Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates

Definitions

• 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.
• enzyme immobilization 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 with the aim of stabilizing the enzymes and reducing costs by making them recyclable.
• 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 (CLEC) and cross-linked enzyme agglomerates (CLEA). Claims to increased specific activity have been made for both of these.
• 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 CLEC where time- consuming crystallization protocols are required.
• both CLEC .and CLEA are limiting in that some active sites of the enzymes are not exposed, and hence processes utilizing either CLEA or CLEC would require excess enzyme catalyst (with an associated increased cost) for a particular function, to compensate for this.
• these processes do not have easy control over particle size and morphology over a large range of particle sizes. ⁇ " '
• 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 interfacial 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 immobilized with a majority of active sites of the enzymes being orientated either internally or externally, are formed from individual droplets.
• 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.
• the individual structures may have openings so that the liquid phases can pass in or out of the structures.
• 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.
• 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.
• 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.
• 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.
• the enzyme can be selected from enzyme classes such as Esterases, Proteases, Nitrilases, Nitrile hydratases, Oxynitrilases, Epoxide hydrolases, Halohydrin dehalogenases, Polyphenoloxidases (eg laccase), Penicillin amidases, Amino acylases, Ureases, Uricases, Lysozymes Asparaginases, Elastases, it is preferably lipase.
• enzyme classes such as Esterases, Proteases, Nitrilases, Nitrile hydratases, Oxynitrilases, Epoxide hydrolases, Halohydrin dehalogenases, Polyphenoloxidases (eg laccase), Penicillin amidases, Amino acylases, Ureases, Uricases, Lysozymes Asparaginases, Elastases, it is preferably lipase.
• the lipase can be chosen from microbial, 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 B, Candida utilis lipase, Thermomyces lanuginosus lipase, Rhizomucor miehei lipase, Aspergillus niger lipase, Aspergillus oryzae lipase, Penicillium sp lipase, Mucor javanicus lipase, Mucor miehei lipase, Rhizopus arrhizus lipase, Rhizopus delemer lipase, Rhizopus japonicus lipase, Rhizopus niveus lipa
• the stabilized lipase structures may, in particular, be used in hydrolysis, acidolysis, alcoholysis, esterification, transesterification, interesterification, ammoniolysis, aminolysis, and perhydrolysis reactions.
• Other enzyme classes will be used in other reaction mechanisms particular to their function.
• the emulsion may be provided by dissolving or solubilizing the enzyme in the hydrophilic phase (herein also referred to as 'the water phase' or simply as 'W'), and forming the emulsion by mixing the enzyme containing hydrophilic phase with the hydrophobic phase (herein also referred to as 'the oil phase' or simply as 'O').
• the emulsion may be of the type O ⁇ /V, ie oil or hydrophobic phase droplets in a continuous water or hydrophilic phase, W/O, ie water or hydrophilic phase droplets in a continuous oil or hydrophobic phase, 0/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 ('salting out').
• the cross-linking of the enzyme molecules may be effected by means of a cross-linking agent.
• the process may include adding the cross-linking agent to the hydrophilic phase and/or to the hydrophobic 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 to form a cross-linked macromolecule, 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.
• 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.
• 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.
• 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 hydrophobicity while modification with aspartic acid would result in improved hydrophilicity 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, centrifugation, 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 hydrophobic liquids) or water (for hydrophilic liquids).
• a suitable solvent such as hexane or supercritical carbon dioxide (for hydrophobic liquids) or water (for hydrophilic liquids).
• this may be effected by contacting the stabilized enzyme capsuies 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.
• the first iiquid phase can then be extracted by supercritical fluid extraction.
• the fluid is then preferably supercritical carbon dioxide.
• 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.
• 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 KH 2 P0 4 /NaOH solution.
• the hydrophilic phase may include or comprise a polyethylene glycol (PEG).
• PEG polyethylene glycol
• a low molecular weight polyethylene glycol such as PEG400 or PEG100
• it may be used on its own, ie the hydrophilic phase will then consist of the low molecular weight polyethylene glycol.
