Indian Patents. 270795:ENZYME PREPARATIONS OBTAINABLE BY ENZYME IMMOBILIZATES

Title of Invention	ENZYME PREPARATIONS OBTAINABLE BY ENZYME IMMOBILIZATES
Abstract	The invention is directed to enzyme preparations which are obtainable by enzyme immobilizates which comprise enzymes or microorganisms containing enzymes immobilized on an inert carrier being provided with a silicone coating obtained by hydrosilylation, to a process for producing such enzyme preparations and to the use of enzyme preparations as an industrial biocatalyst.

Full Text	Enzyme preparations The invention relates to novel enzyme preparations for use as biocatalysts. Microorganisms and isolated enzymes find wide use as a catalyst in the chemical industry or in food production. An overview is offered, for example, by: A. Liese, K. Seelbach, C. Wandrey, Industrial Biotransformations, Wiley-VCH: 2 000, Weinheim, Germany. In order to ensure economic use of such biocatalysts, some conditions have to be satisfied: the biocatalyst has to be active for a sufficiently long time under the reaction conditions, it should be readily removable after the end of the reaction and it should be reusable as often as possible. Ideally, these conditions should be satisfied for a very wide range of reaction conditions (for example temperature range, type of solvents used, pressures, etc), in order to provide as universal as possible a catalyst. In order to satisfy these conditions, it is typically necessary to immobilize the enzymes or microorganisms containing enzymes used. Frequently, the enzymes or microorganisms containing enzymes are immobilized noncovalently on carriers; the carriers used are frequently ion exchange resins or polymer particles which possess suitable particle size distributions. Examples for this purpose are the commercial products Novozym 435, Lipozym RM IM or Lipozym TL IM from Novozymes A/S, Bagsvaerd, Denmark or Amano PS, from Amano, Japan. These examples are immobilized lipases which find wide use, since such immobilizates also exhibit industrially utilizable activities in nonaqueous systems, i.e. those which comprise only organic solvents, if any, as described, for example, in J. Chem. Soc, Chem. Comm. 1989, 934- 935. A disadvantage of the use of such immobilizates is, however, firstly the desorption of the enzyme or of the microorganisms containing enzymes which occurs depending on the reaction system used, for example in the case of use of surfactant components. The loss of activity associated with such a desorption is shown in Comparative Example 1. In addition, such preparations possess inadequate mechanical stability, as a result of which use in simple stirred reactors is possible only with acceptance of significantly restricted reusability, if at all. The mechanical instability of such preparations is shown in Comparative Example 2. In order to enable the repeated use of such enzyme preparations, other reactor designs therefore have to be used. Eur. J. Lipid Sci. Technol. 2003, 105, 601-607 describes, for example, the use of a fixed bed reactor for performing lipase-catalyzed esterifications. A disadvantage of this process is, however, the restriction to low-viscosity homogeneous reaction mixtures, since high-viscosity mixtures or suspensions cannot be conveyed through a fixed bed. K. Faber "Biotransformations in Organic Chemistry", Springer: 2000, Berlin, Germany, describes, on page 3 84 ff., the use of enzymes incorporated into alginates. However, the preparations thus obtained have an exceptionally low mechanical stability and exhibit only low activity in nonaqueous systems. In addition, the subsequent crosslinking of immobilized enzymes with reactive substances, for example glutaraldehyde, is described. However, a disadvantage is the usually significantly reduced specific activity of the crosslinked preparations compared to the activity before the modification. Furthermore, this process does not make any contribution to improving the Likewise described there is the covalent immobilization of enzymes on reactive carriers. A disadvantage here is that suitable functional groups have to be present on the surface of the enzyme, which can react with the carrier; in addition, a loss of enzyme activity is often achieved as the result. J. Am. Chem. Soc. 1999, 121, 9487-9496 describes the incorporation of enzymes into siloxane matrices, known as sol-gels. A disadvantage of sol-gel preparations is the low particle size distribution which complicates efficient removal by filtration, the lack of mechanical stability, the occurrence of desorption, the use of toxic reactants (for the toxicity of TEOS, see, for example, Nippon Sanso Giho 1990, 9, 68-72 and Archives of Toxicology 1994, 68, 277-283; for the toxicity of TMOS, see, for example, Fundamental and Applied Toxicology 1989, 13, 285-295; for the production of nontoxic sol-gels, laborious steps are necessary, for example storage over 6 months or thermal treatment at 350°C, as described in Polimery w Medycynie 2000, 30, 45-54, and also the swelling behaviour which is very highly dependent upon the solvent used and does not permit universal use in different reaction systems (aqueous and nonaqueous). J. Sol Gel Sci. Technol. 2003, 26, 1183-1187 shows, by way of example, the solvent dependence of the enzyme activity observed and hence the lack of satisfaction of the demand for wide usability. Landbauforschung Volkenrode, 2002, special edition 241, 41-46 describes sol-gel preparations in which enzymes are first immobilized onto "fine" silicone particles and then encapsulated into a sol-gel. The problem of mechanical stability is thus solved partially, but the experiments described show that sufficient activities are achieved only in selected solvents; use in a solvent-free system is not described at all. In addition, the preparations are not obtained in a directly usable form, but rather first have to be cut to an appropriate size, which is barely implementable on the industrial scale. J. Mol. Catal. B, 2005, 35, 93-99 describes the immobilization of enzymes by incorporation of aqueous enzyme solutions into mechanically stable silicone spheres, known as static emulsions. The resulting specific activities of the preparations are, though, at up to 33 U/g, much too low compared to the above- described immobilizates on inert carriers, where specific activities of more than 1000 U/g can be achieved easily (U = unit or pmol/min). WO 03/106607 Al likewise describes such static emulsions, but exclusively the use in aqueous systems is described; the application is a washing composition, i.e. not a biocatalysis, and the resulting particle sizes, at approx. 10 ^m, are too small for efficient filtration out of organic reaction mixtures. There is therefore still a need for methods of enzyme immobilization which overcome the disadvantages of the prior art, in order to implement biocatalytic processes which have not been realizable to date. It was therefore an object of the present invention to provide enzyme preparations which do not have one or more of the disadvantages of the prior art preparations. In particular, enzyme preparations shall be provided, which have a high stability with respect to mechanical forces and with respect to desorption and at the same time preferably possess specific activities in different aqueous and nonaqueous reaction mixtures which are high enough to enable industrial use. In terms of their particle size distribution, the enzyme preparations should preferably be capable of being removed from the reaction system in a simple manner and of being reused. Further objects which are not specified explicitly are evident from the context of the description which follows, the examples and the claims. It has been found that, surprisingly, this object is achieved by enzyme preparations which are obtained by immobilizing enzymes or microorganisms comprising enzymes on an inert carrier and then coating with a silicone coating obtained by hydrosilylation. The present invention therefore provides enzyme preparations which are obtainable by enzyme immobilizates which comprise enzymes or microorganisms containing enzymes immobilized on an inert carrier being provided with a silicone coating obtained by hydrosilylation, and their use as an industrial biocatalyst. The present invention also provides a process for preparing the inventive enzyme preparations, which is characterized in that enzyme immobilizates which comprise enzymes or microorganisms comprising enzymes immobilized on an inert carrier are provided with a silicone coating obtained by hydrosilylation. The inventive enzyme preparations have the advantage that they have a high stability with respect to mechanical forces and with respect to desorption. In spite of these improved properties, the inventive enzyme preparations have specific activities in various aqueous reaction mixtures (for example in the hydrolysis of tributyrin) and nonaqueous reaction mixtures (for example in the solvent-free synthesis of propyl laurate), which are high enough to enable industrial use. The inventive enzyme preparations also have the advantage that the selection of the carrier material and of the associated particle size distribution allows the particle size to be adjusted such that simple removal of the enzyme preparations from the reaction system and hence also the reuse of the enzyme preparations is possible. The inventive enzyme preparations and a process for their production are described below by way of example, without any intention that the invention be restricted to these illustrative embodiments. When ranges, general formulae or compound classes are specified below, these shall not only encompass the corresponding ranges or groups of compounds which are mentioned explicitly but also all sub-ranges and sub-groups of compounds which can be obtained by selecting individual values (ranges) or compounds. When documents are cited within the present description, their contents shall be incorporated completely in the disclosure-content of the present invention. When compounds, for example organically modified polysiloxanes which may have different units more than once are described in the context of the present invention, they may occur in these compounds in random distribution (statistical oligomer) or ordered (block oligomer). Information regarding the number of units in such compounds should be interpreted as the mean value, averaged over all appropriate compounds. The inventive enzyme preparations are notable in that they are obtainable by providing enzyme immobilizates which comprise enzymes or microorganisms comprising enzymes immobilized on an inert carrier with a silicone coating, which is obtained by hydrosilylation. To produce the enzyme immobilizates, it is possible to use whole cells, resting cells, purified enzymes or cell extracts which comprise the corresponding enzymes, or mixtures thereof. Preference is given to using hydrolytic enzymes, for example lipases, esterases or proteases, for example lipases from Candida rugosa, Candida antarctica, Pseudomonas sp., Thermomyces langosiosus, porcine pancreas, Mucor miehei, Alcaligines sp., cholesterol esterase from Candida rugosa, esterase from the porcine liver, more preferably lipases. Accordingly, the enzyme immobilizates preferably comprise enzymes from the class of the hydrolases, preferably lipases. The inert carriers used may be inert organic or inorganic carriers. The inert carriers used, or present in the enzyme immobilizate, are preferably those particulate carriers which have a particle size distribution in which at least 90% of the particles have a particle size of 10 to 5000 [jm, preferably of 50 \|im to 2000 Jim. The organic carriers used may especially be those which comprise or consist of polyacrylate, polymethacrylate, polyvinylstyrene, styrene-divinylbenzene copolymers, polypropylene, polyethylene, polyethylene terephthalate, PTFE and/or other polymers. The carrier materials used may, depending on the enzyme to be immobilized, especially be acidic or basic ion exchange resins, for example Duolite A568, Duolite XAD 761, Duolite XAD 1180, Duolite XAD 7HP, Amberlite IR 120, Amberlite IR 400, Amberlite CG 50, Amberlyst 15 (all products from Rohm and Haas) or Lewatit CNP 105 and Lewatit VP OC 1600 (products from Lanxess, Leverkusen, Germany). The inorganic carriers used may be oxidic and/or ceramic carriers known from the prior art. In particular, the inorganic carriers used may, for example, be Celite, zeolites, silica, controlled-pore glass (CPG) or other carriers, as described, for example, in L. Cao, "Carrier-bound Immobilized Enzymes: Principles, Application and Design", Wiley-VCH: 2005, Weinheim, Germany. More preferably, the inert carriers present in the enzyme immobilizate or the inert carriers used to produce the enzyme immobilizates consist of polyvinylstyrene, polymethacrylate or polyacrylate. The immobilization on the particles can, in accordance with the invention, be effected covalently or noncovalently, preferably noncovalently. For noncovalent immobilization, the carrier can be incubated or impregnated, for example, with an aqueous enzyme solution which may optionally comprise further constituents, for example inorganic salts or detergents. This incubation/impregnation can be carried out, for example, at temperatures between 0°C and 50°C, preferably between 0°C and 40°C. Preference is given to effecting the incubation/impregnation over a period of a few minutes to a few hours. The progress of the incubation can be followed by determining the concentration of the enzyme in the solution with the common methods for protein determination. On attainment of the desired degree of immobilization, the carrier can preferably be washed with water and, if desired, dried. An enzyme immobilizate obtained in this way can subsequently be provided with a silicone coating in accordance with the invention. According to the invention, it is, however, also possible to use enzyme immobilizates which are commercially available, for example Novozym 435, Lipozym RM IM or Lipozym TL IM from Novozymes A/S, Bagsvaerd, Denmark, or Amano PS from Amano, Japan. According to the invention, the silicone coating is obtained by hydrosilylation. To this end, preferably Si-H-functional polysiloxanes are reacted in the presence of catalysts, preferably of transition metal catalysts, with organically modified polysiloxanes which possess at least one terminal carbon-carbon double bond, preferably at least two carbon-carbon double bonds. The Si-H-functional polysiloxanes used are preferably SiH polysiloxanes of the general formula I where N = a + b + c + d + 2 = 3to 850, preferably 6 to 160, a = 1 to 800, preferably 2 to 150, b = 0 to 400, preferably 2 to 75, c = 0 to 10, preferably 0, d = 0 to 10, preferably 0, R1 are independently the same or different, and are selected from the group comprising: saturated or unsaturated, optionally branched alkyl groups having 1 to 3 0 carbon atoms, alkaryl radicals having 7 to 3 0 carbon atoms, aryl radicals having 6 to 3 0 carbon atoms, preferably alkyl groups having 1 to 4 carbon atoms or phenyl, especially methyl; R2a are independently hydrogen or R1; R2b are independently hydrogen or R1; R3 are independently identical or different radicals of the general formula Ia where N =a + b + c + d + 2 = 3to850, preferably 6 to 160, a = 1 to 800, preferably 2 to 150, b = 0 to 400, preferably 2 to 75, c = 0 to 10, preferably 0, d = 0 to 10, preferably 0, R1 are independently the same or different, and are selected from the group comprising: saturated or unsaturated, optionally branched alkyl groups having 1 to 30 carbon atoms, alkaryl radicals having 7 to 3 0 carbon atoms, aryl radicals having 6 to 3 0 carbon atoms, preferably alkyl groups