Potential of Secondary Metabolites of Diaporthe Species Associated with Terrestrial and Marine Origins.

Diaporthe species produce versatile secondary metabolites (SMs), including terpenoids, fatty acids, polyketides, steroids, and alkaloids. These structurally diverse SMs exhibit a wide range of biological activities, including cytotoxic, antifungal, antibacterial, antiviral, antioxidant, anti-inflammatory, and phytotoxic activities, which could be exploited in the medical, agricultural, and other modern industries. This review comprehensively covers the production and biological potencies of isolated natural products from the genus Diaporthe associated with terrestrial and marine origins. A total of 275 SMs have been summarized from terrestrial (153; 55%) and marine (110; 41%) origins during the last twelve years, and 12 (4%) compounds are common to both environments. All secondary metabolites are categorized predominantly on the basis of their bioactivities (cytotoxic, antibacterial, antifungal, and miscellaneous activity). Overall, 134 bioactive compounds were isolated from terrestrial (92; 55%) and marine (42; 34%) origins, but about half the compounds did not report any kind of activity. The antiSMASH results suggested that Diaporthe strains are capable of encoding a wide range of SMs and have tremendous biosynthetic potential for new SMs. This study will be useful for future research on drug discovery from terrestrial and marine natural products.

Heterologous Expression, Purification and Characterization of an Alkalic Thermophilic ß-Mannanase CcMan5C from Coprinopsis cinerea.

A endo-1,4-ß-mannanase (CcMan5C) gene was cloned from Coprinopsis cinerea and heterologously expressed in Pichia pastoris, and the recombinant enzyme was purified by Ni-affinity chromatography and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). CcMan5C hydrolyzed only locust bean gum galactomannan (LBG) but not α-mannan from S. cerevisiae or Avicel cellulose, oat spelt xylan, or laminarin from Laminaria digitata. CcMan5C exhibited distinctive catalytic features that were different from previously reported ß-mannanases. (1) CcMan5C is the first reported fungal ß-mannase with an optimal alkalic pH of 8.0-9.0 for hydrolytic activity under assay conditions. (2) CcMan5C is the first reported alkalic fungal ß-mannase with an optimal temperature of 70 °C for hydrolytic activity under assay conditions. (3) The organic solvents methanol, ethanol, isopropanol, and acetone at concentrations of 10% or 20% did not inhibit CcMan5C activity, while 10% or 20% isopropanol and acetone even enhanced CcMan5C activity by 9.20-34.98%. Furthermore, CcMan5C tolerated detergents such as Tween 20 and Triton X-100, and its activity was even enhanced to 26.2-45.6% by 1% or 10% Tween 20 and Triton X-100. (4) CcMan5C solution or lyophilized CcMan5C exhibited unchanged activity and even increasing activity after being stored at -20 °C or -80 °C for 12 months and retained above 50% activity after being stored at 4 °C for 12 months. These features make CcMan5C a suitable candidate for the detergent industry and paper and pulp industry.

The extracellular ß-glucosidase BGL2 has two variants with different molecular sizes and hydrolytic activities in the stipe or pilei of Coprinopsis cinerea.

Two variants of extracellular ß-glucosidase (BGL2) were purified from the stipe and pilei of Coprinopsis cinerea. In the stipe, BGL2 was a monomeric protein with an apparent molecular mass of approximately 220 kDa, representing a mature full-length peptide of BGL2. However, in the pilei, the apparent molecular mass of BGL2 was only approximately 120 kDa, consisting of the 60 kDa N-terminal fragment and 55 kDa C-terminal fragment. The hydrolytic activities of BGL2 purified from the pilei were higher than those of BGL2 purified from the stipe. No mRNA splice variants of bgl2 were detected. Therefore, the different variants of BGL2 in the stipe and pilei were not formed by differential RNA splicing. Furthermore, in vitro experiments showed that full-length BGL2 could be cleaved by endogenous proteases from pilei or commercial trypsin at a similar site to form an oligomeric protein consisting of the N-terminal fragment and C-terminal fragment similar to BGL2 from pilei. The hydrolytic activity of BGL2 increased after cleavage by those proteases in vitro. We conclude that the 120 kDa variant of BGL2 in the pilei of C. cinerea is formed by posttranslational proteolytic cleavage. Posttranslational proteolytic cleavage is an efficient way to regulate the activity of BGL2 to adapt to the needs of different physiological functions in the elongation stipe and expansion pilei of C. cinerea.

