Abstract
Backgroundα-Galactosidases are enzymes that act on galactosides present in many vegetables, mainly legumes and cereals, have growing importance with respect to our diet. For this reason, the use of their catalytic activity is of great interest in numerous biotechnological applications, especially those in the food industry directed to the degradation of oligosaccharides derived from raffinose. The aim of this work has been to optimize the recombinant production and further characterization of α-galactosidase of Saccharomyces cerevisiae.ResultsThe MEL1 gene coding for the α-galactosidase of S. cerevisiae (ScAGal) was cloned and expressed in the S. cerevisiae strain BJ3505. Different constructions were designed to obtain the degree of purification necessary for enzymatic characterization and to improve the productive process of the enzyme. ScAGal has greater specificity for the synthetic substrate p-nitrophenyl-α-d-galactopyranoside than for natural substrates, followed by the natural glycosides, melibiose, raffinose and stachyose; it only acts on locust bean gum after prior treatment with β-mannosidase. Furthermore, this enzyme strongly resists proteases, and shows remarkable activation in their presence. Hydrolysis of galactose bonds linked to terminal non-reducing mannose residues of synthetic galactomannan-oligosaccharides confirms that ScAGal belongs to the first group of α-galactosidases, according to substrate specificity. Optimization of culture conditions by the statistical model of Response Surface helped to improve the productivity by up to tenfold when the concentration of the carbon source and the aeration of the culture medium was increased, and up to 20 times to extend the cultivation time to 216 h.ConclusionsScAGal characteristics and improvement in productivity that have been achieved contribute in making ScAGal a good candidate for application in the elimination of raffinose family oligosaccharides found in many products of the food industry.
Highlights
The raffinose family oligosaccharides (RFOs), consisting mainly of raffinose and stachyose, are complex sugars with one or more galactose residues joined by α-1,6glycosidic bonds to a sucrose
These α-galactosides function as reserve polysaccharides that are stored in the vacuoles of many vegetables, especially in legumes and cereals. α-Galactosidases (α-Gals; EC 3.2.1.22) can catalyse the release of α-d-galactosyl substituents from sugars, such as melibiose, raffinose and stachyose or even polymeric galactomannans
An α-Gal has even been reported that modifies the properties of gum Arabic that is widely used in food and non-food applications [7]. α-Gals can synthesize α-GOS through transglycosylation reactions that occur under supersaturation conditions of a substrate
Summary
The MEL1 gene coding for the α-galactosidase of S. cerevisiae (ScAGal) was cloned and expressed in the S. cerevisiae strain BJ3505. Different constructions were designed to obtain the degree of purification necessary for enzymatic characterization and to improve the productive process of the enzyme. ScAGal has greater specificity for the synthetic substrate p-nitrophenyl-α-d-galactopyranoside than for natural substrates, followed by the natural gly‐ cosides, melibiose, raffinose and stachyose; it only acts on locust bean gum after prior treatment with β-mannosidase. This enzyme strongly resists proteases, and shows remarkable activation in their presence. Optimization of culture conditions by the statistical model of Response Surface helped to improve the productivity by up to tenfold when the concentration of the carbon source and the aeration of the culture medium was increased, and up to 20 times to extend the cultivation time to 216 h
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