HPAEC-PAD and Q-TOF-MS/MS analysis reveal a novel mode of action of endo-ß-1,3(4)-d-glucanase Eng16A from coprinopsis cinerea on barley ß-glucan.

We previously reported that an endo-ß-1,3(4)-d-glucanase, Eng16A, from C. cinerea shows a higher degradation activity toward barley ß-glucan than laminarin. HPAEC-PAD and Q-TOF-MS/MS analyses show that Eng16A-digestion products of barley ß-glucan not only contain some oligosaccharides with (1 → 3)-ß-linkage adjacent to the reducing end, which is consistent with ß-1,3(4)-glucanase-digestion products, but also include some oligosaccharides containing (1 → 4)-ß-linkage adjacent to the reducing end which is consistent with cellulase-digestion products. Thus, Eng16A possesses both cellulase and ß-1,3(4)-glucanase activities. Because Eng16A does not degrade cellulose, we propose that the insertion of a (1 → 3)-ß-linkage among the groups of (1 → 4)-ß-linkages may make these (1 → 4)-ß-linkages prone to cleavage by Eng16A. Furthermore, Eng16A also possesses transglycosylation activity which leads to some products containing one or a few consecutive (1 → 3)-ß-linkages adjacent to the non-reducing end. Therefore, HPAEC-PAD and Q-TOF-MS/MS analyses provide an efficient approach to reveal complicated modes of action of some endo-ß-1,3(4)-d-glucanases on barley ß-glucan.

Endo-ß-1,3-glucanase digestion combined with the HPAEC-PAD-MS/MS analysis reveals the structural differences between two laminarins with different bioactivities.

To resolve the structure of laminarin, the recombinant endo-ß-1,3-glucanase from Coprinopsis cinerea, which has specific activity on ß-1,3 glycosidic bond and could hydrolyze the laminarin with complex structure, was used to hydrolyze laminarin. Then, the structures of enzyme-resistant oligosaccharides were quantitatively and qualitatively analysed by high-performance anion exchange chromatography coupled with mass spectrometry. The laminarin from Laminaria digitata contains 9.51% ß-1,6 glycosidic bonds only in the branches (branch degree 7.68%). The laminarin from Eisenia bicyclis contains more ß-1,6 glycosidic bonds: 19.42% ß-1,6 glycosidic bonds in backbone and more and longer ß-1,6 branches (branch degree 25.99%). The differences in the ratio of glycosidic bonds and branch degree influence their bioactivity: the antioxidant activity and the antimicrobial activity against Gram positive bacteria of the laminarin from E. bicyclis is stronger than the laminarin from L. digitata, but the antimicrobial activity on Gram negative bacteria of the laminarin from E. bicyclis is weaker.

The Modes of Action of ChiIII, a Chitinase from Mushroom Coprinopsis cinerea, Shift with Changes in the Length of GlcNAc Oligomers.

A putative class III endochitinase (ChiIII) was reported previously to be expressed dominantly in fruiting bodies of Coprinopsis cinerea, and its expression levels increased with the maturation of the fruiting bodies. This paper further reports that ChiIII is a novel chitinase with exo- and endoactivities. When the substrate was (GlcNAc)3-5, ChiIII exhibited exoactivity, releasing GlcNAc processively from the reducing end of (GlcNAc)3-5; when the substrate was (GlcNAc)6-7, the activity of ChiIII shifted to an endoacting enzyme, randomly splitting chitin oligosaccharides to various shorter oligosaccharides. This shift in the mode of action of ChiIII may be related to its stronger hydrolytic capacity to degrade chitin in fungal cell walls. The predicted structure of ChiIII shows that it lacks the α+ß domain insertion; however, its substrate binding cleft seems to be deeper than that of common endochitinases but shallower and more open than that of common exochitinases, which may be related to its exo- and endohydrolytic activities.

Comparative Study of Nonautolytic Mutant and Wild-Type Strains of Coprinopsis cinerea Supports an Important Role of Glucanases in Fruiting Body Autolysis.

Autolysis of Coprinopsis cinerea fruiting bodies affects its commercial value. In this study, a mutant of C. cinerea that exhibits pileus expansion without pileus autolysis was obtained using ultraviolet mutagenesis. This suggests that pileus expansion and pileus autolysis involve different enzymes or proteins. Among the detected hydrolytic enzymes, only ß-1,3-glucanase activity increased with expansion and autolysis of pilei in the wild-type strain, but the increase was abolished in the mutant. This suggests that ß-1,3-glucanases plays a major role in the autolysis. Although there are 43 possible ß-1,3-glucoside hydrolases genes, only 4 known genes, which have products that are thought to act synergistically to degrade the ß-1,3-glucan backbone of cell walls during fruiting body autolysis, and an unreported gene were upregulated during pileus expansion and autolysis in the wild-type stain but were suppressed in the mutant. This suggests that expression of these ß-1,3-glucanases is potentially controlled by a single regulatory mechanism.