• a higher molecular weight polyethylene glycol may optionally instead be used, with it then being dissolved in water to form the hydrophilic phase.
• an isocyanate is used as the cross-linking agent in a water-in- oil 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 hydrogei support.
• 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 hydrophobic 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 dioctyl ether, diphenyl ether, or the like; an ester such as triglyceride, isopropyl palmitate or isopropyl myristate; or the like.
• an oil such as mineral, jojoba or avocado oil
• a hydrocarbon such as decane, heptane, hexane or isododecane
• an ether such as dioctyl ether, diphenyl ether, or the like
• an ester such as triglyceride, isopropyl palmitate or isopropyl myristate; or the like.
• the emulsion used in the process of the invention will normally be in the form of a water-in- oil or W/O emulsion; however, as previously indicated, instead a oil-in-water or O/W, oil- in-water-in-oil, ie O/W/O, or water-in-oil-in-water, ie W/O/W, emulsions can be used.
• a wate ' r-in-oil emulsion can be used to ensure that most of the lipase active sites, which are hydrophobic, are oriented outwardly, thus increasing the total effective activity of the structures.
• a second enzyme can advantageously be 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.
• a triglyceride which is hydrolysable by lipase, may be used as the hydrophobic or oil phase, with an O/W emulsion being formed; the dispersed or oil phase, ie the triglyceride, contained within the stabilized cross-linked structures or spheres is hydrolyzed by the lipase during and after the cross- linking reaction.
• the hydrolyzed 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.
• 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 interfacial boundaries to the same extent as the lipase.
• the process may then include, before effecting the cross-linking, centrifuging 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.
• 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.
• the internal cross-linked enzyme sphere morphology can be controlled by modifying the dissolved enzyme concentration in the aqueous phase.
• a hollow enzyme sphere can be formed through using reduced enzyme concentration, and activity by weight will improve due to decreased average diffusional distances for substrates.
• a modifier may be added to the hydrophilic 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.
• an anionic surfactant when used, it may be an alkyl sulphate such as sodium lauryl sulphate or sodium laureth sulphate, or an alky! ether sulphate.
• a cationic surfactant when a cationic surfactant is used, it may be centrimonium chloride.
• non-ionic surfactant when used, it may be an ethoxylated alkyl phenol such as polyoxyethylene(IO) iso-octylcyclohexyl ether (Triton X100) or polyoxyethylene(9) nonylphenyl ether (Nonoxynol-9).
• a zwitterionic or amphiphiliic surfactant when used, it may be decyl betaine.
• a polymeric surfactant when used, it may be an ethylene oxide-propylene oxide-ethylene oxide triblock copolymer, also known as a poloxamer, such as that , available under the trade name Pluronic from BASF, or it may be a propylene oxide- ethylene oxide-propylene oxide triblock copolymer, also known as a meroxapol.
• a precipitator 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.
• 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.
• 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.
• FIGURE 1 is an optical microscope picture of cross-linked lipase 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-iinked 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.
• FIG. 1 shows typical stabilized enzyme spheres or structures obtained according to the method. Particle sized were determined using laser light scattering (Malvem Mastersizer 2000), and an average Sauter mean diameter of 49.4 ⁇ m 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., Kieslich, K. &
• 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-nitrophenol is measured at 410nm.
• the activity obtained was 63 U/g lipase, where U is ⁇ mol/min.
• Lipase spheres were prepared using the following reagents in the following volumes: 200 ⁇ l Candida rugosa lipase solution (as prepared above); 50 ⁇ l nonoxynol-4; 50 ⁇ l tributyrin; 5 ml mineral oil. This mixture was emulsified by stirring for 1 minute at 1500 rpm. To this solution 40 ⁇ l gluteraldehyde was added (25% aqueous solution) and allowed to stir for a further 10 minutes. The emulsion was allowed to stand at 4°C for 12 hours.
