Title of Invention	"A PROCESS OF PRODUCING ENZYME PARTICLES"
Abstract	A process for producing enzyme structures includes providing an emulsion of droplets of a first liquid phase dispersed in a second liquid phase. The one liquid phase is a hydrophilic phase, while the other liquid phase is a hydrophobic phase which is immiscible with the hydrophilic phase. Enzyme molecules are located at or within interfacial boundaries of the droplets and the second liquid phase. The enzyme molecules of the respective droplets are cross-linked so that individual enzyme structures, which are stable and in which the enzymes are immobilized with a majority of active sites of the enzymes being orientated either internally or externally, are formed from individual droplets. Figure 3 is the representative figure.

Title of Invention

"A PROCESS OF PRODUCING ENZYME PARTICLES"

Abstract

A process for producing enzyme structures includes providing an emulsion of droplets of a first liquid phase dispersed in a second liquid phase. The one liquid phase is a hydrophilic phase, while the other liquid phase is a hydrophobic phase which is immiscible with the hydrophilic phase. Enzyme molecules are located at or within interfacial boundaries of the droplets and the second liquid phase. The enzyme molecules of the respective droplets are cross-linked so that individual enzyme structures, which are stable and in which the enzymes are immobilized with a majority of active sites of the enzymes being orientated either internally or externally, are formed from individual droplets. Figure 3 is the representative figure.

