Rodrigo Torres

A Novel Heterofunctional Epoxy‐Amino Sepabeads for a New Enzyme Immobilization Protocol: Immobilization‐Stabilization of β‐Galactosidase from Aspergillus oryzae

The properties of a new and commercially available amino‐epoxy support (amino‐epoxy‐Sepabeads) have been compared to conventional epoxy supports to immobilize enzymes, using the β‐galactosidase from Aspergillus oryzae as a model enzyme. The new support has a layer of epoxy groups over a layer of ethylenediamine that is covalently bound to the support. This support has both a great anionic exchanger strength and a high density of epoxy groups. Epoxy supports require the physical adsorption of the proteins onto the support before the covalent binding of the enzyme to the epoxy groups. Using conventional supports the immobilization rate is slow, because the adsorption is of hydrophobic nature, and immobilization must be performed using high ionic strength (over 0.5 M sodium phosphate) and a support with a fairly hydrophobic nature. Using the new support, immobilization may be performed at moderately low ionic strength, it occurs very rapidly, and it is not necessary to use a hydrophobic support. Therefore, this support should be specially recommended for immobilization of enzymes that cannot be submitted to high ionic strength. Also, both supports may be expected to yield different orientations of the proteins on the support, and that may result in some advantages in specific cases. For example, the model enzyme became almost fully inactivated when using the conventional support, while it exhibited an almost intact activity after immobilization on the new support. Furthermore, enzyme stability was significantly improved by the immobilization on this support (by more than a 12‐fold factor), suggesting the promotion of some multipoint covalent attachment between the enzyme and the support (in fact the enzyme adsorbed on an equivalent cationic support without epoxy groups was even slightly less stable than the soluble enzyme).

Reversible Immobilization of Invertase on Sepabeads Coated with Polyethyleneimine: Optimization of the Biocatalyst's Stability

Invertase from S. cerevisiae has been immobilized by ionic adsorption on Sepabeads fully coated with PEI. The enzyme was strongly adsorbed on the support (no desorption of the invertase was found under conditions in which all of the enzyme was released from conventional anionic exchanger supports (e.g., DEAE‐agarose)). Nevertheless, the enzyme could still be desorbed after its inactivation, and new fresh enzyme could be adsorbed on the supports without detrimental effects on enzyme loading. This is a multimeric enzyme, its minimal oligomerization active state being the dimer, but under certain conditions of pH and concentration it may give larger multimers. Very interestingly, results suggested that the adsorption of the enzyme on this large and flexible polymeric bed was able to freeze some of the different oligomeric structures of the enzyme. Thus, we have found that the enzyme immobilized at certain pH values (pH 8.5) and high enzyme concentration, in which the main enzyme structure is the tetramer, was more stable than immobilized preparations produced in conditions under which oligomerization was not favorable (dimers at low enzyme concentration) or it was too high (e.g., hexamers‐octamers at low pH value). The optimal enzyme preparation remained fully active after a 15‐day incubation at 50 °C and pH 4.5 (conditions of standard industrial use) and presented an optimal temperature approximately 5 °C higher than that of soluble enzyme.

Immobilization of Lactase from Kluyveromyces lactis Greatly Reduces the Inhibition Promoted by Glucose. Full Hydrolysis of Lactose in Milk

The kinetic constants (Km, Vmax, and inhibition constants for the different products) of soluble and different immobilized preparations of β‐galactosidase from Kluyveromyces lactis were determined. For the soluble enzyme, the Km was 3.6 mM, while the competitive inhibition constant by galactose was 45 mM and the noncompetitive one by glucose was 758 mM. The immobilized preparations conserved similar values of Km and competitive inhibition, but in some instances much higher values for the noncompetitive inhibition constants were obtained. Thus, when glyoxyl or glutaraldehyde supports were used to immobilize the enzyme, the noncompetitive inhibition was greatly reduced (Ki ≈ 15 000 and >40 000 mM, respectively), whereas when using sugar chains to immobilize the enzyme the behavior had an effect very similar to the soluble enzyme. These results presented a great practical relevance. While using the soluble enzyme or the enzyme immobilized via the sugar chain as biocatalysts in the hydrolysis of lactose in milk only around 90% of the substrate was hydrolyzed, by using of these the enzyme immobilized via the glyoxyl or the glutaraldehyde groups, more than 99% of the lactose in milk was hydrolyzed.

Reversible and Strong Immobilization of Proteins by Ionic Exchange on Supports Coated with Sulfate-Dextran

