关键词: 枯草芽孢杆菌, 豆豉纤溶酶, 易错PCR 定向进化, 筛选

Abstract:

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.

Key words: Bacillus subtilis, Douchi fibrinolytic enzyme, error-prone PCR, directed evolution, screening