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Stabilization of enzymes via immobilization techniques is a valuable approach in order to convert a necessary protocol (immobilization) into a very interesting tool to improve key enzyme properties (stabilization). Multipoint covalent attachment of each immobilized enzyme molecule may promote a very interesting stabilizing effect. The relative distances among all enzyme residues involved in immobilization have to remain unaltered during any conformational change induced by any distorting agent. Amino groups are very interesting nucleophiles placed on protein surfaces. The immobilization of enzyme through the region having the highest amount of amino groups (Lys residues) is key for a successful stabilization. Glyoxyl groups are small aliphatic aldehydes that form very unstable Schiff’s bases with amino groups, and they do not seem to be useful for enzyme immobilization at neutral pH. However, under alkaline conditions, glyoxyl supports are able to immobilize enzymes via a first multipoint covalent immobilization through the region having the highest amount of lysine groups. Activation of supports with a high surface density of glyoxyl groups and the performance of very intense enzyme–support multipoint covalent attachments are here described.

Commercial epoxy supports may be very useful tools to stabilize proteins via multipoint covalent attachment if the immobilization is properly designed. In this chapter, a protocol to take full advantage of the support’s possibilities is described. The basics of the protocol are as follows: (1) the enzymes are hydrophobically adsorbed on the supports at high ionic strength. (2) There is an “intermolecular” covalent reaction between the adsorbed protein and the supports. (3) The immobilized protein is incubated at alkaline pH to increase the multipoint covalent attachment, thereby stabilizing the enzyme. (4) The hydrophobic surface of the support is hydrophylized by reaction of the remaining groups with amino acids in order to reduce the unfavorable enzyme–support hydrophobic interactions. This strategy has produced a significant increase in the stability of penicillin G acylase compared with the stability achieved using conventional protocols.

The utilization of biomaterials as novel carrier materials for lipase immobilization has been investigated by many research groups over recent years. Biomaterials such as agarose, starch, chitin, chitosan, cellulose, and their derivatives have been extensively studied since they are non-toxic materials, can be obtained from a wide range of sources and are easy to modify, due to the high variety of functional groups on their surfaces. However, although many lipases have been immobilized on biomaterials and have shown potential for application in biocatalysis, special features are required when the biocatalyst is used in non-conventional media, for example, in organic solvents, which are required for most reactions in organic synthesis. In this article, we discuss the use of biomaterials for lipase immobilization, highlighting recent developments in the synthesis and functionalization of biomaterials using different methods. Examples of effective strategies designed to result in improved activity and stability and drawbacks of the different immobilization protocols are discussed. Furthermore, the versatility of different biocatalysts for the production of compounds of interest in organic synthesis is also described.

The synthesis of compounds or chiral building-blocks with the desired configuration is one of the greatest challenges of chemistry and is of great interest in different fields such as analytical chemistry and especially in fine and pharmaceutical chemistry. [...]

The production of xylo-oligosaccharides from xylan using an optimal immobilized catalyst of xylanase from Aspergillus niger is presented. The enzyme extract has several xylanases with different properties doing necessary the development of cheap and simple methods to purify them at industrial scale. The enzyme was successfully purified, immobilized and highly stabilized using a simple protocol. The principal purified xylanase was a 34 KDa protein corresponding with 50% of the endo-xylanase activity of the total strain. Among the different immobilization assayed protocols, the use of aldehyde support allowed the complete immobilization of this fraction keeping 80% of its initial catalytic activity. An optimization of this method promoted a stabilization factor of around 1100-fold more stable than soluble enzyme. The use of the optimal catalyst allowed a maximum hydrolysis degree of 73.4%. The optimization of the reaction conditions (different time and temperature) allowed producing 62% (11.93 mg/mL) of interesting xylooligo-saccharides (XOS2-XOS6) (Figure 1).

Immobilized and stabilized lipases are important biocatalytic tools. In this paper, different tailor-made bifunctional supports were prepared for the immobilization of a new metagenomic lipase (LipC12). The new supports contained hydrophobic groups (different alkyl groups) to promote interfacial adsorption of the lipase and aldehyde groups to react covalently with the amino groups of side chains of the adsorbed lipase. The best catalyst was 3.5-fold more active and 5000-fold more stable than the soluble enzyme. It was successfully used in the regioselective deacetylation of peracetylated d-glucal. The PEGylated immobilized lipase showed high regioselectivity, producing high yields of the C-3 monodeacetylated product at pH 5.0 and 4 °C.

