Study On The Chitin Deacetylase Cda3 And ChiE2 Of Coprinus Chinensis

During the maturation of the fruit body of basidiomycete Coprinopsis cinerea,the stipe grew rapidly and the cell wall restructured within a very short time.When cell wall reconstructed,both rigidity and plasticity are needed to ensure that the cell wall can reconstruct and maintain its shape.Therefore,it is significant to study the chitin hydrolases,which can act on an important component of the cell wall,chitin.Chitin hydrolase mainly includes two large groups,chitinase that breaks glycosidic bonds,and chitin deacetylase that removes the acetyl of chitin.In this paper,two related enzymes were studied respectively,and its enzymatic properties were explored,which provided the basis for studying the expansion mechanism of fungal cell wall.In this study,a chitin deacetylase Cda3 was successfully obtained using a Cda3eukaryotic expression strain stored in the laboratory.The chitin deacetylase Cda3 from C.cinerea deacetylated chitin-oligosaccharides with dp?2.Since Cda3 firstly removed the intermediate acetyl group of?Glc NAc?₄,it was an endo-acting deacetylase.Different from previously reported deacetylation modes,Cda3 deacetylated chitinbiose at either the reducing end or the nonreducing end;Cda3 deacetylated chitintriose at any subsite including the end and the intermediate;Cda3 further removed acetyl groups at any subsite,the intermediate,nonreducing and reducing end of chitintetraose after removal of the first intermediate acetyl group.3D structural analysis showed that Cda3has aromatic amino acids distributing at both the+1 and-1 subsites of the catalytic site,which may be responsible for its distinctive deacetylation mode.Furthermore,Cda3was active on crystalline chitin,its deacetylation activity increased with the DA decreases of chitinous substrates and showed a higher activity towards the cell wall of the basal stipe with the higher molar ratio of Glc N/Glc NAc than that of the apical stipe.Cda3 with distinctive deacetylation mode and activity indicates its function during the maturation of the fruiting bodies of C.cinerea and a potential for preparation of mushroom chitosan for application in the food,cosmetics,and pharmaceutical industries.In order to obtain the chitinase Chi E2,a recombinant expression strain of Chi E2was constructed.Although pure Chi E2 protein was not obtained,it was proved that the fermentation broth indeed contained the recombinant protein Chi E2 by the LC/MS method.The Chi E2 protein in the fermentation broth was concentrated by a salting-out precipitation method,and the substrate specificity was examined.It was found that Chi E2 had no hydrolytic activity on the insoluble chitin powder.3D structure analysis found that there is a ring structure near the substrate binding groove of Chi E2,which may hinder the binding of insoluble substrate with Chi E2.Considering published articles of our laboratory,Chi E2 does not play a major role in the elongation growth of the C.cinerea.