Immobilized Trienzymatic System with Enhanced Stabilization for the Biotransformation of Lactose.

The use of ketohexose isomerases is a powerful tool in lactose whey processing, but these enzymes can be very sensitive and expensive. Development of immobilized/stabilized biocatalysts could be a further option to improve the process. In this work, ß-galactosidase from Bacillus circulans, l-arabinose (d-galactose) isomerase from Enterococcus faecium, and d-xylose (d-glucose) isomerase from Streptomyces rubiginosus were immobilized individually onto Eupergit C and Eupergit C 250 L. Immobilized activity yields were over 90% in all cases. With the purpose of increasing thermostability of derivatives, two post-immobilization treatments were performed: alkaline incubation to favor the formation of additional covalent linkages, and blocking of excess oxirane groups by reacting with glycine. The greatest thermostability was achieved when alkaline incubation was carried out for 24 h, producing l-arabinose isomerase-Eupergit C derivatives with a half-life of 379 h and d-xylose isomerase-Eupergit C derivatives with a half-life of 554 h at 50 °C. Preliminary assays using immobilized and stabilized biocatalysts sequentially to biotransform lactose at pH 7.0 and 50 °C demonstrated improved performances as compared with soluble enzymes. Further improvements in ketohexose productivities were achieved when the three single-immobilizates were incubated simultaneously with lactose in a mono-reactor system.

Synthesis of Oligosaccharides Derived from Lactulose (OsLu) Using Soluble and Immobilized Aspergillus oryzae ß-Galactosidase.

ß-Galactosidase from Aspergillus oryzae offers a high yield for the synthesis of oligosaccharides derived from lactulose (OsLu) by transgalactosylation. Oligosaccharides with degree of polymerization (DP) ≥ 3 have shown to possess higher in vitro bifidogenic effect than di- and tetrasaccharides. Thus, in this work, an optimization of reaction conditions affecting the specific selectivity of A. oryzae ß-galactosidase for synthesis of OsLu has been carried out to enhance OsLu with DP ≥ 3 production. Assays with ß-galactosidase immobilized onto a glutaraldehyde-agarose support were also carried out with the aim of making the process cost-effective and industrially viable. Optimal conditions with both soluble and immobilized enzyme for the synthesis of OsLu with DP ≥ 3 were 50 °C, pH 6.5, 450 g/L of lactulose, and 8 U/mL of enzyme, reaching yields of ca. 50% (w/v) of total OsLu and ca. 20% (w/v) of OsLu with DP 3, being 6'-galactosyl-lactulose the major one, after a short reaction time. Selective formation of disaccharides, however, was favored at 60 °C, pH 4.5, 450 g/L of lactulose and 8 U/mL of enzyme. Immobilization increased the enzymatic stability to temperature changes and allowed to reuse the enzyme. We can conclude that the use, under determined optimal conditions, of the A. oryzae ß-galactosidase immobilized on a support of glutaraldehyde-agarose constitutes an efficient and cost-effective alternative to the use of soluble ß-galactosidases for the synthesis of prebiotic OsLu mixtures.

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, ß-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.

Immobilization of enzymes: a literature survey.

The term immobilized enzymes refers to "enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities, and which can be used repeatedly and continuously." Immobilized enzymes are currently the subject of considerable interest because of their advantages over soluble enzymes. In addition to their use in industrial processes, the immobilization techniques are the basis for making a number of biotechnology products with application in diagnostics, bioaffinity chromatography, and biosensors. At the beginning, only immobilized single enzymes were used, after 1970s more complex systems including two-enzyme reactions with cofactor regeneration and living cells were developed. The enzymes can be attached to the support by interactions ranging from reversible physical adsorption and ionic linkages to stable covalent bonds. Although the choice of the most appropriate immobilization technique depends on the nature of the enzyme and the carrier, in the last years the immobilization technology has increasingly become a matter of rational design. As a consequence of enzyme immobilization, some properties such as catalytic activity or thermal stability become altered. These effects have been demonstrated and exploited. The concept of stabilization has been an important driving force for immobilizing enzymes. Moreover, true stabilization at the molecular level has been demonstrated, e.g., proteins immobilized through multipoint covalent binding.

Reversible covalent immobilization of enzymes via disulfide bonds.

