Galactooligosaccharides formation during enzymatic hydrolysis of lactose: towards a prebiotic enriched milk

The formation of galacto-oligosaccharides (GOS) in skim milk during the treatment with several commercial β-galactosidases (Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae) was analyzed in detail, at 4°C and 40°C. The maximum GOS concentration was obtained at a lactose conversion of approximately 40-50% with B. circulans and A. oryzae -galactosidases, and at 95% lactose depletion for K. lactis -galactosidase. Using an enzyme dosage of 0.1% (v/v), the maximum GOS concentration with K. lactis -galactosidase was achieved in 1 h and 5 h at 40°C and 4°C, respectively. With this enzyme, it was possible to obtain a treated milk with 7.0 g/L GOS −the human milk oligosaccharides (HMOs) concentration is between 5 and 15 g/L−, and with a low content of residual lactose (2.1 g/L, compared with 44-46 g/L in the initial milk sample). The major GOS synthesized by this enzyme were 6-galactobiose [Gal-β(1→6)-Gal], allolactose [Gal-β(1→6)-Glc] and 6´-O-β-galactosyl-lactose [Gal-β(1→6)-Gal-β(1→4)-Glc].The project BIO2010-20508-C04-01 from the Spanish Ministry of Science and Innovation supported this research. B.R.C was supported by a fellowship (FPI program)Peer reviewe

Biocatalizador inmovilizado basado en alginato para la biotransformación de carbohidratos

Biocatalizador inmovilizado basado en alginato para la biotransformación de carbohidratos. Procedimiento de obtención de un biocatalizador, que comprende: inmovilizar una enzima fúngica por inclusión en un gel de alginato cálcico; y el secado posterior el biocatalizador inmovilizado obtenido en el paso (a). La invención también se refiere al biocatalizador obtenido por el procedimiento de la invención y que comprende enzimas fúngicas, preferiblemente fructosiltransferasa o - fructofuranosidasa, inmovilizadas en alginato. Además la invención se refiere al uso de dicho biocatalizador para la biotransformación en las que el sustrato es una disolución concentrada de un carbohidrato y se puede llevar a cabo en un reactor continuo.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Universidad Autónoma de MadridA1 Solicitud de patente con informe sobre el estado de la técnic

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.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (BIO2013-48779-C4-1-R and BIO2016-76601-C3-1-R). We thank the support of COST-Action CM1303 on Systems Biocatalysis. P.S.-M. thanks the Spanish Ministry of Education for FPU Grant (FPU13/01185). We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

Regioselective synthesis of neo-erlose by the beta-fructofuranosidase from Xanthophyllomyces dendrorhous

The beta-fructofuranosidase from the yeast Xanthophyllomyces dendrorhous (Xd-INV) catalyzes the synthesis of neo-fructooligosaccharides (neo-FOS of the 6G-series), which contain a beta(2-6) linkage between a fructose and the glucosyl moiety of sucrose. In this work we demonstrate that the enzyme is also able to fructosylate other carbohydrates that contain glucose, in particular disaccharides (maltose, isomaltulose, isomaltose, trehalose) and higher oligosaccharides (maltotriose, raffinose, maltotetraose), but not monosaccharides (glucose, fructose, galactose). With maltose as acceptor, the reaction in the presence of Xd-INV proceeded with high regioselectivity; the product was purified and chemically characterized, and turned out to be 6’-O--fructosylmaltose (neo-erlose). Using 100 g/L sucrose as fructosyl donor and 300 g/L maltose as acceptor, the maximum concentration of neo-erlose was 38.3 g/L. Thus, novel hetero-fructooligosaccharides with potential applications in the functional food and pharmaceutical industries can be obtained with Xd-INV.Projects BIO2010-20508-C04-01 and BIO2010-20508-C04-04 from Spanish Ministry of Science and Innovation supported this research. We thank Fundación Ramon Areces for an institutional grant to the Centro de Biología Molecular Severo Ochoa.Peer reviewe

Selective Synthesis of Galactooligosaccharides Containing β(1→3) Linkages with β-Galactosidase from Bifidobacterium bifidum (Saphera)

