FI94257B - Process for the preparation of biocatalysts - Google Patents

Abstract

The present invention relates to biocatalysts which are in the form of beads and have high mechanical strength, comprising enzymatically active material, a cationic polyelectrolyte, preferably chitosan, multiply charged anions and silica, and to process for their preparation. This application likewise relates to processes for reacting organic substances, e.g. for preparing alpha -hydroxy carboxylic acids from alpha -keto carboxylic acids, especially 2-(R)-hydroxy- 4-phenylbutyric acid from 2-oxo-4-phenylbutyric acid, using the biocatalysts according to the invention.

Description

94257

Method for the preparation of biocatalysts Förfarande för framställning av biokatalysatorer

The invention relates to bead-shaped, mechanically very strong biocatalysts, to a process for their preparation and to a process for the conversion of organic substances by means of the biocatalysts according to the invention.

The term biocatalysis broadly encompasses all forms of catalysis in which the activating agent is a biological system, e.g. whole cells, cell fragments or cell organelles, or is derived from a biological system, e.g. enzymes. Enzymes are compounds, usually proteins or glycoproteins, that specifically accelerate biochemical reactions by lowering activation energy. In the following, the term biocatalyst not only means the activating agent itself, but may possibly also comprise components used to immobilize, strengthen, regenerate the activating agent, etc.

Immobilized, i.e. biocatalysts with limited mobility are primarily suitable for industrial purposes. Immobilization makes it possible to carry out the catalyzed processes continuously and secondly. to achieve and maintain high biocatalyst densities, to achieve high time-space yields and to reduce the cost of preparing the product from the reaction solution.

For immobilization, the biocatalytic material can be enclosed in, for example, polymeric coatings (matrices), semipermeable membrane capsules or fibers or behind an ultrafiltration membrane, crosslinked with a bi- or multifunctional ionic binder or a synergistic or naturally occurring polymeric or natural A combination of these methods is also possible.

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When immobilizing, it is important, on the one hand, to achieve the highest possible biocatalytic activity and, on the other hand, good mechanical strength and chemical stability, but at the same time good permeability of the biocatalyst. A common method of immobilization is, for example, the "entrapment" of a biologically active substance from natural polymers, such as e.g. cellulose, agar, gelatin or synthetic polymers such as e.g. into a matrix made of polyacrylamide, polyurethane or epoxy resins. Immobilization by ionotropic gelation (ionic cross-linking of matrix materials), for example natural polyanions such as alginate, pectin, carrageenan, etc., is particularly gentle by coating or spraying . Ca2 +, Co2 +, Zn2 +, Fe2 +, Fe3 +, A13 + form chelates and bead-shaped biocatalysts.

Ionotropic gelation by organic polymers generally produces only very soft, mechanically unstable immobilates that tend to swell or deform, for example with difficulties in use in filler reactors often used in enzymatic reactions in industry. Curing methods carried out either by post-treatment of immobilates or by pretreatment of the biologically active substance before the addition of a gelling agent with in each case multifunctional crosslinkers, such as polyaldehydes, e.g. solutes and or due to the detrimental effect of enzymes. An alternative is to use inorganic substances, e.g. as in the method described in German application DE 35 20 001, which describes the immobilization of an enzymatic substance with silica sol and alginate. Drying and shrinkage of biocatalyst beads are also used for curing because the beads do not reach their original moisture volume after rewetting and remain harder. German application DE 28 35 875 describes a process for ion-tropical gelation using polyanions, e.g. sodium alginate, and for example calcium ions as crosslinkers, in which process biocatalyst beads to which enzymatic substances have been added are dried at a temperature of at most 80 ° C. However, such drying steps cannot be used to immobilize highly sensitive enzymes or enzyme systems, especially living cells, due to the high temperatures used in the drying or the dry state inactivating the cells or enzymes.

