Use of crosslinked crystals as a novel form of enzyme immobilization

The invention relates to the process for performing enzyme catalyzed processes using cross-linked enzyme immobilization crystals for the preparation of selected products, which comprises

(a) combining at least one suitably chosen substrate with at least one enzyme acting on the substrate and in the form of cross-linked immobilized crystals; and

b) maintaining the combination produced in operation a) under appropriate conditions so that the enzyme acts on the substrate and yields the desired product.

Also included within the scope of the invention are the process of immobilizing the enzymes in the form of cross-linked, optionally lyophilized crystals as well as devices containing them.

RELATED DEPOSIT REQUESTS

This application is a continuation apart from US Patent Specification No. 2. Serial No. 07/562 280 issued August 3, 1990 β entitled Processes for the Preparation and Use of Cross-linked Crystals as a New Form of Enzyme Immobilization ”. The teachings of the above patent are incorporated herein by reference.

HISTORY OF THE INVENTION

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Enzymes are used as industrial catalysts in the preparation of economical and fine specialty chemicals on a laboratory and industrial scale (Jones, JB, Tetrahedron 42: 3351-3403 (1986)) for the preparation of food products (Zaks et. Al. , Trains in Bioteehnology 6: 272-275 (1988)), and as agents for the synthesis of organic compounds (Wong, C.-H., Science 244: 1145-1152 (1989); CHEMTRACTS-Org. Chem. 3: 91-111 (1990)).

Enzyme-based production can significantly reduce environmental pollution largely implicated in the industrial-scale production of chemical intermediates that would otherwise not be used, as industrial-scale production of acrylamide using the nitrile hydrate enzyme (Nagasawa, T. and Yamada, H., Trends in Bioteehnology 7: 153-158 (1989)).

Enzymes are also used in biosensor applications to detect various substances of clinical, industrial and other interest (Hall, E., Biosensor ”, Open University Press (1990)). In the clinical field, enzymes may be used in extracorporeal therapy, such as hemodialysis and haemofiltration, where enzymes selectively remove toxic waste and blood materials (Klein, M. and Langer, R., Trends in Biotechnology 4: 179). -185 (1986)). Enzymes are used in this area because they function efficiently as catalysts in a wide range of various reaction types at moderate temperatures, and with stereocissiveness and substrate specificity. However, there are disadvantages associated with the use of enzymatic catalysts whose use has been limited to 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 environments where enzymes normally function. Many of the most economically interesting chemical reactions performed in common practice are incompatible with the aqueous environment, where, for example, substrates and products are often insoluble or unstable, and where hydrolysis can compete significantly as a catalyst recovery. Soluble enzymes from the reaction product and unreacted substrate in the feedstock often require the use of costly and complicated separation technologies. Finally, enzymes are difficult to store because it is essential to maintain their activity and functional integrity for commercially reasonable periods of time (months to years) without having to cool to 420 to -80 ° C for temperatures of liquid Ng. ), or keep in aqueous solvents of adequate ionic strength, pH, etc.

Enzyme immobilization processes have in some cases overcome these disadvantages. Immobilization can improve the stability of enzyme catalysts and protect their functional integrity in harsh solvent environments at extreme temperatures characteristic of laboratory and industrial chemical processes (Harrmeier, W. Trends in Biotechnology 3: 149-153 (1985)). Streaming processes can be performed with enzyme particles immobilized on columns, for example, when soluble feed stock passes through the particles and is gradually converted into product.

As used herein, the term "enzyme immobilization" refers to

<img file="PT98564B_D0002.tif" />

refers to the insolubilization of the enzyme catalyst by binding to, encapsulating, or aggregating into macroscopic particles (10^-1 mm).

Numerous useful journals on enzyme immobilization processes have appeared as literature (Maugh, Τ. Η., Science 223: 474-476 (1984); Tremper, J., Trends in Bioteehnology 3: 45-50 (1985)). Maugh describes five major enzyme immobilization processes, such processes include: adsorption onto a solid support (such as ion exchange resins); covalent bonding to supports (such as ion exchange resins, porous ceramics or glass layers); retention in polymer gels; encapsulation; and precipitating soluble proteins by crosslinking them with bifunctional reagents in a random and undefined manner. In addition, all cells (normally dead and permeable) that showed the desired enzyme activity at high levels can be immobilized (eg, Nagasawa,. Β Yamada, H., Trends in Bioteehnology 7: 153-158 (1989 )).

Each of these immobilization techniques have their own advantages and limitations and none of them can be considered optimal or dominant. In most of them, the enzyme catalyst ultimately represents only a small fraction of the total volume of material present in the reactor. chemical. As such, the volume of immobilized medium is prepared from an inert but often expensive carrier. In all of them, the immobilization interactions of the enzyme catalyst molecules with each other and / or with the carrier substance tend to be occasional and undefined. As a result, although these interactions give some added stability to the catalyst molecules; enzyme, its relative non-specificity and irregularity |

<img file="PT98564B_D0003.tif" />

<img file="PT98564B_D0004.tif" />

makes this stabilization suboptimal and irregular. In most cases, access to the active zone of the enzyme catalyst remains poorly defined. In addition, the above described immobilization processes fail because they encounter problems associated with storage and refrigeration, and cannot even conventionally immobilize normally engineered enzymes as being exchanged from one into another solvent of choice without the risk for the structural and functional integrity of the enzyme. In practical terms, except for binding with carrier particles, conventionally immobilized enzymes bear close similarities to soluble enzymes, and share with them the susceptibility to denaturation and loss of function in harsh environments.

In general, immobilization processes lead to the reduction of rates observed in enzyme catalyzed reactions relative to those obtained in solution. This is mainly a consequence of the boundaries of internal diffusion of the substrate and external diffusion of the product with immobilized enzyme particles (Quiocho, EA, and Rchards, Ε. Μ., Biochemistry 5: 4062-4076 (1967)). The necessary presence of the inert carrier in the immobilized enzymatic particles increases the free midline between the outer solvent of the immobilized enzyme particles and the enzyme catalyst activating zone thereby promoting such enzymes. _Λ diffusion problems. When working with immobilized cells, immobilization problems are particularly serious, even if cell walls and membranes have somehow become permeable to substrate and product, which may be related to the multiple enzymatic activities of contamination, metabolites, toxins comprised. cells, and the stability of cells in harsh solvents and environments.

<img file="PT98564B_D0005.tif" />

operating at high temperatures. An improved immobilization technique that avoids the limitations of currently available processes will be useful in promoting the use of enzymes as industrial catalysts, especially if they are presented as useful on a large scale (Daniels, MJ, Methods in Enzymology 136: 371-379 (1987)). ).

SUMMARY OF THE INVENTION

The present invention relates to a process for the immobilization of enzymes by forming enzyme crystals and especially also crosslinking the resulting crystals using a bifunctional reagent; refers to crosslinked and immobilized enzyme crystals (referred to as ClECs or CLIECs) prepared by this process; refers to lyophilization as a means of improving the storage, handling, and handling properties of immobilized enzymes, and to a process for producing a desired product by means of a C1EC or a set of CLECs catalyzed reaction.

In the process of the present invention, small (10-1 mm) protein crystals develop in aqueous solutions or aqueous solutions comprising organic solvents, wherein the enzymatic catalyst is functionally and structurally stable. In a preferred embodiment, the crystals are then crosslinked with a bifunctional reagent, such as glutaraldehyde. This crosslinking results in the stabilization of crystal lattice contact between the specific enzyme catalyst molecules that make up the crystal. As a result of this increased stabilization, immobilized and cross-linked enzymatic crystals may function at elevated temperatures, extreme pH and rigid, organic, or almost anhydrous aqueous media, including mixtures thereof. That is, a CLEC of the present invention may function in environments incompatible with the functional integrity of the uncrystallized and non-cross-linked natural enzyme or conventionally immobilized enzyme catalysts.

Additionally, CLECs prepared from this process can be lyophilized to produce a lyophilized CLEC that can be stored in lyophilized form in a room at non-refrigerating temperatures for long periods of time, and which can easily be reconstituted in aqueous solvents. , organic or in an aqueous-organic solvent mixture of choice, without formation of amorphous suspensions, and with minimal risk of denaturation.

The present invention also relates to the ECCs produced by the present process and their use in large-scale industrial or laboratory production of selected materials, organic chiral molecules, peptides, carbohydrates, lipids, or other chemical species. At present these are typically prepared by conventional chemical processes which may require harsh conditions (e.g. aqueous, organic or near-anhydrous solvents, aqueous / organic solvent mixtures or elevated temperatures) which are incompatible with the functional integrity of natural enzyme catalysts, uncrystallized and non-crosslinked. Other macromolecules with catalytic activity may also be incorporated into the proposed CLEC technology, which may include catalytic antibodies (Lerner et al., RA, Benkovic, SJ, and Sohultz, PG, Scince 252: 659-667 (1991) θ polynucleotide | (Cech, TR, Cell 64: 667-669 (1991); Celander, DW, and Cech.

<img file="PT98564B_D0006.tif" />

.if

RT, 251: 401-407 (1991)).

The present invention also relates to a process for preparing a product chosen by a CLEC catalyzed reaction of the present invention.

In an example of the β process of the present invention, the enzyme zinc thermolysin and metalloprotease was used to synthesize a chiral precursor of the dipeptidyl artificial sweetener, aspartame. The thermolysin enzyme was crystallized from an aqueous solution of dimethyl sulfoxide starter at 45% 1.4M calcium acetate and 0.05M sodium cacodylate, pH 6.5. The resulting crystals were crosslinked with glutaraideide to form an OLEC thermolysin. Thermolysin OLEC was then transferred from the aqueous crystallization solution in which it was prepared, to an ethyl acetate solution comprising the substrates, N- (benzyloxycarbonyl) -I-aspartic acid (ZL-Asp) and L-ester. methyl phenylalanine ((ι-Phe-OMe). Thermolysin OLEO was then used to catalyze the condensation reaction of the two substrates to synthesize the methyl N- (benzyloxycarbonyl) -L-aspartyl-L-phenylananine ester (ZL-Asp-L-Phe-OMe), which is the dipeptidyl precursor. of artificial sweetener aspartame. Using one of the many known techniques (see, for example, Lindeberg, G.,

J. Chem. Ed. 64: 1062-1064 (1987)) the L-aspartic acid in the dipeptidyl precursor can be protected by removal of the benzyloxycarbonyl (Z-) group to yield aspartame (L-Asp-L-Phe-OMe).

In a second example of the process and practice of the present invention, the thermolysin enzyme was used to produce thermolysin CLECs. The activity and stability of thermolysin CLECs

were compared to those of soluble thermolysins under conditions!

<img file="PT98564B_D0007.tif" />

optimal θ extreme pH and temperature conditions, followed by incubation in the presence of organic solvents and subsequently incubation in the presence of exogenous protease.

The thermolysin enzyme was crystallized from a solution of 1.2 M calcium acetate and 30% dimethyl sulfoxide pH

8.0 The resulting crystals were crosslinked with glutaraldehyde to a concentration of 12.5% ​​to obtain a CLEC thermolysin. Thermolysin OLEO was then lyophilized by a standard technique (Cooper et TG, The Tools of Biochemistry, pages 379-380 (John Wiley and Sons, NY (1977)) to obtain a lyophilized thermolysin CLEC enzyme. This lyophilized CLEC was then transformed directly into two solvents, a mixture of an aqueous / organic solvent β an organic solvent, different and chosen without intervening with a solvent exchange technique and without forming amorphous suspensions, and with a minimal risk of denaturation. . These solvents include acetonitrile, dioxane, acetone and tetrahydrofuran, but do not exclude others. Following incubation, activity was tested by cleavage spectrophotometry of the PLAGA dipeptide substrate (furylacryloyl glycyl-L-leu cl.na amide).

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

In a fourth example of the process and practice of the present invention,

<img file="PT98564B_D0008.tif" />

as here unraveled the enzyme. This step (porcine liver) was crystallized from an aqueous solution of 15 mg / ml protein in 0.25 M calcium acetate at pH 5.6 at room temperature. The resulting crystals were crosslinked with glutaraldehyde to a concentration of 12.5% ​​to obtain an OLEO ester. This CLEC step was lyophilized as described in EXAMPLE 2.

In a fifth example of the process and practice of the present invention and as disclosed herein, the lipase enzyme (Geotrichum Candidum) was crystallized from an aqueous 20 mg / ml protein solution in 50 mM Tris at pH 7 at room temperature. The resulting crystals were crosslinked with glutaraldehyde to a concentration of 12.5% ​​to obtain an OLEO lipase. CLEC lipase was lyophilized as described in Example 2.

In a sixth example of the process and practice of the present invention, the enzyme lysazine (egg white) was crystallized from an aqueous 40 mg / ml protein solution in a 40 mM sodium acetate buffer comprising 5% sodium chloride. at pH 7.4 at room temperature (Blake, CCE et al., Nature, 196: 1173 (1962)). The resulting crystals were crosslinked with glutaraldehyde to a concentration of 20% to obtain a CLEC lys: ozone. The lysozyme OLEO was lyophilized as described in Example 2.

In a seventh example of the process and practice of the present invention the asparaginase enzyme (Escherichia coli) was crystallized from an aqueous solution of 25 mg / ml protein in sodium acetate and 33% ethanol at a pH of 5.0 to 4.^δ C. Crystallization is a modification of the technique described by Grabner et al., U.S. Pat. 3,664,926 (1972) As described herein, the resulting crystals were crosslinked with glutaraldehyde to a concentration of 7.5% to obtain a CLBC asparginase. CIEC asparginase was lyophilized as described in Example 2.

