Cesar Mateo

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.

General Trend of Lipase to Self-Assemble Giving Bimolecular Aggregates Greatly Modifies the Enzyme Functionality

Three microbial lipases (those from Candida rugosa, Humicola lanuginosa, and Mucor miehei) have been found to exhibit a tendency to form bimolecular aggregates in solution even at very low enzyme concentrations (44 microg/mL) in the absence of a detergent, as detected by gel filtration. The monomolecular form of the enzymes was found as unique only at low enzyme concentration and in the presence of detergents. However, in the case of the lipase B from Candida antarctica, no bimolecular form could be identified even at enzyme concentrations as high as 1.2 mg/mL in the absence of detergent. It has been stated that bimolecular and monomolecular structures display very different functional properties: (i) the enzyme specific activity decreased when the lipase concentration increased; (ii) the bimolecular form was much more stable than the monomeric one yielding a higher optimal T (increasing between 5 and 10 degrees C) and higher stability in inactivation experiments (the dimer half-life became several orders of magnitude higher than that of the monomer); (iii) the enantioselectivity depended on the enzyme concentration even after immobilization. For example, with use of the lipase from H. lanuginosa, the enantiomeric excess of the remaining ester in the hydrolysis of fully soluble ethyl ester of (R,S)-2-hydroxy-4-phenylbutanoic acid varied from 4 to 57 when the concentrated or diluted enzyme immobilized on PEI support, respectively, was used. It seems that the bimolecular structure of lipases might be formed by two open lipase molecules (interfacially activating each other) in very close contact and hence with a very altered active center.

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.

Coating of Soluble and Immobilized Enzymes with Ionic Polymers: Full Stabilization of the Quaternary Structure of Multimeric Enzymes

This paper shows a simple and effective way to avoid the dissociation of multimeric enzymes by coating their surface with a large cationic polymer (e.g., polyethylenimine (PEI)) by ionic exchange. As model enzymes, glutamate dehydrogenase (GDH) from Thermus thermophilus and formate dehydrogenase (FDH) from Pseudomonas sp. were used. Both enzymes are very unstable at acidic pH values due to the rapid dissociation of their subunits (half-life of diluted preparations is few minutes at pH 4 and 25 degrees C). GDH and FDH were incubated in the presence of PEI yielding an enzyme-PEI composite with full activity. To stabilize the enzyme-polymer composite, a treatment with glutaraldehyde was required. These enzyme-PEI composites can be crosslinked with glutaraldehyde by immobilizing previously the composite onto a weak cationic exchanger. The soluble GDH-PEI composite was much more stable than unmodified GDH at pH 4 and 30 degrees C (retaining over 90% activity after 24 h incubation) with no effect of the GDH concentration in the inactivation course. The composite could be very strongly, but reversibly, adsorbed on cationic exchangers. Similarly, FDH could be treated with PEI and glutaraldehyde after adsorption on cationic exchangers, This permitted a stabilized FDH preparation. In this way, the coating of the enzymes surfaces with PEI is used as a simple and efficient strategy to prevent enzyme dissociation of multimeric enzymes. These composites can be used as a soluble catalyst or reversibly immobilized onto a cationic exchanger (e.g., CM-agarose).

Preparation of a Stable Biocatalyst of Bovine Liver Catalase Using Immobilization and Postimmobilization Techniques

Bovine liver catalase was immobilized on different supports. The tetrameric nature of this enzyme was found to cause its rapid inactivation in diluted conditions due to subunit dissociation, a fact that may rule out its industrial use. Multi‐subunit immobilization using highly activated glyoxyl agarose was not enough to involve all enzyme subunits. In fact, washing the derivative produced a strong decrease in the enzyme activity. Further cross‐linking of previously immobilized enzyme with tailor‐made dextran‐aldehyde permitted the multimeric structure to be fully stabilized using either multisubunit preparations immobilized onto highly activated glyoxyl‐agarose support or one subunit enzymes immobilized onto poorly activated glyoxyl‐agarose. The highest stability of the final biocatalyst was observed using the multisubunit immobilized derivative cross‐linked with dextran‐aldehyde. The optimal derivative retained around 60% of the immobilized activity, did not release any enzyme subunits after boiling in the presence of SDS, and did not lose activity during washing, and its stability did not depend on the dilution. This derivative was used for 10 cycles in the destruction of 10 mM hydrogen peroxide without any decrease in the enzyme activity.