A novel endoglucanase gene was cloned from a cDNA library of the filamentous fungus Rhizopus stolonifer var.reflexus TP-02 and expressed in Escherichia coli BL21.Structure and function of the gene product endoglucanaseⅡ(abbreviated as EGⅡ) were analyzed by a series of bioinformatics software.Mutational analysis of the carbohydrate-binding modules 1(CBM1) and the Glycoside Hydrolase 45(GH45) domain of EGⅡ was conducted to discuss their structural consequences.Results of structure analysis indicated that N39S could influence the formation of CBM1 close horizontal plane,while V136D and E260D had a strong effect on the active center of GH45 domain.The fermentation characteristics of recombinants showed that N39S could shorten the peak time of mutant strains,and V136D and E260D improved the activity significantly.Furthermore,the maximum endoglucanase activity of the mutant EGⅡ-F containing all 6 mutations(N39S,V136D,T251G,D255G,P256S and E260D) was 1.321 IU / mL,which was observed at 21 h during the fermention process,and was increased by 82.7% compared to that of wild type EGⅡ.