Use of crosslinked crystals as a novel form of enzyme immobilization

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DESCRIPTION

Use of cross-linked crystals as a new form of enzyme immobilization.

Background of the invention

Enzymes are used as industrial catalysts for the inexpensive, experimental and large-scale production of fine chemicals and specialty chemicals (Jones, JB, Tetrahedron 42: 3351-3403 (1986)), for food production (Zaks et. Al. , Trends in Biotechnology 6: 272-275 (1988)), and as tools for the synthesis of organic compounds (Wong, C.-H., Science 244: 1145-1152 (1989); CHEMTRACTS-Org. Chem. 3: 91-111 (1990): Klibanov, AM, Acc. Chem. Res. 23: 114-120 (1990)).

Enzyme-based manufacturing can significantly reduce the burden of environmental pollution implicit in the large-scale manufacture of otherwise unusable chemical intermediates, as shown in the large-scale production of acrylamide using the enzyme nitrile hydratase (Nagasawa, T . and Yamada; H., Trends in Biotechnology 7: 153-158 (1989)).

Enzymes are also used in biosensor applications to detect various substances of clinical, industrial or other interest (Hall, E., "Biosensors", Open University Press (1990)). In the clinical area, enzymes can be used in extracorporeal therapies, such as hemodialysis and hemofiltration, where enzymes selectively remove waste and toxic materials from the blood (Klein, M. and Langer, R., Trends in Biotechnology 4: 179-185 (1986)). Enzymes are used in these areas because they function effectively as catalysts for a wide range of reaction types, at moderate temperatures, and with substrate specificity and stereoselectivity. However, there are disadvantages associated with the use of soluble enzyme catalysts, which have limited their use in laboratory and industrial chemical processes (Akiyama et. Al., CHEMTECH 627-634 (1988)).

Enzymes are expensive and relatively unstable compared to most industrial and laboratory catalysts, even when used in aqueous media, where they function normally. Many of the more economically interesting chemical reactions carried out in common practice are incompatible with aqueous media, where, for example, substrates and products are generally insoluble or unstable, and where hydrolysis can compete significantly. Also, the recovery of the soluble enzyme catalyst from the unreacted product and substrate in the base material generally requires the application of complex and expensive separation technology. Finally, it is difficult to store enzymes in such a way that they retain their activity and functional integrity for commercially reasonable periods of time (months to years) without having to resort to refrigeration (temperatures 4 ° C to -80 ° C). ° C to N₂ liquid), or by maintenance in aqueous solvents of appropriate ionic strength, appropriate pH, etc.

In many cases, thanks to enzyme immobilization methods, solutions have been found for these disadvantages. Immobilization can improve the stability of enzyme catalysts and protect their functional integrity in the media of aggressive solvents and extreme temperatures characteristic of laboratory and industrial chemical processes (Hartmeier, W., Trends in Biotechnology 3: 149-153 (1985) ). Continuous flow processes can be operated with enzyme particles immobilized in columns where, for example, soluble base material passes over the particles and is gradually converted to product. As used in the present invention, the term "enzyme immobilization" refers to the insolubilization of the enzyme catalyst by binding to, encapsulating, or agglomerating into macroscopic particles (10^-1 mm).

Several useful reviews of enzyme immobilization methods have appeared in literature sources (Maugh, TH, Science 223: 474-476 (1984); Tramper, J., Trends in Biotechnology 3: 45-50 (1985)). Maugh describes five general approaches to enzyme immobilization. These include: adsorption on solid supports (such as ion exchange resins); covalent attachment to supports (such as ion exchange resins, porous ceramic materials, or glass beads); entrapment in polymeric gels; encapsulation; and precipitation of soluble proteins by crosslinking with bifunctional reagents in an undefined and random manner. EP-A367302 (Soejima et al.) Describes a water soluble cross-linked Achromobacter I protease that can be used in a process for semi-synthesizing human insulin. EP-A-341503 (Visuri) describes a water insoluble glucose isomerase formed by a crystalline enzyme converted to solid form by cross-linking with dialdehyde (glutaraldehyde) in the presence of a compound containing an amino group. Likewise, it is possible to immobilize whole cells (usually dead and made permeable) that have expressed the desired enzymatic activity at high levels (eg Nagasawa, T. and Yamada, H., Trends in Biotechnology 7: 153-158 (1989 )).

Each of these immobilization procedures has its own advantages and limitations, and none of them can be considered optimal or dominant. In most of them, the enzyme catalyst at the bottom represents only a small fraction of the total volume of material present in the chemical reactor. As such, the mass of the immobilized medium is composed of inert, but generally expensive, carrier material. In all of them, the immobilization interactions of the enzyme catalyst molecules with each other and / or with the carrier material tend to be random and undefined. As a result, while these interactions give some improved stability to the enzyme catalyst molecules, their relative nonspecificity and unevenness make this stabilization suboptimal and uneven. In most cases, access to the active site of the enzyme catalyst remains poorly defined. Furthermore, with the immobilization methods described above, it is impossible to solve associated problems.

ES 2 199 933 T3 two with storage and cooling. And enzymes immobilized by conventional means generally cannot be manipulated, such as converting them into one or another solvent of choice, without jeopardizing the functional and structural integrity of the enzyme. In practice, except for binding to the carrier particle, enzymes immobilized by conventional means strongly resemble soluble enzymes, and share with them a susceptibility to denaturation and loss of function in aggressive environments.

Generally speaking, immobilization methods lead to observed enzyme-catalyzed reduction of reaction rates relative to those obtained in solution. This is largely a consequence of the limits of internal diffusion of substrate and external diffusion of product within the immobilized enzyme particle (Quiocho, FA, and Richards, FM, Biochemistry 5: 4062-4076 (1967)). The necessary presence of inert carrier in the immobilized enzyme particles increases the mean free path between the solvent outside the immobilized enzyme particle and the active site of the enzyme catalyst and thus exacerbates these diffusion problems. Being immobilized cells, the diffusion problem is particularly serious, even when the walls and membranes of the cells are permeabilized to the substrate and the product in some way. A further concern would be the multitude of contaminating enzyme activities, metabolites and toxins contained in cells and the stability of cells in harsh solvent and extreme temperature operating environments. An improved immobilization technique that does not contain the limitations of currently available methods would be useful in promoting the use of enzymes as industrial catalysts, particularly if it is shown that it can be used on a large scale (Daniels, MJ, Methods in Enzymology 136: 371- 379 (1987)).

Summary of the invention

The present invention relates to a method of immobilizing an enzyme by forming crystals of the enzyme and also generally by crosslinking the resulting crystals using a bifunctional reagent; to cross-linked immobilized enzyme crystals (referred to as CLEC or CLIEC) produced with this method; to the lyophilization of CLECs as a means of improving the storage, handling and manipulation properties of immobilized enzymes and to a method of manufacturing a desired product through a reaction catalyzed by a CLEC or set of CLECs.

In the method of the present invention, small protein crystals (10^-1 mm) from aqueous solutions or aqueous solutions containing organic solvents, in which the enzyme catalyst is structurally and functionally stable. In a preferred embodiment, the crystals are then cross-linked with a bifunctional reagent, such as glutaraldehyde. This crosslinking process results in the stabilization of the crystal lattice contacts between the individual enzyme catalyst molecules that make up the crystal. As a result of this added stabilization, the cross-linked immobilized enzyme crystals can function at elevated temperatures, extreme pH values, and in aggressive aqueous, organic, or quasi-anhydrous media, including mixtures thereof. That is, a CLEC of the present invention may function in environments incompatible with the functional integrity of the corresponding native, non-cross-linked, non-crystallized enzyme or enzyme catalysts immobilized by conventional means.

On the other hand, CLECs made with this method can be subjected to lyophilization, thereby producing a lyophilized CLEC that can be stored in this lyophilized form at non-refrigerated (room) temperatures for extended periods of time, and that can be easily reconstituted in Aqueous, organic or aqueous-organic solvents mixed of choice without forming amorphous suspensions and with minimal risk of denaturation.

The present invention also relates to CLECs produced by the present method and their use in laboratory and large-scale industrial production of selected materials such as chiral organic molecules, peptides, carbohydrates, lipids, or other chemical species. Currently, the latter are typically prepared using conventional chemical methods that may require aggressive conditions (eg. aqueous, organic or quasi-anhydrous solvents, mixed aqueous / organic solvents, or elevated temperatures) that are incompatible with the functional integrity of the native, non-cross-linked, non-crystallized enzyme catalyst. Other macromolecules with catalytic activity can also be incorporated into the proposed CLEC technology. These macromolecules can include catalytic antibodies (Lerner, R. A., Benkovic, SJ, and Schultz, PG, Science 252: 659-667 (1991)) and catalytic polynucleotides (Cech, TR, Cell 64: 667-669 (1991); Celander, DW, and Cech, TR Science, 251: 401-407 (1991)).

The present invention also relates to a method for making a selected product by a CLEC catalyzed reaction of the present invention.

In an example of the method and practice of the present invention, the enzyme thermolysin, a zinc metalloprotease, was used to synthesize a chiral precursor of the artificial dipeptidyl sweetener, aspartame. The thermosilin enzyme was crystallized from an initial aqueous solution of 45% dimethylsulfoxide and 55% 1.4M calcium acetate, 0.05M sodium cacodylate at pH 6.5. The resulting crystals were cross-linked with glutaraldehyde to form a thermolysin CLEC. The thermolysin CLEC was then transferred from the aqueous crystallization solution in which it was formed to the ethyl acetate solution containing the substrates, N- (benzyloxycarbonyl) -L-aspartic acid (ZL-Asp) and L-methyl ester. -phenylalanine (L-Phe-OMe). The CLEC of thermolysin was then used to catalyze a condensation reaction of the two substrates to synthesize N- (benzyloxycarbonyl) L-aspartyl-L-phenylalanine (ZL-Asp-L-Phe-OMe) methyl ester, which is the dipeptidyl precursor of the artificial sweetener aspartame. Using any of a large number of known techniques (see, for example, Lindeberg, G., J. Chem. Ed. 64:

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1062-1064 (1987)), the L-aspartic acid in the synthesized dipeptidyl precursor can be deprotected by removal of the benzyloxycarbonyl group (Z-) to produce aspartame (L-Asp-L-Phe-OMe).

In a second example of the method and practice of the present invention, the enzyme thermolysin was used to produce CLEC of thermolysin. The activity and stability of thermolysin CLECs were compared with those of soluble thermolysin under optimal conditions and extreme conditions of temperature and pH, followed by an incubation in the presence of organic solvents and then an incubation in the presence of exogenous protease.

The thermolysin enzyme was crystallized from a 1.2 M calcium acetate and 30% dimethylsulfoxide solution at pH 8.0. The resulting crystals were cross-linked with glutaraldehyde at a concentration of 12.5% ​​to form a CLEC of thermolysin. The thermolysin CLEC was then lyophilized by a standard procedure (Cooper, TG, The Tools of Biochemistry, pages 379-380 (John Wiley and Sons, NY (1977)) to form a lyophilized thermolysin CLEC. This lyophilized CLEC was then directly transformed into the various aqueous, organic and aqueous / mixed organic solvents of choice without the need to resort to a solvent exchange procedure, without forming amorphous suspensions, and with minimal risk of denaturation. These solvents included, without limitation, acetonitrile, dioxane, acetone, and tetrahydrofuran. After incubation, the activity was analyzed spectrophotometrically by cleavage of the furyl-acryloyl-glycyl-L-leucine-amide dipeptide substrate (FAGLA).

In a third example of the method and practice of the present invention, the elastase (porcine pancreatic) enzyme was crystallized from an aqueous solution of 5.5 mg / ml of proteins in 0.1 M sodium acetate at pH 5.0 at room temperature (Sawyer, L. et al., J. Mol. Biol. 118: 137-208). The resulting crystals were cross-linked with glutaraldehyde at a concentration of 5% to form an elastase CLEC. The elastase CLEC was lyophilized as described in Example 2.

In a fourth example of the method and practice of the present invention, and as described therein, the enzyme esterase (porcine liver) was crystallized from an aqueous solution of 15 mg / ml of proteins in 0 calcium acetate. 25 M at pH 5.6 at room temperature. The resulting crystals were cross-linked with glutaraldehyde at a concentration of 12.5% ​​to form an esterase CLEC. The esterase CLEC was lyophilized as described in Example 2.

In a fifth example of the method and practice of the present invention, and as described therein, the lipase enzyme (Geotrichum candidum) was crystallized from an aqueous solution of 20 mg / ml of proteins in 50 mM Tris at pH 7 at room temperature. The resulting crystals were cross-linked with glutaraldehyde at a concentration of 12.5% ​​to form a lipase CLEC. The lipase CLEC was lyophilized as described in Example 2.

In a sixth example of the method and practice of the present invention, the enzyme lysozyme (chicken egg white) was crystallized from an aqueous solution of 40 mg / ml of proteins in 40 mM sodium acetate buffer with sodium chloride 5 % at pH 7.4 at room temperature (Blake, TLC et al., Nature, 196: 1173 (1962)). The resulting crystals were cross-linked with glutaraldehyde at a concentration of 20% to form a CLEC of lysozyme. The lysozyme CLEC was lyophilized as described in Example 2.

In a seventh example of the method and practice of the present invention, the enzyme asparaginase (Escherichia coli) was crystallized from an aqueous solution of 25 ng / ml of proteins in 50 mM sodium acetate and 33% ethanol at pH 5.0 at 4 ° C. Crystallization constitutes a modification to the procedure described by Grabner et al. [US Patent 3,664,926) (1972)]. As described herein, the resulting crystals were crosslinked with glutaraldehyde at a concentration of 7.5% to form an asparaginase CLEC. Asparaginase CLEC was lyophilized as described in Example 2.

Other enzymes that can be similarly immobilized and that can be used to catalyze an appropriate reaction include luciferase and urease; Other enzymes, such as those listed in Tables 1-5, can also be crystallized and cross-linked using the present method, to produce a desired CLEC which, in turn, can be used to catalyze a reaction that results in the manufacture of a selected product or to catalyze a reaction that constitutes an intermediate step (i.e., one in a series of reactions) in the manufacture of a selected product. It is known that while crosslinking helps to stabilize most crystals, it is neither necessary nor preferable in all cases. Some crystalline enzymes retain functional and structural integrity in aggressive environments, even when crosslinking is not present. Although in the preferred embodiment the crystal is cross-linked, cross-linking is not always necessary to produce a crystallized enzyme useful in the present method.

