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

The transglycosylation activity of a novel commercial ß-galactosidase from Bifidobacterium bifidum (Saphera) was evaluated. The optimal conditions for the operation of this enzyme, measured with o-nitrophenyl-ß-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 ß-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-ß(1→3)-Gal-ß(1→4)-Glc (3'-O-ß-galactosyl-lactose), Gal-ß(1→6)-Glc (allolactose), Gal-ß(1→3)-Glc (3-galactosyl-glucose), Gal-ß(1→3)-Gal (3-galactobiose), and the tetrasaccharide Gal-ß(1→3)-Gal-ß(1→3)-Gal-ß(1→4)-Glc. In general, B. bifidum ß-galactosidase showed a tendency to form ß(1→3) linkages followed by ß(1→6) and more scarcely ß(1→4).

An efficient continuous flow process for the synthesis of a non-conventional mixture of fructooligosaccharides.

A sustainable and scalable process for the production of a new mixture of fructooligosaccharides (FOS) was developed using a continuous-flow approach based on an immobilized whole cells-packed bed reactor. The technological transfer from a classical batch system to an innovative flow environment allowed a significant improvement of the productivity. Moreover, the stability of this production system was ascertained by up to 7 days of continuous working. These results suggest the suitability of the proposed method for a large-scale production of the desired FOS mixture, in view of a foreseeable use as a novel prebiotic preparation.

Micro-scale procedure for enzyme immobilization screening and operational stability assays.

OBJECTIVE: A simple and inexpensive methodology, based on the use of micro-centrifuge filter tubes, is proposed for establishing the best enzyme immobilization conditions. RESULTS: The immobilized biocatalyst is located inside the filter holder during the whole protocol, thus facilitating the incubations, filtrations and washings. This procedure minimizes the amount of enzyme and solid carrier needed, and allows exploring different immobilization parameters (pH, buffer concentration, enzyme/carrier ratio, incubation time, etc.) in a fast manner. The handling of immobilized enzymes using micro-centrifuge filter tubes can also be applied to assess the apparent activity of the biocatalysts, as well as their reuse in successive batch reaction cycles. The usefulness of the proposed methodology is shown by the determination of the optimum pH for the immobilization of an inulinase (Fructozyme L) on two anion-exchange polymethacrylate resins (Sepabeads EC-EA and Sepabeads EC-HA). CONCLUSION: The micro-scale procedure described here will help to overcome the lack of guidelines that usually govern the selection of an immobilization method, thus favouring the development of stable and robust immobilized enzymes that can withstand harsh operating conditions in industry.

Síntesis enzimática de fructooligosacáridos estimuladores de la microbiología colónica / Enzymatic synthesis of fructooligosaccharides that stimulate the gut microbiota

El control de la microflora intestinal es uno de los objetivos de los alimentos funcionales y nutracéuticos. El equilibrio simbiótico puede lograrse mediante la ingesta de microorganismos vivos (probióticos) o de los denominados prebióticos (oligosacáridos no digeribles). Los prebióticos son fermentados selectivamente por la microbiota generando cambios específicos en su composición que producen un beneficio en la salud del hospedador. Entre los prebióticos los fructooligosacáridos (FOS) constituyen uno de los grupos más importantes. Las levansacarasas (EC 2.4.1.10) son una familia de enzimas que catalizan la transferencia de un grupo fructosilo desde una sacarosa a diferentes aceptores entre ellos otra molécula de sacarosa dando lugar a FOS sobre los que puede transferir otro grupo fructosilo para llegar a formar levano un polímero con aplicaciones en alimentación y biomedicina. Si el grupo fructosilo se transfiere sobre una molécula de agua da lugar a la hidrólisis de la sacarosa. En este trabajo se caracterizó una levansacarasa de Z. mobilis y los productos de reacción con sacarosa como sustrato se analizaron por cromatografía de intercambio aniónico con detector amperométrico de pulsos (HPAEC-PAD). Con objeto de optimizar el proceso biocatalítico la enzima se inmovilizó por atrapamiento en geles de alginato cálcico y las esferas resultantes se deshidrataron para formar DALGEEs (Dry ALGinate Entrapped Enzymes). Se probaron diferentes estrategias de inmovilización para minimizar la pérdida de la enzima por los poros. El efecto de la inmovilización en el comportamiento de la levansacarasa fue analizado

