Phylogenetic position of the ectomycorrhizal basidiomycete Tricholoma dulciolens in relation to species of Tricholoma that produce “matsutake” mushrooms

Tricholoma dulciolens is an ectomycorrhizal basidiomycete that produces “matsutake”-like mushrooms in association with Picea abies in Fennoscandia. The phylogenetic position of T. dulciolens relative to matsutake species is, however, unknown. In the present study, we demonstrated that T. dulciolens is phylogenetically independent from T. matsutake and its allied species. In addition, herbarium specimens identified as T. caligatum from Abies alba forests in Spain were phylogenetically related to but distinct from T. dulciolens. Based on ribosomal internal transcribed spacer DNA sequence data, T. dulciolens appears to exist in North America, as well. This is the first phylogeny of T. dulciolens in Fennoscandia and its related species in southern Europe.     

The (oxalato)aluminate complex as an antimicrobial substance protecting the “shiro” of Tricholoma matsutake from soil micro-organisms

Tricholoma matsutake, a basidiomycete, forms ectomycorrhizas with Pinus densiflora as the host tree. Its fruiting body, “matsutake” in Japanese, is an edible and highly prized mushroom, and it grows in a circle called a fairy ring. Beneath the fairy ring of T. matsutake, a whitish mycelium-soil aggregated zone, called “shiro” in Japanese, develops. The front of the shiro, an active mycorrhizal zone, functions to gather nutrients from the soil and roots to nourish the fairy ring. Bacteria and sporulating fungi decrease from the shiro front, whereas they increase inside and outside the shiro front. Ohara demonstrated that the shiro front exhibited antimicrobial activity, but the antimicrobial substance has remained unidentified for 50 years. We have identified the antimicrobial substance as the (oxalato)aluminate complex, known as a reaction product of oxalic acid and aluminum phosphate to release soluble phosphorus. The complex protects the shiro from micro-organisms, and contributes to its development.     

Genetic mosaics in the massive persisting rhizosphere colony “shiro” of the ectomycorrhizal basidiomycete Tricholoma matsutake

The ectomycorrhizal basidiomycete Tricholoma matsutake produces commercially valuable fruit bodies matsutake on a massive persisting rhizosphere aggregate of mycelia and mycorrhizas called shiro. Using inter-retrotransposon amplified polymorphism analysis, we attempted to explore the potential diversity within the population of T. matsutake isolated from small Pinus densiflora woodlands located in various parts of Japan. In general, random phylogenetic relationship was noted among T. matsutake tested. The population from each limited sampling area was highly heterogeneous. Even some isolates from fruit bodies produced in the same shiro and those from spores in the same fruit bodies were found to be genetically diverse, indicating the occurrence of genetic mosaics in shiro. In a mosaic shiro, heterologous genets produced their fruit bodies concurrently. Data suggested that the dispersal of spores through sexual reproduction may have been more prevalent than generally accepted in T. matsutake to bring mosaicism and coordination of heterologous genets within the shiro. Implementation of management taking such diversity into consideration is urgently needed for the restoration of devastated matsutake fields in Japan. Exploration of individual clones in mosaic fungal resources that promote colonization and fruit body production is necessary for it.     

Purification and properties of an acidic β-glucosidase from Acidobacterium capsulatum

Acidobacterium capsulatum, an acidophilic, mesophilic and chemoorganotrophic bacterium, produced an inducible, acidic β-glucosidase in the cellobiose medium. The enzyme was successively purified 109 times by CM-Sepharose, Sephacryl S-200 chromatography and preparative discontinuous polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis of the purified enzyme gave a single band at pH 4.3. The enzyme had an optimum pH of 3.0 and optimum reaction temperature of 55°C, being stable from pH 1.5 to 6.0 and at temperatures from 20 to 45°C. No activity was detected above pH 6.5 or above 65°C. The molecular weight of 90,000 was estimated by gel filtration and the enzyme had an isoelectric point of 7.0. The enzyme hydrolyzed aryl-β-glycosides and β-linked disaccharides.     