Other enzymes that can be identically immobilized and used to catalyze suitable reactions include luciferase and urease. Other enzymes, such as those mentioned in Tables 1 to 5, may also be crystallized and crosslinked according to the present process to produce a desired CIEC which may in turn be used to catalyze a reaction that results in the production of a chosen product or product. to catalyze a reaction which is an intermediate phase (i.e. one of a series of reactions) in the production of a selected product. It is recognized that although retieulation helps to stabilize most crystals, it is not always necessary or desirable in all cases. Some crystalline enzymes retain functional and structural integrity in harsh environments even in the absence of crosslinking. Although in preferred embodiments, the crystal is crosslinked, crosslinking is not always necessary to produce an enzyme crystal useful in the present process.

CIECs have several key features that confer significant advantages over the currently used conventional enzyme immobilization processes. CIECs underscore the need for a separate inert support structure. The gap of an inert support will improve the diffusion properties of the substrate and product within the CIEGs and will develop enzyme concentrations within the crystal which are concentrations close to the theoretical capacity limit for molecules of such size. High concentrations of enzymes can already lead to significant operational savings through increased use of ex-positive acronym. Giac.0 volume uo caualj-eu.uox ', a solution in the field of conuacco uu oruCRAGjOi with the enzyme and overall reaction in the Aaoric and U. cagi cao-3 rare combinations. — el s, _ .. · j ·, ... SC-, ods in xjnzpixol, lpt&gt;: p / 1 — p / ρ ^ Ipo /) Uniformity through crystal volume and acmíSiitacLH ao ^ ι ^ ζιΧ ^ β. ccrj.xLi &lt;zUx_j.ite 'cm ^ sc

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.-: 1 xt of the obtained enzymatic activity for the corresponding soluble lysozyme OLEO.

Ligature 11 is a representation g; bcLílOS OUuiitOij CLca 8.3uX5zXÍCLClG θϋ21 ^ · 3.ϋΧ ^ δ. Figure 3 illustrates the results of the results. XO * is soluble and the corresponding asparaginase d-, md.

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_L toijp roles cíj.e

Ul and function for a given enzyme or set cp Θ to L / S.u1xj. “*, Enzymes sop l J conuions that are of interest for the preparation sxntsJtxcu which are CtS2._3.ois. &lt;. . Enzymes in the excesses currently available would be very useful. lol

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- · 'ic ·. ', &lt;. -v-. The crosslinking method allows the use of dxmds in atoms, including aqueous, organic solvents. anhydrous mixtures with you

I solvents, let us enter and high temperatures, which are innocuous,

I are compatible with enzyme function using a presently available process. In addition, stabilization of the crystal jelly makes it possible to lyophilize with standard processes. Lyophilized jLLOs can be stored for commercially attractive periods of time (months to years) in the absence of refrigeration and facilitate the rapid and uncomplicated use of ILLLs in industrial laboratory scale processes by simply adding solvents of choice, without requiring the intervention of solvent exchange processes. The CLdbs are too. highly resistant &amp; diges so on the part of synogenous proteases. f. The pressure process facilitates the use of versatile enzyme catalysts in mainstream industrial chemical processes, such as new laboratory compounds for new compounds.

Although crosslinking will contribute to the stability of the enzyme crystal, it is not always necessary or desirable in all cases. Some crystallized teachings maintain functional and structural integrity in harsh environments even in the absence of crosslinking. Preferred embodiment of this process includes crosslinking an enzyme crystal and is described in detail in the following sections. It is understood, however, that crystallized enzymes which are not subsequently cross-linked may be used, or some embodiments may be used. of the present invention.

Normal interactions between the constituent molecules of the teachin in the crystalline jelly of a single crystal result in well-defined pores of limited size which carry the enzyme molecules into the body of the particle (11), the substrates larger than the one. Available pore size does not penetrate the dome particle body as a consequence of limited pore sizeL

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A number of neural reactions to the classical reactions involve various substrates such as pores of the X which would be beyond the scope of the present invention. dal .0

P6S, GAS.

Nucleotides, polysaccharides and other organic polymers, in which the number of polymeric eubunits would be such as to make the polymer larger than the number of crystalline pores in the Oils.

however, the catalysis may be looped over the surface of the present invention. immobilization process, in such cases, no longer. j? 0 b r- XXlâ. CS te- 3 O X .. ^ ci. jp te.X¹ C XC ϋ.-1-ct-bten. -t li kj G dc ^ n.3- p. Âj-b »Χ ^ ίιθ.

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p-nitro-anilide (elastase), 4-ueuylurbiferyl d-acetylchloride (lysozine) and bAd.i (asararine).

I you process d ₌ this invention, a first in the art _zIt is appropriate to adapt a protocol for the desired repair to the catalyzed reaction of the enzyme. The uncertainty strand, when crystallized from the appropriate solution, may be cross-linked with glutaraldehyde or another suitable bifunctional reagent in the crystallization solution._The. In order to prepare a water at this depth, the enzyme of choice may be lyophilized as described in Example d.

There are several advantages to the present use over the processes catalysed by presently available teachings. For example, the matrix of the crosslinked crystal in an eyelet develops its own support, whereby beds, glasses, gels or films of ominous carriers are required in order to bind the assay catalyst as necessary in the available prior immobilization processes. As a result, the concentration of the enzyme in CldO is enhanced for the theoretical storage limit that can be achieved for molecules thus yielding the densities attainable in concentrated solutions. The whole 11.10 consists of the active enzyme (and used in the inactive vehicular subarea), and thus the reduction of the enzyme proportions in the reaction related to diffusion in the field observed as follows. the immobilized conveu-higher teaches with respect to the teaches in the solution should be narrowed, as the average free concuta for the substrate and product between the Liei ensi. u- free solvent will be free reindeer:

• To heal the ddmOs (I compare the particulate matter of conventional immobilized teaching vehicles). Such high-protein conditions will be particularly useful in thiosensory, analytical, and other applications.

J4

H

<img file="PT98564B_D0011.tif" />

amounts of protein in small volumes. In industrial processes, the performance and soundness of the CLECs lead to significant operating savings by increasing the effective activity of a given catalyst volume, thereby reducing the size of the factories as well as capital investment (Daniels et al. MJ, Methods in Enzmol, 136: 371-379 (1987)). CLECs are relatively monodis persian, with a macrostopic size and shape reflecting natural crystal growth characteristics of the 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 retrieved from feed stock by either simple process, including basic economic operations such as filtration, centrifugation, solvent settling, and others.

In addition, the use of lyophilized CLECs allows for routine handling and storage 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 requiring long solvent exchange processes, or the formation of amorphous suspensions. The lyophilized CLEC form encompasses the primary utility of enzymes as catalysts and a wide spectrum of enzymes and functional conditions.

A second advantage of a CLEC is that the crosslinking of crystallized enzymes stabilizes and confers resistance to crystal lattice and the mechanical or chemical enzyme constituent molecules. As a result, a CLEC may be the only means of achieving significant concentrations of the active enzyme catalyst in rigid, organic aqueous solvents.

<img file="PT98564B_D0012.tif" />

and anhydrous, or in mixtures of aqueous-organic solvents. The use of enzymes as catalysts in organic syntheses has been hampered by their tendency to denature in the presence of non-aqueous solvents, and particularly in aqueous and non-aqueous solvent mixtures (Klibanjov,

AR, Trends in Bioch.em.ical Sciences, 14: 141-144 (1989)) ·

Ras OLEOs, the restriction of conformational modality that leads to stability is developed by inter-molecular contacts and cross-linking between the enzyme lattice molecules that form the crystal lattice, rather than the absence of water in the medium. As a result, intermediate water concentrations can be tolerated by enzymes when formulated with OLEOs, as it has not been possible so far (see Table 12). In commercial applications, aqueous-organic solvent mixtures allow manipulation of product formation taking advantage of the relative solubilities of the products and substrates. Even in aqueous media, immobilized or soluble enzyme catalysts are subjected to mechanical forces within a reactor which can lead to denaturation and a shorter average life. GLEO chemical crosslinking develops the necessary mechanical strength (Quiocbo and Ricbards, Proc. Ratl. Acad. Sci. (USA) 52: 833-839 (1964)) resulting from an increased reactor time for the enzyme catalyst.

A third advantage of a GLEO is that as a result of its crystalline nature, it can achieve uniformity throughout its cross-linked crystal volume. Crystalline enzymes as described herein develop and are crosslinked in aqueous environments and consequently the arrangement of molecules within the crystal lattice remains uniform and regular. This uniformity is maintained by inter-molecular contacts and chemical cross-linking between the crystal lattice enzyme molecules, even when

<img file="PT98564B_D0013.tif" />

Even when exchanged between other aqueous, organic or pre-anhydrous media, or other mixed aqueous / organic solvents, in all these solvents, the enzyme molecules maintain a uniform distance between them, forming well-defined stable pores within the OLEOs that facilitate substrate access to enzyme catalysts as well as product removal. Uniformity of enzymatic activity is critical in industrial, medical and analytical applications where reproducibility and consistency are indispensable.

A fourth advantage of using an OLEO is that it has an increased average operating and storage life. Interactions of lattice, even in the absence of cross-linking, are known to stabilize proteins, due in part to restrictions on the degrees of conformational freedom required for protein denaturation. In OLEOs, the interactions of lattice, when fixed by chemical crosslinking, are particularly important in preventing denaturation, especially in aqueous and non-aqueous solvent mixtures; (Klibanov, AM, Trends in BioChemical Sciences 14: 141-144 (1989)). Enzymes that have been in the crystalline state for months or years usually retain a high percentage of their catalytic activity. Crosslinked enzyme crystals immobilized in anhydrous solvents will even be further protected from damage and microbial contamination, which is a serious problem of storing large amounts of protein in a nutrient rich aqueous environment. In the case of a lyophilized OIL, the immobilized enzyme is stored in the absence of solvent. This, and the stabilization achieved by crosslinking, allows storage in the absence of refrigeration for long periods of time.

A fifth advantage of using an OLEO is that it exhibits increased temperature stability as a result of lattice stabilization of the lattice.

<img file="PT98564B_D0014.tif" />

Crystal Performing reactions at higher temperatures than those used in conventional processes will increase the reaction rates for chemical reactions of interest either thermodynamically or by increasing the diffusion rate inside and outside the crystal lattice of the CLEGs. These combined effects may represent a major improvement in reaction efficiency because they maximize the yield of a given amount of enzyme catalyst, which is usually the most expensive component of the reaction process (Da niels, MJ, Methods in Enzymol. 156: 371-579 (1987)) · The temperature stability exhibited by OLECs is remarkable because most enzyme systems require mild reaction conditions. The CLECs will also be stabilized 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 individual enzyme molecules on the bottom surface of the crystal lattice. This restricted solvent accessibility greatly increases the OLEO cofactor or ion retention characteristics compared to conventionally immobilized or solution immobilized enzymes. This property of CLECs will allow the use of economically superior continuous flow processes (See for example Cyama et al., Methods in Enzymol. 136: 503-516 (1987)) in situations where the enzyme could otherwise be inactivated by metal ion bleaching by cofactor. For example, in the conventionally immobilized enzyme ZL-Asp-L-Phe-CMe thermolysin d-&gt; dipeptidyl precursor precursor-mediated synthesis is known to lose catalytic activity in continuous flow column processes, in part by bleaching the compounds. calcium ions essential for thermolysin activity. In practice, calcium ion bleaching forced the use of less batch processes

<img file="PT98564B_D0015.tif" />

(NaKanishi et al., Biotechnology 3: 459-464 (1985)) Bleaching occurs when ionic calcium complexes are formed with the ZL-Asp substrate, in competition with the natural calcium-binding zones on the enzyme surface. resulting in loss of catalytic activity. The high density of the enzyme, and the correspondingly limited solvent-accessible volume in the CLEC interstices, discourages the formation of the L-Asp-0a complex.⁺⁺ responsible for metal ion bleaching.

GLEG Preparation - Enzyme Crystallization

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

Enzyme crystals are developed by controlled precipitation of proteins removed from the aqueous solution, or from the aqueous solution comprising organic solvents.

Conditions to be controlled include, for example, the solvent evaporation rate, the presence of appropriate co-solvents and buffers, and the appropriate pH and temperature. A comprehensive review of the various factors affecting protein crystallization has been published by McPherson (Methods Enzymol. 114: 112 (1985)). In addition, both McPherson and Gi liland (J. Crystal Growth 90: 51-59 (1988)) compiled a comprehensive list of all proteins and nucleic acids that had been recorded as crystallized as well as the conditions that led to their crystallization. A compendium of crystals and crystallization processes, as well as a coordinate erection of solvent-treated proteins and crystalline nucleic acid structures, is maintained by the Protein Data Bank (Bernstein et al., J. Mol. Biol. 112: 555-542 (1977)) at Brookhaven National Laboratory. Such references may be

Ls i

<img file="PT98564B_D0016.tif" />

used to determine the conditions required for the crystallization of a given previously crystallized protein or enzyme, with a prelude to the formation of an appropriate CLEG, and may guide the formulation of a crystallization strategy for proteins that do not possess. Alternatively an intelligent essay and error investigation strategy (See for example Oarter, GW Jr. θ Garter, CW, J. Biol. Chem. 254; 12219-12223 (1979)) can, in most cases, produce suitable crystallization conditions for most proteins, including, but not limited to, those discussed above, by increasing an acceptable level of purity. that can be achieved by these. The level of purity required may vary widely from protein to protein. In the case of lysozyme, for example, the enzyme may be directly generated from its unpurified source, egg white (Gilliland, GL, J. Crystal Growth 90: 51-59 (1988)).