Immobilization of enzymes on glutaraldehyde-activated supports has been largely used on supports previously activated with amine groups. Therefore, the supports are positively charged hence usually the immobilization is promoted through a two step mechanism: in a first step the enzyme is adsorbed on the support via an anionic exchange mechanism and then, the covalent immobilization occurs. In this paper a new glutaraldehyde activated support without a net charge is presented and characterized in immobilizations of trypsin, penicillin acylase G, lipase and E. coli BL21 cell extract. Immobilization mechanism was studied and this was produced without an adsorption step. This support promoted initially a reversible immobilization, converting into irreversible after incubation of the enzyme-support for several days or after a reduction step. In addition the stability of glutaraldehyde groups was studied retaining around 50 and 25% of its immobilization capacity for 24 h at pH 7 and 10 respectively. This fact allows the incubation of the enzyme with the support even at alkaline pH promoting an extra stabilization factor for trypsin on this support.

The hydrolysis of phenolic compounds using an immobilized and highly active and stable derivative of laccase from Trametes versicolor is presented. The enzyme was immobilized on aldehyde supports. For this, the enzyme was enriched in amino groups by chemical modification of its carboxyl groups. The aminated enzyme was immobilized with a high recovered activity (over 60%). Aldehyde derivatives were more stable than soluble or aminated-soluble enzyme and the reference derivatives after incubation in different inactivating conditions (high temperatures, different pH values or presence of organic cosolvents). The most stable derivative was obtained immobilizing the chemically aminated enzyme at pH 10 on aldehyde supports with a stabilization factor approximately 280 fold after incubation at pH 7 and 55 °C. In addition, it was possible to prepare immobilized derivatives with a maximal enzyme loading of 60 mg g−1 of support. This derivative could be reused for 10 reaction cycles with negligible lost of activity.

This Communication presents the development of a novel strategy for the easy conjugation of biomolecules to hydrophobic magnetic microparticles via reversible coating with previously functionalized lipase molecules. First, the ability of lipase to be strongly adsorbed onto hydrophobic surfaces was exploited for the stabilization of microparticles in aqueous medium by the creation of a hydrophilic surface. Second, the surface amino acids of lipase can be tailored to suit biomolecule conjugation. This approach has been demonstrated by amino-epoxy activation of lipase, enabling the conjugation of different biomolecules to the magnetic particle's surface. For example, it was possible to immobilize 70% of Escherichia coli proteins on the recovered particles. Furthermore, this strategy could be extended to other lipase chemical modification protocols, enabling fine control of biomolecule coupling. These conjugation techniques constitute a modular methodology that also permits the recycling of the magnetic carrier following use. © 2013 American Chemical Society.

The production of xylooligosaccharides (XOS) using a packed-bed enzymatic reactor was studied at lab-scale. For this, a xylanase from Aspergillus versicolor was immobilized on different supports. The optimal derivative was xylanase immobilized on glyoxyl-agarose supports. This derivative preserved 85% of its catalytic activity; it was around 700-fold more stable than the soluble enzyme after incubation at 60 C and was able to be reused for at least 10 1 h-cycles retaining full catalytic activity. About 18% of oligosaccharides with prebiotic interest (X2-X6) were produced by the glyoxyl derivative in batch hydrolysis. The production of xylobiose was 2.5-fold higher using the immobilized preparation than with soluble enzyme and small concentrations of xylose (<0.1%) were observed only at the end of the reaction. The derivative was employed on a packed bed reactor, and the continuous operation with no recirculation reached 56% and 70% of the end of reaction with flow rates of 60 mL/h and 12 mL/h, respectively. In continuous operation with recirculation at a flow rate of 60 mL/h, the reaction was completed after four hours. © 2013 Elsevier B.V. All rights reserved.