Full Text	THIS INVENTION relates to the stabilization of .enzymes. More particularly, it relates to a process for producing stabilized enzyme structures, to stabilized enzyme structures, and to the use of such stabilized enzyme structures. ' Enzymes are commonly required as catalysts in various industries, such as in chemical, pharmaceutical and cosmetic industries. However, unlike chemical catalysts, enzymes have limited application and shelf life due to their instability. Enzymes are extremely temperature and pH dependant, making their use in many processes difficult, in addition, soluble enzymes cannot be easily recovered from aqueous media, and' enzyme activity generally decreases during storage or processing, limiting the application of enzymes as catalysts in chemical processing, Commercial application of enzymes a$ catalysts can be enhanced by enzyme immobilization, which provides the dual advantages of increasing enzyme stability by making the enzymes more rigid (by immobilizing them on or in a solid phase), and Increasing the overall size of the catalyst, thereby making recovery simpler, immobilization of enzymes onto solid supports Is therefore commonly practiced wfth the aim of stabilizing the enzymes and reducing costs by making them recyclable. However, immobilized enzymes display limitations, the most important being reduced enzyme activity per unit reactor volume due to only a small fraction of the immobilized volume constituting the active catalyst (enzyme). The Applicant is also aware of self-supported immobilized enzymes in the form of cross-linked enzyme crystals (CLEG) and cross-linked enzyme agglomerates (CLEA). Claims to increased specific activity have been made for both of these. In addition, CLEC and CLEA cross-linked enzymes are stable in reaction media, and can be easily separated and recycled, CLEA appears to provide a less expensive and more efficient method compared to CLEG where time- consuming crystallization protocols are required. However, both ClEC .and GLEA are limiting in that some active sites of the enzymes are not exposed, and hence processes utilizing either GLEA or CLEC would require excess enzyme catalyst (with an associated increased cost) for a particular function, to compensate for thfs. In addition these processes do not have easy control over particle size and morphology over a large range of particle sizes. It is thus an object of this invention to provide a process for producing stabilized enzyme structures suitable for use as a catalyst, whereby these drawbacks are at least reduced, Thus, according to a first aspect of the invention, there is provided a process for producing enzyme structures, which process includes providing an emulsion of droplets of a first liquid phase dispersed in a second liquid phase, with the one liquid phase being a hydrophilic phase and the other liquid phase being a hydrophobic phase which is immiscible with the hydrophilic phase, and with enzyme molecules being located at or within interfacia! boundaries of the droplets and the second liquid phase; and cross-linking the enzyme molecules of the respective droplets so that individual enzyme structures, which are stable and in which the enzymes are immobifized with a majority of active sites of the enzymes being orientated either internaliy or externally, are formed from individual droplets. Since, in an emulsion, the droplets of the immiscible first liquid phase, are normally spherical, the structures will thus normally be of hollow spherical form, with the insides or interiors of the spherical structures being either empty or filled. In other words, each enzyme structure comprises a spherical wall of cross-linked immobilized enzyme molecules, and a hollow centre, core or interior which can either be empty or contain a liquid, i.e. be filled, as hereinafter described. In one embodiment of the invention, the individual structures may have openings so that the liquid phases can pass in or out of the structures. However, in another embodiment of the invention, the structures may be liquid impervious, ie they may be in the form of capsules, with the first liquid phase then being trapped inside the capsules ie filling the hollow cores of the capsules. If such stabilized enzyme capsules are then used in a liquid reaction system, eg to catalyze the reaction system, they can easily be separated from the other components of the reaction system, eg by flotation, by selecting a first liquid phase having an appropriate density. However, when used in such a system, they need not necessarily only be separated by flotation since the fact that the stabilized enzyme structures are self-supporting, means that they can easily be separated from the other components in the reaction system and recycled or re-used. Enzyme molecules often contain both hydrophilic and hydrophobic ends or faces. When such enzymes are used, collection and/or orientation thereof at the interfacial boundaries of the droplets and the second liquid phase, will be enhanced or ensured. Modifications may be made to native enzymes to enhance such properties. Thus, an additive for modifying the hydrophobicity and/or charge of the enzyme may be added to the hydrophilic phase and/or to the hydrophobic phase and/or to the emulsion. Examples of additives or modifiers that can be used for this purpose include specific amino acids; amino compounds; proteins; long chain hydrocarbon aldehydes; and other modifiers which bind covalently or otherwise to the enzymes. While the enzyme can be selected from enzyme classes such as Esterases, Proteases, Nitnlases, Nitrile hydratases, Qxynitrilases, Epoxide hydrolases, Halohydrin dehalogenases, Poiyphenoloxidases (eg laccase), Penicillin amidases, Amino acylases, Ureases, Uricases, Lysozymes Asparaginases, Elastases, it is preferably lipase. The lipase can be chosen from mlcrobial, animal, or plant sources, including any one of the following: Pseudomonas cepacia lipase, Pseudomonas fluorescens lipase, Pseudomonas alcaligenes lipase Candida rugosa lipase, Candida antarctica lipase A, Candida antarctica lipase Bt Candida utilis lipase, Thermomyces tanuglnosus lipase, Rhizomucor miehei lipase, Aspergillus niger lipase, Aspergiltus oryzae lipase, Peniciilium sp lipase, Mucor javanicus lipase, Mucor mlehel lipase, Rhizopus arrhizus lipase, Rhizopus deiemer lipase, Rhizopus japonlcus lipase, Rhizopus niveus lipase, and Porcine Pancreatic lipase. When lipase is used, the stabilized lipase structures may, in particular, be used in hydrolysis, acidolysts, alcohoiysis, esteriflcation, transesteriflcation, interesterification, ammonioiysfs, aminofysis, and perhydrolysis reactions. Other enzyme classes will be used in other reaction mechanisms particular to their function. More particularly, the emulsion may be provided by dissolving or solubiliztng the enzyme in the hydrophiiic phase (herein also referred to as 'the water phase' or simply as W), and forming the emulsion by mixing the enzyme containing hydrophiiic phase with the hydrophobic phase (herein also referred to as 'the oil phase' or simply as 'O'), Thus, the emulsion may be of the type O/W, ie oil or hydrophobic phase droplets in a continuous water or hydrophiiic phase, W/0, ie water or hydrophiiic phase droplets in a continuous oil or hydrophobic phase, O/W/O, W/O/W, or the like, The process may further include selectively force precipitating the enzyme at the interface (for O/W emulsions) or within the droplet volume (for W/O emulsions), for example, by increasing the concentration of a salt present in the water phase ('saiting ouf). The cross-linking of the enzyme molecules may be effected by means of a cross-linking agent. Thus, the process may include adding the cross-linking agent to the hydrophiiic phase and/or to the hydrophobia phase and/or to the emulsion. The cross-linking agent will typically be selected so that the cross-linking is only effected once a sufficient time period has elapsed, after the emulsion formation, for enzyme orientation at the phase interface to take place. The cross-linking agent, when used, is a multifunctional reagent, ie a molecule having two or more functional groups or reactive sites which can react with groups on the enzyme Id form a cross-linked macromoiecule, ie the stabilized structure. The cross-linking agent may be selected from the following: an isocyanate such as hexamethylene diisocyanate or toluene diisocyanate; an aldehyde such as glutaraldehyde, succinaldehyde and glyoxal; an epoxide; an anhydride; or the like. The use of various cross-linking reagents may also allow for modification of the spheres' physical and/or chemical properties. Protection of the active sites of an enzyme from being occupied by, or reacting with, the crosslinking agent may be achieved by the addition of a temporary protectant that can occupy the active sites during cross-linking. In the case of iipase, this protectant may, for example, be tributyrin, Tributyrin, which is water-soluble, can then easily be removed by washing in water. Specific enzymes (even within specific classes) require different protectants to minimise or prevent activity loss during cross-linking, If agglomeration of the stabilized enzyme structures or spheres is a problem, this may be reduced or inhibited through the addition of amino acids after cross-linking. These amino acids may react with any residual free cross-linker groups and thus modify the cross-linked spheres" physical properties. Modification of the spheres by amino acids may also enhance the activity of the enzyme towards a specific substrate by manipulating the surface properties of the spheres. Phenylglycine may, for example, be added to cross-linked spheres to improve sphere hydraphobiciiy while modification with aspartic acid would result in improved hydrophiiicity of the spheres. The process may include recovering or separating the stabilized enzyme structures from the second liquid phase, eg by means of flotation, filtration, centrifugatton, magnetism, or the like. The thus recovered stabilized enzyme structures may be washed, if desired, and thereafter dried, if also desired. Drying of the stabilized enzyme structures may be effected by means of spray drying, vacuum drying or lyophilization (freeze drying). The process may further include, if desired, extracting the first liquid phase from the stabilized enzyme structures, eg by means of drying, freeze drying or extraction with a suitable solvent, such as hexane or supercritical carbon dioxide (for hydrophobia liquids) or water (for hydrophilic liquids). Thus, when it is desired to extract the first liquid phase (normally the oil phase) from the stabilized enzyme capsules, this may be effected by contacting the stabilized enzyme capsules with an organic solvent capable of dissolving the first liquid phase, or by contacting the capsules with a mixture of a suitable surfactant in water. Alternatively, the first liquid phase can then be extracted by supercritical fluid extraction. The fluid is then preferably supercritical carbon dioxide. The critical point for carbon dioxide (31.2°C and 73.8 bar) is sufficiently low so that the extraction process will not damage the stabilized enzyme structure. White the hydrophilic phase in which the enzymes are dissolved may comprise only water, it is believed that improved results may be achieved if it then includes a suitable buffer. The buffer should be selected to facilitate the cross-linking of the enzyme molecules, while ensuring enzyme stability. Thus, for example, the hydrophilic phase may comprise a buffer solution with pH 7-8. Such a buffer may be phosphate buffered saline (PBS) solution, a Tris-(hydroxymethyl)-aminomethane (TRIS) buffer-containing aqueous solution, or a KHzPCVNaOH solution. Alternatively, the hydrophilic phase may include or comprise a polyethylene gtycol (PEG). When a low molecular weight polyethylene glycol, such as PEG400 or PEG100, is used, it may be used on its own, ie the hydrophilic phase will then consist of the low molecular weight polyethylene glycol. However, a higher molecular weight polyethylene glycol may optionally instead be used, with ft then being dissolved in water to form the hydrophilic phase. When an isocyanate is used as the cross-linking agent in a water-in-oii emulsion, the cross-linking agent will react with the PEG as well as with the enzyme, leading to the formation of reinforced stabilized enzyme capsules that contain an enzyme incorporated membrane with an internal hydrogel support. Alternatively, acrylamide may be polymerized to provide a similar support. This can advantageously improve the mechanical strength of the capsules, improving, for example, resistance against shear damage. The water immiscible phase, ie the hydrophobia phase, may comprise an oil such as mineral, jojoba or avocado oil; a hydrocarbon such as decane, heptane, hexane or isododecane; an ether such as dloctyl ether, diphsnyl ether, or the like; an ester such as triglyceride, isopropyf palmitate or isopropyl myristate; or the like. It is believed that the emulsion used in the process of the invention will normally be in the form of a water-in-oii or W/0 emulsion; however, as previously indicated, instead a oil-in~water or O/W, oil- in-water-in-oil, le OAV/O, or water-in-oiMn-water, ie W/0/W, emulsions can be used. •> Thus, for example, when the enzyma is lipase, a water-in-oil emulsion can be used to ensure that most of the llpase active sites, which are hydrophobia, are oriented outwardly, thus increasing the total effective activity of the structures. Furthermore, when a water-ln-oil emulsion is used, a second enzyme can advantageously ba dissolved In the aqueous or hydrophilic phase. If this second enzyme also has the ability to accumulate at the droplet/second liquid phase interfaces, the resultant cross-linked enzyme structures will contain both enzymes. Alternatively, if the second enzyme is selected so that it does not accumulate at the interfaces, a cross-linked enzyme structure will result with one enzyme being a major component of the structure, while the second enzyme is encapsulated or contained inside the structure. Such a combination enzyme structure can advantageously be used, for example, to catalyze multiple reactions in a single reaction step. Moreover, co-factors or reaction mediators, modified or otherwise, may be included in the droplet, e.g. a redox enzyme and suitable mediator may be incorporated in the sphere in order to regenerate a second redox enzyme In the sphere. In a particular embodiment of the invention, a triglyceride, which is hydrolysabte by lipase, may be used as tie hydrophobic or oil phase, with an O/W emulsion being formed; the dispersed or oil phase, ie the trigtyceride, contained within the stabilized cross-linked structures or spheres is hydrolyzed by the lipase during and after the cross-linking reaction. The hydroiyzed products are generally water-soluble, and can thus readily be leached out, thereby minimizing or reducing the number of processing steps required to produce the stabilized structures. In yet another embodiment of the invention, an initial O/W emulsion can be formed. In doing so, a certain degree of purification of the lipase takes place, since impurities present therein will not collect at the interfadal boundaries to the same extent as the lipase, The process may then include, before effecting the cross-linking, centrifugtng the emulsion and separating a concentrated emulsion from a dilute water phase, Thereafter,- a further O/W emulsion can be formed, using the concentrated emulsion. This step can, if desired, be repeated one or more times, to increase lipase purity. After the final such purification step, the emulsion may then be inverted to form a W/O emulsion, by the addition of surfactants with lower HLB values, which may be in the range of 3-10, more preferably 4-6. This ensures preferential orientation of the lipase active sites towards the outside of the dispersed phase droplets. Thereafter, cross-linking of the lipase as hereinbefore described, can be effected. When an enzyme is used that collects at the interface, and a W/O emulsion is used, the Internal cross-linked -enzyme sphere morphology can be controlled by modifying the dissolved enzyme concentration in the aqueous phase. For example, a hollow enzyme sphere can be formed through using reduced enzyme concentration, and activity by weight will improve due to decreased average diffusiona! distances for substrates. To impart specific properties to the stabilized enzyme structures, a modifier may be added to the hydrophiiic phase and/or to the hydrophobic phase and/or to the emulsion. One or more of the following modifiers can be added in this fashion: a surfactant, a precipitator and an additive. A surfactant may be used when it is desired to impart enhanced enzyme activity (as regards its use in a subsequent catalyzed reaction), and improved emulsion stability. The surfactant may be anionic, cationic, non-ionic, zwitterionic, polymeric, or mixtures of two or more of these. When an anionic surfactant is used, it may be an alkyl sulphate such as sodium lauryi sulphate or sodium iaureth sulphate, or an alkyl ether sulphate. When a cationic surfactant is used, It may be centrirnonium chloride. When a non-ionic surfactant is used, it may be an ethoxylated alkyl phenol such as polyoxyethylene(IO) iso-octylcyclohexyl ether (Triton X100) or polyoxyethylene(9) nonylphenyl ether (Monoxynol-9}. When a zwitterionic or amphiphilfic surfactant is used, it may be decyl betaine. When a polymeric surfactant is used, it may be an ethyiene oxlde-propylene oxide-ethylene oxide triblock copolymer, also known as a poioxamer, such as that available under the trade name Pluronic from BASF, or it may be a propylene oxide-ethyfene oxide-propylene oxide triblock copofymer, also known as a meroxapo!. A preclpitator can be used when it is desired to precipitate the enzyme onto the emulsion interfaces. The precipitator, when present, may be an inorganic salt such as ammonium sulphate; an organic solvent such as 1,2-dimethylethane or acetone; or a dissolved polymer. Additives or adjuvants will be used to impart desired properties to the emulsion and/or to the stabilized enzyme structures. Properties that can be modified by use of such additives include pH, by using, for example, a buffer; ionic strength, by using, for example, salts; viscosity, by using, for example, PEG; magnetic properties, by using, for example, iron salts; agglomeration tendency, by using, for example, a surfactant possessing steric hindrance properties; and zeta potential, by using, for example, an anionic surfactant. According to a second aspect of the invention, there is provided an enzyme structure, which comprises cross-linked enzyme molecules so that the structure is stable, with the structure being hollow, and in which the enzymes are immobilized, with a majority of active sites of the enzymes being orientated either internally or externally. The enzyme structure may be as hereinbefore described with reference to the first aspect of the invention. According to a third aspect of the invention, there is provided a method of carrying out a reaction, which includes allowing a reaction medium to undergo a reaction in the presence of a plurality of the enzyme structures as hereinbefore described, with the reaction thus being catalyzed by the enzyme structures. The invention will now be described in more detail with reference to the following non-limiting examples and the accompanying drawings. In the drawings, FIGURE 1 is an optical microscope picture of cross-linked llpase capsules prepared in accordance with Example 1; FIGURE 2 is a particle size distribution of the cross-linked lipase capsules prepared in Example 1; FIGURE 3 is an optical microscope picture of cross-Jinked lipase capsules prepared in accordance with Example 2; and FIGURE 4 is a particle size distribution of the cross-linked lipase capsules prepared in Example 2. EXAMPLE 1 (non-optimized) Cross-linked or stabilized Lipase spheres (structures) from wateNn-oll emulsion 1 g of lipase Amano AK was added to 195 g phosphate buffered saline (PBS) solution (pH 7.8) and 5 g mineral oil (Castrol). This blend was then homogenized for 5 minutes using a Silverson L4R laboratory rotor-stator homogenizer at 6000 rpm, 1,5 g of hexamethylene di-socyanate (Merck Schuchardt) was added to the emulsion. The emulsion was then stored at room temperature for 2 hours. The cross-linked enzyme structures were then recovered by filtration using 0,45 urn filter paper and washed 5 times with 50 ml of PBS each time (total 250 mi PBS), Figure 1 shows typical stabilized enzyme spheres or structures obtained according to the method. Particle sizes were determined using laser light scattering (Maivem Mastersizer 2000), and an average Sauter mean diameter of 49.4 urn was obtained (see Fig. 2). The activity of the stabilized enzyme (lipase) structures was determined using a p-Nitrophenylacetate assay method as described by Vorderwulbecke, T,, Kies!ich, K. & Erdmarsn, H. (1992). 