Levan versus fructooligosaccharide synthesis using the levansucrase from Zymomonas mobilis: effect of reaction conditions

This work addresses the effect of sucrose concentration and temperature on the three activities displayed by the levansucrase from Zymomonas mobilis: formation of levan (polymerization), production of short-chain fructooligosaccharides (FOS), and sucrose hydrolysis. Of the conditions tested, levan formation reached the highest value at 4ºC and 100 g/L sucrose. The increase of temperature (40ºC) and sucrose concentration (600 g/L) caused a significant decrease of the levan concentration and a higher production of FOS. However, an increase of the temperature also caused an enhancement of the undesired hydrolytic activity. Several inulin-type FOS (1-kestose, nystose, 1F-fructosylnystose), neoFOS (blastose, neokestose, neonystose) and levan-type FOS (6-kestose, 6,6-nystose) were synthesized by levansucrase. The latter compound was purified and characterized by mass spectrometry and 2D NMR. Using 600 g/L sucrose at 40°C, the maximum yield of FOS was reached at 85% sucrose conversion; at this point, the reaction mixture contained (in weight basis) 31% glucose, 14% fructose, 15% sucrose and 40% FOS (including a small contribution of levan).This work was supported by a grant from the Spanish Ministry of Economy and Competitiveness (BIO2013-48779-C4-1-R). We thank the support of COST-Action CM1303 on Systems Biocatalysts. We thank Amano Enzyme Inc. (Japan) for the kind donation of the levansucrase. We thank Beneo Iberica for the generous supply of an inulin sample (Raftiline). P. S-M. thanks the Spanish Ministry of Education for FPU Grant.Peer reviewe

Continuous Packed Bed Reactor with Immobilized β-Galactosidase for Production of Galactooligosaccharides (GOS)

The β-galactosidase from Bacillus circulans was covalently attached to aldehyde-activated (glyoxal) agarose beads and assayed for the continuous production of galactooligosaccharides (GOS) in a packed-bed reactor (PBR). The immobilization was fast (1 h) and the activity of the resulting biocatalyst was 97.4 U/g measured with o-nitrophenyl-β-d-galactopyranoside (ONPG). The biocatalyst showed excellent operational stability in 14 successive 20 min reaction cycles at 45 °C in a batch reactor. A continuous process for GOS synthesis was operated for 213 h at 0.2 mL/min and 45 °C using 100 g/L of lactose as a feed solution. The efficiency of the PBR slightly decreased with time; however, the maximum GOS concentration (24.2 g/L) was obtained after 48 h of operation, which corresponded to 48.6% lactose conversion and thus to maximum transgalactosylation activity. HPAEC-PAD analysis showed that the two major GOS were the trisaccharide Gal-β(1→4)-Gal-β(1→4)-Glc and the tetrasaccharide Gal-β(1→4)-Gal-β(1→4)-Gal-β(1→4)-Glc. The PBR was also assessed in the production of GOS from milk as a feed solution. The stability of the bioreactor was satisfactory during the first 8 h of operation; after that, a decrease in the flow rate was observed, probably due to partial clogging of the column. This work represents a step forward in the continuous production of GOS employing fixed-bed reactors with immobilized β-galactosidases

A three-step process for the bioconversion of whey permeate into a glucose-free D-tagatose syrup

We have developed a sustainable three-stage process for the revaluation of cheese whey permeate into D-tagatose, a rare sugar with functional properties used as sweetener. The experimental conditions (pH, temperature, cofactors, etc.) for each step were independently optimized. In the first step, concentrated whey containing 180–200 g/L of lactose was fully hydrolyzed by β-galactosidase from Bifidobacterium bifidum (Saphera®) in 3 h at 45 °C. Secondly, glucose was selectively removed by treatment with Pichia pastoris cells for 3 h at 30 °C. The best results were obtained with 350 mg of cells (previously grown for 16 h) per mL of solution. Finally, L-arabinose isomerase US100 from Bacillus stearothermophilus was employed to isomerize D-galactose into D-tagatose at pH 7.5 and 65 °C, in presence of 0.5 mM MnSO4. After 7 h, the concentration of D-tagatose was approximately 30 g/L (33.3% yield, referred to the initial D-galactose present in whey). The proposed integrated process takes place under mild conditions (neutral pH, moderate temperatures) in a short time (13 h), yielding a glucose-free syrup containing D-tagatose and galactose in a ratio 1:2 (w/w).This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (Grants BIO2016-76601-C3-1-R and C3-2-R) and Fundación Ramón Areces (XIX Call of Research Grants in Life and Material Sciences). We thank Fundación Ramón Areces for an institutional grant to the Center of Molecular Biology Severo Ochoa. F.V. Cervantes thanks CONACYT (Mexico) for her Ph.D. fellowship (Ref. 440242)

Production of galacto-oligosaccharides by the β-galactosidase from kluyveromyces lactis : comparative analysis of permeabilized cells versus soluble enzyme

8 páginas, 5 figuras, 3 tablas -- PAGS nros. 10477-10484The transgalactosylation activity of Kluyveromyces lactis cells was studied in detail. Cells were permeabilized with ethanol and further lyophilized to facilitate the transit of substrates and products. The resulting biocatalyst was assayed for the synthesis of galacto-oligosaccharides (GOS) and compared with two soluble β-galactosidases from K. lactis (Lactozym 3000 L HP G and Maxilact LGX 5000). Using 400 g/L lactose, the maximum GOS yield, measured by HPAEC-PAD analysis, was 177 g/L (44% w/w of total carbohydrates). The major products synthesized were the disaccharides 6-galactobiose [Gal-β(1→6)-Gal] and allolactose [Gal-β(1→6)-Glc], as well as the trisaccharide 6-galactosyl-lactose [Gal-β(1→6)-Gal-β(1→4)-Glc], which was characterized by MS and 2D NMR. Structural characterization of another synthesized disaccharide, Gal-β(1→3)-Glc, was carried out. GOS yield obtained with soluble β-galactosidases was slightly lower (160 g/L for Lactozym 3000 L HP G and 154 g/L for Maxilact LGX 5000); however, the typical profile with a maximum GOS concentration followed by partial hydrolysis of the newly formed oligosaccharides was not observed with the soluble enzymes. Results were correlated with the higher stability of β-galactosidase when permeabilized whole cells were usedProjects BIO2007-67708-C04-01, BIO2007-67708-C04-03, BIO2010-20508-C04-01, and BIO2010-20508-C04-04 from the Spanish Ministry of Science and Innovation supported this research. B.R.-C. and M.A.d.A. were supported by fellowships from the Spanish Ministries of Science and Innovation (FPI program) and Education and Culture (FPU program), respectivelyPeer reviewe