Another disadvantage of calcium alginate immobilates prepared by ionotropic gelation, in addition to their poor mechanical properties, is the limitation of the reaction media that can be used in their further processing. Such immobilizers are, for example, chemically unstable in the presence of ions such as sodium and potassium in conventional buffer solutions. In such electrolyte solutions, especially if they are highly concentrated, the gel-fixing calcium ions in potassium alginate gels are displaced because sodium or potassium ions, respectively, compete for alginate ions. Phosphate ions also gradually lead to the dissolution of calcium alginate biocatalyst beads because phosphates react with calcium and remove the structure-strengthening agent from alginate. Dissolution can be prevented by using polycations, e.g. chitosan, which are cross-linked with polyvalent anions, such as [Fe (CN) 6] 3 ', [Fe (CN) 6)] 4-polyphosphates and the like, as matrix substances. German patents DE 30 05 632 and DE 30 05 633 describe the preparation of biocatalyst beads using 4 94257 chitosan and K3 (Fe (CN) g). The method described in DE 30 05 632 comprises a drying step at a temperature of up to 80 ° C for curing the beads. In order to be able to immobilize highly sensitive enzymatic substances, this drying step is not included in the process according to DE 30 05 633, which, however, results in poorer mechanical strength of the biocatalyst beads.

The object of the present invention is to provide bead-shaped biocatalysts - which are very mechanically strong - whose stability is guaranteed to be maintained even with the use of conventional biological solutions and buffer systems, and - which ensure that the enzymatic activity of a highly sensitive enzymatically active substance is maintained.

It is a further object of the invention to provide suitable methods for preparing such biocatalysts.

Bead-shaped biocatalysts containing an enzymatically active substance, a cationic polyelectrolyte, preferably chitosan, polyvalent anions and silicic acid have proved to be particularly suitable for solving these tasks. Such biocatalysts are prepared by a gentle immobilization method using ionotropic gel formation as well as using silica in solid form.

This invention relates to bead-shaped, mechanically very strong biocatalysts comprising an enzymatically active substance, a cationic polyelectrolyte and polyvalent anions, which are characterized in that the biocatalysts according to the invention contain silicic acid.

The bead-shaped biocatalysts according to the invention are characterized by their good mechanical strength due to the formation of a dual gel matrix structure. Thus, for example, they have good sealing properties when filling the reaction vessel and can be used as a filling material, for example in solid bed reactors. Due to the mild immobilization conditions, which do not include a drying step or treatment with harmful substances, these biocatalysts according to the invention are particularly suitable for highly sensitive enzymatic substances, especially for living cells. Compared to the prior art methods, the preparation of bead-shaped biocatalysts according to the invention using solid silicic acid has the advantage that time-consuming and labor-intensive methods for preparing the crosslinkers used in the curing step can be avoided. In addition, the biocatalysts of the invention can be used in buffer systems containing ions that degrade alginate gels. Thus, the biocatalysts according to the invention can be used, for example, in solutions containing phosphate ions, e.g. in a very low-cost and therefore industrially preferred tripolyphosphate buffer. The bead-shaped biocatalysts according to the invention are suitable for the conversion of organic substances. Since they can be used for reactions which require high electrolyte concentrations, for example, biocatalyst beads according to the invention which contain a bacterium of the genus Proteus * as enzymatically active substance are suitable; or oxidoreductase for the preparation of α-hydroxycarboxylic acids from α-ketocarboxylic acids, e.g. 2- (R) -hydroxy-4-phenylbutyric acid from 2-oxo-4-phenylbutyric acid.

The bead-shaped biocatalysts according to the invention are very mechanically strong. By good mechanical strength is meant, for example, good compressive strength, i.e. that the biocatalyst beads according to the invention, which have an average size of about 3 mm, do not show more than 30 to 50 μπι: η, in particular 40 / m deformation, after being subjected to a reaction of 0.01 to 0.03 N for 1 to 5 minutes; , especially a load of 0.02 N and no deformation increases at all as the load continues for another 10-20 minutes, 6 94257 especially for 15 minutes.

The nature of the enzymatic substance contained in the biocatalysts according to the invention is determined by the reactions for which the biocatalysts are to be used.

It consists primarily of whole cells, cell fragments or cell organelles of the microorganism, enzymes, or any combination of these types of enzymatically active substances.

The microorganism may be of prokaryotic or eukaryotic quality. Suitable microorganisms are, for example, a bacterium, e.g. lactic acid bacterium (Leuconostoc. Streptococcus, Lactobacillus), Corvnebacteriuro. Streptomyces. Pseudomonas. Xanthomonas. Bacillus. Clostridium. Escherichia. Proteus, etc., - fungus, eg Mucor. Aspergillus. Penicillium. especially yeasts such as Saccharomyces. Candida, etc., - algae, eg Chlorella. Curvularia, etc., or - a plant cell, e.g. Catharanthus roseus. Daucus carota, Digitalis lanata. Morinda citrifolia, etc.