'Comparison of iipases by different assays', Enzyme Microb. Techno!,, 14, 631-639; and L6pez~Serrano P., Cao L, van Rarrtwijk & Sheldon RA (2002), 'Cross-linked enzyme aggregates with enhanced activity : application to iipases', Biotechnology Letters,, 24,1379-1383. This assay measures the release of p-nitrophenol from a p-nitrophenyl ester of a fatty acid. The reaction is done at pH 7.4 at 37*C and the liberated p-niirophenol is measured at 410nrrt. The activity obtained was 63 U/g lipase, where U is umof/mm. EXAMPLE 2 Cross-linked or stabilized Lipase spheres .(.structures).from water-tn-oil emujsion A Sipase solution was prepared by resuspending Candida rugosa lipase (Ailus Biologies, Inc.) in 100 fnM Tris-CI (Tris(hydroxyrnethyl)aminomethan0) buffer (pH 8.0) to a final concentration of 100 mg/ml. The enzyme sample was diafiitered using an Amicon ultrafiltratton cell fitted with a 10 K poiyether sulfone membrane (Microsep (Pty) Ltd, PO Box 391647, Bramiey 2018, South Africa) against 3 volumes of 100 mM Tris-Ci buffer (pH 8.0). Lipase spheres were prepared using the following reagents in the following volumes: 200 u! Candida rugosa lipase solution (as prepared above); 50 u! nonoxynoM; 50 ul tribulyrin; 5 ml mineral oil. This mixture was emulsified by stirring for 1 minute at 1500 rpnx To this solution 40 ul gluteraldebyde was added (25% aqueous solution) and allowed to stir for a further 10 minutes. The emuision was allowed to stand at 4eC for 12 hours. After crosslinktng the emuisiorn was centrifuged at 10000 rpm for 5 minutes using a Beckrnan J2-21 ME centrifuge fitted with JA 20.1 rotor, after which the oil phase was removed. The peiiet was washed thrice with 10 ml of 100 mM Tris-Cl buffer (pH 8.0) and pallet was recovered using centrifugation as mentioned above. After washing the peflet was resuspended in 1 mi buffer and assayed for enzyme activity. Rgure 3 shows the enzyme spheres obtained. The spheres had a narrow size distribution between about 10 and 100 urn (Figure 4). The activity of the stabilized enzyme (Iipase) structures was determined using a p-nstrophenylpaimrtate and p-ratrophenytbutyrste assay method as described by Vorderwulbecke, T., Kiesllch, K, & Erdrnann, H. (1992), 'Comparison of iipases by different assays', Enzyme Microb, Technof., 14, 631-639; and L6pez-Serrano P., Cao L., van Rantwijk & Shefdon RA. (2002), 'Cross-linked enzyme aggregates with enhanced activity; application to Upases', Biotechnology Letters., 24,1379-1383. This assay measures the release of p-nitrophenoi from a p-nitrophenyl ester of a fatty acid. The reaction is dons at pH 8.0 at 37°C and the liberated p-mtrophenol is measured at 410nm. The activity without tributyrin as additive was 0.11% (for p-nitrophenylpalmitate) compared to th© original free enzyme in aqueous solution. Surprisingly, the actfv'rty obtained with tributyrin as an additive ranged from about 5% (for p-nitrophenyipaimtete) to 124% (for p-nitrophenylbutyrate) compared to the original free enzyme in aqueous solution. EXAMPLE 3 Dextmn aldehydei as,CTOss~tjnker Example 2 was repeated, except that the cross-linking agent used was acOvated dextran from leuconstoc species, average moiecuiar weight 20 kDa (dextran aldehyde), the oil phase was vegetable oil, the ratio of iipase solution to Tris buffer was 1:1, and no surfactant was used. Dextran aldehyde wss prepared by reacting dextran with excess sodium metaperiodate as described by Hong, T,, Guo, W., Yuan, H.t Li, J., Liu, Y., Ma, L,, Bat, Y., & Li, T. (2004) 'Periodate oxidation of nanoscaled magnetic dextran composites', Journal of Magnetism and Magnetic Materials, 269, 95-100, Activity obtained was 7.5% (for p-nitrophenyipaimitale) compared to the original free enzyme in aqueous solution. EXAMPLE 4 Cross-linked enzvme spheres from oii-in-water emulsion Example 2 was repeated, except that an oii-in-watsr emulsion was generated by changing the ratio of liquid phases and the surfactant. EXAMPLE 5 Different Example 3 was repeated except that the enzyme used was laccase from UD4 species as described by Jordaan, J., Pletschke, B.I. & Leukes, W.D. (2004) "Purification and partial characterization of a thermostable laccase from an unidentified basldtomycete', Enz Microb Tachnol. 34, 635-641 , and the tributyrin was substituted with syringic acid (saturated solution in ethanol). The spheres were equilibrated with tOO mM sucdnate-lactate buffer pH 4.5. The spheres were assayed for laccase activity with ABTS as the substrate at 25°C and the product was followed spectrophotometricalfy at 420 nm according to the method of Jordaan, J. & Leukes, W.D. (2003) Isolation of a thermostable iaccase with DMAS and MBTH oxidatfve coupling activity from a mesophilic white rot fungus'. Enz Microb TechnoL 33(2/3), 212-219. EXAMPLES Protein .coocentratio n Decreasing the lipase concentration of Example 2 (without tributyrin) by half, leads to an increase of more than 1 00% in lipase activity by weight. A possible explanation for the increased activity that was achieved with lower protein concentration (compared to higher protein concentration) is the preferential accumulation of the protein (in this case lipase) at the water-oil interface. This would lead to 'hollow' spheres at lower iipase concentration. On a per weight basis, hollow spheres would be expected to have higher activity compared to ftiled' spheres, due to shorter average dtffusional distances for reaction substrates and products. EXAMPLE 7 Addition of a precipitant Addition of acetone as a precipitant to the emulsion of Example 2 (without tributyrin), leads to an increase in activity of 114%. EXAMPLE 8 Choice of oil phase Substituting vegetable oil for mineral oil in the process of Example 2 (without tributyrin) leads to a four-fold increase in activity, but increased difficulty of recovery from the ^product solution. This is possibly because of the presence of hydrolysed oil. .EXAMPLE.. 9 Addltion of protectant As discussed in Example 2, the addition of tributyrin as protectnt, led to an increase in Candida rugosa lipase activity from 0.14% to 5% (for p-nltrophenylpalmitate) compared to the original free enzyme concentration. This 'protectant ability' does not work for all enzymes. For example, the addition of tributyrin as protectant to lipase from Rhizopus otyzae led to a .twelve-fold decrease Jn activity from 4.17 to 0.35% (for p-nitrophenylbutyrate) compared to the original free enzyme. EXAMPLE 10 Binding of residual free cross-linker groups to ..reduce aggregation Through the addition of an amino acid to the final product, aggregation of spheres was reduced. This is thought to be due to the amino acid binding to the residual crosslinker groups on the sphere surface. Surprisingly, improved activity was also observed compared to controls where an amino acid was not used, specifically when phenylgiyctne was used as the amino acid. An improvement in activity of about 100% for lipase spheres (based on both p-nitrophenylbutyrate and p-nitrophenyipalmitate) was observed compared to the normal method of Example 2 (without tributyrin). EXAMPLE 11 Recycling of laccase cross-linked spheres Laccase spheres were prepared according to the method in Example 5. The 'spheres were reacted six times with 2f2'-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt (ABTS) as a substrate with recovery and washing between each 14 reaction. Laccase activity after these six recycles was comparable to the original activity of the spheres, EXAMPLE12 Recycling. M liease Cross-linked..s[^hereswith.NEE Lipase spheres were prepared according to the method in Example 2. The spheres were reacted three times with naproxen ethyl ester (NEE) as a substrate at 40'C, with recovery and washing between each reaction. Activity decreased by about 70% over three recycles for the cross-linked [ipase spheres (CLECs of the same enzyme showed similar activity losses. Brady, D., Steenkamp, L, Skein, E., Chaplin, J.A, and Reddy, S. (2004) 'Optimisation of the enanttoselective biocatafytic hydrolysis of naproxen ethyl ester using ChiroCLEC-CR. Enz. Microb. Techno!. 34, 283-291). EXAMPLE13 Recycling of (ipase cross-linked spheres, With Lipase spheres were prepared according to the method in Example 2. The spheres were reacted with p-nrtrophenyipatmitate as a substrate, with recovery and washing between each reaction. Activity decreased to 79.6% of original lipase sphere activity in the final recycle. of lipase sphere activity Candida rugosa lipase spheres were prepared according to the method in Example 2. Candida rugosa lipase CLEA's were prepared according to Example 6 of United States Patent Application 20030149172, Cao, L., and Elzinga, J,, with a gkiteraldehyde to ethylene d ia mine ratio of 1 :7. 88 . Activity retention of spheres as compared to CLEA's with p-nitrophenylpalmitate as the substrate was measured as 2.7% for lipase spheres and 3.4% for CLEA's wnije activity with p-nitrophenylbutyrate as trie substrate was measured as 53.7% for lipase spheres and 6.5% for CLEA's. EXAMPL£15 15 Water in Oil Candida rugosa lipase spheres were prepared according to the method in Example 2, Oil in Water Candida rugosa lipase spheres were prepared acoordjng to the method in Example 2 except that the volume of oil was reduced to 0.2 ml and the volume of buffer was increased to 5 ml. Specific activity obtained for the Water in Oil emulsion was 136.0% higher than the Oil in Water emulsion with p-nitrophenylbutyrafe as the substrate, and increase from 8.9 to 26.0 U/mg from the OinW to WinO emulsion, EXAMPLE 16 Sphere size control effected through mechanical agitation Candida rugosa lipase spheres were prepared according to the method in Example 2, except that a Silverson homogenizer was used to create the emulsion rather than stirring. Two experiments were performed varying only in the speed setting of the homogenizer, namely 1000 and 3000 rpm respectively. Particle size distribution was determined and the results indicated a mean diameter of 52.0 urn and 20,6 urn for the spheres produced using 1000 rpm and 3000 rpm respectively while specific activity increased by 28% and 83% for p-nitrophenylpalmitate and p-nitrophenylbutyrate as substrates respectively compared to, The invention thus provides a method of stabilizing an enzyme by means of cross-linking, using emulsions as a vehicle therefor. The invention also relates to exposing maximum surface area of enzyme per unit volume of the structure, for subsequent reaction when the structure is used as a catalyst. Additionally, the stabilized enzyme structures are easily recyclable, less expensive than most immobilized enzyme products, and will find widespread application as catalysts in various processes. Additionally, due to the selective orientation of lipases at the hydrophilic/hydrophobic phase interface, they will be concentrated there. So this method, when applied in the example of an oil in water emulsion, will simultaneously purify the desired lipase from a crude cell lysate. The same would be true of other enzymes with external hydrophobic regions, including many membrane-associated enzymes. The cross-linking of lipases at the phase interface will fix them in the activated {lid open) state. 16 The use of oil-in-water emulsions can permit mono-layer lipase spheres, thereby providing a cross-linking method that provides the maximum surface area to enzyme mass. The use of water-in-oil emulsions would allow for denser, multi-layered enzyme spheres. This form of enzyme immobilization allows for enzyme recovery and recycling, It is believed that the process of the present invention, which provides the stabilized enzyme hollow spherical structures, provides the following advantages when the structures are subsequently used to catalyze reactions: 1. Maximum exposed surface area of catalyst (spherical, hollow capsules). 2. Buoyancy of catalyst can be controlled, eg, floating particles could be separated from the reaction medium with ease. 3. The mean size (diameter) of the immobilized enzyme particle formed can be controlled by controlling the size distribution of the emulsion. 4. Through use of the natural self-orientation of many lipases and some other enzymes at solvent interfaces, the immobilized enzyme sphere may be generated in a controlled manner so as to orientate the majority of active sites either towards the lumen or externally as required. 5. Due to the presence of a hydrophilic/ hydrophobic interface, enzymes such as Iipase are immobilized in the active form. We claim: 1. A process for producing enzyme structures, comprising: providing an emulsion of droplets of a first liquid phase dispersed in a second liquid phase, with the one liquid phase being a hydrophilic phase and the other liquid phase being a hydrophobic phase which is immiscible with the hydrophilic phase, characterized in that enzyme molecules are located at or within interfacial boundaries of the droplets and the second liquid phase; the enzyme molecules of the respective droplets are cross-linked so that enzyme structures, which are stable and in which the enzymes are immobilized with a majority of active sites of the enzymes being orientated either internally or externally, are formed from individual droplets; and the enzyme structures are recovered from the second liquid phase. 2. The process as claimed in Claim 1, wherein the structures have openings so that the liquid phases can pass in or out of the structures. 3. The process as claimed in Claim 1, wherein structures are liquid impervious. 4. The process as claimed in any one of Claims 1 to 3 inclusive, which includes adding to the hydrophilic phase and/or to the hydrophobic phase and/or to the emulsion, a modifier for modifying the hyrophobicity and/or charge of the enzyme. 5. The process as claimed in any one of Claims 1 to 4 inclusive, wherein the enzyme is a lipase. 6. The process as claimed in Claim 5, wherein the lipase is Candida rugosa lipase. 7. The process as claimed in Claim 5 or Claim 6, wherein the provision of the emulsion is effected by dissolving the enzyme in the hydrophilic or water (W) phase and forming the emulsion by mixing the enzyme containing hydrophilic phase with the hydrophobic or oil (0] phase. 8. The process as claimed in Claim 7, which includes selectively precipitating the enzyme at the interface when the emulsion is an oil(O) in water(W) emulsion in which hydrophobic phase droplets are dispersed in a continuous hydrophilic phase, or within the droplet volume, when the emulsion is a water(W) in oil(O) emulsion in which hydrophilic phase droplets are dispersed in a continuous hydrophobic phase. 9. The process as claimed in Claim 7 or Claim 8, wherein the cross-linking of the enzyme molecules is effected by means of a cross-linking agent which is added to the hydrophilic phase and/or to the hydrophobic phase and/or to the emulsion. 10. The process as claimed in Claim 9, which includes adding to the hydrophilic phase and/or to the hydrophobic phase and/or to the emulsion, a temporary protectant that occupies active sites of the enzyme during the cross-linking, thereby inhibiting occupation of or reaction with the active sites by the cross-linking agent. 11. The process as claimed in any one of Claims 7 to 10, which includes adding an amino acid to the emulsion to inhibit agglomeration of the enzyme structures. 12. The process as claimed in any one of Claims 7 to 11, which includes recovering the enzyme structures from the second liquid phase. 13. The process as claimed in any one of Claims 7 to 12, which includes extracting the first liquid phase from the enzyme structures. 14. The process as claimed in any one of Claims 7 to 13, wherein the hydrophilic phase comprises water and, optionally, a buffer in the water. 15. The process as claimed in any one of Claims 7 to 13, wherein the hydrophilic phase comprises a polyethylene glycol and, optionally, water admixed with the polyethylene glycol. 16. The process as claimed in any one of Claims 7 to 15, wherein the hydrophobic phase comprises an oil; a hydrocarbon; an ether; or an ester. 17. The process as claimed in any one of Claims 7 to 16, wherein the emulsion is a water(W) in oil(O) emulsion in which hydrophilic phase droplets are dispersed in a continuous hydrophobic phase, with a second enzyme, co factor and/or mediator being present in the hydrophilic phase. 18. The process as claimed in any one of Claims 7 tol6, wherein a triglyceride, which is hydrolysable by lipase, is used as the hydrophobic phase, with an oil(O) in water(W) emulsion, in which hydrophobic phase droplets are dispersed in a continuous hydrophilic phase, being formed and with the dispersed hydrophobic phase contained within the cross-linked structures being hydrolyzed by the lipase during and after the cross-linking reaction. 19. The process as claimed in any one of Claims 1 to 16, wherein the emulsion is an oil(O) in water(W) emulsion, in which hydrophobic phase droplets are dispersed in a continuous hydrophilic phase, with the process including, before effecting the cross-linking, centrifuging the emulsion and separating a concentrated emulsion from a dilute hydrophilic phase, to increase enzyme purity; and inverting the emulsion to form a water(W) in oil(O) emulsion in which hydrophilic phase droplets are dispersed in a continuous hydrophobic phase, by the addition of a surfactant with a lower hydrophilic-lipophilic balance (HLB) value. 20. The process as claimed in any one of Claims 1 to 19 wherein, to impart specific properties to the enzyme structures, a modifier is added to the hydrophilic phase and/or to the hydrophobic phase and/or to the emulsion. 21. The process as claimed in Claim 20, wherein the modifier is a surfactant, for imparting enhanced enzyme activity and improved emulsion stability. 22. The process as claimed in Claim 20, wherein the modifier is a precipitator for precipitating the enzyme onto the emulsion interfaces. 23. The process as claimed in Claim 20, wherein the modifier is an additive for modifying the pH; ionic strength; viscosity; magnetic properties; agglomeration tendency; and/or zeta potential of the emulsion and/or the enzyme structures.