[EN] The transglycosylation activity of a novel commercial β-galactosidase from Bifidobacterium bifidum (Saphera) was evaluated. The optimal conditions for the operation of this enzyme, measured with o-nitrophenyl-β-d-galactopyranoside, were 40 °C and pH around 6.0. Although at low lactose concentrations the property of this enzyme was basically hydrolytic, an increase of lactose concentration to 400 g/L resulted in a significant formation (107.2 g/L, 27% yield) of prebiotic galactooligosaccharides (GOS). The maximum amount of GOS was obtained at a lactose conversion of approximately 90%, which contrasts with other β-galactosidases, for which the highest GOS yield is achieved at 40-50% lactose conversion. Using high-performance anion-exchange chromatography with pulsed amperometric detection, semipreparative high-performance liquid chromatography-hydrophilic interaction liquid chromatography, mass spectrometry, and 1D and 2D NMR, we determined the structure of most of the GOS synthesized by this enzyme. The main identified products were Gal-β(1→3)-Gal-β(1→4)-Glc (3′-O-β-galactosyl-lactose), Gal-β(1→6)-Glc (allolactose), Gal-β(1→3)-Glc (3-galactosyl-glucose), Gal-β(1→3)-Gal (3-galactobiose), and the tetrasaccharide Gal-β(1→3)-Gal-β(1→3)-Gal-β(1→4)-Glc. In general, B. bifidum β-galactosidase showed a tendency to form β(1→3) linkages followed by β(1→6) and more scarcely β(1→4).This work was supported by a grant from the Spanish Ministry of Economy and Competitiveness (BIO2016-76601-C3-1). The group at CIC bioGUNE thanks Agencia Estatal de Investigación (Spain) for the Severo Ochoa Excellence Accreditation (SEV-2016-0644)

Killer cell immunoglobulin-like receptor genes in Spanish multiple sclerosis patients.

Killer cell immunoglobulin-like receptors (KIRs) are regulators of cytolytic activity of natural killer and certain T cells through interactions with human leukocyte antigen (HLA) class I ligands. KIRs have been shown to contribute to the pathogenesis of several autoimmune diseases, but their role in multiple sclerosis (MS) is still unclear. Here we determined the influence of KIR genes and their HLA class I ligands on susceptibility to MS and on the response to interferon-beta treatment in a Spanish population. KIR and HLA genotyping were performed in 200 MS patients and 200 controls. Significantly higher frequencies were found for KIR2DL5 and KIR3DS1 genes in MS patients and the carriage of the KIR2DL1 gene was associated with a higher progression index. Moreover, the frequency of the HLA-Bw4 motif was significantly reduced in MS patients. The KIR2DL1 and HLA-C2 matches were more frequent in MS patients, whereas the KIR3DL1 and HLA-Bw4 matches were more frequent in healthy controls. Nevertheless, non significant associations were found between all the KIR genes and therapeutic response to interferon-beta. Our results confirm that the carriage of HLA-Bw4 is a protective factor in MS and suggest that KIR2DL5 and KIR3DS1 may have a predisposing role in the disease

Killer cell immunoglobulin-like receptor genes in Spanish multiple sclerosis patients

Killer cell immunoglobulin-like receptors (KIRs) are regulators of cytolytic activity of natural killer and certain T cells through interactions with human leukocyte antigen (HLA) class I ligands. KIRs have been shown to contribute to the pathogenesis of several autoimmune diseases, but their role in multiple sclerosis (MS) is still unclear. Here we determined the influence of KIR genes and their HLA class I ligands on susceptibility to MS and on the response to interferon-beta treatment in a Spanish population. KIR and HLA genotyping were performed in 200 MS patients and 200 controls. Significantly higher frequencies were found for KIR2DL5 and KIR3DS1 genes in MS patients and the carriage of the KIR2DL1 gene was associated with a higher progression index. Moreover, the frequency of the HLA-Bw4 motif was significantly reduced in MS patients. The KIR2DL1 and HLA-C2 matches were more frequent in MS patients, whereas the KIR3DL1 and HLA-Bw4 matches were more frequent in healthy controls. Nevertheless, non significant associations were found between all the KIR genes and therapeutic response to interferon-beta. Our results confirm that the carriage of HLA-Bw4 is a protective factor in MS and suggest that KIR2DL5 and KIR3DS1 may have a predisposing role in the disease

Microbial β-glucosidases from cow rumen metagenome enhance the saccharification of lignocellulose in combination with commercial cellulase cocktail

