摘要： 异甘露聚糖酶是制备益生元甘露寡糖的关键酶。对分泌异甘露聚糖酶枯草芽孢杆菌K-6（Bacillus subtilis K-6）的紫外诱变正突变株K-6—9（Bacillus subtilis K-6—9）的诱变机理进行了研究。通过对出发菌株和正突变株的异甘露聚糖酶基因进行克隆和表达，以生物信息学的方法对异甘露聚糖酶基因序列和蛋白序列进行分析，得到突变株的突变碱基以及突变氨基酸。基因序列研究结果显示，突变体的总碱基突变频率为1．02，碱基突变类型包括碱基的颠换、转换。在检测到的11个碱基突变中，转换的频率（54．5％）是颠换频率（45．5％）的1．2倍，其中，C／T之间的转换所占比例最大，A-G和A—T也具有较高的替换频率，说明胸腺嘧啶（T）具有较高的辐射敏感。通过蛋白序列的比较分析，有2个氨基酸发生突变，由第21位保守氨基酸脯氨酸变异为非保守氨基酸丝氨酸，第23位非保守氨基酸丝氨酸变异为保守氨基酸谷氨酸。综合分析表明：保守区氨基酸与非保守区氨基酸的相互突变可能对其蛋白的正确组装及功能的发挥起重要作用。

关键词: 异甘露聚糖酶, 紫外线诱变, 基因克隆, 突变

Abstract: Isomannanase is the key enzymes of making probiotics-Mannan oligosaccharides （MOS）. A Bacillus subtilis K-6 strain that can secret isomannanase preserved in the laboratory was induced by UV. The mutation mech- anism of a positive mutant K-6-9 was studied. Initial Bacillus subtilis K-6 and positive mutant Bacillus subtilis K-6- 9 was cloned and expressed,then analyzed the gene and protein sequence of isomannanase mutation gene by bioin- fonnatics,the total base mutation and amino acids mutations of the mutants obtained. The results indicated that the total base mutation rate of mutants was 1.02%, the types of base changes included base transition and transversion. Among the 11 base mutations that had detected, the frequency of base transitions （54.5 % ） was 1.2 times higher that the base transversions （45.5 % ）, the transitions between C and T accounted for largest proportion, A-G transi- tions and A-T transversions were also interchanged at high frequency. After of protein sequences, there were two point mutation forms, which were Pro （conservative amino acid）-Ser（ non-conservative amino acid） at 21nt and Ser （non-conservative amino acid）-Glu （conservative amino acid） at 23nt. From a comprehensive and comparative data analysis,the mutual mutations between conservative amino acid and non-conservative amino acid may play an im- portant role in proteins assemble correctly and execute their function effectively.

Key words: Isomannanase, UV-mutation, Cloning Mutantion

中图分类号: 

• Q78