Jose M. Guisan

Epoxy Sepabeads: A Novel Epoxy Support for Stabilization of Industrial Enzymes via Very Intense Multipoint Covalent Attachment

Sepabeads‐EP (a new epoxy support) has been utilized to immobilize‐stabilize the enzyme penicillin G acylase (PGA) via multipoint covalent attachment. These supports are very robust and suitable for industrial purposes. Also, the internal geometry of the support is composed by cylindrical pores surrounded by the convex surfaces (this offers a good geometrical congruence for reaction with the enzyme), and it has a very high superficial density of epoxy groups (around 100 μmol/mL). These features should permit a very intense enzyme‐support interaction. However, the final stability of the immobilized enzyme is strictly dependent on the immobilization protocol. By using conventional immobilization protocols (neutral pH values, nonblockage of the support) the stability of the immobilized enzyme was quite similar to that achieved using Eupergit C to immobilize the PGA. However, when using a more sophisticated three‐step immobilization/stabilization/blockage procedure, the Sepabeads derivative was hundreds‐fold more stable than Eupergit C derivatives. The protocol used was as follows: (i) the enzyme was first covalently immobilized under very mild experimental conditions (e.g., pH 7.0 and 20 °C); (ii) the already immobilized enzyme was further incubated under more drastic conditions (higher pH values, long incubation periods, etc.) in order to “facilitate” the formation of new covalent linkages between the immobilized enzyme molecule and the support; (iii) the remaining epoxy groups of the support were blocked with very hydrophilic compounds to stop any additional interaction between the enzyme and the support. This third point was found to be critical for obtaining very stable enzymes: derivatives blocked with mercaptoethanol were much less stable than derivatives blocked with glycine or other amino acids. This was attributed to the better masking of the hydrophobicity of the support by the amino acids (having two charges).

Reversible enzyme immobilization via a very strong and nondistorting ionic adsorption on support–polyethylenimine composites

New tailor-made anionic exchange resins have been prepared, based on films of large polyethylenimine polymers (e.g., MW 25,000) completely coating, via covalent immobilization, the surface of different porous supports (agarose, silica, polymeric resins). Most proteins contained in crude extracts from different sources have been very strongly adsorbed on them. Ionic exchange properties of such composites strongly depend on the size of polyethylenimine polymers as well as on the exact conditions of the covalent coating of the solids with the polymer. On the contrary, similar coating protocols yield similar matrices by using different porous supports as starting material. For example, 77% of all proteins contained in crude extracts from Escherichia coli were adsorbed, at low ionic strength, on the best matrices, and less than 15% of the adsorbed proteins were eluted from the support in the presence of 0.3 M NaCl. Under these conditions, 100% of the adsorbed proteins were eluted from conventional DEAE supports. Such polyethylenimine-support composites were also very suitable to perform very strong and nondistorting reversible immobilization of industrial enzymes. For example, lipase from Candida rugosa (CRL), beta-galactosidase from Aspergillus oryzae and D-amino acid oxidase (DAAO) from Rhodotorula gracilis, were adsorbed on such matrices in a few minutes at pH 7.0 and 4 degrees C. Immobilized enzymes preserved 100% of catalytic activity and remained fully immobilized in 0.2 M NaCl. In addition to that, CRL and DAAO were highly stabilized upon immobilization. Stabilization of DAAO, a dimeric enzyme, seems to be due to the involvement of both enzyme subunits in the ionic adsorption.

Immobilization of enzymes on heterofunctional epoxy supports

Immobilization of enzymes and proteins on activated supports permits the simplification of the reactor design and may be used to improve some enzyme properties. In this sense, supports containing epoxy groups seem to be useful to generate very intense multipoint covalent attachment with different nucleophiles placed on the surface of enzyme molecules (e.g., amino, thiol, hydroxyl groups). However, the intermolecular reaction between epoxy groups and soluble enzymes is extremely slow. To solve this problem, we have designed “tailor-made” heterofunctional epoxy supports. Using these, immobilization of enzymes is performed via a two-step process: (i) an initial physical or chemical intermolecular interaction of the enzyme surface with the new functional groups introduced on the support surface and (ii) a subsequent intense intramolecular multipoint covalent reaction between the nucleophiles of the already immobilized enzyme and the epoxy groups of the supports. The first immobilization may involve different enzyme regions, which will be further rigidified by multipoint covalent attachment. The design of some heterofunctional epoxy supports and the performance of the immobilization protocols are described here. The whole protocol to have an immobilized and stabilized enzyme could take from 3 days to 1 week.

Activation of Bacterial Thermoalkalophilic Lipases is Spurred by Dramatic Structural Rearrangements.