Full Text

THIS INVENTION relates to the stabilization of .enzymes. More particularly, it relates to a process for producing stabilized enzyme structures, to stabilized enzyme structures, and to the use of such stabilized enzyme structures. '
Enzymes are commonly required as catalysts in various industries, such as in chemical, pharmaceutical and cosmetic industries. However, unlike chemical catalysts, enzymes have limited application and shelf life due to their instability. Enzymes are extremely temperature and pH dependant, making their use in many processes difficult, in addition, soluble enzymes cannot be easily recovered from aqueous media, and' enzyme activity generally decreases during storage or processing, limiting the application of enzymes as catalysts in chemical processing,
Commercial application of enzymes a$ catalysts can be enhanced by enzyme immobilization, which provides the dual advantages of increasing enzyme stability by making the enzymes more rigid (by immobilizing them on or in a solid phase), and Increasing the overall size of the catalyst, thereby making recovery simpler,
immobilization of enzymes onto solid supports Is therefore commonly practiced wfth the aim of stabilizing the enzymes and reducing costs by making them recyclable. However, immobilized enzymes display limitations, the most important being reduced enzyme activity per unit reactor volume due to only a small fraction of the immobilized volume constituting the active catalyst (enzyme). The Applicant is also aware of self-supported immobilized enzymes in the form of cross-linked enzyme crystals (CLEG) and cross-linked enzyme agglomerates (CLEA). Claims to increased specific activity have been made for both of these. In addition, CLEC and CLEA cross-linked enzymes are stable in reaction media, and can be easily separated and recycled, CLEA appears

