枯草杆菌碱性蛋白酶Ki2基因的序列测定及M222A定点突变

Sequencing and M222A Site directed Mutagenesis

含有枯草杆菌碱性蛋白酶Ｋｉ２基因的１９ｋｂＤＮＡ片段用限制酶切成几个小片段，将这些片段分别插入Ｍ１３ｍｐ１８或Ｍ１３ｍｐ１９中，用通用测序引物测得全序列。所得全序列与蛋白酶Ｅ相比较，在结构基因部分仅有８个碱基不同，由此而导致两个氨基酸的差异。此１９ｋｂ的片段插入枯草杆菌大肠杆菌穿梭质粒ｐＢＥ２，得到的重组质粒转化蛋白酶缺陷型的枯草芽孢杆菌ＤＢ１０４，结果表明枯草杆菌碱性蛋白酶Ｋｉ２基因在ＤＢ１０４中能利用自身的调控元件表达并分泌到胞外。将Ｋｉ２蛋白酶的２２２位甲硫氨酸突变成丙氨酸，突变后的Ｋｉ２蛋白酶具有抗氧化性。

The 1.9kb DNA fragment containing subtilisin Ki 2 gene was digested by restriction enzymes into several small fragments.These fragments were inserted into M13mp18 or M13mp19 and sequenced by using universal sequencing primers.Comparing the sequence of subtilisin Ki 2 with subtilisin E,in the structure gene region,only 8 nucleotides difference which deduced two amino acids varying was found .The 1.9kb DNA fragment was recombinated with pBE 2 which is a shuttle vector between E.coli and Bacillus Subtilis. The recombinated plasmid was used to transform B.subtilis DB104 that is a double mutant strain deficient in both alkaline and neutral proteases.The results shew that subtilisin Ki 2 gene could be expressed in DB104 under its own expression elements and secreted into culture medium.The Met 222 of subtilisin Ki 2 was mutated into Ala 222.The mutant subtilisin Ki 2 was oxidation resistant but with lower specific activity.