For use as GLEGs in the process of this invention, larger single crystals that are required for X-ray diffraction analysis are not required, and may in fact be undesirable due to diffusion problems related to crystal size. Microcrystalline pulverized crystals (i.e. crystals whose size / cross section is in the order of 10 mm) are suitable for GLEGs and are often observed to be unusually resilient in X-ray crystallographic literature. Microcrystals are very useful in method of this invention for minimizing diffusion problems (See for example, Quiocho, A.A., and Richards, A.M., Bioohemistry 5: 4062-4076 (1987)).

Normally, the crystals are produced by combining the protein to be crystallized with an appropriate aqueous solvent or an aqueous solvent comprising suitable precipitating agents, such as salts or organic compounds. The solvent is combined with the protein at room temperature.

<img file="PT98564B_D0017.tif" />

experimentally determined to be suitable for induction of crystallization and acceptable for maintaining protein stability and activity. The solvent may optionally include co-solutes, such as bivalent cations, cofactors or quaotropes, as well as buffer species against pH. The need for co-solutes and their concentrations are determined experimentally to facilitate crystallization. In an industrial-scale process controlled precipitation leading to crystallization can be accomplished by simply combining the protein, precipitant, co-solutes, and optionally buffers in a batch process. Alternatively laboratory crystallization processes such as dialysis or vapor diffusion may also be adapted. McPhersons (Methods Enzymol. 114: 112 (1985)), θ Gilliland (J. Orystal Growth 90: 51-59 (1988)) include a comprehensive list of suitable conditions in their journals of sadalization literature. Occasionally, incompatibility between the cross-linking reagent and the crystallization medium may require crystal exchange within a more suitable solvent system.

Many of the proteins for which crystallization conditions have already been described in the literature have e.g. considerable potential as practical enzymatic catalysts in laboratory and industrial chemical processes, and are directly subjected to formulation as OLEGs through the process of the present invention. Table 1 is a sampling of enzymes that have already been crystallized. Note that the conditions recorded in most of these references have been optimized for the development of large diffraction quality crystals, often with great efforts. Some degree of adjustment of conditions for smaller crystals used in the production of CLEOs may be necessary in some cases.

<img file="PT98564B_D0018.tif" />

- 25 TABLE 1

Enzyme

Biological or microbial bridge

References (including those cited herein) Alcohol dehydrogenase horse liver

Eklund et al., J. Mol. Biol. 146: 561-587 (1981) alcohol oxidase

Pichia pastoris

Boys et al., J. Mol. Biol. 208: 211-212 (1989)

Tykarska et al., J. Pro tein Oliem. 9: 83-86 (1990) aldolase (fructose biophosphate) rabbit muscle calf muscle human muscle

Drosophila melanogaster

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 (196.9) Millar et al., Trans.

Roy soc. Lond. B293: 209-214 (1981)

Brenner et al., J. Biol. Oh em. 257: H747-H749 (1982) Aldolase (PKDG)

Pseudomonas putida

Vandlen et al., J. Biol.

Chem. 248: 2251-2253 (197?)

<img file="PT98564B_D0019.tif" />

Enzyme

Biological or microbial source

References (including those cited herein) alkaline phosphatase

Escherichia coli

Sowadski et al., J. Mol Biol. 150: 245-272 (1981) asparaginase

Erwinia carotova

Escherichia coli

Escherichia coli

Proteus vulgaris

North et al., Eature 224: 594-595 (1969).

Epp et al., Eur. J. Bio chem. 20: 452-437 (1971) Yonei et al., J. Mol. Biol. 110: 179-186 (1977)

Tetsuya et al., J. Biol. Chem. 248: 7620-7621 (1972) * carbonic anhydrase human erythrocyte (C) human erythrocyte (B) bovine erythrocyte * horse erythrocyte catalase

Micrococcus luteus

Penicillium vitale

Kannan et al., J. Mol. Biol. 12: 740-760 (1965) Kannan et al., J. Mol. Biol. 63: 601-604 (1972) Oarlsson et al., J. Mol Biol. 80: 375-375 (1975)

Glauser et al., Acta Cryst. 21: 175-177 (1960) Marie et al., J. Mol. Biol. 129; 675-676 (1979) Vainshtein et al., Acta Cryst. A37: 029 (1981)

<td></td><td>tz / Z '·</td><td>- 27 - f- '</td>

<td>TABLE 1 (Gont.)</td><td></td><td></td>

<td>acatalase</td><td>beef liver</td><td>Eventoff et al., J. Mol. Biol. 103: 799-801 (1976)</td>

<td>ereatin kinase</td><td>beef heart muscle of Bunny</td><td>Gilliland et al., J. MoL Biol. 170: 791-793 (1983 McPherson, J. Mol. Biol. 81: 83-86 (1973)</td>

<td>Mglu.tamin.ase</td><td>Actenobacter Glutanimasificans Pseudomonas 7A</td><td>Wlodawer et al., J. Mol. Biol. 99; 295-299 (1975) Wlodawer et al., J. Mol. Biol. 112: 515-519 (197?</td>

<td>Glucose Oxidase</td><td>Aspergillus Niger</td><td>Kalisz et al., J. Mol. Biol. 213: 207-209 (199-</td>

<td>m-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>Mlacate deshydro genase</td><td>pigs chicken fish dog Bacillius stearo thermophilus</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 (198g</td>

<td>Lipase</td><td>Geotrichum can didum</td><td>Hata et al., J. Biochem. 86: 1821-1827 (1979)</td>

(Cont'd)

Enzyme

Biological or microbial source

References (including those cited here) aelipase horse pancreate juice Mucor meibei human pancreatic juice

Lombardo et al., J. Mol Biol. 205: 259-261 (198) Brady et al., Nature 343: 767-770 (1990) Winkler et al., Nature 343: 771-774 (1990) aluciferase

Firefly

Green. AA, et al., Bi chem. Biophys, Ac,

20; 170 (1956)

Gluciferase

Vibrio harveyii

Swanson et al., J. Biol Chem. 260: 1287-1289 (1985) anitrile hydratase

Brevibact er ium R312

Nagasawa et al., Bioche Biophys. Res. Cornun 159: 1305-1512 (1986)

m

«Peroxidase

P. chlororaphis B25

Nagasawa et al., Eur.J. Biochem. 162: 691-698 (1987) Horseradish

Braithwaite et al., J. Mol. Biol. 106: 229-23C (1976) horseradish roots (Type E4)

Aibara et al., J. Chem. 90: 489-496

Bio (19811), ABELA_1 (Cont.)

<td>Enzyme</td><td>Biological or microbial bridge</td><td>References (including those cited here)</td>

<td>«Peroxidase</td><td>horseradish Japanese</td><td>Morita Acta Cryst.A28: S52 (1979)</td>

<td>«Peroxidase</td><td>Caldaromyces</td><td>Rubin et al., J. Biol.</td>

<td>(chloride)</td><td>fumago</td><td>Chem. 257, 7768-7769 (1982)</td>

<td>«Peroxidase</td><td>Sarchomyces</td><td>Poulos et al., J. Biol.</td>

<td>(cytochrome)</td><td>cerevisae</td><td>Ohem 253: 3730-3735 (1978)</td>

<td>«Peroxidase (glutathione)</td><td>bovine erythrocyte</td><td>Ladenstein et al., J. Mol. Biol. 104: 877-882 (1979)</td>

<td>«Subtilisin</td><td>Bacillus subtilis (New) Bacilius amyloliquefaciens (BPN ') Bacilius subtilis (Carisberg)</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 (197G</td>

<td>«Superoxide dismutase</td><td>bovine spinach Sac ch.ar omy ces cerevisiae Esche richia coli</td><td>Eichardson 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 ($ 197</td>

TABLE_1 (Cont'd)

Enzyme «superoxide dismutase ^ thermolysin« urease * xylose isomerase

<img file="PT98564B_D0020.tif" />

Biological or microbial bridge

Bacillus stearothermopbillus

Pseudomonas ovalis

Bacillus thermo proteolyticus green beans

Streptomyces rubiginosus

Arthrobacter

B3728

Streptomyces olivochromogene s

Streptomyces violaceoniger

Actinoplanes missouriensis

References (including those cited here)

Bridgen et al., J. Mol Biol. 105: 353-335 (1976)

Yamakura et al., J. Bibl Chem. 251; 4792-4795 (1976)

Matthews et al., Nature New Biol. 258; 37-41 (1972)

Sumner, JB, J. Biol. Chem. 69: 455 (1926)

Carrell et al., J. Bio Chem. 259; 3230-3236 (1984)

Akins et al., Biochym. Biophys Acta 874; 375-377 (1986)

Paber et al., Protein Engineering 1; 459-466 (1987)

Glasfeld et al., J. Bio Chem. 263; 14612-14613 (1988)

Rey et al., Proteins; Struc.Punc.Genet. 4; 165-172 (1988)

<img file="PT98564B_D0021.tif" />

GLECs Preparation - Crosslinking Reaction

Once the crystals are developed in a suitable medium, they can then be crosslinked. Crosslinking results in stabilization of the crystal lattice by introducing covalent bonds between the constituent enzyme molecules in the crystal, making it possible to transfer the enzyme into an alternate reaction medium that might otherwise be incompatible with the existence of crystal lattice, or even with the existence of the intact non-denaturing protein. Cross-linking can be achieved by a wide variety of bifunctional reagents, although in practice cheap and simple glutaraldehyde has become the reagent of choice. (For a representative listing of other available crosslinking agents, see, for example, the 1990 catalog of Pierce Chemical Company).

Glutaraldehyde crosslinking forms strong covalent bonds between mainly the amino acid residues of lysine within and between the crystal lattice molecules that make up the crystal. Cross-linking interactions prevent the enzyme molecules from crystallizing back into solution by immobilizing or insolubilizing the enzyme molecules within the microcrystalline particles (ideally 10^-1). The immobilized macroscopic, insolubilized crystals can then be readily separated from the unreacted feedstock and substrate stock by simple techniques such as filtration, decantation and the like. Such crystals may also be used on packed columns of GLECs in continuous flow processes, where they exhibit enhanced metal ion retention properties and cofactors.

3,

Through the process of the present invention, GLECs are obtained for use as enzyme catalysts in existing and new environments. The increased stability of the OLECs, which results from the crosslinking reaction, makes it possible to transfer the OLEO into a solvent (eg aqueous, organic, or anhydrous solvents, or a mixture thereof), which would otherwise be incompatible. The operation is in the chemical reactor and at high temperatures with extreme pH. The macrostopic OLEO catalyst particles can also be readily manipulated allowing recovery from the feedstock through simple processes such as filtration, centrifugation or solvent decantation. In addition, these can be used in ballad columns in continuous flow processes.

OLEO Preparation - Lyophilization

A suspension of one volume of cross-linked thermolysin crystals in 10 volumes of sterilized water at pH 7.0 was lyophilized overnight using a VirTis Model / 24 lyophilizer. The lyophilized crystals were stored at room temperature or 4 ° C. O prior to reconstitution, which was completed by the addition of ten volumes of the solvent of choice directly in crystals over crystals removed from storage. The rehydrate crystals were reconstructed in a 10 mM calcium acetate buffer at pH 7.0 for FAGLA cleavage experiments. Reconstituted lyophilized OLEOs were routinely stored at room temperature.

In contrast, soluble enzymes required storage at -70 ° C to maintain specific activity for more than one week. This protocol was used for all enzymes described in the exemplification included herein.

Synthesis of the aspartame precursor with the thermolysin OLEO The process of the present invention, by which crystallized enzymes are produced, is described below and exemplified.

<img file="PT98564B_D0022.tif" />

simplified by preparing crystals of immobilized thermolysin crosslinked enzymes for use in pathway production}? aspartame dipeptidyl in ethyl acetate, which is a substantially anhydrous, organic solvent. Thermolysin, a crystallizable thermolysin whose structure has been resolved to a resolution of 1.6 A (Holmes and Matthews, J. Mol. Biol. 160: 623-639 (1982)), is an example of an enzyme that can be be used as a CLEC in this case. Thermolysin is used in the production of aspartame artificial sweetener (Isowa et al U.S. Patent 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.

156: 503-516 (1987)). Hitherto, most aspartames appear to be produced by conventional synthetic chemical techniques, although the use of conventionally immobilized thermolysins in practically anhydrous media has yielded encouraging results (Oyama et al., J. Org. Chem. 46: 5242-5244 (1981); Nakanishi et al., Biotechnology 3: 459-464 (1985)). Improvements in enzymatic experiments for aspartame production, as possible through the use of the present process, make it competitive with the presently used process, both in terms of convenience and cost (Oyama et al., Methods in Enzymol 136:

503-516 (1987)).

Evaluation of Thermolysin CLECs The process of the present invention was also used to produce thermolysin CLECs which have been evaluated for their pH dependence and stability, stability and high temperatures, resistance to exogenous proteolysis and stability in the presence of an organic solvent. Thermolysin CLECs were compared to soluble thermolysin as described in detail in Example 2 and Figures 1 to 4.

