Search

BACKGROUND: Mushrooms have been widely considered as health foods as their extracts have anti‐hypertensive and anti‐tumor activities. After a thorough literature survey, we hypothesized that enzymes in mushroom extracts play an important role in synthesizing functional molecules. Therefore, in this study, proteins extracted from reishi mushroom (Ganoderma lucidum), which is used in oriental medicine, were identified by the proteomic approach, and appropriate extraction methods for improving angiotensin‐converting enzyme (ACE) inhibitory activities were investigated. RESULTS: Various glycoside hydrolases (GHs), such as β‐N‐acetylhexosaminidase (GH family 20), α‐1,2‐mannosidase (GH family 47), endo‐β‐1,3‐glucanase (GH family 128), and β‐1,3‐glucanase (GH152), that degrade glycans in the fruiting body were identified. The residual glucanase activities generated β‐oligosaccharides. Additionally, the glutamic acid protease of the peptidase G1 family was determined as the major protein in the extract, and the residual peptidase activity of the extracts was found to improve ACE inhibitory activities. Finally, it was observed that extraction at 50 °C is suitable for yielding functional molecules with high ACE inhibitory activities. CONCLUSION: Water extraction is generally believed to extract only functional macromolecules that exist in mushroom fruiting bodies. This study proposed a new concept that describes how functional molecules are produced by enzymes, including proteases and GHs, during extraction. © 2018 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Cellobiohydrolases belonging to glycoside hydrolase family 6 (CBH II, Cel6A) play key roles in the hydrolysis of crystalline cellulose. CBH II from the white-rot fungus Phanerochaete chrysosporium ( PcCel6A) consists of a catalytic domain (CD) and a carbohydrate-binding module connected by a linker peptide, like other known fungal cellobiohydrolases. In the present study, the CD of PcCel6A was crystallized without ligands, and p-nitrophenyl β- d-cellotrioside ( pNPG3) was soaked into the crystals. The determined structures of the ligand-free and pNPG3-soaked crystals revealed that binding of cellobiose at substrate subsites +1 and +2 induces a conformational change of the N-terminal and C-terminal loops, switching the tunnel-shaped active site from the open to the closed form. [ABSTRACT FROM AUTHOR]

ABSTRACT Feruloyl esterase (FAE) catalyzes the hydrolysis of the ferulic and diferulic acids present in plant cell wall polysaccharides, and tannase catalyzes the hydrolysis of tannins to release gallic acid. The fungal tannase family in the ESTHER database contains various enzymes, including FAEs and tannases. Despite the importance of FAEs and tannases in bioindustrial applications, three-dimensional structures of the fungal tannase family members have been unknown. Here, we determined the crystal structure of FAE B from Aspergillus oryzae ( AoFaeB), which belongs to the fungal tannase family, at 1.5 Å resolution. AoFaeB consists of a catalytic α/β-hydrolase fold domain and a large lid domain, and the latter has a novel fold. To estimate probable binding models of substrates in AoFaeB, an automated docking analysis was performed. In the active site pocket of AoFaeB, residues responsible for the substrate specificity of the FAE activity were identified. The catalytic triad of AoFaeB comprises Ser203, Asp417, and His457, and the serine and histidine residues are directly connected by a disulfide bond of the neighboring cysteine residues, Cys202 and Cys458. This structural feature, the 'CS-D-HC motif,' is unprecedented in serine hydrolases. A mutational analysis indicated that the novel structural motif plays essential roles in the function of the active site. Proteins 2014; 82:2857-2867. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The basidiomycete Coprinopsis cinerea produces the glycoside hydrolase family 6 enzyme CcCel6C at low and constitutive levels. CcCel6C exhibits unusual cellobiohydrolase activity; it hydrolyses carboxymethyl cellulose, which is a poor substrate for typical cellobiohydrolases. Here, we determined the crystal structures of CcCel6C unbound and in complex with p-nitrophenyl β-d-cellotrioside and cellobiose. CcCel6C consists of a distorted seven-stranded β/α barrel and has an enclosed tunnel, which is observed in other cellobiohydrolases from ascomecetes Hypocrea jecorina (HjeCel6A) and Humicola insolens (HinCel6A). In HjeCel6A and HinCel6A, ligand binding produces a conformational change that narrows this tunnel. In contrast, the tunnel remains wide in CcCel6C and the conformational change appears to be less favourable than in HjeCel6A and HinCel6A. The ligand binding cleft for subsite −3 of CcCel6C is also wide and is rather similar to that of endoglucanase. These results suggest that the open tunnel and the wide cleft are suitable for the hydrolysis of carboxymethyl cellulose. [ABSTRACT FROM AUTHOR]

