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A novel β-glucosidase gene, bgl1G5, was cloned from Phialophora sp. G5 and successfully expressed in Pichia pastoris. Sequence analysis indicated that the gene consists of a 1,431-bp open reading frame encoding a protein of 476 amino acids. The deduced amino acid sequence of bgl1G5 showed a high identity of 85% with a characterized β-glucosidase from Humicola grisea of glycoside hydrolase family 1. Compared with other fungal counterparts, Bgl1G5 showed similar optimal activity at pH 6.0 and 50 °C and was stable at pH 5.0-9.0. Moreover, Bgl1G5 exhibited good thermostability at 50 °C (6 h half-life) and higher specific activity (54.9 U mg⁻¹). The K (m) and V (max) values towards p-nitrophenyl β-D-glucopyranoside (pNPG) were 0.33 mM and 103.1 μmol min⁻¹ mg⁻¹, respectively. The substrate specificity assay showed that Bgl1G5 was highly active against pNPG, weak on p-nitrophenyl β-D-cellobioside (pNPC) and p-nitrophenyl-β-D-galactopyranoside (ONPG), and had no activity on cellobiose. This result indicated Bgl1G5 was a typical aryl β-glucosidase.

A cellulase-producing mesophilic fungal strain, named G5, was isolated from the acidic wastewater and mud of a tin mine and identified as Phialophora sp. based on the internal transcribed spacer sequence. The volumetric activities and specific activities of cellulase induced by different carbon sources (Avicel, corn cob, wheat bran and corn stover) were compared. The cellulase complex of Phialophora sp. G5 exhibited the optimal activities at 60-65 °C and pH 4.0-5.0, and had good long-term thermostability at 50 °C. Compared with the commercial cellulase (Accellerase 1500, Genencor), the enzyme under study showed 60% and 80% of the capacity to hydrolyze pure cellulose and natural cellulose, respectively. This is the first study to report that a cellulytic enzymes complex from Phialophora genus, and the superior properties of this enzyme complex make strain G5 a potential microbial source to produce cellulase for industrial applications, and the production ability could be improved by mutagenesis.