CONTROLLED DISSOLUTION CROSS-LINKED PROTEIN CRYSTALS

Abstract: The present invention relates to cross-linked protein crystals characterized by the ability to change from insoluble and stable form to soluble and active form upon a change in the environment of said crystals, said change being selected from the group consisting of change in temperature, change in pH, change in chemical composition, change from concentrate to dilute form, change in oxidation-reduction potential of the solution, change in the incident radiation, change in transition metal concentration, change in fluoride concentration, change in free radical concentration, change in metal chelater concentration, change in shear force acting upon the crystals and combinations thereof. According to one embodiment of this invention, such cross-linked protein crystals are capable of releasing their protein activity at a controlled rate. This invention also provides methods for producing such cross-linked protein crystals, methods using them for protein delivery and methods using them in cleaning agents, including detergents, pharmaceutical compositions, vaccines, personal care compositions, including cosmetics, veterinary compositions, foods, feeds, diagnostics and formulations for decontamination.

Description: CONTROLLED DISSOLUTION CROSSLINKED PROTEIN CRYSTALS TECHNICAL FIELD OF THE INVENTION The present invention relates to crosslin ed protein crystals characterized by the ability to change from insoluble and stable form to soluble and active form upon a change in the environment surrounding said crystals, said change being selected from the group consisting of change in temperature, change in pH, change in chemical composition, change from concentrate to dilute form, change in oxidation-reduction potential of the solution, change in the incident radiation, change in transition metal concentration, change in flouride concentration, change in free radical concentration, change in metal chelater concentration, change in shear force acting upon the crystals and combinations thereof. According to one embodiment of this invention, such crosslinked protein crystals are capable of releasing their protein activity at a controlled rate. This invention also provides methods for producing such crosslinked protein crystals, methods using them for protein delivery, methods for using them in cleaning agents, including detergents, therapeutic formulations, pharmaceutical compositions, vaccines, personal care compositions, including cosmetics, veterinary compositions, foods, feeds, diagnostics and formulations for decontamination. BACKGROUND OF THE INVENTION Proteins are used m a wide range of applications m the fields of industrial chemistry, pharmaceuticals, veterinary products, cosmetics and other consumer products, foods, feeds, diagnostics and decontamination. At times, such uses have been limited by constraints inherent m proteins themselves or imposed by the environment or media m which they are used. Such constraints may result m poor stability of the proteins, variability of performance or high cost. In order for proteins to realize their full potential m the fields m which they are used, they must be able to function without excessive intervention by their surrounding environment. In the past, environmental elements have often posed barriers to the widespread use of proteins. Various approaches have been employed to overcome these barriers. However, these approaches have incurred either loss of protein activity or the additional expense of protein stabilizing carriers or formulations . One unique approach to overcoming barriers to the widespread use of proteins is crosslinked enzyme crystal ("CLEC™) technology [N.L. St. Clair and M.A. Navia, J. Am. Chem. Soc. 114, pp. 4314-16 (1992)]. Crosslinked enzyme crystals retain their activity m environments that are normally incompatible with enzyme function. Such environments include prolonged exposure to proteases and other protein digestion agents, high temperature or extreme pH. In such environments, crosslinked enzyme crystals remain insoluble and stable. Protein solubility, leading to controlled release or dissolution of protein, is important m many industrial and medical fields. Such fields include those concerning cleaning agents, including detergents, pharmaceuticals, including therapeutics and vaccines, consumer and personal care products, veterinary products, foods, feeds, diagnostics and decontamination. Various approaches to controlled release have been proposed. These include encapsulation, such as that described m United States patents 4,579,779 and 5,500,223. Other approaches include the use of mechanical or electrical feed devices and osmotic pumps. Controlled release m the pharmaceutical field has been addressed by various means. United States patent 5,569,467 refers to the use of sustained release microparticles comprising a biocompatible polymer and a pharmaceutical agent, which is released as the polymer degrades. United States patent 5,603,956 refers to solid, slow release pharmaceutical dosage units comprising crosslinked amylase, alpha amylase and a pharmaceutical agent. United States patent 4,606,909 refers to oral, controlled-release multiple unit formulations m which homogeneous cores containing particles of sparingly soluble active ingredients are coated with a pH-sensitive erodable coating. United States patent 5,593,697 refers to pharmaceutical or veterinary implants comprising a biologically active material, an excipient comprising at least one water soluble material and at least one water insoluble material and a polymer film coating adapted to rupture at a predetermined period of time after implant. The objective of controlled release of proteins, however, must be balanced with the fact that the protein itself may not be stable under storage conditions. Protein stability may also be adversely affected by other components of the formulation m which it is contained. For example, heavy duty liquid detergents constitute hostile environments for component enzymes. Such problems have been approached through the use of mutant subtilism proteases, which are said to have improved oxidative stability. See United States patent 4,760,025 and PCT patent application O89/06279. Proteins, the enzymes most widely used m detergents, catalyze their own decomposition. Strategies such as the addition of protease inhibitors (e.g., borate with glycols) or the lowering of water activity have been only partially effective . Another approach, described m United States patent 5,385,959, is encapsulation of degradation- sensitive detergent components m capsules of composite emulsion polymers, which permit dilution release thereof. United States patent 5,286,404 refers to a liquid detergent composition said to have improved enzyme solubility while preserving enzyme activity. The improvement is attributed to chemical modification of free primary ammo groups m an enzyme solution via aldehyde treatment, acylation or alkylation. Despite such progress in protein technology generally, the need still exists for proteins which are stable under conditions of storage, while active under conditions of use. DISCLOSURE OF THE INVENTION The present invention relates to crosslinked protein crystals characterized by the ability to change from an insoluble and stable form to a soluble and active form upon a change m the environment surrounding said crystals, said change being selected from the group consisting of change m temperature, change m pH, change m chemical composition, change from concentrate to dilute form, change m oxidation- reduction potential of the solution, change m the incident radiation, change m transition metal concentration, change in flouπde concentration, change m free radical concentration, change in metal chelater concentration, change m shear force acting upon the crystals and combinations thereof. According to one embodiment of this invention, such crosslinked protein crystals are capable of releasing their protein activity at a controlled rate. Advantageously, crosslinked protein crystals according to this invention are insoluble and stable under storage conditions and soluble and active under conditions of use. This invention also provides cleaning agents, including detergents, therapeutic proteins pharmaceutical compositions, vaccines, personal care compositions, including cosmetics, veterinary compositions, foods, feeds, diagnostics and formulations for decontamination. Additionally, this invention includes methods for producing such crosslinked protein crystals and methods for protein delivery using them. „ _ 9 - 6 - BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph representing the stability of various enzymes in Ciba detergent # 16 at 40°C. 5 Figure 2 is a graph representing the washing performance of liquid detergent formulations, including a formulation containing crosslinked subtilism crystals according to the present invention, on fabric soiled with blood, milk and carbon black. 10 Figure 3 is a graph representing the washing performance of liquid detergent formulations, including a formulation containing crosslinked subtilism crystals according to the present invention, after storage at 30 C C, on fabric soiled with cocoa. 15 Figure 4 is a graph representing the washing performance of liquid detergent formulations, including a formulation containing crosslinked subtilism crystals according to the present invention, after storage at 40°C, on fabric soiled with cocoa. 20 Figure 5 is a graph representing the washing performance of liquid detergent formulations, including a formulation containing crosslinked subtilism crystals according to the present invention, after storage at 30°C, on fabric soiled with blood, milk and 25 carbon black. Figure 6 is a graph representing the washing performance of liquid detergent formulations, including a formulation containing crosslinked subtilism crystals according to the present invention, after 30 storage at 40°C, on fabric soiled with blood, milk and carbon black. Figure 7 is a graph representing the washing performance of liquid detergent formulations, including a formulation containing crosslinked subtilism crystals according to the present invention, after storage at 30°C, on fabric soiled with blood. Figure 8 is a graph representing the solubility of crosslinked subtilism crystals according to the present invention at 30°C. Figure 9 is a graph representing the solubility of crosslinked subtilism crystals according to the present invention at 37°C. DETAILED DESCRIPTION OF THE INVENTION In order that the invention herein described may be more fully understood, the following detailed description is set forth. In the description, the following terms or phrases are employed: Aqueous-orσanic solvent mixture -- a mixture comprising n% organic solvent, where n is between 1 and 99 and m% aqueous, where m is 100-n. Biphasic substrate — A solution of a substrate with two distinct phases, either liquid/solid or liquid/liquid phases, one of which contains a substrate for a reaction catalyzed by the protein constituent of a crosslinked protein crystal. An emulsion of olive oil an aqueous solution containing discrete aqueous and organic phases exemplifies a substrate for crosslinked crystals of lipase. Catalytically effective amount -- an amount of crosslinked protein crystals of this invention which is effective to treat, protect, repair or detoxify the area to which they are applied over some period of time. Change m chemical composition — any change m the chemical components of the environment surrounding the crosslinked protein crystals that affects the environment or the crosslmker, including addition of chemical reagents, chemical changes induced by application of energy in the form of light, microwave, or radiation to the environment, chemical events that affect the crosslmker and combinations thereof . Change m shear force acting upon the crystals -- any change m factors of the environment surrounding the crosslinked protein crystals under conditions of use, such as, changes m mechanical pressure, both positive and negative, revolution stirring, centrifugation, tumbling, mechanical agitation and filtration pumping. Controlled dissolution -- dissolution of crosslinked protein crystals or release of the protein constituent from the crystalline state to the soluble state that is (1) triggered by a change the environment surrounding said crystals, said change being selected from the group consisting of change m temperature, change m pH, change chemical composition, change from concentrate to dilute form, change oxidation-reduction potential of the solution, change m the incident radiation, change m transition metal concentration, change in flouride concentration, change in free radical concentration, change m metal chelater concentration, change m shear force acting upon the crystals and combinations thereof and (2) controlled by a factor selected from the group consisting of the following: degree of crosslmkmg of said crosslinked protein crystals, the am o acids residues involved m the crosslinks, whether the crosslmker is homobifunctional or heterobifunctional, the length of time of exposure of protein crystals to the crosslmkmg agent, the rate of addition of crosslmkmg agent to said protein crystals, the nature of the crosslmker, the chain length of the crosslmker, the surface area of said crosslinked protein crystals, the size of said crosslinked protein crystals, the shape of said crosslinked protein crystals and combinations thereof. As used herein, the phrase "controlled dissolution" does not include leaching. Controlled dissolution crosslinked protein crystals — crosslinked protein crystals that slowly dissolve after being exposed a given trigger and release the soluble form of the protein into solution. The activity of controlled dissolution crosslinked protein crystals arises primarily from the soluble form of the protein released from the crystal. Crosslinked crystal form of protein — crosslinked protein crystals that remain insoluble and in the solid state when added to solution. Enhanced protein activity — activity of the crosslinked crystal form of a protein which is enhanced as compared with the soluble form of the protein. According to various embodiments of the present invention, enhanced protein activity is exhibited by any one of the following: a crosslinked crystal form of a protein having activity that is 200-300 times higher than that of the soluble form of the protein; a crosslinked crystal form of a protein having activity that is 100-200 times higher than that of the soluble form of the protein; a crosslinked crystal form of a protein having activity that is 10-100 times higher than that of the soluble form of the protein; a crosslinked crystal form of a protein having activity that is 20-50 times higher than that of the soluble form of the protein; a crosslinked crystal form of a protein having activity that is 10-20 times higher than that of the soluble form of the protein; a crosslinked crystal form of a protein having activity that is 20-30 times higher than that of the soluble form of the protein; a crosslinked crystal form of a protein having activity that is 5-10 times higher than that of the soluble form of the protein; a crosslinked crystal form of a protein having activity that is 2-3 times higher than that of the soluble form of the protein; a crosslinked crystal form of a protein having activity that is at least 3 times higher than that of the soluble form of the protein; a crosslinked crystal form of a protein having activity that is at least 2 times higher than that of the soluble form of the protein; a crosslinked crystal form of a protein having activity that is at least 25-99% higher than that of the soluble form of the protein; a crosslinked crystal form of a protein having activity that is at least 25-30% higher than that of the soluble form of the protein; or a crosslinked crystal form of a protein having activity that is at least 20% higher than that of the soluble form of the protein. Formulations for decontamination — formulations selected from the group consisting of: formulations for decontamination of chemical wastes, herbicides, insecticides, pesticides, environmental hazards and chemical warfare agents. Insoluble and stable form of a protein -- a form of a protein which is insoluble m aqueous solvents, organic solvents or aqueous-organic solvent mixtures and which displays greater stability than the soluble form of the protein. According to an alternate embodiment of this invention, the phrase "insoluble and stable form of a protein" may be a protein which is insoluble m dry formulations but soluble wet formulations. In any embodiment, the crosslinked protein crystals may be active m insoluble form. And m one embodiment, the crosslinked protein crystals may be active m insoluble form, then dissolve or are removed or digested once their function is complete. acromolecular substrate — a large biomolecule, such as a protein or a carbohydrate having a molecular weight of at least 600-700 Daltons, which is also a substrate for a reaction catalyzed by the protein constituent of a crosslinked protein crystal. Organic solvents — any solvent of non- aqueous origin. Pharmaceutically effective amount — an amount of crosslinked protein crystals which is effective to treat a condition an individual to whom they are administered over some period of time. Prophylact cally effective amount — an amount of crosslinked protein crystals which is effective to prevent a condition in an individual to whom they are administered over some period of time. Protein -- a protein or, alternatively, a glycoprote or, alternatively, any peptide having a tertiary structure. The protein constituents of the crosslinked protein crystal formulations of this invention may be naturally or synthetically modified. They may be glycoprotems, phosphoprotems, sulphoprotems, lodoprotems, methylated proteins, unmodified proteins or contain other modifications. The protein constituent of the crosslinked protein crystal formulations of this invention may be any protein including, for example, hormones, such as parathyroid hormone, enzymes, antibodies, viral receptors, viral surface glycoprotems, parasite glycoprotems, parasite receptors, T-cell receptors, MHC molecules, immune modifiers, tumor antigens, mucms, inhibitors, growth factors, trophic factors, cytokines, lymphokmes, cytokines, toxoids, nerve growth hormones, blood clotting factors, adhesion molecules, multidrug resistance proteins, adenylate cyclases, bone morphogenic proteins and lectms. Also included among proteins are glycoprotem hormones and cytokines. Examples of hormones include follicle stimulating hormone, human choπonic gonadotrop , lutemizmg hormone, thyrotrophm and ovme, bovme, porcine, murine and rat alleles of these hormones. Examples of cytokine glycoprotems include α-mterferon, lymphotoxm, and mterleukm-2. Also included are glycoprotem tumor-associated antigens, for example, carcmoembryonic antigen (CEA) , human mucms, her-2/neu, and prostate-specific antigen (PSA) [R.A. Henderson and O.J. Finn, Advances Immunology, 62, pp. 217-56 (1996) ] . Protein activity — an activity selected from the group consisting of binding, catalysis, or activities which generate a functional response withm the environment in which the protein is used, such as the induction of an immune response and immunogenicity or hydrolysis of lipids m lipase deficient individuals, or combinations thereof. Protein activity release rate — the quantity of protein dissolved per unit time. Soluble form of protein — individual protein molecules solution and dissociated from a crystal lattice . Small molecule substrate — molecules having molecular weights generally less than 600 Daltons which are also substrates for reactions catalyzed by the protein constituents of crosslinked protein crystals. Therapeutic protein -- A protein which is administered to a patient in a pharmaceutical formulation and manner. Therapeutic proteins include, for example, hormones, enzymes including lipase, antibodies, viral receptors, T-cell receptors, chemokmes, chemokme receptors, MHC molecules, tumor antigens, mucms, inhibitors, growth factors, trophic factors, cytokines, lymphokines, toxoids, nerve growth hormones, blood clotting factors, adhesion molecules, multidrug resistance proteins, adenylate cyclases and bone morphogenic proteins. Vaccine antigen -- a protein derived from an infectious agent such as a virus, parasite, or tumor antigen. The protein activity of such vaccine antigens is to induce protective immunity against the infectious agent. The crosslinked protein crystals of this invention are particularly advantageous because they are stable m harsh environments imposed by the formulations or compositions m which they are employed or conditions of their storage. At the same time, these crosslinked protein crystals are capable of controlled dissolution or release of their activity when exposed to one or more triggers m their environment. Such triggers may be selected from the group consisting of change m temperature, change m pH, change m chemical composition, change from concentrate to dilute form, change m shear force acting upon the crystals and combinations thereof. Controlled dissolution or release of activity of crosslinked protein crystals according to this invention may also be triggered over a change time. Specific examples of such triggers include an increase or decrease in temperature, for example, an increase m temperature from a low temperature between about 0°C and about 20°C to a high temperature between about 25°C