having 1 to 4 carbon atoms or phenyl, especially methyl; R2a are independently hydrogen or R1; R2b are independently hydrogen or R1; R3 are independently identical or different radicals of the formula la or an R1 radical. Preference is given to using, as the SiH-functional polysiloxane, a polysiloxane of the general formula I where N = a + b + c + d + 2 = 6 to 160, a = 2 to 150, b = 2 to 75, c =0, d =0, R1 are independently the same or different, and are selected from the group comprising: saturated or unsaturated, optionally branched alkyl groups having 1 to 3 0 carbon atoms, alkaryl radicals having 7 to 30 carbon atoms, aryl radicals having 6 to 30 carbon atoms, preferably alkyl groups having 1 to 4 carbon atoms or phenyl, especially methyl; R2a are independently hydrogen or R1; R2b are independently hydrogen or R1. It is well known to the person skilled in the art that the compounds are or may be present in the form of a mixture with a distribution controlled essentially by statistical laws. The values for the indices a, b, c and d are therefore typically mean values. According to the invention, the olefinic reactants, i.e. the polysiloxanes containing a terminal carbon- carbon double bond, are preferably polysiloxanes of the general formula II: N =m + n + o+p + 2 = 3to 1000, preferably 10 to 600, m = 1 to 800, preferably 2 to 600, n = 0 to 20, preferably 0 to 10, more preferably 0, o = 0 to 10, preferably 0, p = 0 to 10, preferably 0, R4 are independently the same or different and are from the following group: saturated or unsaturated, optionally branched alkyl groups having 1 to 3 0 carbon atoms, alkaryl radicals having 7 to 3 0 carbon atoms, aryl radicals having 6 to 3 0 carbon atoms, preferably alkyl groups having 1 to 4 carbon atoms or phenyl, especially methyl; R5 are independently a terminally unsaturated alkyl radical, preferably vinyl, or an alkoxy radical, preferably having 3 to 2 0 carbon atoms, or R4; R6 are independently identical or different radicals of the general formula IIa N = m + n + o+p + 2 = 3to 1000, preferably 10 to 600, m = 1 to 800, preferably 2 to 600, n = 0 to 20, preferably 0 to 10, more preferably 0, o = 0 to 10, preferably 0, p = 0 to 10, preferably 0, R.4 are independently the same or different and are from the following group: saturated or unsaturated, optionally branched alkyl groups having 1 to 3 0 carbon atoms, alkaryl radicals having 7 to 3 0 carbon atoms, aryl radicals having 6 to 30 carbon atoms, preferably alkyl groups having 1 to 4 carbon atoms or phenyl, especially methyl; R5 are independently a terminally unsaturated alkyl radical, preferably vinyl, or an alkoxy radical, preferably having 3 to 2 0 carbon atoms, or R4; R6 are independently identical or different radicals of the general formula Ila or R4 radicals. N =m+n+o+p+2=10to 600, m = 2 to 600, n =0, The polysiloxanes containing a terminal carbon-carbon double bond used are preferably polysiloxanes of the general formula II: o =0, p =0, R4 are independently the same or different and are from the following group: saturated or unsaturated, optionally branched alkyl groups having 1 to 30 carbon atoms, alkaryl radicals having 7 to 3 0 carbon atoms, aryl radicals having 6 to 3 0 carbon atoms, preferably alkyl groups having 1 to 4 carbon atoms or phenyl, especially methyl; R5 are independently a terminally unsaturated alkyl radical, preferably vinyl, or an alkoxy radical, preferably having 3 to 2 0 carbon atoms. It is well known to the person skilled in the art that the compounds of the formula II are or may be present in the form of a mixture with a distribution controlled essentially by statistical laws. The values of the indices m, n, o and p are therefore typically mean values. The hydrosilylation can be carried out by established methods in the presence of a catalyst. It is possible, for example, to use catalysts which are typically used for hydrosilylations, for example platinum, rhodium, osmium, ruthenium, palladium, iridium complexes or similar compounds or the corresponding pure elements or their derivatives immobilized on silica, alumina or activated carbon or similar carrier materials. Preference is given to performing the hydrosilylation in the presence of Pt catalysts such as cisplatin or Karstedt catalyst [tris(divinyltetramethyldisiloxane)- bis-platinum]. The amount of catalyst used is preferably 10~7 to 10-1 mol per mole of olefin or per mole of terminal carbon-carbon double bond, preferably 1 to 100 ppm. The hydrosilylation is carried out preferably at temperatures of 0 to 200°C, preferably of 20 to 120°C. The hydrosilylation can be carried out in the presence or absence of solvent. Generally, solvents are not needed for the performance of the reaction. The reaction can, however, be carried out in suitable solvents, for example aliphatic or aromatic hydrocarbons, cyclic oligosiloxanes, alcohols or esters. Suitable solvents are especially cyclohexane or toluene. According to the invention, based on the mass of the carrier used, preferably 1 to 500% by mass, more preferably 10 to 200% by mass, especially preferably 20 to 150% by mass, of siloxane components are used. The siloxane components are composed especially of the sum total of the compounds of the formula I and II and of their reaction products. The hydrosilylation can be carried out using a wide variety of different ratios of the compounds of the formulae I to compounds of the formulae II. Preference is given to effecting the hydrosilylation at a molar ratio based on the reactive groups of 1:10 to 10:1, more preferably of 1:5 to 5:1, especially preferably of 1:1.1 to 1.1:1 and most preferably of 1:1. Selection of the compounds of the general formula I and II used and variation in their mixing ratios allows the properties of the silicone coating to be tailored in relation to perviousness for substrates and other reaction properties. Selection of the weight ratio of silicone components to enzyme immobilizates allows the layer thicknesses of the silicone coating to be varied and to be adjusted to appropriate requirements. The inventive silicone coating, produced by hydrosilylation, can be obtained by carrying out the hydrosilylation in the presence of the enzyme immobilizates. However, it is also possible to obtain the coatings by applying a siloxane obtained by hydrosilylation subsequently to the enzyme immobilizates. This can be effected, for example, by treating the enzyme immobilizates with a solution of the siloxane, for example a solution of the siloxane in an organic solvent, especially cyclohexane or toluene. Subsequently, the solvent can be removed, for example, by evaporation. The concentration of siloxane in such a solution is preferably 10 to 100% by mass, more preferably 30 to 100% by mass. However, preference is given to obtaining the inventive silicone coating by carrying out the hydrosilylation in the presence of the enzyme immobilizates. The inventive enzyme preparations are preferably prepared by the process according to the invention described below. This process for preparing enzyme preparations is notable in that enzyme immobilizates which comprise enzymes or microorganisms comprising enzymes immobilized on an inert carrier are provided with a silicone coating obtained by hydrosilylation. Preference is given to performing the process according to the invention in such a way that the enzyme immobilizates are provided with a silicone coating by contacting the enzyme immobilizates with a reaction mixture which comprises SiH-functional polysiloxanes, polysiloxanes containing terminal carbon-carbon double bonds and a catalyst which catalyzes the hydrosilylation under hydrosilylation conditions. In particular, the process can be performed in such a way that a hydrosilylation reaction is carried out in the presence of enzyme immobilizates which comprise enzymes or microorganisms comprising enzymes immobilized on an inert carrier. The silicone formed in the hydrosilylation allows the enzyme immobilizate to be provided with a silicone coating. The hydrosilylation can be carried out in a manner known to those skilled in the art. Preference is given to performing the hydrosilylation using the abovementioned parameters/feedstocks/catalysts. In a preferred embodiment of the process according to the invention, a particular amount of enzyme immobilizate is admixed with a mixture (reaction mixture) of the silicone reagents (compounds of the formulae I and II plus catalyst), for example by adding a mixture comprising compounds of the general formula I and of the general formula II and Karstedt catalyst. For example, it is possible to add to 1 g of an enzyme immobilizate a mixture of compounds of the formula I and II in a molar mixing ratio of 10:1 to 1:10, and also Karstedt catalyst, for example 50 ppm based on the amount of silicone components present. For the purpose of optimizing the coating properties, it may be advantageous to dissolve the silicone components including the catalyst, before the addition, in a solvent, for example cyclohexane, toluene or another organic solvent, and then to add the solution to the enzyme immobilizate. When, for example, cyclohexane is used as the solvent, it has been found to be advantageous, after addition of the solution to the enzyme immobilizates, to strongly disperse this mixture for approx. 15 to 3 0 min, for example by means of a vortexer (Ika, level 9), until the bulk of the cyclohexane has evaporated off. Subsequently, the resulting enzyme preparations are dried, i.e. hardened, in a drying cabinet at 50°C, for example for 12 hours. Altering the mixing ratios of the compounds of the general formula I and II allows the properties of the silicone coating to be varied without any problem and adjusted to appropriate requirements. A further embodiment of the process according to the invention differs from the above embodiment in that the enzyme immobilizates to be coated are immersed into the desired reaction mixture, then removed from the reaction mixture and dried. The removal can be effected, for example, using a screen which retains the enzyme immobilizate particles. The immersion time is preferably 1 to 10 minutes. The drying can be effected in a conventional drying cabinet. Preference is given to effecting the drying/hardening at a temperature of 20°C to 80°C, preferably at 40°C to 60°C, more preferably at approx. 50°C. In a further embodiment of the process according to the invention, which is suitable especially for performance on the industrial scale, the hydrosilylation is carried out using a pelletizing pan unit (for example from Erweka or Eirich) . In this case, a defined amount of enzyme immobilizate particles is introduced into the so-called pan unit and stirred. Subsequently, either the mixture comprising compounds of the formulae I and II, and also catalyst and also if appropriate solvent, is added or else, preferably, using a two-substance nozzle (for example from Schlick or others), in which the mixture or the components is/are applied under pressure (for example nitrogen or synthetic air) in the form of a fine droplet mist, in order to ensure a very homogeneous distribution on the particles. After a prolonged coating time, the particles are removed as described above and dried, i.e. hardened, for a few hours at a temperature of 2 0°C to 80°C, preferably of 40°C to 60°C, more preferably of 50°C, in a drying cabinet, and can then be stored at room temperature until further use. In a further embodiment, the particles can be generated in a fluidized bed reactor (for example from Erweka), in which particles and the reaction mixture are applied with high dispersion in appropriate mixing ratios. The inventive enzyme preparations can be used, for example, as biocatalysts, especially as industrial biocatalysts. The present invention is illustrated in detail with reference to Figures 1 and 2, without being restricted thereto. Figures 1 and 2 show images of stirred suspensions in a 100 ml beaker with a diameter of the base of 4 cm. The suspensions have been produced as described in Example 2. Figure 1 shows that the stirred suspension based on untreated NZ435 is cloudy as a result of fine particles. The stirred suspension based on NZ435 treated in accordance with the invention, in contrast, is clear, i.e. comprises no particles or at least no particles in a visible size (Fig. 2). The examples which follow are intended to illustrate the present invention in detail, without restricting the scope of protection which is evident from the description and the claims. Examples Materials and methods: Novozym 435 (NZ435) is a commercial enzyme immobilizate from Novozymes A/S, Bagsvaerd, Denmark, a lipase B from C. antarctica immobilized on a polymethacrylate by adsorption. Hydrolytic activity (tributyrin hydrolysis in aqueous medium): The hydrolytic activity was determined by the so-called pH-stat method. In this method, the acid released in the hydrolysis is titrated against a base, such that the pH of the solution is kept constant. The time dependence of the consumption of base allows the acid released, and hence the enzyme activity, to be quantified. Illustrative procedure: 10-20 mg of catalytically active particles were added to 25 ml of Tris-HCl buffer (1 mM, pH 7.5; additionally contains 0.1 mM NaCl and CaCl2) and 500 jil of tributyrin were added. The hydrolytic activity was quantified on an autotitrator (Tritroline alpha, from Schott) via the amount of base titrated in (50 mM NaOH). Hydrolytic activity (ethyl valerate in aqueous medium): Analogously to the determination of the hydrolysis activity using the example of tributyrin, ethyl valerate can also be used. Illustrative procedure: 10-20 mg of catalytically active particles were added to 25 ml of phosphate buffer (1 mM, pH 8.0) and 500 ul of ethyl valerate. The hydrolytic activity was quantified on an autotitrator (Tritroline alpha, from Schott) via the amount of base titrated in (10 mM NaOH). Synthesis activity in PLU units (propyl laurate synthesis activity in solvent-free system): 10 mg of catalytically active particles were added to 5 ml of equimolar substrate solution (lauric acid and 1-propanol) and incubated while shaking and/or stirring at 60°C. Samples (Vsampie: 50 (0,1) were taken every 5 min over 25 min and transferred into 950 (0,1 of decane (internal standard: 4 mM dodecane). The PLUs were determined with reference to the initial product formation rates. Propyl laurate was detected by gas chromatography (retention time: 9.791 min) (Shimadzu 2010, BTX column from SGE; length 25 m, I.D. 0.22 (am; film: 0.25 (0m; detector type: FID at 300°C; injector temperature 275°C and injection volume 1 1, split ratio 35.0; carrier gas pressure (helium) 150 kPa; temperature programme: start temperature 60°C, hold for 1.5 min, temperature rise 20°C/min, end temperature 250°C, hold for 2.5 min). Determination of laccase activity: To determine laccase activity, catalytically active particles (native or immobilized laccase) are transferred into 19 ml of potassium phosphate buffer (100 mM, pH 6, 37°C) with 1 ml of ABTS solution (ready- to-use solution, 1.8 mM, available from Sigma-Aldrich) and the increased extinction is measured photospectro- metrically at 405 nm. Laccase activity shall be monitored over a period of 2 0 min. The samples are taken at intervals of 5 min. The activity can be determined as follows: activity A Ext. 405 change in extinction as a function of time Vtotai total volume of reaction batch [20 ml] Vsample volume of sample [2 ml] At change in time [min] e extinction coefficient for ABTS at 405 nm [43.2 ml mol-1 cm-1] d path length of cell [1 cm] The activity is reported in units (U/ml or U/g) defined as 1 mol of substrate conversion per minute. Protein determination according to Bradford: The determination of the protein content in the supernatant was carried out according to the method of Bradford (Anal. Chem. 1976, 72, 248-254), which is based on the binding of the triarylme thane dye Coomassie Brilliant Blue G-250 to basic and aromatic amino acid residues in the protein. This binding causes a shift in the absorption maximum from 465 nm to 595 nm. To establish the calibration, the absorbances of BSA were determined in the concentrations of 5-20 g/l. To this end, the particular samples were made up to 800 l with H2O, and 200 l of Bradford reagent (Bio Rad, Munich) were added, and the samples were measured at 595 nm. To determine the leaching behaviour of the catalytically active particles under harsh reaction conditions, the procedure was extended by the following steps: The protein content of Novozym 435 (NZ4356) was determined by the following scheme. The NZ43 5 particles were incubated with shaking at 45°C in acetonitrile/H20 (1:1, v/v) for 3 0 min, and then samples (for example 1 ml) were taken from the supernant, lyophilized and resuspended in H2O (likewise 1 ml). Subsequently, the protein content was determined as described above. The results can be taken from Table 1. Comparative Example 1: Determination of the mechanical stability of conventional enzyme immobilizates For the purpose of determining the mechanical stabilities of the particles, they were incubated in various high-viscosity equimolar substrate solutions (for example polyethylene glycol (molar mass approx. 