to provide a less expensive and more efficient method compared to CLEG where time-
consuming crystallization protocols are required. However, both ClEC .and GLEA are
limiting in that some active sites of the enzymes are not exposed, and hence processes
utilizing either GLEA or CLEC would require excess enzyme catalyst (with an
associated increased cost) for a particular function, to compensate for thfs. In addition
these processes do not have easy control over particle size and morphology over a
large range of particle sizes.
It is thus an object of this invention to provide a process for producing stabilized enzyme structures suitable for use as a catalyst, whereby these drawbacks are at least reduced,
Thus, according to a first aspect of the invention, there is provided a process for producing enzyme structures, which process includes
providing an emulsion of droplets of a first liquid phase dispersed in a second liquid phase, with the one liquid phase being a hydrophilic phase and the other liquid phase being a hydrophobic phase which is immiscible with the hydrophilic phase, and with enzyme molecules being located at or within interfacia! boundaries of the droplets and the second liquid phase; and
cross-linking the enzyme molecules of the respective droplets so that individual enzyme structures, which are stable and in which the enzymes are immobifized with a majority of active sites of the enzymes being orientated either internaliy or externally, are formed from individual droplets.
Since, in an emulsion, the droplets of the immiscible first liquid phase, are normally spherical, the structures will thus normally be of hollow spherical form, with the insides or interiors of the spherical structures being either empty or filled. In other words, each enzyme structure comprises a spherical wall of cross-linked immobilized enzyme molecules, and a hollow centre, core or interior which can either be empty or contain a liquid, i.e. be filled, as hereinafter described.
In one embodiment of the invention, the individual structures may have openings so that the liquid phases can pass in or out of the structures. However, in another embodiment of the invention, the structures may be liquid impervious, ie they may be in the form of capsules, with the first liquid phase then being trapped inside the capsules ie filling the

hollow cores of the capsules. If such stabilized enzyme capsules are then used in a liquid reaction system, eg to catalyze the reaction system, they can easily be separated from the other components of the reaction system, eg by flotation, by selecting a first liquid phase having an appropriate density. However, when used in such a system, they need not necessarily only be separated by flotation since the fact that the stabilized enzyme structures are self-supporting, means that they can easily be separated from the other components in the reaction system and recycled or re-used.
Enzyme molecules often contain both hydrophilic and hydrophobic ends or faces. When such enzymes are used, collection and/or orientation thereof at the interfacial boundaries of the droplets and the second liquid phase, will be enhanced or ensured. Modifications may be made to native enzymes to enhance such properties. Thus, an additive for modifying the hydrophobicity and/or charge of the enzyme may be added to the hydrophilic phase and/or to the hydrophobic phase and/or to the emulsion. Examples of additives or modifiers that can be used for this purpose include specific amino acids; amino compounds; proteins; long chain hydrocarbon aldehydes; and other modifiers which bind covalently or otherwise to the enzymes.
While the enzyme can be selected from enzyme classes such as Esterases, Proteases, Nitnlases, Nitrile hydratases, Qxynitrilases, Epoxide hydrolases, Halohydrin dehalogenases, Poiyphenoloxidases (eg laccase), Penicillin amidases, Amino acylases, Ureases, Uricases, Lysozymes Asparaginases, Elastases, it is preferably lipase.
The lipase can be chosen from mlcrobial, animal, or plant sources, including any one of the following: Pseudomonas cepacia lipase, Pseudomonas fluorescens lipase, Pseudomonas alcaligenes lipase Candida rugosa lipase, Candida antarctica lipase A, Candida antarctica lipase Bt Candida utilis lipase, Thermomyces tanuglnosus lipase, Rhizomucor miehei lipase, Aspergillus niger lipase, Aspergiltus oryzae lipase, Peniciilium sp lipase, Mucor javanicus lipase, Mucor mlehel lipase, Rhizopus arrhizus lipase, Rhizopus deiemer lipase, Rhizopus japonlcus lipase, Rhizopus niveus lipase, and Porcine Pancreatic lipase.
When lipase is used, the stabilized lipase structures may, in particular, be used in hydrolysis, acidolysts, alcohoiysis, esteriflcation, transesteriflcation, interesterification,

ammonioiysfs, aminofysis, and perhydrolysis reactions. Other enzyme classes will be used in other reaction mechanisms particular to their function.
More particularly, the emulsion may be provided by dissolving or solubiliztng the enzyme in the hydrophiiic phase (herein also referred to as 'the water phase' or simply as W), and forming the emulsion by mixing the enzyme containing hydrophiiic phase with the hydrophobic phase (herein also referred to as 'the oil phase' or simply as 'O'), Thus, the emulsion may be of the type O/W, ie oil or hydrophobic phase droplets in a continuous water or hydrophiiic phase, W/0, ie water or hydrophiiic phase droplets in a continuous oil or hydrophobic phase, O/W/O, W/O/W, or the like,
The process may further include selectively force precipitating the enzyme at the interface (for O/W emulsions) or within the droplet volume (for W/O emulsions), for example, by increasing the concentration of a salt present in the water phase ('saiting ouf).
The cross-linking of the enzyme molecules may be effected by means of a cross-linking agent. Thus, the process may include adding the cross-linking agent to the hydrophiiic phase and/or to the hydrophobia phase and/or to the emulsion. The cross-linking agent will typically be selected so that the cross-linking is only effected once a sufficient time period has elapsed, after the emulsion formation, for enzyme orientation at the phase interface to take place.
The cross-linking agent, when used, is a multifunctional reagent, ie a molecule having two or more functional groups or reactive sites which can react with groups on the enzyme Id form a cross-linked macromoiecule, ie the stabilized structure. The cross-linking agent may be selected from the following: an isocyanate such as hexamethylene diisocyanate or toluene diisocyanate; an aldehyde such as glutaraldehyde, succinaldehyde and glyoxal; an epoxide; an anhydride; or the like. The use of various cross-linking reagents may also allow for modification of the spheres' physical and/or chemical properties.
Protection of the active sites of an enzyme from being occupied by, or reacting with, the crosslinking agent may be achieved by the addition of a temporary protectant that can

occupy the active sites during cross-linking. In the case of iipase, this protectant may, for example, be tributyrin, Tributyrin, which is water-soluble, can then easily be removed by washing in water. Specific enzymes (even within specific classes) require different protectants to minimise or prevent activity loss during cross-linking,
If agglomeration of the stabilized enzyme structures or spheres is a problem, this may be reduced or inhibited through the addition of amino acids after cross-linking. These amino acids may react with any residual free cross-linker groups and thus modify the cross-linked spheres" physical properties. Modification of the spheres by amino acids may also enhance the activity of the enzyme towards a specific substrate by manipulating the surface properties of the spheres. Phenylglycine may, for example, be added to cross-linked spheres to improve sphere hydraphobiciiy while modification with aspartic acid would result in improved hydrophiiicity of the spheres.
The process may include recovering or separating the stabilized enzyme structures from the second liquid phase, eg by means of flotation, filtration, centrifugatton, magnetism, or the like. The thus recovered stabilized enzyme structures may be washed, if desired, and thereafter dried, if also desired. Drying of the stabilized enzyme structures may be effected by means of spray drying, vacuum drying or lyophilization (freeze drying).
The process may further include, if desired, extracting the first liquid phase from the stabilized enzyme structures, eg by means of drying, freeze drying or extraction with a suitable solvent, such as hexane or supercritical carbon dioxide (for hydrophobia liquids) or water (for hydrophilic liquids). Thus, when it is desired to extract the first liquid phase (normally the oil phase) from the stabilized enzyme capsules, this may be effected by contacting the stabilized enzyme capsules with an organic solvent capable of dissolving the first liquid phase, or by contacting the capsules with a mixture of a suitable surfactant in water. Alternatively, the first liquid phase can then be extracted by supercritical fluid extraction. The fluid is then preferably supercritical carbon dioxide. The critical point for carbon dioxide (31.2°C and 73.8 bar) is sufficiently low so that the extraction process will not damage the stabilized enzyme structure.

