豆豉纤溶酶纤溶活性的定向进化研究

通过易错PCR方法向来源于枯草芽孢杆菌DC-12的豆豉纤溶酶基因中引入突变并构建突变体库.利用底物H-D-Val-Leu-Lys-pNA以酶活力为指标对突变体库进行筛选,通过三轮易错PCR最终获得一株酶活力提高的突变株M324.序列分析表明突变体M324酶基因发生了六处碱基突变,其中四处突变发生氨基酸取代,另两处为无义突变.通过SWISS-MODEL Repository模拟突变酶的结构显示,三个突变氨基酸位于回环结构上,一个位于α螺旋上.纤维蛋白平板法测定纤溶酶活,结果显示突变酶的比活力是野生型的2.44倍.

Directed Evolution of Douchi Fibrinolytic Enzyme

Random mutagenesis on Douchi fibrinolytic enzyme gene of Bacillus subtilis DC12 was performed by using error-prone PCR strategy. After three cycles of error prone PCR and screening by substrate H-D-Val-Leu-Lys-pNA, M324 , the best mutant with improved activity was obtained. Gene analysis of the M324 mutant showed that the mutant had six nucleotide substitutions and four of them caused amino acid changes. According to the 3D structure of the mutant enzyme mimicked by SWISS-MODEL Repository, three mutated amino acids were located in loop structures and one was located in α-helix.The fibrinolytic activities of the two enzymes were determined by fibrin plate method.The result showed that the specific activity of the mutant M324 was 2.44 fold higher than that of the wild type.