John A. Buswell

Lignocellulolytic enzyme profiles of edible mushroom fungi

One of the most economically-viable processes for the bioconversion of many types of lignocellulosic wastes is represented by edible mushroom cultivation. Lentinula edodes, Volvariella volvacea and Pleurotus sajor-caju are three important commercially cultivated mushrooms which exhibit varying abilities to utilise different lignocellulosics as growth substrate. Examination of the lignocellulolytic enzyme profiles of the three species show this diversity to be reflected in qualitative variations in the major enzymic determinants (i.e. cellulases, ligninases) required for substrate bioconversion. For example, L. edodes, which is cultivated on highly lignified substrates such as wood or sawdust, produces two extracellular enzymes which have been associated with lignin depolymerisation in other fungi, (manganese peroxidase and laccase). Conversely, V. volvacea, which prefers high cellulose-, low lignin-containing substrates produces a family of cellulolytic enzymes including at least five endoglucanases, five cellobiohydrolases and two β-glucosidases, but none of the recognised lignin-degrading enzymes.

Molecular cloning of a new laccase from the edible straw mushroom Volvariella volvacea: possible involvement in fruit body development.

Cloning of a laccase-encoding cDNA from the edible straw mushroom, Volvariella volvacea, was performed using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. The cDNA of the putative laccase gene (lac4) consisted of 1689 bp, including an open reading frame encoding a 23-amino acid signal peptide at the N-terminal end and a 540-amino acid mature protein with a predicted molecular mass of 58173 Da and a pI value of 6.1. The 10 histidine residues and one cysteine residue required to co-ordinate the four copper atoms at the active site of the protein were all conserved. The amino acid sequence of V. volvacea lac4 has a high degree of identity with other basidiomycete laccases. Transcription of the laccase gene was analysed by RT-PCR and, unlike many other laccase genes, shown to be regulated independently of either copper or aromatic compounds under the test conditions. However, the laccase gene is strongly expressed during that part of the mushroom developmental cycle involving fruit body morphogenesis.

β-Glucosidase components of the cellulolytic system of the edible straw mushroom, Volvariella volvacea

The edible straw mushroom, Volvariella volvacea (V-14), produced β-glucosidase when grown in liquid culture on a variety of carbon sources including cellulose, cellobiose, salicin, sorbose, lactose, esculin, cotton wool, and filter paper. Two cell-associated β-glucosidases, BGL-I and BGL-II, were purified 32-fold and 23-fold, respectively, from extracts of cellulose-grown mycelium. The purification procedure included DEAE-Sepharose chromatography, Sephacryl S-200 chromatography, and fast protein liquid chromatography (FPLC) using Mono-Q and Mono-P anion-exchange columns. The enzymes were found to be homogeneous and to have native molecular weights of 158 kDa (BGL-I) and 256 kDa (BGL-II) by gel filtration. The isoelectric points for BGL-I and BGL-II were 5.6 and 5–5.2, respectively. Both isozymes displayed relatively broad pH optima with maximum reaction velocities recorded at pH 7.0 for BGL-I and pH 6.2 for BGL-II, and were rapidly denatured at temperatures of 60°C and above. Purified BGL-I and BGL-II were both active against p-nitrophenyl-β-d-glucopyranoside, p-nitrophenyl-α-l-arabinofuranoside, p-nitrophenyl-α-d-glucopyranoside, cellobiose, salicin, and esculin, but only BGL-I was active against p-nitrophenyl-β-d-xylopyranoside. BGL-I and BGL-II exhibited Km values for p-nitrophenyl-β-d-glucopyranoside of 90 and 500 μm, respectively. Isozyme activities were adversely affected by several reported β-glucosidase inhibitors, various metal ions, and lignin-derived aromatic acids and aldehydes. Glucose production from microcrystalline cellulose by a commercial cellulase preparation was enhanced by 9.7% in reaction mixtures supplemented with BGL-II.

Extracellular cellulolytic enzyme profiles of five lignicolous mangrove fungi

Five lignicolous mangrove fungi (Hypoxlon oceanicum, Julella avicenniae, Lignincola laevis, Savoryella lignicola and Trematosphaeria mangrovei) produced extracellular endoglucanase, cellobiohydrolase and β-glucosidase when cultivated under static conditions in a defined liquid growth medium. Agitation of cultures completely repressed enzyme production. Cellulase production by H. oceanicum was 2–5 fold higher compared to other strains. Increasing salinity generally resulted in reduced enzyme production although responses varied, but J. avicenniae appeared relatively unaffected by salinity within the range 0–3.4%.