White the hydrophilic phase in which the enzymes are dissolved may comprise only water, it is believed that improved results may be achieved if it then includes a suitable buffer. The buffer should be selected to facilitate the cross-linking of the enzyme molecules, while ensuring enzyme stability. Thus, for example, the hydrophilic phase may comprise a buffer solution with pH 7-8. Such a buffer may be phosphate buffered saline (PBS) solution, a Tris-(hydroxymethyl)-aminomethane (TRIS) buffer-containing aqueous solution, or a KHzPCVNaOH solution.
Alternatively, the hydrophilic phase may include or comprise a polyethylene gtycol (PEG). When a low molecular weight polyethylene glycol, such as PEG400 or PEG100, is used, it may be used on its own, ie the hydrophilic phase will then consist of the low molecular weight polyethylene glycol. However, a higher molecular weight polyethylene glycol may optionally instead be used, with ft then being dissolved in water to form the hydrophilic phase. When an isocyanate is used as the cross-linking agent in a water-in-oii emulsion, the cross-linking agent will react with the PEG as well as with the enzyme, leading to the formation of reinforced stabilized enzyme capsules that contain an enzyme incorporated membrane with an internal hydrogel support. Alternatively, acrylamide may be polymerized to provide a similar support. This can advantageously improve the mechanical strength of the capsules, improving, for example, resistance against shear damage.
The water immiscible phase, ie the hydrophobia phase, may comprise an oil such as mineral, jojoba or avocado oil; a hydrocarbon such as decane, heptane, hexane or isododecane; an ether such as dloctyl ether, diphsnyl ether, or the like; an ester such as triglyceride, isopropyf palmitate or isopropyl myristate; or the like. It is believed that the emulsion used in the process of the invention will normally be in the form of a water-in-oii or W/0 emulsion; however, as previously indicated, instead a oil-in~water or O/W, oil-
in-water-in-oil, le OAV/O, or water-in-oiMn-water, ie W/0/W, emulsions can be used.
•> Thus, for example, when the enzyma is lipase, a water-in-oil emulsion can be used to
ensure that most of the llpase active sites, which are hydrophobia, are oriented outwardly, thus increasing the total effective activity of the structures.
Furthermore, when a water-ln-oil emulsion is used, a second enzyme can advantageously ba dissolved In the aqueous or hydrophilic phase. If this second

enzyme also has the ability to accumulate at the droplet/second liquid phase interfaces, the resultant cross-linked enzyme structures will contain both enzymes. Alternatively, if the second enzyme is selected so that it does not accumulate at the interfaces, a cross-linked enzyme structure will result with one enzyme being a major component of the structure, while the second enzyme is encapsulated or contained inside the structure. Such a combination enzyme structure can advantageously be used, for example, to catalyze multiple reactions in a single reaction step. Moreover, co-factors or reaction mediators, modified or otherwise, may be included in the droplet, e.g. a redox enzyme and suitable mediator may be incorporated in the sphere in order to regenerate a second redox enzyme In the sphere.
In a particular embodiment of the invention, a triglyceride, which is hydrolysabte by lipase, may be used as tie hydrophobic or oil phase, with an O/W emulsion being formed; the dispersed or oil phase, ie the trigtyceride, contained within the stabilized cross-linked structures or spheres is hydrolyzed by the lipase during and after the cross-linking reaction. The hydroiyzed products are generally water-soluble, and can thus readily be leached out, thereby minimizing or reducing the number of processing steps required to produce the stabilized structures.
In yet another embodiment of the invention, an initial O/W emulsion can be formed. In doing so, a certain degree of purification of the lipase takes place, since impurities present therein will not collect at the interfadal boundaries to the same extent as the lipase, The process may then include, before effecting the cross-linking, centrifugtng the emulsion and separating a concentrated emulsion from a dilute water phase, Thereafter,- a further O/W emulsion can be formed, using the concentrated emulsion. This step can, if desired, be repeated one or more times, to increase lipase purity. After the final such purification step, the emulsion may then be inverted to form a W/O emulsion, by the addition of surfactants with lower HLB values, which may be in the range of 3-10, more preferably 4-6. This ensures preferential orientation of the lipase active sites towards the outside of the dispersed phase droplets. Thereafter, cross-linking of the lipase as hereinbefore described, can be effected.
When an enzyme is used that collects at the interface, and a W/O emulsion is used, the Internal cross-linked -enzyme sphere morphology can be controlled by modifying the

dissolved enzyme concentration in the aqueous phase. For example, a hollow enzyme sphere can be formed through using reduced enzyme concentration, and activity by weight will improve due to decreased average diffusiona! distances for substrates.
To impart specific properties to the stabilized enzyme structures, a modifier may be added to the hydrophiiic phase and/or to the hydrophobic phase and/or to the emulsion. One or more of the following modifiers can be added in this fashion: a surfactant, a precipitator and an additive.
A surfactant may be used when it is desired to impart enhanced enzyme activity (as regards its use in a subsequent catalyzed reaction), and improved emulsion stability. The surfactant may be anionic, cationic, non-ionic, zwitterionic, polymeric, or mixtures of two or more of these. When an anionic surfactant is used, it may be an alkyl sulphate such as sodium lauryi sulphate or sodium iaureth sulphate, or an alkyl ether sulphate. When a cationic surfactant is used, It may be centrirnonium chloride. When a non-ionic surfactant is used, it may be an ethoxylated alkyl phenol such as polyoxyethylene(IO) iso-octylcyclohexyl ether (Triton X100) or polyoxyethylene(9) nonylphenyl ether (Monoxynol-9}. When a zwitterionic or amphiphilfic surfactant is used, it may be decyl betaine. When a polymeric surfactant is used, it may be an ethyiene oxlde-propylene oxide-ethylene oxide triblock copolymer, also known as a poioxamer, such as that available under the trade name Pluronic from BASF, or it may be a propylene oxide-ethyfene oxide-propylene oxide triblock copofymer, also known as a meroxapo!.
A preclpitator can be used when it is desired to precipitate the enzyme onto the emulsion interfaces. The precipitator, when present, may be an inorganic salt such as ammonium sulphate; an organic solvent such as 1,2-dimethylethane or acetone; or a dissolved polymer.
Additives or adjuvants will be used to impart desired properties to the emulsion and/or to the stabilized enzyme structures. Properties that can be modified by use of such additives include pH, by using, for example, a buffer; ionic strength, by using, for example, salts; viscosity, by using, for example, PEG; magnetic properties, by using, for example, iron salts; agglomeration tendency, by using, for example, a surfactant

possessing steric hindrance properties; and zeta potential, by using, for example, an anionic surfactant.
According to a second aspect of the invention, there is provided an enzyme structure, which comprises cross-linked enzyme molecules so that the structure is stable, with the structure being hollow, and in which the enzymes are immobilized, with a majority of active sites of the enzymes being orientated either internally or externally.
The enzyme structure may be as hereinbefore described with reference to the first aspect of the invention.
According to a third aspect of the invention, there is provided a method of carrying out a reaction, which includes allowing a reaction medium to undergo a reaction in the presence of a plurality of the enzyme structures as hereinbefore described, with the reaction thus being catalyzed by the enzyme structures.
The invention will now be described in more detail with reference to the following non-limiting examples and the accompanying drawings.
In the drawings,
FIGURE 1 is an optical microscope picture of cross-linked llpase capsules prepared in accordance with Example 1;
FIGURE 2 is a particle size distribution of the cross-linked lipase capsules prepared in Example 1;
FIGURE 3 is an optical microscope picture of cross-Jinked lipase capsules prepared in accordance with Example 2; and
FIGURE 4 is a particle size distribution of the cross-linked lipase capsules prepared in Example 2.
EXAMPLE 1 (non-optimized)
Cross-linked or stabilized Lipase spheres (structures) from wateNn-oll emulsion
1 g of lipase Amano AK was added to 195 g phosphate buffered saline (PBS) solution
(pH 7.8) and 5 g mineral oil (Castrol). This blend was then homogenized for 5 minutes
using a Silverson L4R laboratory rotor-stator homogenizer at 6000 rpm, 1,5 g of

hexamethylene di-socyanate (Merck Schuchardt) was added to the emulsion. The emulsion was then stored at room temperature for 2 hours. The cross-linked enzyme structures were then recovered by filtration using 0,45 urn filter paper and washed 5 times with 50 ml of PBS each time (total 250 mi PBS), Figure 1 shows typical stabilized enzyme spheres or structures obtained according to the method. Particle sizes were determined using laser light scattering (Maivem Mastersizer 2000), and an average Sauter mean diameter of 49.4 urn was obtained (see Fig. 2).
The activity of the stabilized enzyme (lipase) structures was determined using a p-Nitrophenylacetate assay method as described by Vorderwulbecke, T,, Kies!ich, K. & Erdmarsn, H. (1992). 'Comparison of iipases by different assays', Enzyme Microb. Techno!,, 14, 631-639; and L6pez~Serrano P., Cao L, van Rarrtwijk & Sheldon RA (2002), 'Cross-linked enzyme aggregates with enhanced activity : application to iipases', Biotechnology Letters,, 24,1379-1383.
This assay measures the release of p-nitrophenol from a p-nitrophenyl ester of a fatty acid. The reaction is done at pH 7.4 at 37*C and the liberated p-niirophenol is measured at 410nrrt. The activity obtained was 63 U/g lipase, where U is umof/mm.
EXAMPLE 2
Cross-linked or stabilized Lipase spheres .(.structures).from water-tn-oil emujsion
A Sipase solution was prepared by resuspending Candida rugosa lipase (Ailus Biologies,
Inc.) in 100 fnM Tris-CI (Tris(hydroxyrnethyl)aminomethan0) buffer (pH 8.0) to a final
concentration of 100 mg/ml. The enzyme sample was diafiitered using an Amicon
ultrafiltratton cell fitted with a 10 K poiyether sulfone membrane (Microsep (Pty) Ltd, PO
Box 391647, Bramiey 2018, South Africa) against 3 volumes of 100 mM Tris-Ci buffer
(pH 8.0).
Lipase spheres were prepared using the following reagents in the following volumes: 200 u! Candida rugosa lipase solution (as prepared above); 50 u! nonoxynoM; 50 ul tribulyrin; 5 ml mineral oil. This mixture was emulsified by stirring for 1 minute at 1500 rpnx To this solution 40 ul gluteraldebyde was added (25% aqueous solution) and allowed to stir for a further 10 minutes. The emuision was allowed to stand at 4eC for 12 hours.

