Characterization of an acidic α-galactosidase from hemp (Cannabis sativa L.) seeds and its application in removal of raffinose family oligosaccharides (RFOs)

  • Weiwei Zhang Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College
  • Fang Du State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University
  • Guoting Tian Institute of Biotechnology and Germplasmic Resource, Yunnan Academy of Agricultural Science, Kunming 650223, China
  • Yongchang Zhao Institute of Biotechnology and Germplasmic Resource, Yunnan Academy of Agricultural Science, Kunming 650223, China
  • Hexiang Wang State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University
  • Tzibun Ng School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China

Abstract

By means of chromatographic procedures which involved chromatography on the cation-exchangers CM-cellulose and SP-Sepharose, chromatography on the anion-exchangers DEAE-cellulose and Q-Sepharose, and gel filtration on Superdex 75 by fast protein liquid chromatography, an acidic α-galactosidase designated as hemp seed α-galactosidase (HSG) was purified from hemp (Cannabis sativa L.) seeds. Results of SDS-PAGE and gel filtration on FPLC Superdex 75 disclosed that the enzyme was a monomeric protein with a molecular weight of 38 kDa. Sequences of the inner peptides of the α-galactosidase obtained by MALDI-TOF-MS showed that HSG was a novel α-galactosidase since there was little similarity to the majority of α-galactosidases recorded in the literature. A pH of 3.0 and a temperature of 50 ℃ were optimal for the activity of  the enzyme. The activity of HSG was inhibited by the chemical modification reagent N-bromosuccinimide (NBS). HSG contained 16 tryptophan residues and two tryptophan residues on the surface, which are crucial to the α-galactosidase activity. The heavy metal ions Cd2+, Cu2+, Hg2+ and Zn2+ ions inhibited its activity. The Km and Vmax for hydrolysis of pNPGal (4-nitrophenyl α-D-galactopyranoside) were respectively 0.008 mM and 68 μM min-1 mg-1. HSG also catalyzed hydrolysis of raffinose and other natural substrates. Hence the α-galactosidase possesses tremendous potential in food and feed industries for elimination of indigestible oligosaccharides from leguminous products.
Published
2018-09-08
Section
Articles