CA2003767A1

CA2003767A1 - Biocatalysts and processes for the manufacture thereof

Info

Publication number: CA2003767A1
Application number: CA002003767A
Authority: CA
Inventors: Pierre F. Fauquex; Gottfried Sedelmeier
Original assignee: Ciba Geigy AG
Current assignee: Novartis AG
Priority date: 1988-11-28
Filing date: 1989-11-24
Publication date: 1990-05-28
Also published as: DK596589D0; FI94257B; EP0371408A3; FI895635A0; FI94257C; ES2055779T3; JPH02190187A; PT92400B; DK596589A; IE63088B1; IE893773L; JP3042691B2; PT92400A; DK173712B1; EP0371408A2; EP0371408B1; DE58907947D1; ATE107691T1

Abstract

4-17341/+

Biocatalysts and processes for the manufacture thereof Abstract The present invention relates to spherical biocatalysts of high mechanical strength, comprising enzymatically active material, a cationic polyelectrolyte, preferably chitosan, polyvalent anions and silicic acid, and to processes for the manufacture thereof. This application also relates to processes for the conversion of organic substances, e.g. for the manufacture of .alpha.-hydroxycarboxylic acids from .alpha.-ketocarboxylic acids, especially 2-(R)-hydroxy-4-phenylbutyric acid from 2-oxo-4-phenyl-butyric acid, using the biocatalysts of the invention.

Description

20933~67 4-17341/+

Biocatalysts and processes for the manufacture thereof The present invention relates to spherical biocatalysts of high mechanical strength, to processes for the manufacture thereof and to processes for the conversion of organic substances using the biocatalysts of the invention.

Background to the invention In its broadest sense, the term "biocatalysis" includes all forms of catalysis in which the activating substance is a biological system, e.g.
whole cells, cell fragments or cell organelles, or originates from a biological system, e.g. enzymes. Enzymes are compounds, generally proteins or glycoproteins, that accelerate specifically biochemical reactions by reducing the activation energy. In the following, the expression "biocatalyst" not only refers to the activating substance itself, but may also include, where applicable, components that have been used for the immobilisation, stabilisation, regeneration, etc. of the activating substance.

Immobilised biocatalysts, that is to say biocatalysts whose mobility has been restricted, are particularly suitable for industrial purposes. As a result of the immobilisation it becomes possible to carry out the catalysed process continuously and repeatedly, create and maintain high biocatalyst densities, achieve high space-time yields and reduce the costs of isolating the product from the reaction solution.

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Z~ 767 For immobilisation, ~he biocatalytic material may, for example, be entrapped in polymeric enclosing substances (matrix), in capsules or fibres consisting of semi-permeable membranes or behind ultra-filtration membranes, or crosslinked with bi- or multi-functional reagents, or fixed to carriers consisting of inorganic material or natural or synthetic polymers by adsorption or by ionic or covalent bonding. A combination of these methods is also possible.

During the immobilisation it is necessary to ensure, on the one hand, the greatest possible preservation of the biocatalytic activity and, on the other hand, high mechanical strength and chemical stability and also, at the same time, good permeability of the biocatalyst. A widely used immobilisation method is, for example, entrapment of the biologically active material in a matrix of natural polymers, such as, inter alia, cellulose, agar or gelatin, or synthetic polymers, such as, inter alia, polyacrylamide, polyurethanes or epoxy resins. An especially gentle immobilisation method is ionotropic gel formation (ionic crosslinking of the matrix substances) for which suitable enclosing materials are, for example, natural polyanions, such as alginate, pectin, carrageenan, etc..
Dropwise introduction or spraying of a suspension of enzymatically active material and an aqueous solution of the matrix substance into an aqueous crosslinker solution containing multivalent counterions, for example, inter alia, Ca , Co , Zn , Fe , Fe , Al , results in chelatisation and the formation of spherical biocatalysts.

As a rule, ionotropic gel formation by organic polymers produces only relatively soft, mechanically unstable immobilisates that show a tendency towards swelling and deformation, with the result that difficulties arise, for example, when they are used in packed reactors like those frequently used industrially for enzymatic reactions. Hardening methods comprising aftertreatment of the immobilisates or pretreatment of the biologically active material before addition to the gelling agent, both of which are carried out with multi-functional crosslinking agents such as polyaldehydes, e.g. glutaraldehyde, or isocyanates, e.g. hexamethylene diisocyanate, can be employed only to a very limited extent owing to 2~337~i7 their cell-damaging and/or enzyme-damaging action. An alternative is the use of inorganic substances, e.g. as in the method described in German Offenlegungsschrift DE 35 20 001 which discloses the immobilisation of enzymatic material by means of silica sol and alginate. Drying and shrinking of the biocatalyst beads are also used for hardening since the beads do not reabsorb their original moisture content when re-moistened and remain harder. German Offenlegungsschrift DE 28 35 875 describes a method of ionotropic gel formation using polyanions, e.g. sodium alginate, and, for example, calcium ions as crosslinker, in which the biocatalyst beads charged with enzymatic substance are dried at a temperature of up to 80C. It is impossible, however, to carry out such drying steps whenever highly sensitive enzymes or enzyme systems, especially living cells, are to be immobilised since the high tempera-tures during the drying process, or the dry state itself, inactivate the cells or enzymes.