After crosslinktng the emuisiorn was centrifuged at 10000 rpm for 5 minutes using a Beckrnan J2-21 ME centrifuge fitted with JA 20.1 rotor, after which the oil phase was removed. The peiiet was washed thrice with 10 ml of 100 mM Tris-Cl buffer (pH 8.0) and pallet was recovered using centrifugation as mentioned above. After washing the peflet was resuspended in 1 mi buffer and assayed for enzyme activity. Rgure 3 shows the enzyme spheres obtained. The spheres had a narrow size distribution between about 10 and 100 urn (Figure 4).
The activity of the stabilized enzyme (Iipase) structures was determined using a p-nstrophenylpaimrtate and p-ratrophenytbutyrste assay method as described by Vorderwulbecke, T., Kiesllch, K, & Erdrnann, H. (1992), 'Comparison of iipases by different assays', Enzyme Microb, Technof., 14, 631-639; and L6pez-Serrano P., Cao L., van Rantwijk & Shefdon RA. (2002), 'Cross-linked enzyme aggregates with enhanced activity; application to Upases', Biotechnology Letters., 24,1379-1383.
This assay measures the release of p-nitrophenoi from a p-nitrophenyl ester of a fatty acid. The reaction is dons at pH 8.0 at 37°C and the liberated p-mtrophenol is measured at 410nm. The activity without tributyrin as additive was 0.11% (for p-nitrophenylpalmitate) compared to th© original free enzyme in aqueous solution. Surprisingly, the actfv'rty obtained with tributyrin as an additive ranged from about 5% (for p-nitrophenyipaimtete) to 124% (for p-nitrophenylbutyrate) compared to the original free enzyme in aqueous solution.
EXAMPLE 3
Dextmn aldehydei as,CTOss~tjnker
Example 2 was repeated, except that the cross-linking agent used was acOvated
dextran from leuconstoc species, average moiecuiar weight 20 kDa (dextran aldehyde),
the oil phase was vegetable oil, the ratio of iipase solution to Tris buffer was 1:1, and no
surfactant was used. Dextran aldehyde wss prepared by reacting dextran with excess
sodium metaperiodate as described by Hong, T,, Guo, W., Yuan, H.t Li, J., Liu, Y., Ma,
L,, Bat, Y., & Li, T. (2004) 'Periodate oxidation of nanoscaled magnetic dextran
composites', Journal of Magnetism and Magnetic Materials, 269, 95-100, Activity
obtained was 7.5% (for p-nitrophenyipaimitale) compared to the original free enzyme in
aqueous solution.

EXAMPLE 4
Cross-linked enzvme spheres from oii-in-water emulsion
Example 2 was repeated, except that an oii-in-watsr emulsion was generated by
changing the ratio of liquid phases and the surfactant.
EXAMPLE 5 Different
Example 3 was repeated except that the enzyme used was laccase from UD4 species as described by Jordaan, J., Pletschke, B.I. & Leukes, W.D. (2004) "Purification and partial characterization of a thermostable laccase from an unidentified basldtomycete', Enz Microb Tachnol. 34, 635-641 , and the tributyrin was substituted with syringic acid (saturated solution in ethanol).
The spheres were equilibrated with tOO mM sucdnate-lactate buffer pH 4.5. The spheres were assayed for laccase activity with ABTS as the substrate at 25°C and the product was followed spectrophotometricalfy at 420 nm according to the method of Jordaan, J. & Leukes, W.D. (2003) Isolation of a thermostable iaccase with DMAS and MBTH oxidatfve coupling activity from a mesophilic white rot fungus'. Enz Microb TechnoL 33(2/3), 212-219.
EXAMPLES
Protein .coocentratio n
Decreasing the lipase concentration of Example 2 (without tributyrin) by half, leads to an
increase of more than 1 00% in lipase activity by weight.
A possible explanation for the increased activity that was achieved with lower protein concentration (compared to higher protein concentration) is the preferential accumulation of the protein (in this case lipase) at the water-oil interface. This would lead to 'hollow' spheres at lower iipase concentration. On a per weight basis, hollow spheres would be expected to have higher activity compared to ftiled' spheres, due to shorter average dtffusional distances for reaction substrates and products.
EXAMPLE 7
Addition of a precipitant

Addition of acetone as a precipitant to the emulsion of Example 2 (without tributyrin), leads to an increase in activity of 114%.
EXAMPLE 8 Choice of oil phase
Substituting vegetable oil for mineral oil in the process of Example 2 (without tributyrin) leads to a four-fold increase in activity, but increased difficulty of recovery from the ^product solution. This is possibly because of the presence of hydrolysed oil.
.EXAMPLE.. 9
Addltion of protectant
As discussed in Example 2, the addition of tributyrin as protectnt, led to an increase in Candida rugosa lipase activity from 0.14% to 5% (for p-nltrophenylpalmitate) compared to the original free enzyme concentration. This 'protectant ability' does not work for all enzymes. For example, the addition of tributyrin as protectant to lipase from Rhizopus otyzae led to a .twelve-fold decrease Jn activity from 4.17 to 0.35% (for p-nitrophenylbutyrate) compared to the original free enzyme.
EXAMPLE 10
Binding of residual free cross-linker groups to ..reduce aggregation
Through the addition of an amino acid to the final product, aggregation of spheres was
reduced. This is thought to be due to the amino acid binding to the residual crosslinker
groups on the sphere surface. Surprisingly, improved activity was also observed
compared to controls where an amino acid was not used, specifically when
phenylgiyctne was used as the amino acid. An improvement in activity of about 100%
for lipase spheres (based on both p-nitrophenylbutyrate and p-nitrophenyipalmitate)
was observed compared to the normal method of Example 2 (without tributyrin).
EXAMPLE 11
Recycling of laccase cross-linked spheres
Laccase spheres were prepared according to the method in Example 5. The 'spheres were reacted six times with 2f2'-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt (ABTS) as a substrate with recovery and washing between each
14

reaction. Laccase activity after these six recycles was comparable to the original activity of the spheres,
EXAMPLE12
Recycling. M liease Cross-linked..s[^hereswith.NEE
Lipase spheres were prepared according to the method in Example 2. The spheres
were reacted three times with naproxen ethyl ester (NEE) as a substrate at 40'C, with
recovery and washing between each reaction. Activity decreased by about 70% over
three recycles for the cross-linked [ipase spheres (CLECs of the same enzyme showed
similar activity losses. Brady, D., Steenkamp, L, Skein, E., Chaplin, J.A, and Reddy, S.
(2004) 'Optimisation of the enanttoselective biocatafytic hydrolysis of naproxen ethyl
ester using ChiroCLEC-CR. Enz. Microb. Techno!. 34, 283-291).
EXAMPLE13
Recycling of (ipase cross-linked spheres, With
Lipase spheres were prepared according to the method in Example 2. The spheres were reacted with p-nrtrophenyipatmitate as a substrate, with recovery and washing between each reaction. Activity decreased to 79.6% of original lipase sphere activity in the final recycle.
of lipase sphere activity
Candida rugosa lipase spheres were prepared according to the method in Example 2. Candida rugosa lipase CLEA's were prepared according to Example 6 of United States Patent Application 20030149172, Cao, L., and Elzinga, J,, with a gkiteraldehyde to ethylene d ia mine ratio of 1 :7. 88 .
Activity retention of spheres as compared to CLEA's with p-nitrophenylpalmitate as the substrate was measured as 2.7% for lipase spheres and 3.4% for CLEA's wnije activity with p-nitrophenylbutyrate as trie substrate was measured as 53.7% for lipase spheres and 6.5% for CLEA's.
EXAMPL£15
15

Water in Oil Candida rugosa lipase spheres were prepared according to the method in Example 2, Oil in Water Candida rugosa lipase spheres were prepared acoordjng to the method in Example 2 except that the volume of oil was reduced to 0.2 ml and the volume of buffer was increased to 5 ml. Specific activity obtained for the Water in Oil emulsion was 136.0% higher than the Oil in Water emulsion with p-nitrophenylbutyrafe as the substrate, and increase from 8.9 to 26.0 U/mg from the OinW to WinO emulsion,
EXAMPLE 16
Sphere size control effected through mechanical agitation
Candida rugosa lipase spheres were prepared according to the method in Example 2,
except that a Silverson homogenizer was used to create the emulsion rather than
stirring. Two experiments were performed varying only in the speed setting of the
homogenizer, namely 1000 and 3000 rpm respectively. Particle size distribution was
determined and the results indicated a mean diameter of 52.0 urn and 20,6 urn for the
spheres produced using 1000 rpm and 3000 rpm respectively while specific activity
increased by 28% and 83% for p-nitrophenylpalmitate and p-nitrophenylbutyrate as
substrates respectively compared to,
The invention thus provides a method of stabilizing an enzyme by means of cross-linking, using emulsions as a vehicle therefor. The invention also relates to exposing maximum surface area of enzyme per unit volume of the structure, for subsequent reaction when the structure is used as a catalyst. Additionally, the stabilized enzyme structures are easily recyclable, less expensive than most immobilized enzyme products, and will find widespread application as catalysts in various processes.
Additionally, due to the selective orientation of lipases at the hydrophilic/hydrophobic phase interface, they will be concentrated there. So this method, when applied in the example of an oil in water emulsion, will simultaneously purify the desired lipase from a crude cell lysate. The same would be true of other enzymes with external hydrophobic regions, including many membrane-associated enzymes.
The cross-linking of lipases at the phase interface will fix them in the activated {lid open) state.
16

The use of oil-in-water emulsions can permit mono-layer lipase spheres, thereby providing a cross-linking method that provides the maximum surface area to enzyme mass.
The use of water-in-oil emulsions would allow for denser, multi-layered enzyme spheres.
This form of enzyme immobilization allows for enzyme recovery and recycling,
It is believed that the process of the present invention, which provides the stabilized enzyme hollow spherical structures, provides the following advantages when the structures are subsequently used to catalyze reactions:
1. Maximum exposed surface area of catalyst (spherical, hollow capsules).
2. Buoyancy of catalyst can be controlled, eg, floating particles could be separated
from the reaction medium with ease.
3. The mean size (diameter) of the immobilized enzyme particle formed can be
controlled by controlling the size distribution of the emulsion.
4. Through use of the natural self-orientation of many lipases and some other
enzymes at solvent interfaces, the immobilized enzyme sphere may be
generated in a controlled manner so as to orientate the majority of active sites
either towards the lumen or externally as required.
5. Due to the presence of a hydrophilic/ hydrophobic interface, enzymes such as
Iipase are immobilized in the active form.