Purification and properties of an extracellular β-glucosidase from Lenzites trabea

Summary When culturing the cellulolytic-active Basidiomycete and brown-rot fungus Lenzites trabea A-419 in submerged culture with glucose and cellulose as a carbon source, the fungus only excreted -glucosidase (EC 3.2.1.21) and an endo-1,4--glucanase (EC 3.2.1.4).No evidence for C₁ activity (EC 3.2.1.91) was found in the culture filtrate or in the ultra concentrate. -Glucosidase could be separated from endoglucanase by chromatography on Sepharose 6-B. Further fractionation of the -glucosidase on DEAE-Sephadex A-25 resulted in a 525-fold purification. The molecular weight of the isolated -glucosidase was determined by co-chromatography on Sephadex G-200 to be 320,000 daltons. The enzyme developed maximum activities at pH 4.5 and 75°C. The enzyme does not act on crystalline cellulose or CMC, but it hydrolyzes cellotriose,-tetraose, and-pentaose to cellobiose and glucose. -glucosidase activity was strongly inhibited by the reaction product, glucose. A K_i value of 2.7×10^–3 (M) for noncompetitive inhibition was found.     

Characterization of an acid-tolerant β-1,4-glucosidase from Fusarium oxysporum and its potential as an animal feed additive

An extracellular β-glucosidase (BGL) from Fusarium oxysporum was purified to homogeneity by a single chromatography step on a gel filtration column. The optimum activity of BGL on cellobiose was observed at pH 5.0 and 60 °C. Under the same conditions, the K _m and V _max values for p-nitrophenyl β-d-glucopyranoside and cellobiose were 2.53 mM, 268 U?mg protein^?1 and 20.3 mM, 193 U?mg protein^?1, respectively. The F. oxysporum BGL enzyme was highly stable at acidic pH (t _1/2?=?470 min at pH 3). A commercial BGL Novo188 (Novozymes) and F. oxysporum BGL were compared in their ability to supplement Celluclast 1.5 L (Novozymes). In comparison with the commercial Novo188 (267 mg?g substrate^?1), F. oxysporum BGL supplementation released more reducing sugars (330 mg?g substrate^?1) from cellulose under simulated gastric conditions. These properties make F. oxysporum BGL a good candidate as a new commercial BGL to improve the nutrient bioavailability of animal feed.     

One-step purification and characterization of an intracellular β-glucosidase from Metschnikowia pulcherrima

A collection of 60 non-Saccharomyces yeasts isolated from grape musts in Uruguayan vineyards was screened for beta-glucosidase activity and Metschnikowia pulcherrima was the best source of this enzyme activity. Its major beta-glucosidase was successfully purified to homogeneity by ion-exchange chromatography on amino-agarose gel. The enzyme exhibited an optimum catalytic activity at 50 degrees C and pH 4.5 and was active against (1 --> 4)-beta and (1 --> 2)-beta glycosidic linkages. In spite of preserving 100% of its activity and stability in the presence of 12% (v/v) ethanol and 5 g glucose/l, the enzyme was unstable below pH 4. We characterized the beta-glucosidase from M. pulcherrima with a view to its potential applications in wine-making.     

Production,purification, and properties of β-glucosidase from Candida wickerhamii

Summary Candida wickerhamii growing on cellobiose produced -glucosidase with high activity against -nitrophenyl glucoside (PNPG) but low activity against cellobiose. -glucosidase production was constitutive, and was repressed by -glucosides and glucose. -glucosides containing an aromatic moiety in the aglycon were the best substrates for -glucosidase indicating that the enzyme is an aryl--glucosidase. A -glucosidase from C. wickerhamii cells was purified by (NH₄)₂SO₄ precipitation, dialysis, ion-exchange chromatography and gel filtration. The purified enzyme was homogeneous as shown by sodium-dodecyl-sulphate polyacrylamide gel electrophoresis and discontinuous gel electrophoresis. The purified enzyme hydrolysed PNPG but not cellobiose. The K_m of the enzyme was 0.185 mM. Glucose inhibited the enzyme competitively and the K_i was 7.5 mM. The apparent molecular mass was 97,000. The optimum pH and temperature for enzyme activity were between pH 7 and 7.4 and 40°C respectively. At temperatures of 45°C and greater the enzyme was inactivated. The activation energy of the enzyme was 29.4 kJ · mol^-1.     

Optimization of β-glucosidase production from Aspergillus niger

本研究对Aspergillus niger Glu05生产β-葡萄糖苷酶的培养基组分及培养条件进行了优化.优化后的培养基组成和培养条件分别为:麸皮4％,tryptone 4％,1μmol MnSO4,1μmol NaCl,KH2PO40.2％,oH自然,摇床转速250 r/min,培养温度30℃,培养周期5d.优化后发酵液中酶活力达到44.11 IU/mL,与初始的产酶水平32.87 IU/mL相比,提高了36％.     