Novel heterofunctional glyoxyl-agarose supports were prepared. These supports contained the maximal concentration of glyoxyl groups to promote maximization of covalent immobilization and groups' capability to adsorb proteins by various mechanisms (e.g., ionic exchange, metal-chelate formation). Immobilization on various supports makes it possible to orientate and rigidify an enzyme in various regions of its surface. The use of different heterofunctional supports allowed for obtaining catalysts with different activity, stability, and selectivity properties. © Springer Science+Business Media, New York 2013.

The soluble lipase from Pseudomonas fluorescens (PFL) forms bimolecular aggregates in which the hydrophobic active centers of the enzyme monomers are in close contact. This bimolecular aggregate could be immobilized by multipoint covalent linkages on glyoxyl supports at pH 8.5. The monomer of PFL obtained by incubation of the soluble enzyme in the presence of detergent (0.5% TRITON X-100) could not be immobilized under these conditions. The bimolecular aggregate has two amino terminal residues in the same plane. A further incubation of the immobilized derivative under more alkaline conditions (e.g., pH 10.5) allows a further multipoint attachment of lysine (Lys) residues located in the same plane as the amino terminal residues. Monomeric PFL was immobilized at pH 10.5 in the presence of 0.5% TRITON X-100. The properties of both PFL derivatives were compared. In general, the bimolecular derivatives were more active, more selective and more stable both in water and in organic solvents than the monomolecular ones. The bimolecular derivative showed twice the activity and a much higher selectivity (100 versus 20) for the hydrolysis of R,S-2-hydroxy-4-phenylbutyric acid ethyl ester (HPBEt) in aqueous media at pH 5.0 compared to the monomeric derivative. In experiments measuring thermal inactivation at 75 °C, the bimolecular derivative was 5-fold more stable than the monomeric derivative (and 50-fold more stable than a one-point covalently immobilized PFL derivative), and it had a half-life greater than 4 h. In organic solvents (cyclohexane and tert-amyl alcohol), the bimolecular derivative was much more stable and more active than the monomeric derivative in catalyzing the transesterification of olive oil with benzyl alcohol. © 2012 Elsevier Ltd. All rights reserved.

An endoxylanase from Streptomyces halstedii was stabilized by multipoint covalent immobilization on glyoxyl-agarose supports. The immobilized enzyme derivatives preserved 65% of the catalytic activity corresponding to the one of soluble enzyme that had been immobilized. These immobilized derivatives were 200 times more stable 200 times more stable than the one-point covalently immobilized derivative in experiments involving thermal inactivation at 60 °C. The activity and stability of the immobilized enzyme was higher at pH 5.0 than at pH 7.0. The optimal temperature for xylan hydrolysis was 10 °C higher for the stabilized derivative than for the non-stabilized derivative. On the other hand, the highest loading capacity of activated 10% agarose gels was 75 mg of enzyme per mL of support. To prevent diffusional limitations, low loaded derivatives (containing 0.2 mg of enzyme per mL of support) were used to study the hydrolysis of xylan at high concentration (close to 1% (w/v)). 80% of the reducing sugars were released after 3 h at 55 °C. After 80% of enzymatic hydrolysis, a mixture of small xylo-oligosaccharides was obtained (from xylobiose to xylohexose) with a high percentage of xylobiose and minimal amounts of xylose. The immobilized-stabilized derivatives were used for 10 reaction cycles with no loss of catalytic activity. © 2013 Elsevier Ltd. All rights reserved.