In addition to having poor mechanical properties, a further disadvantage of the calcium alginate immobilisates produced by ionotropic gel forma-tion is the limited range of possible reaction media for the further processing. Such immobilisates are, for example, chemically unstable in the presence of ions found in conventional buffer solutions, such as sodium or potassium. In such electrolyte solutions, especially when they are highly concentrated, the gel-solidifying calcium ions in calcium alginate gels, for example, are displaced since sodium ions or, as the case may be, potassium ions compete for the alginate anion. Phosphate ions too give rise to the gradual dissolution of calcium alginate bio-catalyst beads, since phosphates react with calcium and deprive the alginate of the structure-strengthening agent. Dissolution can be prevented if, as matrix substances, polycations, e.g. chitosan, are used which are crosslinked with polyvalent anions such as, inter alia, [Fe(CN)6] , [Fe(CN)6] and polyphosphates. German Patent Specifications DE 30 05 632 and DE 30 05 633 describe the manufacture of biocatalyst beads using chitosan and K3(Fe(CN)6). The process described in DE 30 05 632 comprises a drying step at a temperature of up to 80C
for hardening the beads. To enable highly sensitive enzymatic substances 2~)~37~7 to be immobilised, this drying step is not included in the process described in DE 30 05 633, which, however, results in lower mechanical strength of the biocatalyst beads.

Object of the invention It is an object of the present invention to provide spherical (bead--shaped) biocatalysts . that exhibit high mechanical strength, . the stability of which is ensured even when using conventional biological solutions and buffer systems, and . that continue to ensure the enzymatic activity of highly sensitive enzymatically active material.

It is also an object of the invention to provide suitable processes forthe manufacture of such biocatalysts.

There have been found to be especially suitable for achieving these objects spherical biocatalysts that contain enzymatically active material, a cationic polyelectrolyte, preferably chitosan, polyvalent anions and silicic acid. Such biocatalyst beads are manufactured in a gentle immobilisation process by ionotropic gel formation and by using silicic acid in solid form.

Description of the invention The present invention relates to spherical (bead-shaped) biocatalysts of high mechanical strength, comprising enzymatically active material, a cationic polyelectrolyte and polyvalent anions, wherein the biocatalysts of the invention contain silicic acid.

The spherical biocatalysts of the invention are characterised by high mechanical strength, which can be attributed to the formation of a dual-gel-matrix structure. They therefore have good packing properties, for example when used to pack a reaction vessel, and can be used as ;~0~ 7 filling material e.g. for fixed bed reactors. Owing to the mild immobilisation conditions, which do not include any drying step or treat-ment with damaging substances, the biocatalysts of the invention are suitable especially for highly sensitive enzymatic material, especially for living cells. The manufacture of the spherical biocatalysts accord-ing to the invention by using silicic acid in solid form has the advantage over prior a~t processes that time-consuming and expensive processes for the preliminary preparation of the crosslinking agents used in the hardening step are avoided. Furthermore, the biocatalysts of the inventlon can be used in buffer systems containing ions that cause dis-solution of alginate gels. The biocatalysts of the invention can there-fore be used, for example, in solutions containing phosphate ions, e.g.
in tripolyphosphate buffer which is very cheap and therefore advantageous for industrial purposes. The spherical biocatalysts of the invention are suitable for the conversion of organic substances. Since they can be used for reactions requiring high electrolyte concentrations, biocatalyst beads of the invention containing a bacterium of the genus Proteus or an oxido-reductase as enzymatically active material are suitable, for example, for the manufacture of ~-hydroxycarboxylic acids from ~-keto-carboxylic acids, e.g. 2-(R)-hydroxy-4-phenylbutyric acid from 2-oxo-4-phenylbutyric acid.

The spherical biocatalysts of the invention exhibit high mechanical strength. The expression "high mechanical strength" is used to mean, for example, high compression resistance, that is to say the biocatalyst beads of the invention having an average size of about 3 mm exhibit, after from one to five minutes under a load of from 0.01 to 0.03 N, especially 0.02 N, a maximum deformation of from 30 to 50 ~m, especially 40 ~m, and, after from 10 to 20 minutes, especially 15 minutes, do not exhibit any further increase in deformation.

