Lucia Fernandez-Arrojo

Molecular and Biochemical Characterization of a β-Fructofuranosidase from Xanthophyllomyces dendrorhous

ABSTRACT An extracellular β-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°C) and thermostability (with a T50 in the range 66 to 71°C) is higher than that exhibited by most yeast invertases. The enzyme was able to hydrolyze fructosyl-β-(2→1)-linked carbohydrates such as sucrose, 1-kestose, or nystose, although its catalytic efficiency, defined by the kcat/Km ratio, indicates that it hydrolyzes sucrose approximately 4.2 times more efficiently than 1-kestose. Unlike other microbial β-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 β-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 β-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.

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

BackgroundA 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.ResultsIn 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 (p NPbetaG) and p NP-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 p NPbetaG 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 p NPbetaG). 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.ConclusionsThe 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 β-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 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 used.

Biochemical characterization of a β‐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

ABSTRACT An extracellular β-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°C) and thermostability (with a T50 in the range 66 to 71°C) is higher than that exhibited by most yeast invertases. The enzyme was able to hydrolyze fructosyl-β-(2→1)-linked carbohydrates such as sucrose, 1-kestose, or nystose, although its catalytic efficiency, defined by the kcat/Km ratio, indicates that it hydrolyzes sucrose approximately 4.2 times more efficiently than 1-kestose. Unlike other microbial β-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 β-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 β-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.

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.

Ants impact the energy reserves of natural enemies through the shared honeydew exploitation

1. Ants, as well as many species of parasitoids and predators, rely on sugar‐rich foods such as honeydew to fulfill their energetic needs. Thus, ants and natural enemies may interact through the shared honeydew exploitation.

Vinyl sulfone-activated silica for efficient covalent immobilization of alkaline unstable enzymes: application to levansucrase for fructooligosaccharide synthesis

Most methodologies for covalent immobilization of enzymes usually take place at high pH values to enhance the nucleophilicity of protein reactive residues; however, many enzymes inactivate during the immobilization process due to their intrinsic instability at alkaline pH values. Vinyl sulfone (VS)-activated carriers may react with several protein side-chains at neutral pHs. In this work, levansucrase-an alkaline unstable enzyme of technological interest because it forms fructooligosaccharides (FOS) and levan from sucrose-was covalently attached to VS-activated silica at pH 7.0 in a short time (5 h). Theoretical immobilization yields were close to 95% but the apparent activity did not surpass 25%, probably due to random attachment with unproductive orientations and rigidification of the enzyme structure. Due to diffusional hindrance and/or local microenvironmental effects caused by the silica surface, the immobilized levansucrase was unable to produce levan but synthesized a similar amount of FOS than the free enzyme [95 g L−1 in 28 h, with a major contribution of FOS of the β(2 → 1) type]. The VS-activated biocatalysts showed a notable operational stability in batch reactors.

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

ObjectiveA simple and inexpensive methodology, based on the use of micro-centrifuge filter tubes, is proposed for establishing the best enzyme immobilization conditions.ResultsThe 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).ConclusionThe 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.

On the Enzyme Specificity for the Synthesis of Prebiotic Galactooligosaccharides

β-Galactosidases catalyze transgalactosylation reactions in which lactose as well as the glucose and galactose released by hydrolysis serve as galactosyl acceptors yielding a series of galactooligosaccharides (GOS). GOS constitute the major part of oligosaccharides in human milk and are responsible of the formation of a Bifidus microbiota in the intestine of milk-fed babies. The bioactive properties of GOS depend on their chemical composition, structure, and polymerization degree. We have analyzed the product specificity of various β-galactosidases, namely, those from Kluyveromyces lactis, Bacillus circulans, and Aspergillus oryzae. The major products synthesized by B. circulans β-galactosidase contained only β-(1 → 4) bonds, whereas the enzyme from K. lactis synthesized GOS with major presence of β-(1 → 6) linkages. The A. oryzae β-galactosidase formed preferentially β-(1 → 6) bonds, with minor proportion of β-(1 → 3). B. circulans and K. lactis β-galactosidases produce nearly 45–50 % (w/w) GOS, whereas the A. oryzae enzyme produces less than 30 % (w/w). Another difference between the three enzymes was the polymerization degree of products; in particular, for a GOS mixture enriched in disaccharides, K. lactis and A. oryzae β-galactosidases are the best choices. In contrast, the B. circulans enzyme would be preferable for a GOS product with a high trisaccharides and tetrasaccharides content.