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XynII from Volvariella volvacea has high sodium dodecyl sulfate (SDS) resistance, with the potential for industrial applications under harsh conditions. It consists of a single glycoside hydrolase family 10 (GH10) catalytic domain but contains an additional unique 10 and 4 amino acid residues at the N- and C-terminus, respectively. In this study, five XynII derivatives with N- and/or C-terminus deletions were constructed to determine the effects of these regions on enzyme activity, substrate specificity, thermostability, and SDS resistance. Our results revealed that N- and/or C-terminal truncations significantly increased enzyme activity and thermostability, but reduced SDS resistance. Specifically, the XynIIΔNC4 mutant had 2.53-fold more catalytic efficiency ( k/ K) towards beechwood xylan than wild-type and 3.0-fold more thermostability ( t). XynIIΔNC4 displayed 3.33-, 4.38-, 1.37-, and 1.98-fold more activity against xylotriose, xylotetraose, xylopentaose, and xylohexaose, respectively, than XynII did. However, its half-life ( t) in 4 % SDS was only 1.72 h, while that of XynII was 4.65 h. Circular dichroism analysis revealed that deletion of N- and C-terminal segments caused minor changes in secondary structure. Our observations suggest that the extra N- and C-terminal segments in wild-type XynII evolved to strengthen the interaction between these regions of the protein, making the local structure more rigid and reducing structural flexibility. In this way, N- and C-terminal truncations increased the thermostability and activity of XynII on different xylans and linear xylooligosaccharides, but reduced its resistance to SDS. [ABSTRACT FROM AUTHOR]

Abstract: Recombinant Volvariella volvacea endoglucanase 1 (EG1) and its catalytic module (EG1-CM) were obtained by expression in Pichia pastoris, purified by two-step chromatography, and the catalytic activities and binding capacities were compared. EG1 and EG1-CM exhibited very similar specific activities towards the soluble substrates carboxymethyl cellulose, lichenan and mannan, and insoluble H3PO4 acid-swollen cellulose, whereas the specific activities of EG1-CM towards the insoluble substrates α-cellulose, Avicel and filter paper were approximately 58, 43 and 38%, respectively compared to EG1. No increase in reducing sugar release was detected in the reaction mixture supernatants after 50h exposure of filter paper, Avicel or α-cellulose to EG1-CM, whereas increases in the total reducing sugar equivalents (i.e. reducing sugar released into solution together with new reducing ends generated in the cellulosic substrates) in reaction mixtures were observed after 1h. In reaction mixtures containing EG1, soluble reducing sugar equivalents were detected in supernatants after 3h incubation with the insoluble cellulosic substrates. EG1-CM did not adsorb to Avicel, and the binding capacities of EG1-CM towards filter paper and H3PO4 acid-swollen cellulose were 27.9–33.3% and 29.6–60.6%, respectively of values obtained with EG1 within the range of total added protein. In enzymatic deinking experiments, the ink removal rate in EG1-CM-treated samples was only slightly higher (∼8%), than that of untreated controls, whereas that of the EG1-treated samples was 100% higher. Bio-stoning of denim with EG1-CM resulted in increases of 48% and 40% in weight loss and indigo dye removal, respectively compared with untreated controls. These increases were considerably lower than the corresponding values of 219% and 133% obtained when samples were treated with EG1. [Copyright &y& Elsevier]

A β-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 β-glucosidase and two protease-generated peptides were used to generate cDNA fragments encoding a portion of the β-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 β-glucosidase with deduced amino acid sequences of other microbial β-glucosidases showed the highest overall homology with glycoside hydrolase family 3 β-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 α-lactose, β-lactose, cellobiose,d- xylose,l-sorbose, β-gentiobiose, sophorose ord-galactose. Addition ofl-sorbose andd-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. [ABSTRACT FROM AUTHOR]

We used PCR-based methods to clone and sequence four previously unidentified cellulase cDNAs: cbhI-I, cbhI-II, cbhII-I and egII. CbhI-I, cbhI-II and cbhII-I consist of 1710, 1610 and 1453 bp, respectively, and encode for 512, 458 and 442 amino acids, respectively. EgII consists of 1180 bp encoding for 310 amino acids, and belongs to family 61 of the glycosyl hydrolases. CbhI-I, cbhII-I and egII all have a modular structure, with the catalytic domain (CD) and cellulose-binding domain (CBD) located at the C-terminus in cbhI-I and egII, and at the N-terminus in cbhII-I. CbhI-II shows high homology to cbhI-I but lacks a CBD. Northern blotting revealed that cbhI-I, cbhI-II and cbhII-I were coordinately expressed at various stages of the mushroom developmental cycle (substrate colonization to mature fruit body), although the number of cbhI-I transcripts was much smaller. No egII expression was detectable during the substrate colonization phase but transcription levels increased as fruit body morphogenesis progressed. [ABSTRACT FROM AUTHOR]

Abstract: High level expression of axe1, a gene previously cloned from Volvariella volvacea that encodes an acetyl xylan esterase with two potential N-linked glycosylation sites, has been achieved in Pichia pastoris using a codon-optimized axe1 synthesized by the primer extension PCR procedure. The GC content of the codon-optimized axe1 was 48.62% compared with 55.49% in the native gene. Using the codon-optimized construct, AXE1 expression in P. pastoris was increased from an undetectable level to 136.45U/ml six days after induction of yeast cultures grown in BMMY medium. A further increase (to 463U/ml) was achieved when conditions for yeast culture were optimized as follows: 2.8% methanol, 0.63% casamino acids, and pH 8.0. This latter value represented a 3.4-fold and 246-fold increase in the enzyme levels recorded in non-optimized P. pastoris cultures and in rice straw-grown cultures of V. volvacea, respectively. N-linked glycosylation played an essential role in AXE1 secretion but had only a slight effect on the catalytic activity and stability of the recombinant enzyme. [Copyright &y& Elsevier]

A gene encoding cellobiose dehydrogenase (VvCDH) from Volvariella volvacea was successfully expressed in Pichia pastoris with codon optimization using its native signal sequence. VvCDH had optimum pH and temperature at 5.5 and 60 °C respectively and showed a broad range of pH stability between 5 and 8. Kinetic analysis showed that the best substrate is cellobiose and that the K m for celloolgosaccharides increases with substrate length. Moreover, lactose is also efficiently oxidized, but glucose and maltose are poor substrates. A large amount of gluconic acid was generated and the overall hydrolysis yield was increased when adding VvCDH to Trichoderma reesei D-86271 enzymatic cocktail during hydrolysis of cellulose substrates, indicating VvCDH involved in the enzymatic cellulose saccharification. VvCDH shows some different enzymatic properties from basidiomycetous CDHs and can be supplemented to T. reesei cellulase cocktail for commercial application. [ABSTRACT FROM AUTHOR]