<img file="PT98564B_D0023.tif" />

<img file="PT98564B_D0024.tif" />

tion revealed the following;

1. Regarding pH dependence and stability, both forms demonstrated maximum activity at pH 7. ^and demonstrated similar activity in the acidic range. In the alkaline pH range, GLEC maintains maximum activity at pH 10; soluble thermolysin shows 75% of activity at pH 8.5 and only 25% of activity at pH 9 θ θ completely inactive at pH 9.5 ·

2. Further stabilization achieved with GLECs results in enzymatic activity at higher temperatures than is possible with soluble thermolysin. The increased stability of thermolysin OLEO at lower temperatures makes storage easier than the soluble enzyme. Thermal stability and autolysis resistance has also been demonstrated for thermolysin CLEGs, which retain maximum activity after five days of incubation at 65 ° C. In contrast, soluble thermolysin lost 50% of its initial activity two hours after incubation and showed reduced activity after 24 hours incubation at 65 ° C.

5.

The enzymatic activity of thermolysin OLECs was not affected by the four day incubation period in the presence of the powerful Streptococcus protease, Pronase. In contrast, soluble thermolysin was rapidly degraded and lost virtually all activity after 90 minutes of incubation.

4 Thermolysin GLEGs and soluble thermolysin exhibited markedly different stability in the presence of organic solvents, as shown in Table 12. GLECs thermolysins maintained a maximum activity greater than 95% after evaluation and after undergoing an incubation period. in all organic solvents.

These characteristics of thermolysins GLECs and

<img file="PT98564B_D0025.tif" />

other OLEO enzymes make them particularly useful as they are easier to store, more stable, and less easily inactivated or degraded than their corresponding soluble enzymes.

Evaluation of OLEO Esterase The process of the present invention was also used to produce OLEO esterase which have been evaluated for their activity and resistance to exogenous proteolysis. The esterase OLEOs were compared to soluble esterase as described in detail in Example 4 and Figures 7 and 8. The results of the evaluation revealed the following:

1. The esterase OLEOs maintained approximately 50% of activity compared to the soluble enzyme.

2. Soluble esterase was highly susceptible to proteolytic degradation. Soluble esterase activity was reduced to 50% of initial activity after 10 minutes incubation in the presence of protease. One hour after the incubation period the soluble enzyme had lost more than 90% of its initial activity. In contrast, the enzymatic activity of OLEO esterase was not affected by protease incubation.

Evaluation of OLEO Lipases The process of the present invention was also used to produce OLEO lipases which have been evaluated for their activity. OLEO lipases were compared to soluble lipases as described in detail in Example 5 and Figure 9.

The results of the evaluation demonstrated that OLEO lipases retain approximately 90% of activity compared to the soluble enzyme.

Evaluation of OLEOs

The process of the present invention was also used to produce CLECs lysozymes which were evaluated for their activity and resistance to exogenous protease. CLECs lysozymes were compared to soluble lysozymes as described in detail in Example 6 and Figure 10. Evaluation results demonstrated that CLECs lysozymes retain approximately 50% of activity compared to soluble enzyme. Evaluation of CLECs Asparaginases The process of the present invention was also used to produce CLECs asparaginases which were evaluated for their activity. CLECs asparaginases were compared to soluble asparaginases as described in detail in Example 7^and Figure 11. Evaluation results demonstrated that asparaginases CLECs retain approximately 77% of activity compared to soluble enzyme.

CLECs General Applications

μ. ' μι »ι. · ιιιι ..ι i»

As disclosed herein, CLECs represent a new technology of wide use in many areas, including but not limited to industrial scale synthesis, laboratory operations, biosensors and medical applications. Examples of various systems using conventional enzyme immobilization processes in their execution are given in Tables 2 and 5 below. One skilled in the art should be able to adapt them, and adapt systems similar to the CLEC technology disclosed in this specification. To illustrate this fact specific examples are discussed in more detail from the stated categories.

Table 2 below provides examples using enzymes conventionally immobilized in an industrial process. These examples can be readily adapted to technology.

- 37 CLEG described herein.

TABLE 2

Enzyme

Production or Application

Substrates

References (including those cited) 'thermolisi na' aspartame precursor

Z-Asp, L-Phe-OMe

Oyama et al., J. Org. Ghem. 46: 5242-5244 (1981) Nakanishi et al., Trends in Biotechology 3; 459-464 (1985) 'subtilisin' aspartame

L-Asp-L-Phe,

Davino, AA, US Patent 4295648 (1981) 'lipase' cocoa butter substitutes palm oils

Harwood, J., Trenis in Biochemical Sciences 14: 125-126 (1989)

Macrae, AR, J.

Am. Oil Chem. Soc 60: 291-294 (1983) Nitrile hydratase, nitrilase, amidase Acrylonitrile acrylamide

Uagasawa, T. and

Yamada, H., Trend in Bioteehnology

7: 1532158 (1989)

<img file="PT98564B_D0026.tif" />

TABLE_2 (Cont'd)

<td>Enzyme</td><td>Production or Application</td><td>Substrates</td><td>References (including cited)</td>

<td>«Amino acyl</td><td>"resolution</td><td>amino acid</td><td>Schmidt-Kastner,</td>

<td>se (fungal)</td><td>ami in acid</td><td>N-acyl-L, L</td><td>G. &amp; Egerer, P. io</td>

<td>«Esterase</td><td>gives</td><td>esters of</td><td>Biotechnology vol</td>

<td>amino acid</td><td></td><td>amino acids</td><td>6a: 387-421 (1984)</td>

<td>«Subtilisin</td><td></td><td>D, L amides</td><td>and references th3</td>

<td>«Amidases</td><td></td><td>amino acid</td><td>rein</td>

<td>«Hydantionase</td><td></td><td>from D, L</td><td>Eusee, MO, Metho</td>

<td>'Dehydrogena-</td><td></td><td>hydantoins</td><td>ds in Enzymology</td>

<td>specific ones</td><td></td><td>a-hi acids droxycarbo-</td><td> 136: 463 (1987)</td>

<td>'Amino peptide dase «Transaminase</td><td></td><td>xylics</td><td>Pusee, MO, Metho ds in Enzymology 136: 463 (1987)</td>

<td>«Amino acid</td><td>"production:</td><td>keto acids</td><td>Rozzell, JD, Me</td>

<td>dehydrogenase</td><td>amino acid</td><td>or hydroxy</td><td>thods in Enzymolo</td>

<td>+ format-</td><td>general</td><td>fumarate /</td><td>gy 136: 479 (1987)</td>

<td>sidrogenase</td><td>"production:</td><td>/ fumaric</td><td>Enzym.es in Indonesia-</td>

<td>«L-aspartase</td><td>amino acid</td><td>acid, ammo</td><td>try, Ed Gerhartz,</td>

<td>'L-asparta-</td><td>specific</td><td>nium fumarate</td><td>W., VOH Press 199C</td>

<td>if 4-decar-</td><td>L-aspartic</td><td>D, La amino</td><td></td>

<td>boxylase</td><td>L-ala acid</td><td>e-caprolacta</td><td></td>

<td>aspartase +</td><td>nino</td><td>ac (ACL)</td><td></td>

<td>L aspartate-</td><td>L-lysine</td><td>DL-2amino</td><td></td>

<td>4 decarboxyl if «ACL hydrates • if</td><td>L-cysteine L-Isoleucine L-methionine</td><td>2thiazoline 4carboxylium</td><td></td>

<img file="PT98564B_D0027.tif" />

Enzyme

Production or Application

Substrate

References (including those mentioned) * L-ACT hydro lase slyase * L-tryptophan synthetase fumarate hydantoinase

L-Isoleucine

L-methionine

L-phenylalani in

L-Tryptophan

L-valine

L-Malic Acid

D n carbamoyl p-hydroxyl phenyl glycine cinnamate indole, L-serine fumarate

5β-hydroxyhydantoin

Lipases, Esterases * Resolution of racemates of stereoselective sinthesis sinthetic chemistry

Jones, JB, Tetrahedron 42: 3351-3403 (1988)

Butt, S. and Robert s, SM ·, Natural Product Reporjbs 489-503 (1986), and references cited therein for a more comprehensive review of this area * fumarase

Chibata et al.,

Methods in Enzymo logy 136: 455 (19 ^ 7)

<td colspan="2">TABLE 2 (Cont'd)</td><td colspan="2">1 · Zr</td>

<td>Enzyme</td><td>Production or Application</td><td>Substrate</td><td>References (including those mentioned)</td>

<td>'Lactase, β-galactosi- dases</td><td>«Disaccharide synthesis eg galactosyl- -N-aeetyl ga- lactosamine</td><td>lactose &amp; N- -acetyl ga- lactosamine</td><td>Larsson et al., Methods in Enzymology 136: 230 (198 ')</td>

<td>'Lipase, terase</td><td>«L-menthol</td><td>4 isomer mix</td><td>Eukui, S., Tanaka, A., Methods in Enzymology 1556: 293 (1987)</td>

<td>«Amidases</td><td>* D-valine (intermediate for pyre- throid insec ticide fluvinate)</td><td>B, L amino acid amide</td><td>Schmidt-Kastner, G. &amp; Egerer, P. ir Biotechnology vol 6a: 387-421 (1984) and references therein</td>

<td>Lipase (Can dida cylindricea)</td><td>* R (+) 2 phenoxypropionic acids (herhicides)</td><td>2chloro pro pionic acids</td><td>Biocatalysts in Qrganic Syntheses eds Tramper, van de Pias &amp; Linko; Proceedings of International Sympo sium in Netherlan3 1985</td>

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

ΑΊ ·

TABLE_2 (Cont'd)

<td>Enzymes</td><td>Production or Application</td>

<td>«Lipases, es</td><td>'Organic syn-</td>

<td>terases, ami</td><td>single theses</td>

<td>doses,</td><td>glycerides</td>

<td>lases</td><td>peptides</td>

<td>'Proteases,</td><td>'2 (p-chloro-</td>

<td>peptidases</td><td>phenoxy) pro</td>

<td>«Yeast lipase</td><td>pionic acid: herbicide</td>

<td>«Strictodine</td><td>«Alkaloid</td>

<td>synthetase</td><td>production eg stricto- sidine</td>

<td>«Penicillin</td><td>'6-amino</td>

<td>acylase</td><td>nicillinamic</td>

<td>«Penicillin amidase</td><td>acid and 7- -ADCA</td>

<td>'Hydroxyste-</td><td>«Steroid</td>

<td>roid dehydro</td><td>transformed</td>

<td>genase</td><td>tions</td>

Substrate resolution of racemic ester penicillin G or V

References (including cited)

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 comprehensive review of th area is

Pfitzner et al., Methods in Enzyme logy 136: 342 (1987)

Enz.Eng 6: 291 (1982) Enz.Eng.8: 155

Carrea et al., Me thods in Enzymolo gy 136: 150 (1987)

- 42 1ΙΛ / '·

L

TABLE 2 (Cont'd)

<td>Enzymes</td><td>Production or Application</td><td>Substrate</td><td>References (including cited)</td>

<td>* 5'phospho- diesterase, nucleases</td><td>* 5'ribonucleo MM tides</td><td></td><td>Keller et al., He all in Enzymolo gy 136: 517 (1987)</td>

<td>* esterase</td><td>lactam pre cursor (chiral monoesters eg / iamino glutaric acid mono- alkyl ester)</td><td>corresponding ding diesters</td><td>Japanese patent application: 82-159, 493 (1981) Biseibutsu Company</td>

<td>Lipase</td><td>κβ-blockers</td><td></td><td>Kloosterman, M et al., Trends in Biotechnology 6: 251-256 (1988)</td>

Acrylamide production using OLEO technology

The following description is a description of an embodiment of the process of the present invention: The adaptation of acrylamide production from immobilized cells that overproduce nitrile hydrase enzyme (Nagasawa, T. and Yamada, H., Trends in Biotechnology 7: 153- 158 (1989)) is the OLEO technology disclosed herein.

The industrial-scale production of acrylamide, a conveniently important chemical process, has been described by Yamada and colleagues (Nagasawa, T. and Yamada, Η. Trends in Biotechnology 7 · 153-158 (1989)). Acrylamide Kilotons by

- 43 days are produced in induced cell-loaded chemical reactors chosen as overproducers of the enzyme nitrile hydratase. Nitrile hydratase has also been reported to be purified and crystallized from two sources, Brevibacterium R312 (Kagasawa et al., Biochem. Biophys. Res. Commun. 139; 13θ5-1312 (1986) and P. chlororaphis B23 (Nagasawa et al. al., Eur. J. Biochem. 162; 691-698 (1987)). As disclosed herein the crystalline enzymes may each be immobilized by crosslinking with glutaraldehyde or with other suitable crosslinking reagents to produce a CLEC. Nitrile hydratase CLEC can then be used in a conventional reactor instead of commonly used induced cells. Adapting this process to CLEC technology leads to immediate advantages. Such advantages include; reduced plant size improved input flow as a result of increased activity per unit volume implied in the higher concentration of enzymes in the CLECs, and improved product substrate and diffusion rate; reduction of unwanted contamination and side reactions as a result of the higher purity of the CLECs; and reduced sensitivity to microbial contamination in the absence of cells. In addition, there are other benefits available only through a CLEC-based process. These benefits include; operation and higher temperatures to improve reaction rates; The ability to operate in aqueous, organic and practically anhydrous solvents, allowing for an increased average life in both processing and storage resulting from the higher mechanical and physical stability of CLECs, particularly in unconventional solvents.