The type of enzymatically active material comprised in the biocatalystsof the invention is governed by the reaction for which the biocatalysts are to be used. Preferably, it consists of whole cells, cell fragments or cell organelles of a microorganism, or of enzymes or of any combination of these types of enzymatically active material.

2~1V37~,7 The microorganism may be of a prokaryotic or eukaryotic nature. Suitable microorganisms are, for example, - a bacterium, e.g. a lactic acid bacterium (Leuconostoc, Streptococcus, Lactobacillus), Corynebacterium, Streptomyces, Pseudomonas, Xanthomonas, Bacillus, Clostridium, Escherichia, Proteus etc., - a fungus, e.g. Mucor, Asper~illus, Penicillium, especially yeasts such as, for example, Saccharomyces, Candida etc., - an alga, e.g. _lorella, 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, that is to say cell subunits enveloped in membranes or consisting of membrane systems (chloroplasts, thylacoids, mitochondria etc.), as the enzymatically active material is advantageous when the reaction to be catalysed requires a multi-enzyme system, for example when co-factors (pyridine nucleotides or flavine nucleotides etc.) are needed, or when, instead of a catabolic product, a biosynthetic, e.g. a secondary, product is to be manufactured, for which complicated metabolism chains are necessary.

Isolated enzymes also are suitable as enzymatically active material.
Typical examples of such enzymes are oxido-reductases (lactate dehydrogenase, alcohol dehydrogenase, glucose oxidase etc.), transferases (hexokinase, glutamine transaminase etc.), lyases (fumarase, aspartase etc.), isomerases (glucose isomerase, mannose isomerase etc.), ligases (glutathione synthetase, NAD-synthetase etc) and so on.

The biocatalysts of the invention may also comprise as enzymatically active material any combination of the types of enzymatic material described, for example a combination of whole cells of two or more species or strains of microorganisms or a combination of whole cells and 2~)~376'7 enzymes. The fundamental idea is to make the biocatalytic properties of a cell complete using additional enzymes that are not available in the cell or that are available in the cell only in too small a quantity.

Especially preferred as enzymatic material are whole cells of a bacterium or yeast, preferably a bacterium of the genus Proteus, especially a bacterium of the species Proteus vul~aris and/or of the species Proteus mirabilis. Special preference is given to the species Proteus vulgaris, e.g. Proteus vulgaris ATCC 9484. Special preference is also given to enzymatically active material that contains an oxido-reductase or that is an oxido-reductase.

The cationic polyelectrolyte is a natural or synthetic polymer or a natural polymer after chemical modification, for example a polymer having protonated amino groups, preferably chitosan ([2-amino-2-deoxy-(1 ~4)-~-D-glucopyranane; poly-(1,4-~-D-glucopyranosamine]). Chitosan is a linear polymer of high molecular weight consisting of glucosamine subunits, which is isolated from chitin by partial de-acetylation with the aid of a concentrated alkaline solution and heat. Chitin is common in nature, for example in marine invertebrates, insects and fungi. Chitin from crabs, shrimps, lobsters etc. is generally used for the production of chitosan.
The spherical biocatalysts of the invention preferably comprise chitosan having a molecular weight of from about 50,000 to about 3,000,000.

Suitable polyvalent anions are polyvalent inorganic or polyvalent organic anions. The inorganic anions are preferably orthophosphate, metaphos-phates, e.g. hexametaphosphate, pyrophosphates, polyphosphates, e.g.
tri-, tetra- or octa-polyphosphate, or cyanoferrates, e.g. [Fe(CN)6] or [Fe(CN)6] . Tripolyphosphate is especially preferred. The organic anions are preferably polymeric organic carboxylates, sulfonates or hydroxy compounds.

X~ 3~67 Suitable silicic acid is preferably a precipitated silicic acid, more especially a precipitated silicic acid having an average particle size in the range of from 5 to 50 ~m and an average bulk density in the range of from 5 to 100 g/100 ml, especially about from 20 to 50 g/100 ml.

The present invention further relates to a process for the manufacture of the spherical biocatalysts of the invention having high mechanical strength, which process comprises mixing enzymatically active material with precipitated silicic acid in solid form, an aqueous buffer solution and an aqueous solution of a cationic polyelectrolyte to form a suspension while avoiding the formation of foam, introducing the suspension dropwise into a crosslinker bath containing polyvalent anions and shrinking and solidifying the resulting biocatalyst beads in the crosslinker bath, and optionally separating them from the crosslinker bath.

