Mushroom Termitomyces Clypeatus Research Articles

Cloning, characterization and expression of a gene encoding endo-1, 4- β-xylanase from the fungus Termitomyces clypeatus

Enzymatic degradation of hemi-cellulosic substrates has gained plenty of industrial attentions recently. Complete enzymatic degradation of complex and recalcitrant hemicellulose requires an enzymatic cocktail consisting primarily of endo-1,4-β-xylanase (xyl), β-xylosidase, arabinofuranosidase etc. This article reports, for the first time, the identification, cloning, expression and partial characterization of a potent endo-1,4- β-xylanase gene (pxyl) from the mushroom Termitomyces clypeatus (TC) in E. coli and S. cerevisiae. The cDNA for pxyl was found to be 678 bp that in turn gives rise to a precursor protein (Pxyl) of 225 amino acids long when cloned in prokaryotic expression vector. To characterize additionally, the cDNA was also expressed in S. cerevisiae. Bioinformatics study predicted that the Pxyl contains a 19 amino acid long leader peptide that enables post translational modifications including glycosylation as well as its efficient secretion in the medium. The recombinant protein has been found to be a member of GH11 family containing two distant glutamic acids as catalytic residues. This report describes yet another new and potent source of xylanase for commercial exploitation by industry in future.

AkP from mushroom Termitomyces clypeatus is a proteoglycan specific protease with apoptotic effect on HepG2

Termitomyces clypeatus is an edible mushroom, prized for its therapeutic values and as producer of industrially important enzymes. However, the biomedical efficacies of anticancer proteases have not been reported yet. The present study aimed to purify and characterize a serine protease (AkP) from T. clypeatus for investigating cytotoxic potency on HepG2, Hep3B, and compared the effect on normal hepatic L-02 cells. Purification and biochemical characterization of AkP were evaluated by three stage chromatography, 1D/2D-SDS-PAGE, 1D zymography, far-UV CD spectral analysis, N-terminal sequencing, MALDI⿿TOF/MS⿿MS analysis and enzyme kinetics studies. AkP could cleave the growth promoting cell surface proteoglycans of HepG2, corroborated by RP-HPLC analysis. AkP (IC50: 75±1.18nM) mediated anti-proliferative activity solely on HepG2 cells through the induction of apoptosis. Augmentation of apoptosis was attributed to up-regulation of p53 and Bax protein expression succeeded by caspase-3 activation. Serine protease inhibitor phenyl methane sulfonyl fluoride (PMSF) inhibited both its proteolytic activity and cytotoxicity on HepG2. These findings demonstrate that AkP could be an effective biomolecule for killing of cancer cells by p53 restoration and surface proteoglycans cleavage.

Heteroglycan of an edible mushroom Termitomyces clypeatus: structure elucidation and antioxidant properties

A water-soluble heteroglycan (PS) of an average molecular weight ∼1.98 ×105 Da was isolated from the aqueous extract of an edible mushroom Termitomyces clypeatus (R. Heim). The structure of the polysaccharide (PS) was established using total hydrolysis, methylation analysis, Smith degradation, and 1D/2D NMR experiments. Total hydrolysis indicated the presence of d-glucose, d-galactose, d-mannose, and l-fucose in a molar ratio of 4.10:1.95:1.0:0.95, respectively. The chemical and NMR analysis indicated the presence of a repeating unit with a backbone consisting of one each of the residues (1→3)-α-d-galactopyranosyl, (1→3)-α-d-mannopyranosyl, (1→3)-α-d-glucopyranosyl, (1→3)-β-d-glucopyranosyl, (1→6)-β-d-glucopyranosyl, and (1→6)-α-d-galactopyranosyl, respectively. The (1→3)-α-d-mannopyranosyl residue was found branched at O-2 with terminal α-l-fucopyranosyl moiety and (1→3)-β-d-glucopyranosyl residue was branched at O-6 with terminal α-d-glucopyranosyl residue. The PS exhibited antioxidant properties.

Purification and characterisation of κ-casein specific milk-clotting metalloprotease from Termitomyces clypeatus MTCC 5091

Milk-clotting enzymes are valued as chymosin-like protease substitutes for cheese making industries. An extracellular metalloprotease (AcPs) with high milk-clotting activity was purified from edible mushroom Termitomyces clypeatus and characterised. AcPs was preferentially active towards κ-casein, analysed by Urea-PAGE and LC–ESI-MS, whereas the degradation of α and β-casein components by AcPs proceeded slowly justifying its suitability for cheese making. RP-HPLC peptide profiling revealed that the AcPs activity on milk casein was similar to that of a commercial milk coagulant. The enzyme exhibited pH and temperature optima at 5.0 and 45°C, respectively and showed a pI value of 4.6. One- and two dimensional zymographies revealed a single polypeptide band with proteolytic signal. The MALDI-TOF/MS followed by peptide mass fingerprinting revealed homology with a predicted protein of Populus trichocarpa. To our knowledge, this is the first report on a metalloprotease from T. clypeatus, and the results indicate that this enzyme can be considered as a potential substitute for chymosin in cheese manufacturing.