2400) and oleic acid) with high power inputs and at temperatures of > 60°C, and then the integrity of the particles was studied. Using NZ435 (5% by weight in polyethylene glycol (molar mass approx. 2400) and oleic acid), the formation of fine particles could be detected with the naked eye after 24 hours, for example by virtue of clear occurrence of turbidity. Comparative Example 2: Determination of the desorption stability of conventional enzyme immobilizates For the purpose of determining the desorption stability of the particles under harsh reaction conditions, fractions of 50 mg of NZ43 5 were shaken in 2 0 ml of MeCN/H2O (1:1, v/v) solution at 45°C for 30 min. Defined samples (for example 1 ml) were taken from the supernatant and the protein content in the supernatant was determined as described above. The particles were recovered by means of a fluted filter and washed with 100 ml of H2O, and dried at 50°C for 12 h, in order then to determine the hydrolytic activity and the synthesis activity in PLU according to the scheme described above. The results can be taken from Table 2. Example 1: Production of a stable enzyme preparation Illustrative preparation: I g of NZ435 particles were admixed in a metal dish with 1 ml of reaction mixture, consisting of various compositions of compounds of the general formulae I and II (for composition see Table 3; the components of the general formulae I and II were prepared by processes familiar to those skilled in the art, as described, for example, in US 7,196,153 B2, by equilibration), and Karstedt catalyst (Syloff 4000, product from Dow Corning, USA) . The silicone components including the catalyst were in each case dissolved in 3 ml of cyclohexane before the application and then added to the particles in the metal dish. The addition was followed immediately by strong dispersion by means of a vortexer (Ika, level 9) for 15-30 min until the bulk of the cyclohexane had evaporated off. Subsequently, the particles were dried at 50°C in a drying cabinet for about 12 h. The particles produced by this process, compared to the untreated immobilizate, have activity yields in the hydrolysis of up to 73% (Examples 1 i and 1 iii, 0.77 U/mg of NZ435 compared to 1.05 U/mg for untreated NZ435, as described in Comparative Example 1), or 65% (Example 1 i, 0.68 U/mg of NZ435) . In the synthesis, activity yields of 94% (Example 1 i) , 84% (Example 1 iii) or 68% (Example 1 ii) were achieved. Example 2: Determination of the mechanical stability of inventive enzyme preparations 300 mg of particles (untreated, native NZ435 or NZ435 treated according to iii in Table 3) were each stirred vigorously at 60°C in 5 ml of lauric acid for 90 min (magnetic stirrer plate from Ika, RT Power model, level 5, stirrer bar: length 3.1 cm, width 0.6 cm). After the stirrer had been removed, photographs of the stirred suspensions were taken (Figs. 1 and 2). Figure 1 shows clearly that the stirred suspension based on untreated NZ435 is cloudy as a result of fine particles. The stirred suspension based on NZ435 treated in accordance with the invention, in contrast, is clear, i.e. comprises no particles or at least no particles in a visible size (Fig. 2). Subsequently, the particles were removed from the suspensions by means of a fluted filter, rinsed with approx. 10 ml of acetone and dried at 50°C for 12 h. For the purpose of determining the mechanical stability of the particles, a determination of the particle size distribution was carried out. The screen fractions used here had the following exclusion sizes: 800 n, 500 m, 300 m, 150 m and 75 m. By screening, the particle size distributions before and after stirring were determined. The results can be taken from Table 4. Example 3: Determination of the desorption stability of stable enzyme preparations Analogously to Comparative Example 2, the particles obtained from Example 1 were treated with water/acetonitrile and then the hydrolysis activity, the synthesis activity and the amount of protein released were determined. The result of this determination can be taken from Table 5. While untreated native enzyme immobilizate after incubation exhibits no hydrolysis activity whatsoever and 56 g/mg of immobilizate were detectable as free protein, the silicone-coated particles exhibit hydrolysis activities of up to 75% of the starting activity (sample ii, 0.51 U/mg of NZ435 vs. 0.77 U/mg of NZ435 before leaching), synthesis activities of up to 55% of the starting activity (sample ii, 2980 U/g of NZ435 vs. 5400 U/g of NZ435 before leaching), and an enzyme desorption reduced by up to 86% (sample ii). Example 4: Production of an enzyme immobilizate 1 g of Lewatit VPOC1600 (from Lanxess) was stirred at room temperature in 5 ml of CALB solution (Lipozym CALB L, Novozymes A/S, Bagsvaerd, Denmark, hydrolytic activity: 2700 U/ml) for approx. 18 h, removed by means of a fluted filter and rinsed with 250 ml of distilled water, then dried under air for 3 h, rinsed with 1 ml of isopropanol and dried under air once more for 1 h. The immobilizates thus produced were stored in closable reaction vessels at 4°C until further use. The activities of the enzyme immobilizates were measured by processes described above (hydrolytic activity of 1.04 U/mg and a synthesis activity of 6000 PLU/g). The loading density was determined by means of a Bradford test to be about 3 0 gprotein/mgvPOC1600 • The enzyme immobilizates thus generated were coated with silicone in a second step by the process described above (composition as in test i in Tab. 3). The proportion by mass of enzyme immobilizate in the preparation corresponds to 63%. The hydrolytic activity of the coated preparations is 0.43 U/mg and the synthesis activity 2927 PLU/g. Analogously to Comparative Example 2, the uncoated particles and the silicone-coated particles were treated with water/acetonitrile and then the hydrolysis activity, the synthesis activity and the amount of protein released were determined. The result can be taken from Table 6. As can be discerned from Table 6, nine times the amount of protein is desorbed in the case of the untreated immobilizate. At the same time, activity yields of 54% (hydrolysis activity) and 60% (synthesis activity) can be achieved. The suitability of the above-described method for coating enzyme immobilizates with silicones, which has already been shown for commercial finished preparations using NZ435, has accordingly also been demonstrated for self-loaded enzyme immobilizates. Example 5: Determination of enzyme activities in organic solvent The activity of the enzyme preparations of Examples 1 and 4, and also of the corresponding native immobilizates is determined by performing a propyl laurate synthesis in methylcyclohexane (starting concentration of substrates = 10 mM, T = 25°C). It is clear from Table 7 that the activity yield of the enzyme preparations on use in organic solvent is excellent and that the desorption stability likewise becomes clear. Example 6: Production of further lipase preparations Analogously to Example 1 Lipozym RM IM (lipase from M. miehei, immobilized on Duolite A568 from Lanxess, available from Novozymes A/S) is provided with a siloxam coating and the activity yield is determined analogously to Example 5 in organic solvent. It is clear from Table 8 that, based on the amount of Lipozym RM IM used, a quantitative activity yield is achieved within the margin of error. Example 7: Production of further lipase preparations Analogously to Example 4, a lipase from T. lanuginosa (available as Esterase TL01 from Asa Spezialenzyme, comprising a lipase with additional esterase function) is immobilized on Lewatit VPOC1600 and provided with a siloxane coating. Activity is determined by determining the ethyl valerate hydrolysis. Table 9 shows that the native preparation has an activity of 261 U/g, while the coated preparation has an activity of 157 U/g. Based on the content of native immobilizate, this represents an activity of 285 U/g; that is, a quantitative activity yield can be achieved within the margin of error. Example 8: Production of an esterase preparation Analogously to Example 7, an esterase from R. oryzeae is immobilized on Lewatit VPOC1600, coated and its activity determined in the hydrolysis of ethyl valerate. Again, activity yield is quantitative within the margin of error. Example 9: Production of a laccase preparation Analogously to Example 4, a laccase (EC 1.10.3.2) from Myceliophthora Thermophilia (available as Flavorstar from Novozymes A/S) was immobilized on Lewatit VP OC 1600 (4.5 mg of protein on 1 g of Lewatit VPOC 1600), provided with a siloxane coating and tested for activity in ABTS assay. Claims 1. Enzyme preparations, obtainable by enzyme immobilizates which comprise enzymes or microorganisms containing enzymes immobilized on an inert carrier being provided with a silicone coating obtained by hydrosilylation. 2. Enzyme preparations according to Claim 1, characterized in that the enzymes are those from the class of the hydrolases, preferably lipases. 3. Enzyme preparations according to either of Claims 1 and 2, characterized in that the inert carriers have a particle size distribution in which 90% of the particles have a particle size of 10 to 5000 m. 4. Enzyme preparations according to any one of Claims 1 to 3, characterized in that the inert carriers used consist of polyvinylstyrene, polymethacrylate or polyacrylate. 5. Enzyme preparations according to any one of Claims 1 to 4, characterized in that the silicone coating is obtained by hydrosilylating SiH-functional polysiloxanes with polysiloxanes containing terminal carbon-carbon double bonds. 6. Enzyme preparations according to Claim 5, characterized in that the SiH components used are polysiloxanes of the general formula I where N =a+b+c+d+2=3to 850, a = 1 to 800, b = 0 to 400, c = 0 to 10, d = 0 to 10, R1 are independently the same or different, and are selected from the group comprising: saturated or unsaturated, optionally branched alkyl groups having 1 to 30 carbon atoms, alkaryl radicals having 7 to 3 0 carbon atoms, aryl radicals having 6 to 3 0 carbon atoms; R2a are independently hydrogen or R1; R2b are independently hydrogen or R1; R3 are independently identical or different radicals of the general formula la: where N =a + b + c + d + 2 = 3to 850, preferably 6 to 160, a = 1 to 800, b = 0 to 400, c = 0 to 10, d = 0 to 10, R1 are independently the same or different, and are selected from the group comprising: saturated or unsaturated, optionally branched alkyl groups having 1 to 3 0 carbon atoms, alkaryl radicals having 7 to 3 0 carbon atoms, aryl radicals having 6 to 3 0 carbon atoms; R2a are independently hydrogen or R1; R2b are independently hydrogen or R1; R3 are independently identical or different radicals of the formula la or an R1 radical. Enzyme preparations according to Claim 6, characterized in that the SiH components used are polysiloxanes of the general formula I where N =a+b+c+d+2=6to 160, a = 2 to 150, b = 2 to 75, c =0, d =0, R1 are independently the same or different, and are selected from the group comprising: saturated or unsaturated, optionally branched alkyl groups having 1 to 3 0 carbon atoms, alkaryl radicals having 7 to 3 0 carbon atoms, aryl radicals having 6 to 3 0 carbon atoms; R2a are independently hydrogen or R1- R2b are independently hydrogen or R1. Enzyme preparations according to at least one of Claims 5 to 7, characterized in that the polysiloxanes containing a terminal carbon-carbon double bond used are polysiloxanes of the general formula II: where N =m+n+o+p+2=3to 1000, m = 1 to 800, n = 0 to 20, o = 0 to 10, p = 0 to 10, R4 are independently the same or different and are from the following group: saturated or unsaturated, optionally branched alkyl groups having 1 to 3 0 carbon atoms, alkaryl radicals having 7 to 3 0 carbon atoms, aryl radicals having 6 to 30 carbon atoms; R5 are independently a terminally unsaturated alkyl or alkoxy radical or R4; R6 are independently identical or different radicals of the general formula Ha: where N =m+n+o+p+2=3to 1000, m = 1 to 800, n = 0 to 20, o = 0 to 10, p = 0 to 10, R4 are independently the same or different and are from the following group: saturated or unsaturated, optionally branched alkyl groups having 1 to 3 0 carbon atoms, alkaryl radicals having 7 to 3 0 carbon atoms, aryl radicals having 6 to 30 carbon atoms; R5 are independently a terminally unsaturated alkyl or alkoxy radical, or R4; R6 are independently identical or different radicals of the general formula Ila or R4 radicals. Enzyme preparations according to at least one of Claims 5 to 8, characterized in that the polysiloxanes containing a terminal carbon-carbon double bond used are polysiloxanes of the general formula II: wnere N = m + n + o + p + 2 = 10 to 600, m = 2 to 600, n = 0 to 10, o =0, P =0, R4 are independently the same or different and are from the following group: saturated or unsaturated, optionally branched alkyl groups having 1 to 3 0 carbon atoms, alkaryl radicals having 7 to 3 0 carbon atoms, aryl radicals having 6 to 30 carbon atoms; R5 are independently a terminally unsaturated alkyl or alkoxy radical. 10. Process for preparing enzyme preparations according to any one of Claims 1 to 7, characterized in that enzyme immobilizates which comprise enzymes or microorganisms comprising enzymes immobilized on an inert carrier are provided with a silicone coating obtained by hydrosilylation. 11. Process according to Claim 10, characterized in that the enzyme immobilizates are provided with a silicone coating by contacting the enzyme immobilizates with a reaction mixture which comprises SiH-functional polysiloxanes, polysiloxanes containing terminal carbon-carbon double bonds and a catalyst which catalyzes the hydrosilylation under hydrosilylation conditions. 12. Use of enzyme preparations according to any one of Claims 1 to 9 as an industrial biocatalyst. The invention is directed to enzyme preparations which are obtainable by enzyme immobilizates which comprise enzymes or microorganisms containing enzymes immobilized on an inert carrier being provided with a silicone coating obtained by hydrosilylation, to a process for producing such enzyme preparations and to the use of enzyme preparations as an industrial biocatalyst.