and about 70°C. Other triggers include a change from acidic pH to basic pH and a change from basic pH to acidic pH. Examples of triggers of change from concentrate to dilute form include, for example, a change m solute concentration, a change m concentration of all solutes from about 2-fold to about 10, 000-fold, a change m concentration of all solutes from about 2-fold to about 700-fold, an increase or decrease salt concentration, an increase or decrease m water concentration, an increase or decrease in organic solvent concentration, a decrease m protein concentration and a decrease m detergent concentration. Additional triggers involve changes m chemical composition of the environment surrounding the crosslinked protein crystals that affect the environment or the crosslmker itself. Such changes include, for example, addition of chemical reagents, increase or decrease in organic solvent concentration, chemical events that affect the crosslmker, chemical changes induced by application of energy, including light, microwave or radiation. As explained above, any of these triggers may act m combination or m sequence with one or more of the other triggers. Controlled dissolution of crosslinked protein crystals according to the present invention may also be effected by a change time sufficient to permit a protein activity release rate between about 0.1% per day and about 100% per day, a change m time sufficient to permit a protein activity release rate between about 0.01% per hour and about 100% per hour and a change time sufficient to permit a protein activity release rate between about 1% per minute and about 50% per minute . Crosslinked protein crystals according to this invention, therefore, include those capable of releasing their protein activity at a controlled rate upon exposure to a change m their environment, said change being selected from the group consisting of change m pH, change m solute concentration, change m temperature, change m chemical composition, change shear force acting upon the crystals and combinations thereof. Said controlled rate of releasing protein activity may be determined by a factor selected from the group consisting of the following: degree of crosslmkmg of the crosslinked protein crystals, length of time of exposure of protein crystals to the crosslmkmg agent, the rate of addition of crosslmKmg agent to the protein crystals, the nature of the crosslmker, the chain length of the crosslmker, the ammo acids residues involved in the crosslinks, whether the crosslmker is homobifunctional or heterobifunctional, the surface area of the crosslinked protein crystals, the size of the crosslinked protein crystals, the shape of the crosslinked protein crystals and combinations thereof. As a result of their crystalline nature, the crosslinked protein crystals of this invention achieve uniformity across the entire crosslinked crystal volume. This uniformity is maintained by the intermolecular contacts and chemical crosslinks between the protein molecules constituting the crystal lattice. The protein molecules maintain a uniform distance from each other, forming well-defined stable pores withm the crosslinked protein crystals that facilitate access of substrate to the protein, as well as removal of product. In these crosslinked protein crystals, the lattice interactions, when fixed by chemical crosslinks, are particularly important providing stability and preventing denaturation, especially in storage, under conditions including harsh environments created by components of compositions in which the crystals are used. At the same time, the protein crystals are crosslinked m such a way that they dissolve or release their protein activity upon exposure to a trigger in their environment encountered under conditions of use. Thus, they may be substantially insoluble and stable in a composition under storage conditions and substantially soluble and active under conditions of use of said composition. Factors contributing to the release rate of protein activity of crosslinked protein crystals according to this invention include the degree of crosslmkmg of the crosslinked protein crystals, the length of time of exposure of protein crystals to the crosslmkmg agent, the rate of addition of crosslmkmg agent to the protein crystals, the length of time of exposure of protein crystals to the crosslmkmg agent, the nature of the crosslmker, the ammo acids residues involved in the crosslinks, whether the crosslmker is homobifunctional or heterobifunctional, the chain length of the crosslmker, the surface area of the crosslinked protein crystals, the size of the crosslinked protein - n . Λcm - 98/4673 - 17 - crystals, the shape of the crosslinked protein crystals and combinations thereof. In addition to their activity, crosslinked protein crystals according to this invention are 5 particularly stable and insoluble under storage conditions, including the attendant storage temperature, storage pH, storage time, storage concentrate form, storage involving little or no shear force acting upon the crystals, or combinations 10 thereof. Advantageously, these crosslinked protein crystals are soluble and active under conditions of use, including conditions involving change in temperature, change m pH, change m chemical composition, change from concentrate to dilute form, 15 change m shear force acting upon the crystals and combinations thereof. Such properties make the crosslinked protein crystals of this invention particularly useful for delivery of cleaning agents, including detergents, therapeutic proteins, 20 pharmaceuticals, personal care agents or compositions, including cosmetics, vaccines, veterinary compositions, foods, feeds, diagnostics and formulations for decontamination. According to one embodiment, the crosslinked 25 protein crystals of this invention are characterized by a half-life of activity under storage conditions which is greater than at least 2 times that of the soluble form of the protein that is crystallized to form the crystals that are crosslinked and activity similar to 30 that of the soluble form of the protein under conditions of use. According to one embodiment of this invention, crosslinked protein crystals are characterized by activity which is similar to that of their soluble or uncrosslmked crystallized counterparts under conditions of use. Advantageously however, the crosslinked protein crystals of this invention display improved stability under storage conditions, as compared to their soluble or uncrosslmked crystallized counterpart proteins. One advantage of controlled dissolution crosslinked protein crystals is that a trigger is required to release the soluble form of the protein from the crystal lattice. Therefore, controlled dissolution crosslinked protein crystals can be prepared and function as crosslinked protein crystals with protein activity in the solid crystalline state until a trigger is encountered. After the trigger is encountered, soluble protein is released and protein activity derives from both the crosslinked crystal form, as well as the soluble form of the protein. According to one embodiment of this invention, advantageous properties are obtained from crosslinked protein crystals that are prepared as controlled dissolution crosslinked protein crystals, but which are not subsequently exposed to a trigger. In particular, in the absence of an appropriate trigger to initiate dissolution, such crosslinked protein crystals may exhibit enhanced protein activity to macromolecular substrates, biphasic substrates or small molecule substrates as compared with their soluble counterparts. For example, lipase crystals crosslinked with sulfosuccmιmιdyl-6- [ -methyl-α- (2-pyrιdyldιthιo) toluamιdo]hexanoate (Sulfo-LC-SMPT) , which are not subsequently exposed to a trigger to break the -S-S- bonds of the crosslinks, exhibit enhanced hydrolysis activity toward a biphasic olive oil substrate. More particularly, lipase crystals crosslinked with sulfo-LC-SMPT, are characterized by about a five to ten fold higher protein activity for hydrolysis of an olive oil sustrate than the soluble form of the protein that is crystallized to form the crystals that are crosslinked. In addition, lipase crystals crosslinked with l-ethyl-3- [3- dimethylam oproplyl] carbodiimide hydrochloride (EDC) are characterized by about almost two fold higher protein activity for hydrolysis of an olive oil sustrate than the soluble form of the protein that is crystallized to form the crystals that are crosslinked. The protein constituent of the crosslinked protein crystals of this invention may be any protein, including, for example, therapeutic proteins, prophylactic proteins, including antibodies, cleaning agent proteins, including detergent proteins, personal care proteins, including cosmetic proteins, veterinary proteins, food proteins, feed proteins, diagnostic proteins and decontamination proteins. Included among such proteins are enzymes, such as, for example, hydrolases, lsomerases, lyases, ligases, transferases and oxidoreductases . Examples of hydrolases include ther olysm, elastase, esterase, lipase, nitrilase, amylase, pectmase, subtilmase, hydantomase, asparagmase, urease, subtilism and other proteases and lysozyme. Examples of lyases include aldolases and hydroxynitnle lyase. Examples of oxidoreductases include peroxidase, laccase, glucose oxidase, alcohol dehydrogenase and other dehydrogenases . Other enzymes which may be crystallized and crosslinked include cellulases and oxidases. Examples of therapeutic or prophylactic proteins include hormones, enzymes, including lipase, - 20 - antibodies, inhibitors, growth factors, trophic factors, cytokines, lymphok es, toxoids, erythropoietm, Factor VIII, insulin, glucagon like peptide-I (msul otropin) , amylm, TPA, dornase- , 5 -1-antιtπpsm, human growth hormones, nerve growth hormones, parathyroid hormone, bone morphogenic proteins, urease, toxoids, fertility hormones, FSH, LSH, postridical hormones, tetanus toxoid, diptheπa toxoid. 0 The crosslinked protein crystals of this invention may be used in any of a number of chemical processes. Such processes include industrial and research-scale processes, such as organic synthesis of specialty chemicals and pharmaceuticals. Enzymatic 5 conversion processes include oxidations, reductions, additions, including esterifications and transesteπfications, hydrolyses, eliminations, rearrangements, and asymmetric conversions, including stereoselective, stereospecific and regioselective 0 reactions. Thus, crosslinked protein crystals according to this invention may be advantageously used instead of conventional soluble or immobilized proteins m cleaning agents, including detergents, pharmaceuticals, 5 therapeutics, veterinary compounds, personal care compositions, including cosmetics, foods, feeds, vaccines, pulp, paper and textile processing, diagnostics and formulations for decontamination. Crosslinked protein crystals according to 0 this invention may also be used m various environmental applications. They may be used m place of conventional soluble or immobilized proteins for environmental purposes, such wide area decontamination of environmental hazards. Alternatively, the crosslinked protein crystals of this invention may be used m cleaning agents, selected from the group consisting of detergents, such as powdered detergents and liquid detergents, bleaches, household cleaners, hard surface cleaners, industrial cleaners and carpet and upholstery shampoos . Cleaning agents containing crosslinked protein crystals according to the present invention may also comprise compounds conventionally included m such agents. See, for example, Soaps and Detergents, A Theoretical and Practical Review, Louis Spitz (Ed.), AOCS Press (Champlam, Illinois) (1996) . Such compounds include anionic, non-ionic, cationic or zwitterionic surfactants, or mixtures thereof. Anionic surfactants are exemplified by alkyl sulfates, alkyl ether sulfates, alkyl sulfonates, alkylaryl sulfonates, olefin sulfonates, alkyl ether phosphates, alkyl ether phosphates, fatty acid salts, soaps, isothionates and sulfonated unsaturated esters and acids . Non-ionic surfactants are exemplified by products of condensation of an organic aliphatic or alkyl aromatic hydrophobic compound with an alkylene oxide, alkyl polyglucosides and sugar esters. Cationic surfactants are exemplified by quarternary ammonium salts of tertiary alkyl amines, am o amides, ammo esters or lmidazol es containing al least one long chain (C 8 -C 22 ) aliphatic group or an alkyl-aryl group, wherein alkyl comprises about 4 to 12 carbon atoms and aryl is preferably a phenylene group. Zwitterionic surfactants are exemplified by derivatives of quarternary ammonium, quarternary phosphoniu or tertiary sulfonium compounds, derivatives of secondary and tertiary amines and derivatives of heterocyclic secondary and tertiary amines . And crosslinked protein crystals according to this invention may be used as ingredients m personal care compositions, including cosmetics, such as creams, lotions, emulsions, foams, washes, compacts, gels, mousses, slurries, powders, sprays, pastes, ointments, salves, balms, drops, shampoos, and sunscreens. In topical creams and lotions, for example, they may be used as humectants or for skm protection, softening, bleaching, cleaning, deprote ization, lipid removal, moisturizing, decoloration, coloration or detoxification. They may also be used as anti-oxidants in cosmetics. According to this invention, any individual, including humans and other mammals, may be treated m a pharmaceutically acceptable manner with a pharmaceutically effective or a catalytically effective amount of crosslinked protein crystals for a period of time sufficient to treat a condition m the individual to whom they are administered over some period of time. Alternatively, individuals may receive a prophylactically effective or a catalytically effective amount of crosslinked protein crystals of this invention which is effective to prevent a condition m the individual to whom they are administered over some period of time. Such crosslinked protein crystals may be administered alone, as part of a pharmaceutical, personal care or veterinary preparation or as part of a prophylactic preparation, such as a vaccine, with or without adjuvant. They may be administered by parenteral or non-parenteral route. For example, they may be administered by oral, pulmonary, nasal, aural, anal, dermal, ocular, intravenous, intramuscular, mtraarterial, mtraperitoneal, mucosal, sublmgual, subcutaneous, or mtracranial route. In either pharmaceutical, personal care or veterinary applications, crosslinked protein crystals may be topically administered to any epithelial surface. Such epithelial surfaces include oral, ocular, aural, anal and nasal surfaces, to treat, protect, repair or detoxify the area to which they are applied. The present invention also includes controlled release formulations comprising crosslinked protein crystals according to this invention. In such formulations, the crosslinked protein crystals are substantially insoluble under storage conditions and capable of releasing their protein activity m. vivo at a controlled rate. For example, a pharmaceutical controlled release formulation according to this invention, administered by oral route, is characterized m that the component crosslinked protein crystals are substantially insoluble under gastric pH conditions and substantially soluble under small intestine pH conditions. Alternatively, for these and other uses according to this invention, the crosslinked protein crystals may be active m the insoluble form and then dissolve and are removed or digested once their function is complete. Pharmaceutical, personal care, veterinary or prophylactic compositions comprising crosslinked protein crystals according to this invention may also be selected from the group consisting of tablets, liposomes, granules, spheres, microparticles, microspheres and capsules. For such uses, as well as other uses according to this invention, crosslinked protein crystals may be formulated into tablets. Such tablets constitute a liquid-free, dust-free form of crosslinked protein crystal storage which are easily handled and retain acceptable levels of activity. Alternatively, the crosslinked protein crystals may be in a variety of conventional depot forms employed for administration to provide reactive compositions. These include, for example, solid, semi- solid and liquid dosage forms, such as liquid solutions or suspensions, gels, creams, balms, emulsions, lotions, slurries, powders, sprays, foams, pastes, ointments, salves, balms and drops. Compositions or formulations comprising the crosslinked protein crystals of this invention may also comprise any conventional carrier or adjuvant used in pharmaceuticals, personal care compositions or veterinary formulations. These carriers and adjuvants include, for example, Freund's adjuvant, ion exchangers, alumina, aluminum stearate, lecithin, buffer substances, such as phosphates, glyc e, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protam e sulfate, disodium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium, trisilicate, cellulose- based substances and polyethylene glycol. Adjuvants for topical or gel base forms may include, for example, sodium carboxymethylcellulose, polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol and wood wax alcohols. According to one embodiment of this invention, crosslinked protein crystals may be combined with any conventional materials used for controlled release administration. Such materials include, for example, coatings, shells and films, such as enteric coatings and polymer coatings and films. The most effective mode of administration and dosage regimen of formulations or compositions comprising crosslinked protein crystals of this invention will depend on the effect desired, previous therapy, if any, the individual's health status or status of the condition itself and response to the crosslinked protein crystals and the judgment of the treating physician or clinician. The crosslinked protein crystals may be administered m any dosage form acceptable for pharmaceuticals, personal care compositions or veterinary formulations, at one time or over a series of treatments. The amount of the crosslinked protein crystals that may be combined with carrier materials to produce a single dosage form will vary depending upon the particular mode of administration, formulation, dose level or dose frequency. A typical preparation will contain between about 0.01% and about 99%, preferably between about 1% and about 50%, crosslinked protein crystals (w/w) . Upon improvement of the individual's condition, a maintenance dose of crosslinked protein crystals may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced as a function of the symptoms, to a level at which the improved condition is retained. When the condition has been alleviated to the desired level, treatment should cease. Individuals may, however, require intermittent treatment on a long-term basis upon any recurrence of the condition or symptoms thereof. An alternate embodiment of the present invention includes protein delivery systems comprising the crosslinked protein crystals disclosed herein. Such a system may be used to deliver proteins such as those included in cleaning agents, such as detergents, personal care products, such as cosmetics, pharmaceuticals, such as lipase, veterinary compositions, vaccines, foods, feeds, diagnostics and formulations for decontamination. Protein delivery systems of this invention, which may be formulations or devices, such as implantable devices, may be icroparticulate protein delivery systems, wherein the crosslinked protein crystals have a longest dimension between about 0.01 μm and about 500 μm, alternatively between about 0.1 μm and about 50 μm. The crosslinked protein crystal components of such systems may have a shape selected from the group consisting of: spheres, needles, rods, plates, such as hexagons and squares, rhomboids, cubes, bipryamids and prisms. Advantageously, the crosslinked crystal form of the proteins of this invention allow loading of up to between about 50% and about 90% protein per unit of weight. One example of a protected protein system according to this invention is suitable for storage in a medium such as a liquid detergent, prior to use. The crosslinked protein crystal components of such a system are insoluble under storage conditions in said medium — which typically causes degradation of the soluble form of the protein that is crystallized to form said crystal that is crosslinked — and soluble under conditions of use. According to the present invention, preparation of crosslinked protein crystals includes the steps of crystallizing and crosslmkmg the protein. This may be carried out as illustrated below. Preparation of Crosslinked Protein Crystals - Protein Crystallization Protein crystals are grown by controlled crystallization of protein out of aqueous solution or aqueous solution-containing organic solvents. Conditions to be controlled include, for example, the rate of evaporation of solvent, the presence of appropriate co-solutes and buffers, pH and temperature. A comprehensive review of the various factors affecting the crystallization of proteins has been published by McPherson, Methods Enzymol. , 114, pp. 112-20 (1985) . McPherson and Gilliland, J. Crystal Growth, 90, pp. 51-59 (1988) have compiled comprehensive lists of proteins and nucleic acids that have been crystallized, as well as the conditions under which they were crystallized. A compendium of crystals and crystallization