The bacterial thermoalkalophilic lipases that hydrolyze saturated fatty acids at 60–75 °C and pH 8–10 are grouped as the lipase family I.5. We report here the crystal structure of the lipase from Geobacillus thermocatenulatus, the first structure of a member of the lipase family I.5 showing an open configuration. Unexpectedly, enzyme activation involves large structural rearrangements of around 70 amino acids and the concerted movement of two lids, the α6- and α7-helices, unmasking the active site. Central in the restructuring process of the lids are both the transfer of bulky hydrophobic residues out of the N-terminal end of the α6-helix and the incorporation of short side chain residues to the α6 C-terminal end. All these structural changes are stabilized by the Zn2+-binding domain, which is characteristic of this family of lipases. Two detergent molecules are placed in the active site, mimicking chains of the triglyceride substrate, demonstrating the position of the oxyanion hole and the three pockets that accommodate the sn-1, sn-2, and sn-3 fatty acids chains. The combination of structural and biochemical studies indicate that the lid opening is not mediated by temperature but triggered by interaction with lipid substrate.

The coimmobilization of d-amino acid oxidase and catalase enables the quantitative transformation of d-amino acids (d-phenylalanine) into α-keto acids (phenylpyruvic acid)

We have studied the production of phenylpyruvic acid by mild stereospecific oxidation of d-phenylalanine (from d/l racemic mixtures) catalyzed by d-amino acid oxidase (DAAO) from Trigonopsis variabilis. The performance of this reaction requires the continuous bubbling of oxygen that inactivated soluble DAAO. The immobilization of the enzyme inside porous supports avoids the interaction of the enzyme with the hydrophobic oxygen/water interfaces; therefore, this inactivation cause is fully prevented. Hydrogen peroxide (a reaction by-product) exerts deleterious effects on both the enzyme and the desired product (phenylpyruvic acid). These deleterious effects could be reduced by using immobilized DAAO and separately immobilized catalase; however, a much more effective elimination of hydrogen peroxide was achieved when both enzymes were coimmobilized. The effectiveness of coimmobilized catalase in preventing phenylpyruvic destruction by hydrogen peroxide is more than 20-fold higher than that of separately immobilized catalase. In fact, by using DAAO/catalase-coimmobilized derivatives, more than 98% of phenylpyruvic acid was obtained after complete oxidation of d-phenylalanine by using the racemic mixture as substrate. This also prevented enzyme from inactivation. In this way, 40 reaction cycles were performed without apparent loss of enzyme activity. These results open an effective and simple way of using any oxidase where we can expect very similar problems to those described in this paper.

Immobilization-stabilization of penicillin G acylase from Escherichia coli

AbstractWe have developed a strategy for immobilization-stabilization of penicillin G acylase from E.coli, PGA, by multipoint covalent attachment to agarose (aldehyde) gels. We have studied the role of three main variables that control the intensity of these enzyme-support multiinteraction processes:1.surface density of aldehyde groups in the activated support;2.temperature; and3.contact-time between the immobilized enzyme and the activated support prior to borohydride reduction of the derivatives. Different combinations of these three variables have been tested to prepare a number of PGA-agarose derivatives. All these derivatives preserve 100% of catalytic activity corresponding to the soluble enzyme that has been immobilized but they show very different stability. The less stable derivative has exactly the same thermal stability of soluble penicillin G acylase and the most stable one is approximately 1,400 fold more stable. A similar increase in the stability of the enzyme against the deleterious effect of organic solvents was also observed. On the other hand, the agarose aldehyde gels present a very great capacity to immobilize enzymes through multipoint covalent attachment. In this way, we have been able to prepare very active and very stable PGA derivatives containing up to 200 International Units of catalytic activity per mL. of derivative with 100% yields in the overall immobilization procedure.

Modulation of the enantioselectivity of Candida antarctica B lipase via conformational engineering. Kinetic resolution of (±)-α-hydroxy-phenylacetic acid derivatives

Abstract The modulation, via immobilization and engineering the reaction medium, of the enantioselectivity exhibited by the lipase from Candida antarctica B (CABL) in the hydrolysis of α-hydroxy-phenylacetic acid derivatives is shown. The enzyme was purified and immobilized using different protocols to obtain immobilized enzyme preparations with different orientations and micro-environments. The catalytic properties (activity, specificity, enantioselectivity) of the resulting derivatives were found to be quite different from each other. The enantioselectivity ( E value) strongly depends on the type of derivative and the conditions employed. Thus, the enzyme immobilized on cyanogen bromide (CNBr) presented E =7.4, while the PEI derivative yielded E =67 in the hydrolysis of α-hydroxy-phenylacetic acid methyl ester under similar conditions. Moreover, the enantioselectivity of the PEI derivative decreased from 67 to 14 on lowering the reaction temperature from 25 to 4°C at pH 5, while the E of some other derivatives improved significantly under similar experimental changes. Similar changes in the E values were observed in the hydrolysis of ( RS )-2-butyroyl-2-phenylacetic acid. Using this substrate, the interfacially adsorbed enzyme (octadecyl) afforded an E value of only 2 at pH 5, while the glutaraldehyde derivative presented a high enantioselectivity ( E >400) under all conditions studied. The corresponding ( S )-ester and ( R )-acid were obtained with excellent enantiomeric excess using the glutaraldehyde derivative, while using the interfacially immobilized one there was no appreciable enantioselectivity. Thus, using differently immobilized derivatives and different experimental conditions, lipase enantioselectivity could vary from negligible to up to 400. The experimental conditions were also found to have varying effects on the different lipase derivatives.

