选获得了最佳突变菌株37D07,包含4个氨基酸替换,其最适作用温度比野生型提高了13℃,酶活约提高了17%,热稳定性也相应提高。To study the thermostability of alkali- tolerant xylanase gene xylBYG, the directed evolution of alkali -tolerant xylanase gene xyl- BYG was performed by error - prone PCR technology. The product of the mutant gene was obtained after two rounds of error - prone PCR, and was recombined into the expression vector pET -32a （ ＋ ）, and then introduced into E. coli BI221 to construct a mutant library. The results showed that the best mutant strain 371907 was obtained through screening, which contained four amino acid substitutions. Compared with the wild - type strian, its operation temperature increased 13℃, and the enzyme activity increased approximately 17%, and the thermostability was also corresponding increased.

关 键 词：木聚糖酶 易错PCR 最适作用温度 酶活 热稳定性 

分 类 号：Q53[生物学—生物化学]