recipes, as well as a repository of coordinates of solved protein and nucleic acid structures, is maintained by the Protein Data Bank at the Brookhaven National Laboratory [http//www. pdb.bnl.gov; Bernstein et al . , J. Mol . Biol . , 112, pp. 535-42 (1977)]. These references can be used to determine the conditions necessary for crystallization of a protein, as a prelude to the formation of an appropriate crosslinked protein crystal, and can guide the crystallization strategy for other proteins. Alternatively, an intelligent trial and error search strategy can, m most instances, produce suitable crystallization conditions for many proteins, provided - 28 - that an acceptable level of purity can be achieved for them [see, e.g., C.W. Carter, Jr. and C.W. Carter, J. Biol. Chem., 254, pp. 12219-23 (1979)]. For use in crosslinked protein crystals according to this invention, the large single crystals which are needed for X-ray diffraction analysis are not required. Microcrystalline showers are suitable. For example, the crosslinked protein crystals may have a longest dimension between about 0.01 μm and about 500 μm, alternatively, between 0.1 μm and about 50 μm. They may also have a shape selected from the group consisting of: spheres, needles, rods, plates, such as hexagons and squares, rhomboids, cubes, bipryamids and prisms. In general, crystals are produced by combining the protein to be crystallized with an appropriate aqueous solvent or aqueous solvent containing appropriate crystallization agents, such as salts or organic solvents. The solvent is combined with the protein and may be subjected to agitation at a temperature determined experimentally to be appropriate for the induction of crystallization and acceptable for the maintenance of protein activity and stability. The solvent can optionally include co-solutes, such as divalent cations, co-factors or chaotropes, as well as buffer species to control pH. The need for co-solutes and their concentrations are determined experimentally to facilitate crystallization. In an industrial-scale process, the controlled precipitation leading to crystallization can best be carried out by the simple combination of protein, precipitant, co-solutes and, optionally, buffers in a batch process. As another option, proteins may be crystallized by using protein precipitates as the starting material. In this case, protein precipitates are added to a crystallization solution and incubated until crystals form. Alternative laboratory crystallization methods, such as dialysis or vapor diffusion, can also be adopted. McPherson, supra and Gilliland, supra, include a comprehensive list of suitable conditions m their reviews of the crystallization literature. Occasionally, incompatibility between the crosslmkmg agent and the crystallization medium might require exchanging the crystals into a more suitable solvent system. Many of the proteins for which crystallization conditions have already been described, may be used to prepare crosslinked protein crystals according to this invention. It should be noted, however, that the conditions reported in most of the above-cited references have been optimized to yield, m most instances, a few large, diffraction quality crystals. Accordingly, it will be appreciated by those of skill m the art that some degree of adjustment of these conditions to provide a high yielding process for the large scale production of the smaller crystals used making crosslinked protein crystals may be necessary. Preparation of Crosslinked Protein Crystals - Crosslmkmg of Protein Crystals Once protein crystals have been grown a suitable medium they can be crosslinked. Crosslmkmg results in stabilization of the crystal lattice by introducing covalent links between the constituent protein molecules of the crystal. This makes possible transfer of the protein into an alternate environment that might otherwise be incompatible with the existence of the crystal lattice or even with the existence of intact protein. Advantageously, crosslmkmg according to the present invention is carried out m such a way that, under conditions of storage, the crosslmkmg interactions prevent the constituent protein molecules m the crystal from going back into solution, effectively msolubilizmg or immobilizing the protein molecules into microcrystallme particles. Upon exposure to a trigger m the environment surrounding the crosslinked protein crystals, such as under conditions of use rather than storage, the protein molecules dissolve, releasing their protein activity. The rate of dissolution is controlled by one or more of the following factors: the degree of crosslmkmg, the length of time of exposure of protein crystals to the crosslmkmg agent, the ammo acid residues involved the crosslinks, whether the crosslmker is ho obi functional or heterobifunctional, the rate of addition of crosslmkmg agent to the protein crystals, the nature of the crosslmker, the chain length of the crosslmker, the surface area of the crosslinked protein crystals, the size of the crosslinked protein crystals, the shape of the crosslinked protein crystals and combinations thereof. Alternatively, controlled dissolution crosslinked protein crystals function as crosslinked protein crystals m the absence of the specific trigger required for initiating and maintaining dissolution. Crosslmkmg can be achieved using one or a combination of a wide variety of multifunctional reagents, at the same time (m parallel) or m sequence, including bifunctional reagents. Upon exposure to a trigger in the surrounding environment, or over a given period of time, the crosslinks between protein crystals crosslinked with such multifunctional crosslmkmg agents lessen or weaken, leading to protein dissolution or release of activity. Such crosslmkmg agents include glutaraldehyde, succmaldehyde, octanedialdehyde and glyoxal. Additional multifunctional crosslmkmg agents include halo-triazmes, e.g., cyanuric chloride; halo- pyrimidines, e.g., 2, 4, 6-trιchloro/bromo-pyπmιdme; anhydrides or halides of aliphatic or aromatic mono- or di-carboxylic acids, e.g., maleic anhydride, (meth) acryloyl chloride, chloroacetyl chloride; N- methylo compounds, e.g., N-methylol-chloro acetamide; di-isocyanates or di-isothiocyanates, e.g., phenylene- 1, 4-dι-ιsocyanate and aziπdmes. Other crosslmkmg agents include epoxides, such as, for example, di-epoxides, tri-epoxides and tetra-epoxides . According to a preferred embodiment of this invention, the crosslmkmg agent is glutaraldehyde, used alone or m sequence with an epoxide. For a representative listing of other available crosslmkmg reagents see, for example, the 1996 catalog of the Pierce Chemical Company. Such multifunctional crosslmkmg agents may also be used, at the same time (m parallel) or m sequence, with reversible crosslmkmg agents, such as those described below. Crosslmkers useful m various embodiments of this invention are (1) those which create covalent links from one cysteme side chain of a protein to another another cysteme side chain, (2) those which create covalent links from one lysme side chain of a protein to another, or (3) those which create covalent links from one cysteme side chain of a protein to a lysine side chain. Crosslmkmg may occur through intermolecular and intramolecular covalent crosslinks. Protein crystals may be crosslinked with one of the following crosslmkers to produce controlled dissolution crosslinked protein crystals: dimethyl 3, 3' -dithiobispropionimidate-HCl (DTBP) ; dithiobis (succmimidylpropionate) (DSP) ; bismaleimidohexane (BMH) ; bis [Sulfosuccmimidyl] suberate (BS) ; 1,5- dιfluoro-2, 4-dmιtrobenzene (DFDNB) ; dimethylsuberimidate • 2HC1 (DMS); disuccinimidyl glutarate (DSG) ; disulfosuccmimidyl tartarate (Sulfo- DST) ; l-ethyl-3- [3-dιmethylammoproplyl] carbodiimide hydrochloride (EDC) ; ethylene glycolbis [sulfo- succmimidylsuccmate] (Sulfo-EGS) ; N- [γ-maleιmιdo- butyryloxy] succmimide ester (GMBS); N-hydroxysulfo- succmιmιdyl-4-azιdobenzoate (Sulfo-HSAB) ; sulfosuccmιmιdyl-6- [ -methyl-o-- (2-pyrιdyldιthιo) toluamιdo]hexanoate (Sulfo-LC-SMPT) ; bιs-[β-(4- azidosalicylamido) ethyl] disulfide (BASED); and NHS- PEG-Vmylsulfone (NHS-PEG-VS) . According to an alternate embodiment of this invention, crosslmkmg may be carried out using reversible crosslmkers, parallel or sequence. The resulting crosslinked protein crystals are characterized by a reactive multi-functional linker, into which a trigger is incorporated as a separate group. The reactive functionality is involved m linking together reactive ammo acid side chains m a protein and the trigger consists of a bond that can be broken by altering one or more conditions m the surrounding environment (e.g., pH, temperature, or thermodynamic water activity) . This is illustrated diagrammatically as: X-Y-Z + 2 AA residues --> AA -X-Y-Z-AA 2 change in environment —> AA-X+Y—Z-AA 2 -- where X and Z are groups with reactive functionality — where Y is a trigger — where AA α and AA 2 represent reactive amino acid residues on the same protein or on two different proteins. The bond between the crosslinking agent and the protein may be a covalent or ionic bond, or a hydrogen bond. The change in surrounding environment results in breaking of the trigger bond and dissolution of the protein. Thus, the crosslinks between protein crystals crosslinked with such reversible crosslinking agents break, leading to protein crystal dissolution or release of activity. Alternatively, the reactive functionality of the crosslinker and the trigger may be the same, as in: X-Z + 2AA residues —> AA 1 -X-Z-AA 2 change in environment —> AA : + X—Z-AA 2, The crosslinker may be homofunctional (X=Y) or heterofunctional (X is not equal to Y) . The reactive functionality X and Y may be, but not limited to the following functional groups (where R, R', R", and R'" may be alkyl, aryl or hydrogen groups) : I. Reactive acyl donors are exemplified by: carboxylate esters RCOOR', amides RCONHR', Acyl azides RCON 3 , carbodiimides R-N=C=N-R', N-hydroxyimide esters, RCO-O-NR 1 , imidoesters R-C=NH2 + (OR' ) , anhydrides RCO-O- COR', carbonates RO-CO-O-R', urethanes RNHCONHR' , acid halides RCOHal (where Hal=a halogen) , acyl hydrazides RCONNR'R", 0-acylisoureas RCO-0-C=NR' (-NR"R' " ) , II. Reactive carbonyl groups are exemplified by: aldehydes RCHO and ketones RCOR', acetals RCO(H 2 )R', ketals RR'C0 2 R'R". Reactive carbonyl containing functional groups known to those well skilled in the art of protein immobilization and crossl kmg are described m the literature [Pierce Catalog and Handbook, Pierce Chemical Company, Rockford, Illinois (1994); S.S. Wong, Chemistry of Protein Conjugation and Cross-Linking, CRC Press, Boca Raton, Florida (1991)]. III. Alkyl or aryl donors are exemplified by: alkyl or aryl halides R-Hal, azides R-N 3 , sulfate esters RS0 3 R', phosphate esters RP0(0R' 3 ), alkyloxonium salts R 3 0+, sulfonium R 3 S+, nitrate esters RON0 2 , Michael acceptors RCR' =CR' "COR" , aryl fluorides ArF, isonitriles RN+ΞC-, haloammes R 2 N-Hal, alkenes and alkynes . IV. Sulfur containing groups are exemplified by disulfides RSSR', sulfhydryls RSH, epoxides R 2 C ~ CR ' 2 . V. Salts are exemplified by alkyl or aryl ammonium salts R 4 N+, carboxylate RCOO-, sulfate ROS0 3 -, phosphate ROPO ' and amines R 3 N. The table below includes examples of triggers, organized by release mechanism. In the table, R= is a multifunctional crosslmkmg agent that can be an alkyl, aryl, or other chains with activating groups that can react with the protein to be crosslinked. Those reactive groups can be any variety of groups such as those susceptible to nucleophilic, free radical or electrophilic displacement including halides, aldehydes, carbonates, urethanes, xanthanes, epoxides among others. 36 The dissolution of the crosslinked protein crystals of this invention can be controlled by selecting appropriate crosslmkers and the associated triggers. Examples of physical and chemical triggers available include: change m temperature, change in pH, change m chemical composition, change from concentrate to dilute form, change m oxidation-reduction potential of the solution, change the incident radiation, change m transition metal concentration, change m flouπde concentration, change m free radical concentration, change m metal chelater concentration, change m shear force acting upon the crystal and combinations thereof. Additional examples of reversible crosslinkers are described in T.W. Green, Protective Groups in Organic Synthesis, John Wiley & Sons (Eds.) (1981). Any variety of strategies used for reversible protecting groups can be incorporated into a crosslinker suitable for producing crosslinked protein crystals capable of reversible, controlled solubilization. Various approaches are listed, in Waldmann's review of this subject, in Angewante Chemie Inl. Ed. Enσl.. 35, p. 2056 (1996) . Other types of reversible crosslinkers are disulfide bond-containing crosslinkers. The trigger breaking crosslinks formed by such crosslinkers is the addition of reducing agent, such as cysteine, to the environment of the crosslinked protein crystals. Disulfide crosslinkers are described in the Pierce Catalog and Handbook (1994-1995) . Examples of such crosslinkers include: Homobifunctional (Symmetric) DSP - Dithiobis (succinimidylpropionate) , also know as Lomant's Reagent DTSSP - 3-3 ' -Dithiobis (sulfosuccinimidylpropionate) , water soluble version of DSP DTBP - Dimethyl 3, 3 ' -dithiobispropionimidate*HCl BASED - Bis- (β- [4-azidosalicylamido] ethyl ) disulfide DPDPB - 1, 4-Di- ( 3 ' - [2 ' -pyridyldithio] - propionamido ) butane . Heterobifunctional (Asymmetric) SPDP - N-Succinimidyl-3- (2-pyridyldithio) propionate LC-SPDP - Succmimidyl-6- (3- [2-pyrιdyldιthιo] propionate) hexanoate Sulfo-LC-SPDP - Sulfosuccmιmιdyl-6- (3- [2- pyridyldlthio] propionate) hexanoate, water soluble version of LC-SPDP APDP - N- (4- [p-azιdosalιcylamιdo]butyl) -3'- (2'- pyridyldithio) propionamide SADP - N-Succmimidyl (4-azιdophenyl) 1, 3'- dithiopropionate Sulfo-SADP - Sulfosuccmimidyl (4-azidophenyl) 1,3'- dithiopropionate, water soluble version of SADP SAED - Sulfosuccmιmιdyl-2- (7-azιdo-4-methycoumarm-3- acetamide) ethyl-1, 3 ' dithiopropionate SAND - Sulfosuccmιmιdyl-2- (m-azido-o- nitrobenzamido) ethyl-1, 3 ' -dithiopropionate SASD - Sulfosuccmιmιdyl-2- (p-azidosalicylamido) ethyl- 1,3' -dithiopropionate SMPB - Succmιmιdyl-4- (p-maleimidophenyl) butyrate Sulfo-SMPB - Sulfosuccmιmιdyl-4- (p- maleimidophenyl) butyrate SMPT - 4-Succmιmιdyloxycarbonyl-methyl- - (2- pyridylthio) toluene Sulfo-LC-SMPT - Sulfosuccmιmιdyl-6- ( -methyl- - pyridylthio) toluamido) hexanoate . In order that this invention may be better understood, the following examples are set forth. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention m any manner. EXAMPLES Example 1 - Preparation of Crosslinked Subtilism Cryst&ls Crystallization of Subtilism One volume of Alcalase 2.5L (Novo Nordisk Biomdustrials, Franklmton, North Carolina) was added to 2 volumes of a solution of 15% sodium sulfate (pH 5.5) prepared at 30-35°C. The crystallization solution was seeded with 1/2,000-1/500 volume seeds (30 mg/ml slurry of crystals m 15% sodium sulfate (pH 5.5), pH supported at 5.5 by adding NaOH. The seeded crystallization solution was incubated at 30- 35°C, stirring by magnetic stirrer overnight. This yielded 60-80% (by activity) crystal rods, 10-50 μm, m length, 1-3 μm m width, after 24-48 hours. Example 2 - Crosslmkmg of Subtilism Crystals Subtilism crystals were crosslinked using one of a variety of crosslinkers, including: glutaraldehyde, glyoxal, succmaldehyde, octanedialdehyde and epoxides. Glutaraldehyde Crosslmkmg Glutaraldehyde ("GA") (supplied as 50% m aqueous by Aldrich Chemical Co.) was diluted m deionized water at 4°C in the various amounts listed in Table I below. For each ml of subtilism crystals (27 mg/ml) 15% sodium sulfate, 10 μl of the diluted glutaraldehyde was added to the slurry while shaking on a vortex at low speed (for amounts less than 5 ml) or stirring with an overhead stirrer at medium speed (for amounts 25 ml - 500 ml) . After mixing for the allotted crosslmkmg time, the samples were centrifuged for 20 - 4 0 - seconds at maximum speed, the supernatant was discarded and replaced with 15% sodium sulfate. This "washing" was repeated a total of 5 times. The final resuspension was effected with 900 μl of 15% sodium sulfate. Glyoxal Crosslinking Glyoxal (supplied as 40% in aqueous by Aldrich Chemical Co.) ("GY") was added to the crystal suspension to give a final concentration of 0.01-1.0%. For each ml of subtilisin crystals (27 mg/ml) in 15% sodium sulfate 0.25 μl to 25 ml (0.01 to 1%) of the glyoxal was added to the slurry, while magnetically - 4 1 - stirring at ambient temperature. After stirring for 1 hour, the crosslinked crystals were centrifuged and washed, as described for glutaraldehyde crosslinking. Octanedialdehyde Crosslinking Octanedialdehyde ("OA") (100% as supplied by DSM Chemie Linz), in the amounts shown in Table II below, was added undiluted to 1 ml of subtilisin crystal slurry (27 mg/ml in 15% sodium sulfate) while magnetically stirring at ambient temperature. Stirring was continued for the specified time of minutes or hours before the crosslinked crystals were centrifuged and washed, as described for glutaraldehyde crosslinking. Table II - Octanedialdehyde Crosslinking Succinaldehyde Crosslinking Succinaldehyde ("SA") (40% as supplied by DSM Chemie Linz) was added undiluted, in the amounts shown in Table III below, to 1 ml of subtilisin crystal slurry (27 mg/ml in 15% sodium sulfate) while magnetically stirring at ambient temperature. Stirring was continued for the specified time of minutes or hours before the crosslinked crystals were centrifuged and washed, as described for glutaraldehyde crossl kmg. Table III - Succinaldehyde Crosslinking Epichlorohydrin Crosslmkmg A 10 μl aliquot of epichlorohydrin ("EP") (99%, Sigma Chemical Co., St. Louis, Missouri) was added undiluted to 1 ml of subtilism crystal slurry (27 mg/ml m 15 sodium sulfate) while stirring at ambient temperature. Stirring was continued for the specified time of minutes or hours before the crosslinked crystals were centrifuged and washed, as described for glutaraldehyde crosslmkmg. Epoxide Crosslmkmg General Procedure Crosslmkmg of subtilism was carried out individually using one of a variety of epoxides. These included: 1) General name - DENACOL a) DENACOL EX-411 b) DENACOL EX-421 c) DENACOL EX- 614 d) DENACOL EX-201 e) DENACOL EX-202; all obtained from Nagase American Corporation. 2) Obtained from Tokyo Kasei Inc. America: a) Neopentyl Glycol diglycidyl Ether (N448) ("NP") b) Ethylene Glycol diglycidyl Ether (E0342 ) ( "EG" ) . The concentration of the epoxide was varied between 0.01 and 4.0% and the crosslmkmg time was varied from 1 hour to 72 hours. The procedure for addition to and removal of crossl ker from enzyme was as described above for glutaraldehyde crosslmkmg. Subsequent crosslinking with glutaraldehyde (0.01 to 0.2%) for (1 hour to 5 hours) yielded strongly crosslinked enzyme crystals, insoluble m water, but active m the azocasem assay. A sample of 1 ml of subtilism crystal slurry (27 mg/ml 15% sodium sulfate) was mixed by vortexmg at low speed to assure a uniform suspension of crystals. Epoxide (10% solution in DMF) was added to the crystal slurry in the amounts specified m Table IV, and the mixture was shaken at ambient temperature. After the allotted time between 1 and 72 hours at ambient temperature, glutaraldehyde (10% m DMF) was added to the epoxide/crystal mixture and stirring was continued at ambient temperature for the time specified m Table IV. The resulting crosslinked enzyme crystals were washed 2x with 1% (NH 4 ) 2 SO 4 /10mM CaCl 2 then 3x with water and finally lx with 1% (NH 4 ) 2 SO 4 /10mM CaCl 2 before resuspendmg m 1% (NH 4 ) 2 SO 4 /10mM CaCl 2 . Table IV - Epox de/Glutaraldehyde Crosslinking Large Scale Preparation of A Preferred Epoxide Sample Prior to crosslmkmg, a sample of 380 ml of crystalline subtilism in 15% sodium sulfate (27 mg/ml) was mixed by overhead stirring at ambient temperature for 5 minutes to assure a uniform suspension of crystals. Neopentyl glycol diglycidyl ether (3.838 ml of a 10 solution in DMF) was added to the crystal slurry and the mixture was stirred at ambient temperature. After 5 hours at ambient temperature, 3.838 ml of glutaraldehyde (10% in DMF) was added to the epoxide/crystal mixture and stirring was continued at ambient temperature for 1.5 hours. The resulting crosslinked enzyme crystals were washed 2x with 1% (NH 4 ) 2 SO 4 /10mM CaCl 2 then 3x with water and finally lx with 1! (NH l 2 SO 4 /10mM CaCl 2 , before resuspendmg in 1 ! (NH, ) 2 SO 4 /10mM CaCl- Example 3 - Activity Assay In order to test the activity of crosslinked protein crystals according to this invention, as well as other enzyme samples, we developed the following azocasem assay. Material : 2.0M Tris Buffer. 500ppm CaCl 2 0.2M Tris Buffer. 