Homologous Expression, Purification And Functional Mechanisms Of Two Cellobiose Dehydrogenases In Neurospora Crassa

The world’s energy resources and environmental issues become increasingly serious, many researches have carried out to change the most abundant, cheapest plant fiber into energy and useful resources through the application of cellulase. Now, cellulase is widely used in food industry, wine-making, textiles industey, agriculture and other areas. With the increase demand of cellulases, the efficient cellulose decomposing strains and cellulases already became the hot spots of the microbial community all over the world.The ascomycete Neurospora crassa (N. crassa) is a well-known laboratory model, it is also an efficient cellulose degrader and produces a full array of lignocellulose-degrading enzymes. An enzyme called cellobiose dehydrogenase (CDH) is produced along with the cellulases. CDH is a hemo-flavoenzyme that belongs to the glucose-methanol-choline (GMC) oxidoreductase family. The CDH production and characteristic by N.crassa was investigated in this study. N.crassa has two putative cellobiose dehydrogenase genes (cdh) in its genome. CDH was produced only under cellulolytic conditions. Deletion of nc-cdhl eliminated almost all of the strains CDH activity, whereas the deletion of nc-cdh2 had little effect on total extracellular CDH activity, which indicates that NC-CDH1 is a major contributor to overall CDH activity.The homologous expression of nc-cdh1 and nc-cdh2 under the control of the constitutive D-glyceraldehyde-3-phosphate dehydrogenase (gpdA) promoter enabled recombinant CDH production under non-cellulolytic conditions. Both NC-CDH1 and NC-CDH2 produced by N.crassa were successfully purified and characterized. Both enzymes had an optimal reaction pH of 6.0, and a catalytic temperature range of 45-55℃. The pH stability results demonstrated that both enzymes were stable when stored in buffers with pH ranging from 4.0 to 7.0. During the test period (10 h), both enzymes were stable in room temperature ranging from 20 to 30℃. NC-CDH1 and NC-CDH2 have molecular weights of 100 kDa and 130 kDa, respectively. When their N-linked glycans were removed by N-glycosidase F treatment, both enzymes showed a molecular weight of 95 kDa.NC-CDH2 lacks the cellulose-binding domain (CBD), the adsorption abilities of Avicel and filter were much poorer than NC-CDH1 and contributed marginally to total CDH activity in N.crassa. However, NC-CDH2 has specific activity similar to that of NC-CDH1 (7.93 vs.8.89 IU mg-1), even it has a much lower Km value than that of NC-CDH1 (5.79 vs.25.72 mM), all this suggesting that the lower activity contribution of NC-CDH2 in the wild-type strain may result from its lower enzyme production.Addition purified N. crassa CDH-1 to Acdh-1 strain crude enzymes, the cellulase activity could be restored to WT level, the effect of CDH-1 was 6-7 times higher than CDH-2. Meanwhile, by the data of chemical component analyses and structure observation of SEM, we can find out that knockout gene nc-cdhl of N.crassa, the degradation efficiency of rice straw decreased distinctly, suggesting that NC-CDH1 is an efficient enhancer in the breakdown of lignocelluloses with a series of cellulolytic enzymes.