Medical Applications of CLEC Technology - Extracorporeal Treatment The process of the present invention and an appropriately selected CLEC or set of CLECs may also be used for medical applications. A CLEC or set of CLECs may be used, for example, to remove a component from a fluid, such as blood, usually by altering the component, and then converting it into a substance that is not harmful to an individual. be removed by normal organic processes (eg via detoxification, liver degradation, renal excretion). In this specification, an appropriately chosen CLEC or set of CLECs are contacted with a body fluid comprising the component to be altered, or a reagent (product or substrate) of a reaction in which the component participates, on which the enzyme CLEC acts. As a result, the enzyme is capable of acting on the component to be altered or with another substance which is a reaction product in which the component to be altered participates. Enzyme activity results in either direct alteration of the component to be removed or alteration of the reaction product in which the component participates (thus making the continuation of the reaction impossible). This may be accomplished by use of an extracorporeal device including a suitably chosen CLEC or set of CLECs and a retention means which is made of a material: such as a porous material in which a CLEC is retained or a tube in which the CLEC is retained. is present which allows contact between the component Qu itself and the substance in the fluid which is a product of a reaction in which the component to be changed participates.

This can be achieved by inserting an appropriate CLEC into a suitable body compartment such as peritoneum or a lymph node where the CLEC will have access to body fluids. This insertion may be made surgically or by injection of a CLEC mixture. Direct injection of CLEC into the bloodstream will not be appropriate.

<img file="PT98564B_D0028.tif" />

given the high risk of embolism. The use of CLECs apr? In this area, it should serve as an alternative to genetic processes in enzyme replacement therapy to correct a natural deficiency such as, for example, phenylketonory.

Table 3 illustrates some medical applications in which CLECs can be used. For most of these cases, extracorporeal treatment is still under research, but the benefits that CLECs offer can promote new treatments in areas that did not previously have alternative treatments.

TABLE 3

Used enzyme

Removal of

Asparaginase

Asparagine

Treaty / Patients

Leukemia (Aspargine removal, an important nutrient in cancer, damages leukemic cells that cannot produce the essential amino acid asparagine; normal cells can produce asparagine and are therefore unaffected by this treatment)

References

Klein, M. Lan, Ger, R., Tren in Biotechnology 4: 179-185 (1986) and their references

Chang, TMS, Methods in Enzymology 13: 444-457 (1987) and their references

- 46 ^ / * * '• ABELA_J (Cont'd)

<td>Used enzyme</td><td>Removal of</td><td>Disease / Patients Treaties</td>

<td>Heparinase</td><td>Heparin</td><td>Deaparinization for hemo-perfusion of patients eg dialysis if renal</td>

<td>Bilirubin oxidase</td><td>bilirubin</td><td>New jaundice -Christmas</td>

<td>Carboxipepti dase</td><td>Methotrexate</td><td>Sub patients underwent chemotherapy</td>

<td>Tyrosinase</td><td>Aromatic Amino Acids</td><td>Liver deficiencies exhibiting pathological elevations of amino acids</td>

<td>Eenylalanine ammoniasis</td><td>Eenilalani- at</td><td>Eenylketonuria and liver deficiency</td>

References

Langer, R., et al., Science 217; 261-263 (1982)

Lavin et al., Science 230; 545-545 (1985)

Pitt, AM, et al., Appl. Bio chem. Biotechnol. 8: 55-68 (1983)

Chang, TMS, Sem. Liver Dis. Ser. 6; 148 (1986)

Ambrus, et al.,

CM,

J · Phí rm.

&amp; Exp. Ther.

224; 598-602 (1985)

<img file="PT98564B_D0029.tif" />

TABLE (COnt.)

<td>Used enzyme</td><td>Removal of</td><td>Disease / Patients Treaties</td><td>References</td>

<td>System of</td><td>Urea (with</td><td>Detoxification</td><td>Ohang, TMS,</td>

<td>many enzymes</td><td>poured into</td><td>for disability</td><td>Methods in En N »</td>

<td>including;</td><td>glu-</td><td>renal chronic</td><td>zymology 137;</td>

<td>urease glute dehydrated bush Genase, Glucose and Dehydrate nase and one transaminase</td><td>tamic)</td><td>of patients</td><td>444-457 (1987) and their references cias Ohang, TMS, Enzym Eng. 5: 225 (1980)</td>

<td>Arginase</td><td>Arginine</td><td>Hyperarginine is my family</td><td>Kanalas, JJ et al., Biochem. Med. 27; 46-55 (1982)</td>

<td>Glutamate, dehydrogenase and ammonia</td><td>ammonia</td><td>liver deficiency</td><td>Maugh, TH, Science 223: 474-476 (1984)</td>

A particular application of the process of the present invention is the heparinase system for de-heparinization (Bernstein et al., Methods in Enzymology 137; 515-529 (1987), which is discussed below.

All blood-sprinkled extracorporeal devices, such as renal dialysis, continuous arteriovenous hemofiltration, or extracorporeal membrane oxygenators, require heparinization of the patient to prevent blood clotting. However, heparinization of the patient causes bleeding complications and remains a risk to human safety. These problems increase when the time to disperse the blood increases, for example with the membrane oxygenator, and can lead to serious bleeding. Following extracorporeal therapy, heparin can be removed from the blood using a heparinase device in the effluent of the extracorporeal device that eliminates all heparin from the patient's blood flow and thus avoids heparinization problems.

Published research (Langer et al., Science 217 261-263 (1982)); Bernstein et al., Methods in Enzymology 137 515-529 (1987), detail the problems presented by conventionally immobilized enzymes used in extracorporeal devices. The main problem is that conventional immobilization causes low retention of enzymatic activity per unit volume, thus requiring a large volume of the immobilized enzyme to perform the necessary heparinization. This volume is too large to be used on humans. However, the high retention of volume activity in GLEG due to the lack of inert support is this problem and offers a practical solution for human de-heparinization. The increased stability of CLEG reduces enzyme disassociation from the crosslinked crystal, being superior to less stable conventionally immobilized enzymes because the resulting immune response to enzyme cleavage is reduced. The temperature stability of OLEO prevents enzyme denaturation due to high transient temperatures during storage and is similar when GLEG retains high activity even when stored at elevated temperatures. In addition, GLEG is cheaper and more convenient to use than its conventionally immobilized counterparts because of its longer operating and storage life.

Additional Applications of GLEG Technology: Biosensors

<img file="PT98564B_D0030.tif" />

A GLEO or set of CLECs may be used as a component of a sensor by referring to them as biosensors, detecting and / or quantifying an analytical element of interest in a fluid, such as body fluid (eg, blood urine), a medium. laboratory and chemical reaction, organic medium, water, culture medium and beverages. In some cases, the fluid in question may be a gas, such as on an alcohol analysis (Barzana, E., Klibanov A., and Karell, M.,

RASA Tech Briefs 13: 104 (1989)). Upon application an appropriately selected OLEO or set of OLEOs are contacted with a fluid for analysis with respect to the analyte of interest. The analyte of interest may be measured directly (e.g., blood glucose level) or indirectly (eg by detecting or quantifying a substance that is a reagent (prod or substrate) in a reaction in which the analyte of interest participates in ). In either case, OLEO is able to act on the analyte or the substance that is a reagent in a reaction in which the analyte also participates. Enzyme activity results in a detectable exchange (eg, pH change, light production, heat, change in electrical potential) that is detected and / or quantified by appropriate detection means (eg pH electrode, device). heat or light sensor, means for measuring electrical exchange) Janata, J., et al., Anal. Chem. 62: 33R-44R (199θ). Any useful means for detecting the change resulting from the enzyme catalyzed process can be used. A biosensod? of the present invention comprises an OLEO or set of OLEOs and an OLEO retention means which allows contact between the OLEOs and the analyte of interest or the fluid substance which is a reagent in the reaction wherein the element of interest analysis participates.

Table 4 illustrates some biosensor applications in which CLECs can be used. Currently immobilized enzymes are used in these applications but have low stability, low enzymatic density, short lifespan and lack of reproducibility. These examples may be readily adapted to the CLEC technology disclosed herein.

TABLE 4

<td>Used enzyme</td><td>Detection of:</td><td>Application</td><td>References</td>

<td>Oxidated glucose dase</td><td>Glucose</td><td>Diabetes</td><td>Daniles, B., Moss bacb, K., Methods in Enzymology 137 4-7 (1987) Hall, E. “Biosenso Open University Press (Ι99θ) Taylor, E., Proce Biotechnology Cor ference (1989); 275-287 Anthony et al., Biosensors, Fundamentals and Applications, Oxford University Press (1987)</td>

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

<td>Urease</td><td>Urea</td><td>Renal puncture</td><td>Hsuie, GH et al. Polym. Mater. Sci Eng. 57: 825-829 (1987)</td>

<td> 1</td><td colspan="2">THE ·</td><td> - 51 -</td>

<td colspan="2">TkBELk 4 (Cont'd)</td><td></td><td></td>

<td>Used enzyme</td><td>Detection of:</td><td>Application</td><td>References</td>

<td></td><td></td><td></td><td>Kobos, et al., Ana. Chem. 60: 1996-199 (1988)</td>

<td>Oxidated lactate dose and desi- drogenase</td><td>lactate</td><td>applications clinics</td><td>Blaedel, WJ &amp; Jenkins, R.A., Ana Chem. 48 (8): 1240 (1976) Sagaguchi, Y. et al., J. Appl. Bio chem. 3:32 (1981)</td>

<td>Glueose-6-pi- ruvato desi- drogenase</td><td>Glucose-6- -phosphate sa carose and ATI?</td><td>diabetes and Other Medical Applications cas</td><td>ibid as glucose Oxidase</td>

<td>Alcohol dehydrogenase, alcohol dase</td><td>Ethanol and other alcohols; acetic and formic acids</td><td>Breathalyzer and applications industrial</td><td>Romette, JL et al., Methods in Enzymology 137: 217-225 (1987) Ho, MH, Methods in Enzymology 157: 271-288 (1987)</td>

<td>β-fructoside if</td><td>Sucrose</td><td>applications industrial</td><td>Romette, JL, et al., Methods in Enzymology 137; 217-225 (1987)</td>

<td>Cholesterol oxidase</td><td>Cholesterol</td><td>Test of cholesterol</td><td>Satoh, I., Methods in Enzymology 137: 217-225 (1987)</td>

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

<img file="PT98564B_D0031.tif" />

1ABELA_4 (Oont.)

<td>Used enzyme</td><td>Detention of;</td><td>Application</td><td>References</td>

<td>Gatalase</td><td>Uric acid, cholesterol</td><td>ArterOscle- rotic and other medical applications cas</td><td>Satoh I., Methods in Enzymology 137; 217-225 (1987)</td>

<td>Carboxi pep- tidase</td><td>methotrexate</td><td>Oancro</td><td>ibid as glucose oxidase</td>

<td>Carbonic anhydrase</td><td>Dioxide of carbon</td><td>applications industrial, laboratory and environmental</td><td>ibid as glucose oxidase</td>

<td>L-amino acid oxidase</td><td>amino acids</td><td>medical and industrial</td><td>ibid as glucose oxidase</td>

<td>β-lactamase penicillin</td><td>penicillin</td><td>applications medical</td><td>Anzai et al., Bull Chem. Soc. Jpn.60; 4133-4137 (1988)</td>

<td>Posftatase alkaline</td><td>Eosphate</td><td>Goalolyte monitoring</td><td>ibid as glucose Oxidase</td>

<td>Nitrate / nitrite reductase</td><td>Nitrates and nitrite</td><td>Food and metabolite monitoring</td><td>ibid as glucose oxidase</td>

<td>Aryl sulpha- tase</td><td>Sulfate</td><td>Monitoring of tabolitos</td><td>ibid as glucose oxidase</td>

<img file="PT98564B_D0032.tif" />

TABLE_4 (Gont.)

Used enzyme

Dehydrogen Succinate

Bacterial Luciferase

Firefly I.uciferase

<td>Detention of:</td><td>Application</td><td>Efferences</td>

<td>Succinate</td><td>Industrial</td><td>ibid as glucose</td>

<td></td><td></td><td>oxidase</td>

<td>FMNH₂ and reac</td><td>Detection of</td><td>Wannlund J., et al.</td>

<td>coupling</td><td>quantities</td><td>Luminescent assays</td>

<td>of</td><td>IO '¹⁸ in</td><td>Perspectives in</td>

<td></td><td>MH₂ measures</td><td>docrinology and cli</td>

<td></td><td>before</td><td>chemical chemistry¹¹,</td>

<td></td><td>tion of</td><td>Eds Serio, M. and</td>

<td></td><td>bertação</td><td>and Pazzagli 1: 125</td>

<td></td><td>photonics</td><td> (1982)</td>

<td></td><td></td><td>Kurkijarvi et al.,</td>

<td></td><td></td><td>Methods in Enzyme-</td>

<td></td><td></td><td>logy 137: 171-181</td>

<td></td><td></td><td> (1987)</td>

<td>ATP and reac-</td><td>Detection of</td><td>Kurkijarvi et al.,</td>

<td>coupling</td><td>quantities</td><td>Methods in Enzymolo</td>

<td>of</td><td>OH ”¹^ of</td><td>gy 137: 171-181</td>

<td></td><td>ATP by</td><td> (1987)</td>

<td></td><td>set you free</td><td>Murachi et al., Me</td>

<td></td><td>photonation</td><td>thods in Enzymology</td>

<td></td><td>here</td><td> 137: 260-271 (1988)</td>

In the process of the present invention, as performed for sample analysis in a biosensor, it is particularly desirable to produce the strongest detectable signals from the smallest possible amounts of substrate and catalysts. In this sense, the CLEG technology unveiled here is particularly attractive as it is achieved

54 at the highest possible concentrations of the enzyme catalyst in a given volume.