BACKGROUND: A complete saccharification of plant polymers is the critical step in the efficient production of bio-alcohols. Beta-glucosidases acting in the degradation of intermediate gluco-oligosaccharides produced by cellulases limit the yield of the final product. RESULTS: In the present work, we have identified and then successfully cloned, expressed, purified and characterised 4 highly active beta-glucosidases from fibre-adherent microbial community from the cow rumen. The enzymes were most active at temperatures 45–55°C and pH 4.0-7.0 and exhibited high affinity and activity towards synthetic substrates such as p-nitrophenyl-beta-D-glucopyranoside (pNPbetaG) and pNP-beta-cellobiose, as well as to natural cello-oligosaccharides ranging from cellobiose to cellopentaose. The apparent capability of the most active beta-glucosidase, herein named LAB25g2, was tested for its ability to improve, at low dosage (31.25 units g(-1) dry biomass, using pNPbetaG as substrate), the hydrolysis of pre-treated corn stover (dry matter content of 20%; 350 g glucan kg(-1) dry biomass) in combination with a beta-glucosidase-deficient commercial Trichoderma reseei cellulase cocktail (5 units g(-1) dry biomass in the basis of pNPbetaG). LAB25g2 increased the final hydrolysis yield by a factor of 20% (44.5 ± 1.7% vs. 34.5 ± 1.5% in control conditions) after 96–120 h as compared to control reactions in its absence or in the presence of other commercial beta-glucosidase preparations. The high stability (half-life higher than 5 days at 50°C and pH 5.2) and 2–38000 fold higher (as compared with reported beta-glucosidases) activity towards cello-oligosaccharides may account for its performance in supplementation assays. CONCLUSIONS: The results suggest that beta-glucosidases from yet uncultured bacteria from animal digestomes may be of a potential interest for biotechnological processes related to the effective bio-ethanol production in combination with low dosage of commercial cellulases

Procesos biocatalíticos para la producción sostenible de oligosacáridos prebióticos empleando glicosiltransferasas inmovilizadas

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 17-06-2009Prebiotics are selectively fermented ingredients that allow specific changes, in composition and/or activity, in the gastrointestinal microflora that confer benefits upon host well-being and health. Prebiotics are resistant to gastrointestinal acidity, to hydrolysis by mammalian enzymes and to absorption, and therefore reach the colon were they are fermented by the endogenous bacteria. Non digestible oligosaccharides (and a disaccharide, lactulose) are, to date, the only prebiotic (or probable prebiotic) molecules known, and can be obtained by enzymatic synthesis from non activated sugars (i.e sucrose, lactose and maltose) using glycosyltransferases. In this work, several enzymes (β-fructofuranosidases, β-galactosidases and α-glucosidases) from various commercial preparations and from several non-conventional yeast strains were assayed for their transglycosilation activity. A fructosyltransferase (FTase) from Aspergillus aculeatus, present in the commercial preparation Pectinex Ultra SP-L, showed an outstanding transfructosylating/hydrolysis ratio and therefore was selected as a good candidate for inulin-type fructooligosacharide (FOS) production. Inulin-type FOS are fructose oligomers with a terminal glucosyl group, in which fructosyl moieties are linked via β(1→2) glycosidic bonds. This type of bonds confers prebiotic properties to FOS molecules. The FTase from A. aculeatus was purified, characterized and used in transfructosylation reactions with sucrose as substrate (using pure sucrose solutions or low-cost alternatives such as sugar beet syrup and molasses), and with raffinose as substrate. The latter substrate yielded a family of “raffinose fructooligosacharides” that showed promising prebiotic properties according to in vitro experiments with intestinal microbiota from weaning pigs. When sucrose was used as substrate a maximum yield of 62% of FOS was reached with a concomitant 91% sucrose conversion. Glucose, the subproduct of the FOS synthesis reaction, reached concentrations of 28% and showed an inhibitory effect with an apparently mixed non-competitive mechanism. Glucose was successfully depleted using a mixed enzyme reaction combining the FTase with a glucose oxidase. With this system, sucrose conversion reached 100% and, although the yield of FOS was not significantly affected in the studied conditions, the concentration of FOS referred to the total carbohydrates was 89%. The FTase from A. aculeatus was succesfully immobilized in calcium alginate gel beads, and a new type of biocatalyst was developed when drying the gel beads to a final water content of 1.5%. The gel beads reduced 96% of their original volume upon drying, and the resulting particles, named DALGEEs (Dry Alginate Entrapped Enzymes), showed significant advantages such as better storage stability (not needing refrigeration), and higher volumetric activity (40-fold), compared to the gel based biocatalyst. DALGEEs did not rehydrate nor suffer from swelling when used in highly concentrated sucrose solutions (with low water activity) and thus were found to be sustainable and efficient biocatalysts for FOS production. The operational stability of both calcium alginate gel beads and DALGEEs were tested in a fixed-bed reactor operating at 35 ºC with a continuous flow of 600 g/l sucrose (0.1 ml/min) for 4 weeks, and there was no substantial loss of activity perceived. Under these conditions, the volumetric productivities of FOS, obtained for the gel beads-packed reactor and for the DALGEEs-packed reactor, were 180 and 4034 g· L-1· day-1, respectively