The use of whole cells, cell fragments or cell organelles, i.e. cell subunits coated with membranes or consisting of membrane systems (chloroplasts, tylocoids, mitochondria, etc.) as an enzymatically active substance is preferred if the reaction to be catalyzed requires *; a multienzyme system, e.g. if cofactors (pyridine or flavin nucleotides, etc.) are required, or if a biosynthetic, e.g. secondary, product requiring complex metabolic chains is to be prepared instead of a catabolic substance.

Isolated enzymes are also suitable as enzymatically active substances. Typical examples of such enzymes are oxidoreductases (e.g. lactate dehydrogenase, alcohol dehydrogenase, glucose oxidase), transferases, (e.g. hexokinase, glutamine nitranase), lyases (e.g. fumarase, iserase, ispartase), aspartase, ispartase. isomerase), ligases 7 94257 (e.g. glutathione synthetase, NAD synthetase), etc.

As the enzymatically active substance, the biocatalysts of the invention may also contain any combination of the enzymatically active substances described, for example a combination of whole cells of two or more species or strains of microorganisms or a combination of whole cells and enzymes. The main idea is to supplement the biocatalytic properties of the cell with additional enzymes that are either absent or very few in the cell.

Particularly preferred enzymatically active substances are whole cells of a bacterium or yeast, in particular a bacterium of the genus Proteus, preferably of the species Proteus vuloaris and / or Proteus mirabilis. Particularly preferred is the species Proteus vulgaris, e.g. Proteus vulgaris ATCC 9484. Particularly preferred is also an enzymatically active substance which contains oxidoreductase or is oxidoreductase.

The cationic polyelectrolyte is a natural or synthetic polymer or a chemically modified natural polymer, e.g. a polymer containing protonated amino groups, preferably chitosan ([2-amino-2-deoxy- (1- * 4) -O-D-glucopyranane; poly (1,4 Chitosan is a high molecular weight linear polymer composed of glucosamine subunits obtained from chitin by partial deacetylation with concentrated lye and heat. The chitin is widely distributed in the sea, as it is widely distributed in the marine selenium. [3-D-glycopyranose amine]. chitin in crabs, shrimps, lobsters, etc. The bead-shaped biocatalysts of the invention preferably contain chitosan having a molecular weight of about 50,000 to about 3,000,000.

Suitable polyvalent anions are polyvalent inorganic or polyvalent organic anions. Inorganic anions are preferably orthophosphate, metaphosphates, e.g. hexametaphosphate, pyrophosphates, polyphosphates, e.g. tri-, tetra- or octapolyphosphate, or cyano-ferrates, e.g. [Fe (CN) g] 4- or [ Fe (CN) g] 3 '. Tripolyphosphate is particularly preferred. Organic anions are preferably polymeric organic carboxylates, sulfonates or hydroxy compounds.

Suitable silicic acid is preferably precipitated silicic acid, particularly preferably precipitated silicic acid having an average grain size of 5 to 50 μτη and an average bulk density of 5 to 100 g / 100 ml, in particular about 20 to 50 g / 100 ml.

The present invention furthermore relates to a process for preparing the pearl-like, mechanically high-strength biocatalysts according to the invention, which process is characterized in that the enzymatically active substance is mixed with an aqueous suspension of precipitated aqueous silica solution to a spray bath containing polyvalent anions and the biocatalyst beads thus formed are shrunk and reinforced in a crosslinking bath and possibly separated from the crosslinking bath.

Prevention of foaming is crucial in the preparation of a suspension of an enzymatically active substance, a precipitated silica paste in solid form, an aqueous buffer solution and an aqueous solution of a cationic polyelectrolyte, as otherwise catalyst catalysts with poorer mechanical properties are formed. It is preferred to prevent foaming by mixing the various components together in the presence of silicic acid. Otherwise, the order of addition of the substances can be any. It is preferred to first mix the enzymatic substance with the silica in solid form and only then add it to the aqueous buffer solution and finally mix it with the cationic polyelectrolyte solution.

