Francisco Batista-Viera

Immobilization of β-galactosidase from Kluyveromyces lactis on silica and agarose: comparison of different methods

Abstract The covalent immobilization of β -galactosidase from Kluyveromyces lactis ( β -gal) on to two different porous carriers, CPC-silica and agarose, is reported. CPC-silica was silanizated and activated with glutaraldehyde. The activation of agarose via a cyanylating agent (CDAP) was optimized. Gel-bound protein and gel-bound activity were both measured directly, allowing the determination of apparent specific activities (S.A.). Higher amounts of β -gal were immobilized on the activated CPC-silica (maximum capacity, 23 mg ml −1 of packed support) than on the CDAP-activated agarose. For the lower enzyme loading assayed (12.6 mg ml −1 packed support), 100% of the enzyme was immobilized but only 34% of its activity was expressed. This inactivation during immobilization was confirmed by the S.A. values (22–29 EU mg −1 for the CPC-derivatives and 80 EU mg −1 for soluble β -gal). The K app (3.4 mM) for the CDAP-derivative with ONPG as substrate was higher than the K M value for soluble β -gal (2 mM). When the enzyme loading was increased five-fold, the K app increased four-fold, to 13 mM. The V app values for the CPC-derivatives were remarkably lower than the V max for soluble β -galactosidase. CDAP-derivatives showed better thermal stabilities than CPC-derivatives but neither of them enhanced the stability of the soluble enzyme. When stored at 4°C, the activity of both derivatives remained stable for at least 2 months. Both derivatives displayed high percentages of lactose conversion (90%) in packed bed mini-reactors. Glucose production was 3.3-fold higher for the CPC-derivative than for the CDAP-derivative, as a consequence of the higher flow rates achieved.

Polyethylene glycol as a spacer for solid-phase enzyme immobilization

Abstract With the aim to improve the performance of enzyme bound to hydrophilic solid phases, their immobilization with polyethylene glycol (PEG) tether have been studied. Sweet potato β-amylase, which hydrolyses the high molecular weight substrate starch and β-galactosidase, which acts on low molecular weight substrates, were used as model enzymes and beaded thiol–agarose as solid phase. Several two step methods for the introduction of the tether using a bis-oxirane homobifunctional PEG as well as a heterobifunctional derivative with a hydroxysuccinimide ester and a maleimide group have been evaluated. Amino groups, native and de novo thiol groups in the enzymes were utilized for immobilization. The best approach was found to be to first introduce the PEG derivative via one of its reactive groups to the enzyme. Subsequently the formed conjugate was bound to the solid phase by the remaining reactive group. Attempts to first introduce the PEG tether into the solid phase were not successful. A high degree of substitution with PEG chains on the enzyme leads to high immobilization yields for both β-amylase and β-galactosidase, but relatively lower gel-bound activity for the former enzyme which is acting on a high molecular weight substrate and thus more sensitive for steric shielding effects. With optimal degree of PEG substitution (which occurred at five times molar excess of the heterobifunctional reagent) the gel-bound activity of β-amylase was increased from 12% (for the derivative without tether) to 31%.

Preparative purification of soybean agglutinin by affinity chromatography and its immobilization for polysaccharide isolation.

Optimized procedures for the affinity purification of soybean agglutinin (SBA) from soybean flour, and its further immobilization, were developed. Lectin purification on galactosyl-Sepharose yielded 44.5+/-3.5 mg of pure SBA/50 g of flour. To prepare SBA adsorbents, the lectin was immobilized onto 1-cyano-4-(dimethylamino)pyridinium tetrafluoroborate (CDAP) activated Sepharose with high yields (77%). Feasibility of the use of this improved SBA adsorbent for affinity purification of Streptococcus pneumoniae capsular polysaccharides from strain 14 (CPS-14) at laboratory scale was demonstrated. Using SBA-Sepharose adsorbent (7.0 mg lectin per ml), amounts of 6.3 mg of pure CPS-14 per cycle were produced, the adsorbent being reused up to four times without loss of capacity.

beta-Galactosidase from Kluyveromyces lactis immobilized on to thiolsulfinate/thiolsulfonate supports for lactose hydrolysis in milk and dairy by-products.

After reversible immobilization of neutral beta-galactosidase from Kluyveromyces lactis on to thiolsulfinate/thiolsulfonate supports, more than 80 % of the activity was retained. Blocking the remaining reactive groups with glutathione increased the thermal stability of the derivatives almost two-fold. These derivatives achieved a high degree of conversion (85-90 %) of lactose (50g / l) in saline solution, whey, whey permeates, and skimmed milk, either batchwise or in packed beds. They remained fully active upon storage for 10 months in activity buffer at 4 deg C, and when treated with sanitizing agents.

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.