The 1,3(4)- β-d-glucanases of glycoside hydrolase family 16 provide useful examples of versatile yet specific protein–carbohydrate interactions. In the present study, we report the X-ray structures of the 1,3(4)- β-d-glucanase Phanerochaete chrysosporium Laminarinase 16A in complex with β-glucan products from laminarin (1.6 Å) and lichenin (1.1 Å) hydrolysis. The G6G3G3G glucan, in complex with the enzyme, showed a β-1,6 branch in the acceptor site. The G4G3G ligand–protein complex showed that there was no room for a β-1,6 branch in the −1 or −2 subsites; furthermore, the distorted residue in the −1 subsite and the glucose in the −2 subsite required a β-1,3 bond between them. These are the first X-ray crystal structures of any 1,3(4)- β-d-glucanase in complex with glucan products. They provide details of both substrate and product binding in support of earlier enzymatic evidence. [ABSTRACT FROM AUTHOR]

Abstract: The crystal structure of endo-β-(1→4)-glucuronan lyase from Trichoderma reesei (TrGL) has been determined at 1.8Å resolution as the first three-dimensional structure of polysaccharide lyase (PL) family 20. TrGL has a typical β-jelly roll fold, which is similar to glycoside hydrolase family 16 and PL7 enzymes. A calcium ion is bound to the site far from the cleft and appears to contribute to the stability. There are several completely conserved residues in the cleft. Possible catalytic residues are predicted based on structural comparison with PL7 alginate lyase A1–II′. [Copyright &y& Elsevier]

The basidiomycete Phanerochaete chrysosporium produces xyloglucanase Xgh74B, which has the glycoside hydrolase (GH) family 74 catalytic domain and family 1 carbohydrate-binding module, in cellulose-grown culture. The recombinant enzyme, which was heterologously expressed in the yeast Pichia pastoris, had high hydrolytic activity toward xyloglucan from tamarind seed (TXG), whereas other β-1,4-glucans examined were poor substrates for the enzyme. The existence of the carbohydrate-binding module significantly affects adsorption of the enzyme on crystalline cellulose, but has no effect on the hydrolysis of xyloglucan, indicating that the domain may contribute to the localization of the enzyme. HPLC and MALDI-TOF MS analyses of the hydrolytic products of TXG clearly indicated that Xgh74B hydrolyzes the glycosidic bonds of unbranched glucose residues, like other GH family 74 xyloglucanases. However, viscometric analysis suggested that Xgh74B hydrolyzes TXG in a different manner from other known GH family 74 xyloglucanases. Gel permeation chromatography showed that Xgh74B initially produced oligosaccharides of degree of polymerization (DP) 16–18, and these oligosaccharides were then slowly hydrolyzed to final products of DP 7–9. In addition, the ratio of oligosaccharides of DP 7–9 versus those of DP 16–18 was dependent upon the pH of the reaction mixture, indicating that the affinity of Xgh74B for the oligosaccharides of DP 16–18 is affected by the ionic environment at the active site. [ABSTRACT FROM AUTHOR]