In the preparation of the biocatalyst beads according to the invention, an enzymatic substance as described above is used, i.e. whole cells, cell fragments or cell organelles or enzymes of the microorganism or a combination thereof. The whole cells can then be alive, dead or, for example, lyophilized or dehydrated. Culturing the cells in a culture medium containing all the organic and inorganic constituents necessary for reproduction (C-source, N-source, trace elements, growth media, etc.) and cell fragmentation to obtain cell fragments or isolating cell organelles takes place by methods known per se. The immobilizing enzymes are, for example, dissolved, dispersed, suspended, emulsified or dried. Said and more enzymatically active substance as defined above is used in the process according to the invention as a cell, cell fragment and cell organelle sediment, filtrate or suspension or as an enzyme in aqueous solution. In the context of this specification, the term "in aqueous solution" means that the solution may additionally contain inorganic or organic salts and the like. It may also be, for example, a commonly used biological buffer such as acetate, phosphate, Tris buffer.

Suitable aqueous buffer solutions are, for example, a biological buffer having a pH of 3 to 7, preferably about 5, e.g. phosphate, citrate or acetate buffer.

The cationic polyelectrolytes used in the process of the invention have been described above. The most preferred of these is chitosan. When chitosan is used to make biocatalyst beads, it must first be placed in aqueous solution to become an integrated component of the immobilizer. To this end, chitosan, which is insoluble in water in non-protonated form, is added to dilute, aqueous, in particular organic, acids, for example formic acid, acetic acid, pyruvic acid, orenoic acid, citric acid or the like, preferably acetic acid having a high pH or 94257 is less than 7, preferably 4 to 5.5 and is optionally heated to about 60 ° C to support the dissolution process. The chitosan solution is finally filtered to remove insolubles. An aqueous solution of chitosan with a viscosity of 1000 to 20,000 cP and a concentration of 0.5 to 5% (w / w) is preferably used to prepare the biocatalyst beads according to the invention. It is particularly preferred to use an aqueous solution of highly viscous chitosan having a viscosity of about 10,000 cP at a concentration of about 1.4% (w / w).

These substances are preferably used in such a way that the weight ratio of silica to cationic polyelectrolyte is 1.5 to 50: 1, particularly preferably about 15: 1. The final concentrations of silica in the suspension described above are preferably 2 to 15% (w / w ), e.g. 10%. The final concentrations of cationic polyelectrolyte in the suspension described above are preferably 0.3 to 1.5% (w / w), especially about 0.7%.

The silicic acid used to prepare the biocatalyst beads of the invention has been described above. It is used in solid form.

An aqueous solution of a polyvalent anion is used as the crosslinking bath. Possible anions are described above. They are used in the form of acids or salts in a crosslinking bath, e.g. alkali salts, e.g. sodium or potassium salts, but also ammonium salts. An aqueous solution using tripolyphosphoric acid or a salt thereof, e.g. sodium or potassium tripolyphosphate, is preferred. The compounds mentioned in the crosslinking bath are, for example, in concentrations of 0.5 to 10%. (w / v), preferably 1.5 to 3% (w / v). The pH of the crosslinking solution is 5 to 10, preferably about 8.

A suspension of the enzymatically active substance, silicic acid, buffer solution and polycation is added dropwise to the crosslinking solution with stirring, and suitably such that the volume of the crosslinking bath is at least about three times the volume of the suspension. The dropwise addition takes place using a small orifice nozzle, for example a syringe, a syringe-like injection device, a porous plate or a similar suitable device. The drops can be blown from the nozzle tip using compressed air, compressed nitrogen or the like. The suspension can also be injected into the crosslinking solution, for example using a spray device, preferably a pressure spray device.

The biocatalyst beads obtained by the process described above are kept for at least about an hour, for example overnight, in a crosslinking bath for shrinkage and curing. They are optionally separated from the crosslinking bath by conventional methods suitable for separating solid and liquid phases, such as decantation, filtration, etc., and are subjected to a physiological washing process, for example with physiological saline, before further use.

The invention also relates to mechanically very strong bead-shaped biocatalysts which can be prepared by the process according to the invention.

The invention furthermore relates to processes for the conversion of organic substances which are characterized in that bead-shaped biocatalysts of the invention are used for the reaction.

