Bioconversion of Beet Molasses to Alpha-Galactosidase and Ethanol

Abstract

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.

Keywords: Saccharomyces cerevisiae; alpha-galactosidase; beet molasses; bioconversion; ethanol; invertase.

Figure 1

Construction of S. cerevisiae strain BJ3505Δsuc2. (A) The sequence [36,185–40,044] bp of chromosome 9 of S. cerevisiae (Gene ID: 854644; NC_001141.2) containing the SUC2 ORF was chosen to carry out the suc2Δ(266–388) deletion by replacement with the URA3 cassette between sites BamHI and KpnI. SR1 and SR2 are the chromosomal recombination sequences and the primers pairs P5-P6, P7-P8, and P5-P8 are used to obtain the fragments 2,419, 2,110, and 4,845 bp, respectively to check the chromosomal integration. (B) Physical maps of the plasmids constructed to generate strain BJ3505Δsuc2: the URA3 cassette from pSparkURA3 was cloned between the BamHI-KpnI sites of pJETSUC2 to obtain pJETsuc2Δ(266–388)URA3, which was digested with BglII to release the suc2Δ(266–388)URA3 cassette that was integrated through SR1 and SR2 sequences into genome of the origin strain.

Figure 2

Verification of S. cerevisiae strain BJ3505Δsuc2. (A) Negative (yellow color) and positive (orange color) functional assay of extracellular (1) and intracellular invertase activity (2) as described in section Analytical Methods. N, strain BJ3505Δsuc2; P, strain BJ3505 (parental); B, blank. (B) PCR analysis of the “upstream” (2,419 bp, lane 1), “downstream” (2,110 bp, lane2), and between them (4,845 bp, lane 4) of the SUC2 gene that codes for the invertase. Lane 3, GeneRuler 1Kb DNA Ladder (Thermo Fisher Scientific).

Figure 3

Time course of extracellular alpha-galactosidase activity using a conventional culture medium. Cultures in the YPHSM medium were inoculated to an OD₆₀₀ of 0.5 from pre-cultures of strains BJ3505Δsuc2 (empty symbol) and BJ3505 (full symbol) transformed with the plasmids YEpMEL1 (circle), and YEpMEL1His (triangle).

Figure 4

Evaluation of culture media based on beet molasses for ScAGal production. Time course of residual sugar (A) and extracellular alpha-galactosidase activity (B) by BJ3505Δsuc2 (empty symbol) and BJ3505 (full symbol) in culture media YR (triangle) and PR (circle). The solid and dashed lines correspond to the strains transformed with the plasmid YEpMEL1 and YEpFLAG-1, respectively. The media YR and PR were prepared with 8% beet molasses supplemented with 1% yeast extract or 2% peptone and the cultures were inoculated to OD₆₀₀ = 2.

Figure 5

Growth kinetic and conversion of substrates to products of strains BJ3505Δsuc2/YEpMEL1 and BJ3505/YEpMEL1. Biomass (circles), total sugar (triangles), reducing sugar (squares), ethanol (green circles), extracellular alpha-galactosidase activity (blue circles), and extracellular invertase activity (blades) of cultures BJ3505Δsuc2/YEpMEL1 (solid line) and BJ3505/YEpMEL1 (dashed line). The cultures were inoculated in the YR production medium (8% beet molasses, 1% yeast extract) (Phase I). After a period of growth of 40 h, part of each of the cultures was separated and cooled with 1% yeast extract (Phase II) while the rest remained in the same conditions of phase I. The free medium of cells recovered from final cultures of BJ3505Δsuc2/YEpMEL1 was reused as a production medium using strain BJ3505/YEpMEL1 (Phase III). In each phase the same initial cell density was maintained (OD₆₀₀ = 4).

Figure 6

Visualization of cultures of BJ3505Δsuc2/YEpMEL1 and BJ3505/YEpMEL1 by optical microscopy (A) and TEM (B). Va, vacuole; Pc, Cytoplasmic particles; Nu, nucleus; Mi, mitochondria; Vs, secretion vesicles.

Figure 7

Pareto graphic and main interactions on the production of ScAGal. (A) Independent variables on the response at the 95% level of significance (vertical line) before remove the statistically insignificant effects. (B) Interaction Time/Inoculum effect, where the time varies from −1 to +1 while the inoculum remains constant at the value +1 (up line) and −1 (down line).

Figure 8

Response surface-contour plots for extracellular ScAGal production by the strain BJ3505Δsuc2/YEpMEL1 on beet molasses based medium using RSM. Estimated extracellular alpha-galactosidase activity (ScAGal) as function of (A) inoculum size (I) and time (T); (B) beet molasses (M) and time (T); (C) beet molasses (M) and inoculum size (I); (D) beet molasses (M) and yeast extract (YE); (E) yeast extract (YE) and time (T); (F) yeast extract (YE) and inoculum size (I). The values of the third and fourth variables were remained constant at level 0.

Figure 9

Cultivation of strain BJ3505Δsuc2/YEpMEL1 in bioreactors under controlled pH conditions. (A) Time course of cellular biomass (black circles), extracellular alpha-galactosidase activity (blue circles), and pH (black dashed line) in optimized YR (Bioreactor 1 and 2) and YPHSM (Bioreactor 3). The production of ethanol (gray dashed line) is shown in bioreactors 1 and 2. (B) Macroscopic visualization of cell colonies from ended-bioreactors, at pH 9 and pH 6, and re-seeding on selective medium CM-Trp. Re-seeding: 1, BJ3505/YEpMEL1 in YPHSM (pH 7); 2, BJ3505ΔSuc2/YEpMEL1 in YPHSM (pH 7); 3, BJ3505ΔSuc2/YEpMEL1 in YR (pH 6); 4, BJ3505ΔSuc2/YEpMEL1 in YR (pH 9).

Figure 10

Simulation of a two-step bioprocess to obtain ScAGaL and ethanol from beet molasses-based medium. The ended-culture of the fermentation 1 carried out by the invertase-deficient strain BJ3505Δsuc2/YEpMEL1 is centrifuged, and the extracellular medium filtered by TTF to obtain a permeate with the recombinant protein, ScAGal (Step 1). The sucrose recovered in the filtrate is used in the fermentation 2 to produce ethanol by a strain of S. cerevisiae with invertase activity (Step 2). The biomass recovered from both fermentations can be used as yeast extract (YE) supplement after an autolysis treatment. M, beet molasses; YE, yeast extract; TFF, tangential flow filtration. Modificated imagen from SuperPro Designer.