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An endo-β-1,4-glucanase gene, egG5, was cloned from the fungus Phialophora sp. G5. The 1,290-bp open reading frame encodes a bimodular cellulase composed of an N-terminal family 1 carbohydrate-binding module (CBM) and a C-terminal family 5 glycoside hydrolase catalytic module. Recombinant EgG5 produced in Pichia pastoris exhibited maximal activity at pH 4.0-5.0 and 70 °C, retained 40% of the maximal activity at pH 2.0, and was stable at pH 2.0-10.0. When compared with its closest homolog in Trichoderma sp. C-4 (70.6% identity), EgG5 had better thermostability (51.6% activity at 65 °C for 12 h vs 10% activity at 60 °C for 20 min). Sequence-structure analysis indicated that the distinct β-sheet in EgG5 in place of a linking loop in Trichoderma sp. C-4 endoglucanase might be the reason. To verify its function, two mutants, EgG5-Mut (disrupting the β-sheet with four amino acid substitutions) and EgG5-CBM (removing the CBM), were constructed, expressed in P. pastoris, and characterized. Both mutants had similar pH optima (pH 4.0) and temperature optima (70 °C) but varied in pH stabilities (pH 2.0-10.0 and pH 2.0-7.0, respectively) and thermostabilities. The thermostability of EgG5-Mut (13.4% activity vs 52.5% of EgG5 at 65 °C for 12 h) confirmed the effect of β-sheet on enzyme thermostability. EgG5-CBM was more thermostable (94.9% activity at 65 °C for 12 h and 15.5% activity at 80 °C for 30 min) and had higher specific activity (711.6 vs 60.3 U mg(-1) of EgG5). This study presents an excellent endoglucanase with potential use in the bioconversion of lignocellulosic materials and provides good ideas for the improvement of enzyme thermostability.

An endo-β-1,4-xylanase gene, designated xyn10G5, was cloned from Phialophora sp. G5 and expressed in Pichia pastoris. The 1,197-bp full-length gene encodes a polypeptide of 399 amino acids consisting of a putative signal peptide at residues 1-20, a family 10 glycoside hydrolase domain, a short Gly/Thr-rich linker and a family 1 carbohydrate-binding module (CBM). The deduced amino acid sequence of XYN10G5 shares the highest identity (53.4%) with a putative xylanase precursor from Aspergillus terreus NIH2624. The purified recombinant XYN10G5 exhibited the optimal activity at pH 4.0 and 70 °C, remained stable at pH 3.0-9.0 (>70% of the maximal activity), and was highly thermostable at 70 °C (retaining ~90% of the initial activity for 1 h). Substrate specificity studies have shown that XYN10G5 had the highest activity on soluble wheat arabinoxylan (350.6 U mg(-1)), and moderate activity to various heteroxylans, and low activity on different types of cellulosic substrates. Under simulated gastric conditions, XYN10G5 was stable and released more reducing sugars from soluble wheat arabinoxylan; when combined with a glucanase (CelA4), the viscosity of barley-soybean feed was significantly reduced. These favorable enzymatic properties make XYN10G5 a good candidate for application in the animal feed industry.

Based on conserved amino-acid regions predicted for the chitin synthases (Chs) of Saccharomyces cerevisiae, two different primer sets were synthesized and used in polymerase chain reactions (PCRs) to amplify 614-bp and 366-bp sequences from genomic DNA of the zoopathogenic fungus Phialophora verrucosa. DNA-sequencing and Southern-blotting analyses of the 614-bp DNA amplification products suggested that portions of two distinct P. verrucosa chitin synthase genes (PvCHS1, PvCHS2), coding for two different zymogenic-type PvChs isozymes, had been identified. The deduced amino-acid sequence of each fell into different Chs classes, namely class I and class II. In addition, the 366-bp DNA segment was shown to code for a conserved region having homology with the CSD2/CAL1 gene of S. cerevisiae, which encodes a nonzymogenic-type enzyme, Chs3, in that fungus. The amino-acid sequence derived from PvCHS3 exhibits 88.2% similarity and 78.4% identity to the same amino-acid region of the S. cerevisiae enzyme. These results provide a critical first step toward investigating the molecular and pathogenic importance of CHS gene regulation in this fungus and for exploring steps leading to Chs function as potential targets for the design of new therapeutic agents.