CLECs have several key features that give them significant advantages over conventional enzyme immobilization methods in use today. CLECs eliminate the need for a separate inert support structure. The lack of an inert support will improve the diffusion properties of the substrate and the product within the CLECs and offers concentrations of enzymes within the crystal that are close to the theoretical packing limit for molecules of such size. High enzyme concentrations can lead to significant operational savings through increased effective activity of a given volume of catalyst, reduction in substrate contact time with enzyme, and overall reduction in plant size and capital expenditures (Daniels , MJ, Methods in Enzymol., 136: 371-379 (1987)). The uniformity throughout the crystal volume and the improved stability of the constitutive enzyme in CLECs creates new opportunities for the use of

ES 2 199 933 T3 enzymatic catalysis under aggressive conditions, such as elevated temperature, and aqueous, organic or quasi-anhydrous solvents, as well as their mixtures. Also, the restricted solvent access and normal protein environment implicit in a crystal lattice should lead to improved retention of metal ions and cofactors for CLECs relative to conventional immobilized enzyme systems.

Brief description of the drawings

Figure 1 is a graphical representation of the results of the evaluation of the enzymatic activity of a soluble CLEC and one of thermolysin.

Figure 2 is a graphical representation of the results of a comparison of pH dependencies of a CLEC of thermolysin and soluble thermolysin.

Figure 3 is a graphical representation of the measurement of soluble and crystalline thermolysin activity after incubation at 65 ° C.

Figure 4 is a graphical representation of the results of the evaluation of the resistance of a soluble CLEC and a thermolysin to exogenous proteolytic degradation.

Figure 5 is a graphical representation of the results of the evaluation of the enzymatic activity for soluble elastase and the corresponding CLEC of elastase.

Figure 6 is a graphical representation of the resistance of soluble elastase and the corresponding elastase CLEC to exogenous proteolytic degradation.

Figure 7 is a graphical representation of the results of the evaluation of the enzymatic activity for soluble esterase and the corresponding CLEC of esterase.

Figure 8 is a graphical representation of the resistance of soluble esterase and the corresponding CLEC of esterase to exogenous proteolytic degradation.

Figure 9 is a graphical representation of the results of the evaluation of the enzymatic activity for soluble lipase and the corresponding lipase CLEC.

Figure 10 is a graphical representation of the results of the evaluation of enzymatic activity for soluble lysozyme and the corresponding CLEC of lysozyme.

Figure 11 is a graphical representation of the results of the evaluation of the enzymatic activity for soluble asparaginase and the corresponding CLEC of asparaginase.

Detailed description of the invention

It would be extremely useful to have a general and simple procedure that ensures stability and function for a given enzyme or set of enzymes under conditions that are of interest to the synthetic chemist and that are too aggressive for use with enzymes that use chemicals. currently available methods.

Cross-linked immobilized enzyme crystals (referred to as CLEC or CLIEC), as described herein, can serve this purpose. Stabilization of the crystal lattice and constitutive enzyme catalysts in the crystal through the crosslinking reaction allows the use of CLECs in environments including aqueous, organic or quasi-anhydrous solvents, their mixtures, pH values extremes and elevated temperatures, which are incompatible with the function of the enzyme using the currently available methods. Likewise, the stabilization of the crystal lattice in the CLEC allows the lyophilization of the CLEC by standard methods. Freeze-dried CLECs can be stored for commercially interesting periods of time (months to years) without refrigeration, and facilitate quick and simple use of CLECs in laboratory-scale and industrial-scale processes by simply adding solvents. Optionally, without the need for intermediate solvent exchange processes. CLECs are also highly resistant to digestion by exogenous proteases. The method of the present invention facilitates the use of versatile enzyme catalysts in the main industrial chemical processes, as well as in the laboratory synthesis of new compounds for research.

Although crosslinking contributes to the stability of a crystallized enzyme, it is neither necessary nor preferable in all cases. Some crystallized enzymes retain functional and structural integrity in aggressive environments, even in the absence of crosslinking. The preferred embodiment of the present method includes cross-linking of a crystallized enzyme and is described in detail in the sections that follow. However, it is to be understood that crystallized enzymes that are not subsequently cross-linked may be used in some embodiments of the present invention.

Regular interactions between constitutive enzyme molecules in the crystal lattice of a CLEC result in well-defined pores of limited size that lead to enzyme molecules within the body of a

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CLEC. As a consequence, substrates that are larger than the available pore size will not be able to penetrate the body of the CLEC particle.

As a consequence of the limitation in pore size, many enzymatic reactions of academic and commercial interest that comprise substrates larger than the pore size of CLECs would be outside the scope of the present invention. This would include most reactions involving large polymers, such as proteins, polynucleotides, polysaccharides, and other organic polymers, where the number of polymeric subunits would make the polymer larger than the crystal pore size of CLECs. However, in such cases, catalysis can be carried out on the surface of the CLEC.

The present invention constitutes a method of immobilization of a selected protein, in particular an enzyme, by crystallization and cross-linking of the protein, which results in a cross-linked immobilized enzyme crystal (CLEC) that can be used to catalyze the manufacture of a selected product, namely, peptide, carbohydrate, lipid, or chiral organic molecule. The present invention also relates to said CLECs and to the method of manufacturing a selected product by a CLEC-catalyzed reaction or a CLEC-catalyzed step within a series of reactions. In one embodiment of the present invention the aspartame dipeptidyl precursor has been produced in a cross-linked immobilized thermolysin catalyzed condensation reaction performed by the present method. In another embodiment of this invention, the FAGLA reporter substrate has been cleaved to obtain a colorimetric product, the presence of which is indicative of enzymatic activity in a thermolysin CLEC. The hydrolysis of FAGLA has been used as a model reaction to indicate the resistance of thermolysin CLEC in various environments that would normally be incompatible with the activity of said enzyme.

In other embodiments of this invention, the enzymes elastase, esterase, lipase, asparaginase, and lysozyme have been used to cleave various indicated substances, such as p-nitrophenyl acetate (esterase and lipase), succinyl (ala) 3-p-nitroanilide ( elastase), 4-methylumbelliferyl N-acetyl-chitriosidase (lysozyme) and NADH (asparaginase).

By the method of this invention, a person of ordinary skill in the art can adapt a protocol to make a desired product by a reaction catalyzed by an immobilized enzyme. The enzyme of interest, when crystallized from an appropriate solution, can be cross-linked with glutaraldehyde or other suitable bifunctional reagent in the crystallization solution to produce a CLEC of said enzyme. Subsequently, the CLEC of the chosen enzyme can be lyophilized as described in Example 2.

The use of a CLEC offers several advantages compared to currently available enzyme catalyzed methods. For example, the cross-linked crystal matrix in a CLEC is self-supporting. Expensive carrier beads, glasses, films, or gels are not required to bind the enzyme catalyst, unlike currently available immobilization methods. As a result, the enzyme concentration in a CLEC is close to the theoretical packing limit that can be achieved for molecules of a certain size, which far exceeds the densities achievable even in concentrated solutions. The entire CLEC is composed of an active enzyme (and not an inactive carrier) and therefore the diffusion-related reduction in enzyme reaction rates normally observed with conventionally immobilized enzymes relative to enzymes in solution should be minimized, since the mean free path for substrate and product between the active enzyme and the free solvent will be greatly shortened for CLECs (compared to enzyme-bearing particles immobilized by conventional means). These high protein densities will be particularly useful in biosensor, analytical, and other applications that require large amounts of protein in small volumes. In industrial processes, thanks to the superior performance and compactness of CLECs, significant operational savings can be made by increasing the effective activity of a given volume of catalyst, thereby achieving reductions in plant size, as well as also in capital expenditures (Daniels, MJ, Methods in Enzymol. 136: 371: 379 (1987)). CLECs are relatively monodisperse with a macroscopic size and shape that reflect the natural crystal growth characteristics of individual enzyme catalysts. Replacing existing carrier-immobilized enzyme media with CLECs should not be difficult, as both systems are comparable in size and shape, and both can be similarly recovered from the feedstock by any number of simple methods. , including basic economic operations such as filtration, centrifugation, solvent decantation and others.

In addition, the use of lyophilized CLECs allows for routine storage and handling of these materials prior to use (dry storage at room temperature without refrigeration for extended periods of time). Lyophilized CLECs also allow routine formulation by direct addition of solvents and substrates of interest, without lengthy solvent exchange processes or formation of amorphous suspensions. The lyophilized CLEC form extends the general utility of enzymes as catalysts by bringing it to a broader spectrum of enzymes and functional conditions.

A second advantage of CLECs is that the crosslinking of the crystallized enzyme stabilizes and strengthens the crystal lattice and constitutive enzyme molecules, both mechanically and chemically. As a result, a CLEC may be the only means to achieve significant concentrations of active enzyme catalyst in aggressive aqueous, organic, quasi-anhydrous solvents or in aqueous-organic solvent mixtures. The use of enzymes as catalysts in organic synthesis has been hampered by their tendency to denature in the presence of non-aqueous solvents and, in particular, in mixtures of aqueous and non-aqueous solvents (Klibanov, AM, Trends in Biochemical Sciences, 14: 141- 144 (1989)). In CLEC, the conformational mobility restriction that leads to

ES 2 199 933 T3 stability is offered through chemical contacts and inter-molecular crosslinks between the constitutive enzymatic molecules that make up the crystal lattice rather than through the quasi-absence of water in the medium. As a result, enzymes can tolerate intermediate water concentrations when these enzymes are formulated as CLEC, which has not been possible until today (see Table 12). In commercial applications, aqueous-organic solvent mixtures allow manipulation of the product formation by taking advantage of the relative solubilities of the products and substrates. Even in aqueous media, enzyme catalysts, immobilized or soluble, are subjected to mechanical forces within a chemical reactor which can cause denaturation and a shorter half-life. Chemical crosslinks within CLEC offer the necessary mechanical strength (Quiocho and Richards, Proc. Natl. Acad. Sci. (USA) 52: 833-839 (1964)) that results in increased reactor life for the enzyme catalyst.

A third advantage of CLECs is that as a result of their crystalline nature, a CLEC can achieve uniformity throughout the volume of the crosslinked crystal. Crystalline enzymes as described herein grow and crosslink in an aqueous environment, and thus the distribution of molecules within the crystal lattice remains uniform and regular. This uniformity is maintained thanks to the inter-molecular contacts and cross-links between the enzymatic molecules that make up the crystal lattice, even when they are exchanged for other aqueous, organic or quasi-anhydrous media or mixed aqueous / organic solvents. In all these solvents, the enzyme molecules maintain a uniform distance from each other, thereby forming stable, well-defined pores within the CLECs that facilitate substrate access to the enzyme catalysts, as well as product separation. Uniformity of enzyme activity is critical in industrial, medical and analytical applications where reproducibility and consistency are of paramount importance.

A fourth advantage of using a CLEC is that it should exhibit an increased storage and operational half-life. It is known that crystal lattice interactions, even in the absence of crosslinking, stabilize proteins, due, in part, to restrictions in the conformational degrees of freedom necessary for protein denaturation. In CLECs, crystal lattice interactions, when fixed by chemical crosslinks, are of particular importance in preventing denaturation, especially in aqueous and nonaqueous solvent mixtures (Klibanov, AM, Trends in Biochemical Sciences 14: 141-144 ( 1989)). Enzymes that have been in the crystalline state for months and years routinely retain a high percentage of their catalytic activity. Cross-linked immobilized enzyme crystals stored in anhydrous solvents will be further protected against microbial contamination and damage, which is a serious problem when large amounts of protein are stored in a nutrient-rich aqueous environment. In the case of a lyophilized CLEC, the immobilized enzyme is stored in the absence of solvent. Thanks to the latter and to the stabilization achieved by crosslinking, storage without refrigeration for long periods of time is possible.

A fifth advantage of using a CLEC is that it should exhibit improved temperature stability as a consequence of stabilization of the crystal lattice through crosslinking. Carrying out reactions at a temperature higher than that used with conventional methods would increase the reaction rates for the chemical reactions of interest, both thermodynamically and by improving the rate of diffusion when entering and leaving the CLEC crystal lattice. These combined effects would represent a great improvement in the efficiency of the reactions, because they would maximize the productivity of a given amount of enzyme catalyst, which is generally the most expensive component of the reaction process (Daniels, MJ, Methods in Enzymol. 136: 371379 (1987)). The temperature stability exhibited by CLECs is extraordinary, because most enzyme systems require mild reaction conditions. CLECs would also stabilize against denaturation by transient elevated temperatures during storage.

A final advantage of using a CLEC is that pores of regular size and shape are created between the individual enzyme molecules in the underlying crystal lattice. This restricted accessibility of the solvent greatly improves the retention characteristics of the metal ion or cofactor of CLEC compared to enzymes immobilized by conventional means and enzymes in solution. This property of CLECs will allow the use of economically superior continuous flow processes in situations (see, for example, Oyama et al. Methods in Enzymol. 136: 503-516 (1987)) in which the enzyme otherwise it would be inactivated by metal ion or cofactor leaching. For example, in the thermolysin-mediated synthesis of the dipeptidyl precursor of aspartame, ZL-Asp-L-Phe-OMe, it is known that the enzyme immobilized by conventional means loses catalytic activity in flow-through column processes, in part due to leaching. of calcium ions essential for thermolysin activity. In practice, the leaching of calcium ions has forced the use of less efficient batch processes (Nakanishi et al., Biotechnology 3: 459-464 (1985)). Leaching occurs when complexes of calcium ions are formed with the ZL-Asp substrate, competing with the natural calcium binding sites on the surface of the enzyme, which causes the loss of catalytic activity. The high enzyme density and, consequently, the limited volume of access for the solvent in the interstices of the CLEC does not stimulate the formation of the L-Asp-Ca complexes.⁺⁺ responsible for the leaching of the metal ion.

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CLEC preparation - enzyme crystallization

In the method of the present invention, a cross-linked immobilized enzyme crystal (CLEC) is prepared as follows:

Enzyme crystals grow by controlled precipitation of protein from aqueous solution or aqueous solution containing organic solvents. Conditions to be controlled include, for example, the rate of evaporation of the solvent, the presence of appropriate cosolutes and buffers, and the pH and temperature. A complete review of the various factors that affect protein crystallization has been published by McPherson (Methods Enzymol. 114: 112 (1985)). In addition, both McPherson and Gilliland (J. Crystal Growth 90: 51-59 (1988)) have compiled complete lists of all proteins and nucleic acids that were reported to have been crystallized, as well as the conditions leading to their crystallization. There is a compendium of crystals and crystallization recipes, as well as a repository of coordinate data of crystallized structures of nucleic acids and proteins resolved in the Protein Data Bank (Bernstein et al. J. Mol. Biol. 112: 535- 542 (1977)) of the Brookhaven National Laboratory. These references can be used to determine the conditions necessary for the crystallization of a given protein or a previously crystallized enzyme as a prelude to the formation of an appropriate CLEC, and can serve as a guide for the formulation of a crystallization strategy for proteins that do not. have. Alternatively, a smart trial and error search strategy (see, eg, Carter, CW Jr. and Carter, CW, J. Biol. Chem. 254: 12219-12223 (1979)) can, in most cases, produce appropriate crystallization conditions for most proteins, including, without limitation, those mentioned above, as long as an acceptable level can be reached. of purity in them. The level of purity required can vary widely from protein to protein. In the case of lysozyme, for example, the enzyme has been crystallized directly from its unpurified source, the white of the chicken egg (Gilliland, GL, J. Crystal Growth 90: 51-59 (1988)).