Purification and partial characterization of two xylanases that differ in hydrolysis of soluble and insoluble xylan fractions

Abstract Two isoenzymes of xylanase (1,4 β-D-xylan xylanohydrolase, E.C. 3.2.1.8) have been extracted and purified from culture supernatants of Streptomyces sp. A451 by ammonium sulfate fractionation, DEAE- and CM-Sepharose chromatography, and gel permeation chromatography on Sephadex G75. The enzymes were found to be homogeneous and to have molecular weights of 22.8 kDa (xyl I) and 33.1 kDa (xyl II) by SDS-polyacrylamide gel electrophoresis. Isoelectric focusing gave pI values of 8.6 and 8.9 for enzymes I and II, respectively. Both enzymes were found to have similar pH optima (pH 5.6 for I and pH 5.4 for II) and similar temperature optima (50°C). The two enzymes had almost identical temperature stability profiles. With oat spelt xylan, activation energy values of 35.3 kJ mol −1 for enzyme I and 42.7 kJ mol −1 for enzyme II were obtained. Examination of products obtained from soluble and insoluble oat spelt and birch xylans showed that enzyme treatment of birch xylan produced oligomer profiles very different from that with oat spelt xylan, and the difference in oligomer profiles between the two xylanases was much more evident with birch xylan than with oat spelt xylan. Xylotriose was the major product of xylanase I activity; xylotetraose was dominant in hydrolysates with enzyme II. With birch xylan, enzyme II was markedly more effective in depolymerization of the soluble as compared with the insoluble fraction.

Production of cellulases and hemicellulases by the straw mushroom, Volvariella volvacea

Carboxymethylcellulase (CMCase), Avicelase and β-glucosidase production was monitored in submerged cultures of two strains (V 14 and V 34) of Volvariella volvacea grown on Avicel. All three cellulolytic enzymes were detectable in culture supernatants, although levels of CMCase and Avicelase were considerably higher in cultures of V. volvacea V 14. β-Glucosidase activity was also observed in mycelial extracts of both fungal strains. Addition of 0·2% Tween 80 to cultures markedly enhanced extracellular cellulase activity, especially in V. volvacea V 34. Cellulases, together with xylanase and both intra- and extracellular β-xylosidase, were also recorded in cultures of V. volvacea V 34 grown on paddy straw.

Transformation of the edible fungi, Pleurotus ostreatus and Volvariella volvacea

A transformation system is described for the edible mushrooms Pleurotus ostreatus and Volvariella volvacea . The system developed is based on a positive selection strategy using the trp3 iar gene from Coprinus cinereus , which confers resistance to the antimetabolite 5-fluoroindole. Transformation frequencies were low in both species. Southern blot analysis confirmed the integration of transforming DNA into the genome of transformants and indicated the presence of tandemly duplicated copies of the plasmid in some of these transformants. The system has potential for introducing beneficial traits such as enhanced substrate bioconversion and faster sporophore development.

Biomass and extracellular hydrolytic enzyme production by six mushroom species grown on soybean waste

SummarySoybean waste is a good substrate for biomass production, and the expression of amylolytic, xylanolytic and proteolytic enzymes, by selected mushroom fungi. Considerable potential exists for converting soybean wastes into added-value products using systems based on these fungi.

Cloning of the cbhI and cbhII genes involved in cellulose utilisation by the straw mushroom Volvariella volvacea.

Abstract The straw mushroom Volvariella volvacea is cultivated on substrates rich in cellulose and has been shown to produce a family of cellulolytic enzymes. A PCR-based strategy was adopted to clone genes involved in cellulose utilisation, using degenerate primers designed to amplify conserved catalytic domain sequences of cellobiohydrolases (CBHs). PCR with these primers produced two DNA fragments with sequence similarity to the cbhI and cbhII gene families detected in Trichoderma, Phanerochaete and Agaricus species. Full-length clones of these genes were obtained from an EMBL3 genomic library, and RACE-PCR was used to verify the presence of introns. The cbhI homologue has a coding region of 1722 bp, containing two introns, generating a 536 amino acid polypeptide product. The cbhII gene has a coding region of 1693 bp, containing five introns, and gives rise to a 470-amino acid polypeptide product. Northern and PCR analyses were used to study the expression of the genes. These revealed that transcripts of both genes were induced on medium containing cellulose – with cbhI being expressed more strongly than cbhII– but were repressed on medium containing glucose.

Effect of lignin-derived phenolic monomers on the growth of the edible mushrooms Lentinus edodes, Pleurotus sajor-caju and Volvariella volvacea

Pleurotus sajor-caju was generally more tolerant to lignin-related phenolic monomers and tannin derivatives than Lentinus edodes and the straw mushroom, Volvariella volvacea. Several phenols, at up to 5 mM, enhanced mycelial growth of P. sajor-caju. No clear pattern was evident when the effects of phenols and tannins on the growth of V. volvacea and L. edodes were compared, but the lower concentrations of 4-hydroxybenzaldehyde and vanillin which were tested were markedly more toxic to the straw mushroom. The distribution of phenolic monomers and tannin derivatives in the agricultural wastes used for mushroom cultivation may be an important growth determinant. However, the differences in the growth inhibition profiles of L. edodes, P. sajor-caju and V. volvacea suggest that, alone, the effect of these compounds on fungal growth is unlikely to account for the varying abilities of the three mushroom species to grow and fruit on a particular lignocellulosic substrate.