从匍枝根霉TP-02的cDNA文库中克隆得到内切葡聚糖酶基因eg2,在大肠杆菌BL21中成功表达,通过对其结构功能进行初步分析,并对EGⅡ的纤维素结合模块CBM1及催化结构域GH45区进行突变研究。结构分析表明:N39S可影响CBM1亲水平面的形成,V136D及E260D对GH45活性中心的改变较大。突变株的发酵特性显示,N39S可缩短达到峰值的时间,V136D及E260D可显著提高酶活。其中,突变株EGⅡ-F在发酵21 h后,酶活达到最高为1.321 IU/mL,比突变前提高了82.7%。

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.