50ppm CaCl 2 50% urea Azocasem - 45 - 5% trichloroacetic acid ("TCA") Alcalase (2.5L) ChiroCLEC-BL™ (crosslinked subtilis crystals, available from Altus Biologies, Inc., Cambridge, Massachusetts) The assay was carried out, preparing azocasem just prior to use, by dissolving 600 mg azocasem with 10 ml of 50% urea and vortexmg lightly to complete the dissolution. Then 10 ml 2.0M Tris was added and vortexed to mix, increasing the volume to 100 ml by adding deionized water. The stock solutions of the enzyme to be assayed m 0.2M Tris were prepared, to provide 50 μl aliquots to be assayed, as follows: Without detergent: 0.03 mg/ml Alcalase (soluble, uncrosslmked subtilism Carlsberg 80.3 mg/ml) 3.0 mg/ml ChiroCLEC-BL™. With 120 ul detergent/ml solution: 0.03 mg/ml Alcalase 3.0 mg/ml ChiroCLEC-BL™. We added 50 μl aliquots of enzyme to 150 μl of 0.2M Tris and placed the mixtures in 5 ml test tubes with micro-stir bars. We then warmed both the test tubes and the azocasem at 40°C for 1 mmute using a metal heating block. After that, we added 1 ml of the azocasem to each tube and stirred at 40°C for 15 minutes using the heating block at stir speed 4. We then added 2 ml TCA to each tube, mixing by vortex, and placed the tubes m an ice bath immediately, allowing the samples to stand at 0°C for 20 minutes. We microfuged the samples for 5 minutes at maximum rpm and microfiltered, if necessary. We measured absorbance of the expressed activity in abs-units/mg protein-min supernatant at λ390. In this assay, all measurements were done in triplicate. Controls were void of enzyme but contained detergent if it was present in the assay. This time = 0 assay was repeated at time = 15 minutes and other times, if necessary. The detergents used in the various assays included Tide, Wisk and Ciba-Geigy detergents #15, #16 and #44 ("Ciba detergents") . Ciba detergent #15 constitutes a typical European detergent formulation — liquid (aqueous) detergent on the basis of 15% alkylbenzene sulfonate, 14% fatty alcohol ethoxylate and 10% fatty acid salt (soap) . Ciba detergent #16 constitutes a typical United States detergent formulation — liquid (aqueous) detergent on the basis of 7.5% alkyl benzene sulfonate, 10% fatty alcohol ethoxylate and 17% alkyl either sulfate. Ciba detergent #44 constitutes a typical compact detergent formulation — liquid (aqueous) detergent on the basis of 6% fatty alcohol ethoxylate, 23% alkyl ether sulfate and 10% sodium citrate. Ciba detergents #15, #16 and #44 may be obtained upon request from Ciba Specialty Chemicals Corp., Division Consumer Care Chemicals, Greensboro, North Carolina. We prepared assay stock solutions from dilution stocks, and carried out the assays, as follows. Activity Assay - 200x Dilution - Crosslinked Subtilism Crystals and Crystalline Subtilis m Heavy Duty Liquid Detergent (Ciba #15, Ciba #44, Tide and Wisk) Stocks A, B, C and D were prepared m 10 ml neoprene tubes as follows. Stock A : Crosslinked Subtilism Crystals Prepared According to this Invention (-27 mg/ml) We centrifuged 37 μl slurry of crosslinked subtilism crystals (equal to 1 mg crosslinked enzyme crystals) to remove supernatant, added 1 ml detergent and vortexed to mix. A 50 μl aliquot of the resulting mixture was added to 9.95 ml water, to a final concentration of 5 μg/ml. Stock B : Uncrosslmked Subtilism Crystals (-27 mg/ml) We centrifuged 37 μl slurry of subtilism crystals (equal to 1 mg enzyme crystals) to remove supernatant, added 1 ml detergent and vortexed to mix. A 50 μl aliquot of the resulting mixture was added to 9.95 ml water, to a final concentration of 5 μg/ml. Stock C: Alcalase We added 18.75 μl Alcalase (80.3 mg/ml) to 3 ml detergent and vortexed to mix. A 50 μl aliquot of the resulting mixture was added to 9.95 ml water, to a final concentration of 2.5 μg/ml. Stock D : Detergent A 50 μl aliquot of detergent was added to 9.95 ml water . Azocasein stock (6 mg/ml) was prepared as described above. Upon dilution of Stock A and B to 5 μg/ml, the t=0 assay was set up immediately and carried out as described above, except that the amount of stock sample used was 200 μl, instead of 50 μl + 150 μl 0.2M Tris. While the tubes were heating for 1 minute at 40°C, two additional samples of 2 ml each of Stocks A, B and C were placed in 1.5 ml microcentrifuge tubes and heated to 52°C while shaking for further testing after 5 minutes and 15 minutes dilution with heating . Activity Assay - 670x Dilution - Crosslinked Subtilisin Crystals and Crystalline Subtilisin in Detergent Concentrate (Ciba #16) Stocks A, B, C and D were prepared in 10 ml neoprene tubes as follows. Stock A : Crosslinked Subtilisin Crystals Prepared According to this Invention (-27 mg/ml) We centrifuged 124 μl slurry of crosslinked subtilisin crystals (equal to 3.35 mg crosslinked enzyme crystals) to remove supernatant, added 1 ml detergent and vortexed to mix. A 50 μl aliquot of the resulting mixture was added to 33.45 ml water, to a final concentration of 5 μg/ml. Stock B : Uncrosslmked Subtilisin Crystals (-27 mg/ml) We centrifuged 124 μl slurry of subtilisin crystals (equal to 3.35 mg enzyme crystals) to remove supernatant, added 1 ml detergent and vortexed to mix. A 50 μl aliquot of the resulting mixture was added to 33.45 ml water, to a final concentration of 5 μg/ml. Stock C: Alcalase We added 167 μl Alcalase (80.3 mg/ml) to 8 ml detergent and vortexed to mix. A 50 μl aliquot of the resulting mixture was added to 33.45 ml water, to a final concentration of 2.5 μg/ml. Stock D : Detergent A 50 μl aliquot of detergent was added to 33.45 ml water. Azocasein stock (6 mg/ml) was prepared as described above. The t=0 assay was set up immediately and carried out as described above, except that the amount of stock sample used was 200 μl, instead of 50 μl, plus 150 μl 0.2M Tris buffer. While the tubes were heating for 1 minute at 40°C, two additional samples of 2 ml each of Stocks A, B and C were placed in Eppendorf tubes and heated to 40°C while shaking for further testing after 5 minutes and 15 minutes dilution with heating. Example 4 - Stability Study In order to test the stability of crosslinked enzyme crystals according to this invention, as well as other enzyme samples, we developed the following assays . Azocasein Assay - Stability Study 52°C First, we prepared stock solutions of the enzyme samples in detergent in 2 ml Eppendorf tubes with screw caps. After incubating the mixtures in a water bath at 52°C for the appropriate times, we added 1.47 ml of 0.2M Tris buffer to one of each enzyme sample tube, and mixed well. To assay for activity after the appropriate time of incubation followed by dilution, we removed a 50 μl aliquot from each tube and assayed as described below. The remaining samples of enzyme/detergent stocks were placed in a water bath at 52°C, with further aliquots being removed for assay at specific times. The assay was performed by adding 50 μl enzyme sample to 150 μl 0.2M Tris buffer and heating to 40°C for 1 minute. At a constant 40°C temperature, we then added 1.0 ml azocasein stock (as described in Example 1) to each sample, stirring for 15 minutes using a heating block at stir speed 4. We then added 2 ml TCA to each tube, mixing by vortex, and placed the tubes in an ice bath immediately, allowing the samples to stand at 0°C for 20 minutes. We microfuged the samples for 5 minutes at maximum rpm and microfiltered, if necessary. We measured absorbance of the supernatant at λ390 and expressed activity as abs'units/mg protein*min. In this assay, all measurements were done in triplicate. Controls were void of enzyme but contained detergent if it was present in the assay. In order to assess activity as part of stability studies carried out at 52 C C, we prepared assay stock solutions from dilution stocks, and carried out the assays, as follows. For Alcalase Stocks : Stock A: Alcalase (80.3 mg/ml) in commercial detergent (Tide or Wisk, deactivated by heating at 70°C for 4 hours) - final concentration = 0.25 mg/ml. The stock was prepared by adding 31.2 μl Alcalase to 9.97 ml detergent. A 200 μl aliquot of the resulting mixture was placed in each of several 2 ml Eppendorf tubes (3x for t=0, 30 and others) . Stock B: Alcalase (80.3 mg/ml) Ciba detergent (Ciba #15, Ciba #16 or Ciba #44) - final concentration = 0.25 mg/ml. The stock was prepared by adding 31.2 μl Alcalase to 9.97 ml detergent. A 200 μl aliquot of the resulting mixture was placed in each of several 2 ml Eppendorf tubes (3x for t=0, 1 hour and 4-6 hours) . Stock C: Commercial detergent (Tide or Wisk, deactivated by heating at 70°C for 4 hours) 3x (200 μl of the above in 2 ml Eppendorf tubes) . Stock D: Ciba detergent (Ciba #15, Ciba #16 or Ciba #44) 3x (200 μl of the above in 2 ml Eppendorf tubes) . The t=0 assay was performed immediately after 1.47 ml of 0.2 M Tris was added to one of each of tubes containing Stocks A-D and the contents mixed well. Remaining samples of Stocks A-D were placed in a water bath and heated to 52°C. Otherwise, the assays were carried out as described above. For Crosslinked Subtilis Crystals and Crystalline Subtilis Stocks A, B, C and D were prepared m 2 ml Eppendorf tubes with screw caps as follows. Stock A: ChiroCLEC-BL™ commercial detergent (denatured Tide or Wisk) - final concentration = 25 mg/ml. The stock was prepared by centrifuging 3.12 ml enzyme slurry to remove water and then diluting the enzyme to 10 ml with detergent. A 200 μl aliquot of the resulting mixture was placed in each of several 2 ml Eppendorf tubes (4x for t=0, 24 hours, 48 hours and 72 hours) . Stock B: ChiroCLEC-BL™ in Ciba detergent (Ciba #15, Ciba #16 or Ciba #44)- final concentration = 25 mg/ml. The stock was prepared by centrifuging 3.12 ml enzyme slurry to remove water and then diluting the enzyme to 10 ml with detergent. A 200 μl aliquot of the resulting mixture was placed in each of several 2 ml Eppendorf tubes (4x for t=0, 24 hours, 48 hours and 72 hours at 52°C) . Stock C: Commercial detergent (Tide or Wisk, deactivated by heating at 70°C for 4 hours) 4x (200 μl of the above in 2 ml Eppendorf tubes) . Stock D: Ciba-Geigy detergent (Ciba #15, #16 or #44, depending on which detergent was chosen for Stock B) 4x (200 μl of the above in 2 ml Eppendorf tubes) . The t=0 assay was set up immediately after 1.47 ml of 0.2M Tris was added to one of each of tubes containing Stocks A-D and the contents mixed well. Remaining samples of Stocks A-D were placed in a water bath and heated to 52°C. Otherwise, the assays were carried out as described above. For Crosslinked Subtilisin Crystals and Crystalline Subtilisin Stocks A, B and C were prepared in 2 ml Eppendorf tubes with screw caps as follows. Stock A: Uncrosslmked subtilism crystals (~ 27 mg/ml) m Ciba detergent (Ciba #15, #16 or #44) - final concentration = - 1 mg/ml. The stock was prepared by centrifugmg 50 μl crystal slurry to remove supernatant, then adding 1.35 ml detergent (Ciba #15, Ciba #16 or Ciba #44) to a final concentration of 1 mg/ml. An 80 μl aliquot of the resulting mixture was placed m each of several 2.0 ml Eppendorf tubes (3x for t=0, 15 minutes and others) . Stock B: Crosslinked subtilism crystals according to this invention (~ 27 mg/ml) m Ciba detergent (Ciba #15, #16 or #44) - final concentration = - 1 mg/ml. The stock was prepared by centrifugmg 50 μl crystal slurry to remove supernatant, then adding 1.35 ml detergent, to a final concentration of 1 mg/ml. An 80 μl aliquot of the resulting mixture was placed each of several 2.0 ml Eppendorf tubes (3x for t=0, 15 minutes and others) . Stock C: Ciba detergent (Ciba #15, #16 or #44) - 3x (80 μl of the above m 2.0 ml tubes). The t=0 assay was set up immediately after 1.8 ml of water was added to one of each of tubes containing Stocks A-C and the contents mixed well. Remaining samples of Stocks A-C were placed m a water bath and heated to 52°C. Otherwise, the assays were carried out as described above, except for the addition of 150 μl 0.2 M Tris buffer instead of 200 μl . Azocasem Assay - Stability Study 40°C First, we prepared stock solutions of the enzyme samples m detergent in 2 ml Eppendorf tubes with screw caps. To assay stability at t=0, we added 1.8 ml of deionized water to a 25 μl of each sample and mixed well. We removed a 25 μl aliquot from each tube and assayed as described below. The remaining samples of enzyme/detergent stocks were placed m a water bath at 40°C, with further aliquots being removed for assay at specific times. The assay was performed by adding 25 μl of the diluted enzyme sample to 175 μl 0.2M Tris buffer and heating to 40°C for 1 m ute. At a constant 40°C temperature, we then added 1.0 ml azocasem stock (as described m Example 3) to each sample, stirring for 15 minutes using a heating block at stir speed 4. We then added 2 ml TCA to each tube, mixing by vortex, and placed the tubes m an ice bath immediately, allowing the samples to stand at 0°C for 20 minutes. We microfuged the samples for 5 minutes at maximum rpm and icroflltered, if necessary. We measured absorbance of the supernatant at λ390 and expressed activity as abs-units/mg protem-mm. In this assay, all measurements were done in triplicate. Controls were void of enzyme but contained detergent if it was present m the assay. In order to assess activity as part of stability studies carried out at 40°C, we prepared assay stock solutions from dilution stocks, and carried out the assays, as follows. Stock A : Crosslinked Subtilisin Crystals According to this Invention (-27 mg/ml) We centrifuged 124 μl slurry of crosslinked subtilisin crystals (equal to 3.35 mg crosslinked enzyme crystals) to remove supernatant, added 1 ml detergent and vortexed to mix, to a final concentration of 3.35 mg/ml. Stock B : Uncrosslinked Subtilisin Crystals (-27 mg/ml) We centrifuged 124 μl slurry of subtilisin crystals (equal to 3.35 mg enzyme crystals) to remove supernatant, added 1 ml detergent and vortexed to mix, to a final concentration of 3.35 mg/ml. Stock C: Alcalase We added 20.9 μl Alcalase (80.3 mg/ml) to 1 ml Ciba detergent (Ciba #15, Ciba #16 or Ciba #44) and commercial detergent (Tide or Wisk, denatured by heating at 70°C for 4 hours) and vortexed to mix, to a final concentration of 1.67 mg/ml. Stock D : Detergent One ml of commercial detergent (Tide or Wisk, deactivated by heating at 70°C for 4 hours) and Ciba detergents #15, #16 and #44. A 25 μl aliquot of each stock was added to 1.8 ml of deionized water and mixed well. A further 25 μl aliquot of the diluted stock was added to each reaction tube. The t=0 assay was performed immediately after 175 μl of 0.2M Tris was added to each tube containing 25 μl of the various stock samples. Remaining samples of Stocks A-D were placed in a water bath and heated to 40°C. Otherwise, the assays were carried out as described above. Example 5 - Dissolution Studies We also assessed the characteristics of crosslinked enzyme crystals according to this invention, as well as other enzyme samples, with respect to dissolution m concentrate and upon dilution, as detailed below. Stock solutions were prepared and diluted as described above. The resulting dispersions were heated at 40°C and analyzed under a microscope at 250x for dissolution progress. Example 6 - Results of Activity and Stability Assays Crosslinked enzyme crystals of subtilism, as described above, as well as soluble enzymes and other commercial enzymes, alone and in the presence of commercial detergents, were tested for activity m the azocasein assay, as described above. Catalyst concentrations for equivalent activities were determined for Alcalase, ChiroCLEC-BL™, Wisk with active protease and Tide with active protease: ChιroCLEC-BL m : 150 μg/6 μl detergent 0.4 - 0.5 absorbance units Alcalase: 15 μg/6 μl detergent 0.5 - 0.6 absorbance units Tide: 6 μl detergent approximately 0.6 absorbance units Wisk: 6 μl detergent approximately 0.6 absorbance units. The dilution studies (discussed supra) were started by assessing the activities of Alcalase and uncrosslmked crystals of Alcalase m Ciba detergents #15 and #16. Initial activities were comparable and 57 - losses of up to - 50% were seen after 15 minutes at 52°C. Table V summarizes the stability of samples of Alcalase (0.25 mg/ml) and ChiroCLEC-BL™ (25 mg/ml) in denatured Wisk or Tide detergent, or in Ciba detergents #15 and #16 at 52°C. Activity was measured by the azocasein assay. TABLE V Stability of Subtilisin in Detergents at 52°C We also assessed the stability of various enzymes in Ciba detergent #15 at 52°C. The results are depicted in Table VI below: TABLE VI Stability of Subtilisin in Ciba Detergent #15 at 52°C - 58 - All of the crosslinked crystals prepared as described in the table above which had half-lives in detergent concentrate of -30 minutes or more also had good solubility profiles. In addition, we assessed the stability of various enzymes in Ciba detergent #15 versus Ciba detergent #16 at 40 C C. The results are depicted in the Table VII below: TABLE VII Stability of Subtilisin in Ciba Detergent #15 vs. #16 at 40°C We also assessed the effects of crosslinking time on activity and stability of the resulting crosslinked enzyme crystals. These results are - 59 - summarized in the tables below. In Table VIII, an asterisk indicates values measured by incubating 25 μl in a 2 ml tube and "Xs" denotes uncrosslinked protein crystals . In preparing the crosslinked protein crystals described in Tables VIII, IX and X, the protein crystals were crystallized as described in Example 1 and crosslinked with glutaraldehyde as described in Example 2, using the crosslinking times and glutaraldehyde concentrations set forth in that example, or those specified in the tables. TABLE VIII Crosslinkinα Time vs. Concentration of Glutaraldehyde on Stability of Subtilisin in Ciba Detergent #16 at 40°C - 60 - In Table IX, an asterisk indicates that crystals were crushed during crosslinking and dash marks indicate that no measurements were taken at those - 61 - points. All samples were prepared at 1 ml (27 mg) scale . TABLE IX Activity of Glutaraldehyde Crosslinked Subtilisin in Ciba Detergent #16 at 40°C - 62 TABLE X Conditions for Larger Scale Crosslinked Enzyme Crystal Preparation - Stability of Glutaraldehyde Crosslinked Subtilisin in Ciba Detergent #16 at 40°C In Table X, an asterisk indicates that crosslinkings were carried out at a 1-2 g scale, § indicates that crosslinkings were carried out at a 10 g scale on previously crushed crystals and dash marks indicate that no measurements were taken at those points . Figure 1 graphically depicts the stability of 10 g scale preparations of crosslinked subtilisin crystals according to this invention in Ciba detergent #16 at 40°C. In Figure 1, "Altus I" represents crystals crosslinked with 0.25% glutaraldehyde for 2 hours; "Altus II" represents crystals crosslinked with 0.20% glutaraldehyde for 2 hours; "Altus III" represents crystals crosslinked with 0.15% glutaraldehyde for 2 hours and "Altus IV" represents crystals crosslinked with 0.1% neopentyl glycol diglycidyl ether for 5 hours, followed by 0.1% glutaraldehyde for 1.5 hours. All the crosslinked samples were crushed prior to crosslmkmg using a Brmkman Polytron Homogenizer, then prepared on a 10 g scale and monitored by the azocasein assay over one week at 40°C. Example 7 - Results of Dissolution Study The dissolution study demonstrated whether various crosslinked enzyme crystals dissolve concentrate and the extent to which they dissolve upon dilution under conditions of use, for example under wash conditions. Representative results of this test are included the tables below, which "+" indicates that the sample dissolved, "-" indicates that the sample did not dissolve, "-/+" indicates that the sample dissolved somewhat (1 mg/ml in detergent liquid) . In the tables, "GP" denotes crystals crosslinked as described infra for GA crosslmkmg using, instead, ultrapure glutaraldehyde (supplied as an 8% aqueous solution by the Sigma Chemical Co.) which was not diluted prior to addition to the protein crystals. TABLE XI Detergent Liquid Incubation Study - Dissolution Study - Concentrate 14h at 40°C TABLE XII Detergent Liquid Incubation Study - Dissolution Study - 200 fold Dilution 20 minutes at 52 Q C As demonstrated in the tables above, crosslinked enzyme crystals according to this invention are essentially insoluble m concentrated detergent and essentially soluble m diluted detergent under wash conditions . - 66 - Example 8 - Summary of Properties of Crosslinked Enzyme Crystals of This Invention Table XIII below summarizes the overall stability/instability, activity and dissolution properties in Ciba detergent #15 of crosslinked subtilisin crystals prepared according to this invention using dialdehydes . TABLE XIII As demonstrated in Table XIII above, the crosslinked enzyme crystals of the present invention are insoluble and, therefore, stable under storage conditions, while quickly releasing their activity under conditions of use. Advantageously, the crosslinked enzyme crystals of this invention exhibit activity similar to their soluble or uncrosslinked crystallized counterparts under conditions of use, - 67 while displaying 5-6 fold improved stability, as well as favorable dissolution properties. Example 9 - Effect of Change of Chemical Composition On Crosslinked Subtilisin Crystals We crystallized subtilisin as described in Example 1 and crosslinked the resulting crystals as described in Example 2, using GA 1%/1 hour. When 100 μL (2.2 mg) of the resulting crosslinked subtilisin crystals was suspended in 1.5 mL of 33.3% of acetonitrile/phosphate buffer (0.3 M, pH 7.5), the crystals were completely dissolved after 45 minutes at 40°C. Using similar conditions, suspending the crosslinked subtilisin crystals in 1.2 mL of 16.7% acetonitrile/buffer, the crystals were completely dissolved after 5 hours. Activity (U) * crystals were completely dissolved at this time ** crystals were not completely dissolved. Assay: 0.2 mmol (75.8 mg) of TAME in 2.5 mL phosphate buffer was incubated with each crosslinked subtilisin crystal sample (equal to 0.044 mg enzyme crystals) suspension (or solution) at room temperature One unit hydrolyzed 1.0 μmole of TAME per mm. from per mg crosslinked crystals. The results above illustrate the trigger of addition of organic solvent to the environment of crosslinked protein crystals of this invention. Example 10 - Wash Performance of Detergents