• the activity of the stabilized enzyme (lipase) structures was determined using a p- nitrophenylpalmitate and p-nitrophenylbutyrate assay method as described by Vorderwulbecke, T., Kieslich, K. & Erdmann, H. (1992). 'Comparison of lipases by different assays', Enzyme Microb. Technol., 14, 631-639; and L ⁇ pez-Serrano P., Cao L., van Rantwijk & Sheldon R.A. (2002). 'Cross-linked enzyme aggregates with enhanced activity : application to upases', 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 8.0 at 37°C and the liberated p-nitrophenol is measured at 410nm.
• the activity without tributyrin as additive was 0.11 % (for p- nitrophenyipalmitate) compared to the original free enzyme in aqueous solution.
• the activity obtained with tributyrin as an additive ranged from about 5% (for p-nitrophenylpalmitate) to 124% (for p-nitrophenylbutyrate) compared to the original free enzyme in aqueous solution.
• Example 2 was repeated, except that the cross-linking agent used was activated dextran from leuconstoc species, average molecular weight 20 kDa (dextran aldehyde), the oil phase was vegetable oil, the ratio of lipase solution to Tris buffer was 1 :1 , and no surfactant was used.
• Dextran aldehyde was prepared by reacting dextran with excess sodium metaperiodate as described by Hong, T., Guo, W., Yuan, H., Li, J., Liu, Y., Ma, L., Bai, 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-nitrophenylpalmitate) compared to the original free enzyme in aqueous solution.
• EXAMPLE 4 was activated dextran from leuconstoc species, average molecular weight 20
• Example 2 was repeated, except that an oil-in-water emulsion was generated by changing the ratio of liquid phases and the surfactant.
• 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 basidiomycete'. Enz Microb Technol. 34, 635-641 , and the tributyrin was substituted with syringic acid (saturated solution in ethanoi).
• the spheres were equilibrated with 100 mM succinate-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 laccase with DMAB and MBTH oxidative coupling activity from a mesophilic white rot fungus'. Enz Microb Technol. 33(2/3), 212-219.
• Example 2 Decreasing the lipase concentration of Example 2 (without tributyrin) by half, leads to an increase of more than 100% in lipase activity by weight.
• Laccase spheres were prepared according to the method in Example 5. The spheres were reacted six times with 2,2'-Azino-bis-(3-ethylbenzothiazoiine-6-sulfonic acid diammonium salt (ABTS) as a substrate with recovery and washing between each reaction. Laccase activity after these six recycles was comparable to the original activity of the spheres.
• ABTS 2,2'-Azino-bis-(3-ethylbenzothiazoiine-6-sulfonic acid diammonium salt
• 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 lipase 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 enantioselective biocatalytic hydrolysis of naproxen ethyl ester using ChiroCLEC-CR. Enz. Microb. Technol. 34, 283-291).
• Lipase spheres were prepared according to the method in Example 2. The spheres were reacted with p-nitrophenylpalmitate as a substrate, with recovery and washing between each reaction. Activity decreased to 79.6% of original lipase sphere activity in the final recycle.
• Candida rugosa lipase spheres were prepared according to the method in Example 2.
• Candida rugosa lipase CLEA's were prepared according to Example 8 of United States Patent Application 20030149172, Cao, L., and Elzinga, J., with a gluteraldehyde to ethylene diamine ratio of 1 :7.88.
• Oil in Water and Water in Oil Emulsions (orientation of lipase) 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 according 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-nitrophenylbutyrate as the substrate, and increase from 8.9 to 26.0 U/mg from the OinW to WinO emulsion.
• 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 ⁇ m and 20.6 ⁇ m for the spheres produced using 1000 rpm and 3000 rpm respectively while specific activity increased by 28% and 83% for p-nitrophenyipalmitate 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.
• lipases 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.
• 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.
• water-in-oil emulsions would allow for denser, multi-layered enzyme spheres.
• the mean size (diameter) of the immobilized enzyme particle formed can be controlled by controlling the size distribution of the emulsion.
• 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.

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