Bioconversion of Beet Molasses to Alpha-Galactosidase and Ethanol

Molasses are sub-products of the sugar industry, rich in sucrose and containing other sugars like raffinose, glucose, and fructose. Alpha-galactosidases (EC. 3.2.1.22) catalyze the hydrolysis of alpha-(1,6) bonds of galactose residues in galacto-oligosaccharides (melibiose, raffinose, and stachyose) and complex galactomannans. Alpha-galactosidases have important applications, mainly in the food industry but also in the pharmaceutical and bioenergy sectors. However, the cost of the enzyme limits the profitability of most of these applications. The use of cheap sub-products, such as molasses, as substrates for production of alpha-galactosidases, reduces the cost of the enzymes and contributes to the circular economy. Alpha-galactosidase is a specially indicated bioproduct since, at the same time, it allows to use the raffinose present in molasses. This work describes the development of a two-step system for the valuation of beet molasses, based on their use as substrate for alpha-galactosidase and bioethanol production by Saccharomyces cerevisiae. Since this yeast secretes high amounts of invertase, to avoid congest the secretory route and to facilitate alpha-galactosidase purification from the culture medium, a mutant in the SUC2 gene (encoding invertase) was constructed. After a statistical optimization of culture conditions, this mutant yielded a very high rate of molasses bioconversion to alpha-galactosidase. In the second step, the SUC2 wild type yeast strain fermented the remaining sucrose to ethanol. A procedure to recycle the yeast biomass, by using it as nitrogen source to supplement molasses, was also developed.