An alkaline β-glucosidase isolated from an olive brine strain of Wickerhamomyces anomalus

An efficient β-glucosidase (βG)-producing strain, Wickerhamomyces anomalus BS81, was isolated from naturally fermented olive brine and identified based on PCR/restriction fragment length polymorphism of the rDNA internal transcribed spacer and sequence analysis of the D1/D2 region of the 26S rRNA gene. The hydrolytic activity of the βG had an optimum pH of 8.5 and an optimum temperature of 35 °C. The enzyme had high substrate specificity and high catalytic efficiency (K(m) 0.99 mM, V(max) 14 U g(-1) of cells) for p-nitrophenyl-β-d-glucopyranoside. The enzyme was activated by increasing concentrations of NaCl, with maximum activity at 150 g L(-1) NaCl. Although βGs have been purified and characterized from several other sources, the W. anomalusβG is unique among βGs because its relative maximum activity occurs at alkaline pH and 35 °C. Moreover, the yeast strain has esterase activity that acts synergistically with βG to degrade oleuropein to debitter table olives and olive oil.     

Properties of an intracellular β-glucosidase purified from the cellobiose-fermenting yeast Candida wickerhamii

An intracellular -glucosidase was isolated from the cellobiose-fermenting yeast, Candida wickerhamii. Production of the enzyme was stimulated under aerobic growth, with the highest level of production in a medium containing cellobiose as a carbohydrate source. The molecular mass of the purified protein was approximately 94 kDa. It appeared to exist as a dimeric structure with a native molecular mass of about 180 kDa. The optimal pH ranged from 6.0 to 6.5 with p-nitrophenyl -d-glucopyranoside (NpGlc) as a substrate. The optimal temperature for short-term (15-min) assays was 35°C, while temperature-stability analysis revealed that the enzyme was labile at temperatures of 28° C and above. Using NpGlc as a substrate, the enzyme was estimated to have a K _m of 0.28 mM and a V _max of 525 mol product min^–1 mg protein^–1. Similar to the extracellular -glucosidase produced by C. wickerhamii, this enzyme resisted end-product inhibition by glucose, retaining 58% of its activity at 100 mM glucose. The activity of the enzyme was highest against aryl -1,4-glucosides. However, p-nitrophenyl xylopyranoside, lactose, cellobiose, and trehalose also served as substrates for the purified protein. Activity of the enzyme was stimulated by long-chain n-alkanols and inhibited by ethanol, 2-propanol, and 2-butanol. The amino acid sequence, obtained by Edman degradation analysis, suggests that this -glucosidase is related to the family-3 glycosyl hydrolases.     

Kinetic properties of β-glucosidase from Aspergillus ornatus

Summary Kinetic properties of extracellular -glucosidase from Aspergillus ornatus were determined. The pH and temperature optima for the enzyme were found to be 4.6 and 60°C, respectively. Under these conditions, the enzyme exhibited a K _m (p-nitrophenyl--glucoside) value of 0.76±0.11 mM. The activation energy for the enzyme was 11.8 kcal/mol. Several divalent metal ions inhibited -glucosidase activity, some of which showed inhibition of enzyme activity only at higher concentrations. Ag²⁺ was the most potent inhibitor. A metal chelating agent, EDTA, also inhibited -glucosidase activity. Except for trehalose, glucose, glucono--lactone, cellobiose, gentiobiose, laminaribiose, maltose and isomaltose inhibited -glucosidase activity. Glucose was found to be a competitive inhibitor, whereas glucono--lactone and other -linked disaccharides were noncompetitive (mixed) inhibitors of the enzyme.     