To use crystals as CLEC in the method of this invention, the same large, single crystals needed for X-ray diffraction analysis are not required and may actually be undesirable because of diffusion problems associated with the size of the crystal. Rains of microcrystals (i.e. crystals of order 10^-1 mm in size / cross section) are appropriate for CLECs and are frequently seen although rarely covered in the literature on X-ray crystallography. Microcrystals are very useful in the method of this invention to minimize problems with diffusion. (see, for example, Quiocho, FA and Richards, FM, Biochemistry 5: 4062-4076 (1967)).

Generally speaking, crystals are produced by combining the protein to be crystallized with an appropriate aqueous solvent or an aqueous solvent containing appropriate precipitating agents, such as salts or organic compounds. The solvent is combined with the protein at a temperature that according to the experiments carried out is appropriate for the induction of crystallization and is acceptable for the maintenance of the stability and the activity of the protein. Optionally, the solvent can include cosolutes, such as divalent cations, cofactors, or chaotropic agents, as well as buffer species to control pH. The need for cosolutes and their concentrations is determined experimentally to facilitate crystallization. In an industrial scale process, controlled precipitation leading to crystallization is best accomplished by simply combining protein, precipitant, cosolutes, and optionally buffers in a batch process. Alternative laboratory crystallization methods, such as dialysis or vapor diffusion, can also be adapted. McPherson (Methods Enzymol. 114: 112 (1985)) and Gilliland (J. Crystal Growth 90: 51-59 (1988)) provide a complete list of appropriate conditions in their reviews of the crystallization literature. Occasionally, incompatibility between the crosslinking agent and the crystallization medium may require that the crystals be exchanged for a more appropriate solvent system.

Many of the proteins for which crystallization conditions have already been described in the literature have considerable potential as practical enzyme catalysts in laboratory and industrial chemical processes, and are directly subject to being formulated as CLEC within the method of this invention. Table 1 is a sampling of enzymes that have already crystallized. Note that the conditions outlined in most of these references have been optimized for the growth of large diffraction grade crystals, usually through great effort. In some cases a certain degree of adjustment of conditions may be necessary for the smaller crystals used in the manufacture of CLECs.

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TABLE 1

<td>Enzyme</td><td>Microbial or biological source</td><td>References (including those cited within them)</td>

<td>• Alcohol dehydrogenase</td><td>Equine liver</td><td>Eklund et al., J. Mol. Biol. 146: 561-587 (1981)</td>

<td>• Alcohol oxidase</td><td>Pichia pastoris</td><td>Boys et al, J. Mol. Biol .. 208: 211-212 (1989) Tykarska et al., J. Protein Chem. 9: 83-86 (1990)</td>

<td>• Aldolase (fructose-bisphosphate)</td><td>Rabbit muscle Calf muscle Human muscle Drosophila melanogaster</td><td>Eagles et al., J. Mol. Biol. 45: 533-544 (1969) Heidmer et al., Science 171: 677-680 (1971) Goryunov et al., Biofizika 14: 1116-1117 (1969) Millar et al., Trans.Roy.Soc.Lond. B293: 209-214 (1981) Brener et al., J.Biol. Chem. 257: 11747-11749 (1982)</td>

<td>• Aldolase (PKDG)</td><td>Pseudomonas putida</td><td>Vandlen et al., J.Biol. Chem. 248: 2251-2253 (1973)</td>

<td>• Alkaline phosphatase</td><td>Escherichia coli</td><td>Sowadski et al., J.Mol.Biol. 150: 245-272 (1981)</td>

<td>• Asparaginase</td><td>Erwinia carotova Escherichia coli Escherichia coli Proteus vulgaris</td><td>North et al., Nature 224: 594-595 (1969) Epp et al., Eur. J. Biochem. 20: 432-437 (1971) Yonei et al., J.Mol.Biol. 110: 179-186 (1977) Tetsuya et al., J.Biol.Chem. 248: 7620-7621 (1972)</td>

<td>• Carbonic anhydrase</td><td>Human erythrocyte (C) Human erythrocyte (B) Bovine erythrocyte</td><td>Kannan et al., J. Mol. Biol. 12: 740-760 (1965) Kannan et al., J. Mol. Biol. 63: 601-604 (1972) Carlsson et al., J. Mol. Biol. 80: 373-375 (1973)</td>

<td>• Catalase</td><td>Equine erythrocyte Micrococcus luteus Penicillin vitale Bovine liver</td><td>Glauser et al., Acta Cryst. 21: 175-177 (1966) Marie et al., J. Mol. Biol. 129: 675-676 (1979) Vainshtein et al., Acta Cryst. A37: C29 (1981) Eventoff et al., J.Mol. Biol. 103: 799-801 (1976)</td>

<td>• Creatine kinase</td><td>Bovine heart Rabbit muscle</td><td>Gilliland et al., J. Mol. Biol. 170: 791-793 (1983)</td>

<td>• Glutaminase</td><td>Actenobacter glutanimasificans Pseudomonas 7^to</td><td>Wlodawer et al., J. Mol. Biol. 99: 295-299 (1975) Wlodawer et al., J. Mol. Biol. 112: 515-519 (1975)</td>

<td>• Glucose Oxidase</td><td>Aspergillus Niger</td><td>Kalisz et al., J. Mol. Biol. 213: 207.209 (1990)</td>

<td>• β-lactamases</td><td>Staphylococcus aureus Bacillus cereus</td><td>Moult et al., Biochem J. 225: 167-176 (1985) Sutton et al., Biochem J. 248: 181-188 (1987)</td>

<td>• Lactate dehydrogenase</td><td>Porcine Chicken Red painter Bacillus stearothermophilus</td><td>Hackert et al., J. Mol. Biol. 78: 665-673 (1973) Pickles et al., J. Mol. Biol. 9: 598-600 (1964) Adams et al., J. Mol. Biol. 41: 159-188 (1969) Schar et al., J. Mol. Biol. 154: 349-353 (1982)</td>

<td>• Lipase</td><td>Equine Pancreatic Geotrichum candidum Mucor meihei Human pancreatic</td><td>Hata et al., J. Biochem. 86: 1821-1827 (1979) Lombardo et al., J. Mol. Biol. 205: 259-261 (1989) Brady et al., Nature 343: 767-770 (1990). Winkler et al., Nature 343: 771-774 (1990)</td>

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TABLE 1 (continued)

<td>Enzyme</td><td>Microbial or biological source</td><td>References (including those cited within them)</td>

<td>• Luciferase</td><td>Firefly</td><td>Green, A, A., et al., Biochem. Biophys. Minutes 20: 170 (1956)</td>

<td>• Luciferase</td><td>Vibrio harveyii</td><td>Swanson et al., J. Biol. Chem. 260: 1287-1289 (1985)</td>

<td>• Nitrile hydratase</td><td>Brevibacterium R312 P.chlororaphis B23</td><td>Nagasawa et al., Biochem.Biophys.Res. Commun. 139: 1305-1312 (1986) Nagasawa et al., Eur. J. Biochem. 162: 691-698 (1987)</td>

<td>• Peroxidase</td><td>Horseradish Horseradish roots (Type E4) Japanese radish</td><td>Braithwaite et al., J. Mol. Biol. 106: 229-230 (1976) Aibara et al., J.Biochem. 90: 489-496 (1981) Morita, Acta Cryst. A28: S52 (1979)</td>

<td>• Peroxidase (chloride)</td><td>Caldaromyces fumago</td><td>Rubin et al., J.Biol. Chem. 257: 7768-7769 (1982)</td>

<td>• Peroxidase (cytochrome)</td><td>Saccharomyces cerevisae</td><td>Poulos et al., J.Biol. Chem. 253: 3730-3735 (1978)</td>

<td>• Peroxidase (glutathione)</td><td>Bovine erythrocyte</td><td>Landenstein et al., J. Mol. Biol. 104: 877-882 (1979)</td>

<td>• Subtilisin</td><td>Bacillus subtilis (Novo) Bacillus amyloliquefaciens (BPN ') Bacillus subtilis (Carlsberg)</td><td>Drenth et al. J.Mol.Biol. 28: 543-544 (1967) Wright et al., Nature 221: 235-242 (1969) Petsko et al., J. Mol. Biol, 106: 453-456 (1976)</td>

<td>• Superoxide dismutase</td><td>Bovine Spinach Saccharomyces cerevisae, Escherichia coli Bacillus stearothermophillus Pseudomonas ovalis</td><td>Richardson et al., J.Biol. Chem. 247: 6368-6369 (1972) Morita et al., J. Mol. Biol., 86: 685-686 (1974) Beem et al., J. Mol. Biol., 105: 327-332 (1976) Bridgen et al., J. Mol. Biol. 105: 327-332 (1976) Yamakura et al., J.Biol. Chem. 251: 4792-4793 (1976)</td>

<td>• Thermolysin</td><td>Bacillus thermoproteolyticus</td><td>Matthews et al., Nature New Biol. 238: 37-41 (1972)</td>

<td>• Urease</td><td>White bean</td><td>Sumner, JB, J.Biol. Chem. 69: 435 (1926)</td>

<td>• Xylose isomerase</td><td>Streptomyces rubiginosus Arthrobacter B3728 Streptomyces oilvochromogenes Streptomyces violaceoniger Actinoplanes missouriensis</td><td>Carrell et al., J.Biol. Chem. 259: 3230-3236 (1984) Akins et al., Biochym.Biophys.Acta 874: 375-377 (1986) Farber et al., Protein Engineering 1: 459-466 (1987) Glasfeld et al., J.Biol. Chem. 263: 14612-14613 (1988) Rey et al., Proteins: Struc.Func.Genet 4: 165-172 (1988)</td>

CLEC preparation - crosslinking reaction

Once crystals develop in an appropriate medium, they can be crosslinked. Crosslinking results in the stabilization of the crystal lattice by introducing covalent bonds in the crystal between the constitutive enzyme molecules. This allows enzyme transfer to an alternative reaction environment that would otherwise be incompatible with the existence of the crystal lattice, or even the existence of intact undenatured proteins. Crosslinking can be achieved through a wide variety of bifunctional reagents, although in practice glutaraldehyde, inexpensive and simple, has become the reagent of choice. (For a representative listing of other available crosslinking reagents, see, for example, the Pierce Chemical Company 1990 Catalog).

ES 2 199 933 T3

Crosslinking with glutaraldehyde forms strong covalent bonds primarily between lysine amino acid residues within and between enzyme molecules in the crystal lattice that constitutes the crystal. Crosslinking interactions prevent constitutive enzyme molecules within the crystal from going back into solution, thereby effectively insolubilizing and immobilizing enzyme molecules to form microcrystalline particles (ideally 10-1 mm). The macroscopic, immobilized, insolubilized crystals can be easily separated from the feedstock containing product and non-reactive substrate by simple procedures such as filtration, decantation, and others. They can also be used in CLEC loaded columns in continuous flow processes, where they show improved metal ion and cofactor retention properties.

Through the method of this invention, CLECs are obtained to be used as enzyme catalysts in new and existing environments. The improved stability of CLECs, obtained thanks to the crosslinking reaction, allows the CLEC to be transferred to a solvent (eg aqueous, organic or quasi-anhydrous solvents or their mixtures) in which it would otherwise be incompatible, and to activate the operation of the chemical reactor at elevated temperatures or extreme pH values. The macroscopic CLEC catalyst particles can also be easily manipulated, allowing recovery of the feedstock through simple methods, such as filtration, centrifugation, or solvent decantation. They can also be used in loaded columns in continuous flow processes.

CLEC preparation - lyophilization

A one volume suspension of cross-linked thermolysin crystals was lyophilized in ten volumes of demineralized water at pH 7.0 overnight using a VirTis Model # 24 lyophilizer. The lyophilized crystals were stored at room temperature or at 4 ° C before their reconstitution, which was carried out by adding ten volumes of the chosen solvent directly on the crystals taken from storage. The rehydrated crystals were reconstituted in 10 mM calcium acetate buffer at pH 7.0 for the FAGLA cleavage experiments. Lyophilized and reconstituted CLECs were stored at room temperature according to routine procedure. Instead, it was necessary to store soluble enzymes at -70 ° C to maintain their specific activity for more than a week. This protocol was used for all the enzymes described in the exemplifications included in the present invention.

Synthesis of the aspartame precursor with thermolysin CLEC

The method of the present invention, by which cross-linked crystallized enzymes are produced, is described below and exemplified by the production of thermolysin cross-linked immobilized enzyme crystals for use in the production of the dipeptidyl precursor of aspartame, in ethyl acetate. , which is a quasi-anhydrous and organic solvent. Thermolysin, a protein that has been crystallized and whose structure has been resolved in resolution 1.6 A (Holmes and Matthews, J.Mol. Biol. 160: 623-639 (1982)), is an example of an enzyme that it can be used as CLEC in the present method. Thermolysin is used in the production of the artificial sweetener aspartame (Isowa et al. US Patent No. 4,436,925 (1984); Lindeberg, J. Chem. Ed. 64: 1062-1064 (1987); Nakanishi et al., Biotechnology 3: 459-464 (1985); Oyama, et al., Methods in Enzymol, 136: 503-516 (1987)). Currently, most aspartame is produced using a conventional synthetic chemistry approach, although the use of thermolysin immobilized by conventional means in quasi-anhydrous media has given encouraging results (Oyama et al., J. Org. Chem. 46: 5242-5244 (1981); Nakanishi et al., Biotechnology 3: 459464 (1985)). Improvements in the enzymatic approach to the production of aspartame, such as those that can be achieved through the use of the present method, would make it compete with the method currently used, both from the point of view of convenience and cost. (Oyama, et al., Methods in Enzymol. 136: 503-516 (1987)).

