淀粉液化芽孢杆菌BS5582中性蛋白酶基因的克隆表达及其酶学性质研究

Cloning and expression of the neutral protease gene from Bacillus amyloliquefaciens BS5582 and characterization of the enzyme

以一株高产中性蛋白酶的Bacillus amyloliquefaciens BS5582基因组DNA为模板,PCR扩增获得结构基因npr,构建质粒pPIC9K-npr,并转化到表达菌株毕赤酵母GS115中得到重组菌。经甲醇诱导发酵,测定中性蛋白酶活性。结果表明,目的基因大小为1566bp,在宿主中得到了成功表达,重组菌酶活力为9.17×103U/g,酶学性质分析表明,重组中性蛋白酶的最适反应温度为50℃,最适反应pH为7,在40℃中保温1h后仍能保持85%左右活性,在pH4～9的范围内稳定性较好,Ca2+、Mg2+、Mn2+离子对该酶有激活作用。

The neutral protease gene was amplified from Bacillus amyloliquefaciens BS5582 by PCR.The gene was then expressed using pPIC9K as the vector in Pichia pastoris.It showed an activity of 9.17×103U/g with the inducement by methanol.The recombinant enzyme was optically active at pH 7.0 and 50℃.It kept about 85% activity after 1h incubation at 40℃,and showed excellent stability between pH 4 ～ 9.Furthermore,Ca2＋,Mg2＋,Mn2＋ could activate the enzyme activity.