Cloning, expression and functional identification of a α-galactosidase gene from marine-derived Bacillus cereus
JIANG Chaofeng1,2, LI Kuikui1, LI Tang1, CHE Jia1,3, YIN Heng1*
1. Liaoning Provincial Key Laboratory of Carbohydrates, Dalian Engineering Research Central for Carbohydrate Agriculture Preparations, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian 116023, China; 2.School of Chemical Science，University of Chinese Academy of Sciences, Beijing 100049, China; 3.College of Food Science and Engineering，Dalian Ocean University, Dalian 116023, China
Abstract: A new α-galactosidase gene (BcgalA) was cloned from a marine derived Bacilluscereus using genetic engineering technology, and the open reading frame (ORF) of the gene encoding 441 amino acids was subcloned into Escherichiacoli BL21 (DE3) cells for expression through vector pET-28a in order to obtain highly efficient α-galactosidase and perform functional identification. The purified enzyme showed an apparent molecular weight of 50 380 on SDS-PAGE. Enzyme activity tests revealed that BcGalA against 4-nitrophenyl-α-D-galactopyranoside (pNP-α-Gal) had the specific activity of (1.498±0.034)U/mg and against raffinose (0.261±0.012)U/mg at the condition of 37 ℃ and pH 8.0. The enzyme has no activity toward galactomannan, indicating that it only worked on low molecular weight substrates. In conclusion, a new α-galactosidase gene (BcgalA) cloned and expressed here belonged to GH4 α-galactosidase that only worked on low molecular weight substrates. The finding provides a sample for exploring the relationship between the structure and function of GH4 α-galactosidase.
2021, Vol. 36 
