Efficiency Analysis and Mechanism Insight of that Whole-Cell Biocatalytic Production of Melibiose from Raffinose with Saccharomyces cerevisiae

Abstract

Melibiose is widely used as a functional carbohydrate. Whole-cell biocatalytic production of melibiose from raffinose could reduce its cost. However, characteristics of strains for whole-cell biocatalysis and mechanism of such process are unclear. We compared three different Saccharomyces cerevisiae strains (liquor, wine, and baker's yeasts) in terms of concentration variations of substrate (raffinose), target product (melibiose), and by-products (fructose and galactose) in whole-cell biocatalysis process. Distinct difference was observed in whole-cell catalytic efficiency among three strains. Furthermore, activities of key enzymes (invertase, α-galactosidase, and fructose transporter) involved in process and expression levels of their coding genes (suc2, mel1, and fsy1) were investigated. Conservation of key genes in S. cerevisiae strains was also evaluated. Results show that whole-cell catalytic efficiency of S. cerevisiae in the raffinose substrate was closely related to activity of key enzymes and expression of their coding genes. Finally, we summarized characteristics of producing strain that offered advantages, as well as contributions of key genes to excellent strains. Furthermore, we presented a dynamic mechanism model to achieve some mechanism insight for this whole-cell biocatalytic process. This pioneering study should contribute to improvement of whole-cell biocatalytic production of melibiose from raffinose.

Metabolite Changes in Indonesian Tempe Production from Raw Soybeans to Over-Fermented Tempe

“…Invertase and α-galactosidase hydrolyze raffinose to produce melibiose. Thus, in FF (24 h), melibiose suddenly increased and then gradually decreased as microorganisms utilized it [ 35 ]. In this study, glucose, fructose, and galactose were also detected in the process, similar to the previous report that glucose was the main substrate for fermenting microorganism [ 31 ].…”

Enzymatic Production of Melibiose from Raffinose by the Levansucrase from Leuconostoc mesenteroides B-512 FMC

“…Dietary melibiose effectively suppresses the Th2 response and improves the induction of oral tolerance . Particularly, melibiose can be used as a high-value additive to human functional foods and pharmaceuticals to maintain and promote good health because of its strong ability to function as a prebiotic …”

“…That is to say, levansucrase could play an important role in synthesizing kinds of fructo-oligosaccharide according to different fructosyl acceptors, including levan (β-2,6-fructan), melibiose, raffinose, lactosucrose, and so on. However, the costs of enzymatically producing melibiose from raffinose through hydrolysis are too high, , and although the proposed solution of using whole-cell catalytic technology instead of enzymatic hydrolysis can reduce the cost, detailed knowledge of the reaction mechanism of whole-cell catalysis and effective methods to evaluate production by the strain remained unclear …”

“…6 Particularly, melibiose can be used as a high-value additive to human functional foods and pharmaceuticals to maintain and promote good health because of its strong ability to function as a prebiotic. 7 Difficult to obtain by other methods, melibiose is mainly produced through an enzymatic transglycosylation reaction with raffinose and lactose or galactose as substrates (Figure 1). Among these enzymes, β-fructofuranosidase (also called invertase, EC 3.2.1.26), a member of glycoside hydrolase family 68 (GH68) that catalyzes the hydrolysis and transfructosylation of sucrose, has been reported to catalyze the hydrolysis of raffinose (α-D-galactopyranosyl-(1→6)-α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside) to produce melibiose.…”

“…However, the costs of enzymatically producing melibiose from raffinose through hydrolysis are too high, 22,23 and although the proposed solution of using whole-cell catalytic technology instead of enzymatic hydrolysis can reduce the cost, detailed knowledge of the reaction mechanism of whole-cell catalysis and effective methods to evaluate production by the strain remained unclear. 7 L. mesenteroides, belonging to Lactobacillaceae, is a Grampositive, facultative anaerobe, chemoorganotroph, and catenation-shaped bacterium. Many Leuconostoc strains can produce polymers, such as dextrans and levans, which have a wide variety of commercial applications, such as dextransucrase from L. mesenteroides NRRL B-512F 24 and L. mesenteroides IBT-PQ, 25 alternansucrases from L. mesenteroides B-1355, 26 and levansucrase from L. mesenteroides B-512 FMC.…”