CBH Overexpresses The Degradation Of Cellulose And Its Enzymatic Properties

Cellulase is a kind of combined hydrolytic enzymes system,mainly includes endoglucanase,exoglucanase and β-glucosidase.The three cellulases play different roles during the period of cellulose degradation from polysaccharides to disaccharides and finnaly breaking into glucose.N.crassa as an erukaryotic model organism,which has the ability to produce cellulose.In prior period of work,a strain 60 of N.crassa had been successfully constructed by transformed the plasmid of qa-2.5 myc.6HisdH9680,which enabled the strain to over-express exoglucanase.In this article we further explored its degrading efficency on cellulose,and also the properties of exoglucanase were analyzed after separated and purified from the broth fermented by strain 60.By the method of plate culture and measurement of enzyme activity,the exoglucanase activity of strains 60 was proved increased by 16%compared to the wild strain87-3,which reached the highest level after seven days fermentation.The rate of mycelial growth and the ability of degrading the CMC-Na were all higher than strain WT87-3.In the further step,the strain 60 was investigated on exoglucanase expression by adding different types of carbon source,nitrogen source,inducer,and metal ion.The optimum condition was:straw 1g,5g bran basal,peptone 0.02g,cellulose 0.025g and Cac120.03g in vogel’s medium.The enzyme activity of strain 60 was increased from 19.14U/mL to 24.78 U/ml under the optimum conditions.Next we seeded the strain 60 into the acid-treated bran to test its degrading effiency.The results showed that the rate of cellulose degradation was 30.2%after 7 days solid fermentation,and exoglucanase to reached 26.4 U/mL at the same time.SEM pictures showed that the cellulose structure was mostly fractured and broken by compared with pre-degraded bran.In order to analyze the properties of exoglucanase,we used nickel column affinity chromatography method to separate and purify the exoglucanase of strain 60.The result of SDS-PAGE electrophoresis showed the protein size was 62KDa,The enzymatic properties of exoglucanase showed the optimum reacting conditioin was at 50℃,pH 5.Metal ions Mg2+,Na+,Ca2+,and Mn2+ had promoting effects on enzyme activity,while Pb2+ and Li+ had very strong inhibition.N-heptane and isobutanol increased the enzyme activity saperately by 8.62%and 4.61%,but EDTA,Urea,DTT,H2O2 decreased enzyme activity.Surface active agents Tween-20 and Triton X-100 also could increase activity of exoglucanase.By catalyzing the substrate of microcrystalline cellulose,the kinetic parameters was determined,the Km value was 1.826 mg/ml,the Vmax was2.39 × 10’3.