Considerable efforts have often been made to couple an ultimate enzyme reaction of interest either directly or through suitable intermediates for the production of light through luciferase-like enzymes (Kurkijarvi et al., Methods in Enzymol. 157: 171- 181 (1988)). This is done to take advantage of the unparalleled sensitivity and efficiency of the proton detection equipment, which allows for the detection of femtomeric concentrations of enzyme reaction products under appropriate conditions. Following this principle, biosensor systems were designed using conventionally immobilized enzymes to detect various substrates of clinical or other interest. Light production reactions were coupled to assay for substrate detection reactions similar to D-glucose, L-lactate, L-glutamate and ethanol, among others, at extremely low concentrations.

With respect to this application, the lucife rase enzyme obtained from Vibrio harveyii was recorded as crystallized (Swanson et al., J. Biol. Ghem. 260: 1287-1289 (1985)). The crystals of this luciferase may be crosslinked with glutaraldehyde or another suitable reagent to obtain a luciferase GLEC. Rare biosensor or analytical uses, a luciferase OLEO offers many advantages over conventionally immobilized enzymes. In an OLEO, the total volume of OLEO luciferase consists of the light-emitting enzyme. In a conventionally immobilized enzyme system, however, 95% of the total volume is seized by the inert carrier, the function of which resembles the absorption of light emitted by the enzyme. In addition, the increased stability of CLEGs can facilitate storage at room temperature, and also make possible new sensor applications in harsh environments.

-55Ζ * elevated peratures.

Additional Applications of CLEC Technology - Laboratory Reactions

CLECs can be used as laboratory reagents or in large scale processes that can be used for laboratory reactions. Some of the broader categories of reactions are presented in Table 5.

TABLE 5

Used enzyme

Lipases, phospholipases

Catalyzed reaction type

Stereoselective synthesis; including esterification, transesterification, aminolysis, lactonization, polyolization, acylation, oxymolysis and resolution of fresh mixtures

References

Zaks, A. Banov, The Proc.Nat &amp;

M.

Aca &lt;

I know.USA. 82:

3192-5196 (1985)

KlibanoVjA.B

Acc.Chem.Ees

23: 114-120 (1990) and re ferences the-rein

Esterases

Stereoselective synthesis and resolution

Wong, CHChent ract-Organ; c Chemistry 3: 91-111 (1990) and reference therein

Kobayashi et al., Tetrahedron Letters

<img file="PT98564B_D0033.tif" />

TABLE_5 (Confc.)

<td>Used enzyme</td><td>Catalyzed reaction type</td><td>References</td>

<td></td><td></td><td>Vol 25, # 24: 2557-2560 (1984) Schneider et al., Agnew. Chem.Int.Ed. Engl.23 (# 1): 64-68 (1984)</td>

<td>Tyrosinase</td><td>Oxidation of phenols for quinone production</td><td>Kazandjian, RZ and Kli banov.AMJ Am. Chem.Soc. 110; 584-589 (1986)</td>

<td>proteases, for example subtili fate</td><td>stereoselective hydrocarbon acylation</td><td>Riva et al., J.Am.Chem.Sos 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 Γ3 ferences the- rein</td>

<td>Other enzymes which require -factors: iso- merases, liases,</td><td>Stereoselective synthesis</td><td>Wong, CH, Chetracts-Organiz Chemistry 3: 91-111 (1990)</td>

<img file="PT98564B_D0034.tif" />

<td>Used enzyme</td><td>Catalyzed Reaction Type</td><td>References</td>

<td>Aldolases, Glico sil transferases and glycosidases</td><td></td><td>and referen- dogs therein</td>

<td>Other enzymes that do not require Added cofactors: flavo enzymes, pyrido- xal phosphate enzyme but enzyme metal but</td><td>Stereoselective synthesis</td><td>Wong, CH, Obe tracts-Organi Ghemistry 3: 91-111 (1990) and reference therein</td>

<td>Enzymes that require co-factor kinases (ATP), oxireductases (RAD / P), methyl transferases (SArn), CoA-requiring enzymes, sulfurases (PAPS)</td><td>Stereoselective synthesis</td><td>W0ng, GH, Ghe tracts-Organi Ghemistry 3: 91-111 (1990) and reference therein</td>

Schneider et al., (Agnew. Ghem. Int. Ed. Engl. 23 (RS.l); 64-68 (1984)) illustrates how enzymes can be used in Organic syntheses. Pork liver esterase was used in the mesoester transformation into a chiral monoester in an aqueous phosphate buffer.

The advantages of GLEG catalyzed reactions for laboratory use are three. First, GLEG retains high

Activity in harsh environments (e.g., practically anhydrous aqueous, organic solvents and mixtures thereof, and at high temperatures) which are typical of laboratory chemistry experiments. Second, CLEC exhibits high operational and storage stability that is appropriate for intermittent laboratory experiments. Third, its useful activity per unit volume allows for shorter reaction times and requires smaller enzyme volumes (for unit of activity). Thus, the advantage that CLECs offer over immobilized or free enzymes develops organic chemistry processes with a highly selective and alternative synthetic web.

In all of these cases described below, to which we are not limited, the process of this invention may be adapted by one of skill in the art for converting a process using a conventionally immobilized enzyme catalyst for the use of an appropriate enzyme CLEC. CLECs can not only replace conventionally immobilized enzymes but can also be used in intermediate cell transformations. The present invention will be illustrated from the following examples, which are not intended to limit it in any way possible.

Example 1

Crystallization and crosslinking of thermolysin for synthesis of aspartame precursor, Z-Asp-Phe-OMe.

Crystallization

25 mg of thermolysin obtained from Bacillus termoproteolyticus were purchased from Boehringer-mannheim Gmbh and dissolved in 4 ml of 45% dimethyl sulfoxide (DMSO) and 1.40 M to 55% calcium acetate, θ cacodilate.

Sodium 0.50 Μ at pH 6.5 These initiation conditions are similar to those described by Matthews et al. For the production of thermolysin crystals with diffraction qualities (see, for example, Rolmes and Matthews, J. Mol. Biol., 160; 625-659 (1982)). The protein solution was then concentrated to one microliter in an Oentricon 10 micro-concentrator. Good microcrystalline yield was obtained by a light crystallization process described herein, wherein 1 ml of water, or 1.40 M calcium acetate, was rapidly injected into the above-described DMSO-thermolysin solutions. 0.50 M sodium cacodylate at pH 6.5 · In this process hexagonal microcrystals approximately^-¼ ^ in length).

Crosslinking of the thermolysin microcrystals protocol used in this specific example of the process of the present invention is an adaptation of that described by LTakanishi et al. (Biotechnology 3: 459-464 (1985)), in which protocol thermolysin was first adsorbed on the basis of a carrier composed of an Amberlite Xad-7 ion exchange resin, and subsequently immobilized by glutaraldehyde crosslinking (Quicho and Richards, Proc. Natl Acad. Sci. (USA) 52: 833-839 (1964)). In this example, the thermolysin microcrystals obtained above were centrifuged and pelleted, and the supernatant was discharged, 5 ml of 17.5% technical grade glutaraldehyde in 2.5% PASO, 0.05 Μ calcium acetate. θ 0.025 M sodium cacodylate at pH 6.5 were then added to the single crystals. The mixture was incubated with gentle shaking at 37 ° C for 4 hours. The cross-linking reaction was terminated by repeatedly washing the crystals with 10 ml aliquots of water to remove the glutaraldehyde solution. Washed cross-linked thermolysin crystals constitute the OLEO thermolysin used below as a catalyst.

<img file="PT98564B_D0035.tif" />

Synthesis of Z-Asp-Phe-Ome in an aqueous solution and ml of an OLEO thermolysin suspension were added to a continuous stirring batch reactor and incubated at 37 ° C. After centrifugation and decantation of the supernatant, an aqueous reaction mixture was added to the HPLC. This solution was prepared by mixing 80 mg ZL-Asp and 80 mg L-Phe-Ome-Hcl in 1 ml water with added acetic acid to give a pH of 7&gt; θ. HPLC analysis. Table 6 shows the peak HPL peak height of the ZL-Asp substrate after the indicated reaction time, normalized to 1 at time t = 0. Because ZL-Asp is limited in this reaction and measuring its emptying is equivalent to measuring the appearance of ZL-Asp-L-Phe-OMe product (Nakaniehi et al., Biotechnology 3: 459-464 (1985)) · Table 6 also comprises the height of the standardized peak of the remaining limiting substrate ZL-Asp, and an estimate of the final reaction grade. It is evident that the reaction proceeds to about 20% of completion within the first 30 seconds and is stabilized therein. These results are consistent with the observations by Nakanishi et al. (Biotechnology 3; 459-464 (1985) when conventionally immobilized thermolysin is used in an aqueous reaction mixture as described above and is attributed to the solubility of 'ZL-1'.

-Asp-L-Phe-OMe in water.

TABLE 6

<td>Reaction Time (s)</td><td>Peak Height (normalized)</td><td>% of Completion</td>

<td> 0</td><td> 1.000</td><td></td>

<td> 30</td><td> 0.727</td><td> 27,3%</td>

<img file="PT98564B_D0036.tif" />

TABLE. 6 (Cont'd)

<td>Reaction Time (s)</td><td>Peak Height (Normalized)</td><td>% of Completion</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>

<td>Synthesis of Z-Asp-Phe-</td><td colspan="2">Me in a partially anhydrous solution</td>

ml of a thermolysin CLEC suspension were added to a continuous stirring batch reactor and incubated at 37 ° C. After counterfugation and decantation of the supernatant, a practically anhydrous organic reaction mixture was added to the CLECs. This solution was prepared by mixing 80 mg ZL-Asp and 240 mg L-Phe-OMe in 1 ml 99% ethyl acetate · 1% water. Samples were removed for HPLC analysis. Table 7 shows the peak HPLC height of the ZL-Asp substrate peak after the indicated reaction time, normalized to 1 at time t = 0. Due to the fact that ZL-Asp is limited in this reaction the measurement of its emptying is equivalent to the measurement of the appearance of the product ZL-Asp-L-Phe-OMe (Nakanishi et al. Biotechnology 3; 459-464 (1985)) · Table 7 also comprises the height of the normalized peak of the remaining limiting substrate ZL-Asp, and an estimate of the degree of completion of the reaction. In this case the reaction proceeds to about 70% of completion within the first 30 seconds and stabilizes there. These results are consistent with the observations of Nakanishi et al. (Biotechnology 3: 459-464 (1985)) when using conventionally immobilized thermolysin in a substantially anhydrous reaction mixture, and are attributed to inhibition of the enzyme product.

TABLE 7

- 62 .. ,, - 4-,. Λ, / / ​​· U

Reaction Time (s)

120

180

Peak Height (normalized)

1.000

0.323

0.314

0.305

0.272% of Completion

67,7%

68,6%

69,%

72,%

Example ...... 2

Crystallization, cross-linking and lyophilisation of thermolysin and assessment of the characteristics of the resulting product eMWTfWWWWBWf -; '· * Τί * ί, ι' ·. - · “λ- · * · '' ·» '- - * »·· - · - * · j4» ”· - · m.- ·» »· - - · τ ^ β · ^ r · ​​ew ι^- τι .ίμημ * »

Thermolysin crystallization

.. IIIII III |_MIH »1W — WÍWMI — IULHHII — Μΐ ·· ιrnini &lt;í * lll

Thermolysin (Diawa Kasei KK, Japan) was dissolved in 10 mM calcium acetate (Sigma), pH 10.0 to a concentration of 1% (w / v), pH of the solution was maintained at 10.0 by titration with After 2 M NaQH. After complete solubilization, the protein solution was titrated to pH 8.0 with 2 M HCl. Solid calcium acetate was added to 1.2 M. Then 30% dimethyl sulfoxide (Sigma) was added. . The protein was concentrated to 100 mg / ml by ultrafiltration in an Amicon shake cell (10,000 IJVCO membrane). The concentrated enzyme was aliquoted and stored at -70 ° C. Thermolysin was crystallized by the addition of 9 volumes of sterile water to one volume of concentrated protein solution (100 mg / ml). The solution was briefly centrifuged and allowed to stand overnight at room temperature. The crystals were washed with 10 volumes of 10 ml calcium acetate at pH 7, θ ^and It was recovered by low speed centrifugation (10 minutes at 1500 χ G, Beckmar centrifuge GPFi).

The rapid addition of water to a concentrated (100 mg / ml) solution of thermolysin induces the formation of crystals that become visible under low power magnification within 10 minutes. Crystal size is reproducible as a function of final protein concentration. 3 volumes of water to one volume of thermolysin concentrate (100 mg / ml) will produce 0.5 mm long Z-ray diffraction capacity hexagonal rods corresponding to the crystals previously described by Oolman et al., (Oolman, P .ffi., Jansonius,

JN and Matthews, BW, J. Mol. Biol. 70: 701-724 (1972)), as confirmed by us by diffraction analysis. Adding 10 volumes of water to one of the concentrates; Protein yields the resulting crystal length to 0.05 mM. These microcrystals are preferred in OLEO applications because they tend to minimize diffusion problems related to crystal size (see, for example, Quiocho, ρ. A. and Richards, Ρ. M. Biochemistry 5: 4062-4076 (1967)) Within a given protein batch the size of the crLs was consistently uniform. (Crystals of 0.05 - 0.10 mM in length were used in this study to facilitate accurate pipetting of crystal suspensions). Densiometric views of the SDS-PAGE revealed a sixfold higher purification of the crystallizing enzyme significantly increasing the specific activity of the CLECs. Crystallization resulted in a 20% decrease in total CLEC protein activity compared to soluble thermolysin when tested by spectrophotometric cleavage of the furylacrylyl glycyl-L-leucine amide dipeptide substrate (PAGLA) as described below.