Accumulation and cross-linkage of ß-1,3/1,6-glucan lead to loss of basal stipe cell wall extensibility in mushroom Coprinopsis cinerea.

The mature basal stipe of mushroom Coprinopsis cinerea loses wall extensibility. We found that an endo-ß-1,3-glucanase ENG from C. cinerea could restore mature basal stipe wall extensibility via pretreatment such that the ENG-pretreated basal stipe walls could be induced to extend by chitinase ChiIII. ENG pretreatment released glucose, laminaribiose, and 3-O-D-gentiobiose-D-glucose from the basal stipe walls, consistent with ENG-digested products of ß-1,6-branched ß-1,3-glucan. Different effects of endo-ß-1,3-glucanase ENG and exo-ß-1,3-glucanase EXG pretreatment on the structure, amount and ratio (ß-1,3-glucoside bonds to ß-1,6-glucoside bonds) of products from the basal stipe and the apical stipe cell walls, respectively, and on the cell wall extensibility and the cell wall ultra-architecture of the basal stipes were analyzed. All results demonstrate that the more accumulation and cross-linkage of ß-1,6-branched ß-1,3-glucan with wall maturation lead to loss of wall extensibility of the basal stipe regions compared to the apical stipe cell walls.

Molecular Mechanism by Which the GATA Transcription Factor CcNsdD2 Regulates the Developmental Fate of Coprinopsis cinerea under Dark or Light Conditions.

Coprinopsis cinerea has seven homologs of the Aspergillus nidulans transcription factor NsdD. Of these, CcNsdD1 and CcNsdD2 from C. cinerea show the best identities of 62 and 50% to A. nidulans NsdD, respectively. After 4 days of constant darkness cultivation, CcnsdD2, but not CcnsdD1, was upregulated on the first day of light/dark cultivation to induce fruiting bodies, and overexpression of CcnsdD2, but not CcnsdD1, produced more fruiting bodies under a light/dark rhythm. Although single knockdown of CcnsdD2 did not affect fruiting body production due to upregulation of its homolog CcnsdD1, the double-knockdown CcNsdD1/NsdD2-RNAi transformant showed defects in fruiting body formation under a light/dark rhythm. Knockdown of CcnsdD1/nsdD2 led to the differentiation of primary hyphal knots into sclerotia rather than secondary hyphal knots under a light/dark rhythm, similar to the differentiation of primary hyphal knots into sclerotia of the wild-type strain under darkness. The CcNsdD2-overexpressing transformant produced more primary hyphal knots, secondary hyphal knots, and fruiting bodies under a light/dark rhythm but only more primary hyphal knots and sclerotia under darkness. RNA-seq revealed that some genes reported previously to be involved in formation of hyphal knots and primordia, cyclopropane-fatty-acyl-phospholipid synthases cfs1-3, galectins cgl1-3, and hydrophobins hyd1-3 were downregulated in the CcNsdD1/NsdD2-RNAi transformant compared to the mock transformant. ChIP-seq and electrophoretic mobility shift assay demonstrated that CcNsdD2 bound to promoter regulatory sequences containing a GATC motif in cfs1, cfs2, cgl1, and hyd1. A molecular mechanism by which CcNsdD2 regulates the developmental fate of C. cinerea under dark or light conditions is proposed. IMPORTANCE The model mushroom Coprinopsis cinerea exhibits remarkable photomorphogenesis during fruiting body development. This study reports that the C. cinerea transcription factor CcNsdD2 promotes primary hyphal knot formation by upregulating cfs1, cfs2, cgl1, and hyd1. Although the induction of CcnsdD2 is not under direct control of light and photoreceptors, the CcNsdD2-mediated developmental fates of the primary hyphal knots depend on the following light/dark rhythm cultivation or dark cultivation after full growth of mycelia in the constant dark cultivation. This study provides new insight into the molecular mechanism by which CcNsdD2 regulates the developmental fate of C. cinerea under dark or light conditions. In addition, the result that overexpression of CcnsdD2 induced more secondary hyphal knots, primordia, and fruiting bodies under light/dark rhythm cultivation conditions has potential applied value in the edible mushroom industry.