Containing Crosslinked Subtilism Crystals We assessed the activity and storage stability of crosslinked enzyme crystals of this invention m liquid detergent, using a washing assay designed to test the ability of the detergent to remove stains from a fabric. Washing Assay Preparation of fabric Cloth samples of the same size and weight were cut from the same bolt: 5 g of soiled test cloth and 5 g of cotton ballast with no soil (Ciba No. 1-3005) . Prior to washing the samples, we measured the light intensity (= lightness) remitted by the soiled fabric samples (as described below) . Preparation of detergent solution The sample of liquid detergent to be tested was heated m a flask for two hours at 20°C. The sample was then homogenized by vigorous shaking and 0.8 g of the detergent was removed from the flask and added to 200 ml of tap water (20°C) a metallic beaker. The aqueous detergent solution was stirred for 60 seconds. ΛIΛ „ 8/467 - 69 - Washing A sample of soiled test cloth and a sample of unsoiled ballast were placed together into the beaker containing the aqueous detergent solution. The beaker 5 was closed tightly and immediately inserted into a preheated (40°C) washing machine (Unitest, manufactured by Hereus, Switzerland) . During the washing process, the beaker was rotated constantly in a water bath heated to 40°C. As a result, the contents of the beaker 0 continuously warmed, up to a temperature of 40°C. Exactly 20 minutes after the fabric was placed in the detergent solution, washing was stopped and the washed fabric was immediately removed from the detergent solution and rinsed for 30 seconds with cold 5 tap water (13 - 15°C) . The wet fabric was centrifuged and ironed to remove wrinkles and dried at the same time . Measurement of washing performance Each sample of the washed and dried fabric 0 was examined for stain removal by remission measurements (lightness Y) between 460 and 700 nm using a Spectraflash 500 (Datacolor) . A cut off filter was used to eliminate potential interference by contamination with UV-absorbing materials. The 5 lightness value of each test cloth was measured 5x and an average calculated. With increasing washing performance, the lightness of the fabric increases. Washing performance is thus defined as a difference in lightness, ΔY: 0 ΔY = Lightness of fabric after washing - Lightness of fabric before washing 70 - Example 11 - Effect of Concentration of Crosslinked Subtilisin Crystals on Washing Performance of Detergents Containing Them Washing performance of crosslinked enzyme crystals according to this invention was examined as a function of their concentration in the liquid detergent, using the materials described below. Test fabric: EMPA (Eidgenδssische Materialprύfungs und Forschungsanstalt, St. Gallen, Switzerland) #116 soiled with a combination of blood, milk and carbon black . Liquid detergent: Ciba detergent #16. Enzyme : - Crosslinked enzyme crystals; sample Altus IV (as described in Example 6) Uncrosslinked enzyme (Alcalase) . Concentration of enzyme in liquid detergent: enzyme concentrations were between 0.05 and 0.9 w% (dry matter weight). Table XIV provides further details. TABLE XIV - 71 Preparation of liquid detergent with enzyme Specific aliquots of the suspension of enzyme crystals (see Table XIV) were added to a flask and centrifuged to separate the crystals from the liquid. The liquid was discarded and the crystals were suspended and homogenized m the liquid detergent (for quantities see Table XIV) . The resulting preparations were used m the washing tests. Washing tests to evaluate the performance of the enzyme detergent formulations were carried out as described m the assay above. The results of the study are depicted m Figure 2. The figure demonstrates that at enzyme concentrations > 0.1 w%, the washing effect of Ciba liquid detergent #16 formulated with Altus IV exceeds that of the formulation with uncrosslmked Alcalase. The efficacy of both crosslinked and uncrosslmked enzymes was reduced at enzyme concentrations below 0.1 w% . Example 12 - Storage Stability and Washing Performance of Detergents Containing Crosslinked Subtilism Crystals Detergents formulated with crosslinked and uncrosslmked enzymes were stored at a constant temperature, m order to examine enzyme stability concentrated liquid detergent. The detergent - 72 - formulations (150 g each) were prepared by the same procedure as the samples in Example 10. Liquid detergent: Ciba detergent #16. Enzyme : - Crosslinked enzyme crystals: sample Altus IV 5 (Example 6) - Uncrosslinked enzyme (Alcalase) Enzyme concentration: 0.3 w% (dry matter) in liquid detergent . Storage temperature for stability studies: 10 All samples were stored at 30°C for between 0 and 7 days. After 7 days, the samples were divided after 7 days into two equal portions, in order to study stability at elevated temperature. One portion continued to be stored at 30°C, while the other was 15 stored at 40°C. Test fabric: Three different soiled fabrics were used. All of them were standard test materials available from EMPA: EMPA #112: cocoa soiled fabric 20 • EMPA #116: blood, milk and carbon black soiled fabric EMPA #111: blood soiled fabric. Washing performance on cocoa soiled fabric Washing performance of various enzyme 25 formulated liquid detergents was studied with respect to removal of cocoa stains from a cocoa soiled test fabric, using the washing assay described above. Storage stability was determined by assessing washing performance periodically during the detergent storage 30 time, thus monitoring the impact of storage temperature on enzyme performance in the liquid detergent. In this 32 - 73 - assay, the effectiveness of the liquid detergent decreases as enzyme stability degrades. The results of this assay, shown in Figures 3 and 4, are discussed below. Storage Stability at 30°C As demonstrated m Figure 3, both Alcalase and Altus IV formulated detergents exhibited an improved performance after 2 days of storage (compared to initial values) . However, as storage time increased, the performance of the Alcalase formulation decreased continuously over time, while the Altus IV formulated detergent exhibited no degradation, even after 28 days of storage. Storage Stability at 40°C As demonstrated m Figure 4, when the temperature was raised from 30 to 40°C, Alcalase formulated detergent lost activity withm 2 days, while the Altus IV formulated detergent degraded slightly, while removing the cocoa soil from the test fabric significantly, even after 21 days of storage at 40°C. Washing performance on fabric soiled by a combination of blood, milk and carbon black (EMPA #116 test fabric) The experimental conditions and detergents were the same (except the stained fabric) as for washing of cocoa stains. The results of the washing tests are depicted Figures 5 and 6. Storage Stability at 30°C Figure 5 clearly illustrates the decay of washing performance of the Alcalase formulated detergent after 2 days of storage at 30°C. However, liquid detergent containing Altus IV enzyme maintained its original washing performance, even after 28 days of storage . Storage Stability at 40°C As demonstrated in Figure 6, when the storage temperature was raised from 30 to 40°C, Alcalase formulated detergent lost nearly all of its washing performance withm 2 days. In contrast, detergent containing Altus IV retained its washing power for an additional 14 days. Washing performance on fabric soiled with blood Washing performance on blood stains was tested with enzyme containing detergents stored at 30°C. The detergent composition, washing conditions were the same as m washing of cocoa stains. The results of the washing test are illustrated Figure 7. The assays show that the washing effect on blood stain by Ciba #16 liquid detergent formulated with Alcalase was low m comparison to detergent without enzyme. On the other hand, the Altus IV formulation was more active m washing conditions and more stable storage. Storage Stability at 30°C The washing effect of Alcalase formulated detergent decreased rapidly with storage time, whereas Altus IV formulated detergent retained almost completely its full capacity after 28 days of storage. Example 13 - Solubility of Crosslinked Subtilisin Crystals at 30°C and 37°C We studied the solubility of various subtilisin crystals, which had been crosslinked with glutaraldehyde (GA) , octanedialdehyde (OA) , neopentyl glycol diglycidyl ether (NP) followed by glutaraldehyde, or DENACOL EX-411 (411) followed by glutaraldehyde . In 1.5 ml Eppendorf tubes, samples of uncrosslinked subtilisin crystals and crosslinked subtilisin crystal slurry, equal to 37.5 mg of enzyme, were microfuged at 5, 000 rpm for 5 min and the supernatant liquid was removed. A 1.5 ml aliquot of PBS buffer (0.01 M phosphate, 0.0027 M potassium chloride, 0.137 M sodium chloride, pH 7.4) was added to each sample, bringing the concentration of subtilisin to 25 mg/ml. The samples were transferred to 2 ml glass vials with screw caps and magnetic stir bars then were incubated at 30°C or at 37°C. Samples were studied for dissolution by periodically removing 50 μl of the slurry, microfugmg at 13,000 rpm for 5 mins, removing 20 μl of the aliquot and placing it in 980 μl of deionized water, then measuring UV absorbance at 280 nm. The following samples were studied: Crosslinker Crosslinker Concentration Crosslmkmg Time GA 1.0% 1.5h GA 0.25% 2h GA 0.2% 2h GA 0.15% 2h NP/GA 0, .1%/0.1% 5h/1.5h 411/GA 0, .015%/0.035% 16h/lh OA 0.2% 16h OA 0.1% 16h OA 0.05% 16h The solubility profiles of the samples, shown m Figures 8 and 9, illustrate different rates of dissolution for the crosslinked crystals. Example 14 - Reversible Crosslmkers - Disulfide Crosslinked Subtilis Crystals We prepared subtilism crystals (30-40 μm average, 27 mg/ml in Na 2 S0 4 ) as previously described for subtilism crystallization. We then crosslinked the crystals using one of the following crosslmkers: 1) Dimethyl 3, 3 ' -dιthιobιspropιonιmιdate » HCl - (DTBP) (Pierce) 2) Dithiobis (succinimidylpropionate) - (DSP) (Pierce) 3) 3, 3'- Dithiobis (sulfosuccmimidylpropionate) - (DTSSP) (Pierce) . Crosslmkmg was carried out m 15 ml neoprene screw cap tubes by placing 740 μl of subtilism crystal slurry (20 mg) m 9.26 ml of buffer (25 mM NaC0 3 /50 M NaHC0 3 , pH 8.0). One crosslmker was added to each tube as follows: A) 93 mg DTBP (30 mM) B) 100 mg DTSSP (16 mM) C) 120 mg DSP (30 mM) . The tubes were tumbled at ambient temperature (24-26°C) until all samples were determined to be insoluble m 32 mM NaOH (5 days) - 100 μl sample in 300 μl NaOH. Uncrosslmked samples were readily soluble m 32 mM NaOH at the same concentrations. Crosslmkmg was stopped by the addition of 1 ml of 1 M Tris, pH 7.5. The samples were centrifuged at 3,000 rpm for 5 minutes, the supernatant removed and replaced by 5 ml of 100 mM Tris, pH 7.5. Centπfugation at 3,000 rpm, for 5 mm, followed by replacement of supernatant with 5 ml of 100 mM Tris (pH 7.5) was repeated 3x . Example 15 - Dissolution of Disulfide Bond-Containing Crosslinked Subtilism Crystals A 200 mM solution of cysteme was prepared by dissolving 121 mg cysteme m 5 ml 100 mM Tris (pH 7.5) . A 400 μl aliquot of the cysteme solution was added to 3 x 750 μl vials. A 400 μl aliquot of 100 mM Tris (pH 7.5) was added to another 3 x 750 μl vials. Each crosslinked sample (100 μl) was added to one vial containing cysteme and one vial without cysteme. All samples were incubated at 37°C and monitored for dissolution of crosslinked enzyme crystals (direct visual and microscopic observation) . After incubation for 3 hrs at 37°C, the DTBP sample appeared to be fully soluble m the presence of cysteme and insoluble m its absence. The DTSSP sample appeared to be nearly fully soluble the presence of cysteme and insoluble m its absence. The DSP sample was barely soluble in the presence of cysteme and insoluble m its absence. _ - 78 - Example 16 - Crystallization of Candida Ruσosa Lipase A 5 kg aliquot of Candida rugosa lipase ("CRL") m powder form (Meito) was mixed with 5 kg celite and dissolved in 102 L distilled deionized water and the volume brought to 200 L with the deionized water. The suspension was mixed m an Air Drive Lightning Mixer for 2 hours at room temperature and then filtered through a 0.5 micron filter to remove celite. The mixture was then ultraflltered and concentrated to 14 L (469 g) using a 3K hollow fiber filter membrane cartridge. Solid calcium acetate was added to a concentration of 5mM Ca(CH 3 COO) 2 . The pH was adjusted to pH 5.5 with concentrated acetic acid as necessary. A 7 litre aliquot was crystallized by either addition of 1.75 litres of 2-methyl-2, 4- pentanediol ("MPD") or by addition of 3.5 litres of a 30% solution of PEG-8000. The resulting solution was mixed and crystallization allowed to proceed overnight at ambient temperature for about 17-20 hrs . The crystal yield was about 70%. Recrystallization The Candida rugosa lipase crystals were solubilized by the addition of 50 mM sodium phosphate (pH 5.2). Soluble protein concentration of the crystallization solution was adjusted to 20 mg/ml. MPD was added gradually with stirring over a 6-hour period, to a final concentration of 25%. The resulting solution was mixed and crystallization allowed to proceed at ambient temperature for 20 hours. Example 17 - Crystallization of Candida Rugosa Lipase Candida rugosa lipase crystals prepared as described Example 16, prior to the solubilization and recrystallization steps, were solubilized by the addition of 50 mM sodium acetate (pH 6.5). Soluble protein concentration of the crystallization solution was adjusted to 20 mg/ml. MPD was added gradually with stirring over a 6-hour period to a final concentration of 20%. The resulting solution was mixed and crystallization allowed to proceed at ambient temperature for 20 hours. Example 18 - Crosslinking of Candida Rugosa Lipase Crystals Candida rugosa lipase crystals, prepared as described in Example 16, were crosslinked by addition of untreated neat glutaraldehyde (Sigma) by adding 2 ml of 20% glutaraldehyde stepwise in a 40.5 ml volume over one hour to 8 ml of stirred lipase crystals (25 mg/ml) , at ambient temperature. The final crosslinker concentration was 4.0%. Crosslinking was allowed to proceed over 24 hours. Crystals were recovered by low speed centrifugation and washed with 25% MPD in 50 mM sodium phosphate (pH 5.2) . Example 19 - Crosslinking of Candida Rugosa Lipase Crystals Candida rugosa lipase crystals, prepared as described in Example 16, were crosslinked by addition of untreated neat glutaraldehyde by adding 2 ml of 20% glutaraldehyde gradually over a one hour period. Crystals were crosslinked and processed as described in Example 18. _ . - 80 - Example 20 - Crosslmkmg of Candida Rugosa Lipase Crystals Candida rugosa lipase crystals, prepared as described m Example 16, were crosslinked as described 5 m m Example 19, except that the reaction was allowed to proceed for 24 hours. The crystals were then processed as described m Example 18. Example 21 - Crosslmkmg of Candida Rugosa Lipase Crystal? 10 Candida rugosa lipase crystals, prepared as described m Example 17, were crosslinked by addition of glutaraldehyde to a final concentration of 4.0%. Crosslmkmg was allowed to proceed for 3 hours. The crystals were processed as described m Example 18. 15 Example 22 - Crosslmkmg of Candida Rugosa Lipase Crystals Candida rugosa lipase crystals, prepared as described m Example 17, were crosslinked m neat glutaraldehyde at a concentration of 6.5% for 1 hour. 20 Crosslmkmg and processing were performed as described m Example 18. Example 23 - Crosslmkmg of Candida Rugosa Lipase Crystals Candida rugosa lipase crystals, prepared as 25 described in Example 17, were crosslinked neat glutaraldehyde at a concentration of 6.0% for 1 hour. Crossl kmg and processing were performed as described Example 18. Example 24 - pH Controlled Solubility of Crosslinked Candida Rugosa Lipase Crystals Solubility of various crosslinked Candida rugosa lipase crystals was studied following an increase in pH from 6.5 to 9.0. The crystals were incubated at 1 mg/ml in 50 mM sodium phosphate (pH 9) containing 25% MPD. Aliquots were removed after 3 hour and 24 hour incubation at 25°C with stirring. Activity and soluble protein concentration were measured as described in Example 25. The results are described in the table below. Crosslinked Crystal Time (hr) Preparation 3 24 Activity (U) [Prot. ] Activity (U) [Prot. ] (mg/ml) (mg/ml) Example 18 7.5 0.47 20 1 Example 19 10.8 0.60 11.3 0.63 Example 20 7.5 0.42 8.8 0.49 Example 25 - pH Solubility of Crosslinked Candida Rugosa Lipase Crystals Solubility of various crosslinked Candida rugosa lipase crystals was studied following an increase in pH from 5.2 to 7.5. The crystals were incubated at 1 mg/ml in 50 mM sodium phosphate (pH 7.5) containing 25% MPD. Aliquots were removed after 3 hour and 24 hour incubation at 25°C with stirring. Insoluble material was removed by filtration (0.25 micron) . Activity in solution was measured spectrophotometrically by monitoring the hydrolysis of para nitrophenyl acetate (Fluka) at 400 nm. Substrate concentration was 1 mM. The assay was performed at 25°C in a 1 ml volume of 50 mM sodium acetate (pH 6.5) . Soluble protein concentration was measured by absorbance at 280 mm. Results are presented in the table below. Crosslinked Crystal Time (hr) Preparation 3 24 Activity(U) [Prot.] Activity(U) [Prot. ] (mg/ml) (mg/ml) Example 21 2.4 0.12 15 0.91 Example 22 10.0 0.63 15 1.0 Example 23 2.5 0.17 11 0.69 Example 26 - Crystallization of Human Serum Albumin Human serum albumin ("HSA") was purchased from Sigma Chemical Company as a lyophilized powder. We added 10 grams of protein powder to a 75 ml stirred solution of 100 mM phosphate buffer pH 5.5 at 4°C. Final protein concentration was 120 mg/ml (determined from OD 280 , extinction coefficient for serum albumin was assumed to equal 1) . Saturated ammonium sulfate solution (767 g/1) prepared in deionized water was added to the protein solution to a final concentration of 50% saturation (350 g/1). The crystallization solution was "seeded" with 1 ml of albumin crystals (50 mg/ml) in 50% ammonium sulfate (pH 5.5). Seed crystals were prepared by washing a sample of crystals free of precipitate with a solution of 50% saturated ammonium sulfate in 100 mM phosphate buffer (pH 5.5) . The seeded crystallization solution was incubated at 4°C overnight on a vigorously rotating platform. Crystal rods (20 μ) appeared in the solution overnight (16 hr) . SUBSTITUTE SHEET (RULE 261 Example 27 - Crosslinking of Human Serum Albumin Crystals We crosslinked human serum albumin crystals, prepared as described in Example 18, at 4°C in a 10 ml stirred solution of crystals and mother liquor containing 50% saturated ammonium sulfate, as described above. The crystals, which were not washed prior to crosslinking, were crosslinked with glutaraldehyde as supplied by the manufacturer (Sigma) . Glutaraldehyde ("GA") (20%) was added to the stirred crystallization solution in 4 equal volumes (62.5 μl) at 15 minute intervals to a final concentration of 0.5% (250 μl GA) . The crystals were then incubated at 4°C. Aliquots were removed at incubation times 0, 30 min, 60 min and 4 hours incubation. Crosslinked albumin crystals were collected by low speed centrifugation and washed repeatedly with pH 7.5, 100 mM Tris HC1, 4°C. Washing was stopped when the crystals could be centrifuged at high speed without aggregation. Example 28 - Crosslinking of Human Serum Albumin Crystals We crosslinked human serum albumin crystals as described in Example 27 above, with one modification; glutaraldehyde (20%) was added to the crystallization solution in 4 equal volumes (131.3 μl) at 15 minute intervals to a final concentration of 1% (525 μl GA) . Example 29 - Solubility of Human Serum Albumin Crystals Crosslinked in 0.5% GA, time: 0 minutes incubation. Dissolution Induced by Elevated Temperature Human serum albumin, crystallized as described in Example 26 and crosslinked for 0 minutes - 84 - in 0.5% glutaraldehyde, as described in Example 27, was assayed for solubility by incubating the crystals (20 mg/ml) with stirring, in phosphate buffered saline solution (pH 7.5) at room temperature ("RT") or at 37°C. Aliquots were removed for assay at times 0.5, 1, 4 and 24 hours. Insoluble material was removed from the solution by centrifugation and the soluble protein concentration was measured spectrophotometrically at 280 nm, as indicated in Table XV. TABLE XV Example 30 - Solubility of Human Serum Albumin Crystals Crosslinked in 0.5% GA, time: 30 minutes incubation. Dissolution Induced by Elevated Temperature Human serum albumin, crystallized as described in Example 26 and crosslinked in 0.5% glutaraldehyde, as described in Example 27, was assayed for solubility by incubating the crystals (20 mg/ml) in phosphate buffered saline solution (pH 7.5) at room temperature or at 37°C. Aliquots were removed for assay at times 0.5, 1, 4 and 24 hours. Insoluble material was removed from the solution by centrifugation and the soluble protein concentration was measured spectrophotometrically at 280 nm, as indicated in Table XVI. 15 - TABLE XVI Example 31 - Solubility of Human Serum Albumin Crystals Crosslinked m 0.5% GA, time: 60 minutes incubation. Dissolution Induced by Elevated Temperature Human serum albumin, crystallized as described m Example 26 and crosslinked with 0.5% glutaraldehyde, as described Example 27, was assayed for solubility by mcubatmg the crystals (20 mg/ml) m phosphate buffered salme solution (pH 7.5) at room temperature or at 37°C. Aliquots were removed for assay at times 0.5, 1, 4 and 24 hours. Insoluble