The enantioselectivity of a lipase from Geobacillus thermocatenulatus (BTL2) has been altered by a site-directed immobilization on tailor-made disulfide supports. The enzyme was genetically modified introducing a unique cysteine into different positions of the protein surface. These new enzyme variants (φ-BTL2) maintained the catalytic properties of the native enzyme. The φ-BTL2 was immobilized by different orientation on disulfide support at pH 7 via disulfide-exchange and additionally rigidification was introduced by the immobilization on disulfide-aldehyde support through the interaction of the reactive amino groups on the protein near to the position of the introduced cysteine with aldehydes on the support. The oriented immobilization of the φ-BTL2-S334C variant on disulfide-aldehyde supports generated a fully enantioselective biocatalyst (ee > 99%) in the kinetic resolution of rac-2-O-butyryl-2-phenylacetic acid and the asymmetric hydrolysis of phenylglutaric acid dimethyl diester. The simple orientation by the introduced cysteine on the lipase immobilization on disulfide supports was enough for good selectivity (ee > 99%) in the asymmetric hydrolysis but not in the kinetic resolution (ee = 27%). The rigidification of the oriented immobilized Q40C, T94C or S334C variants on disulfide-aldehyde support was key in the lipase selectivity improvement in the kinetic resolution process (ee from 27-50 to >99%). In this case, the oriented immobilization did not alter significantly the selectivity. A similar result was obtained for S196C variants in the asymmetric hydrolysis, which it selectivity was greatly altered depending on the immobilization method, no selectivity was observed after immobilization on disulfide support but a fully selective catalyst (ee > 99%) was achieved after immobilization on heterofunctional disulfide-aldehyde support. © 2012 Elsevier B.V.

La presente invención se refiere a soportes heterofuncionales activados con grupos capaces de adsorber proteínas a pH neutro vía diferentes mecanismos y grupos aldehído muy reactivos capaces de inmovilizar las enzimas previamente adsorbidas de modo covalente multipuntual. El uso de este tipo de soportes permite la inmovilización y rigidificación de enzimas a través de diferentes regiones de su superficie. Mediante la inmovilización de diferentes proteínas de interés industrial sobre diferentes soportes glioxil heterofuncionales se obtuvieron derivados con diferentes actividades, estabilidades y selectividades.

An endo-1,5-arabinanase (abnA) encoding gene from Aspergillus niveus was identified, cloned and successfully expressed in Aspergillus nidulans strain A773. Based on amino acid sequence comparison, the 34-kDa enzyme could be assigned to CAZy GH family 43. Characterization of purified recombinant endo-1,5-arabinanase (AbnA) revealed that it is active at a wide pH range (pH 4.0-7.0) and an optimum temperature at 70 °C. The immobilization of the AbnA was performed via covalent binding onto agarose-modified supports: glyoxyl iminodiacetic acid-Ni 2+, glyoxyl amine, glyoxyl (4% and 10%) and cyanogen bromide activated sepharose. The yield of immobilization was similar on glyoxyl amine and glyoxyl (96%), and higher than glyoxyl iminodiacetic acid-Ni 2+ (43%) support. The thermal inactivation of these immobilized preparations showed that the stability of the AbnA immobilized on glyoxyl 4 and 10% was improved by 4.0 and 10.3-fold factor at 70 °C. The half-life of glyoxyl 4% derivative at 60 °C was >48 h (pH 5), 9 h (pH 7) and 88 min (pH 9). The major hydrolysis product of debranched arabinan or arabinopentaose by glyoxyl agarose-immobilized AbnA was arabinobiose. © 2012 Elsevier B.V. All rights reserved.

Commercial glucoamylase immobilizes very slowly on highly activated glyoxyl-agarose supports. The resulting derivatives were only 6-fold more stable than soluble enzyme. The unmodified glucoamylase, highly glycosylated, seems to have a low number of Lys able to react with glyoxyl groups on the support. Thus, the enzyme surface was highly enriched in amino groups by chemical modification of carboxyl groups (activated with carbodiimide) with ethylenediamine. The aminated enzyme preserves a good percentage of activity (80%) and it exhibits the same stability than unmodified enzyme. The aminated enzyme was immobilized very rapidly on highly activated glyoxyl-agarose support. The new resulting derivatives preserved 50% of activity and were more than 500-fold more stable than soluble enzyme in experiments of thermal inactivation. © 2010 Elsevier Ltd. All rights reserved.