This enzyme immobilization approach involves the formation of disulfide (-S-S-) bonds with the support. Thus, enzymes bearing exposed nonessential thiol (SH) groups can be immobilized onto thiol-reactive supports provided with reactive disulfides or disulfide oxides under mild conditions. The great potential advantage of this approach is the reversibility of the bonds formed between the activated solid phase and the thiol-enzyme, because the bound protein can be released with an excess of a low-molecular-weight thiol (e.g., dithiothreitol [DTT]). This is of particular interest when the enzyme degrades much faster than the adsorbent, which can be reloaded afterwards. The possibility of reusing the polymeric support after inactivation of the enzyme may be of interest for the practical use of immobilized enzymes in large-scale processes in industry, where their use has often been hampered by the high cost of the support material. Disulfide oxides (thiolsulfinate or thiolsulfonate groups) can be introduced onto a wide variety of support materials with different degrees of porosity and with different mechanical resistances. Procedures are given for the preparation of thiol-activated solid phases and the covalent attachment of thiol-enzymes to the support material via disulfide bonds. The possibility of reusing the polymeric support is also shown.

Development and characterization of a solid-phase biocatalyst based on cyclodextrin glucantransferase reversibly immobilized onto thiolsulfinate-agarose.

Reduction of disulfide bonds and introduction of "de novo" thiol groups in cyclodextrin glucantransferase from Thermoanaerobacter sp. were assessed in order to perform reversible covalent immobilization onto thiol-reactive supports (thiolsulfinate-agarose). Only the thiolation process dramatically improved the immobilization yield, from 0 % for the native and reduced enzyme, up to nearly 90 % for the thiolated enzyme. The mild conditions of the immobilization process (pH 6.8-7.0 and 22 °C) allowed the achievement of 100 % coupling efficiencies when low loads were applied. Ionic strength was a critical parameter for the immobilization process; for high activity recoveries, 50 mM phosphate buffer supplemented with 0.15 M NaCl was required. The kinetic parameters, pH and thermal stabilities for the immobilized biocatalyst were similar to those for the native enzyme. For ß-cyclization activity, optimal pH range and temperature were 4.0-5.4 and 85 °C. The possibility of reusing the support was demonstrated by the reversibility of enzyme-support binding.

Characterization of galactosyl derivatives obtained by transgalactosylation of lactose and different polyols using immobilized beta-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.

Covalent immobilization of tobacco-etch-virus NIa protease: a useful tool for cleavage of the histidine tag of recombinant proteins.

Addition of tags [such as His (histidine) tags] is extremely helpful for the affinity purification of recombinant proteins. In several cases, these tags must be removed before performing functional and structural studies. The enzyme most frequently used to cleave tags of recombinant proteins is the TEV-protease (tobacco-etch-virus NIa protease). The continuous production of this enzyme in soluble form is quite an expensive process and not easily accessible to many laboratories. Thus an interesting alternative is the use of TEV-protease in an immobilized form, which may be reutilized several times. The main objective of the present study was to obtain a TEV-protease in an immobilized form, by covalent immobilization on to solid supports through selective use of different amino acid residues, lysine or cysteine. High protein immobilization yields (75-97%) were obtained with both strategies. The TEV-protease immobilized through its exposed cysteine thiol groups maintained its ability for cleaving a 20 kDa substrate. While the activity of the immobilized TEV-protease maintained only 30% of the activity of the enzyme in soluble form, its stability at 4 degrees C was improved three times. Moreover, this enzyme could be reutilized in at least five cycles of cleavage without loss of performance. The present results indicate that the use of a TEV-protease in an immobilized form is a potentially useful tool for the cleavage of His tags of recombinant proteins and may be useful for reducing the cost of the total process of cleavage.

One-step purification and characterization of an intracellular beta-glucosidase from Metschnikowia pulcherrima.

A collection of 60 non-Saccharomyces yeasts isolated from grape musts in Uruguayan vineyards was screened for beta-glucosidase activity and Metschnikowia pulcherrima was the best source of this enzyme activity. Its major beta-glucosidase was successfully purified to homogeneity by ion-exchange chromatography on amino-agarose gel. The enzyme exhibited an optimum catalytic activity at 50 degrees C and pH 4.5 and was active against (1 --> 4)-beta and (1 --> 2)-beta glycosidic linkages. In spite of preserving 100% of its activity and stability in the presence of 12% (v/v) ethanol and 5 g glucose/l, the enzyme was unstable below pH 4. We characterized the beta-glucosidase from M. pulcherrima with a view to its potential applications in wine-making.

Thiopropyl-agarose as a solid phase reducing agent for chemical modification of IgG and F(ab')2.