A novel endo-ß-1,6-glucanase from the mushroom Coprinopsis cinerea and its application in studying of cross-linking of ß-1,6-glucan and the wall extensibility in stipe cell walls.

We previously reported that chitinases reconstituted heat-inactivated stipe cell wall extension in a steady and continuous extension profile by cleaving chitins cross-linked to various polysaccahrides, whereas, endo-ß-1,3-glucanases reconstituted heat-inactivated stipe wall extension in a profile of an initially fast extension and subsequent termination of extension due to its degradation of ß-1,3-glucan but not other polysaccharides such as ß-1,6-glucans cross-linked to chitins. Thus, a novel endo-ß-1,6-glucanase, GH30A, from Coprinopsis cinerea was cloned and characterized to study cross-linking of ß-1,6-glucan and wall extensibility in stipe walls. GH30A had higher activity and better thermophilicity than reported ß-1,6-glucanases. GH30A hydrolyzed pustulan having ß-1,6-linkages but not other polysaccharides without ß-1,6-linkages; GH30A did not cleave gentiobiose and single ß-1,6-linkage branches in laminarin from Laminaria digitata but cut consecutive ß-1,6-linkage branches in laminarin from Eisenia bicyclis. GH30A reconstituted heated-inactivated stipe cell wall extension with release of glucose and gentiobiose, indicating that ß-1,6-glucans were present and cross-linked to chitins in stipe walls, and cleaving ß-1,6-glucans cross-linked to chitins by GH30A led to wall loosening for extension. However, GH30A individually or in combination with endo-ß-1,3-glucanase reconstituted-stipe wall extension profile was similar to individual endo-ß-1,3-glucanase's, exploring that chitins were also cross-linked to other polysaccharides besides ß-1,3-glucans and ß-1,6-glucans.

Comparative study of ß-glucan-degrading enzymes from Coprinopsis cinerea for their capacities to induce stipe cell wall extension.

We previously reported endo-ß-1,3-glucanase ENG in combination with ß-glucosidase BGL2 at low concentration induced stipe cell wall extension. This study further explored ENG could be replaced by endo-ß-1,3(4)-glucanase ENG16A in combination with BGL2 to induce stipe cell wall extension; similarly, BGL2 could be replaced by ß-glucosidase BGL1 to cooperate with ENG to induce stipe cell wall extension. However, ENG could not be replaced by exo-ß-1,3-glucanase EXG in combination with BGL2 to induce stipe cell wall extension, although EXG alone released higher level of soluble sugars from the stipe cell walls during the reconstituted wall extension than that released from the stipe cell walls by a combination of ENG16A or ENG and BGL2 or BGL1, which was different from chitinase-mediated stipe cell wall extension. These results indicate endo-ß-1,3-glucanases loosen the stipe cell wall, whereas exo-ß-1,3-glucanases and ß-glucosidases play a synergistic role to maintain a low and efficient concentration of endo-ß-1,3-glucanases for stipe cell wall extension. Furthermore, ENG was expressed at a very high level in the matured pilei, in contrast, ENG16A was expressed at a very high level in the elongating apical stipe. Therefore, ENG16A might be involved in stipe elongation growth, while ENG might participate in autolysis of pilei.

Heterologous expression and characterization of a novel chitin deacetylase, CDA3, from the mushroom Coprinopsis cinerea.

The chitin deacetylase CDA3 from C. cinerea deacetylated chitin-oligosaccharides with dp ≥ 2. Since CDA3 firstly removed the intermediate acetyl group of (GlcNAc)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 CDA3 has 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, CDA3 was 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 GlcN/GlcNAc than that of the apical stipe with the lower molar ratio of GlcN/GlcNAc. 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.