The avoidance of foam formation is of decisive importance in the manufacture of the suspension from enzymatically active material, precipitated silicic acid in solid form, aqueous buffer solution and an aqueous solution of a cationic polyelectrolyte, since the formation of foam results in catalyst beads having less good mechanical properties being produced. Foam formation is avoided preferably by mixing the various components in the presence of silicic acid. The sequence in which the substances are combined is otherwise arbitrary. Preferably, the enzymatic material is first mixed with the silicic acid in solid form and only then is it taken up in aqueous buffer solution and subsequently mixed with a solution of the cationic polyelectrolyte.

The process of the invention for the manufacture of the biocatalyst beads uses enzymatic material of the type described above, i.e. whole cells, cell fragments or cell organelles of a microorganism or enzymes or a combination thereof. Whole cells may be living, dead or, for example, in lyophilised or dehydrated form. Culturing of the cells in a nutrient medium containing all the organic and inorganic constituents necessary for maintaining metabolism and for multiplication (carbon source, nitrogen source, trace elements, growth substances etc.), and cell - ' ~ ' .

2~)~33767 _ 9 _ fragmentation to produce cell fragments and isolation of cell organelles, respectively, are carried out by methods that are known per se. Enzymes to be immobilised are, for example, in dissolved, dispersed, suspended, emulsified or dried form. The enzymatically active material mentioned, which has been defined in detail hereinabove, is used for the process of the invention, for example, in the form of a sediment, filtrate or suspension of cells, cell fragments or cell organelles or in the form of an enzyme in aqueous solution. In the context of this description, the expression "in aqueous solution" means that the solution may additionally contain inorganic or organic salts and the like. The solution may be, for example, a conventional biological buffer such as acetate buffer, phosphate buffer, tris buffer and the like.

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

Cationic polyelectrolytes that can be used in the process of the invention have been described hereinabove. Chitosan is preferred. For use in the manufacture of biocatalyst beads, chitosan must first be brought into aqueous solution in order for it to become an integral component of the immobilisate. For that purpose, chitosan, which is water-insoluble in non-protonated form, is taken up in a dilute aqueous, especially organic, acid, for example in formic acid, acetic acid, pyruvic acid, malic acid, citric acid or the like, preferably acetic acid, at a pH of < 7, preferably pH 4 to 5.5, and optionally heated to about 60C to promote the dissolution process. The chitosan solution is then filtered to remove insoluble constituents. For the manufacture of the biocatalyst beads of the invention, there is preferably used an aqueous solution of chitosan having a viscosity in the range of from 1,000 to 20,000 cP, in a concentration in the range of from 0.5 to 5 % (w/w). Especially preferred is the use of an aqueous solution of high viscosity chitosan having a viscosity of about 10,000 cP in a concentration of about 1.4 % (w/w).

2~33'7~i'7 The substances used are preferably employed in such amounts that the ratio by weight of silicic acid to cationic polyelectrolyte is within a range of from 1.5 to 50 : 1, more especially approximately 15 : 1. Pre-ferred final concentrations of silicic acid in the suspension described above are in a range of from 2 to 15 % (w/w), e.g. 10 %. Preferred final concentrations of cationic polyelectrolyte in the above-described sus-pension are from 0.3 to 1.5 % (w/w), especially around 0.7 %.

The silicic acid used for the manufacture of the biocatalyst beads of the invention has been described above. It is employed in solid form.

The crosslinker bath is an aqueous solution of a polyvalent anion. The possible anions have been described hereinabove. They are used for the crosslinker bath in the form of the acids or in the form of salts, e.g.
in the form of alkali metal salts, for example sodium or potassium salts, and also in the form of ammonium salts. Preference is given to an aqueous solution for which tripolyphosphoric acid or a salt thereof, e.g. sodium or potassium tripolyphosphate, is used. The compounds mentioned are present in the crosslinker bath, for example, in a concentration in the range of from 0.5 to 10 % (w/v), preferably from 1.5 to 3 % (w/v). The pH
of the crosslinker solution is in a range of from pH 5 to 10, and is preferably about pH 8.

The suspension consisting of enzymatically active material, silicic acid, buffer solution and polycation is introduced dropwise into the cross-linker solution with stirring, advantageously in such an amount that the volume of the crosslinker bath is at least about three times the volume of the suspension. The dropwise addition is performed using a nozzle having a small outlet, for example an injection syringe, a syringe-like injection device, a porous plate or similar suitable means. The droplets are optionally blown off the tip of the nozzle with compressed air, compressed nitrogen or the like. The suspension may also be sprayed into the crosslinker solution, for example using an atomising device, preferably a pressurised atomising device.