The crystalline polymorphic form of cellulose (cellulose Iα-rich) of the green alga, Cladophora, was converted into cellulose IIII and Iβ by supercritical ammonium and hydrothermal treatments, respectively, and the hydrolytic rate and the adsorption of Trichoderma viride cellobiohydrolase I (Cel7A) on these products were evaluated by a novel analysis based on the surface density of the enzyme. Cellobiose production from cellulose IIII was more than 5 times higher than that from cellulose I. However, the amount of enzyme adsorbed on cellulose IIII was less than twice that on cellulose I, and the specific activity of the adsorbed enzyme for cellulose IIII was more than 3 times higher than that for cellulose I. When cellulose IIII was converted into cellulose Iβ by hydrothermal treatment, cellobiose production was dramatically decreased, although no significant change was observed in enzyme adsorption. This clearly indicates that the enhanced hydrolysis of cellulose IIII is related to the structure of the crystalline polymorph. Thus, supercritical ammonium treatment activates crystalline cellulose for hydrolysis by cellobiohydrolase. [ABSTRACT FROM AUTHOR]

Abstract: The white-rot fungus Phanerochaete chrysosporium has two intracellular β-glucosidases (BGL1A and BGL1B) belonging to glycoside hydrolase (GH) family 1. BGL1B effectively hydrolyzes cellobiose and cellobionolactone, but BGL1A does not. We have determined the crystal structure of BGL1A in substrate-free and gluconolactone complexed forms. The overall structure and the characteristic of subsite −1 (glycone site) were similar to those of other known GH1 enzymes. The loop regions covering on the (β/α)8 barrel was significantly deviated, and they form a unique subsite +1 (aglycone site) of BGL1A. [Copyright &y& Elsevier]

Laminarinase Lam16A from Phanerochaete chrysosporium was recombinantly expressed in Pichia pastoris, crystallized and the structure was solved at 1.34 Å resolution using native sulfur SAD X-ray crystallography. It is the first structure of a non-specific 1,3(4)-β-d-glucanase from glycoside hydrolase family 16 (GH16). P. chrysosporium is a wood-degrading basidiomycete fungus and Lam16A is the predominant extracellular protein expressed when laminarin is used as the sole carbon source. The protein folds into a curved β-­sandwich homologous to those of other known GH16 enzyme structures (especially κ-carrageenase from Pseudoalteromonas carrageenovora and β-agarase from Zobelia galactanivorans). A notable likeness is also evident with the related glycoside hydrolase family 7 (GH7) enzymes. A mammalian lectin, p58/ERGIC, as well as polysaccharide lyase (PL7) enzymes also showed significant similarity to Lam16A. The enzyme has two potential N-glycosylation sites. One such site, at Asn43, displayed a branched heptasaccharide sufficiently stabilized to be interpreted from the X-ray diffraction data. The other N-glycosylation motif was found close to the catalytic centre and is evidently not glycosylated. [ABSTRACT FROM AUTHOR]

The enzymatic kinetics of glycoside hydrolase family 7 cellobiohydrolase (Cel7A) towards highly crystalline celluloses at the solid–liquid interface was evaluated by applying the novel concept of surface density (ρ) of the enzyme, which is defined as the amount of adsorbed enzyme divided by the maximum amount of adsorbed enzyme. When the adsorption levels of Trichoderma viride Cel7A on cellulose Iα from Cladophora and cellulose Iβ from Halocynthia were compared, the maximum adsorption of the enzyme on cellulose Iβ was ∼1.5 times higher than that on cellulose Iα, although the rate of cellobiose production from cellulose Iβ was lower than that from cellulose Iα. This indicates that the specific activity ( k) of Cel7A adsorbed on cellulose Iα is higher than that of Cel7A adsorbed on cellulose Iβ. When k was plotted versus ρ, a dramatic decrease of the specific activity was observed with the increase of surface density (ρ-value), suggesting that overcrowding of enzyme molecules on a cellulose surface lowers their activity. An apparent difference of the specific activity was observed between crystalline polymorphs, i.e. the specific activity for cellulose Iα was almost twice that for cellulose Iβ. When cellulose Iα was converted to cellulose Iβ by hydrothermal treatment, the specific activity of Cel7A decreased and became similar to that of native cellulose Iβ at the same ρ-value. These results indicate that the hydrolytic activity (rate) of bound Cel7A depends on the nature of the crystalline cellulose polymorph, and an analysis that takes surface density into account is an effective means to evaluate cellulase kinetics at a solid–liquid interface. [ABSTRACT FROM AUTHOR]