ß-Glucosidase BGL1 from Coprinopsis cinerea Exhibits a Distinctive Hydrolysis and Transglycosylation Activity for Application in the Production of 3-O-ß-d-Gentiobiosyl-d-laminarioligosaccharides.

We previously reported that ß-glucosidase BGL1 at low concentration (15 µg mL-1) from Coprinopsis cinerea exhibited hydrolytic activity only toward laminarioligosaccharides but not toward cellooligosaccharides and gentiobiose. This study shows that BGL1 at high concentration (200 µg mL-1) also hydrolyzed cellobiose and gentiobiose, which accounted for only 0.83 and 2.05% of its activity toward laminaribiose, respectively. Interestingly, BGL1 at low concentration (1.5 µg mL-1) showed transglycosylation but BGL1 at high concentration (200 µg mL-1) did not. BGL1 utilizes only laminarioligosaccharides but not glucose, gentiobiose, and cellobiose to synthesize the higher oligosaccharides. BGL1 transferred one glucosyl residue from substrate laminarioligosaccharide to another laminarioligosaccharide as an acceptor in a ß(1 → 3) or ß(1 → 6) fashion to produce higher laminarioligosaccharides or 3-O-ß-d-gentiobiosyl-d-laminarioligosaccharides. The BGL1-digested laminaritriose exhibited approximately 90% enhancement in the anti-oxidant activity compared to that of untreated laminaritriose, implying a potential application of BGL1-based transglycosylation for the production of high value-added rare oligosaccharides.

Glucanase-Induced Stipe Wall Extension Shows Distinct Differences from Chitinase-Induced Stipe Wall Extension of Coprinopsis cinerea.

This study reports that a high concentration of the endo-ß-1,3-glucanase ENG (200 µg ml-1) induced heat-inactivated stipe wall extension of Coprinopsis cinerea, whereas a high concentration of the extracellular ß-glucosidase BGL2 (1,000 µg ml-1) did not; however, in combination, low concentrations of ENG (25 µg ml-1) and BGL2 (260 µg ml-1) induced heat-inactivated stipe cell wall extension. In contrast to the previously reported chitinase-reconstituted stipe wall extension, ß-1,3-glucanase-reconstituted heat-inactivated stipe cell wall extension initially exhibited a fast extension rate that quickly decreased to zero after approximately 60 min; the stipe cell wall extension induced by a high concentration of ß-1,3-glucanase did not result in stipe breakage during measurement, and the inner surfaces of glucanase-reconstituted extended cell walls still remained as amorphous matrices that did not appear to have been damaged. These distinctive features of the ß-1,3-glucanase-reconstituted wall extension may be because chitin chains are cross-linked not only to the nonreducing termini of the side chains and the backbones of ß-1,6 branched ß-1,3-glucans but also to other polysaccharides. Remarkably, a low concentration of either the ß-1,3-glucanase ENG or of chitinase ChiE1 did not induce heat-inactivated stipe wall extension, but a combination of these two enzymes, each at a low concentration, showed stipe cell wall extension activity that exhibited a steady and continuous wall extension profile. Therefore, we concluded that the stipe cell wall extension is the result of the synergistic actions of glucanases and chitinases.IMPORTANCE We previously reported that the chitinase could induce stipe wall extension and was involved in stipe elongation growth of the mushroom Coprinopsis cinerea In this study, we explored that ß-1,3-glucanase also induced stipe cell wall extension. Interestingly, the extension profile and extended ultra-architecture of ß-1,3-glucanase-reconstituted stipe wall were different from those of chitinase-reconstituted stipe wall. However, ß-1,3-glucanase cooperated with chitinase to induce stipe cell wall extension. The significance of this synergy between glucanases and chitinases is that it enables a low concentration of active enzymes to induce wall extension, and the involvement of ß-1,3-glucanases is necessary for the cell wall remodeling and the addition of new ß-glucans during stipe elongation growth.

