Improvement Of The Character Of The F-10 Endoglucanase Gene From Bacillus Subtilis By Using Site-directed Mutagenesis

Endoglucanase is one of the most important cellulose enzymes, which cooperates with cellobihydrolase and β-1,4-glucosidase to translate cellulose to glucose. In recent years, along with cellulose enzyme in cotton washing finishing process and the successful application of detergent industry, Bacterial cellulose enzyme preparation has shown a good performance and great economic value.however,The existing of cellulase is low thermostability and low activity and high cost,these have acutely hampered industrialize-tion of cellulose, therefore,it is necessary to perform functional improvement to cellulo-se.So,this paper studying on molecule evolution of an endoglucanase using site-directed mutagenesis has important scientific and practical value.This study useing site-directed mutation technology to genetic engineering bacteria cellulose endoglucanase f-10. The cellulose of endoglucanase 91 lysine (AAA) replace by glutamic acid (the GAA) (K91E),369 lysine (AAA) is replaced by arginine (AGA) (K369R) and(K91E/K369R), Mutation plasmid transformation to e. coli, the mutation screening for endoglucanase gene engineering strains of e. coli A8, B7 and B7A8.Wild type and mutation of endoglucanase gene engineering strains of E. coli by IPTG induction training and separate and purify the expression product. Results show as follows: (1) the expression product of SDS-page analysis showed that the mutation and the wild type endoglucanase are 53KD. (2) the wild and the mutation of endoglucanase optimum reaction pH unchanged, are in pH6.8. (3) mutant A8, B7A8 optimum temperature are not changed, are 50 ℃, and the mutant B7 optimum temperature changed, to 40℃. (4) mutant A8, B7, B7A8 after IPTG induction training for 5 h, the specific activity reached 162.8 U/mg,77.9 U/mg and 202 U/mg. (5) the research results show that the thermal stability of the three mutants increased, when the temperature reached 70 ℃, the activity of f-10 fell sharply, surplus energy is only 12%, A8 is 30%, B7 is 36%and B7A8 is 41%, when the temperature reached 80℃, the residual activity of B7 is still about 22%.By site-directed mutation technique, we obtain the efficient expression in E. coli and good thermal stability of endoglucanase gene engineering strains, endoglucanase structure prediction analysis to explore the relationship between structure and function of endoglucanase, in order to further reform endoglucanase gene engineering bacteria into production to lay a solid foundation.