Full Text

Enzyme preparations
The invention relates to novel enzyme preparations for
use as biocatalysts.
Microorganisms and isolated enzymes find wide use as a
catalyst in the chemical industry or in food
production. An overview is offered, for example, by:
A. Liese, K. Seelbach, C. Wandrey, Industrial
Biotransformations, Wiley-VCH: 2 000, Weinheim, Germany.
In order to ensure economic use of such biocatalysts,
some conditions have to be satisfied: the biocatalyst
has to be active for a sufficiently long time under the
reaction conditions, it should be readily removable
after the end of the reaction and it should be reusable
as often as possible. Ideally, these conditions should
be satisfied for a very wide range of reaction
conditions (for example temperature range, type of
solvents used, pressures, etc), in order to provide as
universal as possible a catalyst.
In order to satisfy these conditions, it is typically
necessary to immobilize the enzymes or microorganisms
containing enzymes used.
Frequently, the enzymes or microorganisms containing
enzymes are immobilized noncovalently on carriers; the
carriers used are frequently ion exchange resins or
polymer particles which possess suitable particle size
distributions. Examples for this purpose are the
commercial products Novozym 435, Lipozym RM IM or
Lipozym TL IM from Novozymes A/S, Bagsvaerd, Denmark or
Amano PS, from Amano, Japan. These examples are
immobilized lipases which find wide use, since such
immobilizates also exhibit industrially utilizable
activities in nonaqueous systems, i.e. those which