Cellobiose dehydrogenase (CDH) is an extracellular flavocytochrome containing flavin andb-type heme, and plays a key role in cellulose degradation by filamentous fungi. To investigate intermolecular electron transfer from CDH to cytochrome c, Phe166, which is located in the cytochrome domain and approaches one of propionates of heme, was mutated to Tyr, and the thermodynamic and kinetic properties of the mutant (F166Y) were compared with those of the wild-type (WT) enzyme. The mid-point potential of heme in F166Y was measured by cyclic voltammetry, and was estimated to be 25 mV lower than that of WT at pH 4.0. Although presteady-state reduction of flavin was not affected by the mutation, the rate of subsequent electron transfer from flavin to heme was halved in F166Y. When WT or F166Y was reduced with cellobiose and then mixed with cytochrome c, heme re-oxidation and cytochrome creduction occurred synchronously, suggesting that the initial electron is transferred from reduced heme to cytochrome c. Moreover, in both enzymes the observed rate of the initial phase of cytochrome creduction was concentration dependent, whereas the second phase of cytochrome creduction was dependent on the rate of electron transfer from flavin to heme, but not on the cytochrome cconcentration. In addition, the electron transfer rate from flavin to heme was identical to the steady-state reduction rate of cytochrome cin both WT and F166Y. These results clearly indicate that the first and second electrons of two-electron-reduced CDH are both transferred via heme, and that the redox reaction of CDH involves an electron-transfer chain mechanism in cytochrome creduction. [ABSTRACT FROM AUTHOR]

Reports on the evidence that endo-1,4-beta-glucanases act on cellulose in suspension-cultured poplar cells. Release of an endo-1,4,-beta-glucanase in the extracellular culture medium and the other localized in their walls; Levels of cell DNA clones; Accumulation of the mRNA correlated with the solubilization of cello-oligosaccharides.

Several catalytic properties of the FAD enzyme cellobiose: quinone oxidoreductase (CBQ) and the heine/FAD enzyme, cellobiose oxidase (CBO) have been investigated and compared. Dichlorophenolindophenol was found to be a very good electron acceptor for cellobiose oxidation by both enzymes. The optimal pH value for this oxidation with dichiorophenol-indophenol as a co-substrate was observed around pH 4 for both enzymes. The turnover numbers of this reaction were also very similar. The Km values for cellobiose oxidation were identical, whereas the Km for CBO with dichlorophenolindophenol is lower than that of CBQ.
Atmospheric oxygen is a very poor electron acceptor for both CBO and CBQ, however, CBO can utilize cytochrome c as an effective electron acceptor, while CBQ cannot. The specific activity of CBO for cytochrome c is thus about 200-times higher than for oxygen. Thus, one way to distinguish the two enzymes is by the cytochrome-c-reducing ability of CBO. Therefore, we propose that the nomenclature for CBO is tentatively changed to cellobiose:cytochrome c oxidoreductase until a rational name can be installed. Both enzymes have radical-reducing activities. The cation radical, derived from 1,2,4,5 tetramethoxybenzene, was reduced by both enzymes at almost the same reaction rate. The phenoxyradical produced by lignin peroxidase, catalyzing the oxidation of acetosyringon, was also reduced by both enzymes. The reduction of phenoxyradicals formed by phenoloxidases (lignin peroxidases, as well as laccases) may be important in preventing repolymerization reactions which we suggest would significantly facilitate lignin degradation. [ABSTRACT FROM AUTHOR]