Depending on the type of immobilized enzymatically active substance used, it is possible to produce a wide range of products resulting from fermentation processes, primary metabolism, microbial transformation, etc., for example - chemicals, e.g. citric acid with Aspergillus niaer. ethanol Zvmomonas mobile. L-malic acid with fumarate, bridge, H2 with chlorella-clostridial coimmobilates, - foods, eg soy sauce with Pediococcus halo-philus.

- enzymes such as amylases in Aspergillus niger.

- drugs, e.g. penicillins with Penicillium chrvsogenum or 12 94257 - amino acids, e.g. L-aspartic acid with L-aspartase.

Preferred are processes for the preparation of α-hydroxycarboxylic acids from α-ketocarboxylic acids by means of biocatalysts according to the invention, the enzymatically active substance of which contains oxidoreductase or is oxidido reductase, or consists of whole cells of Proteus, especially Proteus vulgaris and Proteus vulgaris. vulgaris. The reduction of the substrate takes place by a so-called final reductase, e.g. a substrate-specific dehydrogenase. In general, the reduction equivalents required for final reductase are obtained from a coenzyme, e.g., pyridine nucleotides such as nicotinamide adenine dinucleotide (phosphate) (NADH, NADPH) or flavin nucleotides such as flavin mononucleotide (FMNH) or flavin adenine ducin Reduced nucleotides, in turn, are generated by electron transfer, e.g., from natural or synthetic electron mediators with suitable reduction-oxidation potential, such as ferredoxin or bipyridinium derivatives, e.g., 4,4'-bipyridinium derivative (viogen) or 2,2'-bipyridine. (for example diquat dications such as diquat dibromide or diquat dichloride).

Particularly preferred is a process for the preparation of 2-hydroxy-4-phenylbutyric acid, in particular 2- (R) -hydroxy-4-phenylbutyric acid, from 2-oxy-4-phenylbutyric acid by means of biocatalysts according to the invention whose enzymatically active substance contains or consists of oxidoreductase or oxidoreductase. From whole cells of the genus Proteus, in particular Proteus vulgaris and / or Proteus mirabilis, preferably • Proteus vulgaris.

2- (R) -Hydroxy-4-phenylbutyric acid is a valuable intermediate in the preparation of ACE (angiotensin converting enzyme) inhibitors or their precursors. This class of active substances has received increasing attention in recent years. It expands the potential of available antihypertensive agents and thus the treatment options for hypertension. Of particular importance is the ACE inhibitor 3 - [[1-ethoxycarbonyl-3-phenyl- (1S) -propyl] amino] -2,3,4,5-tetrahydro-2-oxo-1H-1- (3S) -benzazepine-1-acetic acid monohydrochloride (= Benazepril).

This invention relates in particular to a process for the preparation of 2- (R) -hydroxy-4-phenylbutyric acid from 2-oxo-4-phenylbutyric acid, which process is characterized in that the bead-shaped biocatalysts according to the invention, the enzymatically active substance of which belongs to the genus Proteus, species Proteus vulgaris and / or Proteus mirabilis. preferably Proteus vulgaris, from whole cells of the bacterium, is placed in a solid bed or fluidized bed reactor, preferably a solid bed reactor through which an aqueous solution of a 2-oxo-4-phenylbutyric acid substrate at a concentration of, for example, 50 to 200 mM, formate, e.g. alkali metal formate, is continuously passed. aqueous formate, for example at a concentration of 100 to 500 mM, and an electron mediator, e.g. a 4,4'-bipyridinium derivative such as methyl viologene, carbamoylmethyl viologene or benzyl viologene, or a 2,2'-bi-pyridinium derivative such as an aqueous solution of diquat dication at a concentration of, for example, 0.5 to 10 mM. Substrate reduction is catalyzed, for example, by 2-oxocarboxylic acid reductase (2-hydroxycarboxylate viivene oxidoreductase) contained in Proteus vulgaris. Since this enzyme has good enantioselectivity and the product has an enantiomeric excess of more than 99.8% when formed, the use of the product obtained by the process of the invention has the particular advantage that an enantiomerically pure compound can be used at a relatively early stage in the ACE inhibitor synthesis. . With the method described above, it is possible to obtain yields of more than 99%.

The following examples are intended to illustrate the invention, but are not limited to the examples shown.