Complejos covalentes de lipasas con proteínas, sondas de ADN, cofactores u otras biomoléculas. Complejos que comprenden lipasas unidas covalentemente, a través de regiones no involucradas en su centro activo, a biomoléculas de interés (proteínas, como enzimas, anticuerpos o proteínas A o G, cofactores o sondas de ADN). Los complejos se pueden inmovilizar a través del centro activo intacto de las lipasas sobre todo tipo de soportes hidrofóbicos sin necesidad de realizar modificaciones o activaciones en los mismos. La unión de la lipasa a los soportes hidrofóbicos es reversible, lo que permite la inmovilización reversible de los complejos. La invención proporciona dos métodos, químico y biológico, de preparación de estos complejos y un método de inmovilización de los mismos. Estos complejos inmovilizados pueden tener aplicaciones en la industria, en el laboratorio o en diagnóstico, debido a su uso potencial como biocatalizadores o como biosensores. El método químico comprende la adsorción de la lipasa a un soporte hidrofóbico poroso, la conjugación de la lipasa así adsorbida con la biomolécula y desorber este complejo del soporte mediante el uso de detergentes. El método biológico comprende la inserción del gen de la biomolécula en un plásmido conteniendo el gen de la lipasa, en un microorganismo, para que éste exprese la lipasa fusionada a la biomolécula.#La inmovilización reversible de los complejos creados a partir de cualquiera de los dos métodos se puede realizar en soportes hidrofóbicos no porosos.

A new anion exchanger support has been designed for the selective adsorption of small proteins. This has been achieved activating an aminated support with glutaraldehyde and further coating the support surface with bovine serum albumin (BSA). In this support, " wells" are generated by two neighborhoods BSA molecules, on the bottom of those " wells" glutaraldehyde groups are exposed out ready to react with small molecules that have a size small enough to be accommodated between two BSA molecules on the pre-existing support. However, the BSA surface was not inert enough adsorbing many proteins, thereby reducing the selectivity of the system. A further solution was coating the immobilized BSA molecules with dextran, reducing the adsorption of protein on the BSA surface. This new matrix has been evaluated in the selective adsorption of the very small beta-lactoglobulins and alpha-lactalbumin from dairy whey, achieving the selective adsorption of both small proteins while other larger proteins from dairy whey remained in the supernatant. Moreover, a protein crude extract has been offered to the new matrix, and only small proteins could be adsorbed on the support (as probed by gel filtration).Thus this amino-glutaraldehyde-BSA-dextran-Sepharose is a matrix that may be used to selectively ionically adsorb proteins that were smaller than BSA (62. kDa). This strategy may be used for any other kind of adsorbing groups (chelating agents, boronic acid, etc.), or using proteins with different sizes to coat the support, designing tailor-made supports that may permit the fractioning of proteins following their sizes and by adsorption/desorption on different matrices. © 2010 Elsevier B.V.

Novel heterofunctional glyoxyl-agarose supports were prepared. These supports contain a high concentration of groups (such as quaternary ammonium groups, carboxyl groups, and metal chelates) that are capable of adsorbing proteins, physically or chemically, at neutral pH as well as a high concentration of glyoxyl groups that are unable to immobilize covalently proteins at neutral pH. By using these supports, a two-step immobilization protocol was developed. In the first step, enzymes were adsorbed at pH 7.0 through adsorption of surface regions, which are complementary to the adsorbing groups on the support, and in the second step, the immobilized derivatives were incubated under alkaline conditions to promote an intramolecular multipoint covalent attachment between the glyoxyl groups on the support and the amino groups on the enzyme surface. These new derivatives were compared with those obtained on a monofunctional glyoxyl support at pH 10, in which the region with the greatest number of lysine residues participates in the first immobilization step. In some cases, multipoint immobilization on heterofunctional supports was much more efficient than what was achieved on the monofunctional support. For example, derivatives of tannase from Lactobacillus plantarum on an amino-glyoxyl heterofunctional support were 20-fold more stable than the best derivative on a monofunctional glyoxyl support. Derivatives of lipase from Geobacillus thermocatenulatus (BTL2) on the amino-glyoxyl supports were two times more active and four times more enantioselective than the corresponding monofunctional glyoxyl support derivative

