Study On High Activity Exo-β-glucanase Production By Recombinant Trichoderma Reesei

Increasing the activities of exo-β-glucanases, one of the important components of cellulase, is one of the key technologies to strengthening the synergistic degradation of cellulase and reducing the cost of cellulolytic hydrolysis. The methods of MCC-agar plate screening and liquid culture medium screening of filter paper were used in this work respectively to screen Trichoderma reesei transformants, obtaining6superior transformants with higher filter paper collapsing rate and growing rate on MCC-agar plates. Further screening experiment was conducted under shaking flask condition, gaining the high exo-(3-glucanase (C1)-producing transformant T. reesei ZU-101whose C1activity was18.24U·mL-1after48h liquid culture,2.16-fold higher than the original strain. Analysis results demonstrated that filter paper activity (FPA) of the cellulase system of the transformant, representing total activity, increased by61.9%.Further research on cellulase production by transformant T. reesei ZU-101was conducted. Exo-β-glucanase activity was34.66U·mL-1after120h liquid culture,1.26-fold higher than the original strain. As exo-β-glucanase activity ascended significantly, the filter paper activity was significantly improved which filter paper activity was7.35IU-mL-1, increased by40%than the original strain. Although endo-β-glucanase (CMC) and cellobiase activities varied insignificantly from the original strain.The study on fed-batch fermentation process of recombinant T. reesei ZU-101was conducted. The results showed that, after fermentation for48h,2%(Ⅴ/Ⅴ) feeding solution which lactose consistency is15%and the C/N is7was added every48h. The exo-β-glucanase activity of the recombinant T. reesei ZU-101in fed-batch fermentation process is up to55.689U·mL-1,2.11-fold higher than that of recombinant T. reesei ZU-101in batch process.Further study on characteristic of cellulse produced by T. reesei ZU-101in enzymatic hydrolysis when2%sodium hydroxide pretreated corn stover was the substrate. The enzymatic hydrolysis condition as follow:the dosage of cellulase (FPA)20FPIU·g-1substrate, substrate concentration10%, the enzymatic hydrolysis yield of T. reesei ZU-101increased by43%than that of the original strain. The ratio f activities of C1and CMC, C1and filter paper enzyme in the cellulose produced by T. reesei ZU-101is1:23and4:1respectively. While that of the original strain is1:24and3:1, the increase of the percentage of C1in cellulase is available to the synergistic hydrolazion of cellulose with high crystallinity such as corn stover. Further study showed that,1) The enzymatic hydrolysis yield ascended significantly as the dosage of cellobiase increased. At above condition, the enzymatic hydrolysis yield was up to95%when the dosage of xylanase were300U·g-1substrate, the ratio of activities of FPA and CB is1:15;2) The additjion of xylanase can promote the enzymatic hydrolysis of corn srover. When substrate concentration was10%, the dosages of cellulose (FPA) and cellobiase were20IU and100U every substrate respectively, the addition of xylanase (300IU·g-1) increased the enzymatic hydrolysis yield, up to94.4%.The results of the present work have promising application prospects in the bioconversion and utilization of renewable cellulosic materials.