Evaluation of thermolysin CLECs

The method of the present invention has also been used to produce thermolysin CLEC in which pH dependence, stability at elevated temperatures, resistance to exogenous proteolysis and stability in the presence of an organic solvent have been evaluated. Thermolysin CLECs were compared to soluble thermolysin, as described in detail in Example 2 and Figures 1-4. The results of the evaluation showed the following:

1. Regarding pH dependence and stability, both forms demonstrate maximum activity at pH 7 and demonstrate similar activity in the acidic range. In the alkaline pH range, CLEC maintains maximum activity up to pH 10; soluble thermolysin has 75% activity at pH 8.5, only 25% activity at pH 9, and is completely inactive at pH 9.5.

two. The additional stabilization achieved in CLECs results in enzymatic activity at higher temperatures than is possible with soluble thermolysin. Due to the improved stability of CLEC thermolysin at lower temperatures, storage is simpler than for the soluble enzyme. Thermal stability and resistance to autolysis were also demonstrated for thermolysin CLECs, which retained their maximum activity after five days of incubation at 65 ° C. In contrast, soluble thermolysin lost 50% of its initial activity after two hours of incubation and showed negligible activity after 24 hours of incubation at 65 ° C.

ES 2 199 933 T3

3. The enzymatic activity of thermolysin CLECs was not affected by four days of incubation in the presence of the potent streptococcal protease Pronase.^®. In contrast, soluble thermolysin was rapidly degraded and lost all activity after 90 minutes of incubation.

Four. Thermolysin CLECs and soluble thermolysin showed marked differences in stability in the presence of organic solvents, as shown in Table 12. Thermolysin CLECs retained more than 95% of their maximum activity after incubation with all organic solvents evaluated. .

These characteristics make thermosilin CLECs and other enzyme CLECs particularly useful as they are easier to store, more stable, and less easily inactivated or degraded than their corresponding soluble enzymes.

Elastase CLEC Evaluation

The method of the present invention has also been used to produce elastase CLECs in which their activity and resistance to exogenous proteolysis have been evaluated. Elastase CLECs were compared to soluble elastase, as described in detail in Example 3 and Figures 5 and 6. The results of the evaluation demonstrated the following:

1. The elastase CLECs retain approximately 50% of their activity compared to the soluble enzyme.

two. Soluble elastase was rapidly degraded by protease. The activity of the soluble elastase was reduced to 50% with respect to the initial activity after ten minutes of incubation in the presence of protease. After one hour of incubation, the soluble enzyme had lost more than 90% of its initial activity. In contrast, the enzymatic activity of elastase CLEC was not affected by incubation with protease.

Evaluation of esterase CLECs

The method of the present invention has also been used to produce thermolysin CLECs in which their activity and resistance to exogenous proteolysis have been evaluated. The esterase CLECs were compared to soluble esterase, as described in detail in Example 4 and Figures 7 and 8. The evaluation results showed the following:

1. The esterase CLECs retain approximately 50% of their activity compared to the soluble enzyme.

two. Soluble esterase was highly susceptible to proteolytic degradation. The soluble esterase activity was reduced to 50% with respect to the initial activity after ten minutes of incubation in the presence of protease. After one hour of incubation, the soluble enzyme had lost more than 90% of its initial activity. In contrast, the enzymatic activity of elastase CLEC was not affected by incubation with protease.

Assessment of lipase CLECs

The method of the present invention has also been used to produce lipase CLEC in which its activity was evaluated. The lipase CLECs were compared to soluble lipase, as described in detail in Example 5 and Figure 9. The results of the evaluation demonstrated that the lipase CLECs retain approximately 90% of the activity compared to the soluble enzyme.

Evaluation of Lysozyme CLECs

The method of the present invention has also been used to produce lysozyme CLEC in which its activity and resistance to exogenous proteolysis were evaluated. The lysozyme CLECs were compared to soluble lysozyme, as described in detail in Example 6 and Figure 10. The results of the evaluation demonstrated that the lysozyme CLECs retain approximately 50% of the activity compared to the soluble enzyme.

Asparaginase CLEC Assessment

The method of the present invention has also been used to produce CLEC of asparaginase in which its activity was evaluated. Asparaginase CLECs were compared to soluble asparaginase, as described in detail in Example 7 and Figure 11. The evaluation results demonstrated that asparaginase CLECs retain approximately 77% of the activity compared to soluble enzyme.

General applicability of the CLEC

As described herein, CLECs represent a new technology in wide use in many fields, including, but not limited to, industrial-scale synthesis, laboratory tools, biosensors, and medical applications. Tables 2-5 below show examples of various systems that utilize conventionally immobilized enzyme methods in their performance. An expert in the art should be

ES 2 199 933 T3 capable of adapting these systems and similar systems to the CLEC technology described in this application. By way of illustration, specific examples of the categories listed are described in greater detail.

Table 2 below lists examples that use enzymes immobilized by conventional means in an industrial process, examples that can easily be adapted to the CLEC technology described herein.

TABLE 2

<td>Enzyme</td><td>production or application</td><td>Substrates</td><td>References (including those mentioned in them)</td>

<td>• Thermolysin</td><td>• Aspartame precursor</td><td>Z-Asp, L-Phe-OMe</td><td>Oyama et al., J.Org. Chem. 46: 5242-5244 (1981) Nakanishi et al., Trends in Biotechnology 3: 459-464 (1985)</td>

<td>• Subtilisin</td><td>• Aspartame</td><td>L-Asp-L-Phe, OMe</td><td>Davino, AA, US Patent 4,293,648 (1981)</td>

<td>• Lipase</td><td>• Cocoa fat substitutes</td><td>Palm oils</td><td>Harwood, J., Trends in Biochemical Sciences 14: 125-126 (1989) Macrae, AR, JAm. Oil Chem. Soc., 60: 291-294 (1983)</td>

<td>• Nitrile hydratase, nitrilase, amidase</td><td>• Acrylamide</td><td>Acrylonitrile</td><td>Nagasawa, T. and Yamada, H., Trends in Biotechnology 7: 153-158 (1989)</td>

<td>• Amino acylase (fungal)</td><td>• Amino acid</td><td>N-acyl-D, L amino acids</td><td>Schmidi- Kastner, G. and Egerer, P. in Biotechnology vol 6a: 387-421 (1984) and references therein</td>

<td>• Amino acid esterase</td><td>• Redissolution</td><td>Esters of D, L-amino acids</td><td></td>

<td>• Subtilisin</td><td></td><td></td><td></td>

<td>• Amidases</td><td></td><td>Amides of D, L-amino acids</td><td></td>

<td>• Hydantoinases • Specific dehydrogenases</td><td></td><td>Hydantoins</td><td>Fusee, MC, Methods in Enzymology 136: 463 (1987)</td>

<td>• Aminopeptidase</td><td></td><td>Xhydroxycarboxylic acids</td><td></td>

<td>• Transaminase</td><td></td><td></td><td>Fusee, MC, Methods in Enzymology 136: 463 (1987)</td>

<td>• Amino Acid Dehydrogenase + formate dehydrogenase</td><td>• Amino acid production: general</td><td>Keto or hydroxy acids</td><td>Rozzeli, JD, Methods in Enzymology 136: 479 (1987)</td>

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TABLE 2 (continued)

<td>Enzyme</td><td>production or application</td><td>Substrates</td><td>References (including those mentioned in them)</td>

<td>• L-aspartase</td><td>• Production of amino acid: acid L-aspartic specific</td><td>Fumarate / acid fumaric</td><td>Enzymes in Industry; Ed Gerhartz.W., VCH Press 1990</td>

<td>• L-aspartate 4-decarboxylase</td><td>L-alanine</td><td>L-aspartic acid, ammonium fumarate</td><td></td>

<td>aspartase + L aspartate 4 decarboxylase</td><td>L-lysine</td><td>D, The amino e-caprolactam (ACL)</td><td></td>

<td>• ACL hydrolase • L-ACT hydrolase</td><td>L-cysteine L-isoleucine L-methionine</td><td>DL-2- acid amino2-thiazoline- 4-carboxylic</td><td></td>

<td>• Liasa</td><td>L-phenylalanine</td><td>Indole cinnamate, L-serine</td><td></td>

<td>• L-tryptophan synthetase</td><td>L-tryptophan</td><td></td><td></td>

<td>• Fumarasa</td><td>L-valine L-malic acid</td><td>Fumarate</td><td></td>

<td>• Hydantoinase</td><td>Dn-carbamoyl- p-hydroxy- phenylglycine</td><td>5p-hydroxy-hydantoin</td><td></td>

<td>• Lipases, esterases</td><td>• Redissolution of racemates by synthesis stereoselective</td><td>synthetic chemistry</td><td>Jones, JB, Tetrahedron 42: 3351-3403 (1988) Butt, S. and Roberts, SM, Natural Product Reports 489-503 (1986), and references cited therein for a more complete review of this area</td>

<td>• Fumarasa</td><td>• Acid L-malic</td><td>Fumaric acid</td><td>Chibata et al., Methods in Enzymology 136: 455 (1987)</td>

<td>• Lactase β-galactosidases</td><td>• Synthesis of disaccharides, p. ex. galactosylN-acetyl galactosamine</td><td>Lactose and N-acetyl galactosamine</td><td>Larsson et al., Methods in Enzymology 136: 230 (1987)</td>

<td>• Lipase, esterase</td><td>• L-menthol</td><td>Mix of 4 isomers</td><td>Fukui, S., Tanaka, A., Methods in Enzymology 136: 293 (1987)</td>

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TABLE 2 (continued)

<td>Enzyme</td><td>production or application</td><td>Substrates</td><td>References (including those mentioned in them)</td>

<td>• Amidases</td><td>• D-valine (product chemical intermediate for fluvinato insecticide pyrethroid)</td><td>DL-amino acid amide</td><td>Schmidt-Kastner, G. and Egerer, P. in Biotechnology vol 6a: 387-421 (1984) and references therein</td>

<td>• Lipase (Candida cylindricea)</td><td>• R (+) 2-phenoxypropionic acids (herbicides)</td><td>2-chloropropionic acids</td><td>Biocatalysts in Organic Syntheses eds Tramper, van de Plas &amp; Linko; Proceedings of International Symposium in Netherlands 1985</td>

<td>• Lipases, esterases, amidases, aldolases</td><td>• Organic Synthesis Monoglyceride Peptides</td><td></td><td>Jones, JB, Tetrahedron 42: 3351-3403 (1988) Butt, S. &amp; Roberts, SM, Natural Product Reports 489-503 (1986), and references cited therein for a more complete review of this area</td>

<td>• Proteases, peptidases</td><td>• Acid 2 (pchlorophenoxy)</td><td>Ester redissolution racemic</td><td></td>

<td>• Yeast lipase</td><td>propionic: herbicide</td><td></td><td></td>

<td>• Strictosidin synthase</td><td>• Production of alkaloids eg strictosidine</td><td></td><td>Pfitzner et al., Methods in Enzymology 136: 342 (1987)</td>

<td>• Penicillin acylase • Penicillin amidase</td><td>• 6-aminopenicillanic acid and 7-ADCA</td><td>Penicillin G or V</td><td>EnzEng 6: 291 (1982) EnzEng 8: 155</td>

<td>• Hydroxyste- roid-deshi- drugases</td><td>• Steroid transformations</td><td></td><td>Carrea et al., Methods in Enzymology 136: 150 (1987)</td>

<td>• 5'phosphodiesterase, nucleases</td><td>• 5'-ribonucleotides</td><td></td><td>Keller et al, Methods in Enzymology 136: 517 (1987)</td>

<td>• Esterase</td><td>• Forerunner β-lactam (monoesters chiral p. ex. ester monoalkyl acid eamino glutaric)</td><td>Diesters corresponding</td><td>Japan Patent Application: 82-159, 493 (1981) Biseibutsu Company</td>

<td>• Lipases</td><td>• β-blockers</td><td></td><td>Kloosterman, M et al, Trends in Biotechnology 6: 251-256 (1988)</td>

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Acrylamide production using CLEC technology

The following is a description of one use of the method of the present invention: the adaptation of acrylamide production from immobilized cells that overproduce the enzyme nitrile hydratase (Nagasawa, T. and Yamada, H., Trends in Biotechnology 7 : 153-158 (1989)) to the CLEC technology described above in the present invention.

The production of acrylamide on an industrial scale, an important generic chemical, has been described by Yamada et al. (Nagasawa, T. and Yamada, H., Trends in Biotechnology 7: 153-158 (1989)). Each year kilotonnes of acrylamide are produced in chemical reactors loaded with trapped cells selected as over-producers of the enzyme nitrile hydratase. Nitrile hydratase is also reported to be purified and crystallized from two sources. Brevibacterium R312 (Nagasawa et al., Biochem. Biophys. Res. Commun. 139: 1305-1312 (1986) and P. chlororaphis B23 (Nagasawa et al., Eur. J. Biochem. 162: 691-698 (1987)) As described herein, these crystalline enzymes can be immobilized in each case by crosslinking with glutaraldehyde or other appropriate crosslinking reagent to produce a CLEC. The nitrile hydratase CLECs can then be used in a conventional reactor, instead of the trapped cells used today. Adapting this process to CLEC technology translates into immediate benefits. These advantages include: smaller plant size and improved yield as a result of the improved activity per unit volume implicit in the higher concentration of enzymes in CLECs, and improved product and substrate diffusion rates; reduction of contamination and unwanted side reactions, as a result of the higher purity of CLECs; and reduced sensitivity to microbial contamination in the absence of cells. There are also other benefits only available for a CLEC-based method. These benefits include: elevated temperature operation to improve reaction rates; the ability to operate in aqueous, organic and quasi-anhydrous solvents, thereby making it possible to optimize the acrylamide production reaction; and improved half-life in operation and storage, as a result of the greater mechanical and chemical stability of CLECs, particularly in non-conventional solvents.

CLEC technology medical applications - extracorporeal treatment

The method of the present invention and an appropriately selected CLEC or set of CLECs can also be used for medical applications. A CLEC or set of CLECs can be used to, for example, separate a component of a fluid, such as blood, usually by altering the component and thus making it a substance that is not deleterious to an individual or that can be separated by normal body processes (eg. via detoxification, or degradation in the liver, excretion via the kidney). In this application, an appropriately selected CLEC or set of CLEC is contacted with body fluid, which contains the component to be altered, or a reagent (product or substrate) of a reaction in which the component participates, after which acts the enzyme in the CLEC. As a result, the enzyme can act on the component to be altered or with another substance that is a product of a reaction in which the component to be altered participates. The activity of the enzyme causes the direct alteration of the component to be separated or the alteration of the reaction product in which the component participates (whereby it is impossible to continue the reaction). The latter can be accomplished through the use of an extracorporeal device that includes an appropriately selected CLEC or set of CLECs and retention means made of a material such as a porous material on which the CLEC is retained or a tube in which it is held. present a CLEC, which allows contact between the component itself or the substance in the fluid that results from a reaction in which the component to be altered participates.

The latter can also be achieved by inserting an appropriate CLEC into a suitable body compartment, such as the peritoneum or a lymph node, where the CLEC would have access to body fluids. Insertion could be accomplished by surgery or by injection of a thick aqueous solution of CLEC. Direct injection of CLEC into the bloodstream would not be appropriate due to the high risk of embolism that this entails.

