作　　者： ; ; ; ;

机构地区： 华南理工大学生物科学与工程学院

出　　处： 《华南理工大学学报（自然科学版）》 2010年第9期138-141,146,共5页

摘　　要： 为提高豆豉纤溶酶的药用价值,通过易错PCR方法对来源于Bacillus subtilis DC-12的豆豉纤溶酶DFE基因进行突变并构建突变体文库,利用底物H-D-Val-Leu-Lys-pNA对突变体文库进行筛选.通过3轮易错PCR最终获得催化效率提高的突变酶mDFE3.序列分析表明突变酶mDFE3基因发生了6处碱基突变,其中4处突变发生氨基酸取代,2处为同义突变;通过swiss-model repository模拟突变酶mDFE3的结构,显示3个突变氨基酸位于回环结构上,1个位于α螺旋上;酶动力学测定结果表明突变酶mDFE3的Km值由0.58mmol/L降低至0.45mmol/L,突变酶mDFE3的催化效率(kcat)是野生型的2.57倍. In order to improve the medical value of Douchi fibrinolytic enzyme（DFE）,random mutagenesis on DFE gene of Bacillus subtilis DC-12 was performed by using the error-prone PCR strategy to construct a mutant library.After three cycles of error-prone PCR and a screening using substrate H-D-Val-Leu-Lys-pNA,the best mutant enzyme mDFE3 with improved catalytic efficiency was obtained.Gene sequence analysis of mDFE3 shows that the mutant is of six nucleotide substitutions,four of which cause amino acid substitution and the other two cause synonymous mutation.According to the three-dimension structure of mDFE3 mimicked by swiss-model repository,three mutated amino acids are found to locate in loop structures and one is in α-helix.Moreover,the enzyme kine-tic analysis indicates that the Km value of mDFE3 decreases from 0.58mol/L to 0.45mmol/L and that the catalytic efficiency（kcat） of mDFE3 is 2.57 times that of DFE.

关 键 词： 枯草芽孢杆菌 豆豉纤溶酶 易错 定向进化 筛选

领　　域： [生物学]