20~3~67 The biocatalyst beads obtained by the process described above remain inthe crosslinker bath for at least about an hour, e.g. overnight, for shrinking and hardening. They are optionally separated from the cross-linker bath by the customary methods suitable for separating solid and liquid phases, such as decanting, filtration and so on, and, before further use, subjected to a physiological washing process, for example with physiological saline solution.

The present invention also relates to spherical biocatalysts having high mechanical strength that are manufactured by the process of the invention.

The invention relates, in addition, to processes for the conversion of organic substances, which processes comprise using for the conversion spherical biocatalysts according to the present invention.

Depending on the type of immobilised enzymatically active material, it is possible to manufacture a broad range of products that are produced by fermentation processes, in primary metabolism, by microbial transforma-tion etc. of, for example, - chemicals, e.g. citric acid by Asper~illus n ger, ethanol by Zymomonas mobilis, L-malic acid by fumarase, h2 by Chlorella-clostridia co-immobilisates, - foodstuffs, e.g. soy sauce by Pediococcus haloPhilus, - enzymes, e.g. amylases by Asper~illus n ~er, - medicaments, e.g. penicillins by Penicillium chrysogenum, or - amino acids, e.g. L-aspartic acid by L-aspartase.

Preference is given to processes for the manufacture of -hydroxy-carboxylic acids from ~-ketocarboxylic acids using the biocatalysts of the invention the enzymatically active material of which contains an oxido-reductase, is an oxido-reductase or consists of whole cells of a bacterium of the genus Proteus, especially of the species Proteus vul~aris and/or Proteus mirabilis, preferably Proteus vul~aris.
In these processes, the reduction of the substrate is effected by the so-called final reductase, e.g. a substrate-specific dehydrogenase. The Z~)03767 reduction equivalents required by the final reductase are generally supplied by a co-enzyme, e.g. by pyridine nucleotides such as nicotin-amide adenine dinucleotide (phosphate) (NADH, NADPH) or by flavine nucleotides such as flavine mononucleotide (FMNH) or flavine adenine dinucleotide (FADH). The reduced nucleotides are in their turn produced, for example, by electron transfer via natural or synthetic electron mediators having a suitable redox potential, such as ferredoxin or bi-pyridilium derivatives, e.g. 4,4'-bipyridilium derivatives (viologens) or 2,2'-bipyridilium derivatives (for example diquat dications such as diquat dibromide or diquat dichloride). Also known are final reductases that are able to accept the electrons directly from the mediators.

Special preference is given to a process for the manufacture of 2-hydroxy-4-phenylbutyric acid, preferably 2-(R)-hydroxy-4-phenylbutyric acid, from 2-oxo-4-phenylbutyric acid using biocatalysts of the invention the enzymatically active material of which contains an oxido-reductase, is an oxido-reductase or consists of whole cells of a bacterium of the genus Proteus, especially of the species Proteus vul~aris and/or Proteus mirabilis, preferably Proteus vul~aris. 2-(R)-hydroxy-4-phenylbutyric acid is a valuable intermediate in the manufacture of ACE
(angiotensin converting enzyme)-inhibitors or their precursors. This class of active substances has been the subject of growing interest in recent years. It broadens the potential of the available anti-hyper-tensive agents and, therewith, the range of possible therapies in the control of high blood pressure. Of special interest in this connection is the manufacture of the ACE-inhibitor 3-~[1-ethoxycarbonyl-3-phenyl-(lS)-propyl]amino~-2,3,4,5-tetrahydro-2-oxo-lH-1-(3S)-benzazepine-1-acetic acid monohydrochloride (= benazepril).

The present invention relates especially to a process for the manufacture of 2-(R)-hydroxy-4-phenylbutyric acid from 2-oxo-4-phenylbutyric acid, which process comprises using spherical biocatalysts of the invention the enzymatically active material of which consists of whole cells of a bacterium of the genus Proteus, especially of the species Proteus vul~aris and/or Proteus mirabilis, preferably Proteus vul~aris, in a fixed-bed or fluidised-bed reactor, preferably in a fixed-bed .
~, .