We claim:
1. A process for producing enzyme structures, comprising:
providing an emulsion of droplets of a first liquid phase dispersed in a second liquid phase, with the one liquid phase being a hydrophilic phase and the other liquid phase being a hydrophobic phase which is immiscible with the hydrophilic phase, characterized in that
enzyme molecules are located at or within interfacial boundaries of the droplets and the second liquid phase;
the enzyme molecules of the respective droplets are cross-linked so that enzyme structures, which are stable and in which the enzymes are immobilized with a majority of active sites of the enzymes being orientated either internally or externally, are formed from individual droplets; and
the enzyme structures are recovered from the second liquid phase.
2. The process as claimed in Claim 1, wherein the structures have openings so that the liquid phases can pass in or out of the structures.
3. The process as claimed in Claim 1, wherein structures are liquid impervious.
4. The process as claimed in any one of Claims 1 to 3 inclusive, which includes adding to the hydrophilic phase and/or to the hydrophobic phase and/or to the emulsion, a modifier for modifying the hyrophobicity and/or charge of the enzyme.
5. The process as claimed in any one of Claims 1 to 4 inclusive, wherein the enzyme is a lipase.
6. The process as claimed in Claim 5, wherein the lipase is Candida rugosa lipase.
7. The process as claimed in Claim 5 or Claim 6, wherein the provision of the emulsion is effected by dissolving the enzyme in the hydrophilic or water (W) phase and forming the emulsion by mixing the enzyme containing hydrophilic phase with the hydrophobic or oil (0] phase.
8. The process as claimed in Claim 7, which includes selectively precipitating the enzyme at the interface when the emulsion is an oil(O) in water(W) emulsion in which hydrophobic phase droplets are dispersed in a continuous hydrophilic phase, or within the droplet volume, when the emulsion is a water(W) in oil(O) emulsion in which hydrophilic phase droplets are dispersed in a continuous hydrophobic phase.
9. The process as claimed in Claim 7 or Claim 8, wherein the cross-linking of the enzyme molecules is effected by means of a cross-linking agent which is added to the hydrophilic phase and/or to the hydrophobic phase and/or to the emulsion.
10. The process as claimed in Claim 9, which includes adding to the hydrophilic phase and/or to the hydrophobic phase and/or to the emulsion, a temporary protectant that occupies active sites of the enzyme during the cross-linking, thereby inhibiting occupation of or reaction with the active sites by the cross-linking agent.
11. The process as claimed in any one of Claims 7 to 10, which includes adding an amino acid to the emulsion to inhibit agglomeration of the enzyme structures.
12. The process as claimed in any one of Claims 7 to 11, which includes recovering the enzyme structures from the second liquid phase.
13. The process as claimed in any one of Claims 7 to 12, which includes extracting the first liquid phase from the enzyme structures.
14. The process as claimed in any one of Claims 7 to 13, wherein the hydrophilic phase comprises water and, optionally, a buffer in the water.
15. The process as claimed in any one of Claims 7 to 13, wherein the hydrophilic phase comprises a polyethylene glycol and, optionally, water admixed with the polyethylene glycol.
16. The process as claimed in any one of Claims 7 to 15, wherein the hydrophobic phase comprises an oil; a hydrocarbon; an ether; or an ester.
17. The process as claimed in any one of Claims 7 to 16, wherein the emulsion is a water(W) in oil(O) emulsion in which hydrophilic phase droplets are dispersed in a continuous hydrophobic phase, with a second enzyme, co factor and/or mediator being present in the hydrophilic phase.
18. The process as claimed in any one of Claims 7 tol6, wherein a triglyceride, which is hydrolysable by lipase, is used as the hydrophobic phase, with an oil(O) in water(W) emulsion, in which hydrophobic phase droplets are dispersed in a continuous hydrophilic phase, being formed and with the dispersed hydrophobic phase contained within the cross-linked structures being hydrolyzed by the lipase during and after the cross-linking reaction.
19. The process as claimed in any one of Claims 1 to 16, wherein the emulsion is an oil(O) in water(W) emulsion, in which hydrophobic phase droplets are dispersed in a continuous hydrophilic phase, with the process including, before effecting the cross-linking, centrifuging the emulsion and separating a concentrated emulsion from a dilute hydrophilic phase, to increase enzyme purity; and inverting the emulsion to form a water(W) in oil(O) emulsion in which hydrophilic phase droplets are dispersed in a continuous hydrophobic phase, by the addition of a surfactant with a lower hydrophilic-lipophilic balance (HLB) value.
20. The process as claimed in any one of Claims 1 to 19 wherein, to impart specific properties to the enzyme structures, a modifier is added to the hydrophilic phase and/or to the hydrophobic phase and/or to the emulsion.
21. The process as claimed in Claim 20, wherein the modifier is a surfactant, for imparting enhanced enzyme activity and improved emulsion stability.
22. The process as claimed in Claim 20, wherein the modifier is a precipitator for precipitating the enzyme onto the emulsion interfaces.
23. The process as claimed in Claim 20, wherein the modifier is an additive for modifying the pH; ionic strength; viscosity; magnetic properties; agglomeration tendency; and/or zeta potential of the emulsion and/or the enzyme structures.

Documents:

4123-delnp-2006-Abstract-(18-11-2010).pdf

4123-delnp-2006-abstract.pdf

4123-delnp-2006-Claims-(18-11-2010).pdf

4123-DELNP-2006-Claims-(20-04-2012)..pdf

4123-DELNP-2006-Claims-(20-04-2012).pdf

4123-delnp-2006-claims.pdf

4123-DELNP-2006-Correspondence Others-(20-04-2012)..pdf

4123-DELNP-2006-Correspondence Others-(20-04-2012).pdf

4123-delnp-2006-Correspondence-Others-(18-11-2010).pdf

4123-DELNP-2006-Correspondence-Others-(20-09-2010).pdf

4123-delnp-2006-correspondence-others-1.pdf

4123-delnp-2006-correspondence-others.pdf

4123-delnp-2006-description (complete).pdf

4123-delnp-2006-drawings.pdf

4123-delnp-2006-Form-1-(18-11-2010).pdf

4123-delnp-2006-form-1.pdf

4123-delnp-2006-form-18.pdf

4123-delnp-2006-Form-2-(18-11-2010).pdf

4123-delnp-2006-form-2.pdf

4123-delnp-2006-form-26.pdf

4123-DELNP-2006-Form-3-(20-09-2010).pdf

4123-delnp-2006-form-3.pdf

4123-delnp-2006-form-5.pdf

4123-delnp-2006-GPA-(18-11-2010).pdf

4123-delnp-2006-pct-210.pdf

4123-delnp-2006-pct-304.pdf

4123-delnp-2006-pct-409.pdf

4123-delnp-2006-Petition 137-(18-11-2010).pdf

4123-delnp-2006-Petition 138-(18-11-2010).pdf

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Patent Number

252100

Indian Patent Application Number

4123/DELNP/2006

PG Journal Number

17/2012

Publication Date

27-Apr-2012

Grant Date

25-Apr-2012

Date of Filing

17-Jul-2006

Name of Patentee

CSIR

Applicant Address

SCIENTIA, 0002 PRETORIA, SOUTH AFRICA

Inventors:

#	Inventor's Name	Inventor's Address
1	CHETTY (nee SEWLALL), AVASHNEE, SHAMPARKESH	76 MOUNT GRACE, 255 ALBERTUS STREET, LA MONTAGNE, 0184 PRETORIA, SOUTH AFRICA (PREVIOUSLY UNIT 23 LAKEVIEW, HAYMEADOW STREET, FAIRIE GLEN, 0043 PRETORIA, SOUTH AFRICA)
2	ROLFES, HEIDI	408 STONEWALL LANE, FARIE GLEN EXT. 8, 0043 PRETORIA, SOUTH AFRICA
3	JORDAAN, JUSTIN	7 SABIE SANDS, 86 WEBBER ROAD, KLIPPOORTJIE, 1401 GERMISTON, SOUTH AFRICA.
4	MOOLMAN, FRANCIS, SEAN	CRAIG ST. 929, MORELETA PARK, 0044 PRETORIA, SOUTH AFRICA (PREVIOUSLY VLEILOERIE 79, 22 WILKINSON STREET, KILNERPARK, 0186 PRETORIA, SOUTH AFRICA)
5	BRADY, DEAN	19 LIEBENBERG ROAD, NOORDWYK, MIDRAND, 1687 JOHANNESBURG, SOUTH AFRICA

PCT International Classification Number

C12N 9/96

PCT International Application Number

PCT/IB2005/000192

PCT International Filing date

2005-01-27

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	2004/0685	2004-01-28	South Africa