Acta Entomologica Sinica ›› 2020, Vol. 63 ›› Issue (9): 1039-1047.doi: 10.16380/j.kcxb.2020.09.001

• RESEARCH PAPERS •     Next Articles

Cloning of the β-glucosidase gene RpBg7 from Reticulitermes perilucifugus (Blattariae: Rhinotermitidae) and its expression in Pichia pastoris

SU Li-Juan1, XIAO Yuan-Xi1, LI Yan1, WU Zhi-Wei1, ZHAO Peng-Fei1, CHU Jun-Peng1, WEI Ji-Zhen2, AN Shi-Heng2, YIN Xin-Ming2, SONG An-Dong1,*   

  1. (1. College of Life Sciences of Henan Agricultural University, Zhengzhou 450002, China; 2. College of Plant Protection, Henan Agricultural University, Zhengzhou 450002, China)
  • Online:2020-09-20 Published:2020-09-30

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 pastorislignocellulose