The control of the intestinal flora is one of the targets of the functional foods and nutraceuticals. A symbiotic equilibrium can be achieved by the intake of live microorganisms (probiotics) or by the so-called prebiotics (non-digested oligosaccharides). Prebiotics are selectively fermented by the human microbiota allowing specific changes and conferring benefits upon host well-being and health. Among prebiotics fructooligosaccharides (FOS) constitute one of the most established groups. Levansucrases (EC 2.4.1.10) are a family of enzymes that catalyse the transfer of the fructosyl moiety from sucrose to different acceptors such as: (1) sucrose -yielding FOS that can be further fructosylated forming levan a polymer with food and biomedical applications -; (2) water resulting in sucrose hydrolysis. In this work a levansucrase from Zymomonas mobilis was characterized and the reaction products using sucrose as substrate were analysed by High-Performance Anion-Exchange Chromatography (HPAEC-PAD). The number of FOS synthesized by the soluble enzyme was significantly higher compared with previous reports. In order to optimize the biocatalytic process the enzyme was further immobilized by entrapment in calcium alginate gel and the resulting beads were dehydrated to obtain DALGEEs (Dry ALGinate Entrapped Enzymes). Different immobilization strategies were studied to minimize enzyme loss (lixiviation) throughout the pores. The effect of enzyme immobilization on levansucrase behaviour was also analysed

Heterologous overproduction of ß-fructofuranosidase from yeast Xanthophyllomyces dendrorhous, an enzyme producing prebiotic sugars.

The ß-fructofuranosidase Xd-INV from the yeast Xanthophyllomyces dendrorhous is the largest microbial enzyme producing neo-fructooligosaccharides (neo-FOS) known to date. It mainly synthesizes neokestose and neonystose, oligosaccharides with potentially improved prebiotic properties. The Xd-INV gene comprises an open reading frame of 1995 bp, which encodes a 665-amino acid protein. Initial N-terminal sequencing of Xd-INV pointed to a majority extracellular protein of 595 amino acids lacking the first 70 residues (potential signal peptide). Functionality of the last 1785 bp of Xd-INV gene was previously proved in Saccharomyces cerevisiae but only weak ß-fructofuranosidase activity was quantified. In this study, different strategies to improve this enzyme level in a heterologous system have been used. Curiously, best results were obtained by increasing the protein N-terminus sequence in 39 amino acids, protein of 634 residues. The higher ß-fructofuranosidase activity detected in this study, about 15 U/mL, was obtained using Pichia pastoris and represents an improvement of about 1500 times the level previously obtained in a heterologous organism and doubles the best level of activity obtained by the natural producer. Heterologously expressed protein was purified and characterized biochemically and kinetically. Except by its glycosylation degree (10 % lower) and thermal stability (4-5 °C lower in the 60-85 °C range), the properties of the heterologous enzyme, including ability to produce neo-FOS, remained unchanged. Interestingly, besides the neo-FOS referred before blastose was also detected (8-22 g/L) in the reaction mixtures, making Xd-INV the first yeast enzyme producing this non-conventional disaccharide reported to date.

