Beatriz M. Brena

Characterization of Galactosyl Derivatives Obtained by Transgalactosylation of Lactose and Different Polyols Using Immobilized β-Galactosidase from Aspergillus oryzae

The synthesis of novel galactosides is interesting because of their important role in several biological processes. Their properties greatly depend upon the configuration and type of galactoside. Therefore, to study biological activity, it is essential to elucidate the structure of the products. Glycosidases are capable of catalyzing glycosidic linkages with absolute stereoselectivity of the anomeric center. We report the enzymatic synthesis of galactosyl-ethylene glycol, galactosyl-glycerol, and galactosyl-erythritol by immobilized beta-galactosidase from Aspegillus oryzae. The obtained galactosides were isolated and fully characterized by an extensive nuclear magnetic resonance (NMR) study. Complete structure elucidation and full proton and carbon assignments were carried out using 1D ((1)H and (13)C) and 2D (gCOSY, TOCSY, multiplicity-edited gHSQC, and gHMBC) NMR experiments. The beta-galactosidase from A. oryzae showed a strong preference for primary alcohols. For galactosyl-glycerol and galactosyl-erythritol, this preference generated one and two chiral centers, respectively, and a mixture of stereoisomers was obtained as a consequence.

Chromatographic methods for amylases

This review surveys recent developments in chromatographic methods for the separation of amylases from complex extracts, including the separation of isozymes. It contains two tables with the properties and molecular characteristics of alpha-and beta-amylases from different sources as well as an updated review of methods for the determination of amylase activity. The main subject of this review is a detailed evaluation of the application of newly developed chromatographic methods for the purification of amylases.

Thiolation and reversible immobilization of sweet potato δ-amylase on thiolsulfonate-agarose☆

Abstract Among the different methods for obtaining immobilized biocatalysts, those based on thioldisulfide exchange reactions are unique because, simultaneously, they show a stable covalent bond and the possibility of eluting the protein by reduction when the enzymatic activity decays. The adsorbent can thus be reloaded. In this paper we report the use of the recently developed thiolreactive adsorbent thiolsulfonate-agarose, for the immobilization of sweet potato β-amylase. Since native, β-amylase thiol groups were not reactive towards the adsorbent, the enzyme was provided with ‘de novo’ thiol groups by reaction with the heterobifunctional reagent N-succinimidyl-3- (2-pyridyldithio)propionate (SPDP). When the SPDP/β-amylase molar ratio was changed between 3 and 100, up to sixteen exposed thiol groups per mol of enzyme were introduced. This was achieved without affecting the amylolytic activity. The immobilization yield for the intermediate thiolation level was 98%. However, only 19% of the applied enzyme activity was found in the gel suspension. Comparative studies were made on thiolsulfonate-agarose and on a commercial thiol-activated adsorbent (2-pyridyldisulfide-agarose). The immobilization of the thiolated enzyme through reversible disulfide bonds on both adsorbents showed similar results. A close analysis reveals that immobilization of proteins on thiolsulfonate-agarose is a very promising technique.

Enzymatic synthesis of galactosyl-xylose by Aspergillus oryzae β-galactosidase

In aqueous medium, the reaction catalyzed by Aspergillus oryzae β-galactosidase with O-nitrophenyl-β-D-galactopyranoside (ONPG) in the presence of an acceptor leads to the synthesis of transglycosylation compounds in addition to the hydrolysis products (ONP and galactose). Our goal was to develop a simple system for the synthesis of galactosyl-xylose, a disaccharide of possible application to diagnostics. To maximize synthesis yields, we have studied the effect of several conditions: increase of acceptor concentration (0.05-2.7 M xylose), organic co-solvents (dimethylformamide, acetone) and reaction time. In the absence of co-solvents ONPG was completely consumed in 2 h; with 0.5 M xylose the maximum yield of galactosyl-xylose (16%) was attained at 60 min, while with 2.7 M xylose the yield reached 21%. Both co-solvents tested decreased the kinetics of ONPG convertion into products and 50% (v/v) dimethylformamide was deleterious to the synthesis. However, in 50% (v/v) acetone the synthesis yield was 12% and interestingly, the proportion of transglycosylation with respect to the reacted substrate was higher than in buffer. The synthesis of galactosyl-ethyleneglycol was also studied; it was achieved with extremely high yield and no detectable hydrolysis products. This proves that other acceptor alcohols can be preferred over water in some conditions.

Aroma enhancement in wines using co-immobilized Aspergillus niger glycosidases

A major fraction of monoterpenes and norisoprenoids in young wines is conjugated to sugars representing a significant reservoir of aromatic precursors. To promote their release, β-glucosidase, α-arabinosidase, and α-rhamnosidase from a commercial Aspergillus niger preparation, were immobilized onto acrylic beads. The aim of this work was the development and application of an immobilized biocatalyst, due to the well-known advantages over soluble enzyme preparations: control of the reaction progress and preparation of enzyme-free products. In addition, the obtained derivative showed increased stability in simile wine conditions. After the treatment of Muscat wine with the biocatalyst for 20days, free monoterpenes increased significantly (from 1119 to 2132μg/L, p<0.01) with respect to the control wine. Geraniol was increased 3,4-fold over its flavor thresholds, and accordingly its impact on sensorial properties was very relevant: nine of ten judges considered treated wine more intense in fruit and floral notes.

