Edible Straw Mushroom Research Articles

The serine/threonine protein kinase VSKM1 promotes cold stress resistance of the postharvest Volvariella volvacea by enhancing mitoribosome activity

The edible straw mushroom Volvariella volvacea has a desirable taste and diverse effects on health; however, it undergoes rapid autolysis at 4 °C. Spray treatment with the β-glucosidase inhibitor DGlucono-1,5-lactone (DGase) at 100 µmol/L maintained the quality of V. volvacea during storage at 4 °C for 72 h. Enhanced alkaline protease and neutral protease activities can act as indicators of cold stress resistance (CSR) in V. volvacea, as well as the stable ATP level. The total sugar and amino acid content is negatively correlated with CSR in V. volvacea. DGase treatment enhanced the CSR of V. volvacea by inducing the overexpression of genes in the mitochondrial ribosome (mitoribosome). Overexpression of a serine/threonine-protein kinase gene in V. volvacea (VSKM1), which promotes mitoribosome activity in yeast, enhanced freezing tolerance. Our findings indicated that overexpression of VSKM1 promoted CSR by enhancing mitoribosome activity for the quality retention of postharvest V. volvacea.

Lysine and threonine supplementation promotes postharvest quality of Volvariella volvacea mushroom during low-temperature preservation via maintenance of the citrate cycle

Volvariella volvacea, the edible straw mushroom, is popular among consumers; however, its freshness is difficult to maintain at 4 °C. Spray treatment with the ubiquitin-binding enzyme E2 (UBEV2) inhibitor L345-0044 maintained the freshness of V. volvacea during storage at 4 °C for 48 h. Inhibitor treatment enhanced the ability of V. volvacea to resist cold stress by inducing overexpression of genes involved in protein translation, protein turnover, N-glycan biosynthesis, and lysine metabolism. Overexpression of saccharopine dehydrogenase genes, which participate in lysine metabolism in yeast, enhanced freezing tolerance. Anti-freezing experiments showed that the addition of l-lysine or l-threonine improved the quality of V. volvacea at 4 °C with stable soluble sugar levels, as lysine or threonine metabolism produced acetyl-CoA for maintaining the citrate cycle. Our findings revealed that supplementation with l-lysine or l-threonine prolonged the preservation time of V. volvacea for more than 24 h via maintenance of the citrate cycle.

Chilling stress reduced protein translation by the ubiquitination of ribosomal proteins in Volvariella volvacea

In Volvariella volvacea, an important edible mushroom species, cryogenic autolysis is a typical part of abnormal metabolism; however, the underlying mechanisms remain unclear. Ubiquitylome analysis revealed that chilling stress (CS) affected protein translation and degradation by ubiquitination. Comparative proteomics analysis showed that CS downregulated protein expression in V. volvacea V23 instead of VH3 (improved chilling stress resistance strain). The integrative ubiquitylome, proteomics, and transcriptome analyses indicated that CS reduced protein translation by the ubiquitination of ribosomal proteins. An activity assay of the 20S proteasome showed that CS decreased the degradation efficiency of the ubiquitin-proteasome system. UBEV2, one type of ubiquitin-conjugating enzyme E2 (UBE2) in V. volvacea, was upregulated after cold stress treatment using western blot analysis. GST pull-down experiments of UBEV2 provided evidence that CS affected protein translation by the ubiquitination of ribosomal proteins. Co-IP experiments confirmed that UBEV2 bound to the ubiquitinated SSB2, a ribosome-associated molecular chaperone. An anti-freezing experiment demonstrated that the UBE2 inhibitor could improve the cold stress resistance of V. volvacea. Our observations revealed that CS triggered ubiquitination-mediated autolysis associated with a decrease in protein translation and highlighted the mechanistic role of UBEV2 in facilitating cryogenic autolysis in V. volvacea. SignificanceVolvariella volvacea, the edible straw mushroom, is a highly nutritious food source widely cultivated on a commercial scale in tropical and subtropical regions. The challenges associated with the cryogenic autolysis preservation of V. volvacea have limited its marketability. This issue of cryogenic autolysis is both an interesting scientific problem to solve and a practical economic matter. Integrative ubiquitylome, proteomics, and transcriptome analyses, together with GST pulldown and Co-IP experiments, indicated that chilling stress reduced protein translation by the ubiquitination of ribosomal proteins in V. volvacea. This study significantly contributes to our understanding of ubiquitination-mediated autolysis associated with a decrease in protein translation in V. volvacea. Our data highlight the mechanistic role of UBEV2 in facilitating the cryogenic autolysis of V. volvacea. We provided a new idea for the preservation of V. volvacea by inhibiting UBEV2 to increase its marketability.