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

The formation of galactooligosaccharides (GOS) in skim milk during treatment with several commercial ß-galactosidases (Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae) was analysed in detail, at 4 and 40°C. The maximum GOS concentration was obtained at a lactose conversion of approximately 40-50% with B. circulans and A. oryzae ß-galactosidases, and at 95% lactose depletion for K. lactis ß-galactosidase. Using an enzyme dosage of 0.1% (v/v), the maximum GOS concentration with K. lactis ß-galactosidase was achieved in 1 and 5h at 40 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 (2.1g/L, compared with 44-46 g/L in the initial milk sample). The major GOS synthesised by this enzyme were 6-galactobiose [Gal-ß(1 → 6)-Gal], allolactose [Gal-ß(1 → 6)-Glc] and 6'-O-ß-galactosyl-lactose [Gal-ß(1 → 6)-Gal-ß(1 → 4)-Glc].

Detailed analysis of galactooligosaccharides synthesis with ß-galactosidase from Aspergillus oryzae.

The synthesis of galactooligosaccharides (GOS) catalyzed by ß-galactosidase from Aspergillus oryzae (Enzeco) was studied. Using 400 g/L of lactose and 15 U/mL, maximum GOS yield, measured by HPAEC-PAD, was 26.8% w/w of total carbohydrates, obtained at approximately 70% lactose conversion. No less than 17 carbohydrates were identified; the major transgalactosylation product was 6'-O-ß-galactosyl-lactose, representing nearly one-third (in weight) of total GOS. In contrast with previous reports, the presence of at least five disaccharides was detected, which accounted for 40% of the total GOS at the point of maximum GOS concentration (allolactose and 6-galactobiose were the major products). A. oryzae ß-galactosidase showed a preference to form ß(1→6) bonds, followed by ß(1→3) and ß(1→4) linkages. Results were compared with those obtained with ß-galactosidases from Kluyveromyces lactis and Bacillus circulans. The highest GOS yield and specific productivity were achieved with B. circulans ß-galactosidase. The specificity of the linkages formed and distribution of di-, tri-, and higher GOS varied significantly among the three ß-galactosidases.

Microbial ß-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.

Production of Galacto-oligosaccharides by the ß-Galactosidase from Kluyveromyces lactis : comparative analysis of permeabilized cells versus soluble enzyme.

The 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 ß-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-ß(1→6)-Gal] and allolactose [Gal-ß(1→6)-Glc], as well as the trisaccharide 6-galactosyl-lactose [Gal-ß(1→6)-Gal-ß(1→4)-Glc], which was characterized by MS and 2D NMR. Structural characterization of another synthesized disaccharide, Gal-ß(1→3)-Glc, was carried out. GOS yield obtained with soluble ß-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 ß-galactosidase when permeabilized whole cells were used.

Metagenomic era for biocatalyst identification.

Microbial enzymes have many known applications as biocatalysts. However, only a few of them are currently employed for biocatalysis even though an annotated collection of more than 190 billion bases is available in metagenome sequence databases from uncultured and highly diverse microbial populations. This review aims at providing conceptual and technical bases for the translation of metagenome data into both experimental and computational frameworks that facilitates a comprehensive analysis of the biocatalysts diversity space. We will also briefly present the status of the current capabilities that assess and predict catalytic potential of environmental sites and track its diversity and evolution in large-scale biocatalysis process resulting from studies applying metagenomics in association with gene fingerprinting, catabolic arrays and complementary '-omics'.

Biochemical characterization of a beta-fructofuranosidase from Rhodotorula dairenensis with transfructosylating activity.

An extracellular beta-fructofuranosidase from the yeast Rhodotorula dairenensis was characterized biochemically. The enzyme molecular mass was estimated to be 680 kDa by analytical gel filtration and 172 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, of which the N-linked carbohydrate accounts for 16% of the total mass. It displays optimum activity at pH 5 and 55-60 degrees C. The enzyme shows broad substrate specificity, hydrolyzing sucrose, 1-kestose, nystose, leucrose, raffinose and inulin. Although the main reaction catalyzed by this enzyme is sucrose hydrolysis, it also exhibits transfructosylating activity that, unlike other microbial beta-fructofuranosidases, produces a varied type of prebiotic fructooligosaccharides containing beta-(2-->1)- and beta-(2-->6)-linked fructose oligomers. The maximum concentration of fructooligosaccharides was reached at 75% sucrose conversion and it was 87.9 g L(-1). The 17.0% (w/w) referred to the total amount of sugars in the reaction mixture. At this point, the amounts of 6-kestose, neokestose, 1-kestose and tetrasaccharides were 68.9, 10.6, 2.6 and 12.7 g L(-1), respectively.