The use of appropriate CLECs in this area could serve as an alternative to genetic methods in enzyme replacement therapies to correct a natural deficiency, such as, for example, phenylketonuria.

Table 3 illustrates some of the medical applications in which CLECs could be used. For most of these cases, extracorporeal treatment is still in the investigational phase, but the benefits offered by CLECs could provide new treatments in areas where there was no prior alternative treatment.

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TABLE 3

<td>Enzyme used</td><td>Separation from:</td><td>Diseases / patients treated</td><td>References</td>

<td>• Asparaginase</td><td>• Asparagine</td><td>• Leukemia (separation of an asparagine, an important cancer nutrient, damages leukemic cells that cannot make the essential amino acid, asparagine; normal cells can make asparagine and are therefore not affected by this treatment).</td><td>Klein, M., Langer, R., Trends in Biotechnology 4: 179-185 (1986) and references included therein. Chang, TMS, Methods in Enzymology 137: 444-457 (1987) and references therein</td>

<td>• Heparinase</td><td>• Heparin</td><td>• Deheparinization for hemoperfusion patients, eg. ex. kidney dialysis</td><td>Langer, R., et al., Science 217: 261-263 (1982)</td>

<td>• Bilirubin oxidase</td><td>• Bilirubin</td><td>• Neonatal jaundice</td><td>Levin, A., et al., Science 230: 543-545 (1985)</td>

<td>• Carboxypeptidase</td><td>• Methotrexate</td><td>• Chemotherapy patients</td><td>Pitt, AM, et al., Appl.Biochem.Biotechnol. 8: 55-68 (1983)</td>

<td>• Tyrosinase</td><td>• Aromatic amino acids</td><td>• Hepatic failure with pathological amino acid elevations</td><td>Chang, TMS, Sem.Liver Dis.Ser. 6: 148 (1986)</td>

<td>• Phenylalanine ammonium lyase</td><td>• Phenylalanine</td><td>• Phenylketonuria and insufficiency liver</td><td>Ambrus, CM, et al., J. Pharm. Exp. Ther. 224: 598-602 (1983)</td>

<td>• Multienzyme system that includes: urease, glutamate dehydrogenase, glucose, dehydrogenase and transaminase</td><td>• Urea (converted to glutamic amino acid and other amino acids, via ammonia)</td><td>• Detoxification for patients with kidney failure chronicle</td><td>Chang, TMS, Methods in Enzymology 137: 444-457 (1987) references included therein. Chang, TMS, Enzyme Eng 5: 225 (1980)</td>

<td>• Arginase</td><td>• Arginine</td><td>• Familial hyperargininemia</td><td>Kanalas, JJ, et al., Biochem. Med. 27: 46-55 (1982)</td>

<td>• Glutamate dehydrogenase and ammonia</td><td>• Ammonia</td><td>• Renal insufficiency</td><td>Maugh, TH, Science 223: 474-476 (1984)</td>

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A particular application of the present method is the heparin lyase system for deheparinization of blood (Bernstein et al., Methods in Enzymology 137: 515-529 (1987)) discussed below.

All blood-perfused extracorporeal devices, such as renal dialysis, continuous arteriovenous hemofiltration, or extracorporeal membrane oxygenators, require heparinization of the patient to prevent the blood from clotting. However, heparinization of the patient causes bleeding complications and continues to be a threat to human health. These problems increase as infusion times increase, for example with membrane oxygenator, and can lead to severe bleeding. After extracorporeal therapy, heparin can be separated from the blood using a heparinase device in the effluent from the extracorporeal device that removes all heparin from the blood returning to the patient, thus avoiding current heparinization problems.

Published research (Langer et al. Science 217: 261-263 (1982); Bernstein et al., Methods in Enzymology 137: 515-529 (1987)) explain in detail the problems presented by conventionally immobilized enzymes used in devices extracorporeal. The main problem is that conventional immobilization causes low retention of enzyme activity per unit volume, thus requiring a large volume of immobilized enzyme to perform the necessary heparinization. This volume is not practical for use in humans due to its large size. However, the high retention of activity per unit volume in CLECs due to the lack of inert support solves this problem and offers a practical solution to the deheparinization of humans. The improved stability of CLECs will reduce the dissociation of the enzyme from the cross-linked crystal. This characteristic makes CLECs superior to less stable enzymes immobilized by conventional means, because immune responses resulting from enzyme loss will be reduced. The temperature stability of CLEC prevents denaturation of the enzyme due to transient high temperatures during storage; CLECs are likely to retain their high activity even when stored at room temperature. Furthermore, CLECs will be more economical and convenient to use than their counterpart enzymes immobilized by conventional means due to their longer storage and operational lives.

Additional applications of CLEC technology: biosensors

It is possible to use a CLEC or set of CLECs as a component of a sensor, referred to as a biosensor, that serves to detect and / or quantify an analyte of interest in a fluid, such as body fluid (eg blood, urine), media of chemical and laboratory reaction, organic media, water, culture media and drinks. In some cases, the fluid in question may be gas, as in an electronic alcohol consumption analyzer (Barzana, E., Klibanov, A., and Karell, M., NASA Tech Briefs 13: 104 (1989)). In this Application an appropriately selected CLEC or set of CLECs are contacted with a fluid to be analyzed for the analyte of interest. The analyte of interest can be measured directly (eg blood glucose level) or indirectly (eg. detecting or quantifying a substance that is reactant (product or substrate) in a reaction in which the analyte of interest participates). In either case, CLEC can act on the analyte or the substance that is a reactant in a reaction in which the analyte also participates. Enzyme activity causes a detectable change (eg. change in pH, light generation, heat, change in electrical potential) that is detected and / or quantified through appropriate detection means (e.g. pH electrode, light or heat detection device, means for measuring electrical charges) (Janata, J., et al., Anal. Chem. 62: 33R-44R (1990)). Any useful means can be used to detect the change that results from the enzyme catalyzed method. A biosensor of the present invention includes a CLEC or set of CLEC and retention means for the CLEC that allows contact between the CLEC (s) and the analyte of interest or the substance in the fluid that is a reagent in the reaction in which the analyte of interest participates.

Table 4 illustrates some of the biosensor applications in which CLECs could be used. Currently immobilized enzymes are used in these applications, but these have low stability, low enzyme density, short lives, and lack of reproducibility. These examples can easily be adapted to the CLEC technology described herein.

TABLE 4

<td>Enzyme used</td><td>Detection of:</td><td>App</td><td>Reference</td>

<td>• Glucose oxidase</td><td>• Glucose</td><td>• Diabetes</td><td>• Daniles, B., Mossbach, K, Methods in Enzymology 137: 4-7 (1987) • Hall, E “Biosensors” Open University Press (1990) • Taylor, R., Proceed. Biotechnology Conference 1989; 275-287 • Anthony et al., "Biosensors, Fundamentals and Applications", Oxford University Press (1987)</td>

ES 2 199 933 T3

TABLE 4 (continued)

<td>Enzyme used</td><td>Detection of:</td><td>App</td><td>Reference</td>

<td>• Creatinine deiminase</td><td>• Creatinine</td><td>• Renal function</td><td>• Tabata, M., et al., Anal.Biochem. 134: 44 (1983)</td>

<td>• Urease</td><td>• Urea</td><td>• Renal function</td><td>• Hsuie, GH, et al., Polym. Mater. Sci. Eng. 57: 825-829 (1987) • Kobos, et al., Anal.Chem. 60: 1996-1998 (1988)</td>

<td>• Lactase oxidase and dehydrogenase</td><td>• Lactate</td><td>• Chemical applications</td><td>• Blaedel, WJ Jenkins, RA, Anal. Chem. 48 (8): 1240 (1976) • Sagaguchi, Y., et al., J. Applic. Biochem 3:32 (1981)</td>

<td>• Glucose-6- pyruvate dehydrogenase</td><td>• Glucose-6 phosphate, sucrose and adenosine triphosphate (ATP)</td><td>• Diabetes and others Applications medical</td><td>• In the same literature as glucose oxidase</td>

<td>• Alcohol- dehydrogenase, alcohol oxidase</td><td>• Ethanol and other alcohols: acetic, formic acids</td><td>• Electronic analyzers for alcohol consumption and industrial applications</td><td>• Romette, JLet al., Methods in Enzymology 137: 217-225 (1987) • Ho, MH, Methods in Enzymology 137: 271-288 (1987)</td>

<td>• β-fructosidase</td><td>• Sucrose</td><td>• Industrial applications</td><td>• Romette, JLet al., Methods in Enzymology 137: 217-225 (1987)</td>

<td>• Cholesteroloxidase</td><td>• Cholesterol</td><td>• Cholesterol analysis</td><td>• Satoh, I. Methods in Enzymology 137: 217-225 (1987)</td>

<td>• Catalase</td><td>• Uric acid, cholesterol</td><td>• Atherosclerosis and other medical applications</td><td>• Satoh, I. Methods in Enzymology 137: 217-225 (1987)</td>

<td>• Carboxypeptidase</td><td>• Methotrexate</td><td>• Cancer</td><td>• In the same literature as glucose oxidase</td>

<td>• Carbonic anhydrase</td><td>• Carbon dioxide</td><td>• Industrial, laboratory and environmental applications</td><td>• In the same literature as glucose oxidase</td>

<td>• L-amino acid oxidase</td><td>• Amino acids</td><td>• Medical and industrial applications</td><td>• In the same literature as glucose oxidase</td>

<td>• β-lactamasapenicillinase</td><td>• Penicillin</td><td>• Medical applications</td><td>• Anzal et al., Bull.Chem.Soc.Jpn. 60: 4133-4137 (1988)</td>

<td>• Phosphatase alkaline</td><td>• Phosphate</td><td>• Monitoring of metabolites</td><td>• In the same literature as glucose oxidase</td>

<td>• Nitrate / nitrile reductase</td><td>• Nitrates and nitrites</td><td>• Monitoring of metabolites and food</td><td>• In the same literature as glucose oxidase</td>

ES 2 199 933 T3

TABLE 4 (continued)

<td>Enzyme used</td><td>Detection of:</td><td>App</td><td>Reference</td>

<td>• Arylsulfatase</td><td>• Sulfate</td><td>• Monitoring of metabolites</td><td>• In the same literature as glucose oxidase</td>

<td>• Succinate dehydrogenase</td><td>• Succinate</td><td>• Industrial applications</td><td>• In the same bibliography as glucose oxidase</td>

<td>• Luciferase bacterial</td><td>FMNH₂ and coupled reactions</td><td>• Detection of molar quantities 10^-18 by FMNH₂ through photon measurement</td><td>• Wannlund, J., et al., "Luminiscent assays: Perspectives in endocrinology and clinical chemistry" Eds Serio, M. and Pazzagli, M. 1: 125 (1982) • Kurkijarvi et al., Methods in Enzymology 137: 171-181 (1987)</td>

<td>• Firefly Luciferase</td><td>Adenosine triphosphate (ATP) and coupled reactions</td><td>• Detection of molar quantities 10^-12 of ATP through the measurement of photons</td><td>• Kurkijarvi et al., Methods in Enzymology 137: 171-181 (1987) • Murachi et al., Methods in Enzymology 137: 260-271 (1988)</td>

In the method of the present invention, as carried out for sample analysis in a biosensor, it is particularly preferred that the largest possible detectable signal is produced from the least amount of substrate and catalyst. In this regard, the CLEC technology described herein is of special interest, since it achieves the highest possible concentration of enzyme catalyst in a given volume.

Considerable efforts are often made to couple a fundamental enzymatic reaction of interest, either directly or via appropriate chemical intermediates, to the generation of light by enzymes such as luciferase (Kurkijarvi et al., Methods in Enzymol. 137: 171-181 (1988)). The latter is done in order to take advantage of the unprecedented sensitivity and efficiency of photon detection equipment, which allows the detection of femtomolar concentrations of enzymatic reaction products under appropriate conditions. Following this principle, biosensor systems have been designed using enzymes immobilized by conventional means to detect various substrates of clinical or other interest. Light generation reactions have been coupled to analyze reactions that detect substrates such as D-glucose, L-lactate, L-glutamate, and ethanol, among others, at extremely low concentrations.

With regard to this application, the luciferase enzyme from Vibrio harveyii has been reported to be crystallized (Swanson et al., J. Biol. Chem. 260: 1287-1289 (1985)). Crystals of this luciferase can be cross-linked with glutaraldehyde or other appropriate reagent to form a luciferase CLEC. For biosensor and analytical uses, a luciferase CLEC offers a host of advantages compared to enzymes immobilized by conventional means. In a CLEC, the entire volume of the luciferase CLEC would consist of a light emitting enzyme. In an enzyme system immobilized by conventional means, however, at most 95% of the total volume is absorbed by an "inert" carrier material, which most likely functions as an absorber for the light emitted by the enzyme. Also, the improved stability of CLECs should facilitate storage at room temperature and also allow the use of new detection applications in aggressive media and elevated temperatures.

Additional applications of CLEC technology - laboratory reactions

CLECs can be used as laboratory reagents in small columns or in batch processes, which can be used to carry out laboratory reactions. Some of the broad categories of reactions are listed in Table 5.

ES 2 199 933 T3

TABLE 5

<td>Enzyme used</td><td>Type of catalyzed reaction</td><td>Reference</td>

<td>• Lipases, phospholipases</td><td>• Stereoselective synthesis including esterification, transesterification, aminolysis, lactonizations, polycondensations, acylation, oxymolysis and redissolution of mixtures racemic</td><td>• Zaks, A. and Klibanov, AM Proc.Nat.Acad. Sci.USA. 82: 3192-3196 (1985) • Klibanov, AMAcc. Chem. Res. 23: 114-120 (1990) and references therein. • Wong, CH, Chemtracts-Organic Chemistry 3: 91-111 (1990) and references therein.</td>

<td>• Esterases</td><td>• Stereoselective synthesis and redissolution</td><td>• Kobayashi et al., Tetrahedron Letters Vol 25, N ° 24: 2557-2560 (1984) • Schneider et al., Angew.Chem.Int.Ed.Ensl. 23 (N ° 1): 64-68 (1984)</td>

<td>• Tyrosinase</td><td>• Oxidation of phenols to produce quinones</td><td>• Kazandjian, RZ and Klibanov, AMJAm.Chem.Soc. 110: 584-589 (1986)</td>

<td>• Proteases, p. ex. subtilisin</td><td>• Stereoselective acylation of carbohydrates</td><td>• Riva et al., JMChem.Soc. 110: 584-589 (1988)</td>

<td>• Oxidases</td><td>• Selective oxidation of hydrocarbons</td><td>• Klibanov, AM Acc.Chem. Res. 23: 114-120 (1990) and references therein.</td>

<td>• Other enzymes that do not require cofactors: isomerases, lyases, aldolases, glycosyl, transferases, glycosidases</td><td>• Stereoselective synthesis</td><td>• Wong, CH, Chemtracts-Organic Chemistry 3: 91-111 (1990) and references therein.</td>

<td>• Other enzymes that do not require cofactors aggregates: flavoenzymes, pyrodoxal enzymes phosphate, metalloenzymes</td><td>• Stereoselective synthesis</td><td>• Wong, CH, Chemtracts-Organic Chemistry 3: 91-111 (1990) and references therein.</td>

<td>• Enzymes that require cofactors: kinases (adenosine triphosphate-ATP), oxidoreductases (NAD / P), methyl transferases (SAM), enzymes that require CoA, sulfurases (PAPS)</td><td>• Stereoselective synthesis</td><td>• Wong, CH, Chemtracts-Organic Chemistry 3: 91-111 (1990) and references therein.</td>

ES 2 199 933 T3

Schneider et al. (Angew. Chem. Int. Ed. Engl. 23 (No. 1): 64-68 (1984)) illustrates how to use enzymes in organic synthesis. Porcine liver esterase was used in the transformation from meso-ester to chiral mono-ester in an aqueous phosphate buffer.