Influence of the immobilization chemistry on the properties of immobilized β-galactosidases

Neutral b-galactosidases from E. coli and K. lactis were bound to glutaraldehyde-agarose Glut-agarose through . amino groups, and to thiolsulfinate-agarose TSI-agarose through thiol groups. In general, TSI-gels exhibited higher yields . . after immobilization 60-85% than Glut-gels 36-40% . The kinetic parameters of the enzymes bound to TSI-gels .particularly those with lower concentration of active groups were less affected than those of the Glut-gels. This might indicate that the binding to TSI-agarose is more conservative of the protein conformation. However, the Glut-derivatives exhibited in general better thermal and solvent stabilities than TSI-derivatives. The stability of the derivatives was studied in . the presence of ethanol, dioxane and acetone 18% vrv . The stabilization of the immobilized enzymes, for some of the solvents assayed, was evidenced by the existence of final very stable enzyme states with high residual activities, thus allowing the utilization of the derivatives in the presence of organic cosolvents. q 2001 Elsevier Science B.V. All rights reserved.

Immobilization of β-galactosidase on thiolsulfonate-agarose

The preparation and properties of Escherichia coli β-galactosidase conjugates based on a recently developed immobilization method are presented. Enzyme immobilization is performed by reaction of its native thiol groups with agarose-bound thiolsulfonates under mild conditions. Depending on the amount of enzyme applied, 8–114 mg of protein per gram of dried gel derivative was immobilized on the thiolsulfonate agarose. With low protein loadings, the immobilization yield reached 100%. The thiolsulfonate-agarose gels exhibited high selectivity toward active enzyme. Because of this, the specific activity of the immobilized β-galactosidase was up to 50% higher than that of the applied, native enzyme. The method also provides the possibility to design the microenvironment of the immobilized enzyme by blocking residual thiolsulfonate groups with different reagents. The low-load derivatives blocked with glutathione, as well as the high-load derivatives without blocking, had better thermal stability than the soluble enzyme, and also showed excellent long-term stability at low temperatures. Thus, no decrease in enzymic activity was observed after storage of the derivatives for 18 months at +4°C as suspensions in 0.1 m potassium phosphate, pH 7.5.

Activity and stability of Escherichia coli β-galactosidase in cosolvent systems

The kinetic parameters of E.coli β-galactosidase were not altered by the addition of 2-propanol or ethyl acetate (1.6% v/v). While ethylene glycol (1.6% v/v) doubled the values of both KM (0.29 mM) and kcat (1393 s−), tetraethyleneglycol-dimethylether (Tetraglyme,1.6% v/v) preserved KM, but decreased kcat. At 50°C all the cosolvents dramatically shortened the enzymatic half life, and so did Tetraglyme and 2-propanol at 28°C. At 28°C, both ethyl acetate and ethylene glycol stabilised the enzyme 9- and 6-fold respectively. This fact, together with the activation effect of ethylene glycol may lead to practical applications.

Chemical thiolation strategy: A determining factor in the properties of thiol-bound biocatalysts

Immobilization of enzymes on thiolsulphinate-agarose, a thiol-reactive support, is a unique method which allows reversible covalent immobilization under mild conditions, so excellent immobilization and activity yields are obtained. It allows both the formation of stable bonds as well as enzyme desorption and matrix regeneration. The impact of the source of the enzymes thiol group involved in the immobilization (native, reduced disulphide or chemically introduced) on the properties of the resulting biocatalysts was studied using three β-galactosidases from Escherichia coli, Kluyveromices lactis and Aspergillus oryzae as a model. Chemical thiolation, which generates changes at surface exposed lysines, produced derivatives similar to their soluble counterparts. However, the reduction of native disulphide bonds prior to immobilization lead to very variable activity and stability of the derivatives depending on the accessibility and location of the disulphide bonds in the enzyme structure.

Polyethylenimine coated agarose supports, for the reversible immobilisation of β-galactosidase from Aspergillus oryzae

Immobilised enzymes for industrial applications should be very active and operationally stable. Reversible binding, if possible, is desirable to allow matrix reuse. Usually these aims come into conflict: strong binding may imply low expressed activity and poor reversibility. Immobilisation of Aspergillus oryzae β-galactosidase to conventional ionic exchangers is efficient but the derivatives exhibit noticeable enzyme leakage when used for the hydrolysis of lactose from whey. This problem can be avoided by covalent cross-links to the matrix, at the expense of losing reversibility. We have optimised the conditions for polyethylenimine (PEI)-coating of agarose supports to achieve a β-galactosidase derivative that allows a high lactose conversion from whey in a steady bed-reactor with no enzyme leakage, together with good elution properties. Therefore, PEI-coating appears to be a very suitable strategy to convert the agarose surface to an adequate support for a reversible, but strong and non-distorting, ionic protein immobilisation.