Molecular and biochemical characterization of a beta-fructofuranosidase from Xanthophyllomyces dendrorhous.

An extracellular beta-fructofuranosidase from the yeast Xanthophyllomyces dendrorhous was characterized biochemically, molecularly, and phylogenetically. This enzyme is a glycoprotein with an estimated molecular mass of 160 kDa, of which the N-linked carbohydrate accounts for 60% of the total mass. It displays optimum activity at pH 5.0 to 6.5, and its thermophilicity (with maximum activity at 65 to 70 degrees C) and thermostability (with a T(50) in the range 66 to 71 degrees C) is higher than that exhibited by most yeast invertases. The enzyme was able to hydrolyze fructosyl-beta-(2-->1)-linked carbohydrates such as sucrose, 1-kestose, or nystose, although its catalytic efficiency, defined by the k(cat)/K(m) ratio, indicates that it hydrolyzes sucrose approximately 4.2 times more efficiently than 1-kestose. Unlike other microbial beta-fructofuranosidases, the enzyme from X. dendrorhous produces neokestose as the main transglycosylation product, a potentially novel bifidogenic trisaccharide. Using a 41% (wt/vol) sucrose solution, the maximum fructooligosaccharide concentration reached was 65.9 g liter(-1). In addition, we isolated and sequenced the X. dendrorhous beta-fructofuranosidase gene (Xd-INV), showing that it encodes a putative mature polypeptide of 595 amino acids and that it shares significant identity with other fungal, yeast, and plant beta-fructofuranosidases, all members of family 32 of the glycosyl-hydrolases. We demonstrate that the Xd-INV could functionally complement the suc2 mutation of Saccharomyces cerevisiae and, finally, a structural model of the new enzyme based on the homologous invertase from Arabidopsis thaliana has also been obtained.

Characterization of a beta-fructofuranosidase from Schwanniomyces occidentalis with transfructosylating activity yielding the prebiotic 6-kestose.

beta-Fructofuranosidases are powerful tools in industrial biotechnology. We have characterized an extracellular beta-fructofuranosidase from the yeast Schwanniomyces occidentalis. The enzyme shows broad substrate specificity, hydrolyzing sucrose, 1-kestose, nystose and raffinose, with different catalytic efficiencies (k(cat)/K(m)). Although the main reaction catalysed by this enzyme is sucrose hydrolysis, it also produces two fructooligosaccharides (FOS) by transfructosylation. A combination of (1)H, (13)C and 2D-NMR techniques shows that the major product is the prebiotic trisaccharide 6-kestose. The 6-kestose yield obtained with this beta-fructofuranosidase is, to our concern, higher than those reported with other 6-kestose-producing enzymes, both at the kinetic maximum (76gl(-1)) and at reaction equilibrium (44gl(-1)). The total FOS production in the kinetic maximum was 101gl(-1), which corresponded to 16.4% (w/w) referred to the total carbohydrates in the reaction mixture.

Biochemical and structural features of a novel cyclodextrinase from cow rumen metagenome.