comprise only organic solvents, if any, as described,
for example, in J. Chem. Soc, Chem. Comm. 1989, 934-
935. A disadvantage of the use of such immobilizates
is, however, firstly the desorption of the enzyme or of
the microorganisms containing enzymes which occurs
depending on the reaction system used, for example in
the case of use of surfactant components. The loss of
activity associated with such a desorption is shown in
Comparative Example 1. In addition, such preparations
possess inadequate mechanical stability, as a result of
which use in simple stirred reactors is possible only
with acceptance of significantly restricted
reusability, if at all. The mechanical instability of
such preparations is shown in Comparative Example 2.
In order to enable the repeated use of such enzyme
preparations, other reactor designs therefore have to
be used. Eur. J. Lipid Sci. Technol. 2003, 105, 601-607
describes, for example, the use of a fixed bed reactor
for performing lipase-catalyzed esterifications. A
disadvantage of this process is, however, the
restriction to low-viscosity homogeneous reaction
mixtures, since high-viscosity mixtures or suspensions
cannot be conveyed through a fixed bed.
K. Faber "Biotransformations in Organic Chemistry",
Springer: 2000, Berlin, Germany, describes, on page
3 84 ff., the use of enzymes incorporated into
alginates. However, the preparations thus obtained have
an exceptionally low mechanical stability and exhibit
only low activity in nonaqueous systems.
In addition, the subsequent crosslinking of immobilized
enzymes with reactive substances, for example
glutaraldehyde, is described. However, a disadvantage
is the usually significantly reduced specific activity
of the crosslinked preparations compared to the
activity before the modification. Furthermore, this
process does not make any contribution to improving the

Likewise described there is the covalent immobilization
of enzymes on reactive carriers. A disadvantage here is
that suitable functional groups have to be present on
the surface of the enzyme, which can react with the
carrier; in addition, a loss of enzyme activity is
often achieved as the result.
J. Am. Chem. Soc. 1999, 121, 9487-9496 describes the
incorporation of enzymes into siloxane matrices, known
as sol-gels. A disadvantage of sol-gel preparations is
the low particle size distribution which complicates
efficient removal by filtration, the lack of mechanical
stability, the occurrence of desorption, the use of
toxic reactants (for the toxicity of TEOS, see, for
example, Nippon Sanso Giho 1990, 9, 68-72 and Archives
of Toxicology 1994, 68, 277-283; for the toxicity of
TMOS, see, for example, Fundamental and Applied
Toxicology 1989, 13, 285-295; for the production of
nontoxic sol-gels, laborious steps are necessary, for
example storage over 6 months or thermal treatment at
350°C, as described in Polimery w Medycynie 2000, 30,
45-54, and also the swelling behaviour which is very
highly dependent upon the solvent used and does not
permit universal use in different reaction systems
(aqueous and nonaqueous). J. Sol Gel Sci. Technol.
2003, 26, 1183-1187 shows, by way of example, the
solvent dependence of the enzyme activity observed and
hence the lack of satisfaction of the demand for wide
usability.
Landbauforschung Volkenrode, 2002, special edition 241,
41-46 describes sol-gel preparations in which enzymes
are first immobilized onto "fine" silicone particles
and then encapsulated into a sol-gel. The problem of
mechanical stability is thus solved partially, but the
experiments described show that sufficient activities
are achieved only in selected solvents; use in a

solvent-free system is not described at all. In
addition, the preparations are not obtained in a
directly usable form, but rather first have to be cut
to an appropriate size, which is barely implementable
on the industrial scale.
J. Mol. Catal. B, 2005, 35, 93-99 describes the
immobilization of enzymes by incorporation of aqueous
enzyme solutions into mechanically stable silicone
spheres, known as static emulsions. The resulting
specific activities of the preparations are, though, at
up to 33 U/g, much too low compared to the above-
described immobilizates on inert carriers, where
specific activities of more than 1000 U/g can be
achieved easily (U = unit or pmol/min).
WO 03/106607 Al likewise describes such static
emulsions, but exclusively the use in aqueous systems
is described; the application is a washing composition,
i.e. not a biocatalysis, and the resulting particle
sizes, at approx. 10 ^m, are too small for efficient
filtration out of organic reaction mixtures.
There is therefore still a need for methods of enzyme
immobilization which overcome the disadvantages of the
prior art, in order to implement biocatalytic processes
which have not been realizable to date.
It was therefore an object of the present invention to
provide enzyme preparations which do not have one or
more of the disadvantages of the prior art
preparations. In particular, enzyme preparations shall
be provided, which have a high stability with respect
to mechanical forces and with respect to desorption and
at the same time preferably possess specific activities
in different aqueous and nonaqueous reaction mixtures
which are high enough to enable industrial use. In
terms of their particle size distribution, the enzyme
preparations should preferably be capable of being

removed from the reaction system in a simple manner and
of being reused.
Further objects which are not specified explicitly are
evident from the context of the description which
follows, the examples and the claims.
It has been found that, surprisingly, this object is
achieved by enzyme preparations which are obtained by
immobilizing enzymes or microorganisms comprising
enzymes on an inert carrier and then coating with a
silicone coating obtained by hydrosilylation.
The present invention therefore provides enzyme
preparations which are obtainable by enzyme
immobilizates which comprise enzymes or microorganisms
containing enzymes immobilized on an inert carrier
being provided with a silicone coating obtained by
hydrosilylation, and their use as an industrial
biocatalyst.
The present invention also provides a process for
preparing the inventive enzyme preparations, which is
characterized in that enzyme immobilizates which
comprise enzymes or microorganisms comprising enzymes
immobilized on an inert carrier are provided with a
silicone coating obtained by hydrosilylation.
The inventive enzyme preparations have the advantage
that they have a high stability with respect to
mechanical forces and with respect to desorption. In
spite of these improved properties, the inventive
enzyme preparations have specific activities in various
aqueous reaction mixtures (for example in the
hydrolysis of tributyrin) and nonaqueous reaction
mixtures (for example in the solvent-free synthesis of
propyl laurate), which are high enough to enable
industrial use. The inventive enzyme preparations also
have the advantage that the selection of the carrier

material and of the associated particle size
distribution allows the particle size to be adjusted
such that simple removal of the enzyme preparations
from the reaction system and hence also the reuse of
the enzyme preparations is possible.
The inventive enzyme preparations and a process for
their production are described below by way of example,
without any intention that the invention be restricted
to these illustrative embodiments. When ranges, general
formulae or compound classes are specified below, these
shall not only encompass the corresponding ranges or
groups of compounds which are mentioned explicitly but
also all sub-ranges and sub-groups of compounds which
can be obtained by selecting individual values (ranges)
or compounds. When documents are cited within the
present description, their contents shall be
incorporated completely in the disclosure-content of
the present invention. When compounds, for example
organically modified polysiloxanes which may have
different units more than once are described in the
context of the present invention, they may occur in
these compounds in random distribution (statistical
oligomer) or ordered (block oligomer). Information
regarding the number of units in such compounds should
be interpreted as the mean value, averaged over all
appropriate compounds.
The inventive enzyme preparations are notable in that
they are obtainable by providing enzyme immobilizates
which comprise enzymes or microorganisms comprising
enzymes immobilized on an inert carrier with a silicone
coating, which is obtained by hydrosilylation.
To produce the enzyme immobilizates, it is possible to
use whole cells, resting cells, purified enzymes or
cell extracts which comprise the corresponding enzymes,
or mixtures thereof. Preference is given to using
hydrolytic enzymes, for example lipases, esterases or

proteases, for example lipases from Candida rugosa,
Candida antarctica, Pseudomonas sp., Thermomyces
langosiosus, porcine pancreas, Mucor miehei,
Alcaligines sp., cholesterol esterase from Candida
rugosa, esterase from the porcine liver, more
preferably lipases. Accordingly, the enzyme
immobilizates preferably comprise enzymes from the
class of the hydrolases, preferably lipases.
The inert carriers used may be inert organic or
inorganic carriers. The inert carriers used, or present
in the enzyme immobilizate, are preferably those
particulate carriers which have a particle size
distribution in which at least 90% of the particles
have a particle size of 10 to 5000 [jm, preferably of
50 |im to 2000 Jim. The organic carriers used may
especially be those which comprise or consist of
polyacrylate, polymethacrylate, polyvinylstyrene,
styrene-divinylbenzene copolymers, polypropylene,
polyethylene, polyethylene terephthalate, PTFE and/or
other polymers. The carrier materials used may,
depending on the enzyme to be immobilized, especially
be acidic or basic ion exchange resins, for example
Duolite A568, Duolite XAD 761, Duolite XAD 1180,
Duolite XAD 7HP, Amberlite IR 120, Amberlite IR 400,
Amberlite CG 50, Amberlyst 15 (all products from Rohm
and Haas) or Lewatit CNP 105 and Lewatit VP OC 1600
(products from Lanxess, Leverkusen, Germany). The
inorganic carriers used may be oxidic and/or ceramic
carriers known from the prior art. In particular, the
inorganic carriers used may, for example, be Celite,
zeolites, silica, controlled-pore glass (CPG) or other
carriers, as described, for example, in L. Cao,
"Carrier-bound Immobilized Enzymes: Principles,
Application and Design", Wiley-VCH: 2005, Weinheim,
Germany. More preferably, the inert carriers present in
the enzyme immobilizate or the inert carriers used to
produce the enzyme immobilizates consist of
polyvinylstyrene, polymethacrylate or polyacrylate.

