Abstract:Endoglucanase is one of the most important cellulose enzymes, However, the large-scale industrialized application of these enzymes was restricted by the low activity and high cost price. Improving their activity through genetic engineering and protein engineering was considered an efficient approach to cut down the cost price. In this study the site-directed mutation technology was used to improve the activity of the F-10 endoglucanase gene. The cellulose of endoglucanase 91 lysine(AAA) was replaced by glutamic acid(GAA)(K91E), 369 lysine(AAA) was replaced by arginine(AGA)(K369R) and the 91 and 369 were replaced by arginine(AGA). Mutation plasmids were transfored into E. coli, the expression product was induced by IPTG. The result showed that protein molecular weight was 53 000. The optimum reaction pH unchanged, was pH 6.8. The optimum temperature of K369R and K91E/K369R were 50 ℃, the optimum temperature of the mutant K91E have great change, is 40 ℃. The specific activity reached 162.8、77.9、202 U/mg. 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%, 36%, 30% and 41%, when the temperature reached 80 ℃, the residual activity of K91E 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. This research can provide the basis for the further investigations of cellulose.