A novel enzyme, RA.04, belonging to the alpha-amylase family was obtained after expression of metagenomic DNA from rumen fluid (Ferrer et al.: Environ. Microbiol. 2005, 7, 1996-2010). The purified RA.04 has a tetrameric structure (280 kDa) and exhibited maximum activity (5000 U/mg protein) at 70 degrees C and was active within an unusually broad pH range from 5.5 to 9.0. It maintained 80% activity at pH 5.0 and 9.5 and 75 degrees C. The enzyme hydrolyzed alpha-D-(1,4) bonds 13-fold faster than alpha-D-(1,6) bonds to yield maltose and glucose as the main products, and it exhibited transglycosylation activity. Its preferred substrates, in the descending order, were maltooligosaccharides (C3-C7), cyclomaltoheptaose (beta-CD), cyclomaltohexaose (alpha-CD), cyclomaltooctaose (gamma-CD), soluble starch, amylose, pullulan and amylopectin. The biochemical properties and amino acid sequence alignments suggested that this enzyme is a cyclomaltodextrinase. However, despite the similarity in the catalytic module (with Glu359 and Asp331 being the catalytic nucleophile and substrate-binding residues, respectively), the enzyme bears a shorter N-terminal domain that may keep the active site more accessible for both starch and pullulan, compared to the other known CDases. Moreover, RA.04 lacks the well-conserved N-terminal Trp responsible for the substrate preference typical of CDases/MAases/PNases, suggesting a new residue is implicated in the preference for cyclic maltooligosaccharides. This study has demonstrated the usefulness of a metagenomic approach to gain novel debranching enzymes, important for the bread/food industries, from microbial environments with a high rate of plant polymer turnover, exemplified by the cow rumen.

Purification and kinetic characterization of a fructosyltransferase from Aspergillus aculeatus.

A fructosyltransferase present in Pectinex Ultra SP-L, a commercial enzyme preparation from Aspergillus aculeatus, was purified to 107-fold and further characterised. The enzyme was a dimeric glycoprotein (20% (w/w) carbohydrate content) with a molecular mass of around 135 kDa for the dimer. Optimal activity/stability was found in the pH range 5.0-7.0 and at 60 degrees C. It was stable or slightly activated (upto 1.4-fold) in the presence of reducing agents, such as dithiothreitol and 2-mercaptoethanol, and detergents, such as sodium dodecylsulphate and Tween 80. The enzyme was able to transfer fructosyl groups from sucrose as donor producing the corresponding series of fructooligosaccharides: 1-kestose, nystose and fructosylnystose. Using sucrose as substrate, the k(cat) and K(m) values for transfructosylating activity were 1.62+/-0.09 x 10(4)s(-1) and 0.53+/-0.05 M, whereas for hydrolytic activity the corresponding values were 775+/-25s(-1) and 27+/-3 mM. At elevated sucrose concentrations, the fructosyltransferase from A. aculeatus showed a high transferase/hydrolase ratio that confers it a great potential for the industrial production of prebiotic fructooligosaccharides.

Beet sugar syrup and molasses as low-cost feedstock for the enzymatic production of fructo-oligosaccharides.

Sugar syrup and molasses from beet processing containing 620 and 570 mg/mL sucrose, respectively, were assayed as low-cost and available substrates for the enzymatic synthesis of fructo-oligosaccharides (FOSs). A commercial pectinase (Pectinex Ultra SP-L, from Aspergillus aculeatus) characterized by the presence of a transfructosylating activity was used as a biocatalyst. The FOS production increased when lowering the initial pH value of syrup (7.5) and molasses (8.9) to 5.5. Sugar syrup and molasses were diluted in order to reduce substrate viscosity; interestingly, the percentage of FOS with regards to total sugars remained almost constant, which indicated a high transferase-to-hydrolase ratio for this enzyme. Kinetics of FOS production was analyzed. Using approximately 10 U transfructosylating activity per g sucrose, the FOS concentration reached a maximum of 388 mg/mL after 30 h using syrup and 235 mg/mL in 65 h with molasses. These values corresponded to approximately 56 and 49% (w/w), respectively, of the total amount of carbohydrates in the mixture. The enzyme was also covalently immobilized on an epoxy-activated polymethacrylate-based polymer (Sepabeads EC-EP5). We found that immobilized Pectinex Ultra SP-L can be efficiently applied to the synthesis of FOS using syrup and molasses as substrates.