The immobilization on the particles can, in accordance
with the invention, be effected covalently or
noncovalently, preferably noncovalently. For
noncovalent immobilization, the carrier can be
incubated or impregnated, for example, with an aqueous
enzyme solution which may optionally comprise further
constituents, for example inorganic salts or
detergents. This incubation/impregnation can be carried
out, for example, at temperatures between 0°C and 50°C,
preferably between 0°C and 40°C. Preference is given to
effecting the incubation/impregnation over a period of
a few minutes to a few hours. The progress of the
incubation can be followed by determining the
concentration of the enzyme in the solution with the
common methods for protein determination. On attainment
of the desired degree of immobilization, the carrier
can preferably be washed with water and, if desired,
dried. An enzyme immobilizate obtained in this way can
subsequently be provided with a silicone coating in
accordance with the invention.
According to the invention, it is, however, also
possible to use enzyme immobilizates which are
commercially available, for example Novozym 435,
Lipozym RM IM or Lipozym TL IM from Novozymes A/S,
Bagsvaerd, Denmark, or Amano PS from Amano, Japan.
According to the invention, the silicone coating is
obtained by hydrosilylation. To this end, preferably
Si-H-functional polysiloxanes are reacted in the
presence of catalysts, preferably of transition metal
catalysts, with organically modified polysiloxanes
which possess at least one terminal carbon-carbon
double bond, preferably at least two carbon-carbon
double bonds.
The Si-H-functional polysiloxanes used are preferably
SiH polysiloxanes of the general formula I

where
N = a + b + c + d + 2 = 3to 850, preferably 6 to
160,
a = 1 to 800, preferably 2 to 150,
b = 0 to 400, preferably 2 to 75,
c = 0 to 10, preferably 0,
d = 0 to 10, preferably 0,
R1 are independently the same or different, and are
selected from the group comprising: saturated or
unsaturated, optionally branched alkyl groups
having 1 to 3 0 carbon atoms, alkaryl radicals
having 7 to 3 0 carbon atoms, aryl radicals having
6 to 3 0 carbon atoms, preferably alkyl groups
having 1 to 4 carbon atoms or phenyl, especially
methyl;
R2a are independently hydrogen or R1;
R2b are independently hydrogen or R1;
R3 are independently identical or different radicals
of the general formula Ia

where
N =a + b + c + d + 2 = 3to850, preferably 6 to
160,
a = 1 to 800, preferably 2 to 150,
b = 0 to 400, preferably 2 to 75,
c = 0 to 10, preferably 0,
d = 0 to 10, preferably 0,
R1 are independently the same or different, and are

selected from the group comprising: saturated or
unsaturated, optionally branched alkyl groups
having 1 to 30 carbon atoms, alkaryl radicals
having 7 to 3 0 carbon atoms, aryl radicals having
6 to 3 0 carbon atoms, preferably alkyl groups
having 1 to 4 carbon atoms or phenyl, especially
methyl;
R2a are independently hydrogen or R1;
R2b are independently hydrogen or R1;
R3 are independently identical or different radicals
of the formula la or an R1 radical.
Preference is given to using, as the SiH-functional
polysiloxane, a polysiloxane of the general formula I

where
N = a + b + c + d + 2 = 6 to 160,
a = 2 to 150,
b = 2 to 75,
c =0,
d =0,
R1 are independently the same or different, and are
selected from the group comprising: saturated or
unsaturated, optionally branched alkyl groups
having 1 to 3 0 carbon atoms, alkaryl radicals
having 7 to 30 carbon atoms, aryl radicals having
6 to 30 carbon atoms, preferably alkyl groups
having 1 to 4 carbon atoms or phenyl, especially
methyl;
R2a are independently hydrogen or R1;
R2b are independently hydrogen or R1.

It is well known to the person skilled in the art that
the compounds are or may be present in the form of a
mixture with a distribution controlled essentially by
statistical laws. The values for the indices a, b, c
and d are therefore typically mean values.
According to the invention, the olefinic reactants,
i.e. the polysiloxanes containing a terminal carbon-
carbon double bond, are preferably polysiloxanes of the
general formula II:

N =m + n + o+p + 2 = 3to 1000, preferably 10 to
600,
m = 1 to 800, preferably 2 to 600,
n = 0 to 20, preferably 0 to 10, more preferably 0,
o = 0 to 10, preferably 0,
p = 0 to 10, preferably 0,
R4 are independently the same or different and are
from the following group: saturated or
unsaturated, optionally branched alkyl groups
having 1 to 3 0 carbon atoms, alkaryl radicals
having 7 to 3 0 carbon atoms, aryl radicals having
6 to 3 0 carbon atoms, preferably alkyl groups
having 1 to 4 carbon atoms or phenyl, especially
methyl;
R5 are independently a terminally unsaturated alkyl
radical, preferably vinyl, or an alkoxy radical,
preferably having 3 to 2 0 carbon atoms, or R4;
R6 are independently identical or different radicals
of the general formula IIa

N = m + n + o+p + 2 = 3to 1000, preferably 10 to
600,
m = 1 to 800, preferably 2 to 600,
n = 0 to 20, preferably 0 to 10, more preferably 0,
o = 0 to 10, preferably 0,
p = 0 to 10, preferably 0,
R.4 are independently the same or different and are
from the following group: saturated or
unsaturated, optionally branched alkyl groups
having 1 to 3 0 carbon atoms, alkaryl radicals
having 7 to 3 0 carbon atoms, aryl radicals having
6 to 30 carbon atoms, preferably alkyl groups
having 1 to 4 carbon atoms or phenyl, especially
methyl;
R5 are independently a terminally unsaturated alkyl
radical, preferably vinyl, or an alkoxy radical,
preferably having 3 to 2 0 carbon atoms, or R4;
R6 are independently identical or different radicals
of the general formula Ila or R4 radicals.
N =m+n+o+p+2=10to 600,
m = 2 to 600,
n =0,
The polysiloxanes containing a terminal carbon-carbon
double bond used are preferably polysiloxanes of the
general formula II:

o =0,
p =0,
R4 are independently the same or different and are
from the following group: saturated or
unsaturated, optionally branched alkyl groups
having 1 to 30 carbon atoms, alkaryl radicals
having 7 to 3 0 carbon atoms, aryl radicals having
6 to 3 0 carbon atoms, preferably alkyl groups
having 1 to 4 carbon atoms or phenyl, especially
methyl;
R5 are independently a terminally unsaturated alkyl
radical, preferably vinyl, or an alkoxy radical,
preferably having 3 to 2 0 carbon atoms.
It is well known to the person skilled in the art that
the compounds of the formula II are or may be present
in the form of a mixture with a distribution controlled
essentially by statistical laws. The values of the
indices m, n, o and p are therefore typically mean
values.
The hydrosilylation can be carried out by established
methods in the presence of a catalyst. It is possible,
for example, to use catalysts which are typically used
for hydrosilylations, for example platinum, rhodium,
osmium, ruthenium, palladium, iridium complexes or
similar compounds or the corresponding pure elements or
their derivatives immobilized on silica, alumina or
activated carbon or similar carrier materials.
Preference is given to performing the hydrosilylation
in the presence of Pt catalysts such as cisplatin or
Karstedt catalyst [tris(divinyltetramethyldisiloxane)-
bis-platinum].
The amount of catalyst used is preferably 10~7 to
10-1 mol per mole of olefin or per mole of terminal
carbon-carbon double bond, preferably 1 to 100 ppm. The
hydrosilylation is carried out preferably at
temperatures of 0 to 200°C, preferably of 20 to 120°C.

The hydrosilylation can be carried out in the presence
or absence of solvent. Generally, solvents are not
needed for the performance of the reaction. The
reaction can, however, be carried out in suitable
solvents, for example aliphatic or aromatic
hydrocarbons, cyclic oligosiloxanes, alcohols or
esters. Suitable solvents are especially cyclohexane or
toluene.
According to the invention, based on the mass of the
carrier used, preferably 1 to 500% by mass, more
preferably 10 to 200% by mass, especially preferably 20
to 150% by mass, of siloxane components are used. The
siloxane components are composed especially of the sum
total of the compounds of the formula I and II and of
their reaction products.
The hydrosilylation can be carried out using a wide
variety of different ratios of the compounds of the
formulae I to compounds of the formulae II. Preference
is given to effecting the hydrosilylation at a molar
ratio based on the reactive groups of 1:10 to 10:1,
more preferably of 1:5 to 5:1, especially preferably of
1:1.1 to 1.1:1 and most preferably of 1:1. Selection of
the compounds of the general formula I and II used and
variation in their mixing ratios allows the properties
of the silicone coating to be tailored in relation to
perviousness for substrates and other reaction
properties. Selection of the weight ratio of silicone
components to enzyme immobilizates allows the layer
thicknesses of the silicone coating to be varied and to
be adjusted to appropriate requirements.
The inventive silicone coating, produced by
hydrosilylation, can be obtained by carrying out the
hydrosilylation in the presence of the enzyme
immobilizates. However, it is also possible to obtain
the coatings by applying a siloxane obtained by
hydrosilylation subsequently to the enzyme

immobilizates. This can be effected, for example, by
treating the enzyme immobilizates with a solution of
the siloxane, for example a solution of the siloxane in
an organic solvent, especially cyclohexane or toluene.
Subsequently, the solvent can be removed, for example,
by evaporation. The concentration of siloxane in such a
solution is preferably 10 to 100% by mass, more
preferably 30 to 100% by mass. However, preference is
given to obtaining the inventive silicone coating by
carrying out the hydrosilylation in the presence of the
enzyme immobilizates.
The inventive enzyme preparations are preferably
prepared by the process according to the invention
described below. This process for preparing enzyme
preparations is notable in that enzyme immobilizates
which comprise enzymes or microorganisms comprising
enzymes immobilized on an inert carrier are provided
with a silicone coating obtained by hydrosilylation.
Preference is given to performing the process according
to the invention in such a way that the enzyme
immobilizates are provided with a silicone coating by
contacting the enzyme immobilizates with a reaction
mixture which comprises SiH-functional polysiloxanes,
polysiloxanes containing terminal carbon-carbon double
bonds and a catalyst which catalyzes the
hydrosilylation under hydrosilylation conditions. In
particular, the process can be performed in such a way
that a hydrosilylation reaction is carried out in the
presence of enzyme immobilizates which comprise enzymes
or microorganisms comprising enzymes immobilized on an
inert carrier. The silicone formed in the
hydrosilylation allows the enzyme immobilizate to be
provided with a silicone coating.
The hydrosilylation can be carried out in a manner
known to those skilled in the art. Preference is given
to performing the hydrosilylation using the

abovementioned parameters/feedstocks/catalysts.
In a preferred embodiment of the process according to
the invention, a particular amount of enzyme
immobilizate is admixed with a mixture (reaction
mixture) of the silicone reagents (compounds of the
formulae I and II plus catalyst), for example by adding
a mixture comprising compounds of the general formula I
and of the general formula II and Karstedt catalyst.
For example, it is possible to add to 1 g of an enzyme
immobilizate a mixture of compounds of the formula I
and II in a molar mixing ratio of 10:1 to 1:10, and
also Karstedt catalyst, for example 50 ppm based on the
amount of silicone components present. For the purpose
of optimizing the coating properties, it may be
advantageous to dissolve the silicone components
including the catalyst, before the addition, in a
solvent, for example cyclohexane, toluene or another
organic solvent, and then to add the solution to the
enzyme immobilizate. When, for example, cyclohexane is
used as the solvent, it has been found to be
advantageous, after addition of the solution to the
enzyme immobilizates, to strongly disperse this mixture
for approx. 15 to 3 0 min, for example by means of a
vortexer (Ika, level 9), until the bulk of the
cyclohexane has evaporated off. Subsequently, the
resulting enzyme preparations are dried, i.e. hardened,
in a drying cabinet at 50°C, for example for 12 hours.
Altering the mixing ratios of the compounds of the
general formula I and II allows the properties of the
silicone coating to be varied without any problem and
adjusted to appropriate requirements.
A further embodiment of the process according to the
invention differs from the above embodiment in that the
enzyme immobilizates to be coated are immersed into the
desired reaction mixture, then removed from the
reaction mixture and dried. The removal can be
effected, for example, using a screen which retains the