Bioremediation by alkaline protease (AkP) from edible mushroom Termitomyces clypeatus: optimization approach based on statistical design and characterization for diverse applications

AbstractBACKGROUNDBioremediation using enzymes has become an attractive approach for removing hazardous chemicals from the environment. The present study investigated the production, optimization and applications of an extracellular alkaline protease (AkP) from the edible mushroom Termitomyces clypeatus focusing on bioremediation.RESULTSFor optimized enzyme production, the variables significantly influencing the protease activity were screened through the 11 factorial Plackett–Burman design. The optimum values of the selected variables and their mutual interactions were determined through response surface methodology using the Box–Behnken experimental design. Overall a 10‐fold increase in enzyme activity (1830 U mL‐1) was obtained which closely matched the yield predicted by the statistical model (1778.63 U mL‐1) with R2 = 0.9819. AkP could efficiently dehair the goat skin, detached bird feather vanes from its shaft and reduce BOD, COD and pH of tannery waste effluent. The AkP also demonstrated bactericidal property against Staphylococcus aureus as detected by MIC and flow cytometry analyses.CONCLUSIONAs an ecofriendly alternative, the enzyme showed significant promise for bioremediation and industrial applications through time‐saving bioprocesses. This is first report of alkaline protease from T. clypeatus or from a fungal source with wide‐ranging application potential. © 2014 Society of Chemical Industry

Purification and Characterization of a Low Molecular Weight Endo-xylanase from Mushroom Termitomyces clypeatus

A low molecular weight endo-xylanase (EC 3.2.1.8) was purified from an edible mushroom Termitomyces clypeatus grown in submerged medium with oat spelt xylan. Xylanase was purified to apparent homogeneity by ammonium sulfate fractionation and gel filtration chromatography. Its molecular weight was determined by gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 12 kDa. The enzyme was found to be most active at 50 degrees C and pH 5.0, being most stable at pH 6.5. The K(m) for oat spelt xylan was determined to be 10.4 mg/ml. The specificities of the enzyme was observed to be highly specific towards oat spelt xylan and was inhibited by mercuric chloride (HgCl(2)), N-bromosuccinimide, and trans-1,2-diaminocyclohexane-N',N',N',N'-tetraacetic acid strongly. The inhibitory action of N-bromosuccinimide on enzyme confirmed the presence of one tryptophan residue in its substrate-binding site. Amino acid analysis for xylanase showed the presence of high amount of hydrophobic serine, glycine, threonine, and alanine residues. The N-terminal sequencing study for the previously purified and characterized 56 kDa xylanolytic amyloglucosidase reveal the presence of 33.30% identity with glucoamylase chain A from Aspergillus awamori. The N-terminal sequence analysis of the present 12 kDa enzyme showed highest similarity (72.22% identity) towards xylanase from Neurospora crassa.

Fungal biotechnology in food and feed processing

Fungi are of excellent value nutritionally, and of great importance to vegetarians. Edible mushrooms are excellent sources of protein, have low-fat content and are free of cholesterol. They are easily cultivable and are consumed either in fresh or processed form. Yeasts and filamentous fungi secrete a plethora of important enzymes in the growth medium together with other secondary metabolites. Most of these are hydrolytic in nature being employed in different food processing industries as well as in refinement of fodder quality. Edible filamentous fungi producing these enzymes present an added advantage for their use in food and feed. In this article these aspects will be discussed along with the results from edible mushroom Termitomyces clypeatus, producing a wide variety of hydrolytic enzymes and products, from our laboratory. It is likely that the functional understanding of different enzyme classes will provide new applications within the food industry in the future. 2009 Published by Elsevier Ltd.

Cellobiose dehydrogenase production by the mycelial culture of the mushroom Termitomyces clypeatus

Termitomyces clypeatus produces cellobiose dehydrogenase (CDHtc) in cellulose medium with the highest yield (55.88 U mL −1) among all the reported fungal species. The enzyme has been isolated and purified from the culture filtrate. CDHtc was found to be a very thermolabile enzyme with the temperature optimum at 30 °C, while it exhibited a wide range of pH stability from pH 2.0 to 8.0. Lactose was efficiently converted to lactobionic acid in presence of the enzyme. Addition of glucose in the cellulose medium on the first day of growth induced a lag period in enzyme production but ultimately facilitated earlier CDHtc production and the yield was also comparable to that achieved in the cellulose medium.