2~7~i7 reactor, through which there is continuously passed an aqueous solution of the substrate 2-oxo-4-phenylbutyric acid, for example in a concentra-tion in the range of from 50 to 200 mM, formate, e.g. an alkali metal formate, such as potassium or sodium formate, for example in a concentra-tion in the range of from 100 to 500 mM, and an electron mediator, e.g. a 4,4'-bipyridinium derivative, such as methylviologen, carbamoylmethyl-viologen or benzylviologen, or a 2,2'-bipyridilium derivative, such as diquat dication, for example in a concentration in the range of from 0.5 to 10 mM. The reduction of the substrate is catalysed, for example, by the 2-oxocarboxylic acid reductase (2-hydroxycarboxylate viologen oxido-reductase) present in Proteus vulgaris. Since this enzyme has a high enantioselectivity and the product is obtained with an enantiomeric excess of more than 99.8 ~0, the particular advantage of using the product obtained by the process of the invention is that, in the synthesis of ACE-inhibitors, which proceeds via numerous steps, it is possible to use an enantiomerically pure compound at a relatively early stage of the synthesis. The process described above renders possible high conversion rates, of more than 99 %.

The following Examples are intended to illustrate the invention withoutimplying any limitation thereof, for example, to the scope of the Examples.

Abbreviations ee enatiomeric excess HPLC high pressure liquid chromatography rpm revolutions per minute Examples xample 1: Manufacture of spherical biocatalysts containing Proteus vulgaris Proteus vulgaris (ATCC 9484) is sultured in a complete medium (yeast extract 5 g/l, glucose monohydrate 5 g/l, KzHP04 5 g/l, meat peptone 20 g/l, pH 7.0) for 24 hours at 37C while gently gassing with Nz. The 2~1~37~i7 cells so obtained are harvested at 12C in a CEPA centrifuge type Z 41 G
(throughput 300 l/hour) at a speed of 20,000 rpm (16,950 g) while flush-ing with N2. 400 litres of culture liquid yield 580 g of bacterial sedi-ment having a moisture content of about 80 %. 1 part by weight of moist bacterial mass is suspended together with 1 part by weight of silicic acid (~aker Product No. 254; particle size about 20 ~m, 95 % > 3 ~m, 95 %
< 60 ~m; bulk density about 50 g/100 ml) in 3 parts by weight of 50 mM
sodium acetate buffer pH 5.0, and then mixed, with vigorous stirring, with 5 parts by weight of a 1.4 % (w/w) high viscosity aqueous chitosan acetate solution (viscosity about 10,000 cP at 20C and 10 rpm; pH 4.5 adjusted with acetic acid; insoluble constituents removed by sieving).
This suspension (viscosity about 2,000 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; pH 8.1 adjusted with phosphoric acid). For this there is used a syringe having a needle open-ing of 1.4 mm diameter and a flow rate of 1 ml/min. The droplets are blown off the needle (falling distance 10-15 cm) with compressed nitrogen. Spherical particles having a diameter of 2-3 mm are obtained, which remain in the sodium tripolyphosphate crosslinker bath for 2 hours for shrinking and hardening. 100 g of suspension yield approximately 40 g of beads (bulk volume about 80 ml) having a cell concentration of 0.25 g of moist bacterial mass or 0.05 g of dry cells per g of beads which, having regard to the concentration of the silicic acid in the syringed suspension, are referred to hereinafter as "10 % silicic acid biocatalyst beads".

"Control biocatalyst beads" are prepared without the addition of silicic acid for comparison purposes. For this, 1 part by weight of moist bacterial mass is suspended in 4 parts by weight of sodium acetate buffer pH 5.0 and then mixed with 5 parts by weight of the chitosan acetate solution described above. The suspension is added dropwise to sodium tripolyphosphate solution in the manner described above in order to produce spherical particles.

2~1037G7 In the manufacture of the "control biocatalyst beads" considerable foamformation occurs during the suspension process; this does not occur in the manufacture of the "10 % silicic acid biocatalyst beads" since the silicic acid acts as an anti-foaming agent. Owing to the foam formation, the "control biocatalyst beads" are lighter than the beads to which silicic acid has been added, and tend to rise to the surface of the crosslinker bath.
xample 2: Determination of the compression resistance of the bio-catalyst beads The compression resistance of the biocatalyst beads is determined after one day's storage at 4C in the sodium tripolyphosphate solution pH 8.1 using a measuring system for thermomechanical analyses (TA 3000 system with gauge TMA 40, Mettler Instrumente AG, Greifensee, Switzerland).
Groups of three biocatalyst beads (diameter 3 mm) in the moist state are placed in a triangular arrangement, touching one another, on the test bench and a ceramic disc (diameter 6 mm, thickness 0.7 mm, dry weight 70 mg), which has been impregnated with sodium tripolyphosphate solution and on which the sensor is positioned, is placed centrally over them. The deformation of the sample (decrease in the thickness of the beads in ~m) is measured as a function of time by pressing down the ceramic disc under a load of 0.02 N isothermally at 30C. The results are set forth in Table 1.