Root endophyte interaction between ectomycorrhizal basidiomycete Tricholoma matsutake and arbuscular mycorrhizal tree Cedrela odorata, allowing in vitro synthesis of rhizospheric “shiro”

The ectomycorrhizal basidiomycete Tricholoma matsutake associates with members of the Pinaceae such as Pinus densiflora (red pine), forming a rhizospheric colony or “shiro,” which produces the prized “matsutake” mushroom. We investigated whether the host specificity of T. matsutake to conifers is innately determined using somatic plants of Cedrela odorata, a tropical broad-leaved tree (Meliaceae) that naturally harbors arbuscular mycorrhizal fungi. We found that T. matsutake could form in vitro shiro with C. odorata 140 days after inoculation, as with P. densiflora. The shiro was typically aromatic like that of P. densiflora. However, this was a root endophytic interaction unlike the mycorrhizal association with P. densiflora. Infected plants had epidermal tissues and thick exodermal tissues outside the inner cortex. The mycelial sheath surrounded the outside of the epidermis, and the hyphae penetrated into intra- and intercellular spaces, often forming hyphal bundles or a pseudoparenchymatous organization. However, the hyphae grew only in the direction of vascular bundles and did not form Hartig nets. Tricholoma fulvocastaneum or “false matsutake” naturally associates with Fagaceae and was also able to associate with C. odorata as a root endophyte. With T. matsutake, C. odorata generated a number of roots and showed greatly enhanced vigor, while with T. fulvocastaneum, it generated a smaller number of roots and showed somewhat lesser vigor. We argue that the host–plant specificity of ectomycorrhizal matsutake is not innately determined, and that somatic arbuscular mycorrhizal plants have a great potential to form mutualistic relationships with ectomycorrhizal fungi.     

Ectomycorrhization of Tricholoma matsutake and two major conifers in Finland—an assessment of in vitro mycorrhiza formation

This study aimed to test the ability of Tricholoma matsutake isolates to form mycorrhizas with aseptic seedlings of Pinus sylvestris L. and Picea abies (L.) Karst. Germinated seedlings of Scots pine and Norway spruce were separately inoculated with either isolates originating from Finland or Japan. Eight months after inoculation, the Finnish isolate had formed a sheath and Hartig net on both host species. Ectomycorrhizal Scots pine seedlings inoculated with the Finnish isolate showed the same shoot height and dry mass as the controls. Ectomycorrhizal Norway spruce seedlings inoculated with the Finnish isolate had similar shoot height but slightly less dry mass than the control seedlings. For both tree species, inoculation with the Finnish isolate resulted in reduced total nitrogen content per seedling, but carbon content was unaffected. Inoculation with the Japanese isolate resulted in an initial Hartig net-like structure in pine but not in spruce. No typical Hartig net was observed on either tree species. Furthermore, seedlings of both species inoculated with the Japanese isolate showed significantly reduced growth, dry mass, nitrogen, and carbon content per seedling and shoot height (in spruce) compared to the controls. This study documents and describes the in vitro ectomycorrhization between T. matsutake and Scots pine or Norway spruce and the variable mycorrhizal structures that matsutake isolates can form.     

Location and contribution of individual β-glucosidase from Neurospora crassa to total β-glucosidase activity

This study investigated the cellular location and the contribution of individual β-glucosidase (BGL) to total BGL activity in Neurospora crassa. Among the seven bgl genes, bgl3, bgl5, and bgl7 were transcribed at basal levels, whereas bgl1, bgl2, bgl4, and bgl6 were significantly up-regulated when the wild-type strain was induced with cellulose (Avicel). BGL1 and BGL4 were found to be contributors to intracellular BGL activity, whereas the activities of BGL2 and BGL6 were mainly extracellular. Sextuple bgl deletion strains expressing one of the three basally transcribed bgls did not produce any detectable BGL activity when they were grown on Avicel. BGL6 is the major contributor to overall BGL activity, and most of its activity resides cell-bound. The sextuple bgl deletion strain containing only bgl6 utilized cellobiose at a rate similar to that of the wild type, while the strain with only bgl6 deleted utilized cellobiose much slower than that of the wild type.     

Short Communication: Purification and characterization of an intracellular β-glucosidase from Cellulomonas biazotea

World Journal of Microbiology and Biotechnology -     

Purification and properties of an extracellular β-glucosidase from the cellulolytic thermophile Clostridium stercorarium