Studies on Different Cultural Parameters on Vegetative Growth of Straw Mushroom (Volvariella volvacea)

Volvariella volvacea is an edible straw mushroom of tropics and subtropics. The optimum temperature and moisture for the growth of this mushroom are 35°C and 57–60%, respectively. Mushrooms grow on natural or semi synthetic compost and absorb nutrients for their survival. The maintenance and revival of pure culture mycelium with magnificent quality is the first critical stage towards the success of spawn preparation. To maintain any microorganism in artificial conditions, the former has to be cultured on a suitable nutrient medium. All microbes require a set of conditions under which they can grow and sporulate best and culture medium is the major factor influencing fungal cultivation. Five different culture media both in liquid and solid phase, five different pH levels and five temperature regimes were evaluated for the vegetative growth of four different strains of Volvariella volvacea i.e. DMR-484, DMR-463, DMR-819 and DMR-820. Among five liquid media studied, malt extract was found to be the best medium for the growth of all the strains of V. volvacea both in solid as well as in liquid phase. Out of five different pH levels evaluated for the growth of V. volvacea pH7.0 was observed to be the best pH for the growth of all the strains of the test fungus. Out of five different temperature regimes evaluated, 30°C was observed to be the ideal temperature for the growth of V. volvacea.

Identification and expression analysis of a new glycoside hydrolase family 55 exo-β-1,3-glucanase-encoding gene in Volvariella volvacea suggests a role in fruiting body development

The edible straw mushroom Volvariella volvacea is an important crop in South East Asia and is predominantly harvested in the egg stage. Rapid stipe elongation and cap expansion result in a swift transition from the egg to elongation and maturation stage, which are subjected to fast senescence and deterioration. In other mushrooms, β-1,3-glucanases have been associated with degradation (softening) of the cell wall during stipe elongation and senescence. We present a new glycoside hydrolase family 55 (GH55) exo-β-1,3-glucanase gene, exg2, and highly conserved deduced EXG2 protein. The 3D model and presumed catalytic residues of V. volvacea EXG2 are identical to Lentinula edodes EXG2 and Phanerochaete chrysosporium Lam55A, supporting similar enzymatic functions. In addition to previous association to stipe elongation and senescence, our data clearly indicates a role for cap (pileus) expansion. Digital gene expression, quantitative PCR and isobaric tags for relative and absolute quantification analysis showed low exg2 and EXG2 levels in primordia, button, egg and elongation stages and significantly increased levels in the maturation stage. Subsequent relative quantitative PCR analysis designated expression of exg2 to the stipe in the elongation stage and to the pileus and stipe in the maturation stage. EXG2 cell wall softening activity, close correlation of exg2 expression with the principal expanding mushroom tissues and a strong conservation of expression patterns and protein sequences in other mushrooms, make V. volvacea exg2 an important candidate for future studies on mechanisms of fruiting body expansion and senescence causing commodity value loss.

Sequencing and comparative analysis of the straw mushroom (Volvariella volvacea) genome.