Abbreviations ee enantiomeric excess HPLC high performance liquid chromatograph rpm rpm

Example 1: Preparation of bead-shaped biocatalysts containing Proteus vulgaris

Proteus vulgaris (ATCC 9484) is cultured in complete medium (yeast extract 5 g / l, glucose simohohydrate 5 g / l, K2HPO4 5 g / l, meat-derived peptone 20 g / l, pH 7) for 24 hours at 37 ° In C by gently treating it with N2 gas. The cells thus obtained are collected at 12 ° C in a Z 41 G-type CEPA centrifuge (throughput 300 l / h) by rinsing with N 2 at 20,000 rpm (16,950 g). From 400 liters of culture medium, 580 g of bacterial sediment with a moisture content of about 80% are obtained. 1 part by weight of moist bacterial pulp and 1 part by weight of silicic acid (Baker product no. 254; grain size approx. 20 [mu] m, 95%> 3 [mu] m, 95% <60 [mu] m; vol. Weight approx.

50 g / 100 ml) is slurried in 3 parts by weight of 50 mM sodium acetate drop buffer, pH 5, and finally 5 parts by weight of a 1.4% (w / w) highly viscous chitosan acetate solution (viscosity n) are added with vigorous stirring. 10,000 cP, at 20 ° C, 10 rpm, pH adjusted to 4.5 with acetic acid; insoluble components filtered off).

! This suspension (viscosity approx. 2000 cP) is added dropwise with gentle stirring to a 1.5% (w / v) sodium tripolyphosphate solution (at least three times the volume of the suspension; the pH has been adjusted to 8.1 with phosphoric acid). A syringe with a cannula orifice diameter of 1.4 mm and a throughput of 1 ml / min is used for this.) The drops are blown into the cannula with pressurized nitrogen (drop angle 10-15 cm). This produces bead-shaped particles with a diameter of 2-3 mm, which are left to shrink and cure for 2 hours in a sodium tripolyphosphate crosslinking bath. About 40 g of beads are obtained from 100 g of suspension (volume weight approx.

80 ml) with a cell content of 0.25 g of moist bacterial mass or 0.05 g of dry cells per gram of beads, hereinafter referred to as "10% silicic acid biocatalyst beads" in terms of the silica content of the injected suspension.

For comparison, "control biocatalyst beads" are prepared without silicic acid. To this end, 1 part by weight of moist bacterial mass is slurried in 4 parts by weight of sodium acetate buffer having a pH of 5 and then mixed with 5 parts by weight of the chitosan acetate solution described above. The suspension is added dropwise to the sodium tripolyphosphate solution as described above to prepare bead-like particles.

In the slurry step of the preparation of "control biocatalyst beads", a large amount of foam is formed, which does not occur in the preparation of "10% silicic acid biocatalyst beads" because silicic acid acts as a defoamer. Due to foam formation, "control biocatalyst beads" are lighter than beads with added silicic acid and tend to rise to the surface of the crosslinking bath.

Example 2: Determination of the pressure novelty of biocatalyst beads

The compressive strength of the biocatalyst beads is determined after storage for one day at 4 ° C in sodium tripolyphosphate solution, pH 8,1, using a measuring system for thermomechanical analyzes (TA 3000 system with measuring photocell TMA 40, Mettler Instrumente AG , Greifensee, Switzerland). In each case, three wet biocatalyst beads (diameter 3 mm) are placed on the test table at right angles to each other and covered centrally with a ceramic plate (diameter 6 mm, 6 mm, strength. The deformation of the test piece (decrease in bead thickness indicated in μηκηΗ) is measured as a function of time by pressing the ceramic plate under a load of 0,02 N at a constant temperature at 30 ° C. The results are shown in Table 1.

Table 1: Determination of the pressure novelty of biocatalyst beads - deformation of 3 mm biocatalyst beads Γμΐηΐ__

Time [min] Control biocatalyst 10% silica exchange beads biocatalyst beads 0.5 40 10 1 80 20 2 130 30 4 200 40

Table 1 shows that "10% silica biocatalyst beads are substantially more pressure resistant than" control libocatalyst beads. "After 15 minutes, the deformation of 10% silica biocatalyst beads hardly increases at all.