Regulation of protein secretion by mycelial culture of the mushroom Termitomyces clypeatus

Termitomyces clypeatus secreted a 24-kDa xylanase constitutively in xylan medium, but required a gluconeogenic amino acid or Krebs cycle acid for the secretion of a 56-kDa amyloglucosidase in dextrin medium. Aspartate, glutamate, succinate and fumarate all increased secretion of amyloglucosidase from 50% to >90% and enzyme production by 10-fold with little effect on xylanase production. Glutamate or succinate stimulated in vitro release of intracellular amyloglucosidase from washed mycelia in the presence of cycloheximide. Amyloglucosidase accumulated in the absence of glutamate was a high-molecular-mass protein that did not migrate in PAGE. Cellular regulation by the fungus of the secretion of amyloglucosidase is indicated.

Regulation of protein secretion by mycelial culture of the mushroom Termitomyces clypeatus

Termitomyces clypeatus secreted a 24-kDa xylanase constitutively in xylan medium, but required a gluconeogenic amino acid or Krebs cycle acid for the secretion of a 56-kDa amyloglucosidase in dextrin medium. Aspartate, glutamate, succinate and fumarate all increased secretion of amyloglucosidase from 50% to >90% and enzyme production by 10-fold with little effect on xylanase production. Glutamate or succinate stimulated in vitro release of intracellular amyloglucosidase from washed mycelia in the presence of cycloheximide. Amyloglucosidase accumulated in the absence of glutamate was a high-molecular-mass protein that did not migrate in PAGE. Cellular regulation by the fungus of the secretion of amyloglucosidase is indicated.

Saccharification of xylan by an amyloglucosidase of Termitomyces clypeatus and synergism in the presence of xylanase

An amyloglucosidase from a mycelial culture of the mushroom Termitomyces clypeatus hydrolysed larch wood xylan independently and synergistically with an endo-β(1→4) xylanase of the same fungus. The glucoamylase saccharified xylan predigested with xylanase at a faster rate compared to that of xylanase acting on amylase-digested xylan. However, overall saccharification of xylan in both cases was the same. Only glucose was liberated from xylan by amylase digestion whereas xylose, xylobiose and other oligosaccharides were liberated during xylanase digestion. The synergistic response of enzyme combinations was reflected in the liberation of glucose from xylan, rather than xylose. Glucoamylase and xylanase activities on soluble and insoluble fractions of larch wood xylan with different xylose and glucose contents suggested that synergism in xylanolysis by the presence of glucoamylase was dependent on the activity of the participating xylanase on the xylan preparation. It is suggested that possibly α-glucosidic linkages are present in xylan and that amyloglucosidase might be involved in xylanolysis.

Purification and characterisation of a β-glucosidase (cellobiase) from a mushroom Termitomyces clypeatus

A β-glucosidase with cellobiase activity was purified to homogeneity from the culture filtrate of the mushroom Termtomyces clypeatus. The enzyme had optimum activity at pH 5.0 and temperature 65°C and was stable up to 60° C and within pH 2–10. Among the substrates tested, p-nitrophenyl-β- d-glucopyranoside and cellobiose were hydrolysed best by the enzyme. K m and V m values for these substrates were 0.5, 1.25 mM and 95, 91 μmol/min per mg, respectively. The enzyme had low activity towards gentiobiose, salicin and β-methyl- d-glucoside. Glucose and cellobiose inhibited the β- d-glucosidase (PNPGase) activity competitively with K i of 1.7 and 1.9 mM, respectively. Molecular mass of the native enzyme was approximated to be 450 kDa by HPLC, whereas sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated a molecular mass of 110 kDa. The high molecular weight enzyme protein was present both intracellularly and extracellularly from the very early growth phase. The enyme had a p I of 4.5 and appeared to be a glycoprotein.

β-Glucosidase production by the mycelial culture of the mushroom Termitomyces clypeatus

Extracellular cellobiase activity was detected in the mycelial culture of the mushroom T. clypeatus with different mono-, di-, and polysaccharides as carbon source. Higher carbohydrate (2–5%) in the medium strongly repressed enzyme production without inhibiting growth rates. On the other hand, nonglucose monosaccharides also could not improve extracellular enzyme activity. Casein hydrolysate (CH) in the medium at 1% (w/v) concentration largely improved enzyme titer irrespective of carbon source (glucose, xylose, cellobiose, starch) used. Extracellular activity also appeared in high carbohydrate media in the presence of casein hydrolysate. The kinetics of extra- and intracellular production of the enzyme in cellobiose (CB) medium, with or without CH, indicated extracellular and growth-dependent production of the enzyme. A maximum intracellular level of 8% of the total cellobiase was measured at the late phase of growth in CB medium. CH had no effect on pH, temperature optima, and thermal stability of the enzyme produced in different carbohydrate-containing media. T. clypeatus did not liberate any proteinase in the presence or the absence of CH. Thus CH appeared not to improve enzyme titer by repressing any proteinase or stabilizing enzyme activity liberated in CH-free medium. It was therefore suggested that the constitutive production of cellobiase by T. clypeatus was under catabolic repression and CH probably released the repression to some extent. The β-glucosidase activity of the culture filtrate on p- nitrophenyl-β- d-glucoside (pNPG), β-methyl- d-glucoside, and cellobiose had identical pH and temperature optima at 5°C and 65°C, respectively. The enzyme had higher affinity for aryl-β- d-glucoside, while β-CH 3- d-glucoside was a very poor substrate for the enzyme. The activity of the enzyme was readily inhibited by glucose, whereas glucose analogues or any other related sugars did not have any appreciable inhibitory activity.