Volvariella volvacea, the edible straw mushroom, is a highly nutritious food source that is widely cultivated on a commercial scale in many parts of Asia using agricultural wastes (rice straw, cotton wastes) as growth substrates. However, developments in V. volvacea cultivation have been limited due to a low biological efficiency (i.e. conversion of growth substrate to mushroom fruit bodies), sensitivity to low temperatures, and an unclear sexuality pattern that has restricted the breeding of improved strains. We have now sequenced the genome of V. volvacea and assembled it into 62 scaffolds with a total genome size of 35.7 megabases (Mb), containing 11,084 predicted gene models. Comparative analyses were performed with the model species in basidiomycete on mating type system, carbohydrate active enzymes, and fungal oxidative lignin enzymes. We also studied transcriptional regulation of the response to low temperature (4°C). We found that the genome of V. volvacea has many genes that code for enzymes, which are involved in the degradation of cellulose, hemicellulose, and pectin. The molecular genetics of the mating type system in V. volvacea was also found to be similar to the bipolar system in basidiomycetes, suggesting that it is secondary homothallism. Sensitivity to low temperatures could be due to the lack of the initiation of the biosynthesis of unsaturated fatty acids, trehalose and glycogen biosyntheses in this mushroom. Genome sequencing of V. volvacea has improved our understanding of the biological characteristics related to the degradation of the cultivating compost consisting of agricultural waste, the sexual reproduction mechanism, and the sensitivity to low temperatures at the molecular level which in turn will enable us to increase the industrial production of this mushroom.

Comparison of endoglucanase-1 (EG1) induction in the edible straw mushroom Volvariella volvacea by lactose and/or cellobiose with or without added sorbose

We have compared the induction of an endoglucanase (EG1) by α-lactose and/or cellobiose, with or without added L-sorbose, in submerged cultures of Volvariella volvacea, to better understand the mechanism whereby cellulase formation is triggered by these soluble disaccharides. EG1 levels induced by α-lactose and cellobiose were 28.6% and 6.7%, respectively of the highest levels recorded with crystalline cellulose. Sorbose did not induce EG1 and strongly repressed enzyme levels when added to α-lactose but not cellobiose-containing cultures. EG1 levels in cultures containing all three saccharides were similar to those recorded with sorbose and cellobiose although enzyme induction was delayed by 12 h. When V. volvacea was pre-grown for 24 h in medium containing sorbose as the sole carbon source, followed by addition of α-lactose or cellobiose or a mixture of the two, EG1 levels recorded in the α-lactose-supplemented cultures were again markedly lower compared with cultures containing only α-lactose. Maximal enzyme levels in cultures with added cellobiose or cellobiose and α-lactose were not affected although appearance of EG1 in culture supernatants was again delayed by 12 h. Semi-quantitative RT-PCR revealed that higher, more prolonged, levels of eg1 transcription occurred in V. volvacea cultures induced with α-lactose compared with cellobiose- or α-lactose + cellobiose-induced cultures. However, eg1 transcription levels in cultures induced with cellobiose or with cellobiose + lactose, and the corresponding cultures with added sorbose, were not markedly different.

Molecular characterization of a new acetyl xylan esterase (AXEII) from edible straw mushroomVolvariella volvaceawith both de-O-acetylation and de-N-acetylation activity

A new Volvariella volvacea gene encoding a carbohydrate esterase (CE) family 4 acetyl xylan esterase (AXE) (designated as VvaxeII) was cloned and characterized. The coded polypeptide had 253 amino acid residues, with the first 19 serving as a secretion signal peptide. The VvaxeII transcript levels were high when the fungus was grown on oat spelt xylan, cellobiose, microcrystalline cellulose, carboxymethyl-cellulose, lactose, galactose, and chitin from crab as carbon sources. The recombinant VvAXEII produced by expression of VvaxeII in Pichia pastoris exhibited activity toward acetylated oat spelt xylan and various chitinous substrates, but was totally inactive against artificial aromatic acetates such as beta-nitrophenyl, 4-methylumbelliferyl, and alpha-naphthyl acetates. Enzyme-catalyzed hydrolysis was maximal at pH 7.0 and 60 degrees C, and reciprocal plots revealed an apparent K(m) value of 1.42 mg mL(-1) and a V(max) value of 833 IU micromol(-1) protein using glycol chitin as a substrate. The recombinant VvAXEII requires activation by bivalent cations such as Co2+ and Mg2+. Interestingly, the recombinant VvAXEII showed no deacetylation activity to fully acetylated monosaccharides such as xylose tetraacetate.

