摘要
利用重叠延伸法对嗜热细菌(Thermus thermphilus)的木糖异构酶基因xylA进行体外P137G定点突变,通过酿酒酵母表达载体XM204将突变基因xylA137引入酿酒酵母H158中,得到重组菌株H158-XI137.酶活分析表明,H158-XI137在中温条件下的酶活有很大的提高,与对照菌株H158-XI相比,30℃时的酶活提高1倍,并且拓宽了最适反应pH,但是其热稳定性大大降低.结果表明,嗜热细菌木糖异构酶137位的Pro对维持其热稳定性起到重要的作用,并且对其酶学性质有很大的影响.
By using overlap extension PCR, a P137G site mutant named xylA137 was introduced in the xylose isomerase gene xylA from Thermus thermphilus. The recombinant plasmid XM204-XI137 containing P137G was expressed in Saccharomyces eerevisiae H158 to produce the H158-XI137 strain. The results of enzymatic activity show that H158-XI137 is twice of the contrast ＂H158-XI at 30℃. In addition, the range of optimum pH become broader, while the thermostability decreases obviously. The result indicates that the proline at the position 137 of the xylose isomerase is not only very important for the enzymatic activity, but essential for maintaining the thermostability.
出处
《山东大学学报（理学版）》
CAS
CSCD
北大核心
2006年第6期145-148,156,共5页
Journal of Shandong University(Natural Science)
基金
国家自然基金资助项目(50273019)
国家重点基础研究发展计划(973计划)资助项目(2003CB716006
2004CB719702)
关键词
木糖异构酶
定点诱变
热稳定性
脯氨酸
xylose isomerase
site-directed mutagenesis
theermostability
proline