Chitinases Play a Key Role in Stipe Cell Wall Extension in the Mushroom Coprinopsis cinerea.

The elongation growth of the mushroom stipe is a characteristic but not well-understood morphogenetic event of basidiomycetes. We found that extending native stipe cell walls of Coprinopsis cinerea were associated with the release of N-acetylglucosamine and chitinbiose and with chitinase activity. Two chitinases among all detected chitinases from C. cinerea, ChiE1 and ChiIII, reconstituted heat-inactivated stipe wall extension and released N-acetylglucosamine and chitinbiose. Interestingly, both ChiE1 and ChiIII hydrolyze insoluble crystalline chitin powder, while other C. cinerea chitinases do not, suggesting that crystalline chitin components of the stipe cell wall are the target of action for ChiE1 and ChiIII. ChiE1- or ChiIII-reconstituted heat-inactivated stipe walls showed maximal extension activity at pH 4.5, consistent with the optimal pH for native stipe wall extension in vitro; ChiE1- or ChiIII-reconstituted heat-inactivated stipe wall extension activities were associated with stipe elongation growth regions; and the combination of ChiE1 and ChiIII showed a synergism to reconstitute heat-inactivated stipe wall extension at a low action concentration. Field emission scanning electron microscopy (FESEM) images showed that the inner surface of acid-induced extended native stipe cell walls and ChiE1- or ChiIII-reconstituted extended heat-inactivated stipe cell walls exhibited a partially broken parallel microfibril architecture; however, these broken transversely arranged microfibrils were not observed in the unextended stipe cell walls that were induced by neutral pH buffer or heat inactivation. Double knockdown of ChiE1 and ChiIII resulted in the reduction of stipe elongation, mycelium growth, and heat-sensitive cell wall extension of native stipes. These results indicate a chitinase-hydrolyzing mechanism for stipe cell wall extension.IMPORTANCE A remarkable feature in the development of basidiomycete fruiting bodies is stipe elongation growth that results primarily from manifold cell elongation. Some scientists have suggested that stipe elongation is the result of enzymatic hydrolysis of cell wall polysaccharides, while other scientists have proposed the possibility that stipe elongation results from nonhydrolytic disruption of the hydrogen bonds between cell wall polysaccharides. Here, we show direct evidence for a chitinase-hydrolyzing mechanism of stipe cell wall elongation in the model mushroom Coprinopsis cinerea that is different from the expansin nonhydrolysis mechanism of plant cell wall extension. We presumed that in the growing stipe cell walls, parallel chitin microfibrils are tethered by ß-1,6-branched ß-1,3-glucans, and that the breaking of the tether by chitinases leads to separation of these microfibrils to increase their spacing for insertion of new synthesized chitin and ß-1,3-glucans under turgor pressure in vivo.

Characteristics, transcriptional patterns and possible physiological significance of glycoside hydrolase family 16 members in Coprinopsis cinerea.

The glycoside hydrolase (GH) 16 family of Coprinopsis cinerea includes 15 members distributed in four subgroups (A1, A2, B and D) by phylogenetic analysis. The expression patterns match well with the requirement of wall-softening in the germination of basidiospores, hyphal growth and branching, primordium formation, stipe elongation, pileus expansion and autolysis. Remarkably, expression levels of different GH16 members varied with different morphogenetic events. Like orthologs of Aspergillus fumigatus GH16 glucanases (ENG2-5), which were expressed in the dormant conidia and conidiogenesis, and essential for segregation of conidia, some members such as ENG in the subgroup A1 in C. cinerea were also predominantly expressed in dormant basidiospores, primordia and maturing pilei during basidiosporogenesis. In contrast, other members in subgroup A2, subgroup B or D were dominantly expressed in the germinating basidiospores, the growing mycelia, and the elongating stipes. We did not find the members of the GH81 or GH55 family in C. cinerea genome, which was different from A. fumigatus. However, C. cinerea contains an extra three subgroups (A2, B and D) compared with A. fumigatus. These extra subgroups of GH16 family members may function as those endo-ß-1,3-glucanases belonging to other GH families in the development and growth of C. cinerea.