Molecular Cloning, Sequencing, and Expression of a l-Glutamine d-Fructose 6-Phosphate Amidotransferase Gene from Volvariella volvacea

Using 3'-RACE and 5'-RACE, we have cloned and sequenced the genomic gene and complete cDNA encoding L: -glutamine D: -fructose 6-phosphate amidotransferase (GFAT) from the edible straw mushroom, Volvariella volvacea. Gfat contains five introns, and encodes a predicted protein of 697 amino acids that is homologous to other reported GFAT sequences. Southern hybridization indicated that a single gfat gene locus exists in the V. volvacea genome. Recombinant native V. volvacea GFAT enzyme, over-expressed using Escherichia coli and partially purified, had an estimated molecular mass of 306 kDa and consisted of four equal-sized subunits of 77 kD. Reciprocal plots revealed K (m) values of 0.55 and 0.75 mM for fructose 6-phosphate and L: -glutamine, respectively. V. volvacea GFAT activity was inhibited by the end-product of the hexosamine pathway, UDP-GlcNAc, and by the glutamine analogues N (3)-(4-methoxyfumaroyl)-L: -2,3-diaminopropanoic acid and 2-amino-2-deoxy-D: -glucitol-6-phosphate.

Highly efficient Agrobacterium-mediated transformation of Volvariella volvacea

Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied to the edible straw mushroom, Volvariella volvacea. Mycelium pellets were transformed to cold stress resistance using the afp gene as both a selective marker and a reporter gene, under the control of a heterologous Lentinula edodes gpd promoter. The efficiency of transformation is over 100 times higher than that previously reported in V. volvacea. Stable integration of the afp gene with 1–4 copy numbers was confirmed in all 10 randomly selected transgenic events by Southern blot analysis. The mitotic stability of the transformants was demonstrated after five successive transfers on PDA medium without selection pressure and the PCR analysis of basidiospores harvested from transformants.

Molecular cloning and transcriptional expression analysis of an intracellular β-glucosidase, a family 3 glycosyl hydrolase, from the edible straw mushroom,Volvariella volvacea

A beta-glucosidase, with a molecular mass of 95 kDa, was isolated from extracts of Volvariella volvacea mycelium grown on crystalline cellulose. Degenerate primers based on the N-terminal sequences of purified beta-glucosidase and two protease-generated peptides were used to generate cDNA fragments encoding a portion of the beta-glucosidase gene (bgl), and rapid amplification of cDNA ends was used to obtain full-length cDNA clones. The cDNA of bgl contained an ORF of 2586 bp coding for 862 amino acids. Alignment of the deduced amino-acid sequence of beta-glucosidase with deduced amino acid sequences of other microbial beta-glucosidases showed the highest overall homology with glycoside hydrolase family 3 beta-glucosidases from fungi. Transcripts of bgl were detected in total RNA extracted from mycelium grown on cellulose and cellobiose, and from mycelium pre-grown for 72 h in basal medium containing 1% (w/v) sorbitol following addition of alpha-lactose, beta-lactose, cellobiose, d- xylose, l-sorbose, beta-gentiobiose, sophorose or d-galactose. Addition of l-sorbose and d-glucosamine to mycelium grown on 1% (w/v) crystalline cellulose greatly increased the level of bgl expression. bgl Was expressed at various stages of the mushroom developmental cycle (substrate colonization to mature fruit body), although the number of bgl transcripts in pinhead and button stages was slightly smaller.

Expression, Purification and Characterization of a Recombinant β-Glucosidase from Volvariella Volvacea

For the first time, a beta-glucosidase gene from the edible straw mushroom, Volvariella volvacea V1-1, has been over-expressed in E. coli. The gene product was purified by chromatography showing a single band on SDS-PAGE. The recombinant enzyme had a molecular mass of 380 kDa with subunits of 97 kDa. The maximum activity was at pH 6.4 and 50 degrees C over a 5 min assay. The purified enzyme was stable from pH 5.6-8.0, had a half life of 1 h at 45 degrees C. The beta-glucosidase had a K(m) of 0.2 mM: for p-nitrophenyl-beta-D: -glucopyranoside.