material was removed from the solution by centrifugation and the soluble protein concentration was measured spectrophotometrically at 280 nm, as indicated m Table XVII. TABLE XVII Example 32 - Solubility of Human Serum Albumin Crystals Crosslinked in 0.5% GA, time: 240 minutes incubation. Dissolution Induced by Elevated Temperature Human serum albumin, crystallized as described in Example 26 and crosslinked with 0.5% glutaraldehyde, as described in Example 27, was assayed for solubility by incubating the crystals (20 mg/ml) in phosphate buffered saline solution (pH 7.5) at room temperature or at 37°C. Aliquots were removed for assay at times 0.5, 1, 4 and 24 hours. Insoluble material was removed from the solution by centrifugation and the soluble protein concentration was measured spectrophotometrically at 280 nm, as indicated in Table XVIII. TABLE XVIII Example 33 - Solubility of Human Serum Albumin Crystals Crosslinked in 1.0% GA, time: 0 minutes incubation. Dissolution Induced by Elevated Temperature Human serum albumin, crystallized as described in Example 26 and crosslinked as described in Example 27, was assayed for solubility by incubating the crystals (20 mg/ml) in phosphate buffered saline solution (pH 7.5) at room temperature or at 37°C. Aliquots were removed for assay at times 0.5, 1, 4 and 24 hours. Insoluble material was removed from the π - solution by centrifugation and the soluble protein concentration was measured spectrophotometrically at 280 nm, as indicated m Table XIX. TABLE XIX Example 34 - Solubility of Human Serum Albumin Crystals Crosslinked in 1.0% GA, time: 30 minutes incubation. Dissolution Induced by Elevated Temperature Human serum albumin, crystallized as described m Example 26 and crosslinked as descrioed m Example 27, was assayed for solubility by mcubatmg the crystals (20 mg/ml) m phosphate buffered salme solution (pH 7.5) at room temperature or at 37°C. Aliquots were removed for assay at times 0.5, 1, 4 and 24 hours. Insoluble material was removed from the solution by centrifugation and the soluble protein concentration was measured spectrophotometrically at 280 nm, as indicated m Table XX. TABLE XX Example 35 - Solubility of Human Serum Albumin Crystals Crosslinked in 1.0% GA, time: 60 minutes incubation. Dissolution Induced by Elevated Temperature Human serum albumin, crystallized as described in Example 26 and crosslinked as described in Example 27, was assayed for solubility by incubating the crystals (20 mg/ml) in phosphate buffered saline solution (pH 7.5) at room temperature or at 37°C. Aliquots were removed for assay at times 0.5, 1, 4 and 24 hours. Insoluble material was removed from the solution by centrifugation and the soluble protein concentration was measured spectrophotometrically at 280 nm, as indicated in Table XXI. TABLE XXI 19 - Example 35 - Solubility of Human Serum Albumin Crystals Crosslinked 1.0% GA, time: 240 minutes incubation. Dissolution Induced by Elevated Temperature Human serum albumin, crystallized as described m Example 26 and crosslinked as described m Example 27, was assayed for solubility by mcubatmg the crystals (20 mg/ml) m phosphate buffered salme solution (pH 7.5) at room temperature or at 37 C C. Aliquots were removed for assay at times 0.5, 1, 4 and 24 hours. Insoluble material was removed from the solution by centrifugation and the soluble protein concentration was measured spectrophotometrically at 280 nm, as indicated m Table XXII. TABLE XXII Example 36 - Crystallization of Thermolysm Thermolysm was purchased from Diawa (Japan) as a lyophilized powder. Fifteen grams of protein powder were added to a 100 ml stirred solution of 10 mM calcium acetate (pH 11) at ambient temperature. The pH was maintained at 11 by addition of 2 N NaOH, until the thermolysm was completely solubilized. The pH was then adjusted to pH 7.5 by addition of 2 N acetic acid. Crystallization was allowed to proceed overnight at 4°C. Final protein concentration was 40 mg/ml - 90 - (determined from OD 280 , extinction coefficient for thermolysm was assumed to equal 1.8). Crystals were recovered by centrifugation and recrystallized to obtain a more uniform crystal size. Recrystallization 5 was performed m a manner nearly identical to that described for the initial crystallization. Crystals (40 mg/ml protein) were dissolved by addition of base at room temperature. The pH of the crystallization solution was adjusted to 6.5 and crystallization was 10 permitted to proceed at ambient temperature. Crystal rods (50 μ) appeared the solution overnight (16 hr) . Example 37 - Crossl kmg of Thermolysm Crystals Thermolysm crystals, prepared as described m Example 36, were suspended (50 mg/ml) a 50 mM 15 solution of sodium acetate (pH 6.5). Crystals were crosslinked with glutaraldehyde as supplied by the manufacturer (Sigma) . Ten milliliters of glutaraldehyde (10%) were added gradually over a 1 hour period with stirring to a 10 ml suspension of crystals. 20 After all of the glutaraldehyde was added, the crystallization solution incubated at ambient temperature. Aliquots were removed at incubation times 0.5, 1 and 3 hr. Crosslinked crystals were collected by low speed centrifugation and washed exhaustively 25 with pH 7.5 50 mM Tris HCl, containing 10 mM calcium acetate . Example 38 - Solubility of Thermolysm Crystals Crosslinked for 0.5 hr . Dissolution Induced by Removal of Calcium Ions by EDTA 30 Thermolysm, crystallized as described Example 36 and crosslinked for 0.5 hr as described m Example 37, was assayed for solubility by mcubatmg 2 - 91 - the crystals (1 mg/ml) with stirring, m 10 mM Tris HCl (pH 7.2) containing 1 mM EDTA (Sigma) 40°C. One ml aliquots were removed for assay at times 0.5, 3 and 24 hours. Insoluble crystals were removed from the solution by filtration. One ml of 500 mM calcium acetate (pH 7.2) was added to each aliquot. Soluble protein concentration was measured spectrophotometrically at 280 nm. Enzymatic activity was measured spectrophotometrically by monitoring the hydrolysis of a dipeptide substrate, FAGLA (Feder) . Substrate concentration was 1.67 mM. One unit is defined as the amount of enzyme required to hydrolyze 1 μmole of substrate in one mmute at pH 7.2, 40°C. The activity of soluble thermolysm was 27 U/mg protein. Data is presented m Table XXIII. TABLE XXIII Example 39 - Solubility of Thermolysm Crystals Crosslinked for 1 hr. Dissolution Induced by Removal of Calcium Ions by EDTA Thermolysm, crystallized as described m Example 36 and crosslinked for 1 hr as described m Example 37, was assayed for solubility by cubatmg the crystals (1 mg/ml) with stirring, m 10 mM TrisHCl (pH 7.2) containing 1 mM EDTA 40°C. One ml aliquots were removed for assay at times 0.5, 3 and 24 hours. Insoluble crystals were removed from the solution by 92 - filtration. One ml of 500 mM calcium acetate (pH 7.2) was added to each aliquot. Soluble protein concentration was measured spectrophotometrically at 280 nm. Enzymatic activity was measured spectrophotometrically by monitoring the hydrolysis of a dipeptide substrate, FAGLA (Feder) . Substrate concentration was 1.67 mM. One unit is defined as the amount of enzyme required to hydrolyze 1 μmole of substrate m one m ute at pH 7.2, 40°C. The activity of soluble thermolysm was 27 U/mg protein. Data is presented m Table XXIV. TABLE XXIV Example 40 - Solubility of Thermolysm Crystals Crosslinked for 3 hr . Dissolution Induced by Removal of Calcium Ions by EDTA Thermolysm, crystallized as described Example 36 and crosslinked for 3 hr as described m Example 37, was assayed for solubility by mcubatmg the crystals (1 mg/ml) with stirring, m 10 mM TrisHCl (pH 7.2) containing 1 mM EDTA 40°C. One ml aliquots were removed for assay at times 0.5, 3 and 24 hours. Insoluble crystals were removed from the solution by filtration. One ml of 500 mM calcium acetate (pH 7.2) was added to each aliquot. Soluble protein concentration was measured spectrophotometrically at 280 nm. Enzymatic activity was measured - 93 - spectrophotometrically by monitoring the hydrolysis of a dipeptide substrate, FAGLA (Feder) . Substrate concentration was 1.67 mM. One unit is defined as the amount of enzyme required to hydrolyze 1 μmole of substrate in one m ute at pH 7.2, 40°C. The activity of soluble thermolysm was 27 U/mg protein. Data is presented m Table XXV. TABLE XXV Example 41 - Solubility of Thermolysm Crystals Crosslinked for 3 hr . Dissolution Induced by Removal of calcium ions by dilution Thermolysm crystals, prepared as described m Example 36 and crosslinked for 3 hr as described m Example 37, were washed free of calcium containing buffer and assayed for solubility by mcubatmg the crystals (1 mg/ml) with stirring m deionized water. One ml aliquots were removed for assay at times 0.5, 3 and 24 hours. Insoluble crystals were removed from the solution by filtration. One ml of 500 mM calcium acetate (pH 7.2) was added to each aliquot. Soluble protein concentration was measured spectrophotometrically at 280 nm. Enzymatic activity was measured spectrophotometrically by monitoring the hydrolysis of a dipeptide substrate, FAGLA (Feder) . Substrate concentration was 1.67 mM. One unit is defined as the amount of enzyme required to hydrolyze 1 94 - μmole of substrate m one mmute at (pH 7.2), 40°C, The activity of soluble thermolysm was 27 U/mg protein. Data is presented m Table XXVI. TABLE XXVI Example 42 - Crystallization of Glucose Isomerase Glucose isomerase ("GA") was supplied by Cultor (Finland) as a crystal slurry. The enzyme was recrystallized by solubilizmg a 50 ml volume of the crystal slurry at 50°C with stirring for 15 minutes. The solution was clarified by filtration and allowed to cool slowly at room temperature. Fifty micron crystals appeared withm 5 hours. Crystals were recovered by low speed centrifugation and washed with 166 mM magnesium sulfate. Example 43 - Crosslmkmg of Glucose Isomerase Crystals Five hundred milligrams of glucose isomerase crystals, prepared as described m Example 42, were suspended m a 50 ml solution of 166 mM magnesium sulfate. The crystals were crosslinked with glutaraldehyde as supplied by the manufacturer (Sigma) . Five milliliters of glutaraldehyde (10%) were added gradually over a 1 hour period with stirring to the 50 ml suspension. After all of the glutaraldehyde was added, the crystallization solution incubated at ambient temperature. Aliquots were removed at incubation times 1, 3 and 24 hr . Crosslinked crystals were collected by low speed centrifugation and washed exhaustively with 50 mM Tris HCl (pH 7.0) . Example 44 - Solubility of Glucose Isomerase Crystals Crosslinked for 1 hr . Dissolution Induced by Removal of Calcium Ions by Dilution at 50°C Glucose isomerase crystals, prepared as described m Example 42 and crosslinked for 1 hr as described m Example 43, assayed for solubility by mcubatmg the crystals (1 mg/ml) with stirring m deionized water. One ml aliquots were removed for assay at times 1, 3 and 24 hours. Soluble protein concentration was measured spectrophotometrically at 280nm (OD280) (extinction coefficient for GI was assumed to equal 1) . Enzymatic activity was measured spectrophotometrically by monitoring the conversion of fructose to glucose. Glucose concentration was quantitated spectrophotometrically using a coupled enzyme assay containing hexokmase and glucose-6-phosphate dehydrogenase. The dehydrogenase uses NADP as a cofactor and the amount of NADPH formed m the reaction is stoichiometric with the concentration of substrate (glucose) . The assay was purchased as a kit from Boehringer Mannheim and was used according to the manufacturer's instructions. One unit is defined as the amount of enzyme required to convert 1 μmole fructose to glucose in one m ute at pH 7.0, 60°C. The activity of soluble glucose isomerase was 51 U/mg protein. Data is presented Table XXVII. - 96 - TABLE XXVII Example 45 - Solubility of Glucose Isomerase Crystals Crosslinked for 3 hr. Dissolution Induced by Removal of Calcium Ions by Dilution at 50°C Glucose isomerase crystals, prepared as described in Example 42 and crosslinked for 3 hr as described in Example 43, were assayed for solubility by incubating the crystals (1 mg/ml) with stirring in deionized water. One ml aliquots were removed for assay at times 1, 3 and 24 hours. Soluble protein concentration was measured spectrophotometrically at 280nm (OD280) (extinction coefficient for GI was assumed to equal 1). Enzymatic activity was measured spectrophotometrically by monitoring the conversion of fructose to glucose. Glucose concentration was quantitated spectrophotometrically using the coupled enzyme assay containing hexokinase and glucose-6-phosphate dehydrogenase, as described in Example 44. Data is presented in Table XXVIII. - 97 - TABLE XXVIII Example 46 - Solubility of Glucose Isomerase Crystals Crosslinked for 24 hr . Dissolution Induced by Removal of Calcium Ions by Dilution at 50°C Glucose isomerase crystals, prepared as described in Example 42 and crosslinked for 1 hr as described in Example 43, were assayed for solubility by incubating the crystals (1 mg/ml) with stirring in deionized water. One ml aliquots were removed for assay at times 1, 3 and 24 hours. Soluble protein concentration was measured spectrophotometrically at 280nm (OD280) (extinction coefficient for glucose isomerase was assumed to equal 1) . Enzymatic activity was measured spectrophotometrically by monitoring the conversion of fructose to glucose. Glucose concentration was quantitated spectrophotometrically using the coupled enzyme assay containing hexokinase and glucose-6-phosphate dehydrogenase, as described in Example 44. Data is presented in Table XXIX. 98 - TABLE XXIX Example 47 - Preparation of Tablets Containing Crosslinked Protein Crystals According to this Invention Tablets containing crosslinked protein crystals according to this invention may be prepared as follows. A suspension of crosslinked protein crystals is placed m 0.1 M sodium acetate, 20 mM calcium chloride and buffer (pH 7) and dried at 35°C. The resulting dried material may be mixed with sorbitol 50:50 by weight and granulated with Eudragit NE 30D (a neutral copolymer based on ethyl- and methylacrylate) or Eudagit RL 30D (an ammonio-methacrylate copolymer) . The granules are dried (for example, for 16 hours at 40°C) and compressed to round tablets of about 5 mm diameter and weight of about 125 mg. The content of crosslinked protein crystals m such solid preparations is about 45% by weight. If the above-described preparation is made without using sorbitol, the resulting tablets contain about 63% by weight crosslinked protein crystals. When introduced into water or aqueous buffer (such as the above-described acetate buffer) all the tablets disintegrate m a matter of 10 minutes under mild shaking at room temperature) producing particles less than 100 μm in size, the majority in the range of 4-10 μm. Microscopic examination reveals polymer-free singular protein crystals, as the predominant species. The slurry obtained by disrupting the tablets is assayed titrimetπcally using hydrolysis of N(α)-p- tosyl L-argmme methyl ester (TAME) at 25°C (pH 8) . Activity corresponding to between about 50% and 80% of activity of an equal amount of crosslinked protein crystals (counting the indicated weight of the crosslinked crystals, rather than of the whole tablets) results . Example 48 - Crystallization of Candida Ruσosa Lipase Candida rugosa lipase was prepared as described m Example 16. After the addition of solid calcium acetate to 5 mM, however, the pH was adjusted to 4.8 instead of pH 5.5 with concentrated acetic acid as necessary. Next, a seven liter aliquot was crystallized by the addition of 1.4 liters of MPD. The resulting solution was mixed and crystallization was allowed to proceed for 72 hours at 4°C. In the following examples, unless otherwise indicated, lipase crystals were prepared according to this example. Example 49 - Crosslmkmg of Lipase Crystals Lipase crystals were crosslinked using the following crosslinkers: dimethyl 3, 3'- dithiobispropionimidate-HCl (DTBP); dithiobis (succmimidylpropionate) (DSP) ; bismaleimidohexane (BMH) ; bis [Sulfosuccmimidyl] suberate (BS) ; 1,5- dιfluoro-2, -dιnιtrobenzene (DFDNB) ; dimethylsuberimidate- 2HC1 (DMS) ; disucc imidyl glutarate (DSG) ; disulfosuccmimidyl tartarate (Sulfo- DST) ; l-ethyl-3- [3-dιmethylammoproplyl] carbodnmide hydrochloride (EDC) ; ethylene glycolbis [sulfo- succmimidylsuccmate] (Sulfo-EGS) ; N- [γ-maleιmιdo- butyryloxy] succmimide ester (GMBS); N-hydroxysulfo- succmιmιdyl-4-azιdobenzoate (Sulfo-HSAB) ; sulfosuccmιmιdyl-6- [α-methyl-α- (2-pyrιdyldιthιo) toluamido] hexanoate (Sulfo-LC-SMPT) ; bιs-[β-(4- azidosalicylamido) ethyl] disulfide (BASED); NHS-PEG- Vmylsulfone (NHS-PEG-VS) ; and glutaraldehyde (GA) . Dimethyl 3, 3' -dithiobisoropiommidate • HCl (DTBP) Crossl kmg A dimethyl 3, 3' -dithiobispropionimidate ■ HCl (DTBP) solution was prepared by dissolving 27.9 mg of DTBP m 60 μl of water. Next, 40 μl of this solution was added to 21 mg of lipase crystals m 1.5 ml of 10 mM HEPES buffer, pH 8.5 and containing 10 mM calcium chloride and 20% MPD. The crosslmkmg reaction was carried out at ambient temperature for 24 hours with tumbling. After 24 hours, the slurry was centrifuged at 3000 rpm for 5 minutes and the supernatant was discarded. The pellet was then suspended 10 mM HEPES buffer, pH 7.5 and containing 10 mM calcium chloride and 20% MPD. An additional amount (20 μl ) of DTBP solution was added and crosslmkmg was continued for another 24 hours. The crosslmkmg reaction was terminated by washing off the excess reagent with 10 mM sodium acetate buffer, pH 4.8 and containing 10 mM calcium chloride and 20% MPD (five washes with 1 ml of buffer) . Dithiobis (succmimidylpropionate) (DSP) Crosslmkmg A dithiobis (succmimidylpropionate) (DSP) solution was prepared by dissolving 36 mg of DSP m 60 μl of dimethyl sulfoxide (DMSO) . Next, 40 μl of this solution was added to 21 mg of lipase crystals m 1.5 ml of 10 mM HEPES buffer, pH 8.5 and containing 10 mM calcium chloride and 20% MPD. The crosslmkmg reaction was carried out at ambient temperature for 24 hours with tumbling. After 24 hours, the slurry was centrifuged at 3000 rpm for 5 minutes and the supernatant was discarded. The pellet was then suspended m 10 mM HEPES buffer, pH 7.5 and containing 10 mM calcium chloride and 20% MPD. An additional amount (20 μl) of DSP solution was added and crosslmkmg was continued for another 24 hours. The crosslmkmg reaction was terminated by washing off excess reagent with 10 mM sodium acetate buffer, pH 4.8 and containing 10 mM calcium chloride and 20% MPD (five washes with 1 ml of buffer) . Bis Maleim dohexane (BMH) Crosslmkmg A bis maleimidohexane (BMH) solution was prepared by dissolving 12 mg of BMH in 40 μl of dimethyl sulfoxide (DMSO) . Next, 40 μl of this solution was added to 21 mg of lipase crystals m 1.5 ml of 10 mM HEPES buffer, pH 7.5 and containing 10 mM calcium chloride and 20% MPD. The crosslmkmg reaction was carried out at ambient temperature for 24 hours with tumbling. After 24 hours, the slurry was centrifuged at 3000 rpm for 5 minutes and the supernatant was discarded. The pellet was then suspended m 10 mM HEPES buffer, pH 7.5 containing and 10 mM calcium chloride and 20% MPD. An additional amount (20 μl) of BMH solution was added and crosslmkmg was continued for another 24 hours. The crosslmkmg reaction was terminated by washing off the excess reagent with 10 mM sodium acetate buffer, pH 4.8 and containing 10 mM calcium chloride and 20% MPD (five washes with 1 ml of buffer) . Bis rsulfosuccinimidyll uberate (BS) Crosslinking A bis [sulfosuccinimidyl] suberate (BS) solution was prepared by dissolving 29 mg of BS in 50 μl of water. Next, 40 μl of this solution was added to 21 mg of lipase crystals in 1.5 ml of 10 mM HEPES buffer, pH 8.5 and containing 10 mM calcium chloride and 20% MPD. The crosslinking reaction was carried out at ambient temperature for 24 hours with tumbling. After 24 hours, the slurry was centrifuged at 3000 rpm for 5 minutes and the supernatant was discarded. The pellet was then suspended in 10 mM HEPES buffer, pH 7.5 and containing 10 mM calcium chloride and 20% MPD. An additional amount (20 μl) of BS solution was added and crosslinking was continued for another 24 hours. The crosslinking reaction was terminated by washing off the excess reagent with 10 mM sodium acetate buffer, pH 4.8 and containing 10 mM calcium chloride and 20% MPD (five washes with 1 ml of buffer) . 