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

Abstract

Background α-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. Results 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 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. Conclusions ScAGal 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.

Development of a whole-cell biocatalyst for diisobutyl phthalate degradation by functional display of a carboxylesterase on the surface of Escherichia coli

“…Surface display systems mediated by the full length or truncated INP anchors from P. syringae have been extensively exploited in E. coli, Pseudomonas sp., and other species. In the future, other anchors, such as outer membrane protein A (OmpA), OmpC, or OmpF, and so on, and other surface-displayed microorganisms, Saccharomyces cerevisiae, for example, are deserved to be determined [33,34]. In addition, the green fluorescence was concentrated at both poles or on outer membrane of E. coli BL21(DE3) strain carrying pET-28a(+)/inpn/ carEW/gfp plasmid, while GFP distributed evenly for E. coli BL21(DE3) strain carrying pET-28a(+)/carEW/ gfp plasmid, without the INP anchor motif (Fig.…”

Intra-Laboratory Validation of Alpha-Galactosidase Activity Measurement in Dietary Supplements

“…gut proteases should be excluded and highly efficient protease-resistant α-galactosidases are thus of great interest in the food industry. However, to date only a few α-galactosidases have been identified with such properties [ 15 , 17 , 18 , 19 ] and have mainly been isolated from fungi [ 22 , 23 , 24 , 25 ]. As most of alpha-galactosidases in the food industry are obtained from probiotic bacteria like bifido and lactic acid bacteria [ 16 , 17 , 19 , 21 , 22 ], and due to the fact that a large number of these are sensitive to gut proteases, the maximum activity of commercial α-galactosidases should be assured to the customer.…”

“…However, to date only a few α-galactosidases have been identified with such properties [ 15 , 17 , 18 , 19 ] and have mainly been isolated from fungi [ 22 , 23 , 24 , 25 ]. As most of alpha-galactosidases in the food industry are obtained from probiotic bacteria like bifido and lactic acid bacteria [ 16 , 17 , 19 , 21 , 22 ], and due to the fact that a large number of these are sensitive to gut proteases, the maximum activity of commercial α-galactosidases should be assured to the customer. To date, few studies have been carried out on α-galactosidase determination in dietary supplements [ 16 , 17 , 18 , 21 , 23 , 24 ].…”

“…However, only a few protease-resistant α-galactosidases have been identified, and most of them are isolated from fungi [ 16 , 17 , 18 , 19 , 20 , 21 ]. A typical example of a commercial α-galactosidase is that derived from Aspergillus niger mold, which is able to enact its function in the gastrointestinal tract by breaking down specific non-absorbable oligosaccharides before they are metabolized by colonic bacteria [ 15 , 17 , 18 , 19 , 22 , 23 , 24 , 25 ].…”

“…A typical example of a commercial α-galactosidase is that derived from Aspergillus niger mold, which is able to enact its function in the gastrointestinal tract by breaking down specific non-absorbable oligosaccharides before they are metabolized by colonic bacteria [15,[17][18][19][22][23][24][25].…”