Example 3: Preparation of 2-fR) -hydroxy-4-phenylbutyric acid with beaded biocatalysts: on a laboratory scale, 48 ml (pour volume) of "10% silica biocatalyst beads prepared from a suspension of 60 g of the suspension according to Example 1 are separated and added to 200 ml of a nitrogen gasified solution of 50 mM (1.84% (w / v)) sodium tripolyphosphate-17 94257 drop buffer pH 7, 100 mM potassium formate and 1 mM carbamoylmethyl violin (1.1 l). The beads are kept in this reduction solution under vacuum until gas evolution occurs until they turn completely purple due to the formate dehydrogenase of Proteus vulgaris. , e.g. to a glass column with a jacket (inner diameter 1.6 cm, wed height 24 cm, 25 ° C). A nitrogen pre-gasified substrate solution (containing 50 mM sodium tripolyphosphate buffer pH 6.7, 100 mM 2-oxo-4-phenylbutyric acid, 300 mM potassium formate, 3 mM carbamoylmethyl violin) is continuously applied to the column by means of a dosing pump 2, At an overpressure of 0-3.0 bar, with a throughput of 120 ml / h and then 24 ml / h. Reaction result from reduction of 2-oxo-4-phenylbutyric acid to 2- (R) hydroxy-4-phenylbutyric acid catalyzed by 2-oxocarboxylic acid reductase in Proteus vulgaris (2-hydroxycarboxylate viivene analysis) by oxidation reduction column: Nucleosil C18, particle size 5 [mu] m, length 12.5 cm, internal diameter 4.6 mm, throughput 1.0 ml / min, propellant: 3 volumes of acetonitrile / 8 volumes of 100 mM potassium phosphate buffer, pH the value is 3).

Similarly, "control biocatalyst beads" (pour volume 49 ml, from 60 g of the suspension according to Example 1) are used for the reduction of 2-oxo-4-phenylbutyric acid. In this case, it is necessary to shrink the bead layer with a column adapter, since most of the beads float, creating voids in the column.

The results of the productivity definition are shown in Table 2.

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Table 2: Preparation of 2- (R) -hydroxy-4-phenylbutyric acid in a solid bed reactor using bead-shaped biocatalysts (yield)

Yield in a fixed bed reactor at 25 ° C Feed throughput control 10% silica [ml / h] biocatalyst beads biocatalyst. ___, _ beads_ 120 60% 70% 24 96%> 99.5%

Table 2 shows that "control biocatalyst beads" have clearly lower productivity than "10% silicic acid biocatalyst beads".

Due to the increasing condensation of the beads, continuous operation of the control enzyme reactor is not possible, as the column becomes clogged after only one day of use at a throughput of 24 ml / h. In contrast, when "10% silica biocatalyst beads" are used, continuous use under the same conditions does not present any problems.

• Example 4: Preparation of 2- (R) -hydroxy-4-phenylbutyric acid using bead-shaped biocatalysts on a pilot scale and isolation of the product The preparation of "10% silicic acid biocatalyst beads" takes place from 573 g of the bacterial cement according to Example 1, however, instead of a syringe, a device with a gear pump (throughput 1.4 l / h; pressure approx. 0.5 bar) and a 7-spray jet with a capillary size of 0.6 mm is used for the formation. To increase the drip frequency or adjust the drip size, each capillary has a separate duct. After shrinkage and curing in 57% (w / v) sodium tripolyphosphate solution, 573 g of bacterial sediment give about 4 l (pour volume) of beads.

2- (R) -Hydroxy-4-phenylbutyric acid is prepared in the same manner as in Example 3. The resulting 4 L (pour volume) of “10% silicic acid biocatalyst beads” is separated from a sodium tripolyphosphate crosslinking bath and added to 6 L of a nitrogen-treated solution. is 100 mM (3.68%) sodium tripolyphosphate at pH 7, 100 mM potassium formate and 1 mM carbamoylmethyl violin. The beads are kept in this reduction solution under vacuum until the gas escapes until the beads have completely turned purple. Finally, the beads are applied to a chromatography column (inner diameter 11.3 cm, bottom area 100 cm 2, length 60 cm, layer height 40 cm). The nitrogen gas-treated substrate solution (100 mM sodium tripolyphosphate buffer pH 6.7, 112.2 mM 2-oxo-4-phenylbutyric acid, 300 mM potassium formate, 1 mM carbamoylmethyl violin) is sterile filtered and passed continuously through a column with a dosing pump 2.0, At an overpressure of 0 bar, the throughput first being 2.5 1 / h. The yield measured by HPLC is> 99.5%. The column is in operation at room temperature, i. at about 23-26.5 ° C, without interruption overnight.

