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

• Published in: Acta biochimica Polonica Vol. 65; no. 3; p. 383
• Main Authors: Zhang, Weiwei, Du, Fang, Tian, Guoting, Zhao, Yongchang, Wang, Hexiang, Ng, Tzi Bun
• Format: Journal Article
• Language: English
• Published: Poland 01-01-2018
• Subjects: alpha-Galactosidase - antagonists & inhibitors; alpha-Galactosidase - chemistry; alpha-Galactosidase - isolation & purification; Bromosuccinimide - chemistry; Cannabis - enzymology; Chromatography, Liquid - methods; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors - pharmacology; Hot Temperature; Hydrogen-Ion Concentration; Hydrolysis; Metals, Heavy - pharmacology; Molecular Weight; Nitrophenylgalactosides - chemistry; Raffinose - chemistry; Raffinose - isolation & purification; Seeds - enzymology; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tryptophan - analysis; raffinose family oligosaccharides; Cannabis sativa L; purification; α-galactosidases
• ISSN: 0001-527X; 1734-154X
• DOI: 10.18388/abp.2017_1535

An acidic α-galactosidase designated as hemp seed α-galactosidase (HSG) was purified from hemp (Cannabis sativa L.) seeds. 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 using fast protein liquid chromatography, HSG was purified to electrophoretic homogeneity. Results of SDS-PAGE and gel filtration on FPLC Superdex 75 revealed 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 a little similarity to the majority of α-galactosidases recorded in the literature. A pH of 3.0 and a temperature of 50°C were optimal for the activity of the enzyme. The activity of HSG was inhibited by the chemical modification with N-bromosuccinimide (NBS) reagent. HSG contained 16 tryptophan residues and two tryptophan residues on the surface, which were crucial to the α-galactosidase activity. The heavy metal ions Cd
, Cu
, Hg
and Zn
inhibited its activity. The K
and V
for the hydrolysis of pNPGal (4-nitrophenyl α-D-galactopyranoside) were respectively 0.008 mM and 68 μM min
mg
. HSG also catalyzed the hydrolysis of raffinose and other natural substrates. Hence the α-galactosidase possesses a tremendous potential for food and feed industries in the elimination of indigestible oligosaccharides from leguminous products.