Table 1: Determination of the compression resistance of the biocatalyst beads Deformation of the 3 mm biocatalyst beads [~m]
. . . _ __ time [min] control bio- 10% silicic acid catalyst beads biocatalys beads _ 0.5 40 10 2~ i7 Table 1 makes it clear that the "10 % silicic acid biocatalyst beads"
have a considerably higher compression resistance than that of the "control biocatalyst beads". After 15 minutes the "10 % silicic acid biocatalyst beads" exhibit almost no further increase in deformation.

Example 3: Manufacture of 2-(R)-hydroxy-4-phenylbutYric acid on a laboratory scale using spherical biocatalYsts 48 ml (bulk volume) of "10 % silicic acid biocatalyst beads" manufactured from 60 g of suspensior according to Example 1 are sepa.ated from the sodium tripolyphosphate crosslinker bath and added to 200 ml of a solu-tion of 50 mM (1.84 % w/v) sodium tripolyphosphate buffer pH 7.0, 100 mM
potassium formate and 1 mM carbamoylmethylviologen (1,1'-dicarbamoyl-methyl-4,4'-dipyridinium dication) which has been pregassed with nitrogen. The beads remain in this reducing solution, under vacuum for the purpose of degassing, until a completely violet colour, resulting from the reduction of the carbamoylmethylviologen by the formate de-hydrogenase present in Proteus vulgaris, is obtained. The beads are then introduced into an enzyme fixed bed reac~or, i.e. into a glass column provided with a jacket (internal diameter 1.6 cm, bed height 24 cm, 25C). The substrate solution (50 mM sodium tripolyphosphate buffer pH
6.7, 100 mM 2-oxo-4-phenylbutyric acid, 300 mM potassium formate, 3 mM
carbamoylmethylviologen) which has been pregassed with nitrogen is then passed continuously through the column by means of a metering pump at 2.0 to 3.0 bar excess pressure at a flow rate of 120 ml/hour initially and at 24 ml/hour subsequently. The degree of reduction of the 2-oxo-4-phenyl-butyric acid to 2-(R)-hydroxy-4-phenylbutyric acid which is catalysed by the 2-oxocarboxylic acid reductase (2-hydroxycarboxylate viologen oxido-reductase) present in Proteus vulgaris is measured by HPLC analysis (column: Nucleosil C18, particle size 5 ,um, length 12.5 cm, internal diameter 4.6 mm; flow rate 1.0 ml/min.; eluant: 3 parts by volume aceto-nitrile/8 parts by volume 100 mM potassium phosphate buffer pH 3.0).

.

2(~376'~

The "control biocatalyst beads" (bulk volume 49 ml from 60 g of suspension according to Example 1) are used for the reduction of 2-oxo-4-phenylbutyric acid in an analogous manner. For this it is necessary to restrict the bead bed with a column adapter because the majority of the beads float and, therefore, empty zones are produced in the column.

The results of the productivity analysis are set forth in Table 2.

Table 2: Manufacture of 2-(R)-hydroxy-4-phenylbutyric acid in a fixed bed reactor on a laboratory scale usin~ spherical biocatalYsts (degree of conversion) degree of conversion in fixed-bed reactor at 25C
low rate [ml/h] control biocatalyst 10% silicic acid beads biocatalyst beads 120 60 % 70 %
24 96 % >99.5 %

It can be seen from Table 2 that the "control biocatalyst beads" exhibit a markedly poorer productivity than that of the "10 % silicic acid bio-catalyst beads".

Owing to the increasing compaction of the beads, continuous operation of the control enzyme reactor is not possible since the column is blocked after only one day at a flow rate of 24 ml/hour. In contrast, continuous operation can be carried out under the same conditions with the "10 %
silicic acid biocatalyst beads" without any problem.
xample 4: Manufacture of 2-~R)-hydroxy-4-phenylbutyric acid on a pilot scale usin~ spherical biocatalysts, and isolation of the product "10 % silicic acid biocatalyst beads" are manufactured from 573 g of bacterial sediment in accordance with Example 1 except that, instead of a syringe, a device consisting of a gear pump (flow rate 1.4 l/hour;

Z~37ti'7 pressure approximately 0.5 bar) and a 7-jet shower having 0.6 mm capillaries is used for forming the droplets. In order to increase the drop-off frequency and to influence the size of the droplets, each capillary has a separate air delivery channel. After shrinking and hardening in a 3 % (w/v) sodium tripolyphosphate solution, approximately 4 litres (bulk volume) of beads are obtained from 573 g of bacterial sediment.