Selective reduction of native disulfide bonds in immunoglobulins is one of the best methods for introducing reactive groups on to the protein surface. Additionally, the thiol groups so generated may allow oriented conjugation at a specific site of the immunoglobulin. Solid-phase reducing agents have many advantages over soluble ones (including ease of separation of excess reagent from reduced protein by filtration, and the potential for regeneration and multiple reuse). In this work we report a comparative study of the reduction of rabbit IgG and its F(ab')(2) fragments, with mercaptohydroxypropylether-agarose (thiopropyl-agarose), a solid phase reducing agent, and dithiothreitol. The effect of different parameters on the process, such as the amount of reducing agent, incubation period, and temperature, was assessed by titration of thiol groups and SDS-PAGE analysis. Optimized reduction with thiopropyl-agarose introduced six thiol groups in the F(ab')(2) fragment (mol/mol). Native IgG was less reactive, probably due to steric effects, as only an average of three thiol groups were introduced. However, by increasing reaction temperature from 22 to 37 degrees C, six thiol groups could be introduced in native IgG (mol/mol). Reduction with dithiothreitol also introduced six thiol groups in F(ab')(2) fragments (mol/mol) but led to higher thiol content for the whole IgG. These results demonstrated that thiopropyl-agarose can be a very useful tool for exercising more precise control over the reduction treatment, and for selecting which disulfide bridges are to be broken. After 6 h incubation with reducing agent containing 8 and 16 mumoles SH per mg of protein, the resulting reduced IgG retained the same biological activity as the native immunoglobulin. The controlled modification of native disulfides achieved with thiopropyl-agarose will be useful for the development of soluble and insoluble immunoglobulin conjugates.

Preparation of high-density Concanavalin A adsorbent and its use for rapid, high-yield purification of peroxidase from horseradish roots.

Preparation of Concanavalin A-adsorbents by immobilization on Sepharose activated with 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate (CDAP-reagent) is reported. High immobilization yields of lectin (above 90%) were attained using an optimized CDAP-activating protocol. The effect of ligand density on the performance of the adsorbent for specific binding of glycoproteins was studied using horseradish peroxidase (HRP) as a model. Adsorption yields of pure HRP exceeding 90% were obtained with Con A-derivatives containing not < 20 mg of immobilized Con A/ml of packed gel. With lectin content of 2 mg/(ml of packed gel), only 20% of HRP was adsorbed. Purification of peroxidase from horseradish roots extract was successfully accomplished on Con A-Sepharose with high Con A content.

Solid-phase reducing agents as alternative for reducing disulfide bonds in proteins.

Disulfide reduction of Kluyveromyces lactis and Aspergillus oryzae beta-galactosidases and beta-lactoglobulin was assessed. Reduction was performed using one of two thiol-containing agents: dithiothreitol (DTT) or thiopropyl-agarose with a high degree of substitution (1000 micromol of SH groups/g of dried gel). Both reductants allowed an increase of three- (for K. lactis beta-galactosidase) and fourfold (for A. oryzae beta-galactosidase) in the initial content of SH groups in the lactases. Nearly sevenfold fewer micromoles of SH groups per milligram of protein were needed to perform the reduction of K. lactis beta-galactosidase with thiopropyl-agarose than for the same reduction with DTT. However, for A. oryzae beta-galactosidase, nearly twice as many micromoles of SH groups per milligram of protein were needed with thiopropylagarose than with DTT. Disulfide bonds in beta-lactoglobulin were not accessible to thiopropyl-agarose, since this reduction was only possible in the presence of 6 M urea. These results proved that highly substituted thiopropyl-agarose is as good a reducing agent as DTT, for the reduction of disulfide bonds in proteins. Moreover, excess reducing agent was very simply separated from the reduced protein by filtration, making it easier to control the reaction and providing reduced protein solutions free of reductant. All these advantages substantially cut down the time required and therefore the cost of the overall process.

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.

Generating favorable nano-environments for thermal and solvent stabilization of immobilized beta-galactosidase.

beta-Galactosidase (Escherichia coli) was immobilized through its thiol groups on thiolsulfinate-agarose gel. After enzyme immobilization, different nano-environments were generated by reacting the excess of gel-bound thiolsulfinate moieties with 2-mercaptoethanesulfonic acid (S-gel), glutathione (G-gel), cysteamine (C-gel), and mercaptoethanol (M-gel). Concerning thermal stability at 50 degrees C, the G-gel and the M-gel derivatives were the most stable with residual activity values of 67% and 45%, respectively. The stability in several solvent systems was studied: ethyl acetate (1.6% vol/vol), ethylene glycol (50% vol/vol), and 2-propanol (50% vol/vol). In ethyl acetate, both the M-gel and S-gel were highly stabilized; the time required for activity to decay to 80% of the initial activity was increased 29-fold for the M-gel and 20-fold for the S-gel with respect to the soluble enzyme. The G-gel was the least stable of all the derivatives. The different behaviors of the derivatives in thermal and solvent stability studies suggest that each nano-environment contributes differently to the enzyme stability, depending on the denaturing conditions. Therefore, it may be possible to tailor the matrix surface to maximize enzyme stability in particular applications.