New and strong ionic exchange resins have been prepared by the simple and rapid ionic adsorption of anionic polymers (sulfate‐dextran) on porous supports activated with the opposite ionic group (DEAE/MANAE). Ionic exchange properties of such composites were strongly dependent on the size of the ionic polymers as well as on the conditions of the ionic coating of the solids with the ionic polymers (optimal conditions were 400 mg of sulfate‐dextran 5000 kDa per gram of support). Around 80% of the proteins contained in crude extracts from Escherichia coli and Acetobacter turbidans could be adsorbed on these porous composites even at pH 7. This interaction was stronger than that using conventional carboxymethyl cellulose (CMC) and even others such as supports coated with aspartic‐dextran polymer. By means of the sequential use of the new supports and supports coated with polyethyleneimine (PEI), all proteins from crude extracts could be immobilized. In fact, a large percentage (over 50%) could be immobilized on both supports. Finally, some industrially relevant enzymes (β‐galactosidases from Aspergillus oryzae, Kluyveromyces lactis, and Thermus sp. strain T2, lipases from Candida antarctica A and B, Candida rugosa, Rhizomucor miehei, and Rhyzopus oryzae and bovine pancreas trypsin and chymotrypsin) have been immobilized on these supports with very high activity recoveries and immobilization rates. After enzyme inactivation, the protein could be fully desorbed from the support, and then the support could be reused for several cycles. Moreover, in some instances the enzyme stability was significantly improved, mainly in the presence of organic solvents, perhaps as a consequence of the highly hydrophilic microenvironment of the support.

Reversible immobilization of glucoamylase by ionic adsorption on sepabeads coated with polyethyleneimine.

Glucoamylase (GA) from Aspergillus niger was immobilized via ionic adsorption onto DEAE‐agarose, Q1A‐Sepabeads, and Sepabeads EC‐EP3 supports coated with polyethyleneimine (PEI). After optimization of the immobilization conditions (pH, polymer size), it was observed that the adsorption strength was much higher in PEI‐Sepabeads than in Q1A‐Sepabeads or DEAE‐supports, requiring very high ionic strength to remove glucoamylase from the PEI‐supports (e.g., 1 M NaCl at pH 5.5). Thermal stability and optimal temperature was marginally improved by this immobilization. Recovered activity depended on the substrate used, maltose or starch, except when very low loading was used. The optimization of the loading allowed the preparation of derivatives with 750 IU/g in the hydrolysis of starch, preserving a high percentage of immobilized activity (around 50%).

A simple strategy for the purification of large thermophilic proteins overexpressed in mesophilic microorganisms: application to multimeric enzymes from Thermus sp. strain T2 expressed in Escherichia coli.

The heating of protein preparations of mesophilic organism (e.g., E. coli) produces the obliteration of all soluble multimeric proteins from this organism. In this way, if a multimeric enzyme from a thermophilic microorganism is expressed in these mesophilic hosts, the only large protein remaining soluble in the preparation after heating is the thermophilic enzyme. These large proteins may be then selectively adsorbed on lowly activated anionic exchangers, enabling their full purification in just these two simple steps. This strategy has been applied to the purification of an α‐galactosidase and a β‐galactosidase from Thermus sp. strain T2, both expressed in E. coli, achieving the almost full purification of both enzymes in only these two simple steps. This very simple strategy seems to be of general applicability to the purification of any thermophilic multimeric enzyme expressed in a mesophilic host.

Simple purification of immunoglobulins from whey proteins concentrate

We have developed a new protocol with only two steps for purification of immunoglobulins (Ig) from a protein concentrate of whey. Following this protocol, we have an 80% recovery of immunoglobulins, fairly pure. The purification was achieved by eliminating the BSA, via a strong adsorption on DEAE‐agarose. Full desoprtion of the other serum proteins could be achieved without contamination with BSA. Thus, a protein solution containing only Ig and very small proteins (e.g., β‐lactoglobulins and α‐lactalbumin) was obtained. Offering this protein mixture to a lowly activated aminated support, only Ig adsorbed on the support. It has been shown that BSA is able to interact with other proteins (including Ig and lactalbumins). This ability to form complexes with other proteins prevented the success of the direct adsorption of Ig on this mildly activated support, even although Ig should be the largest protein presented in dairy whey.

Very Strong But Reversible Immobilization of Enzymes on Supports Coated With Ionic Polymers

In this chapter, the properties of tailor-made anionic exchanger resins based on films of large polyethylenimine polymers (e.g., molecular weight 25,000) as supports for strong but reversible immobilization of proteins are shown. The polymer is completely coated, via covalent immobilization, the surface of different porous supports. Proteins can interact with this polymeric bed, involving a large percentage of the protein surface in the adsorption. Different enzymes have been very strongly adsorbed on these supports, retaining enzyme activities. On the other hand, adsorption is very strong and the derivatives may be used under a wide range of pH and ionic strengths. These supports may be useful even to stabilize multimeric enzymes, by involving several enzyme subunits in the immobilization.

Stabilization of Multimeric Enzymes Via Immobilization and Further Cross-Linking With Aldehyde-Dextran

Subunit dissociation of multimeric proteins is one of the most important causes of inactivation of proteins having quaternary structure, making these proteins very unstable under diluted conditions. A sequential two-step protocol for the stabilization of this protein is proposed. A multisubunit covalent immobilization may be achieved by performing very long immobilization processes between multimeric enzymes and porous supports composed of large internal surfaces and covered by a very dense layer of reactive groups. Additional cross-linking with polyfunctional macromolecules promotes the complete cross-linking of the subunits to fully prevent enzyme dissociation. Full stabilization of multimeric structures has been physically shown because no subunits were desorbed from derivatives after boiling them in SDS. As a functional improvement, these immobilized preparations no longer depend on the enzyme.