enzyme immobilizate particles. The immersion time is
preferably 1 to 10 minutes. The drying can be effected
in a conventional drying cabinet. Preference is given
to effecting the drying/hardening at a temperature of
20°C to 80°C, preferably at 40°C to 60°C, more
preferably at approx. 50°C.
In a further embodiment of the process according to the
invention, which is suitable especially for performance
on the industrial scale, the hydrosilylation is carried
out using a pelletizing pan unit (for example from
Erweka or Eirich) . In this case, a defined amount of
enzyme immobilizate particles is introduced into the
so-called pan unit and stirred. Subsequently, either
the mixture comprising compounds of the formulae I and
II, and also catalyst and also if appropriate solvent,
is added or else, preferably, using a two-substance
nozzle (for example from Schlick or others), in which
the mixture or the components is/are applied under
pressure (for example nitrogen or synthetic air) in the
form of a fine droplet mist, in order to ensure a very
homogeneous distribution on the particles. After a
prolonged coating time, the particles are removed as
described above and dried, i.e. hardened, for a few
hours at a temperature of 2 0°C to 80°C, preferably of
40°C to 60°C, more preferably of 50°C, in a drying
cabinet, and can then be stored at room temperature
until further use.
In a further embodiment, the particles can be generated
in a fluidized bed reactor (for example from Erweka),
in which particles and the reaction mixture are applied
with high dispersion in appropriate mixing ratios.
The inventive enzyme preparations can be used, for
example, as biocatalysts, especially as industrial
biocatalysts.
The present invention is illustrated in detail with

reference to Figures 1 and 2, without being restricted
thereto.
Figures 1 and 2 show images of stirred suspensions in a
100 ml beaker with a diameter of the base of 4 cm. The
suspensions have been produced as described in
Example 2. Figure 1 shows that the stirred suspension
based on untreated NZ435 is cloudy as a result of fine
particles. The stirred suspension based on NZ435
treated in accordance with the invention, in contrast,
is clear, i.e. comprises no particles or at least no
particles in a visible size (Fig. 2).
The examples which follow are intended to illustrate
the present invention in detail, without restricting
the scope of protection which is evident from the
description and the claims.

Examples
Materials and methods:
Novozym 435 (NZ435) is a commercial enzyme immobilizate
from Novozymes A/S, Bagsvaerd, Denmark, a lipase B from
C. antarctica immobilized on a polymethacrylate by
adsorption.
Hydrolytic activity (tributyrin hydrolysis in aqueous
medium):
The hydrolytic activity was determined by the so-called
pH-stat method. In this method, the acid released in
the hydrolysis is titrated against a base, such that
the pH of the solution is kept constant. The time
dependence of the consumption of base allows the acid
released, and hence the enzyme activity, to be
quantified. Illustrative procedure: 10-20 mg of
catalytically active particles were added to 25 ml of
Tris-HCl buffer (1 mM, pH 7.5; additionally contains
0.1 mM NaCl and CaCl2) and 500 jil of tributyrin were
added. The hydrolytic activity was quantified on an
autotitrator (Tritroline alpha, from Schott) via the
amount of base titrated in (50 mM NaOH).
Hydrolytic activity (ethyl valerate in aqueous medium):
Analogously to the determination of the hydrolysis
activity using the example of tributyrin, ethyl
valerate can also be used. Illustrative procedure:
10-20 mg of catalytically active particles were added
to 25 ml of phosphate buffer (1 mM, pH 8.0) and 500 ul
of ethyl valerate. The hydrolytic activity was
quantified on an autotitrator (Tritroline alpha, from
Schott) via the amount of base titrated in (10 mM
NaOH).
Synthesis activity in PLU units (propyl laurate
synthesis activity in solvent-free system):
10 mg of catalytically active particles were added to
5 ml of equimolar substrate solution (lauric acid and

1-propanol) and incubated while shaking and/or stirring
at 60°C. Samples (Vsampie: 50 (0,1) were taken every 5 min
over 25 min and transferred into 950 (0,1 of decane
(internal standard: 4 mM dodecane). The PLUs were
determined with reference to the initial product
formation rates. Propyl laurate was detected by gas
chromatography (retention time: 9.791 min) (Shimadzu
2010, BTX column from SGE; length 25 m, I.D. 0.22 (am;
film: 0.25 (0m; detector type: FID at 300°C; injector
temperature 275°C and injection volume 1 1, split
ratio 35.0; carrier gas pressure (helium) 150 kPa;
temperature programme: start temperature 60°C, hold for
1.5 min, temperature rise 20°C/min, end temperature
250°C, hold for 2.5 min).
Determination of laccase activity:
To determine laccase activity, catalytically active
particles (native or immobilized laccase) are
transferred into 19 ml of potassium phosphate buffer
(100 mM, pH 6, 37°C) with 1 ml of ABTS solution (ready-
to-use solution, 1.8 mM, available from Sigma-Aldrich)
and the increased extinction is measured photospectro-
metrically at 405 nm. Laccase activity shall be
monitored over a period of 2 0 min. The samples are
taken at intervals of 5 min.
The activity can be determined as follows:
activity
A Ext. 405 change in extinction as a function of time
Vtotai total volume of reaction batch [20 ml]
Vsample volume of sample [2 ml]
At change in time [min]
e extinction coefficient for ABTS at 405 nm
[43.2 ml mol-1 cm-1]
d path length of cell [1 cm]
The activity is reported in units (U/ml or U/g) defined

as 1 mol of substrate conversion per minute.
Protein determination according to Bradford:
The determination of the protein content in the
supernatant was carried out according to the method of
Bradford (Anal. Chem. 1976, 72, 248-254), which is
based on the binding of the triarylme thane dye
Coomassie Brilliant Blue G-250 to basic and aromatic
amino acid residues in the protein. This binding causes
a shift in the absorption maximum from 465 nm to 595
nm. To establish the calibration, the absorbances of
BSA were determined in the concentrations of 5-20 g/l.
To this end, the particular samples were made up to
800 l with H2O, and 200 l of Bradford reagent (Bio
Rad, Munich) were added, and the samples were measured
at 595 nm.
To determine the leaching behaviour of the
catalytically active particles under harsh reaction
conditions, the procedure was extended by the following
steps:
The protein content of Novozym 435 (NZ4356) was
determined by the following scheme. The NZ43 5 particles
were incubated with shaking at 45°C in acetonitrile/H20
(1:1, v/v) for 3 0 min, and then samples (for example
1 ml) were taken from the supernant, lyophilized and
resuspended in H2O (likewise 1 ml). Subsequently, the
protein content was determined as described above. The
results can be taken from Table 1.

Comparative Example 1: Determination of the mechanical
stability of conventional enzyme immobilizates
For the purpose of determining the mechanical
stabilities of the particles, they were incubated in
various high-viscosity equimolar substrate solutions
(for example polyethylene glycol (molar mass approx.
2400) and oleic acid) with high power inputs and at
temperatures of > 60°C, and then the integrity of the
particles was studied. Using NZ435 (5% by weight in
polyethylene glycol (molar mass approx. 2400) and oleic
acid), the formation of fine particles could be
detected with the naked eye after 24 hours, for example
by virtue of clear occurrence of turbidity.
Comparative Example 2: Determination of the desorption
stability of conventional enzyme immobilizates
For the purpose of determining the desorption stability
of the particles under harsh reaction conditions,
fractions of 50 mg of NZ43 5 were shaken in 2 0 ml of
MeCN/H2O (1:1, v/v) solution at 45°C for 30 min.
Defined samples (for example 1 ml) were taken from the
supernatant and the protein content in the supernatant
was determined as described above. The particles were
recovered by means of a fluted filter and washed with
100 ml of H2O, and dried at 50°C for 12 h, in order
then to determine the hydrolytic activity and the

synthesis activity in PLU according to the scheme
described above. The results can be taken from Table 2.

Example 1: Production of a stable enzyme preparation
Illustrative preparation:
I g of NZ435 particles were admixed in a metal dish
with 1 ml of reaction mixture, consisting of various
compositions of compounds of the general formulae I and
II (for composition see Table 3; the components of the
general formulae I and II were prepared by processes
familiar to those skilled in the art, as described, for
example, in US 7,196,153 B2, by equilibration), and
Karstedt catalyst (Syloff 4000, product from Dow
Corning, USA) . The silicone components including the
catalyst were in each case dissolved in 3 ml of
cyclohexane before the application and then added to
the particles in the metal dish. The addition was
followed immediately by strong dispersion by means of a
vortexer (Ika, level 9) for 15-30 min until the bulk of
the cyclohexane had evaporated off. Subsequently, the
particles were dried at 50°C in a drying cabinet for
about 12 h.

The particles produced by this process, compared to the
untreated immobilizate, have activity yields in the
hydrolysis of up to 73% (Examples 1 i and 1 iii,
0.77 U/mg of NZ435 compared to 1.05 U/mg for untreated
NZ435, as described in Comparative Example 1), or 65%
(Example 1 i, 0.68 U/mg of NZ435) . In the synthesis,
activity yields of 94% (Example 1 i) , 84% (Example
1 iii) or 68% (Example 1 ii) were achieved.
Example 2: Determination of the mechanical stability of
inventive enzyme preparations
300 mg of particles (untreated, native NZ435 or NZ435
treated according to iii in Table 3) were each stirred
vigorously at 60°C in 5 ml of lauric acid for 90 min
(magnetic stirrer plate from Ika, RT Power model, level
5, stirrer bar: length 3.1 cm, width 0.6 cm). After the
stirrer had been removed, photographs of the stirred
suspensions were taken (Figs. 1 and 2).
Figure 1 shows clearly that the stirred suspension
based on untreated NZ435 is cloudy as a result of fine
particles. The stirred suspension based on NZ435
treated in accordance with the invention, in contrast,
is clear, i.e. comprises no particles or at least no
particles in a visible size (Fig. 2).
Subsequently, the particles were removed from the
suspensions by means of a fluted filter, rinsed with
approx. 10 ml of acetone and dried at 50°C for 12 h.
For the purpose of determining the mechanical stability
of the particles, a determination of the particle size
distribution was carried out. The screen fractions used
here had the following exclusion sizes: 800 n, 500 m,
300 m, 150 m and 75 m. By screening, the particle
size distributions before and after stirring were
determined. The results can be taken from Table 4.

Example 3: Determination of the desorption stability of
stable enzyme preparations
Analogously to Comparative Example 2, the particles
obtained from Example 1 were treated with
water/acetonitrile and then the hydrolysis activity,
the synthesis activity and the amount of protein
released were determined. The result of this
determination can be taken from Table 5.