Summary Clostridium stercorarium cultures grown on cellobiose contain both an extracellular and a cell-bound -glucosidase activity. A substantial portion of the cell-bound enzyme could be extracted by osmotic shock, suggesting a periplasmic localization. The -glucosidase present in culture supernatants was purified to homogeneity. It was found to be identical in all aspects tested with the cell-bound -glucosidase. The enzyme exists as a monomer with an apparent molecular weight of 85.000 (SDS-PAGE) and a pI of 4.8. It shows optimal activity as pH 5.5 and 65° C. Thiol groups are essential for enzyme activity. In the presence of reducing agents and divalent cations the half-life of the purified enzyme was more than 5 h at 60°C. The enzyme hydrolyses at different rates a wide range of substrates including aryl--glucosides, cellobiose, and disordered cellulose. K _m values were determined as 0.8 mM for p-nitrophenyl--glucoside (PNPG) and 33 mM for cellobiose. The cellular localization and the substrate specificity pattern are consistent with a dual role of the C. stercorarium -glucosidase in cellulose saccharification: (1) Cleavage of cellobiose formed by exoglucanase and (2) degradation of cellodextrins produced by endoglucanase action.     

Immobilization of β-glucosidase from Scytalidium lignicola on Chitosan

Chitosan was found to be a better support than alginate beads for immobilization of β-glucosidase from Scytalidium lignicola. The optimum concentration of glutaraldehyde for enzyme immobilization was 0.2%. Immobolized β-glucosidase was more able in the pH range of 3–6. Immobilized β-glucosidase retained about 70% of its activity at 50%C after 72 h of incubation while free enzyme lost most of its activity. The log of activity retained vs time was a straight line with free enzyme but was curved for immnobilized enzyme. Lineweaver-Burk plots of free and immoblized β-glucosidase gave K_m values of 2 × 10⁻⁴ M and 5.5 × 10⁻⁴ M for p-nitrophenyl β-d-glucopyranoside, respectively. Addition of immobilized β-glucosidase to a saccharification system gave a 30% increase in reducing sugar availability compared to free enzyme addition and was at least 4 times reusable without appreciable loss in enzyme activity.     

Kinetics of improved production and thermostability of an intracellular β-glucosidase from a mutant-derivative of Cellulomonas biazotea

The highest productivity (20 IU l(-1) h(-1)) of beta-glucosidase by a mutant of Cellulomonas biazotea was 2.5-fold more than that of the parent organism. The enzyme had a lower activation energy (57 kJ mol(-1)) than the native enzyme (68 kJ mol(-1)). The enzyme from the mutant had enthalpy and entropy values for irreversible intactivation of 95.6 kJ mol(-1) and 60 J.mol(-1) K(-1) compared with 108 kJ mol(-1) and 86 J mol(-1) K(-1) for the native enzyme suggesting that the mutation had stabilized the enzyme.     

Rhamnolipids as affinity foaming agent for selective collection of β-glucosidase from cellulase enzyme mixture

Selective and effective separation can potentially be achieved with affinity foam fractionation using simple foaming setup and operation. In this study the use of affinity foam fractionation for selective collection and enrichment of β-glucosidase from a cellulase enzyme mixture was evaluated. Rhamnolipids, a group of glycolipids produced most commonly by Pseudomonas aeruginosa, were used as the affinity foaming agent, because of their foaming property and the presence of dirhamnose moiety (a potential substrate analog for β-glucosidase) in some rhamnolipids. The effects of aeration rate, medium pH, cellulase concentration and rhamnolipid concentration on the foam fractionation performance were examined. Among the pH studied (3.1, 5.0, 7.0 and 9.0), pH 5 was clearly the optimal for selective enrichment of β-glucosidase, presumably corresponding to the high binding affinity between the enzyme and the substrate analog (dirhamnose). With adequate rhamnolipid concentrations (≥0.1 g/L), the aeration rate of 0.1L/min (i.e., 2VVM) for 50 ml test samples was found to give the highest enrichment; higher aeration rates produced wetter foam and, thus, lower (diluted) enzyme activity in the foamate. The enrichment increased with the increasing rhamnolipids-to-cellulase ratio, in the range of 0-2 (w/w) investigated in this study. The finding indicated that rhamnolipids were the limiting compounds in these systems so that the amount of surfactant-enzyme complexes formed and removed into the foam phase would increase when more rhamnolipids were added. At the rhamnolipids-to-cellulase ratio of 2, the β-glucosidase activity in the foamate was about 9 times as high as the activity in the original sample of cellulase mixture and about 17 times the activity in the remaining solution (after foaming). The overall FPU (filter paper unit, a measurement of total cellulase activity) and the activities of endo- and exo-glucanases were only enriched 70-150%. The feasibility of affinity foam fractionation was demonstrated.