The advantages of CLEC catalyzed reactions for laboratory use are threefold. First, CLECs retain high activity in aggressive environments (eg aqueous, organic, quasi-anhydrous solvents and their mixtures, and at elevated temperatures), typical in laboratory chemical synthesis experiments. Second, CLECs exhibit high storage and operational stability, which is appropriate for intermittent laboratory experiments. Third, its high activity per unit volume will allow shorter reaction times and will require smaller volumes of enzyme (per unit of activity). Thus, the advantages that CLECs offer compared to free or immobilized enzymes provide organic chemists with a highly selective alternative synthetic tool.

One of ordinary skill in the art can adapt the method of this invention in all the cases described above, without limitation, to convert a process using a catalyst of enzymes immobilized by conventional means into the use of a CLEC of the appropriate enzyme . CLECs can not only replace enzymes immobilized by conventional means, but can also be used in cell-mediated transformations. The present invention will now be illustrated with the following examples, which are not intended to be limiting in any way.

Example 1

Thermolysin crystallization and crosslinking for the synthesis of the aspartame precursor, Z-Asp-Phe-OMe

Crystallization

250 mg of Bacillus thermoprotecolyticus thermolysin were purchased from Boehringer-Mannheim GmbH and then dissolved in 4 ml of 45% dimethylsulfoxide (DMSO) and 55% 1.40M calcium acetate, 0.50M sodium cacodylate at pH 6 ,5. These initial conditions are similar to those described by Matthews et al. for the production of diffractory grade thermolysin crystals (see, for example, Holmes and Matthews, J. Mol. Biol. 160: 623-639 (1982)). The protein solution was then concentrated to 1 ml in a Centricon 10 micro-concentrator. Good microcrystallization production was obtained by a flash crystallization process, now described in the present invention, in which 1 ml of water or 1.40 M of calcium acetate and 0.50 M of sodium cacodylate at pH 6.5 within any of the thermolysin-DMSO solutions described above. This process results in a shower of hexagonal microcrystals of approximately uniform dimensions (approximately 10^-1 mm in length).

Crosslinking of thermolysin microcrystals

The protocol used in this specific example of the method of this invention is an adaptation of that described by Nakanishi et al. (Biotechnology 3: 459-464 (1985), in which thermolysin was first adsorbed on a carrier bead composed of the ion exchange resin Amberlite XAD-7 and later immobilized by crosslinking with glutaraldehyde (Quiocho and Richards, Proc. Natl. Acad. Sci. (USA) 52: 833-839 (1964)). In this exemplification, the thermolysin microcrystals obtained above were centrifuged and granulated and the supernatant was discarded. Next, 5 ml of 17.5% technical grade glutaraldehyde in 2.5% DMSO, 0.05 M calcium acetate and 0.025 M sodium cacodylate were added to the microcrystals at pH 6.5. The mixture was incubated with gentle shaking at 37 ° C for 4 hours. The crosslinking reaction was stopped by repeatedly washing the crystals with 10 ml aliquots of water to remove the glutaraldehyde solution. The washed cross-linked thermolysin crystals constitute the thermolysin CLEC used as a catalyst below.

Synthesis of Z-Asp-Phe-OMe in an aqueous solution

5 ml of a thermolysin CLEC suspension was added to a continuously stirred batch reactor incubated at 37 ° C. After centrifugation and decantation of the supernatant, an aqueous reaction mixture was added to the CLECs. This solution was prepared by mixing 80 mg of ZL-Asp and 80 mg of L-Phe-OMe-HCl in 1 ml of water with added acetic acid to obtain a pH of 7.0. Samples were taken to be analyzed by HPLC. Table 6 shows the maximum height of the HPLC of the peak of the substrate ZL-Asp after the indicated reaction time, normalized to 1 at time t = 0. Since ZL-Asp is rate limiting in this reaction, measuring its depletion is equivalent to measuring the appearance of the ZL-Asp-L-Phe-OMe product (Nakanishi et al. Biotechnology 3: 459-464 (1985)). Table 6 also includes the maximum normalized height of the limiting substrate ZL-Asp that remains, and an estimate of the degree of completion of the reaction. It is apparent that the reaction reached approximately 20% completion within the first 30 seconds and stalled at that point. These results coincide with the observations of Nakanishi et al. (Biotechnology 3: 459-464 (1985)) when using thermolysin immobilized by conventional means in an aqueous reaction mixture as described above, and are attributed to the poor solubility of the product ZL-Asp-L-Phe-OMe in water.

ES 2 199 933 T3

TABLE 6

<td>Reaction time (seconds)</td><td>Maximum height (normalized)</td><td>Percentage of completion</td>

<td> 0</td><td> 1,000</td><td></td>

<td> 30</td><td> 0,727</td><td> 27,3%</td>

<td> 60</td><td> 0,857</td><td> 14,3%</td>

<td> 120</td><td> 0,940</td><td> 6,0%</td>

<td> 180</td><td> 0,797</td><td> 20,3%</td>

Synthesis of Z-Asp-Phe-OMe in a quasi-anhydrous solution

5 ml of a thermolysin CLEC suspension was added to a continuously stirred batch reactor incubated at 37 ° C. After centrifugation and decantation of the supernatant, a quasi-anhydrous organic reaction mixture was added to the CLECs. This solution was prepared by mixing 80 mg of ZL-Asp and 240 mg of L-Phe-OMe in 1 ml of 99% ethyl acetate and 1% of water. Samples were taken to be analyzed by HPLC. Table 7 shows the maximum height of the HPLC of the peak of the substrate ZL-Asp after the indicated reaction time, normalized to 1 at time t = 0. Since ZL-Asp is a rate limiter in this reaction, measuring its decrease is equivalent to measuring the appearance of the ZL-Asp-L-Phe-OMe product (Nakanishi et al. Biotechnology 3: 459-464 (1985)) . Table No. 7 also includes the maximum normalized height of the limiting substrate ZL-Asp that remains, and an estimate of the degree of completion of the reaction. In this case the reaction reached approximately 70% completeness within the first 30 seconds and stalled at that point. These results also coincide with the observations of Nakanishi et al. (Biotechnology 3: 459-464 (1985)) with thermolysin immobilized by conventional means in a quasi-anhydrous reaction mixture and attributed to enzyme inhibition of the product.

TABLE 7

<td>Reaction time (seconds)</td><td>Maximum height (normalized)</td><td>Percentage of completion</td>

<td> 0</td><td> 1,000</td><td></td>

<td> 30</td><td> 0,323</td><td> 67,7%</td>

<td> 60</td><td> 0,314</td><td> 68,6%</td>

<td> 120</td><td> 0,305</td><td> 69,5%</td>

<td> 180</td><td> 0,272</td><td> 72,8%</td>

Example 2

Crystallization, crosslinking and lyophilization of thermolysin and evaluation of the characteristics of the resulting product

Thermolysin crystallization

Thermolysin (Diawa Kasei KK, Japan) was dissolved in 10 Mm calcium acetate (Sigma), pH = 10.0 until a concentration of 10% (w / v) was reached. The pH of the solution was maintained in 10.0 titration with 2M NaOH. After solubilization was complete, the protein solution was titrated to pH 8.0 with 2M HCl. Solid calcium acetate was added to 1.2M. Dimethylsulfoxide (Sigma) was then added to reach 30%. The protein was concentrated to 100 mg / ml by ultrafiltration in an Amicon shaking cell (10,000 MWCO membrane). The concentrated enzyme was aliquoted and stored at -70 ° C. Thermolysin was crystallized by adding 9 volumes of demineralized water to 1 volume of concentrated protein solution (100 mg / ml). The solution was vortexed for a short time and allowed to stand overnight at room temperature. The crystals were washed with 10 volumes of 10 mM calcium acetate pH 7.0 and recovered by low speed centrifugation (10 minutes at 1,500 x G, Beckman GPR centrifuge).

The rapid addition of water to a concentrated thermolysin solution (100 mg / ml) induces the formation of crystals that become visible with low amplification within ten minutes. The crystal size reproducibly depends on the final protein concentration. The ratio of three volumes of water to one volume of thermolysin concentrate (100 mg / ml) will produce X-ray diffractory quality hexagonal rods 0.5 mm in length corresponding to the crystals previously described by Colman et al. (Colman, PM, Jansonius, JN and Matthews, BW, J. Mol. Biol. 70: 701-724 (1972)), as confirmed by diffraction analysis. The addition of ten volumes of water to one of the protein concentrates reduces the length of the resulting crystals to 0.5 mm. These microcrystals are preferable in CLEC applications as they tend to minimize diffusion problems related to crystal size (see, for example, Quiocho, FA and Richards, FM Biochemistry 5: 4062-4076 (1967)). Within a given batch of protein, the crystal size always remained uniform. (I know

ES 2 199 933 T3 used crystals 0.05 - 0.10 mm in length in this study to facilitate accurate pipetting of crystalline suspensions.) SDS-PAGE densitometer scans showed six-fold greater purification of the crystallizing enzyme , with which the specific activity of CLEC is significantly increased. The crystallization caused a 20% reduction in the total activity of the CLEC protein compared to soluble thermolysin, when it was analyzed by spectrophotometric subcellular fractionation of the dipeptide substrate of furyl-acryloylglycyl-L-leucine-amide (FAGLA), according to outlined below.

Crosslinking of thermolysin crystals

The thermolysin crystals were cross-linked for 3 hours at room temperature in a solution of 12.5% ​​glutaraldehyde (Sigma), 5% DMSO and 50 mM Tris pH 6.5. The cross-linked crystals were washed 3 times in demineralized water and recovered by low speed centrifugation, as described with respect to thermolysin crystallization. Chemical crosslinking of enzyme crystals stabilizes the crystal lattice and constitutive enzyme molecules in the crystal sufficiently to allow the practical use of CLECs in environments that would otherwise be incompatible with enzyme function. No measurable difference in enzyme activity was found between cross-linked and non-cross-linked crystals when analyzed (spectrophotometrically) by monitoring subcellular cleavage of the dipeptide substrate FAGLA (described below). On the other hand, crosslinking stabilizes CLECs to the point that they can be lyophilized, retaining the entire enzymatic activity when reconstituted in aqueous, organic and aqueous / mixed organic solvents, as shown in Figure 1 and Table 8. While crystallization caused a 30% decrease in specific activity of CLEC protein compared to soluble thermolysin, cross-linking and lyophilization of CLEC did not further decrease specific activity.

TABLE 8

<td colspan="4">Thermolysin activity</td>

<td></td><td></td><td colspan="2">Absorbance 345 nm</td>

<td></td><td>Time (minutes)</td><td>CLEC</td><td>Soluble enzyme</td>

<td> 1</td><td> 0,0</td><td> 0,314</td><td> 0,315</td>

<td> 2</td><td> 1,0</td><td> 0,272</td><td> 0,271</td>

<td> 3</td><td> 3,0</td><td> 0,235</td><td> 0,218</td>

<td> 4</td><td> 5,0</td><td> 0,204</td><td> 0,198</td>

<td> 5</td><td> 10,0</td><td> 0,184</td><td> 0,185</td>

<td> 6</td><td> 15,0</td><td> 0,183</td><td> 0,184</td>

CLEC Soluble Thermolysin and Thermolysin Enzyme Activity

The catalytic activity of soluble thermolysin and thermolysin of CLEC (Feder, J. and Schuck, JM, Biochemistry 9: 2784-2791 (1970)) was analyzed by hydrolysis of the blocked dipeptide substrate of furylacryloyl-glycyl-L-leucinaamide (FAGLA) (Schweizerhall). The cleavage of the amide bond was measured spectrophotometrically by a decrease in absorbance at 345 nm. The initial enzyme concentration was 10^-7 M by densitometric scanning and protein determination by the Bradford method (Pharmacia LKB UltroScan XL) of SDSS-PAGE gels stained with Coomassie. CLEC enzyme is defined as reconstituted lyophilized cross-linked thermolysin crystals. Soluble enzyme is defined as thermolysin concentrated at 100 mg / ml. Enzyme was added to a 5 ml reaction volume containing substrate. Aliquots of the reaction mixture were removed at the indicated times and the absorbance at 345 nm was measured. The CLEC thermolysin was separated from the reaction mixture by brief centrifugation (Beckman, microcentrifuge E) before taking an absorbance reading. The absorbance was fitted to a pseudo-first degree velocity equation and the kcat / km was calculated by dividing the adjusted value by the enzyme concentration (Multifit 2.0 Curve Fitting for Apple Macintosh Computer; Day Computing PO Box 327, Milton, Cambridge CB4 6WL, UK (1990)).

PH dependence and stability

Optimum pH and stability of the enzyme were compared with those of thermolysin CLECs by cleavage of the dipeptide substrate FAGLA. The results are shown in Figure 2 and Table 9. Both soluble and crystalline forms of the enzyme show maximum activity at pH 7. CLEC and soluble thermolysin also showed similar activity in the acid range, and the bell-shaped pH profile generated by the soluble enzyme was consistent with published data (Feder, J. and Schuck, JM, Biochemistry 9: 2784- 2791 (1970)). However, in the alkaline pH range, the crystalline enzyme maintains a maximum activity, at pH 10, while the soluble enzyme presents activity of 75% at pH 8.5 and only 25% at pH 9. At pH 9.5 the soluble enzyme is completely inactive.

