Characterization of an acidic α-galactosidase from hemp (<i>Cannabis sativa</i> 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
  • Yongchang Zhao
  • Hexiang Wang State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University
  • Tzibun Ng

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