Retieulation of thermolysin crystals

The thermolysin crystals were crosslinked for hours at room temperature in a glutaraldehyde solution.

<img file="PT98564B_D0037.tif" />

12.5% ​​(Sigma), DMSO% and Tris 50 ml pH 6.5 · Cross-linked crystals were washed three times in sterile water and recovered by low speed centrifugation as described for thermolysin crystallization. Chemical crosslinking of the enzymatic crystals stabilizes the crystalline jelly and the constituent enzyme molecules in the crystal sufficiently to permit practical use of the CLECs in environments that would otherwise be incompatible with enzymatic function. There was no measurable difference in enzymatic activity between cross-linked and non-cross-linked crystals when tested (by spectrophotometry) by monitored cleavage of their FAGLA dipeptide substrate (described below). In addition, crosslinking stabilizes the CLECs to the point that they can be lyophilized, with retention of enzyme activity in its entirety upon reconstitution in aqueous, organic solvents, and aqueous-organic mixtures as shown in Figure 1 and Table 8. Although crystallization results in a 30% decrease in CLEC protein specific activity compared to soluble thermolysin, crosslinking and lyophilization of CLECs did not subsequently decrease specific activity.

TABLE 8 - Thermolysin Activity

<td rowspan="2"></td><td rowspan="2">Time (min)</td><td colspan="2">Absorbance 345 nm</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>

<td colspan="2">Enzymatic activity</td><td>of thermolysin</td><td>CLEC and soluble</td>

<td></td><td>The activity</td><td>catalytic</td><td>thermolysin CLEC</td>

<img file="PT98564B_D0038.tif" />

was tested (Feder, J. and Schuck, J. h., Biochemistry 9: 2784-2791 (197θ)) by hydrolysis of the furylacrylyl-glycyl-L-leucine-amide (FAGLA) blocked dipeptide substrate (Schw | ei zerhall) ). Amide bond cleavage was measured spectrophotometrically by a decrease in absorbance at 345 nm.

The initial concentration of the enzyme was 10 ¹ M by Bradford protein determination and densiometric visualization (Pharmacia LKB UltroScan XL) of Coomassie stain SDS-PAGE gel.

The enzyme CLEC is defined as reconstituted lyophilized cross-linked thermolysin crystals and the soluble enzyme is defined as concentrated thermolysin to 100 mg / ml. The enzyme was added to a reaction volume of 5^mI understood the subtract. Aliquots of the reaction mixture were removed at the indicated times, and absorbance at 345 nm was measured. Thermolysin CLEC was separated from the reaction mixture by brief centrifugation (microcentrifuge E, Beckman) prior to absorbance reading. The absorbance was established for a pseudo first order rate equation and the Kcat / Km was calculated by dividing the value established by the enzymatic concentration (Hultifit 2.0 Curve Eitting for the Macintosh Computer App, Day Computing P. 0. Box 327, Milton, Cam Bridge CB4 6YL, UK (1990)) · n

le

Optimal pH dependence and stability and soluble enzyme stability were compared to those of CLEC thermolysin by cleavage of the FAGLA dipeptide substrate. Results are shown in Figure 2 and Table 9. Both the soluble enzyme and the crystalline enzyme showed maximum activity at ph 7. Soluble CL1 and thermolysins also demonstrated similar activity in the acidic range and the bell-shaped pH profile generated by the soluble enzyme was in agreement with published data (Feder, J. and Schuck, J. Μ., Biochemistry 9:

<img file="PT98564B_D0039.tif" />

2784-2791 (1970)). In the alkaline pH range, however, the crystalline enzyme maintains the maximum activity at pH 10, whereas the soluble enzyme showed 75% activity after

8.5, θ only 25% activity at pH 9 · At pH 9.5, the soluble enzyme is completely inactive.

TABLE 9 ~ pH urchin Thermolysin% Maximum activity

<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> 8,5</td><td> 85,000</td><td> 74,610</td>

<td> 5</td><td>7, the</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> 1</td><td> 10,0</td><td> 90,000</td><td> 0,000</td>

<td colspan="3">High temperature stability</td><td></td>

<td></td><td>Can reach</td><td>reaction rates</td><td>highest</td>

lower diffusion times for substrates and products performing the operation of a given chemical process and higher temperature, where we are usually limited by the temperature stability of substrates and products. In enzyme-based catalysis, however, the loss of enzymatic activity often sets the practical temperature limit at which the process can take place. Additional stabilization achieved in OLECs allows enzymatic activity at higher temperatures than possible with β

<img file="PT98564B_D0040.tif" />

soluble enzyme.

Increased stability at lower temperatures simplifies long-term routine storage of CLECs. For example, concentrated solutions (50 mg / ml) of soluble thermolysin at -80 ° C had to be stored to maintain maximum specific activity. At room temperature, activity was usually lost at the end of the first day. In contrast, rehydrated CLECs thermolysins can be routinely stored for months at room temperature with no apparent loss of activity. Unconstituted lyophilized thermolysin CLECs appear to be viable indefinitely.

Stability and thermal resistance to autolysis were demonstrated with CLECs thermolysins after incubation at 65 ° C for five consecutive days (Pigura 3 θ Table 10). Thermolysins CLECs maintained their maximum activity after five days of incubation at elevated temperatures. Conversely, soluble thermolysin lost 50% of its initial activity after two hours of incubation and showed poor activity after 24 hours of incubation at 65 ° C.

TABLE 10 - Thermal Stability of Thermolysin at 65 ° C

<td></td><td>Time</td><td>CLEC</td><td>Soluble Enzyme,</td>

<td></td><td>(days)</td><td></td><td></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>

<img file="PT98564B_D0041.tif" />

TABLE 10 (Cont'd) - Thermolysin Thermal Stability at 65 ° C

<td> 9</td><td>Time (days) 5,000</td><td>CLEG Soluble Enzyme 94.0</td>

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

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

<td>measures</td><td>The activity of post incubation at</td><td>GLEG and soluble thermolysins were 65 ° C. Soluble thermolysin was</td>

incubated in 10 mM calcium acetate, 50 mM Tris pH 7.0 in a 65 ° C water bath. The reaction volume was 5θ0 nl. The final protein concentration was 10 mg / ml. Aliquots were removed at time 0, 1, 2, 4, 6, 10 and 18 hours. Samples were assayed by SL3-PAGE and PAGLA cleavage at room temperature as described above. For CLECs thermolysins, 250 µl of a crystal suspension in 10 mM calcium acetate and 50 mM Tris were also incubated in a 65 ° C water bath. Activity was assayed at times 0, 1, 6, 24, 48, 72, 96. and 120 hours by PAGLA cleavage.

Resistance to exogenous proteolysis

Evaluation of the resistance of CLEG thermolysin to the action of an exogenous protease was also performed. SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis gel) analyzes suggested that commercial enzymes may comprise a substantial percentage of contaminants, some of which may have proteolytic activity against major soluble enzyme species. By storing the enzyme molecules storage in the form of a crystal lattice it can be assumed that the enzyme molecules within a GLEG will be protected from proteolysis. To test this possibility the thermolysins GLnGs and a

<img file="PT98564B_D0042.tif" />

Preparation of a soluble enzyme were incubated in the presence of a streptococcus protease, Pronase (E) a specific protease capable of digesting most free amino acid proteins (Galbiochem. 1990 Gatalog; LaJolla, GA) ·

GLEG and soluble thermolysins were incubated in Tris 50 mu, pH 7.5? at 40 ° C in the presence of protease pronase^-1· '(Galbiochem). The ratio of Pronase ^) to thermolysin was 1/40. To inhibit thermolysin autolysis and prevent proteolytic destruction of pronase by thermolysin, EDTA was added to the soluble enzyme reaction to a final concentration of 100 mM (EDTA inhibits the activity of thermolysin but not Pronase®). At the indicated times aliquots were removed from the reaction mixture and activity was assayed by cleavage spectrophotometry of FAGLA dipeptide substrates. For inhibition of established thermolysin due to the presence of EDTA, the spectrophotometric assay of soluble enzyme activity was performed on a 0.5 M calcium acetate tablet at pH 7.0 and the enzyme concentration was doubled. The crosslinked crystalline enzyme was tested as described above.

As can be seen from Figure 4 and Table 11, soluble molycin teres were rapidly degraded and lost all activity after 9 minutes of incubation. Alternatively, thermolysin GLEC activity was unaffected by four days of incubation in the presence of protease. This inaccessibility to proteolysis is of particular interest in diagnostic bioserver applications where a suitable OLEO may be called upon to act in the presence of an unknown cocktail of naturally occurring proteolytic enzymes.

TABLE 11

Protease Resistance

- 70 TABLE 11

Protease Resistance

<td rowspan="2"></td><td rowspan="2">Time (days)</td><td colspan="2">% Maximum activity</td><td rowspan="2">Time (min)</td>

<td>GLEG</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> “7 »*»</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 an organic solvent

In order for enzymes to gain optimal acceptance as viable industrial catalysts, they must be able to function without excessive intervention in the practical environment of the preparation process. Particularly, this comprises the use of aqueous, polar and non-polar organic solvents and mixtures thereof. In commercial applications, aqueous-organic solvent mixtures allow manipulation of product formation taking advantage of the relative solubilities of the products and substrates.

Soluble thermolysins and GLEO thermolysins exhibited markedly different stability in the presence of organic solvents (Table 12). Soluble enzyme concentrations that could be incubated in organic solvents were limited to a maximum of 10 mg / ml. Concentration values ​​higher than this resulted in instantaneous precipitation.

<img file="PT98564B_D0043.tif" />

of thermolysin under addition of organic solvent. Conversely, the concentrations of CLEGs thermolysins were limited only by the volume occupied by the crystals. Soluble thermolysin retained its highest activity (75%) after incubation in acetone, and its lowest activity (36%) in tetrahydrofuran. After one hour incubation in the presence of acetonitrile or di? xane the soluble enzyme lost approximately 50% in its initial activity. Thermolysin GLEO more than 95% of its maximum activity after incubation with any of the organic solvents tested.

TABLE 12% Maximum Activity

<td></td><td>Enzyme Soluble</td><td>GLEG</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

GLEGs or soluble thermolysins preparations were incubated in 50% (v / v) indicated organic solvent solutions. 100 µl of a paste of GLEGs thermolysins (10 mg / ml) in 10 mM Tris pH 7 placed in a 1/2 dram glass ampoule. An equal volume of the indicated organic solvent was added and the mixture strongly stirred for a short time. 20 of soluble thermolysin (100 mg / ml) was diluted in 80 ul of a 0.01? h pH 7. θ in a 1/2 dram glass ampoule. A 100 µl volume of the organic solvent was then added to the protein solution and was then stirred vigorously for a short time. The CLEC enzyme and the soluble enzyme were then incubated in the presence of the organic solvent for one hour at 40 ° C. Following incubation, enzymatic activity was assayed by cleavage of the described FAGLA dipeptide substrate.

Low concentrations of water are thought to disadvantage the non-bending of intermediate states in conducting enzymatic denaturation. In CLECs, this restriction of conformational mobility is developed by intermolecular contacts and cross-linking between the constituent molecules of the crystal lattice enzymes rather than the near-absence of water in the medium. As a result, concentrations of the intermediate water-organic solvent are readily tolerated by enzymes when formulated as CLECs, something previously not observed with enzymes (See Table 12). This discovery opens a set of new areas of organic chemistry for exploration using enzymatic catalysis.

Even in virtually anhydrous organic solvents, however, routine use of enzymes has been hampered by their tendency to form ill-defined suspensions that are subject to aggregation and accumulation problems. This property makes these preparations inherently unattractive for industrial scale processes. In contrast, CLECs and crystal lattice enzymes remain monodispersed in all such solvents.

Comparison with other immobilization processes i'WMUWumi II Wll ... -. 4. ... ..u. - —llll HM- W Mi wny · * -

Numerous useful publications on enzyme immobilization processes have appeared in the literature (Maugh, E., Science, 223: 474-476 (1984); Tramper, J., Trends in Biotechnoiogy 3: 45-5θ (1985)). Naps reviewed, the enzyme represents

<img file="PT98564B_D0044.tif" />

There is always a small fraction of the total volume of the immobilized particle the mass of which is an inert carrier. The carrier enhances the free medium conduit between the outer solvent of the immobilized enzyme particle and the active enzymes of the enzyme by activating diffusion problems (Chi cho, FA, and Pichards, F., Biochemistry 5: 4062-4076 (1957)).

In an OLEO, the crosslinked crystal matrix develops its own support, eliminating the need for a vehicular substance. As a result, the enzyme concentration in an OLEO is closed to the theoretical packaging limit that can be reached by molecules of a given size, greatly exceeding the densities achievable even in concentrated solutions. Whole OLEO consists of the active enzyme, and hence the reduction related to the diffusion of the enzymatic reaction proportions normally observed with conventionally immobilized enzymes relative to the enzymes in. The solution is minimized (See Figure 1), as the free medium conduit to the substrate and product between the enzyme and the free solvent will be greatly reduced by the CLECs (compared to conventionally immobilized enzyme carrier particles). Importantly, the constituent enzyme of the CLECs is intrinsically monodisperse, and can be recovered by simple manipulations of the CLEC particles, such as filtration, centrifugation or decantation of the solvent.