ES 2 199 933 T3

TABLE 9

<td colspan="4">Thermolysin pH curve</td>

<td></td><td></td><td colspan="2">% of maximum activity</td>

<td></td><td>PH</td><td>CLEC</td><td>Soluble enzyme</td>

<td> 1</td><td> 5,0</td><td> 10,250</td><td> 5,170</td>

<td> 2</td><td> 5,5</td><td> 9,750</td><td> 6,070</td>

<td> 3</td><td> 6,0</td><td> 52,500</td><td> 39,100</td>

<td> 4</td><td> 6,5</td><td> 85,000</td><td> 74,610</td>

<td> 5</td><td> 7,0</td><td> 97,500</td><td> 100,000</td>

<td> 6</td><td> 7,5</td><td> 100,000</td><td> 98,650</td>

<td> 7</td><td> 8,0</td><td> 97,500</td><td> 82,920</td>

<td> 8</td><td> 8,5</td><td> 95,000</td><td> 71,910</td>

<td> 9</td><td> 9,0</td><td> 96,250</td><td> 24,720</td>

<td> 10</td><td> 9,5</td><td> 95,000</td><td> 0,000</td>

<td> 11</td><td> 10,0</td><td> 90,000</td><td> 0,000</td>

Stability at elevated temperature

Higher reaction rates and shorter diffusion times can be achieved for substrates and products by running a given chemical process at a higher temperature, where limiting the temperature stability of substrates and products generally occurs. However, in enzyme-based catalysis the loss of enzyme activity normally sets the practical limit to the temperature at which a process can be run. The additional stability achieved in CLECs allows enzymatic activity at temperatures much higher than those tolerated in the soluble enzyme.

Improved stability at lower temperatures simplifies routine long-term storage of CLEC catalysts. For example, it was necessary to store concentrated solutions (&gt; 50 mg / ml) of soluble thermolysin at -80 ° C to retain a specific maximum activity. At room temperature, activity was usually lost within a day. In contrast, rehydrated thermolysin CLECs could be routinely stored for months at room temperature without apparent loss of activity. Non-reconstituted lyophilized thermolysin CLECs appear to be viable indefinitely.

Thermal stability and resistance to autolysis were demonstrated in thermolysin CLEC after incubation at 65 ° C for five consecutive days (Figure 3 and Table 10). Thermolysin CLECs retained maximum activity after five days of incubation at elevated temperature. In contrast, soluble thermolysin lost 50% of its initial activity only after 2 hours of incubation and showed negligible activity after 24 hours of incubation at 65 ° C.

TABLE 10

<td colspan="4">Thermal stability of thermolysin at 65 ° C% of maximum activity</td>

<td></td><td>Time (days)</td><td>CLEC</td><td>Soluble enzyme</td>

<td> 1</td><td> 0,000</td><td> 100,000</td><td> 100,000</td>

<td> 2</td><td> 0,041</td><td></td><td> 70,000</td>

<td> 3</td><td> 0,083</td><td> 96,000</td><td> 50,000</td>

<td> 4</td><td> 0,164</td><td></td><td> 32,000</td>

<td> 5</td><td> 0,246</td><td></td><td> 17,000</td>

<td> 6</td><td> 0,410</td><td> 97,0</td><td> 10,000</td>

<td> 7</td><td> 1,000</td><td> 101,0</td><td> 2,000</td>

<td> 8</td><td> 2,000</td><td> 97,0</td><td></td>

<td> 9</td><td> 3,000</td><td> 94,0</td><td></td>

<td> 10</td><td> 4,000</td><td> 96,0</td><td></td>

<td> 11</td><td> 5,000</td><td> 92,0</td><td></td>

ES 2 199 933 T3

The soluble thermolysin and thermolysin activity of CLEC was measured after incubation at 65 ° C. Soluble thermolysin was incubated in 10 mM calcium acetate, 50 mM Tris at pH 7.0 in a 65 ° C water bath. The reaction volume was 500 µί. The final protein concentration was 10 mg / ml. Aliquots were removed at 0, 1, 2, 4, 6, 10, and 18 hours. Samples were analyzed by SDS-PAGE and FAGLA cleavage at room temperature as described above. For thermolysin CLECs, a 250 µl crystalline suspension was also incubated in 10 mM calcium acetate and 50 mM Tris in a 65 ° C water bath. Activity was analyzed at 0, 1, 6, 24, 48, 72, 96 and 120 hours by FAGLA cleavage.

Resistance to exogenous proteolysis

The evaluation of the resistance of thermolysin CLEC to the action of an exogenous protease was also carried out. SDS-PAGE (sodium docecyl sulfate polyacrylamide gel electrophoresis) analysis suggests that commercial enzymes may contain a substantial percentage of contaminants, some of which may exert proteolytic activity against major soluble enzyme species. Taking into account the packaging of enzyme molecules in a crystal lattice, it could be assumed that the interior enzyme molecules of a CLEC would be protected against proteolysis. To verify this possibility, the CLECs of thermolysin and a soluble enzyme preparation were incubated in the presence of the streptococcal protease Pronase.^®, a nonspecific protease capable of digesting most proteins to release amino acids (Calbiochem 1990 Catalog; LaJolla, CA).

Soluble thermolysin and CLEC thermolysin were incubated in 50 mM Tris at pH 7.5 at 40 ° C in the presence of the protease Pronase^® (Calbiochem). The Pronase relationship^® athermolysin was 1/40. To inhibit thermolysin autolysis and prevent proteolytic destruction of pronase by thermolysin, EDTA was added to the soluble enzyme reaction to reach a final concentration of 100 mM (EDTA inhibits thermolysin activity but not Pronase^®. Aliquots of the reaction mixture were removed at the times indicated and the activity was analyzed spectrophotometrically through cleavage of the FAGLA dipeptide substrates. To counteract the inhibition of thermolysin due to the presence of EDTA, the spectrophotometric analysis of the soluble enzyme activity was carried out in 0.5 M calcium acetate buffer at pH 7.0 and the enzyme concentration was doubled. . The cross-linked crystalline enzyme was analyzed as described above.

As seen in Figure 4 and Table 11, soluble thermolysin degraded rapidly and lost all activity after 90 minutes of incubation. In contrast, the CLEC activity of thermolysin was not affected by four days of incubation in the presence of protease. This quasi-impermeability to proteolysis is particularly interesting in biosensor diagnostic applications where an appropriate CLEC can be sought to act in the presence of an unknown cocktail of naturally present proteolytic enzymes.

TABLE 11

<td colspan="5">Protease resistance</td>

<td rowspan="2"></td><td rowspan="2">Time (days)</td><td colspan="2">% of maximum activity</td><td rowspan="2">Time (minutes)</td>

<td>CLEC</td><td>Soluble enzyme</td>

<td> 1</td><td> 0,000</td><td> 100,0</td><td> 100,0</td><td> 0,000</td>

<td> 2</td><td> 0,003</td><td></td><td> 25,0</td><td> 5,000</td>

<td> 3</td><td> 0,010</td><td></td><td> 17,5</td><td> 15,000</td>

<td> 4</td><td> 0,021</td><td></td><td> 9,5</td><td> 30,000</td>

<td> 5</td><td> 0,042</td><td> 98,0</td><td> 3,0</td><td> 60,000</td>

<td> 6</td><td> 0,063</td><td></td><td> 1,0</td><td> 90,000</td>

<td> 7</td><td> 0,084</td><td> 101,0</td><td> 0,0</td><td></td>

<td> 8</td><td> 1,000</td><td> 97,0</td><td></td><td></td>

<td> 9</td><td> 2,000</td><td> 99,0</td><td></td><td></td>

<td> 10</td><td> 3,000</td><td> 98,0</td><td></td><td></td>

<td> 11</td><td> 4,000</td><td> 96,0</td><td></td><td></td>

Stability in the presence of organic solvent

For enzymes to gain ideal acceptance as viable industrial catalysts, they must be able to function without undue interference in the practical environment of manufacturing processes. In particular, the latter would include the use of polar and non-polar aqueous and organic solvents and mixtures thereof. In commercial applications, aqueous-organic solvent mixtures allow manipulation of product formation by taking advantage of the relative solubilities of products and substrates.

ES 2 199 933 T3

Soluble thermolysin and thermolysin CLECs showed marked differences in stability in the presence of organic solvents. (Table 12). The soluble enzyme concentrations that could be incubated in organic solvent were limited to a maximum of 10 mg / ml. Concentrations greater than this value resulted in instantaneous precipitation of thermolysin upon addition of organic solvent. In contrast, thermolysin CLEC concentrations were limited only by the volume occupied by the crystals. Soluble thermolysin retained the highest activity (75%) after incubation in acetone and the lowest (36%) in tetrahydrofuran. After one hour of incubation in the presence of acetonitrile or dioxane, the soluble enzyme lost approximately 50% of its initial activity. CLEC thermolysin retained more than 95% of maximum activity after incubation with all organic compounds tested.

TABLE 12

<td rowspan="2"></td><td colspan="2">% of maximum activity</td>

<td>Soluble enzyme</td><td>CLEC</td>

<td>Acetonitrile</td><td> 42</td><td> 102</td>

<td>Dioxane</td><td> 66</td><td> 97</td>

<td>Acetone</td><td> 75</td><td> 99</td>

<td>THF *</td><td> 36</td><td> 96</td>

* Tetrahydrofuran

Stability in organic solvents

Thermolysin CLECs or soluble thermolysin preparations were incubated in 50% (volume / volume) solutions of the indicated organic solvents. 100 µl of a CLEC suspension of thermolysin (10 mg / ml) in 10 mM Tris at pH 7 was placed in a 0.9 g glass bottle. An equal volume of the indicated organic solvent was added and the mixture was briefly vortexed. 20 µl of soluble thermolysin (100 mg / ml) was diluted in 80 µl of 0.015 M Tris buffer at pH 7.0 in a 0.9 g glass bottle. A 100 µl volume of organic solvent was then added to the protein solution and vortexed briefly. The soluble and CLEC enzymes were incubated in the presence of the organic solvent for one hour at 40 ° C. After incubation, enzymatic activity was analyzed by cleavage of the dipeptide substrate FAGLA as described above.

It is believed that low concentrations of water do not favor the unfolding of intermediate states in the process towards denaturation of the enzyme. In CLEC this conformational mobility restriction is offered through intermolecular contacts and cross-links between the constitutive enzymatic molecules that make up the crystal lattice and not through the quasi-absence of water in the medium. As a result, intermediate water-organic solvent concentrations are easily tolerated by enzymes when formulated as CLEC, which has not previously been observed with enzymes (see Table 12). This discovery introduces entirely new areas of synthetic chemistry to be exploited using enzymatic catalysis.

However, even in quasi-anhydrous organic solvents the regular use of enzymes has been hampered by their tendency to form ill-defined suspensions that exhibit coagulation and other agglutination problems. This property makes these preparations inherently unattractive for large-scale industrial processes. In contrast, CLECs and constitutive enzymes within the crystal lattice remain monodisperse in all the solvents mentioned.

Comparison with other immobilization methods

Several useful reviews have appeared in the literature on enzyme immobilization methods (Maugh, TH, Science 223: 474-476 (1984); Tramper, J., Trends in Biotechnology 3: 45-50 (1985)). In this literature, the enzyme always represents a small fraction of the total volume of the immobilized particle, the mass of which is an inert carrier material. The carrier increases the mean free path between the solvent exterior of the immobilized enzyme particle and the active sites of the enzyme, thereby exacerbating diffusion problems (Quiocho, FA, and Richards, FM, Biochemistry 5: 4062-4076 ( 1967)).

In a CLEC, the matrix of a cross-linked crystal supports itself, making the carrier unnecessary. As a result, the enzyme concentration in a CLEC is close to the theoretical packing limit that can be achieved in molecules of a given size, greatly exceeding achievable densities even in concentrated solutions. The entire CLEC consists of an active enzyme and thus the diffusion-related reduction in enzymatic reaction rates generally observed with enzymes immobilized by conventional means is minimized compared to enzymes in solution (see Figure 1), as free path medium for substrate and product between active enzyme and free solvent will be much shorter for CLECs (in

ES 2 199 933 T3 comparison with enzyme-bearing particles immobilized by conventional means). Notably, the constitutive enzyme in CLECs is inherently monodisperse and can be recovered by simple manipulations of the CLEC particles, such as filtration, centrifugation, or solvent decantation.

Example 3

Crystallization, cross-linking and lyophilization of elastase and evaluation of the characteristics of the resulting product

Elastase crystallization

Lyophilized porcine pancreatic elastase (Serva) was dissolved in 0.1M sodium acetate at pH 5.0 with a concentration of 5 mg / ml (weight / volume) at room temperature. Rod-shaped elastase crystals were visualized within the first minute of complete protein solvation. The crystallization solution was transferred to 4 ° C and crystallization was completed overnight. The crystals were recovered via centrifugation as described above.

Elastase crystal crosslinking

A volume of 200 µl of elastase crystals was added to 1.3 ml of 5.77% glutaraldehyde solution and 1.5 M sodium acetate at pH 5.0. The crystals were cross-linked for one hour with mild agitation (shake plate). After crosslinking the crystals were washed with three 15 ml volumes of 0.2M Tris at pH 8.0. The elastase CLEC was lyophilized as described in Example 2.

CLEC Elastase and Soluble Elastase Enzyme Activity

The catalytic activity of soluble elastase and CLEC elastase was analyzed spectrophotometrically by measuring the hydrolysis of the substrate succinyl- (Ala) 3-p-nitroanilide (Bachem) [Bieth, et al. Biochem. Med 11: 350-357 (1974)] (Table 13, Figure 5). Cleavage was monitored by increasing absorbance at 410 nm. The initial concentration of the substrate was 2 x 10^-4. The enzyme concentration was 2.8 x 10^-7M. CLEC or soluble enzyme was added to a reaction volume of 5 ml containing substrate in 0.2 M Tris at pH 8.0. As described above, the CLEC enzyme was separated from the reaction mixture before absorbance was measured.

TABLE 13

<td colspan="4">Elastase activity</td>

<td></td><td></td><td colspan="2">Absorbance 400 nm</td>

<td></td><td>Time (minutes)</td><td>CLEC</td><td>Soluble enzyme</td>

<td> 1</td><td> 0,0</td><td> 0,000</td><td> 0,000</td>

<td> 2</td><td> 0,5</td><td> 0,103</td><td> 0,205</td>

<td> 3</td><td> 1,0</td><td> 0,195</td><td> 0,390</td>

<td> 4</td><td> 2,0</td><td> 0,366</td><td> 0,672</td>

<td> 5</td><td> 3,0</td><td> 0,523</td><td> 0,923</td>

<td> 6</td><td> 4,0</td><td> 0,657</td><td> 1,098</td>

<td> 7</td><td> 5,0</td><td> 0,780</td><td> 1,227</td>

<td> 8</td><td> 6,0</td><td> 0,888</td><td> 1,326</td>

<td> 9</td><td> 7,0</td><td> 0,974</td><td> 1,393</td>

<td> 10</td><td> 10,0</td><td> 1,170</td><td> 1,512</td>

<td> 11</td><td> 15,0</td><td> 1,365</td><td> 1,586</td>

Resistance to exogenous proteolysis

Evaluation of the resistance of elastase CLEC to protease action was also carried out under conditions identical to those described for thermolysin (Example 2). The soluble enzyme and CLEC enzyme activity after incubation with protease was analyzed by hydrolysis of the nitroanilide substrate as described above (Table 14 and Figure 6).