The yield is monitored daily by HPLC analysis • and is maintained by controlling the pump speed at yield values> 99.5%. After 25 days, a total of about 800 l of reaction solution has reacted (average yield 99.6% and throughput capacity about 1 1 / h.

1160 l of the reaction solution formed in the reaction are mixed with 250 l of ethyl acetate and 114 l of 42.5% * orthophosphoric acid are adjusted to pH 2.6. 2- (R) -Hydroxy-4-phenylbutyric acid is extracted into the ethyl acetate in 94% yield. The 2- (R) -hydroxy-4-phenylbutyric acid remaining in the aqueous phase is extracted twice more with 120 l of ethyl acetate each time. After these three extraction steps, the extraction of 2- (R) -hydroxy-4-phenylbutyric acid is complete (99.8%). After combining the three ethyl acetate phases 20 94257 (total volume 450 l) and adding 250 l of ethyl acetate and distilling off 470 l of solvent, the solution is filtered clear with a single-plate filter. 50 L of vinegar ester is used to rinse the filter and added to the clear solution. After distilling off 200 l of solvent, the final volume of the solution is 80 1. To this solution is added 400 l of cyclohexane and 200 l of solvent are distilled off. After centrifugation of the crystallized material and drying in vacuo at 20-25 ° C to constant weight, 23.4 kg of crystalline 2- (R) -hydroxy-4-phenylbutyric acid are obtained.

To control the enantiomeric purity, a sample of the crystalline acid is dissolved in absolute ethanol and reacted with hydrogen chloride gas for 24 hours at room temperature. After distillation of the alcohol and brief degassing under high vacuum, a pale yellow oil remains which is analyzed on a chiral column (inner diameter 250 x 4.6 mm, throughput 1 ml / min, solid phase Chiralcel OD [Stehelin, Basel], type OD-5-1520925, mobile phase: 90% hexane - 10% isopropanol - 0.1% diethylamine) by HPLC analysis at 25 ° C, 32 bar. The substances to be analyzed are present in concentrations of 1 mg / ml in a vortex bath (injection volume 10 μΐ). Measurement (scanning) is performed at 210 nm, analysis by area comparison using an external standard. The ee value obtained is> 99.8%.

4

Claims (7)

A process for the preparation of a bead-shaped biocatalyst with high mechanical pour strength, comprising enzyme-active material consisting of whole cells, cell fragments or cell organelles from a microorganism, or of enzymes or of any combination of these kinds of - zymactive material, chitosan, polyhydric anions and silicic acid, characterized in that said enzyme active material is mixed with precipitated silica in a solid form, an aqueous buffer solution and an aqueous solution of chitosan, avoiding foaming, to a suspension, the suspension is added dropwise to an aqueous: crosslinking bath containing orthophosphate, meta, pyro or polyphosphate, cyanoferrate or polymeric organic carboxylates, sulfonates or hydroxy compounds, and the resulting biocatalyst beads are shrunk and crosslinked and crosslinked in the event bath. 94257 2. A process according to claim 1, characterized in that foam formation is avoided by mixing in the presence of silicic acid. Process according to claim 1 or 2, characterized in that in the suspension, which is the enzyme active material, the silicic acid, the buffer solution and the chitosan, the final concentration of the silicic acid is 2-15% (w / w) and the chitosan is 0.3 - 1.5% (w / w). 4. A process according to claim 1 for the preparation of bead-shaped biocatalyst, characterized in that the enzyme-active material of the biocatalyst is made up of whole cells of bacteria of species Proteus vulgaris and Proteus mirabilis. 5. A process according to claim 1 for the preparation of bead-shaped biocatalyst, characterized in that the biocatalyst contains as polyvalent anions tripolyphosphate. Use of a biocatalyst prepared by the method of claim 4 for the preparation of 2-hydroxy-4-phenylbutyric acid from 2-oxo-4-phenylbutyric acid. A process for the preparation of 2- (R) -hydroxy-4-phenylbutyric acid from 2-oxophenylbutyric acid, characterized in that the bead-shaped biocatalysts prepared according to claim 4 are put into a fixed bed reactor, through which a water solution of the substrate 2-oxo-4-phenylbutyric acid, of formate and of an electron mediator.