EXAMPLE 3

Crystallization, cross-linking and lyophilization of the elastase and evaluation of the characteristics of the resulting product ι .1, 1 ιιΐ | '1ί! · Ί 1¹ 1¹1.1 .im, i IIP — wwwnwi — yWWIWIJWWRIWH

Elastase Crystallization

Freeze-dried pancreatic pig elastase (Serva)

74 µg was dissolved in 0.1 M sodium acetate pH 5.0 to a concentration of 5 mg / ml (w / v) at room temperature. Elastase crystals in the form of sticks became visible within 10 minutes of complete protein dissolution. The crystallization solution was transferred to 4 ° C and crystallization was completed overnight. The crystals were recovered by centrifugation as previously described.

Elastase crystallization

A 200 µl volume of elastase crystals was added to 1.3 ml of a 5.77% glutaraldehyde solution θ 1.5 M sodium acetate pH 5.0. The crystals were crosslinked for one hour with gentle stirring (stir plate). After crosslinking the crystals were washed three times with 15 ml volume of 0.2 M Tris pH 8.0. CLEC elastase was lyophilized as described in Example 2.

Enzymatic activity of soluble CLEC elastase

The catalytic activity of soluble CLEC elastase was assayed by spectrophotometry by measuring the hydrolysis of the succinyl- (Ala) -p-nitroanilide substrate (Bachem) (Bieth, et al., Biochem. Med. 11: 350-357 (1974)) (Table 13 Figure 5). Cleavage was monitored by increasing absorbance at 410 nm. Initial substrate concentration was 2 x 10 ". Enzyme concentration was 2.8 x 10"%. The CLEC or soluble enzyme was added to a reaction volume of 5 ml comprising the substrate in 0.2 Tris pH 8.0. As described above the OLEC enzyme was removed from the reaction mixture prior to measurement of absorption.

TABLE 13 ~ Elastase Activity

Absorbance at 400 nm

Soluble Enzyme

Time (min)

CLEC

- 75 -4 /7^·'

-Γ -Λ

<td>TABLE</td><td>15 - Activity of</td><td>Elastase</td><td></td>

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

<td></td><td>Time (min)</td><td>OIL</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> 7</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>

<td colspan="3">Resistance to exogenous protolysis</td><td></td>

<td></td><td colspan="2">The resistance assessment</td><td>of elastase OLEO</td>

<td colspan="2">protease was performed</td><td colspan="2">under the same conditions as 1</td>

<td colspan="3">for thermolysin (Example 2). THE</td><td>activity of </td>

<td>OLEO and</td><td colspan="2">soluble after incubation with</td><td>the protease was,</td>

<td>by</td><td colspan="3">hydrolysis of nitroanilide substrate as above</td>

<td colspan="2">(Table 14 and Figure 6).</td><td></td><td></td>

<td>TABLE</td><td>14 - Resistance of</td><td>Elastase to</td><td>Protolysis</td>

<td></td><td></td><td colspan="2">% Maximum activity</td>

<td></td><td>Time</td><td>OIL</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>

<img file="PT98564B_D0045.tif" />

TABLE. 14 (Cont.) - Protastant Slastase Resistance% Maximum Activity

<td></td><td>Time</td><td>OIL</td><td>Soluble Enzyme</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> 5,0</td>

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

EXAMPLE 4

Crystallization, freeze-drying esterase cross-linking and evaluation of the resulting product characteristics

Esterase stabilization

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

Cross-linking of False Esterase Crystals As disclosed herein, a 300 µl volume of esterase crystals was added to 5 ml of a 12.5% ​​glutaraldehyde solution © sodium acetate 0.5 to pH 5.5 · The crystals were cross-linked for one hour with shaking your ve (stir plate). After crosslinking 03 crystals were washed three times with 15 ml volumes of 0.5 E calcium acetate, pH 6.3. OLEO esterase was lyophilized as described above in Example 2.

Enzymatic activity of soluble CLEC esterases

The catalytic activity of soluble CLEC esterases were assayed spectrophotometrically by monitoring the hydrolysis of the p-nitrophenyl acetate substrate (Fluka) (Table 15 θ Figure 7). Cleavage was monitored by increased absorption at 400 nm. The initial substrate concentration was 0.001; The enzyme concentration was 1 χ 10 ~ ^ Μ. The CLEC enzyme or soluble enzyme was added to a reaction volume of 5 ml comprising the 0.25% calcium acetate substrate 6.3. As previously described in Example 2, CLEC enzyme was removed from the reaction mixture by centrifugation prior to measurement of absorption.

• TABLE 15 - Esterase Activity

Absorbance at 400 nm

<td></td><td>Time (min)</td><td>CLmC</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>

<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>

<td>.stencia</td><td>to proteolysis</td><td>exogenous</td><td></td>

The evaluation of CLEC esterase resistance to protease action was also performed under conditions identical to those described for thermolysin (Example 2). The activity of the CLEC enzyme and soluble after protease incubation was assayed by hydrolysis of the p-nitrophenyl acetate substrate as described above (Table 16 and Figure 8).

TABLE 16 .V

L.

<img file="PT98564B_D0046.tif" />

- Esterase hesitancy to protolysis% Maximum activity

<td colspan="2">Time (min)</td><td></td><td>OIL</td><td colspan="2">Soluble Enzyme</td>

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

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

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

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

<td>n P</td><td> 45,0</td><td></td><td></td><td> 20,0</td><td></td>

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

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

<td> 8</td><td> 180,0</td><td></td><td> 91,0</td><td>θ, Ο</td><td></td>

<td>MKiPLO _5</td><td></td><td></td><td></td><td></td><td></td>

<td>Gristalization,</td><td colspan="2">crosslinking</td><td colspan="2">and lipase lyophilization and</td><td>evaluated</td>

<td colspan="2">tion of characteristics</td><td>of</td><td colspan="2">resulting product</td><td></td>

<td></td><td>¹¹¹ · ^ml......</td><td></td><td></td><td></td><td></td>

<td>Gristalization</td><td>of lipase</td><td></td><td></td><td></td><td></td>

<td>Such</td><td>like here</td><td>is</td><td>unveiled,</td><td>the lipase enzyme</td><td>(Geo-</td>

trichum candidum) was crystallized by vapor diffusion from a 20 mg / ml aqueous solution of the protein in Tris 50 ni ph 7 comprising 8% ammonium sulfate. Bipyramidal crystals became visible 20 to 30 days after incubation at room temperature. The crystals were removed by centrifugation as previously described in Example 2.

Ejaculation of lipase crystals

As described herein, lipase crystals were added to a 12.3% glutaraldehyde solution. ^andTris 50 mli ph 5.6. The crystals were crosslinked for one hour. After crosslinking the crystals were washed three times with 15 ml volumes of Tris 5θ ηύχ ph 7.0 · GLEC lipase was lyophilized as previously described in Example

2.

<img file="PT98564B_D0047.tif" />

Soluble lipase and CLEC lipase enzymatic activity * ww * a ** kfciV * miWTr Mimr - .. · ί. ·) ^ .— t-ii.-th.ttith—. * -------— rr . '- ·. ... _.

The catalytic activities of soluble lipases and

CLEC were assayed by spectrophotometry by monitoring the hydrolysis of the p-nitrophenyl acetate substrate (Table 17 and Figure 9). Cleavage was monitored by increasing absorption at 400 nm. The initial substrate concentration was —8 0.005%. The enzyme concentration was 1.5 x 10 Μ. The CLEC enzyme or soluble enzyme was added to a reaction volume comprising the substrate in 0.2 Tris at pH 7.0 at room temperature. As previously described in Example 2, the CLEC enzyme was removed from the reaction mixture by centrifugation prior to measurement of absorption.

<td colspan="4">TABLE 17 - Lipase Activity</td>

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

<td></td><td>Time (min)</td><td>CLEC</td><td>soluble mnzima</td>

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

<td></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>

<td>mxEKLPLC</td><td>Read</td><td></td><td></td>

<td colspan="2">QPist / âliSHÇHO, STRUCTURE</td><td colspan="2">and lysozyme freeze drying</td>

<td>liation</td><td>of the characteristics</td><td>of product</td><td>re sultant</td>

<td colspan="2">Crystallization of lysozyme</td><td></td><td></td>

<td colspan="4">Following the Blake process, 0. 0. F. et</td>

- 80 f

V, &lt;&amp;

<img file="PT98564B_D0048.tif" />

Ilature 196: 1173 (1962) 200 mg of egg white lysozyme (Boehringer Manhheim) was dissolved in 2.5 ml of a 0.04 M sodium acetate buffer pH 4.7 at room temperature. After dissolution of the protein, 2.5 ml of 10% sodium chloride were carefully added dropwise to the stirred lysozyme solution, the crystallization solution was allowed to stand overnight at room temperature, and the crystallization was completed. in * 48 hours. The crystals were recovered by centrifugation as described above in Example 2.

Crosslinking of lipase crystals

As described herein, a volume of 500 µl of lysozyme crystals was added to 10 ml of 24% gLuteraldehyde and 50 mM Tris pH 5.6 comprising 20% ​​sodium chloride. The crystals were crosslinked for 20 minutes with gentle stirring (stir plate). After crosslinking The crystals were washed three times with 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.

Enzymatic activity of soluble lysozyme GLEG

The catalytic activities of soluble and GLEG lysozymes were assayed by measuring the proportion of hydrolysis of the 4-methylumbelliferyl-N-acetyl chitrioside substrate (Pluka) (Yang, Ϊ. And Hamaguchi, K., J. Biochem. 8: 1003-1014 ( 1980) (Table 18 and Figure 10) The release of 4-methylumbeliferons was fluorimetrically followed (Perkin Elmer Model LS-50) The initial substrate concentration was 1.42 x 10 Λ The enzyme concentration was 3 x 10 ? GLEG enzyme or soluble eryme were added to a reaction volume of 2 ml which comprised the substrate in 20 mM calcium acetate and

<img file="PT98564B_D0049.tif" />

50 mM potassium chloride, mp 5.3 at 42 ° C. The amount of 4-methylumeliferone was determined fluorimetrically by measuring fluorescence intensity at 450 nm with excitation at 360 nm. The narrow thickness for both excitation and emission was 10 nm. As previously described in Example 5, the OLEO enzyme was removed from the reaction mixture by centrifugation prior to fluorescence measurement.

TABLE 18 - Lisozima Activity

Fluorescence

<td></td><td>Time (min)</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>

<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> 51,500</td>

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

EMPLOYMENT 7

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

Crystallization of asparagines and

As a modification of the technique described by Grabner et al. (U.S. Patent No. 3,664,926 (1972) 25 mg of lyophilized asparaginase (Worthington) was dissolved in 500 µl of a 50 mM sodium phosphate buffer, pH 7.2. The solution was cooled to 4 ° C and the pH adjusted to 5.0 with 1 M acetic acid. Cold (-20 ° C) ethanol was then carefully added dropwise to the asparaginase solution to a final concentration of 33%. The solution was incubated at 4 ° C. Crystallization was completed within 48 hours.

<img file="PT98564B_D0050.tif" />

The crystals were recovered by centrifugation as described above.

Cross-linking of asparaginase crystals

As disclosed herein, the asparin gaseous crystals were cross-linked in a 7.5 glutaraldehyde collision in a 50 mM sodium phosphate buffer pH 5.6. After crosslinking the crystals were washed 5 times with 15 ml volumes of Tris 5θ mA / I, pH 7-0. The asparaginase OLEO was lyophilized as previously described in Example 2.

Soluble asparaginase and asparaginase enzymatic activity GLEG

The catalytic activities of soluble asparaginases and GLEG were assayed spectrophotometrically by measuring the evolution of ammonium ion in the coupled enzyme reaction described below (all reagents were provided by Boebringer Mannheim) Table 19 and Eigura 11).

asparaginase

L-asparagine --------------&gt; asparate + dehydrogenase glutamate

NH4 + + ΞΑΌΗ + gc ketoglutarate ---------------— --------— -&gt; glutamic acid + NAD +

The oxidation of 3SADH was measured by decreasing adsorption at 340 nm. The initial NA3B concentration was 1.4 -r mg / ml. The concentration of asparaginase was ΙΟ^-^ Μ. The concentration of alpha ketoglutarate was 10% there. The concentration) of glutamate dehydrogenase was ΙΟ^-? Μ The asparaginase ox concentration was 2.3 x 10M. As previously described in Example 2, GLEG enzyme was removed from the reaction mixture by centrifugation prior to measuring absorbance.

TABLE 19 - Asparaginase Activity

Absorbance at 340 nm

<td></td><td>Time (min)</td><td>OIL</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> 5,0</td><td> 0,759</td><td> 0,684</td>

<td> 4</td><td> 5,0</td><td> 0,603</td><td> 0,558</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> 0,558</td>

<td> 7</td><td> 50,0</td><td> 0,328</td><td> 0,199</td>

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

Air equivalents

Those skilled in the art will recognize that they will be able to adapt using more than routine experimentation, many equivalent to this specific material and components described herein. Such equivalents are understood to fall within the spirit of the following claims.