1 , 5-Difluoro-2, 4-dinitrobenzene (DFDNB) Crosslinking A 1, 5-Difluoro-2, 4-dinitrobenzene (DFDNB) solution was prepared by dissolving 10 mg of DFDNB in 40 μl of acetone. Next, 40 μl of this solution was added to 21 mg of lipase crystals in 1.5 ml of 10 mM HEPES buffer, pH 8.5 and containing 10 mM calcium chloride and 20% MPD. The crosslinking reaction was carried out at ambient temperature for 24 hours with tumbling. After 24 hours, the slurry was centrifuged at 3000 rpm for 5 minutes and the supernatant was discarded. The pellet was then suspended in 10 mM HEPES buffer, pH 7.5 and containing 10 mM calcium chloride and 20% MPD. An additional amount (20 μl) of DFDNB solution was added and crosslmkmg was continued for another 24 hours. The crosslmkmg reaction was terminated by washing off the excess reagent with 10 mM sodium acetate buffer, pH 4.8 and containing 10 mM calcium chloride and 20% MPD (five washes with 1 ml of buffer^ . Dimethylsuberimidate- 2HC1 (DMS) Crossl kmg A dimethylsuberimidate- 2HC1 (DMS) solution was prepared by dissolving 33 mg of DMS m 40 μl of dimethyl sulfoxide (DMSO) . Next, 40 μl of this solution was added to 21 mg of lipase crystals m 1.5 ml of 10 mM HEPES buffer, pH 8.5 and containing 10 mM calcium chloride and 20% MPD. The crosslmkmg reaction was carried out at ambient temperature for 24 hours with tumbling. After 24 hours, the slurry was centrifuged at 3000 rpm for 5 minutes and the supernatant was discarded. The pellet was then suspended in 10 mM HEPES buffer, pH 7.5 and containing 10 mM calcium chloride and 20% MPD. An additional amount (20 μl) of DMS solution was added and crosslmkmg was continued for another 24 hours. The crossl kmg reaction was terminated by washing off the excess reagent with 10 mM sodium acetate buffer, pH 4.8 and containing 10 mM calcium chloride and 20% MPD (five washes with 1 ml of buffer) . Disucc imidyl glutarate (DSG) Crosslmkmg A disuccmimidyl glutarate (DSG) solution was prepared by dissolving 17 mg of DSG 50 μl of dimethyl sulfoxide (DMSO) . Next, 40 μl of this solution was added to 21 mg of lipase crystals m 1.5 ml of 10 mM HEPES buffer, pH 8.5 and containing 10 mM - 104 - calcium chloride and 20% MPD. The crosslmkmg reaction was carried out at ambient temperature for 24 hours with tumbling. After 24 hours, the slurry was centrifuged at 3000 rpm for 5 minutes and the 5 supernatant was discarded. The pellet was then suspended m 10 mM HEPES buffer, pH 7.5 containing 10 mM calcium chloride and 20% MPD. An additional amount of DSG solution (20 μl ) was added and crosslmkmg was continued for another 24 hours. The crosslmkmg 10 reaction was terminated by washing off the excess reagent with 10 mM sodium acetate buffer, pH 4.8 and containing 10 mM calcium chloride and 20% MPD (five washes with 1 ml of buffer) . Disulfosuccmimidyl tartarate (Sulfo-DST) Crossl kmg 15 A disulfosuccmimidyl tartarate (Sulfo-DST) solution was prepared by dissolving 27 mg of Sulfo-DST m 50 μl of water. Next, 40 μl of this solution was added to 21 mg of lipase crystals in 1.5 ml of 10 mM HEPES buffer, pH 8.5 and containing 10 mM calcium 20 chloride and 20% MPD. The crosslmkmg reaction was carried out at ambient temperature for 24 hours with tumbling. After 24 hours, the slurry was centrifuged at 3000 rpm for 5 minutes and the supernatant was discarded. The pellet was then suspended m 10 mM 25 HEPES buffer, pH 7.5 and containing 10 mM calcium chloride and 20% MPD. An additional amount (20 μl) of Sulfo-DST solution was added and crosslmkmg was continued for another 24 hours. The crosslmkmg reaction was terminated by washing off the excess 30 reagent with 10 mM sodium acetate buffer, pH 4.8 and containing 10 mM calcium chloride and 20% MPD (five washes with 1 ml of buffer) . l-Ethyl-3- r3-Dimethylaminopropyπ carbodiimide hydrochloride (EDO Crosslinking A l-Ethyl-3- [3-Dimethylaminopropyl] carbodiimide hydrochloride (EDC) solution was prepared by dissolving 10 mg of EDC in 1 ml of water. Next, 200 μl of this solution and 5 mg of solid Sulfo-NHS was added to 21 mg of lipase crystals in 1.5 ml of 10 mM HEPES buffer, pH 8.5 and containing 10 mM calcium chloride and 20% MPD. The crosslinking reaction was carried out at ambient temperature for 24 hours with tumbling. After 24 hours, the slurry was centrifuged at 3000 rpm for 5 minutes and the supernatant was discarded. The pellet was then suspended in 50 mM MEΞ buffer, pH 6 and containing 10 mM calcium chloride and 20% MPD. An additional amount of an EDC + Sulfo-NHS solution (200 μl + 5 mg Sulfo-NHS) was added and crosslinking was continued for another 24 hours. The crosslinking reaction was terminated by washing off the excess reagent with 10 mM sodium acetate buffer, pH 4.8 and containing 10 mM calcium chloride and 20% MPD (five washes with 1 ml of buffer) . Ethylene glycolbis \ sulfosuccinimidylsuccinate 1 (Sulfo- EGS) Crosslinking An ethylene glycolbis [sulfosuccinimidyl succinate] (Sulfo-EGS) solution was prepared by dissolving 33 mg of Sulfo-EGS in 40 μl water. Next, 40 μl of this solution was added to 21 mg of lipase crystals in 1.5 ml of 10 mM HEPES buffer, pH 8.5 and containing 10 mM calcium chloride and 20% MPD. The crosslinking reaction was carried out at ambient temperature for 24 hours with tumbling. After 24 hours, the slurry was centrifuged at 3000 rpm for 5 minutes and the supernatant was discarded. The pellet was then suspended in 10 mM HEPES buffer, pH 7.5 and containing 10 mM calcium chloride and 20% MPD. An additional amount (20 μl) of Sulfo-EGS solution was added and crosslmkmg was continued for another 24 hours. The crossl kmg reaction was terminated by washing off the excess reagent with 10 mM sodium acetate buffer, pH 4.8 and containing 10 mM calcium chloride and 20% MPD (five washes with 1 ml of buffer) . N- . γ-maleιιt-idobutyryloxy1 succmimide ester (GMBS) Crosslmkmg An N- [γ-maleιmιdobutyryloxy] succmimide ester (GMBS) solution was prepared by dissolving 23 mg of GMBS m 50 μl of dimethyl sulfoxide (DMSO) . Next, 40 μl of this solution was added to 21 mg of lipase crystals m 1.5 ml of 10 mM HEPES buffer, pH 8.5 and containing 10 mM calcium chloride and 20% MPD. The crossl kmg reaction was carried out at ambient temperature for 24 hours with tumbling. After 24 hours, the slurry was centrifuged at 3000 rpm for 5 minutes and the supernatant was discarded. The pellet was then suspended in 10 mM HEPES buffer, pH 7.5 and containing 10 mM calcium chloride and 20% MPD. An additional amount (20 μl ) of GMBS solution was added and crosslmkmg was continued for another 24 hours. The crosslmkmg was terminated by washing off the excess reagent with 10 mM sodium acetate buffer, pH 4.8 and containing 10 mM calcium chloride and 20% MPD (five washes with 1 ml of buffer) . N-hydroxysulfosuccιnιmιdyl-4-az dobenzoate (Sulfo-HSAB) Crosslmkmg An N-hydroxysulfosuccmιmιdyl-4-azιdobenzoate (Sulfo-HSAB) solution was prepared by dissolving 5 mg of Sulfo-HSAB in 40 μl of water. Next, 40 μl of this solution was added to 21 mg of lipase crystals m 1.5 ml of 10 mM HEPES buffer, pH 8.5 and containing 10 mM calcium chloride and 20% MPD. The crosslmkmg reaction was carried out at ambient temperature for 24 hours with tumbling. After 24 hours, the slurry was centrifuged at 3000 rpm for 5 minutes and the supernatant was discarded. The pellet was then suspended m 10 mM HEPES buffer, pH 8.5 and containing 10 mM calcium chloride and 20% MPD. A second crosslmkmg reaction was carried out at ambient temperature for 10 minutes with shaking and using a 254 nm UV light. The UV lamp was kept 2.5 cm away from the sample. After 10 minutes, the slurry was centrifuged at 3000 rpm for 5 minutes and the supernatant was discarded. The crosslmkmg reaction was terminated by washing off the excess reagent with 10 mM sodium acetate buffer, pH 4.8 and containing 10 mM calcium chloride and 20% MPD (five washes with 1 ml of buffer). SulfosuccιnιmιdyI-6- fα -methyl- - (2-pyrιdyldιthιo) toluamidol hexanoate (Sulfo-LC-SMPT) Crosslmkmg A sulfosuccmιmιdyl-6- fα-methyl- - (2- pyridyldithio) toluamido] hexanoate (Sulfo-LC-SMPT) solution was prepared by dissolving 12 mg of Sulfo-LC- SMPT m 60 μl of water. Next, 40 μl of this solution was added to 21 mg of lipase crystals m 1.5 ml of 10 mM HEPES buffer, pH 8.5 and containing 10 mM calcium chloride and 20% MPD. The crosslmkmg reaction was carried out at ambient temperature for 24 hours with tumbling. After 24 hours, the slurry was centrifuged at 3000 rpm for 5 minutes and the supernatant was discarded. The pellet was then suspended m 10 mM HEPES buffer, pH 7.5 and containing 10 mM calcium chloride and 20% MPD. An additional amount (20 μl) of Sulfo-LC-SMPT solution was added and crosslinking was continued for another 24 hours. The crosslmkmg reaction was terminated by washing off the excess reagent with 10 mM sodium acetate buffer, pH 4.8 and containing 10 mM calcium chloride and 20% MPD (five washes with 1 ml of buffer) . Bis- TB- (4-azιdosalιcylamιdo) ethyll disulfide (BASED) Crosslmkmg A bis [ β- (4azιdosalιcylamιdo) ethyl] disulfide (BASED) solution was prepared by dissolving 3 mg of BASED in 40 μl of dimethyl sulfoxide (DMSO) . Next, 40 μl of this solution was added to 21 mg of lipase crystals m 1.5 ml of 10 mM HEPES buffer, pH 8.5 and containing 10 mM calcium chloride and 20% MPD. The crosslmkmg reaction was carried out at ambient temperature for 30 minutes with shaking under a 365 nm UV light. The UV lamp was shown on the sample from 2.5 cm away. After 30 minutes, the slurry was centrifuged at 3000 rpm for 5 minutes and the supernatant was discarded. The crosslmkmg reaction was terminated by washing off the excess reagent with 10 mM sodium acetate buffer, pH 4.8 and containing 10 mM calcium chloride and 20% MPD (five washes with 1 ml of buffer) . NHS-PEG-Vmylsulfone (NHS-PEG-VS) Crosslmkmg An NHS-PEG-Vmylsulfone (NHS-PEG-VS) solution was prepared by dissolving 20 mg of NHS-PEG-VS 50 μl m water. Next, 50 μl of this solution was added to 34 mg of lipase crystals prepared as m Example 16 m 1.5 ml of 10 mM HEPES buffer, pH 8.5 and containing 10 mM calcium chloride and 20% MPD. The crosslmkmg reaction was carried out at ambient temperature for 24 hours with tumbling. After 24 hours, the slurry was centrifuged at 3000 rpm for 5 minutes and the supernatant was discarded. The pellet was then suspended m 10 mM HEPES buffer, pH 7.5 and containing 10 mM calcium chloride and 20% MPD. An additional amount (25 μl) of GMBS solution was added and crossl kmg was continued for another 24 hours. The crosslmkmg was terminated by washing off the excess reagent with 10 mM sodium acetate buffer, pH 4.8 and containing 10 mM calcium chloride and 20% MPD (five washes with 1 ml of buffer) . Glutaraldehyde (GA) Crosslmkmg Candida rugosa lipase crystals were prepared as described in Example 16. The crosslmkmg reaction was initiated by the addition of untreated, neat glutaraldehyde (Sigma) to the crystal solution to achieve a final crossl ker concentration of 0.3% or 0.5%. The crosslmkmg reaction was then allowed to proceed for 1 hour. The crosslinked crystals were recovered by low speed centrifugation and washed with 10 mM sodium acetate buffer, pH 4.8 and containing 10 mM calcium acetate and 20% MPD. Example 50 - pH Solubility of Crosslinked Candi da Rugosa Lipase Crystals at 37°C The solubility of Candida rugosa lipase crystals crosslinked as described m Example 49 was evaluated. Crystals crosslinked with the following crosslmkers were included dimethyl 3, 3'- dithiobispropiommidate-HCl (DTBP), dithiobis (succmimidylpropionate) (DSP) , bismaleimidohexane (BMH), bis [Sulfosuccmimidyl] suberate (BS) , 1,5- difluoro-2, 4-dmιtrobenzene (DFDNB) , dimethylsuberimidate -2HC1 (DMS), disuccmimidyl glutarate (DSG) , disulfosuccmimidyl tartarate (Sulfo- DST) , l-ethyl-3- [3-dιmethylammopropyl] carbodiimide hydrochloride (EDC), ethylene glycolbis [sulfo- succimmidylsuccmate] (Sulfo-EGS), N- [γ-maleιmιdo- butyryloxy] succmimide ester (GMBS), N-hydroxysulfo- succmιmιdyl-4-azιdobenzoate (Sulfo-HSAB) , sulfosuccmιmιdyl-6- [ -methyl- - (2-pyrιdyldιthιo) toluamido] hexanoate (Sulfo-LC-SMPT), bιs-[β-(4- azidosalicylamido) ethyl ] disulfide (BASED) and glutaraldehyde (GA) . Samples of uncrosslinked lipase crystals prepared as m Example 16 and crosslinked lipase crystal slurry equal to 2.8 mg of crystalline enzyme were dispensed into 1.5 ml Eppendorf tubes and microfuged at 3000 rpm for 5 minutes. The supernatant liquid was removed and the solubility of the resulting crystals was evaluated at pH 7.4 and pH 2.0. For pH 7.4, a 200 μl aliquot of a 0.01 M phosphate buffer with 0.0027 M potassium chloride and 0.137 M sodium chloride at pH 7.4 (PBS) was added to each sample. Under these conditions the concentration of lipase was 14 mg/ml. Next, the samples were incubated at 37°C for 24 hours. For the pH 2.0 dissolution measurement, the crosslinked crystal samples were initially equilibrated m a 10 mM glycme-HCl buffer. The equilibration buffer was prepared by mixing 80% of 10 mM glycme-HCl buffer with 10 mM calcium chloride at pH 2.0, and 20% of MPD, for a final pH of 4.8. Equilibration was carried out overnight at 25°C with tumbling. The equilibrated samples were then microfuged at 300 rpm - I l l - for 5 minutes and the supernatant liquids discarded. The samples were centrifuged at 3000 rpm for 5 minutes and the pellet was suspended in 200 μl of the Glycine -HCl buffer, pH 2.0. Under these conditions, the concentration of lipase was 14 mg/ml. Finally, the samples were incubated at 37°C for 5 hours. The amount of soluble protein in the supernatant after the 5 hour incubation at pH 2.0 or the 24 hour incubation at pH 7.4 was measured using the Bio-Rad Micro-protein assay. After the incubation period, the samples were centrifuged at 14,000 rpm for 5 minutes. Next, the supernatant was filtered through 0.22 μm cellulose acetate filter (Sigma Chemical Co.). The soluble protein concentration was then measured by diluting 2 μl of the filtered supernatant into 798 μl of deionized water. Next, 200 μl of Bio-Rad Protein assay reagent was added to this sample and incubated at ambient temperature for 5 minutes. The absorbance was measured at 595 nm wavelength and compared to a protein standard curve of 0-20 μg bovine serum albumin from Pierce . Different triggers alone or in combination determine the kinetics of protein release. The following samples were studied and the results are described in the Table XXX below. Table XXX Trigger-* Agitation Agitation Aσitation (PBS) and Acid and Pronase pH 2.0 Protein in filtrate in mg Crosslinker (24 hours) (5 hours) (2 hours) DTBP 0.82 0.73 0.81 DSP 0.74 0.84 0.68 BMH 0.86 1.74 0.53 DSG 0.94 0.79 0.68 SULFO-DST 1.13 0.85 0.75 DFDNB 0 0.01 0.06 BASED 0.89 0.99 0.63 GMBS 0.9 0.86 0.83 BS 0.02 0 0.03 SULFO-HSAB 0.24 0.22 0.22 SULFO-EGS 1.29 0.46 0.64 SULFO-SMPT 0.4 0.09 0.06 EDC 0.07 0.16 0.02 DMS 1.09 0.83 0.81 GA (0.3%) 0.98 0.97 0.13 GA (0.5%) 0.5 0.09 0.02 Soluble 2.8 2.8 2.8 Example 51 - Lipase Activity using olive oil as The activity of various crosslinked enzyme crystals was assessed by measuring the hydrolysis of a substrate containing olive oil. During the reaction, the pH was held constant by titrating with 0.05 M NaOH. The reaction was followed with a pH-Stat (Titralab™) electrode from Radiometer, controlled by a VIT90 Video Titrator and a ABU91 Autoburette. Procedure : First, 20 ml of olive oil emulsion was added to the reaction vessel and the reaction mixture was equilibrated to 37°C with stirring. Next, the pH was adjusted to 7.7 using 0.05 M NaOH and an aliquot (34 mg) of crosslinked crystals was added. The pH of the mixture was maintained at 7.7 by titrating with NaOH. The volume of base consumption vs. time (ml/mm) was recorded and plotted. The slope of the initial linear portion of the curve was used to determine the initial reaction rate. a) Temperature of reaction: assay vessel was equilibrated and maintained m 37°C water bath during course of reaction. b) Calculation: Initial rate = base consumption = ml/mm (NaOH) X mm. c) Specific activity (μ moles/mm/mg protein) = initial rate x 1000 x concentration of the titrant/the amount of enzyme (mg) . d) Blank: Without enzyme - Buffer used m the place of enzyme m the reaction mixture. Reagents : a) 3.0 M NaCl (solution A): 34.8 grams of NaCl was added to 150 ml of distilled water and stired. Final volume was made up to 200 ml. b) 75 mM CaCI 2 -2H 2 0 (solution B) : 220 mg of CaCI 2 -2H 2 0 was added to 150 ml of distilled water with stirring. Once the CaCI 2 -2H 2 0 dissolved, the volume was made up to 200 ml. c) Mix: Solution A (40 ml) was added to solution B (20 ml) and H 2 0 (100 ml) . d) 0.5% Albumin: 500 mg of albumin/ 100 ml distilled water, was prepared by dissolving 1 gm of bov e serum albumin Fraction V (Sigma) in water with gentle stirring (avoid forming foam) . After dissolving, the volume was made up to 200 ml. e) An olive oil emulsion was prepared by first dissolving 16.5 gm of gum arable (Sigma) m 130 ml of reagent grade water. Once the gum had dissolved, the volume was increased to 180 ml with distilled water and the solution was filtered through cotton. Next, 20 ml of olive oil (Sigma) was added and an emulsion was generated by mixing in a Quick Prep mixer for 3 minutes . f) Substrate: 50 ml of olive oil emulsion was added to 40 ml of Mix (c) and 10 ml of 0.5% albumin. - 115 - Example 52 - Activity of Crosslinked Candida Rugosa Lipase Crystals The activity of Candida rugosa lipase crystals crosslinked as described in Example 51 was assayed using olive oil as substrate (Table XXXI) . Crystals crosslinked with the following crosslinkers were assayed: dithiobis (succinimidylpropionate) (DSP) ; bismaleimidohexane (BMH) ; NHS-PEG-Vinylsulfone (NHS- PEG-VS); disuccinimidyl glutarate (DSG); l-ethyl-3- [3- dimethylarninopropyl] carbodiimide hydrochloride (EDC) ; sulfosuccinimidyl-6- [α-methyl-α- (2-pyridyldithio) toluamido] exanoate (sulfo-LC-SMPT); and glutaraldehyde (GA) . Table XXXI Crosslinked Crystal Volume Specific Activity Preparation (34 g) added μl units/mg DSP 40 921 BMH 40 1623 NHS-PEG-VS 50 948 DSG 20 933 DSG 40 687 DSG 80 419 EDC 50 1420 EDC 100 1271 EDC 150 686 sulfo-LC-SMPT 20 5756 sulfo-LC-SMPT 100 5624 GA 0.3% 614 GA 0.5% 322 SOLUBLE LIPASE 732 Table XXXI above demonstrates the effects of various crosslinkers and degrees of crosslinking on Candi da rugosa lipase crystals. The amino/sulfo reactive heterobifunctional crosslinker sulfo-LC-SMPT dramatically enhanced the activity of lipase (greater than 7 fold) to an olive oil substrate when compared to the soluble enzyme. The amino reactive (DSP) or the sulfhydryl reactive (BMH) homobifunctional crosslinkers showed slightly enhancing and approximately 2-fold enhancement of activity, respectively, when compared to soluble enzyme. Example 53 - Reversible Crosslinkers - Disulfide Crosslinked Candida Rugosa Lipase Crystals Candida rugosa lipase crystals were prepared as described in Example 48. Samples containing crystals of 20-30 uM in average size and a total protein of 42 mg/ml were crosslinked using either of the reversible crosslinkers: (1) Dimethyl 3, 3' -dithiobispropioni idate -HCl (DTBP) (Pierce) , or (2) Dithiobis (succinimidylpropionate) (DSP) (Pierce), (3) Bis [2- (sulfosuccinimidooxycarbonyloxy) - ethyl] sulfone (sulfo-BSOCOES ) (Pierce) . The crosslinking reaction was carried out in duplicate 1.5 ml microcentrifuge tubes (USA/Scientific) by placing 250 μl of lipase crystal slurry (11.5 mg) into 500 μl of buffer containing 10 mM HEPES, 10 mM calcium acetate, 20% MPD at pH 8.5. Next, the crosslinking reaction was initiated by adding one crosslinker solution to each tube as follows: 2 - 1 17 - A) 50 mM DTBP - 27.9 mg of DTBP was dissolved m 60 μl of water and 20 μl of this solution was added to one tube with the crystals; and B) 14.8 mM DSP - 35.0 mg of DSP was dissolved m 120 μl of DMSO and then 10 μl of this solution was added to one tube with the crystals. C) 7.5 mM Sulfo-BSOCOES - 7.2 mg of sulfo-BSOCOES was dissolved m 60 μl of water and then 20 μl of this solution was added to one tube with the crystals. The tubes with DTBP and DSP were tumbled at ambient temperature (24-26°C) for approximately 2 days or until the sample was determined to be insoluble m 32 mM NaOH. The tube with sulfo-BSOCOES was tumbled at ambient temperature (24-26°C) for approximately 2 days. The solubility test consisted of adding 50 μl of sample to 150 μl of 32 mM NaOH. In this test, uncrosslmked samples were readily soluble m 32 mM NaOH at the same concentrations. The crosslmkmg reaction was terminated by centrifugmg the sample at 3000 rpm for 5 minutes. Next, the supernatant was discarded and washed 3 times with 1 ml of 10 mM Tris -HCl buffer containing 10 mM calcium chloride and 20% MPD at pH 7.0. Example 54 - Dissolution of Disulfide Bond - Containing Crosslinked Candida Rugosa Lipase Crystals A 200 mM solution of cysteme was prepared by dissolving 242 mg of cysteme m 10 ml of 10 mM TRIS HCl buffer containing 10 mM calcium chloride and 20% MPD at pH 7. A 200 μl sample of crosslinked crystal slurry was centrifuged at 3000 rpm for 5 minutes and the supernatant was discarded. The pellet was suspended 200 μl of cysteme containing TRIS buffer. Another 200 μl of crosslinked sample was taken and centrifuged at 3000 rpm for 5 minutes and the supernatant was discarded. This pellet was then suspended in 200 μl of 10 mM Tris -HCl buffer at pH 7.0 without cysteme. All samples were incubated at 37°C for 1 hour and monitored by direct visual and microscopic observation for dissolution m 32 mM NaOH. The sample exposed to DTBP was fully soluble the presence of 200 mM cysteme and insoluble m its absence after incubation for 1 hour at 37°C. The DSP sample was slightly soluble after 1 hour in the presence of cysteme and insoluble its absence. Example 55 - Dissolution by Base Cleavable Crosslinked Candida Rugosa Lipase Crystals A 200 μl sample of crosslinked crystal slurry was centrifuged at 3000 rpm for 5 minutes and the supernatant was discarded. The pellet was suspended 200 μl of Tris buffer and 600 μl of 32 mM NaOH. Another 200 μl of crosslinked sample was taken and centrifuged at 3000 rpm for 5 minutes and the supernatant was discarded. This pellet was then suspended in 200 μl of 10 mM Tπs-HCl buffer at pH 7.0. All samples were incubated at 37°C for 1 hour and monitored by direct visual and microscopic observation for dissolution m 32 mM NaOH. The sample exposed to sulfo-BSOCOES was fully soluble m the presence of NaOH and insoluble m its absence . While we have hereinbefore described a number of embodiments of this invention, it is apparent that our basic constructions can be altered to provide other embodiments which utilize the processes and compositions of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the claims appended hereto rather than by the specific embodiments which have been presented hereinbefore by way of example.

