二硫键和Ca2+结合残基对重组酱油曲霉碱性蛋白酶的影响  

Effects of disulfide bond and Ca2+ binding residue on recombinant alkaline protease from Aspergillus sojae

作　　者：柯野[1] 谢涛 卫孟瑶 樊银凤 余健 KE Ye;XIE Tao;WEI Mengyao;FAN Yinfeng;YU Jian(School of Life Sciences,Shaoguan University,Shaoguan,Guangdong 512005,China)

机构地区：[1]韶关学院英东生命科学学院

出　　处：《福建农林大学学报（自然科学版）》2019年第6期813-818,共6页Journal of Fujian Agriculture and Forestry University:Natural Science Edition

基　　金：广东省科技创新战略专项资金(基础与应用基础研究方向)项目(2018A0303130108);广东省教育厅特色创新项目(2015KTSCX126);韶关学院大学生科技创新培育项目(pdjh2018b0458);韶关学院大学生创新创业训练计划国家级项目(201810576007)

摘　　要：以PDB code:3f7o.1.A蛋白酶作为模板对酱油曲霉碱性蛋白酶(Alkaline protease,Ap)进行同源建模,对模拟的Ap结构分析可知,Ap中无二硫键,结合了1个Ca^2+.分别对预测的Ap二硫键和Ca^2+结合残基进行突变,获得增加二硫键的Y34C-R123C、L178C-T252C、Y34C-R123C/L178C-T252C和Ca^2+结合位点的T179G、D200G、T179G-D200G共6个突变.分别将这些突变基因电转至毕赤酵母KM71中,获得重组菌株;对重组菌株诱导表达,分离纯化突变型Ap,并对其稳定性进行分析.结果表明,Y34C-R123C突变型、L178C-T252C突变型与野生型Ap的表达量差异不显著;在40℃以下,Y34C-R123C突变型比野生型Ap具有更强的热稳定性;T179、D200被突变为甘氨酸后,不利于Ap正确折叠,不能成功表达.Alkaline protease(Ap)structure was homologously modeled referring to a proteinase(PDB code:3f7o.1.A)as the crystal coordinate.The results showed that Ap had no disulfide bond,but bonded a Ca^2+.Then the mutants with added disulfide bonds(Y34C-R123C,L178C-T252C,Y34C-R123C/L178C-T252C),and the mutants with Ca^2+ binding residue(T179G,D200G,T179G-D200G)were respectively constructed using site-directed mutation technology.Subsequently,these mutated genes were respectively transformed into Pichia pastoris KM71 for the recombinant strains.Then the Ap mutants were separated and purified from fermentation broth,and their thermal stability were determined.The results showed that the expression yields of the Y34C-R123C mutant,the L178C-T252C mutant and the wild-type Ap were not significantly different.Under 40℃,the Y34C-R123C mutant was more stable than the wild-type Ap.After replacing glycine with T179 and D200 on the Ca^2+ binding residue site,the Ap was not expressed due to incorrect protein folding.

关 键 词：酱油曲霉碱性蛋白酶 二硫键 Ca^2+结合残基 定点突变 表达 热稳定性 

分 类 号：TS202.3[轻工技术与工程—食品科学]