ES 2 199 933 T3

TABLE 14

<td colspan="4">Elastase resistance to proteolysis</td>

<td></td><td></td><td colspan="2">% of maximum activity</td>

<td></td><td>Weather</td><td>CLEC</td><td>Soluble enzyme</td>

<td> 1</td><td> 0,0</td><td> 100,0</td><td> 100,0</td>

<td> 2</td><td> 10,0</td><td></td><td> 53,0</td>

<td> 3</td><td> 20,0</td><td></td><td> 32,0</td>

<td> 4</td><td> 30,0</td><td> 101,0</td><td> 18,0</td>

<td> 5</td><td> 45,0</td><td></td><td> 11,0</td>

<td> 6</td><td> 60,0</td><td> 102,0</td><td> 8,0</td>

<td> 7</td><td> 120,0</td><td> 101,0</td><td> 3,0</td>

<td> 8</td><td> 180,0</td><td> 103,0</td><td> 2,0</td>

Example 4

Crystallization, crosslinking and lyophilization of esterase and evaluation of the characteristics of the resulting product

Esterase crystallization

As described herein, 30 mg / ml suspension of porcine liver esterase ammonium sulfate (Fluka) was dissolved in 0.25 M calcium acetate at pH 5.6 at room temperature. The esterase crystals became visible within a few minutes after the addition of the calcium acetate solution. The crystallization solution was allowed to stand at room temperature and crystallization was completed overnight. The crystals were recovered by centrifugation as described in Example 2.

Crosslinking of esterase crystals

As described herein, a 300 µΐ volume of esterase crystals was added to a 5 ml solution of 12.5% ​​glutaraldehyde and 0.5 M sodium acetate at pH 5.5. The crystals were cross-linked for one hour with mild agitation (shake plate). After crosslinking, the crystals were washed with three 15 ml volumes of 0.5M calcium acetate at pH 6.3. The esterase CLEC was lyophilized as previously described in Example 2.

Enzymatic activity of soluble esterase and CLEC esterase

The catalytic activity of soluble esterase and CLEC esterase was analyzed spectrophotometrically by monitoring the hydrolysis of the p-nitrophenyl acetate substrate (Fluka) (Table 15, Figure 7). Cleavage was monitored by increasing absorbance to 400 nm. The initial concentration of the substrate was 0.001%. The enzyme concentration was 1 x 10^-8M. CLEC or soluble enzyme was added to a 5 ml reaction volume containing substrate in 0.25M calcium acetate at pH 6.3. As described above in Example 2, the CLEC enzyme was separated from the reaction mixture by centrifugation before absorbance was measured.

TABLE 15

<td colspan="4">Esterase activity</td>

<td></td><td></td><td colspan="2">Absorbance 400 nm</td>

<td></td><td>Time (minutes)</td><td>CLEC</td><td>Soluble enzyme</td>

<td> 1</td><td> 0,0</td><td> 0,000</td><td> 0,000</td>

<td> 2</td><td> 0,5</td><td> 0,770</td><td> 0,252</td>

<td> 3</td><td> 1,0</td><td> 0,128</td><td> 0,297</td>

<td> 4</td><td> 2,0</td><td> 0,208</td><td> 0,337</td>

<td> 5</td><td> 3,0</td><td> 0,260</td><td> 0,346</td>

<td> 6</td><td> 5,0</td><td> 0,324</td><td> 0,353</td>

ES 2 199 933 T3

TABLE 15 (continued)

<td colspan="4">Esterase activity</td>

<td></td><td></td><td colspan="2">Absorbance 400 nm</td>

<td></td><td>Time (minutes)</td><td>CLEC</td><td>Soluble enzyme</td>

<td> 7</td><td> 7,0</td><td> 0,353</td><td> 0,359</td>

<td> 8</td><td> 10,0</td><td> 0,369</td><td> 0,368</td>

Resistance to exogenous proteolysis

The evaluation of the resistance of the esterase CLEC to the action of the protease was also carried out under conditions identical to those described for thermolysin (Example 2). The soluble enzyme and CLEC activity after incubation with protease was analyzed by hydrolysis of the p-nitrophenyl acetate substrate as described above (Table 15 and Figure 8).

TABLE 16

<td colspan="4">Esterase resistance to proteolysis</td>

<td></td><td></td><td colspan="2">% of maximum activity</td>

<td></td><td>Time (minutes)</td><td>CLEC</td><td>Soluble enzymes</td>

<td> 1</td><td> 0,0</td><td> 100,0</td><td> 100,0</td>

<td> 2</td><td> 10,0</td><td></td><td> 68,0</td>

<td> 3</td><td> 20,0</td><td></td><td> 47,0</td>

<td> 4</td><td> 30,0</td><td> 99,0</td><td> 25,0</td>

<td> 5</td><td> 45,0</td><td></td><td> 20,0</td>

<td> 6</td><td> 60,0</td><td> 97,0</td><td> 16,0</td>

<td> 7</td><td> 120,0</td><td> 94,0</td><td> 10,0</td>

<td> 8</td><td> 180,0</td><td> 91,0</td><td> 6,0</td>

Example 5

Crystallization, cross-linking and lyophilization of lipase and evaluation of the characteristics of the resulting product

Lipase crystallization

As described herein, the lipase enzyme (Geotrichum candidum) was crystallized by vapor diffusion from a 20 mg / ml aqueous protein solution in 50 mM Tris at pH = 7 with 8% ammonium sulfate. Dipyramidal crystals became visible after 20 to 30 days of incubation at room temperature. The crystals were recovered by centrifugation as described above in Example 2.

Crosslinking of lipase crystals

As described herein, lipase crystals were added to a solution of 12.5% ​​glutaraldehyde and 50 mM Tris at pH 5.6. The crystals were cross-linked for one hour. After crosslinking, the crystals were washed with three 15 ml volumes of 50 mM Tris at pH 7.0. The lipase CLEC was lyophilized as previously described in Example 2.

Soluble lipase and lipase enzymatic activity of CLEC

The catalytic activity of soluble lipase and CLEC lipase was analyzed spectrophotometrically by monitoring the hydrolysis of the p-nitrophenyl acetate substrate (Table 17, Figure 9). Cleavage was monitored by increasing absorbance at 400 nm. The initial substrate concentration was 0.005%. The enzyme concentration was 1.5 x 10^{- 8} M. CLEC or soluble enzyme was added to a reaction volume of 5 ml containing substrate in 0.2 M Tris at pH 7.0 at room temperature. As described above in Example 2, the CLEC enzyme was separated from the reaction mixture by centrifugation before absorbance was measured.

ES 2 199 933 T3

TABLE 17

<td colspan="4">Lipase activity</td>

<td></td><td></td><td colspan="2">Absorbance 400 nm</td>

<td></td><td>Time (minutes)</td><td>CLEC</td><td>Soluble enzyme</td>

<td> 1</td><td> 0,0</td><td> 0,000</td><td> 0,000</td>

<td> 2</td><td> 1,0</td><td> 0,013</td><td> 0,021</td>

<td> 3</td><td> 5,0</td><td> 0,094</td><td> 0,116</td>

<td> 4</td><td> 10,0</td><td> 0,164</td><td> 0,186</td>

<td> 5</td><td> 15,0</td><td> 0,248</td><td> 0,258</td>

<td> 6</td><td> 30,0</td><td> 0,346</td><td> 0,357</td>

<td> 7</td><td> 45,0</td><td> 0,407</td><td> 0,420</td>

<td> 8</td><td> 60,0</td><td> 0,461</td><td> 0,459</td>

<td> 9</td><td> 90,0</td><td> 0,497</td><td> 0,502</td>

Example 6

Crystallization, crosslinking and lyophilization of lysozyme and evaluation of the characteristics of the resulting product

Crystallization of lysozyme

Following the method of Blake, TLC et al., Nature 196: 1173 (1962), 200 mg of lysozyme from lyophilized chicken egg white (Boehringer Mannheim) was dissolved in 2.5 ml of 0.04 M sodium acetate buffer at pH 4 , 7 at room temperature. After solvation of the protein, 2.5 ml of 10% sodium chloride was added dropwise and with stirring to the lysozyme solution. The crystallization solution was allowed to stand overnight at room temperature and crystallization was complete in 48 hours. The crystals were recovered by centrifugation as described above in Example 2.

Crosslinking of lysozyme crystals

As described herein, a 500 µl volume of lysozyme crystals was added to 10 ml of 24% glutaraldehyde and 50 mM Tris pH 5.6 with 20% sodium chloride. The crystals were cross-linked for 20 minutes with mild agitation (shake plate). After crosslinking, the crystals were washed with three 50 ml volumes of 20 mM calcium acetate and 50 mM potassium chloride pH 5.3. The lysozyme CLEC was lyophilized as previously described in Example 2.

CLEC lysozyme and soluble lysozyme enzymatic activity

The catalytic activity of the soluble lysozyme and lysozyme of CLEC was analyzed by measuring the hydrolysis rate of the substrate 4-methylumbelliferyl-N-acetyl-chitrioside (Fluka) (Yang, Y. and Hamaguchi, KJ Biochem. 8: 1003-1014 (1980 )) (Table 18, Figure 10). The release of 4-methylumbelliferone (Perkin Elmer Model LS-50) was followed fluorimetrically. The initial concentration of the substrate was 1.42 x 10^-3. The enzyme concentration was 3 x 10^-7M. CLEC or soluble enzyme was added to a 2 ml reaction volume containing substrate in 20 mM calcium acetate and 50 mM potassium chloride at pH 5.3 at 42 ° C. The amount of 4-methylumbelliferone was determined fluorimetrically by measuring fluorescence intensities at 450 nm with excitation at 360 nm. The width of the aperture for both excitation and emission was 10 mm. As described above in Example 2, the CLEC enzyme was separated from the reaction mixture by centrifugation before measuring fluorescence.

TABLE 18

<td colspan="4">Lysozyme activity</td>

<td></td><td></td><td colspan="2">Fluorescence</td>

<td></td><td>Time (minutes)</td><td>CLEC</td><td>Soluble enzyme</td>

<td> 1</td><td> 0,000</td><td> 0,000</td><td> 0,000</td>

<td> 2</td><td> 10,000</td><td> 4,400</td><td> 18,900</td>

<td> 3</td><td> 30,000</td><td> 10,500</td><td> 29,400</td>

ES 2 199 933 T3

TABLE 18 (continued)

<td colspan="4">Lysozyme activity</td>

<td></td><td></td><td colspan="2">Fluorescence</td>

<td></td><td>Time (minutes)</td><td>CLEC</td><td>Soluble enzyme</td>

<td> 4</td><td> 60,000</td><td> 27,500</td><td> 44,800</td>

<td> 5</td><td> 90,000</td><td> 33,800</td><td> 51,700</td>

<td> 6</td><td> 120,000</td><td> 45,900</td><td> 59,800</td>

Example 7

Crystallization, crosslinking and lyophilization of asparaginase and evaluation of the characteristics of the resulting product

Asparaginase crystallization

As a modification to the procedure described by Grabner et al. [US Patent 3,664,926 (1972)], 25 ml of lyophilized asparaginase (Worthington) was dissolved in 500 µl of 50 mM sodium phosphate buffer at pH 7.2. The solution was cooled to 4 ° C and the pH was adjusted to 5.0 with 1M acetic acid. Cold ethanol (-20 ° C) was then added dropwise to the asparaginase solution until reaching a final concentration of 33 %. The solution was incubated at 4 ° C. Crystallization was completed in forty-eight hours. The crystals were recovered by centrifugation as described above.

Crosslinking of asparaginase crystals

As described herein, asparaginase crystals were crosslinked in a 7.5% glutaraldehyde solution in 50 mM sodium phosphate buffer at pH 5.6. After crosslinking, the crystals were washed with five 15 ml volumes of 50 mM Tris at pH 7.0. Asparaginase CLECs were lyophilized as described in Example 2.

CLEC Soluble Asparaginase and Asparaginase Enzyme Activity

The catalytic activity of CLEC soluble asparaginase and asparaginase was analyzed spectrophotometrically by measuring the evolution of the ammonium ion in the coupled enzymatic reaction described below (all reagents were purchased from Boehringer Mannheim) (Table 19 and Figure 11).

L-asparagine - -asparaginase ---&gt; aspartate + NH₄ + NH₄ + + NADH + α ketoglutarate

- -glutamate dehydrogenase ---&gt; glutamic acid + NAI

The oxidation of NADH was measured by decreasing the absorbance at 340 nm. The initial concentration of NADH was 1.4 mg / ml. The asparagine concentration was 10^-3M. The alpha ketoglutarate concentration was 10^-4M. The glutamate dehydrogenase concentration was 10^-7M. The asparaginase concentration was 2.3 x 10^-8M. As described above in Example 2, the CLEC enzyme was separated from the reaction mixture by centrifugation before absorbance was measured.

TABLE 19

<td colspan="4">Asparaginase activity</td>

<td></td><td></td><td colspan="2">Absorbance 340 nm</td>

<td></td><td>Time (minutes)</td><td>CLEC</td><td>Soluble enzyme</td>

<td> 1</td><td> 0,0</td><td> 1,000</td><td> 1,000</td>

<td> 2</td><td> 1,0</td><td> 0,867</td><td> 0,825</td>

<td> 3</td><td> 3,0</td><td> 0,739</td><td> 0,684</td>

<td> 4</td><td> 5,0</td><td> 0,603</td><td> 0,538</td>

<td> 5</td><td> 10,0</td><td> 0,502</td><td> 0,406</td>

<td> 6</td><td> 15,0</td><td> 0,449</td><td> 0,338</td>

ES 2 199 933 T3

TABLE 19 (continued)

<td colspan="4">Asparaginase activity</td>

<td></td><td></td><td colspan="2">Absorbance 340 nm</td>

<td></td><td>Time (minutes)</td><td>CLEC</td><td>Soluble enzyme</td>

<td> 7</td><td> 30,0</td><td> 0,328</td><td> 0,199</td>

<td> 8</td><td> 45,0</td><td> 0,211</td><td> 0,187</td>