Claims: We Claim : 1. A crosslinked protein crystal, said protein crystal being capable of change from insoluble and stable form to soluble and active form upon a change in the environment surrounding said crystal, said change being selected from the group consisting of: change in temperature, change in pH, change in chemical composition, change from concentrate to dilute form, change in oxidation-reduction potential of the solution, change in the incident radiation, change in transition metal concentration, change in flouride concentration, change in free radical concentration, change in metal chelater concentration, change in shear force acting upon the crystal and combinations thereof. 2. The crosslinked protein crystal according to claim 1, wherein said change from concentrate to dilute form comprises a change in solute concentration. 3. The crosslinked protein crystal according to claim 2, wherein said change in solute concentration comprises an increase or decrease in salt concentration. 4. The crosslinked protein crystal according to claim 3, wherein said change in solute concentration comprises a decrease in salt concentration. 5. The crosslinked protein crystal according to claim 2, wherein said change in solute concentration comprises an increase or decrease in water concentration. 6. The crosslinked protein crystal according to claim 5, wherein said change solute concentration comprises an increase m water concentration. 7. The crosslinked protein crystal according to claim 2, wherein said change m solute concentration comprises an increase or decrease m organic solvent concentration. 8. The crosslinked protein crystal according to claim 2, wherein said change in solute concentration comprises a decrease detergent concentration. 9. The crosslinked protein crystal according to claim 2, wherein said change m solute concentration comprises a decrease in protein concentration . 10. The crosslinked protein crystal according to claim 1, wherein said change from concentrate to dilute form comprises a change m concentration of all solutes from about 2-fold to about 10, 000-fold. 11. The crosslinked protein crystal according to claim 10, wherein said change from concentrate to dilute form comprises a change concentration of all solutes from about 2-fold to about 700-fold. 12. The crosslinked protein crystal according to claim 1, wherein said change in pH comprises a change from acidic pH to basic pH. 13. The crosslinked protein crystal according to claim 1, wherein said change in pH comprises a change from basic pH to acidic pH. 14. The crosslinked protein crystal according to claim 1, wherein said change in temperature comprises an increase or decrease m temperature. 15. The crosslinked protein crystal according to claim 14, wherein said change m temperature is an increase m temperature from a low temperature between about 0°C and about 20°C to a high temperature between about 25°C and about 70°C. 16. The crosslinked protein crystal according to claim 1, wherein said active form of said protein is a form which is active against macromolecular substrates. 17. A crosslinked protein crystal, said protein crystal having a half-life of activity under storage conditions which is greater than at least 2 times that of the soluble form of the protein that is crystallized to form said crystal that is crosslinked and activity similar to that of the soluble form of the protein under conditions of use. 18. A crosslinked protein crystal, said protein crystal being capable of releasing its protein activity at a controlled rate upon exposure to a change m the environment surrounding said crystal, said change being selected from the group consisting of change m pH, change in solute concentration, change m temperature, change m chemical composition, change m shear force acting upon the crystals and combinations thereof . 19. A crosslinked protein crystal, said protein crystal having about a 5 to 10 fold higher protein activity for any one of a macromolecular substrate, a biphasic substrate or a small molecule substrate, as compared with the soluble form of the protein that is crystallized to form the crystals that are crosslinked. 20. A crosslinked protein crystal, said protein crystal having about a 2 to 3 fold higher protein activity for any one of a macromolecular substrate, a biphasic substrate or a small molecule substrate, as compared with the soluble form of the protein that is crystallized to form the crystals that are crosslinked. 21. A crosslinked protein crystal, said protein crystal having about at least 1.2 times higher protein activity for a macromolecular substrate, a biphasic substrate or a small molecule substrate, as compared with the soluble form of the enzyme that is crystallized to form the crystals that are crosslinked. - 124 - 22. A crosslinked lipase crystal, said lipase crystal having about a 5 to 10 fold higher enzyme activity for hydrolysis of a biphasic olive oil substrate, as compared with the soluble form of the 5 lipase that is crystallized to form the crystals that are crosslinked. 23. The crosslinked lipase crystal according to claim 22, said lipase crystal having about a 2 to 3 fold higher enzyme activity for hydrolysis of a 10 biphasic olive oil substrate, as compared with the soluble form of the lipase that is crystallized to form the crystals that are crosslinked. 24. The crosslinked lipase crystal according to claim 23, said lipase crystal having about at least 15 2 times higher protein activity for hydrolysis of a biphasic olive oil substrate, as compared with the soluble form of the lipase that is crystallized to form the crystals that are crosslinked. 25. The crosslinked lipase crystal according 20 to any one of claims 22, 23 or 24, wherein said protein is crosslinked by sulfosuccinimidyl-6- [α-methyl-α- (2- pyridyldithio) toluamido] hexanoate (Sulfo-LC-SMPT) . 26. The crosslinked lipase crystal according to any one of claims 22, 23 or 24, wherein said protein 25 is crosslinked by l-ethyl-3- [3- dimethylaminoproplyl] carbodiimide hydrochloride (EDC). 27. The crosslinked lipase crystal according to any one of claims 22, 23 or 24, wherein said lipase is crosslinked by sulfosuccmιmιdyl-6- [ -methyl- - (2- pyπdyldithio) toluamido] hexanoate (Sulfo-LC-SMPT) . 28. The crosslinked lipase crystal according to any one of claims 22, 23 or 24, wherein said lipase is crosslinked by a crosslinker selected from the group consisting of l-ethyl-3- [3-dιmethylammopropyl ] carbodiimide hydrochloride (EDC) , dithiobis (succmimidylpropionate) (DSP) , bismaleimidohexane (BMH), NHS-PEG-Vmylsulfone (NHS-PEG-VS) and disucc imidyl glutarate (DSG) . 29. The crosslinked protein crystal according to any one of claims 1, 17, 18, 19, 20 or 21, wherein said protein is crosslinked by a homobifunctional crosslmker. 30. The crosslinked protein crystal according to any one of claims 1, 17, 18, 19, 20, or 21, wherein said protein is crosslinked by a heterobifunctional crosslinker. 31. The crosslinked protein crystal according to claim 18, wherein said controlled rate of releasing protein activity is determined by a factor selected from the group consisting of: the degree of crosslmkmg of said crosslinked protein crystal, the length of time of exposure of protein crystal to the crosslmker, the ammo acids residues involved m the crosslinks, whether the crossl ker is homobifunctional or heterobifunctional, the rate of addition of the crosslmkmg agent to said protein crystal, the nature of the crosslinker, the chain length of the crosslmker, the surface area of said crosslinked protein crystal, the size of said crosslinked protein crystal, the shape of said crosslinked protein crystal and combinations thereof. 32. The crosslinked protein crystal according to claim 18, wherein said crystal has a protein activity release rate of between about 0.1% per day and about 100% per day. 33. The crosslinked protein crystal according to claim 18, wherein said crystal has a protein activity release rate between about 0.01% per hour and about 100% per hour. 34. The crosslinked protein crystal according to claim 18, wherein said crystal has a protein activity release rate between about 1% per mmute and about 50% per mmute. 35. The crosslinked protein crystal according to any one of claims 1, 17, 18, 19, 20 or 21, said protein crystal being substantially insoluble and stable m a composition under storage conditions and substantially soluble and active under nditions of use of said composition. 36. The crosslinked protein crystal according to claim 35, wherein said composition is selected from the group consisting of cleaning agents, detergents, personal care compositions, cosmetics, pharmaceuticals, veterinary compounds, vaccines, foods, feeds, diagnostics and formulations for decontamination . 37. The crosslinked protein crystal according to claim 36, wherein said detergent is selected from the group consisting of powdered detergents, liquid detergents, bleaches, household cleaners, hard surface cleaners, industrial cleaners, carpet shampoos and upholstery shampoos. 38. The crosslinked protein crystal according to claim 36, wherein said cosmetic is selected from the group consisting of creams, emulsions, lotions, foams, washes, gels, compacts, mousses, sunscreens, slurries, powders, sprays, foams, pastes, ointments, salves, balms, shampoos, sunscreens and drops . 39. The crosslinked protein crystal according to any one of claims 1, 17, 18, 19, 20 or 21, wherein said protein is an enzyme. 40. The crosslinked protein crystal according to claim 39, wherein said enzyme is selected from the group consisting of hydrolases, lsomerases, lyases, ligases, transferases and oxidoreductases. 41. The crosslinked protein crystal according to claim 40, wherein said enzyme is selected from the group consisting of proteases, amylases, cellulases, lipases and oxidases. 42. The crosslinked protein crystal according to any one of claims 1, 17, 18, 19, 20 or 21, wherein said protein is selected from the group consisting of therapeutic proteins, cleaning agent proteins, personal care proteins, veterinary proteins, - 128 - food proteins, feed proteins, diagnostic proteins and decontamination proteins . 43. The crosslinked protein crystal according to any one of claims 1, 17, 18, 19, 20 or 21, wherein said protein is selected from the group consisting of hormones, antibodies, inhibitors, growth factors, trophic factors, cytokines, lymphokmes, toxoids, growth hormones, nerve growth hormones, bone morphogenic proteins, toxoids, vitamins and nutrients. 44. The crosslinked protein crystal according to any one of claims 1, 17, 18, 19, 20 or 21, wherein said protein is selected from the group consisting of insulin, amylm, erythropoietm, Factor VIII, TPA, dornase- , -1-antιtrιpsm, urease, fertility hormones, FSH, LSH, postridical hormones, tetanus toxoid and diptheπa toxoid. 45. The crosslinked protein crystal according to any one of claims 1, 17, 18, 19, 20 or 21, said crystal having a longest dimension of between about 0.01 μm and about 500 μm. 46. The crosslinked protein crystal according to any one of claims 1, 17, 18, 19, 20 or 21, said crystal having a longest dimension of between about 0.1 μm and about 50 μm. 47. The crosslinked protein crystal according to any one of claims 1, 17, 18, 19, 20 or 21, said crystal having a shape selected from the group consisting of: spheres, needles, rods, plates, rhomboids, cubes, bipryamids and prisms. 48. A composition comprising a crosslinked protein crystal according to any one of claims 1, 17, 18, 19, 20, 21, 22, 23, or 24 said composition being selected from the group consisting of cleaning agents, detergents, personal care compositions, cosmetics, pharmaceuticals, veterinary compounds, vaccines, foods, feeds, diagnostics and formulations for decontamination . 49. The composition according to claim 48, wherein said detergent is selected from the group consisting of powdered detergents, liquid detergents, bleaches, household cleaners, hard surface cleaners, industrial cleaners, carpet shampoos and upholstery shampoos . 50. The composition according to claim 48, wherein said cosmetic is selected from the group consisting of creams, emulsions, lotions, foams, washes, gels, compacts, sunscreens, slurries, powders, sprays, foams, pastes, ointments, salves, balms, shampoos, sunscreens and drops. 51. A protein delivery system, said system comprising crosslinked protein crystals according to any one of claims 1, 17, 18, 19, 20, 21, 22, 23, or 24. 52. The protein delivery system according to claim 51, wherein said protein is selected from the group consisting of: detergent enzymes, cosmetic proteins, pharmaceutical proteins, agricultural proteins, vaccine proteins and decontamination proteins . 53. The protein delivery system according to claim 52, said protein delivery system being a microparticulate protein delivery system. 54. The protein delivery system according to claim 53, wherein said microparticulate protein delivery system comprises crosslinked protein crystals having a longest dimension between about 0.01 μm and about 500 μm. 55. The protein delivery system according to claim 54, wherein said microparticulate protein delivery system comprises crosslinked protein crystals having a longest dimension of between about 0.1 μm and about 50 μm. 56. The protein delivery system according to claim 53, wherein said microparticulate protein delivery system comprises crosslinked protein crystals having a shape selected from the group consisting of: spheres, needles, rods, plates, rhomboids, cubes, bipryamids and prisms. 57. A detergent formulation comprising a crosslinked protein crystal according to any one of claims 1, 17, 18, 19, 20 or 21. 58. A controlled release formulation comprising a crosslinked protein crystal according to any one of claims 1, 17, 18, 19, 20 or 21. 59. A pharmaceutical controlled release formulation comprising a crosslinked protein crystal according to any one of claims 1, 17, 18, 19, 20 or 21. 60. A pharmaceutical controlled release formulation comprising a crosslinked protein crystal, said crystal being substantially insoluble under storage conditions and capable of releasing its protein activity m. vivo at a controlled rate. 61. The pharmaceutical controlled release formulation according to claim 59, said pharmaceutical being capable of administration by parenteral or non- parenteral routes . 62. The pharmaceutical controlled release formulation according to claim 61, said pharmaceutical being capable of administration by oral, pulmonary, nasal, aural, anal, dermal, ocular, intravenous, intramuscular, mtraarterial, mtraperitoneal, mucosal, sublmgual, subcutaneous or mtracranial route. 63. The pharmaceutical controlled release formulation according to claim 59, wherein said pharmaceutical is capable of administration by oral route and said crosslinked protein crystal is substantially insoluble under gastric pH conditions and substantially soluble under small intestine pH conditions . 64. A vaccine comprising a crosslinked protein crystal according to any one of claims 1, 17, 18, 19, 20 or 21. 65. A formulation comprising a crosslinked protein crystal according to any one of claims 1, 17, 18, 19, 20, 21, 22, 23, or 24 said formulation being selected from the group consisting of tablets, 7 - 132 - liposomes, granules, spheres, microspheres, microparticles and capsules. 66. A method for producing crosslinked protein crystals comprising the step of reacting protein crystals with a first crosslmkmg agent, or a first crossl kmg agent and at least a second crosslmkmg agent, under conditions sufficient to induce crosslmkmg of said crystals to the extent that the resulting crosslinked crystals are characterized by the ability to change from insoluble and stable form to soluble and active form upon a change m their environment, said change being selected from the group consisting of change temperature, change m pH, change m chemical composition, • change from concentrate to dilute form, change in oxidation-reduction potential of the solution, change in the incident radiation, change m transition metal concentration, change m flouπde concentration, change m free radical concentration, change m metal chelater concentration, change m shear force acting upon the crystals and combinations thereof. 67. A method for producing crosslinked protein crystals comprising the step of reacting protein crystals with a first crossl kmg agent, or a first crosslmkmg agent and at least a second crosslmkmg agent, under conditions sufficient to induce crosslmkmg of said crystals to the extent that the resulting crosslinked crystals are characterized by a half-life of activity under storage conditions which is greater than at least 2 times that of the soluble form of the protein that is crystallized to form said crystals that are crosslinked and activity similar to that of the soluble form of the protein under conditions of use. 68. A method for producing crosslinked protein crystals comprising the step of reacting protein crystals with a first crosslmkmg agent, or a first crosslmkmg agent and at least a second crosslmkmg agent, under conditions sufficient to induce crossl kmg of said crystals to the extent that the resulting crosslinked crystals are characterized by being capable of releasing their protein activity at a controlled rate upon exposure to a change in their environment, said change being selected from the group consisting of change pH, change m soluble concentration, change in temperature, change m chemical composition, change in oxidation-reduction potential of the solution, change the incident radiation, change transition metal concentration, change m flouride concentration, change m free radical concentration, change m metal chelater concentration, change m shear force acting upon the crystals and combinations thereof. 69. The method for producing crosslinked protein crystals according to any one of claims 66, 67 or 68, comprising the step of reacting said protein crystals with said first crosslmkmg agent and said at least a second crosslmkmg agent at the same time or m sequence. 70. The method for producing crosslinked protein crystals according to any one of claims 66, 67 or 68, wherein, prior to reacting protein crystals with said crosslmkmg agent, said method further comprises the step of crystallizing said protein. 71. The method for producing crosslinked protein crystals according to any one of claims 66, 67 or 68, wherein the conditions sufficient to induce crosslmkmg are dependent upon a factor selected from the group consisting of: the degree of crosslmkmg of said crosslinked protein crystals, the length of time of exposure of protein crystals to the crosslmkmg agent, the rate of addition of the crosslmkmg agent to said protein crystal, the nature of the crosslmker, the chain length of the crosslmker, the surface area of said crosslinked protein crystals, the size of said crosslinked protein crystals, the shape of said crosslinked protein crystals and combinations thereof. 72. The method for producing crosslinked protein crystals according to claim 68, wherein said controlled rate of releasing protein activity is determined by a factor selected from the group consisting of: the degree of crosslmkmg of said crosslinked protein crystals, the length of time of exposure of protein crystals to the crosslmkmg agent, the rate of addition of the crosslmkmg agent to said protein crystals, the nature of the crosslinker, the chain length of the crosslinker, the surface area of said crosslinked protein crystals, the size of said crosslinked protein crystals, the shape of said crosslinked protein crystals and combinations thereof. 73. The method for producing crosslinked protein crystals according to any one of claims 66, 67 or 68, wherein said crossl kmg agent is a multifunctional crossl kmg agent. 74. The method for producing crosslinked protein crystals according to claim 73, wherein said crosslmkmg agent is a bifunctional crosslmkmg agent . 75. The method for producing crosslinked protein crystals according to claim 73, wherein said crosslmkmg agent is selected from the group consisting of: glutaraldehyde, succinaldehyde, octanedialdehyde and glyoxal. 76. The method for producing crosslinked protein crystals according to claim 73, wherein said crosslmkmg agent is selected from the group consisting of: halo-triazmes, halo-pyrimidmes, anhydrides of aliphatic or aromatic mono- or di- carboxylic acids, halides of aliphatic or aromatic mono- or di-carboxylic acids, N-methylol compounds, di- lsocyanates, di-isothiocyanates and aziridmes. 77. The method for producing crosslinked protein crystals according to any one of claims 66, 67 or 68, wherein said crosslmkmg agent is an epoxide. 78. The method for producing crosslinked protein crystals according to claim 77, wherein said epoxide is selected from the group consisting of: neopentyl glycol diglycidyl ether, ethylene glycol diglycidyl ether, di-epoxides, tri-epoxides and tetra- epoxides . _ - 136 - 79. The method for producing crosslinked protein crystals according to any one of claims 66, 67 or 68, wherein said crossl kmg agent is 0.0076% to 0.5% glutaraldehyde and wherein the conditions 5 sufficient to induce crosslmkmg include reacting protein crystals with a crosslmkmg agent for a period of time between about 3 minutes and about 120 minutes. 80. The method for producing crosslinked protein crystals according to claim 79, wherein said 0 crosslmkmg agent is 0.005% glutaraldehyde and wherein the conditions sufficient to induce crosslmkmg include reacting protein crystals with a crossl kmg agent for a period of time between about 10 minutes and about 30 minutes. 5 81. The method for producing crosslinked protein crystals according to claim 79 wherein, prior to reaction with said protein crystals, said glutaraldehyde is pretreated by incubation at 60°C with a buffer for 1 hour. 0 82. The method for producing crosslinked protein crystals according to any one of claims 66, 67 or 68, wherein said crosslmkmg agent is 0.01% to 1% glyoxal and wherein the conditions sufficient to induce crosslmkmg include reacting protein crystals with a 5 crosslmkmg agent for a period of time between about 30 minutes and about 60 minutes. 83. The method for producing crosslinked protein crystals according to any one of claims 66, 67 or 68, wherein said crosslmkmg agent is 0.05% to 1% 0 octanedialdehyde and wherein the conditions sufficient „,_, 8/4 - 1 37 - to induce crosslmkmg include reacting protein crystals with a crosslinking agent for a period of time between about 30 minutes and about 16 hours. 84. The method for producing crosslinked 5 protein crystals according to claim 83, wherein said crossl kmg agent is 1% octanedialdehyde and wherein the conditions sufficient to induce crossl kmg include reacting protein crystals with a crosslmkmg agent for a period of time between about 1 hour and 0 about 3 hours. 85. The method for producing crosslinked protein crystals according to any one of claims 66, 67 or 68, wherein said crosslmkmg agent is 1% succinaldehyde and wherein the conditions sufficient to 5 induce crosslmkmg include reacting protein crystals with a crosslmkmg agent for a period of time between about 30 minutes and about 3 hours. 86. The method for producing crosslinked protein crystals according to any one of claims 66, 67 0 or 68, wherein said first crosslmkmg agent is 0.01% to 4% epoxide and said second crosslmkmg agent is 0.1% to 0.2% glutaraldehyde and wherein the conditions sufficient to induce crosslmkmg include reacting said protein crystals with said first crosslmkmg agent for 5 a period of time between about 1 hour and about 72 hours and reacting said protein crystals with said second crosslmkmg agent for a period of time between about 1 hour and about 5 hours. 87. The method for producing crosslinked 0 protein crystals according to claim 86, wherein said ,_.„_,„ 6732 - 138 - first crosslinking agent is 0.01% epoxide and said second crosslinking agent is 0.1% glutaraldehyde and wherein the conditions sufficient to induce crosslinking include reacting said protein crystals with said first crosslinking agent for about 5 hours and reacting said protein crystals with said second crosslinking agent for about 1.5 hours. 88. The method for producing crosslinked protein crystals according to any one of claims 66, 67 or 68, wherein said protein is an enzyme. 89. The method for producing crosslinked protein crystals according to any one of claims 66, 67 or 68, wherein said crosslinking agent is a reversible crosslinking agent. 90. The method for producing crosslinked protein crystals according to claim 89, wherein said reversible crosslinking agent is a disulfide crosslinking agent. 91. The method for producing crosslinked protein crystals according to claim 90, wherein said disulfide crosslinking agent is a homobifunctional crosslinking agent or a heterobifunctional crosslinking agent . 92. The method for producing crosslinked protein crystals according to any one of claims 66, 67 or 68, wherein said protein is an enzyme. 93. The method for producing crosslinked protein crystals according to claim 92, wherein said ,„.,„ 6732 - 139 - enzyme is selected from the group consisting of hydrolases, isomerases, lyases, ligases, transferases and oxidoreductases. 94. The method for producing crosslinked protein crystals according to claim 93, wherein said enzyme is from the group consisting of proteases, amylases, cellulases, upases and oxidases. 95. The method for producing crosslinked protein crystals according to any one of claims 66, 67 or 68, wherein said protein is selected from the group consisting of therapeutic proteins, cleaning agent proteins, personal care proteins, veterinary proteins, food proteins, feed proteins, diagnostic proteins and decontamination proteins. 96. The method for producing crosslinked protein crystals according to claim 95, wherein said protein is selected from the group consisting of hormones, antibodies, inhibitors, growth factors, trophic factors, cytokines, lymphokines, toxoids, growth hormones, nerve growth hormones, bone morphogenic proteins, toxoids, vitamins and nutrients.

Inventors: KHALAF NAZER K, MARGOLIN ALEXEY L, PERSICHETTI ROSE A, SHENOY BHAMI C, ST CLAIR NANCY L, KHALAF, NAZER, K., MARGOLIN, ALEXEY, L., PERSICHETTI, ROSE, A., SHENOY, BHAMI, C., ST. CLAIR, NANCY, L.
Applicants: ALTUS BIOLOG INC, KHALAF NAZER K, MARGOLIN ALEXEY L, PERSICHETTI ROSE A, SHENOY BHAMI C, ST CLAIR NANCY L, ALTUS BIOLOGICS INC., KHALAF, NAZER, K., MARGOLIN, ALEXEY, L., PERSICHETTI, ROSE, A., SHENOY, BHAMI, C., ST. CLAIR, NANCY, L.
Application year: 1998
Main classification: C12N11
IPC codes: A23K1, A61K8, A61K9, A61P31, A61Q5, A61Q19, C07K1, C07K14, C11D3, C11D17, C12N9, C12N11, C12S13