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.

A novel thermophilic exochitinase ChiEn3 from Coprinopsis cinerea exhibits a hyperhydrolytic activity toward 85% deacetylated chitosan and a significant application to preparation of chitooligosaccharides from the chitosan.

ChiEn3 from Coprinopsis cinerea was characterized as an exo-acting chitinase with a processivity. ChiEn3 hydrolyzed only soluble chitin and exhibited a hyperhydrolytic activity toward 85% deacetylated chitosan which was 33.6-fold higher than its hydrolytic activity toward glycol chitin. Its maximum hydrolytic activity was observed at 60 °C and retained 66.2% of hydrolytic activity after 60 min incubation at 60 °C. Commercial 85% deacetylated chitosan was degraded by ChiEn3 to a series of COSs with a DP of 2-20 in which COSs with a DP of 3-6 were dominant, whereas, lab-prepared chitosan (FA = 0.65) was degraded by ChiEn3 to COSs with a DP of 2-10 in which the AA dimer was dominant. DPPH-radical-scavenging activity of ChiEn3-digested products of 85% deacetylated chitosan was 3.32-fold higher than that of undigested 85% deacetylated chitosan. Therefore, ChiEn3 shows a valuable advantage for application to the preparation of COSs from commercial 85% deacetylated chitosan.

ChiE1 from Coprinopsis cinerea is Characterized as a Processive Exochitinase and Revealed to Have a Significant Synergistic Action with Endochitinase ChiIII on Chitin Degradation.

Fruiting bodies that exhibit strong autolysis of Coprinopsis cinerea are a good resource for the chitinolytic system. In this study, a new Chitinase ChiE1 from C. cinerea was cloned, heterologously expressed, and characterized. Biochemical analysis demonstrated that ChiE1 is an exochitinase with a processive mode of action. Although ChiE1 contains only a single catalytic domain without a binding domain, it can bind to and degrade insoluble chitin powder and colloidal chitin. The combination of ChiE1 and C. cinerea endochitinase ChiIII could increase the amount of reducing sugar released from chitin powder by approximately 120% compared to using ChiE1 and ChiIII alone. The synergistic action of ChiE1 and ChiIII on degradation of chitin powder is higher than all previously reported synergism of chitinases. The recombinant Chitinase ChiE1 expressed in Pichia pastoris may be used as a synergistic chitinase for a reconstituted chitinolytic system for agricultural, biological, and environmental applications.

Gene cloning, heterologous expression and characterization of a Coprinopsis cinerea endo-ß-1,3(4)-glucanase.

A gene coding endo-ß-1,3(4)-glucanase (ENG16A) was cloned from Coprinopsis cinerea and heterologously expressed in Pichia pastoris. ENG16A only acts on substrates containing ß-1,3 glycosidic bonds but not on substrates containing only ß-1,4- or ß-1,6-glycosidic bonds. Interestingly, compared to the activity of this enzyme towards carboxymethyl (CM)-pachyman containing only ß-1,3-glycosidic bonds, its activity towards barley ß-glucan containing both ß-1,3-glycosidic and ß-1,4-glycosidic bonds was increased by 64.72 %,, its activity towards laminarin containing both ß-1,3-glycosidic and ß-1,6-glycosidic bonds was decreased by 50.83 %. In addition, ENG16A has a higher Km value and Vmax for barley ß-glucan than laminarin, which may be related to the fact that barley ß-glucan contains mainly ß-1,4-glycosidic bonds mixed with a few ß-1,3-glycosidic bonds, whereas laminarin mainly contains ß-1,3-glycosidic bonds with a few ß-1,6-branched glucose residues. The adjacent ß-1,4-glycosidic bond promotes ENG16A to hydrolyse ß-1,3-glycosidic bonds, leading to an increased Vmax; the nearby ß-1,6-glycosidic bonds inhibited its hydrolysis of ß-1,3-glycosidic bonds, resulting in a decreased Vmax. This property is suggested to be related to the mechanism that C. cinerea uses to degrade and utilize hemicellulose in straw medium and to protect its cell wall during the mycelium growth stage.

