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

Abstract

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.

A statistical strategy for optimizing the production of α-galactosidase by a newly isolated Aspergillus niger NRC114 and assessing its efficacy in improving soymilk properties

“…Furthermore, fungi are well-suited for the creation of natural food additives such as colorants and stabilizers, which pose fewer health concerns than synthetic food additives, as well as bioactive metabolites for pharmaceutical usage such as enzymes, statins, and anticancer agents [10]. In addition, α-galactosidase could be obtained by extraction and purification from germinating guar seeds [11], Cannabis sativa L. seeds [12], in bacteria, Pseudomonas sp. [13], and Sulfolobus solfataricus [14].…”

Engineering a Trypsin-Resistant Thermophilic α-Galactosidase to Enhance Pepsin Resistance, Acidic Tolerance, Catalytic Performance, and Potential in the Food and Feed Industry

“…α-Galactosidases can catalyze the removal of α-linked terminal galactose residues from galactooligosaccharides and polysaccharides and have potential applications in food and feed industries . α-Galactosidase production and activity have been reported in fungi, bacteria, plants, and insects. − Most known α-galactosidases belong to glycoside hydrolase (GH) families 27 and 36 . Some α-galactosidases are specific for 4-nitrophenyl α-galactopyranoside ( p NPG), 2-nitrophenyl β-D-galactopyranoside, 4-nitrophenyl β-D-glucuronide, melibiose, maltose, raffinose, or stachyose, , and some had high enzyme activity for polymers guar gum, locust bean gum, carob galactomannan, or gum arabic. , …”

Optimization of Saccharomyces cerevisiae α-galactosidase production and application in the degradation of raffinose family oligosaccharides

“…There are many references to α-Gals isolated from different organisms; many of them have been identified in fungi [22, 23, 32–36], bacteria [29, 37–40], plants [41], and even in the gut of insects [42], and have been characterized because of the big industrial potential for the hydrolysis of RFOs and/or galactomannans. However, there are more current works that have informed α-Gals from original sources [22, 23, 29, 33–36, 41] than the use of expression hosts [32, 37–40, 42] for the enhanced production of enzyme. Moreover, in most cases, the enzyme purification process requires many steps, but there are few studies that have optimized overexpression α-Gals production conditions for cost-effective use on an industrial scale [43–45].…”