关键词: 近暗散白蚁, β-葡糖苷酶, 纤维素酶, 毕赤酵母, 木质纤维素

Abstract:  【Aim】 Termites are the organisms with the high efficiency to use lignocellulose in nature, and are the natural resource reservoir of cellulases. This study aims to explore cellulase genes of new sources and to provide new natural enzymes for efficient utilization of biomass energy. 【Methods】 Based on the previous proteome analysis, the full-length cDNA sequence of β-glucosidase 7 gene (RpBg7) was cloned from Reticulitermes perilucifugus by using RCR and RACE technology, and the RpBg7 sequence was analyzed by bioinformatic software. The RpBg7 protein was expressed with expression vector pPICZαA in Pichia pastoris X-33, and the enzyme activity of the expressed RpBg7 protein was assayed with 4-nitrophenyl β-d-glucopyranoside (4pNPG) as the substrate. 【Results】 We obtained an endogenous β-glucosidase 7 gene, RpBg7 (GenBank accession no: MN944395), from R. perilucifugus. Its open reading frame is 1 485 bp in length, encoding 495 amino acids. The RpBg7 protein has the predicated molecular weight of 57 kD and belongs to glycoside hydrolase 1 (GH1) family with conservative basic amino acid residues Glu187 and Glu394. The RpBg7 protein was successfully expressed in the P. pastoris expression system. The enzyme activity assay results showed that the enzyme activity of RpBg7 in the crude solutions of extracellular secretion proteins and intracellular proteins of P. pastoris was 4.43 and 7.47 U/mL, respectively. 【Conclusion】 The β-glucosidase 7 gene of GH1 in R. perilucifugus was cloned and expressed in P. pastoris, providing conditions for the modification and application of cellulases afterwards.

Key words: Reticulitermes perilucifugus, β-glucosidase, cellulase; Pichia pastoris; lignocellulose