Corrigendum

European Journal of BiochemistryVolume 271, Issue 5 p. 1063-1063 Free Access Corrigendum First published: 17 February 2004 https://doi.org/10.1111/j.1432-1033.2004.04030.xCitations: 1AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL The paper by S. Chen et al. [1] contains errors on page 321. Under the section ‘Enzyme characterization’, the sentence ‘Guaiacol, catechol and 2,6-DMP were poor substrates for the enzyme (< 5% compared with ABTS) and no activity was detected with tyrosine, FA, l-3,4-dimethoxyphenol and dihydroxyphenylalanine (l-DOPA).’ should read: ‘Guaiacol, catechol and 2,3-DMP were poor substrates for the enzyme (< 5% compared with ABTS) and no activity was detected with tyrosine, FA, 3,4-dimethoxyphenol and l-3,4-dihydroxyphenylalanine (l-DOPA)’. Reference 1 Chen, S., Wei, G., Buswell, J.A. (2004) Biochemical and molecular characterization of a laccase from the edible straw mushroom, Volvariella volvacea. Eur. J. Biochem. 271, 318– 328. Citing Literature Volume271, Issue5March 2004Pages 1063-1063 ReferencesRelatedInformation

Biochemical and molecular characterization of a laccase from the edible straw mushroom, Volvariella volvacea.

We have isolated a laccase (lac1) from culture fluid of Volvariella volvacea, grown in a defined medium containing 150 micro m CuSO4, by ion-exchange and gel filtration chromatography. Lac1 has a molecular mass of 58 kDa as determined by SDS/PAGE and an isoelectric point of 3.7. Degenerate primers based on the N-terminal sequence of purified lac1 and a conserved copper-binding domain were used to generate cDNA fragments encoding a portion of the lac1 protein and RACE was used to obtain full-length cDNA clones. The cDNA of lac1 contained an ORF of 1557 bp encoding 519 amino acids. The amino acid sequence from Ala25 to Asp41 corresponded to the N-terminal sequence of the purified protein. The first 24 amino acids are presumed to be a signal peptide. The expression of lac1 is regulated at the transcription level by copper and various aromatic compounds. RT-PCR analysis of gene transcription in fungal mycelia grown on rice-straw revealed that, apart from during the early stages of substrate colonization, lac1 was expressed at every stage of the mushroom developmental cycle defined in this study, although the levels of transcription varied considerably depending upon the developmental phase. Transcription of lac1 increased sharply during the latter phase of substrate colonization and reached maximum levels during the very early stages (primordium formation, pinhead stage) of fruit body morphogenesis. Gene expression then declined to approximately 20-30% of peak levels throughout the subsequent stages of sporophore development.

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 58 173 Da and a p I 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.

Induction of laccase activity in the edible straw mushroom, Volvariella volvacea

Volvariella volvacea, strain V14, produces multiple forms of extracellular laccase when grown in submerged culture in a defined medium with glucose as sole carbon source, and on cotton waste ‘compost’ representative of the conditions used for industrial-scale mushroom cultivation. In liquid culture, enzyme synthesis is associated with the onset of secondary growth, and is positively regulated by copper (up to 200 μM CuSO4) and by various aromatic compounds. In solid-state systems, only low levels of laccase are detectable during the vegetative growth phase but enzyme activity increases sharply at the onset of fruiting and during sporophore development.

Secretion, purification and characterisation of a recombinant Volvariella volvacea endoglucanase expressed in the yeast Pichia pastoris

An endo-β-1,4-glucanase (EG1) from the edible straw mushroom, Volvariella volvacea, has been expressed in Pichia pastoris and the catalytic activity of the recombinant enzyme confirmed. Secretion was obtained using Saccharomyces cerevisiae α-factor, and a two-step purification protocol yielded EG1 in pure form. Recombinant EG1 had a molecular mass (45 kDa) slightly higher than the native enzyme (42 kDa) and an isoelectric point of 6.2. Recombinant and native EG1 also exhibited similar properties with respect to pH (pH 7.5) and temperature optima (55 °C), temperature and pH stability, substrate specificity, specific activities towards carboxymethylcellulose (344 and 324 U mg −1, respectively) and sensitivity to various metal ions, chelating agents and detergents. The P. pastoris system represents an attractive means of generating large quantities of endoglucanase for both research and industrial purposes.

