Characterization of two cellobiose dehydrogenases and comparison of their contributions to total activity in Neurospora crassa

• Two cellobiose dehydrogenases (CDH), which were involved in cellulose degradation, were identified in Neurospora crassa.
• The contribution of both CDHs was compared through the deletion of the cdh genes.
• Homologous expression and purification of both CDHs were achieved for the first time.
• NC-CDH1 and NC-CDH2 have different glycosylation patterns.

Cellobiose dehydrogenase production by Neurospora crassa was investigated in this study. N. crassa has two putative cellobiose dehydrogenase (CDH) genes (cdh) in its genome. CDH was produced only under cellulolytic conditions. Deletion of nc-cdh1 eliminated almost all of the strain’s 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 for the first time. 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. Although NC-CDH2 lacks the cellulose-binding module and contributed marginally to total CDH activity in N. crassa, NC-CDH2 has specific activity similar to that of NC-CDH1 (7.93 vs. 8.89 IU mg−1), and it has a much lower Km value than that of NC-CDH1 (5.79 vs. 25.72 μM). The lower activity contribution of NC-CDH2 in the wild-type strain may results from its lower enzyme production.