Purification, characterization and physiological significance of a chitinase from the pilei of Coprinopsis cinerea fruiting bodies.

We purified a chitinase from pilei extractions of Coprinopsis cinerea fruiting bodies by ammonium sulfate precipitation and CM Sepharose cation exchange chromatography. MALDI-TOF/TOF MS analysis characterized this purified chitinase as a putative class V chitinase, ChiB1. ChiB1 hydrolyzed colloidal chitin and chitosan, whereas it did not hydrolyze chitin powder. ChiB1 cleaved only pNP-(GlcNAc)2, rather than pNP-GlcNAc or pNP-(Glc-NAc)3, to release nitrophenol. ChiB1 preferably and progressively released (GlcNAc)2 from (GlcNAc)6 and digested (GlcNAc)6 to two molecules of (GlcNAc)3 in a small proportion, but did not split (GlcNAc)2, so it is an exochitinase. ChiB1 has an optimum temperature range of 35°C to 40°C and an optimum pH of 5.0. ChiB1 exhibited Km and Vmax values of 2.63 mg ml(-1) and 2.31 µmol min(-1) mg protein(-1) for colloidal chitin, respectively. The ChiB1 gene, along with another putative endochitinase (class III chitinase gene), was expressed dominantly among eight predicted chitinase genes in the genome, and its expression level increased with the maturation of fruiting bodies. ChiB1 incubation released a large amount of soluble ß-glucan fractions from alkali-insoluble cell wall fractions of C. cinerea fruiting bodies, thereby it may promote the degradation of cell walls in synergy with the ß-1,3-glucanases during pileus autolysis.

Purification, characterization and function analysis of an extracellular ß-glucosidase from elongating stipe cell walls in Coprinopsis cinerea.

A ß-glycoside hydrolase was isolated from cell walls material in Coprinopsis cinerea elongating stipes. By analysis of SDS-PAGE, MALDI-TOF/TOF MS and substrate specificity, this enzyme was characterized as an extracellular ß-glucosidase which is a trimer consisting of three homosubunits. ß-Glucosidase did not degrade ß-glucans with modified ends, whereas it hydrolyzed various ß-glucans with free ends and related oligosaccharides with ß-1,3-, ß-1,4- or ß-1,6-linkages. Although this ß-glucosidase possesses glycosyltransferase activity on laminarioligosaccharides, it did not transfer glucose residues from laminaritriose to ß-glucan in stipe cell walls to produce larger ß-glucan molecules; instead, it caused a decrease in the molecular size of stipe wall ß-glucan by removing glucose. Relatively, the molecular size of wall ß-glucans in the elongating apical stipe was less than that found in the non-elongating basal stipes, and this ß-glucosidase was more highly expressed in the elongating apical stipe than in non-elongating basal regions. Therefore, we propose that ß-glucosidase functions by trimming or cutting the ß-glucan side chains on the ß-1,3-glucan backbone to prevent them from forming longer branches, keeping the wall plastic to promote diffuse wall growth.

Silica-supported sulfonic acid-functionalized ionic liquid coated with [bmim][PF6] as a scavenger for the synthesis of amides.

The methodology of using a silica gel-supported functionalized ionic liquid as a scavenger in the purification of parallel synthesis products was demonstrated. Silica-supported sulfonic acid-functional ionic liquid was synthesized by etherification, aminate, and quaternary aminate from activated silica gel and 3-chloropropyl trimethoxysilane, imidazole, and 1,4-butanesultone, which was followed by acidification using trifluoromethanesulfonic acid and anion exchange with potassium hexafluorophosphate. A conventional ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate was then used to coat the surface of the silica gel. The silica-supported functionalized ionic liquid was used as a scavenger in the removal of excess amine in the parallel synthesis of amides. Desired products were obtained in excellent yields and purity with a sequestration time of less than 100 min at room temperature. After scavenging, the scavenger was easily filtered out and regenerated.