While untreated native enzyme immobilizate after
incubation exhibits no hydrolysis activity whatsoever
and 56 g/mg of immobilizate were detectable as free
protein, the silicone-coated particles exhibit
hydrolysis activities of up to 75% of the starting
activity (sample ii, 0.51 U/mg of NZ435 vs. 0.77 U/mg
of NZ435 before leaching), synthesis activities of up
to 55% of the starting activity (sample ii, 2980 U/g of
NZ435 vs. 5400 U/g of NZ435 before leaching), and an
enzyme desorption reduced by up to 86% (sample ii).
Example 4: Production of an enzyme immobilizate
1 g of Lewatit VPOC1600 (from Lanxess) was stirred at
room temperature in 5 ml of CALB solution (Lipozym
CALB L, Novozymes A/S, Bagsvaerd, Denmark, hydrolytic
activity: 2700 U/ml) for approx. 18 h, removed by means
of a fluted filter and rinsed with 250 ml of distilled

water, then dried under air for 3 h, rinsed with 1 ml
of isopropanol and dried under air once more for 1 h.
The immobilizates thus produced were stored in closable
reaction vessels at 4°C until further use.
The activities of the enzyme immobilizates were
measured by processes described above (hydrolytic
activity of 1.04 U/mg and a synthesis activity of
6000 PLU/g). The loading density was determined by
means of a Bradford test to be about
3 0 gprotein/mgvPOC1600 •
The enzyme immobilizates thus generated were coated
with silicone in a second step by the process described
above (composition as in test i in Tab. 3). The
proportion by mass of enzyme immobilizate in the
preparation corresponds to 63%. The hydrolytic activity
of the coated preparations is 0.43 U/mg and the
synthesis activity 2927 PLU/g.
Analogously to Comparative Example 2, the uncoated
particles and the silicone-coated particles were
treated with water/acetonitrile and then the hydrolysis
activity, the synthesis activity and the amount of
protein released were determined. The result can be
taken from Table 6.

As can be discerned from Table 6, nine times the amount
of protein is desorbed in the case of the untreated
immobilizate. At the same time, activity yields of 54%
(hydrolysis activity) and 60% (synthesis activity) can
be achieved. The suitability of the above-described
method for coating enzyme immobilizates with silicones,
which has already been shown for commercial finished
preparations using NZ435, has accordingly also been
demonstrated for self-loaded enzyme immobilizates.
Example 5: Determination of enzyme activities in
organic solvent
The activity of the enzyme preparations of Examples 1
and 4, and also of the corresponding native
immobilizates is determined by performing a propyl
laurate synthesis in methylcyclohexane (starting
concentration of substrates = 10 mM, T = 25°C).

It is clear from Table 7 that the activity yield of the
enzyme preparations on use in organic solvent is
excellent and that the desorption stability likewise
becomes clear.
Example 6: Production of further lipase preparations
Analogously to Example 1 Lipozym RM IM (lipase from
M. miehei, immobilized on Duolite A568 from Lanxess,

available from Novozymes A/S) is provided with a
siloxam coating and the activity yield is determined
analogously to Example 5 in organic solvent.

It is clear from Table 8 that, based on the amount of
Lipozym RM IM used, a quantitative activity yield is
achieved within the margin of error.
Example 7: Production of further lipase preparations
Analogously to Example 4, a lipase from T. lanuginosa
(available as Esterase TL01 from Asa Spezialenzyme,
comprising a lipase with additional esterase function)
is immobilized on Lewatit VPOC1600 and provided with a
siloxane coating. Activity is determined by determining
the ethyl valerate hydrolysis.

Table 9 shows that the native preparation has an
activity of 261 U/g, while the coated preparation has
an activity of 157 U/g. Based on the content of native
immobilizate, this represents an activity of 285 U/g;
that is, a quantitative activity yield can be achieved
within the margin of error.
Example 8: Production of an esterase preparation
Analogously to Example 7, an esterase from R. oryzeae
is immobilized on Lewatit VPOC1600, coated and its
activity determined in the hydrolysis of ethyl
valerate.

Again, activity yield is quantitative within the margin
of error.
Example 9: Production of a laccase preparation
Analogously to Example 4, a laccase (EC 1.10.3.2) from
Myceliophthora Thermophilia (available as Flavorstar
from Novozymes A/S) was immobilized on Lewatit VP OC
1600 (4.5 mg of protein on 1 g of Lewatit VPOC 1600),
provided with a siloxane coating and tested for
activity in ABTS assay.

Claims
1. Enzyme preparations, obtainable by enzyme
immobilizates which comprise enzymes or
microorganisms containing enzymes immobilized on
an inert carrier being provided with a silicone
coating obtained by hydrosilylation.
2. Enzyme preparations according to Claim 1,
characterized in that the enzymes are those from
the class of the hydrolases, preferably lipases.

3. Enzyme preparations according to either of Claims
1 and 2, characterized in that the inert carriers
have a particle size distribution in which 90% of
the particles have a particle size of 10 to
5000 m.
4. Enzyme preparations according to any one of Claims
1 to 3, characterized in that the inert carriers
used consist of polyvinylstyrene, polymethacrylate
or polyacrylate.
5. Enzyme preparations according to any one of Claims
1 to 4, characterized in that the silicone coating
is obtained by hydrosilylating SiH-functional
polysiloxanes with polysiloxanes containing
terminal carbon-carbon double bonds.
6. Enzyme preparations according to Claim 5,
characterized in that the SiH components used are
polysiloxanes of the general formula I

where
N =a+b+c+d+2=3to 850,
a = 1 to 800,
b = 0 to 400,
c = 0 to 10,
d = 0 to 10,
R1 are independently the same or different, and
are selected from the group comprising:
saturated or unsaturated, optionally branched
alkyl groups having 1 to 30 carbon atoms,
alkaryl radicals having 7 to 3 0 carbon atoms,
aryl radicals having 6 to 3 0 carbon atoms;
R2a are independently hydrogen or R1;
R2b are independently hydrogen or R1;
R3 are independently identical or different
radicals of the general formula la:

where
N =a + b + c + d + 2 = 3to 850, preferably 6
to 160,
a = 1 to 800,
b = 0 to 400,
c = 0 to 10,
d = 0 to 10,
R1 are independently the same or different, and
are selected from the group comprising:
saturated or unsaturated, optionally branched
alkyl groups having 1 to 3 0 carbon atoms,
alkaryl radicals having 7 to 3 0 carbon atoms,
aryl radicals having 6 to 3 0 carbon atoms;
R2a are independently hydrogen or R1;
R2b are independently hydrogen or R1;

R3 are independently identical or different
radicals of the formula la or an R1 radical.
Enzyme preparations according to Claim 6,
characterized in that the SiH components used are
polysiloxanes of the general formula I

where
N =a+b+c+d+2=6to 160,
a = 2 to 150,
b = 2 to 75,
c =0,
d =0,
R1 are independently the same or different, and
are selected from the group comprising:
saturated or unsaturated, optionally branched
alkyl groups having 1 to 3 0 carbon atoms,
alkaryl radicals having 7 to 3 0 carbon atoms,
aryl radicals having 6 to 3 0 carbon atoms;
R2a are independently hydrogen or R1-
R2b are independently hydrogen or R1.
Enzyme preparations according to at least one of
Claims 5 to 7, characterized in that the
polysiloxanes containing a terminal carbon-carbon
double bond used are polysiloxanes of the general
formula II:

where
N =m+n+o+p+2=3to 1000,
m = 1 to 800,
n = 0 to 20,
o = 0 to 10,
p = 0 to 10,
R4 are independently the same or different and
are from the following group: saturated or
unsaturated, optionally branched alkyl groups
having 1 to 3 0 carbon atoms, alkaryl radicals
having 7 to 3 0 carbon atoms, aryl radicals
having 6 to 30 carbon atoms;
R5 are independently a terminally unsaturated
alkyl or alkoxy radical or R4;
R6 are independently identical or different
radicals of the general formula Ha:

where
N =m+n+o+p+2=3to 1000,
m = 1 to 800,
n = 0 to 20,
o = 0 to 10,
p = 0 to 10,
R4 are independently the same or different and
are from the following group: saturated or
unsaturated, optionally branched alkyl groups
having 1 to 3 0 carbon atoms, alkaryl radicals
having 7 to 3 0 carbon atoms, aryl radicals
having 6 to 30 carbon atoms;
R5 are independently a terminally unsaturated
alkyl or alkoxy radical, or R4;
R6 are independently identical or different

radicals of the general formula Ila or R4
radicals.
Enzyme preparations according to at least one of
Claims 5 to 8, characterized in that the
polysiloxanes containing a terminal carbon-carbon
double bond used are polysiloxanes of the general
formula II:

wnere
N = m + n + o + p + 2 = 10 to 600,
m = 2 to 600,
n = 0 to 10,
o =0,
P =0,
R4 are independently the same or different and
are from the following group: saturated or
unsaturated, optionally branched alkyl groups
having 1 to 3 0 carbon atoms, alkaryl radicals
having 7 to 3 0 carbon atoms, aryl radicals
having 6 to 30 carbon atoms;
R5 are independently a terminally unsaturated
alkyl or alkoxy radical.
10. Process for preparing enzyme preparations
according to any one of Claims 1 to 7,
characterized in that enzyme immobilizates which
comprise enzymes or microorganisms comprising
enzymes immobilized on an inert carrier are
provided with a silicone coating obtained by
hydrosilylation.

11. Process according to Claim 10, characterized in
that the enzyme immobilizates are provided with a
silicone coating by contacting the enzyme
immobilizates with a reaction mixture which
comprises SiH-functional polysiloxanes,
polysiloxanes containing terminal carbon-carbon
double bonds and a catalyst which catalyzes the
hydrosilylation under hydrosilylation conditions.
12. Use of enzyme preparations according to any one of
Claims 1 to 9 as an industrial biocatalyst.

The invention is directed to enzyme preparations which
are obtainable by enzyme immobilizates which comprise
enzymes or microorganisms containing enzymes
immobilized on an inert carrier being provided with a
silicone coating obtained by hydrosilylation, to a
process for producing such enzyme preparations and to
the use of enzyme preparations as an industrial
biocatalyst.

Documents:

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Patent Number

270795

Indian Patent Application Number

1163/KOL/2008

PG Journal Number

04/2016

Publication Date

22-Jan-2016

Grant Date

20-Jan-2016

Date of Filing

03-Jul-2008

Name of Patentee

EVONIK GOLDSCHMIDT GMBH

Applicant Address

GOLDSCHMIDTSTRASSE 100, 45127 ESSEN

Inventors:

#	Inventor's Name	Inventor's Address
1	DR. ANDREAS BUTHE	KRAFTWERKSTRAßE 70 79636 GRENZACH-WYHLEN
2	DR. OLIVER THUM	MEYGNER BUSCH 14 40880 RATINGEN
3	LARS WIEMANN	BIRKENSTRAßE 6 10559 BERLIN
4	DR. MARION ANSORGE-SCHUMACHER	HAHNER STRASSE 6B 52159 ROETGEN

PCT International Classification Number

A61K39/29

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	10 2007 031 689.7	2007-07-06	Germany