Carboxymethyl xylan-a specific substrate directly differentiating backbone-hydrolysing and side chain-reacting β- d-(1→4)-xylanases of the mushroom Termitomyces clypeatus

The mushroom Termitomyces clypeatus produces two endoxylanases (D) and (X) when grown in media containing dextrin and xylan as carbon sources, respectively. Endoxylanase (D) showed wide variation in its activity on different lots of xylan preparations, and its activity was found to be dependent upon the composition of xylans. The xylose-liberating endoxylanase (X) did not discriminate between different xylans. The activity of xylanase (D) was found to decrease as the proportion of xylose in different xylan preparations increased. The dialyzable oligosaccharides from the digestion of xylan by enzyme (D) contained constituent sugars of xylan, whereas xylose was the main constituent sugar of the undialyzable fraction. Enzyme (D) also could not liberate any reducing group from carboxymethyl xylan (CMX), a suitable substrate for viscometric and colorimetric assays of endolytic activity of xylanases. CMX was found to be modified preferentially at the substituent sugars of xylan rather than at backbone residues. Thus CMX proved to be a specific substrate for colorimetric assay of true endoxylanase activity.

An Inducible Xylanase of the Mushroom Termitomyces clypeatus Differing from the Xylanase/Amylase Produced in Dextrin Medium

Summary: The mushroom Termitomyces clypeatus produces a single endoxylanase (1,4-β-D-xylan xylano-hydrolase, EC 3.2.1.8) in the presence of either dextrin or xylan as sole source of carbon. The enzymes produced in the two conditions are different. The enzyme induced by xylan has been purified 67-fold from the culture filtrate of T. clypeatus. The enzyme preparation gave a single protein band on SDS-PAGE, corresponding to a molecular weight of about 24000. The enzyme has an isoelectric point at pH 4·0 and acts on arabinoxylan and arabinogalactan, but not amylopectin or galactomannan. It shows maximum activity on xylan (1,4-β-linked D-xylopyranose units) at pH 3·5 and 55°C and is fairly stable up to 60°C. The K m for xylan is 4 mg ml-1. Hg2+, Fe2+ and Ag+ are the most potent inhibitors of the enzyme. The pH optimum and molecular weight of this inducible xylanase differ from those of the enzyme produced by the same organism grown in dextrin medium.

Saccharification of untreated agrowastes during mycelial growth of mushroom Termitomyces clypeatu on solid beds

Low-cost production of fermentable sugar from renewable cellulosic biomass is a highly desired technology for future planning. Although a number of active cellulase systems have been studied from various microorganism~,'-~ in vitro saccharification of agrowastes by these enzymes has not yet been found economical. Mushroom fruit bodies which grow excellently on cellulosic wastes have received little attention in this context. Chang and Steinkraus have only recently reported saccharification of agrowaste bed during mycelial growth of mushroom Volvariella v~lvacea.~ A few mushroom strains5-' growing under submerged conditions in our laboratory have been reported as potent producers of different carbohydrase enzyme^.^,^ The present study describes the in vivo saccharification of cellulosic biomass during the mycelial growth of mushroom Termitomyces clypeatus on various agrowastes.

Purification and properties of xylan hydrolase from mushroom Termitomyces clypeatus

The endoxylanase (1,4-β- d-xylan xylanohydrolase, EC 3.2.1.8) from the culture filtrate of a mushroom, Termitomyces clypeatus, has been purified 93-fold by ammonium sulphate precipitation, ion-exchange chromatography (DEAE-Sephadex) and gel permeation chromatography (Bio-Gel P-200). The enzyme preparation gave a single protein band on disc gel electrophoresis at pH 9.5, and has a molecular weight of approx. 90 000. It also acts on amylopectin, arabinoxylan and arabinogalactan. The enzyme shows maximum activity on xylan (1,4-β-linked d-xylopyranose units) at pH 5.5 and at 55° C and is fairly stable between pH 3 and 10 and temperatures up to 60° C. The K m is 4 mg of xylan/ml. Hg 2+ is the most potent inhibitor, whereas Fe 2+, Ag +, iodoacetate and phosphate moderately inhibit the enzyme activity.