Endoglucanase I from the edible straw mushroom, Volvariella volvacea.

We isolated an endoglucanase, EG1, from culture fluid of Volvariella volvacea grown on crystalline cellulose by ion-exchange and gel filtration chromatography, and preparative PAGE. EG1 has a molecular mass of 42 kDa as determined by SDS/PAGE and an isoelectric point of 7.7. Enzyme-catalysed hydrolysis of carboxymethyl-cellulose (CM-cellulose) is maximal at pH 7.5 and 55 degrees C. EG1 also hydrolysed phosphoric acid-swollen cellulose and filter paper (at rates of 29% and 6%, respectively, compared with CM-cellulose), but did not hydrolyse crystalline cellulose, cotton, oat spelt xylan, and birchwood xylan. Degenerate primers based on the N-terminal sequences of purified EGI and a protease-generated fragment were used to generate cDNA fragments encoding a portion of the EG1 gene (eg1), and RACE was used to obtain full-length cDNA clones. The cDNA of eg1 contained an ORF of 1167 bp encoding 389 amino acids. The amino-acid sequence from Ala24 to Thr40 corresponded to the N-terminal sequence of the purified protein. The first 23 amino acids are presumed to be a signal peptide. V. volvacea EG1 has been assigned to glycoside hydrolase family 5 according to the classification of glycohydrolases based on amino-acid sequence similarities. Transcripts of eg1 were detected in total RNA from mycelium grown on cellulose but not from mycelium grown on glucose. Cellobiose also induced eg1 expression in 1- to 4-day-old cultures but the signal intensity was lower than that obtained with cellulose. Catabolite repression was observed 24 h after addition of 1% (w/v) glucose, alpha-lactose, beta-lactose, xylose, mannose, sorbose or fructose to medium containing 1% (w/v) crystalline cellulose. Eg1 was expressed at a high level in the yeast, Pichia pastoris, and the catalytic activity of the recombinant EG1 was confirmed.

Production and distribution of endoglucanase, cellobiohydrolase, and beta-glucosidase components of the cellulolytic system of Volvariella volvacea, the edible straw mushroom.

The edible straw mushroom, Volvariella volvacea, produces a multicomponent enzyme system consisting of endo-1,4-beta-glucanase, cellobiohydrolase, and beta-glucosidase for the conversion of cellulose to glucose. The highest levels of endoglucanase and cellobiohydrolase were recorded in cultures containing microcrystalline cellulose (Avicel) or filter paper, while lower but detectable levels of activity were also produced on carboxymethyl cellulose, cotton wool, xylitol, or salicin. Biochemical analyses of different culture fractions in cultures exhibiting peak enzyme production revealed that most of the endoglucase was present either in the culture filtrate (45.8% of the total) or associated with the insoluble pellet fraction remaining after centrifugation of homogenized mycelia (32.6%). Cellobiohydrolase exhibited a similar distribution pattern, with 58.9% of the total enzyme present in culture filtrates and 31.0% associated with the pellet fraction. Conversely, most beta-glucosidase activity (63.9% of the total) was present in extracts of fungal mycelia whereas only 9.4% was detected in culture filtrates. The endoglucanase and beta-glucosidase distribution patterns were confirmed by confocal laser scanning microscopy combined with immunolabelling. Endoglucanase was shown to be largely cell wall associated or located extracellularly, with the highest concentrations being present in a region 1 to 2 microm wide immediately adjacent to the outer surface of (and possibly including) the hyphal wall and extending 60 to 70 microm from the hyphal tip. Immunofluorescence patterns indicated little if any intracellular endoglucanase. Most beta-glucosidase was located intracellularly in the apical area extending 60 to 70 microm below the hyphal tip, although enzyme was also evident in the extracellular region extending approximately 15 microm all around the hyphal tip and trailing back along the length of the hypha. The regions of the hypha located some distance from the apical region appeared to be devoid of intracellular beta-glucosidase, and the enzyme appears to be associated almost exclusively with, or located on the outside surface of, the hyphal wall.

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 K m values for p-nitrophenyl-β- d-glucopyranoside of 90 and 500 μ m, 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.