A novel approach is proposed to prepare a set of immobilized derivatives of a enzyme covalently rigidified through different regions of its surface. Six different variants of penicillin G acylase (PGA) from Escherichia coli (which lacks Cys) were prepared by introducing a unique Cys residue via site-directed mutagenesis in six different enzyme regions which were rich in Lys residues. All variants exhibited a similar activity and stability compared to those of the native enzyme. Each variant was immobilized on supports having a low concentration of reactive disulfide moieties and a high concentration of poorly reactive epoxy groups. After immobilization at pH 7.0 by site-directed thiol-disulfide intermolecular exchange, derivatives were further incubated at pH 10.0 for 48 h to promote an additional intramolecular reaction between Lys residues of enzyme and epoxy groups of the support. The establishment of at least three covalent attachments per PGA molecule was determined for all immobilized enzyme variants. The different derivatives exhibited diverse stability against several distorting agents and different selectivity in two interesting reactions. The derivative of the PGA variant obtained by replacement of GlnB380 by Cys was the most stable against heat and organic cosolvents: it preserved 90% of the initial activity and was 30-fold more stable than soluble PGA. This derivative also exhibited an improved enantioselectivity in the hydrolysis of chiral esters (E was improved from 8 to 16) and in kinetically controlled synthesis of amides (synthetic yields were increased from 31 to 49%).

[EN] The invention relates to complexes that incIude lipases covalently bonded, by means of regions not involved in the active centre thereof, to biomolecules of interest (proteins such as enzymes, antibodies or proteins A or G, cofactors or DNA probes). The complexes can be immobilised via the intact active centre ofthe lipases on all types ofhydrophobic media, without requiring any modifications or activations thereof. The bonding of the lipase to the hydrophobic media is reversible, allowing the reversible immobilisation of the complexes. The invention provides two methods, chemical and biological, for preparing said complexes, as well as a method for immobilising said complexes. Said immobilised complexes can be used in industry, in laboratories or in diagnostics, given the potential use thereof as biocatalysts or as biosensors. The chemical method incIudes adsorbing the lipase to a porous hydrophobic medium, combining the lipase thus adsorbed with the biomolecule, and desorbing said complex from the medium using detergents. The biological method incIudes inserting the gene of the biomolecule in a plasmid containing the gene of the lipase, in a microorganism, in order for the latter to express the lipase fused with the biomolecuIe. The reversible immobilisation ofthe complexes generated using either ofthe two methods can be carried out on nonporous hydrophobic media., [ES] Complejos que comprenden lipasas unidas covalentemente, a través de regiones no involucradas en su centro activo, a biomoléculas de interés (proteinas, como enzimas, anticuerpos o proteinas A o G, cofactores o sondas de ADN). Los complejos se pueden inmovilizar a través del centro activo intacto de las lipasas sobre todo tipo de soportes hidrofóbicos sin necesidad de realizar modificaciones o activaciones en los mismos. La unión de la lipasa a los soportes hidrofóbicos es reversible, lo que permite la inmovilización reversible de los complejos. La invención proporciona dos métodos, quimico y biológico, de preparación de estos complejos y un método de inmovilización de los mismos. Estos complejos inmovilizados pueden tener aplicaciones en la industria, en el laboratorio o en diagnóstico, debido a su uso potencial como biocatalizadores o como biosensores. El método quimico comprende la adsorción de la lipasa a un soporte hidrofóbico poroso, la conjugación de la lipasa asi adsorbida con la biomolécula y desorber este complejo del soporte mediante el uso de detergentes. El método biológico comprende la inserción del gen de la biomolécula en un plásmido conteniendo el gen de la lipasa, en un microorganismo, para que éste exprese la lipasa fusionada a la biomolécula. La inmovilización reversible de los complejos creados a partir de cualquiera de los dos métodos se puede realizar en soportes hidrofóbicos no porosos.

Procedimiento para la inmovilización covalente orientada de anticuerpos, anticuerpos así obtenidos y sus aplicaciones. La invención describe un procedimiento para la inmovilización covalente orientada de anticuerpos sobre un soporte activado con grupos glioxil que dirigen la inmovilización por la región de la superficie del anticuerpo opuesta al fragmento Fab del anticuerpo. Este anticuerpo inmovilizado obtenido por el procedimiento de la invención puede ser utilizado en distintos procesos biotecnológicos como por ejemplo en cromatografía de afinidad o de detección de antígenos utilizado como inmunosensor.

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 -amino acid oxidase from Rhodotorula gracilis was increased 7.000 fold after stabilization of its quaternary structure.