Stabilization of multimeric enzymes via immobilization and post-immobilization techniques

Abstract Controlled and directed immobilization plus post-immobilization techniques are proposed to get full stabilization of the quaternary structure of most multimeric industrial enzymes. The sequential utilization of two stabilization approaches is proposed: (a) Multi-subunit immobilization: a very intense multi-subunit covalent immobilization has been achieved by performing very long immobilization processes between multimeric enzymes and porous supports composed by large internal surfaces and covered by a very dense layer of reactive groups secluded from the support surface through very short spacer arms. (b) Additional cross-linking with poly-functional macromolecules: additional chemical modification of multi-subunit immobilized derivatives with polyfunctional macromolecules promotes an additional cross-linking of all subunits of most of multimeric enzymes. A number of homo and hetero-dimeric enzymes has been stabilized by the simple application of multi-subunit immobilization but more complex multimeric enzymes (e.g., tetrameric ones) were only fully stabilized after the sequential application of both strategies. After such stabilization of the quaternary structure these three features were observed: no subunits were desorbed from derivatives after boiling them in SDS, thermal inactivation becomes independent from enzyme concentration and derivatives became much more stable than soluble enzymes as well as than non-stabilized derivatives. For example, thermal stability of d -amino acid oxidase from Rhodotorula gracilis was increased 7.000 fold after stabilization of its quaternary structure.

Structural and functional stabilization of L-asparaginase via multisubunit immobilization onto highly activated supports

A new protocol for the stabilization of the quaternary structure of multimeric enzymes has been attempted using as model enzyme (tetrameric) L‐asparaginase from Escherichia coli. Such strategy is based upon multisubunit covalent immobilization of the enzyme onto activated supports (agarose‐glutaraldehyde). Supports activated with different densities of reactive groups were used; the higher the density of groups, the higher the stabilization attained. However, because of the complexity of that enzyme, even the use of the highest densities of reactive groups was not enough to encompass all four subunits in the immobilization process. Therefore, a further chemical intersubunit cross‐linking with aldehyde‐dextran was pursued; these derivatives displayed a fully stabilized multimeric structure. In fact, boiling the modified enzyme derivative in the presence of sodium dodecyl sulfate and β‐mercaptoethanol did not lead to release of any enzyme subunit into the medium. Such a derivative, prepared under optimal conditions, retained ca. 40% of the intrinsic activity of the free enzyme and was also functionally stabilized, with thermostabilization enhancements of ca. 3 orders of magnitude when compared with its soluble counterpart. This type of derivative may be appropriate for extracorporeal devices in the clinical treatment of acute leukemia and might thus bring about inherent advantages in that all subunits are covalently bound to the support, with a longer half‐life and a virtually nil risk of subunit release into the circulating blood stream.

Affinity chromatography of polyhistidine tagged enzymes: New dextran-coated immobilized metal ion affinity chromatography matrices for prevention of undesired multipoint adsorptions

New immobilized metal ion affinity chromatography (IMAC) matrices containing a high concentration of metal-chelate moieties and completely coated with inert flexible and hydrophilic dextrans are here proposed to improve the purification of polyhistidine (poly-His) tagged proteins. The purification of an interesting recombinant multimeric enzyme (a thermoresistant beta-galactosidase from Thermus sp. strain T2) has been used to check the performance of these new chromatographic media. IMAC supports with a high concentration (and surface density) of metal chelate groups promote a rapid adsorption of poly-His tagged proteins during IMAC. However, these supports also favor the promotion of undesirable multi-punctual adsorptions and problems may arise for the simple and effective purification of poly-His tagged proteins: (a) more than 30% of the natural proteins contained in crude extracts from E. coli become adsorbed, in addition to our target recombinant protein, on these IMAC supports via multipoint weak adsorptions; (b) the multimeric poly-His tagged enzyme may become adsorbed via several poly-His tags belonging to different subunits. In this way, desorption of the pure enzyme from the support may become quite difficult (e.g., it is not fully desorbed from the support even using 200 mM of imidazole). The coating of these IMAC supports with dextrans greatly reduces these undesired multi-point adsorptions: (i) less than 2% of natural proteins contained in crude extracts are now adsorbed on these novel supports; and (ii) the target multimeric enzyme may be fully desorbed from the support using 60 mM imidazole. In spite of this dramatic reduction of multi-point interactions, this dextran coating hardly affects the rate of the one-point adsorption of poly-His tagged proteins (80% of the rate of adsorption compared to uncoated supports). Therefore, this dextran coating of chromatographic matrices seems to allow the formation of strong one-point adsorptions that involve small areas of the protein and support surface. However, the dextran coating seems to have dramatic effects for the prevention of weak or strong multipoint interactions that should involve a high geometrical congruence between the enzyme and the support surface.