The interaction of cellobiose dehydrogenase (CDH) with cellobiohydrolase I (CBH I) in cellulose-grown cultures of Phanerochaete chrysosporium was investigated to clarify the role of CDH in cellulose degradation. Decomposition of bacterial microcrystalline cellulose by CBH I was enhanced significantly in the presence of the CDH/ferricyanide redox-system compared with CBH I alone. To explain this phenomenon, a model system, using p-nitrophenyl-β-D-cellobioside as a substrate, was elaborated for measurement of CBH I activity with and without the CDH redox-system. The activity of CBH I for hydrolysis of p-nitrophenyl-β-D-cellobioside was also enhanced in the presence of the redox system. It was found that Km for hydrolysis of p-nitrophenyl-β-D-cellobioside by CBH I was lower in the presence than in the absence of the CDH/ferricyanide redox-system, 142 μM and 384 μM, respectively, while no significant difference was observed between the kcat values. These results indicate that cellulase activity is enhanced by an increased affinity for p-nitrophenyl-β-D-cellobioside, rather than by an increased hydrolysis rate. This shows that cellobiose, the hydrolysis product, acts as a competitive inhibitor of the interaction between CBH I and p-nitrophenyl-β-D-cellobioside. This was confirmed by addition of cellobiose, which was found to competitively inhibit hydrolysis of p-nitrophenyl-β-D-cellobioside by CBH I in the absence of the CDH redox system, and the Ki value for cellobiose inhibition was estimated to be 65 μM. However, this inhibition did not occur if cellobiose was incubated with CDH before addition of CBH I. It was concluded from these results that the reason for the enhancement of CBH I activity in the presence of the CDH redox system was that it relieves competitive inhibition of cellobiose by its oxidation to cellobionolactone. [ABSTRACT FROM AUTHOR]

The enzyme cellobiose dehydrogenase (CDH), produced by many wood‐degrading fungi has, in recent years, attracted considerable interest for its possible role in both cellulose and lignin degradation. To characterize the enzyme better and to identify its role in the degradation of wood and wood components, it is desirable to produce it in higher amounts. We report here that the addition of bovine calf serum to cellulose‐grown cultures of Phanerochaete chrysosporium enhances the production of certain enzymes, CDH in particular. The highest CDH production was obtained with 45 ml of serum/litre of medium added on day 3 or 4. The resultant CDH yield was approx. 700Ő800 units/litre, which was 3.5Ő4 times higher than that in cultures without serum. Serum addition also enhanced the production of b‐glucosidase. However, the impact on CDH production was the most dramatic. The enhanced enzyme production cannot be explained by increased rates of spore germination, simple nutrient effects or cofactor effects. Fractionation of serum by Cohn's fractionation technique showed that the albumin (BSA) fraction had almost the same effect as whole serum. However, purified BSA had less effect than crude BSA (fraction V of Cohn's fractions), suggesting that an additional factor, probably a protease inhibitor in serum, also contributed to the effect of serum. [ABSTRACT FROM AUTHOR]

Summary To study the role of cellulose and cellulase in plant growth, we expressed poplar cellulase (PaPopCel1 ) constitutively in Arabidopsis thaliana . Expression increased the size of the rosettes due to increased cell size. The change in growth was accompanied by changes in biomechanical properties due to cell wall structure indicative of decrease in xyloglucan cross-linked with cellulose microfibrils by chemical analysis and nuclear magnetic resonance (NMR) spectra. The result supports the concept that the paracrystalline sites of cellulose microfibrils are attacked by poplar cellulase to loosen xyloglucan intercalation and this irreversible wall modification promotes the enlargement of plant cells. [ABSTRACT FROM AUTHOR]

Exo-beta-1,3-galactanase from Phanerochaete chrysosporium (Pc1,3Gal43A) consists of a glycoside hydrolase family 43 catalytic domain and a substrate-binding domain that belongs to carbohydrate-binding module family 35. It catalyzes the hydrolysis of beta-1,3-galactan, which is the backbone of the arabinogalactan proteins; the C-terminal carbohydrate-binding module family 35 domain increases the local concentration of the enzyme around beta-1,3-galactan by its high affinity for the substrate. To enable phase determination using the multiwavelength anomalous dispersion method, selenomethionyl Pc1,3Gal43A was crystallized at 298 K using the hanging-drop vapour-diffusion method. The presence of selenium in the crystals was confirmed from the X-ray absorption spectrum. The crystals belonged to space group P2(1) and diffracted to 1.8 A resolution.