摘要
用定点突变和随机突变的方法，对枯草杆菌碱性蛋白酶Ｅ基因进行改造。突变后的基因插入大肠杆菌枯草杆菌穿梭质粒ｐＢＥ２中，在碱性和中性蛋白酶缺陷型的枯草杆菌ＤＢ１０４中进行表达，得到突变种的碱性蛋白酶，它们的突变位点分别是（Ｍ２２２Ａ）、（Ｍ２２２Ａ、Ｎ１１８Ｓ）、（Ｍ２２２Ａ、Ｎ１１８Ｓ、Ｑ１０３Ｒ）、（Ｍ２２２Ａ、Ｎ１１８Ｓ、Ｑ１０３Ｒ、Ｄ６０Ｎ）。各突变种酶的性质测定结果表明，Ｍ２２２Ａ突变使酶抗氧化，Ｎ１１８Ｓ突变使酶增加热稳定性，Ｑ１０３Ｒ和Ｄ６０Ｎ突变虽然能增加酶的比活，但使酶的热稳定性大大下降，尤其是Ｄ６０Ｎ突变使酶变得极不稳定。野生型碱性蛋白酶与（Ｍ２２２Ａ）突变种的等电点均为８９２，而（Ｍ２２２Ａ，Ｎ１１８Ｓ），（Ｍ２２２Ａ，Ｎ１１８Ｓ，Ｑ１０３Ｒ）和（Ｍ２２２Ａ，Ｎ１１８Ｓ，Ｑ１０３Ｒ，Ｄ６０Ｎ）突变酶分别为８８８，９１０和９１７。用ＮｓｕｃＡＡＰＦｐＮＡ作为底物时酶反应最适ｐＨ值为７５～９５，而用酪蛋白作底物时最适ｐＨ值为１０～１２。
Protein engineering was carried out by site directed and random mutagenesis on subtilisin E gene. Four mutants were obtained. They are M222A;M222A,N118S;M222A,N118S,Q103R and M222A,N118S, Q103R,D60N.The mutant genes were recombined in pBE 2 and E. coli B. subtilis shuttle vector,and transformed into B.subtilis DB104,an alkline and neutral proteinase deficient strain. The mutant subtilisin E obtained from their gene expression were purified.The properties of those mutants showed that the M222A mutation led the enzyme become oxidation resistant,N118S mutation increased the heat stability,while Q103R and D60N mutations enhanced specific activity of the enzyme but decreased the heat stability,especially D60N mutation caused the enzyme quite unstable. The IEF PAGE showed that the wild type and M222A mutant had the same pI of 8.92,while those of double mutant,triple mutant and tetra mutant were 8.88,9.10 and 9.17 respectively.The optimum pH range was 7.5～9.5 for substrate suc AAPF pNA and was 10～12 for substrate casein
出处
《生物工程学报》
CAS
CSCD
北大核心
1997年第1期13-17,共5页
Chinese Journal of Biotechnology
基金
863-03主题资助
