短小芽孢杆菌碱性蛋白酶基因的随机突变  

Random Mutation of Alkaline Protease Gene in Bacillus pumilus

作　　者：杨锦[1] 冯红[1] 万民远[1] 焦惠科[1] 张晓露[1] 张义正[1] 

机构地区：[1]四川大学生命科学学院四川省分子生物学及生物技术重点实验室,成都610064

出　　处：《应用与环境生物学报》2010年第1期96-99,共4页Chinese Journal of Applied and Environmental Biology

基　　金：国家高技术研究发展计划(No.2006AA02Z221)资助~~

摘　　要：碱性蛋白酶(DHAP)属于丝氨酸蛋白酶家族(S8A),是由275个氨基酸残基组成的单链多肽,没有二硫键,以Asp32、His64、Ser221作为活性中心.采用易错PCR方法,对短小芽孢杆菌碱性蛋白酶基因(GenBank登录号:AY458140)进行随机突变.在大肠杆菌中建立随机突变文库,然后转化枯草芽孢杆菌WB600,构建枯草芽孢杆菌突变文库,筛选出酶活性提高或最适反应温度和pH值有所降低的突变体.通过3轮的随机突变和筛选,获得2个阳性突变体pP43AP-M166和pP43AP-M375.其中pP43AP-M166在不同的温度和pH条件下,与出发菌株相比,酶活都有不同程度提高,特别是当反应温度为50℃、pH为10时,突变蛋白酶酶活性提高1.23倍,表现出很好的热稳定性.测序结果表明,该突变体含有一个氨基酸置换,位于碱性蛋白酶催化三联体活性中心His64旁,与其只有一个氨基酸残基相隔.De-hair alkaline protease（DHAP）,belonging to serine protease family（S8A）,is composed of 275 amino acid residues with Asp32,His64 and Ser221 as activity centers,and has no disulfide bond.Random mutation of DHAP gene（NCBI No：AY458140） from Bacillus pumilus was carried out with modified prone-error PCR.A random mutation library was constructed in Escherichia coli DH5α and then in B.subtilis WB600.The capacity of the random mutation library is 4×105.After three sequential error-prone PCR followed by screening,pP43AP-M166,the expected mutant with improved properties was obtained.Compared with the wild type,pP43AP-M166 has improved its activity and stability under different pH and temperatures.Moreover,its activity was determined,and it was 1.23 fold higher than that of wild type under optimal reaction temperature at 50 ℃ and pH 10,while it showed excellent thermal stability.The gene sequence analysis indicated that the mutant enzyme had F62C.The position of mutant AA has only one AA separated from His64,which is the most significant composition of catalytic activity center.

关 键 词：碱性蛋白酶 短小芽孢杆菌 定向进化 易错PCR 序列分析 基因表达 

分 类 号：Q93[生物学—微生物学]