2-(R)-hydroxy-4-phenylbutyric acid is prepared analogously to the method indicated in Example 3. The 4 litres (bulk volume) of "10 % silicic acid biocatalyst beads" are separated from the sodium tripolyphosphate cross-linker bath and added to 6 litres of a solution of 100 mM (3.68 % w/v) sodium tripolyphosphate pH 7.0, 100 mM potassium formate and 1 mM
carbamoylmethylviologen which has been pregassed with nitrogen. The beads remain in this reducing solution, under vacuum for the purpose of degassing, until a completely violet colour is obtained. The beads are then introduced into a chromatography column (internal diameter 11.3 cm, base area 100 cm2, length 60 cm, bed height 40 cm). The 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-viologen), which has been pregassed with nitrogen, is sterile-filtered and passed continuously through the column by means of a metering pump at 2.0 to 3.0 bar excess pressure at an initial flow rate of 2.5 l/hour. The degree of conversion, measured by HPLC, is > 99.5 %. The column is operated day and night, without interruption, at room temperature, i.e.
at about 23 to 26.5C.

The degree of conversion is monitored by daily HPLC analysis and maintained at conversion values of > 99.5 % by regulating the pump speed.
After 25 days, a total of about 800 litres of reaction solution have been converted (average degree of conversion 99.6 %) and the flow rate is still 1 litre/hour.

1160 litres of converted reaction solution are mixed with 250 litres of ethyl acetate and adjusted to pH 2.6 with 114 litres of 42.5 % ortho-phosphoric acid. The 2-(R)-hydroxy-4-phenylbutyric acid is extracted into ' ,:
~, .

2~)Q37G7 _ 19 _ ethyl acetate in a yield of 94 %. The 2-(R)-hydroxy-4-phenylbutyric acid that has remained in the aqueous phase is further extracted with two 120 litres portions of ethyl acetate. After the three extraction steps, the extraction of the 2-(R)-hydroxy-4-phenylbutyric acid is complete (99.8 %). After combining the three ethyl acetate phases (total volume 450 litres), addition of 250 litres of ethyl acetate and removal of 470 litres of the solvent by distillation, the solution is filtered clear through a one-plate filter. 50 litres of ethyl acetate are used for rinsing the filter and are added to the clear solution. After removing 200 litres of the solvent by distillation, the final volume of the solu-tion is oO litres. 400 litres of cyclohexane are added to that solution and 200 litres of the solvent are removed by distillation. Centrifuging of the product, which has crystallised, and drying in vacuo at 20 to 25C
until constant weight is obtained yields 23.4 kg of crystalline 2-(R)-hydroxy-4-phenylbutyric acid.

In order to verify the enantiomeric purity, a sample of the crystallineacid is dissolved in absolute ethanol and reacted with hydrogen chloride gas for 24 hours at room temperature. After removing the alcohol by distillation and briefly degassing under a high vacuum, a light-yellow oil remains which is analysed by HPLC at 25C/32 bar over a chiral column (250 x 4.6 mm internal diameter, flow rate 1 ml/min, stationary phase Chiralcel OD [Stehelin, Basle] type OD-5-15-20925, mobile phase: 90 %
hexane - 10 % isopropanol - 0.1 % diethylamine). The substances to be analysed are present in the solvents in a concentration of 1 mg/ml (quantity injected 10 ~l). Scanning is carried out at a wavelength of 210 nm, and evaluation by surface area comparison with an external standard. The ee value found is > 99.8 %.

Claims

1. A spherical biocatalyst of high mechanical strength, comprising enzymatically active material, a cationic polyelectrolyte and polyvalent anions, wherein the biocatalyst contains silicic acid.

2. A spherical biocatalyst according to claim 1, wherein the enzymatical-ly active material consists of whole cells, cell fragments or cell organelles of a microorganism, or of enzymes or of any combination of these types of enzymatically active material.

3. A spherical biocatalyst according to claim 1, wherein the enzymatical-ly active material consists of whole cells of a bacterium or yeast.

4. A spherical biocatalyst according to claim 3, wherein the bacterium belongs to the genus Proteus.

5. A spherical biocatalyst according to claim 4, wherein the bacterium belongs to the species Proteus vulgaris and/or the species Proteus mirabilis.

6. A spherical biocatalyst according to claim 1, wherein the enzymatical-ly active material contains an oxido-reductase or is an oxido-reductase.

7. A spherical biocatalyst according to claim 1, wherein the cationic polyelectrolyte is chitosan.

8. A spherical biocatalyst according to claim 7, wherein the chitosan has a molecular weight of from approximately 50,000 to approximately 3,000,000.

9. A spherical biocatalyst according to claim 1, wherein the polyvalent anions are polyvalent inorganic anions.

10. A spherical biocatalyst according to claim 9, wherein the inorganic anions are orthophosphate, metaphosphates, pyrophosphates, polyphosphates or cyanoferrates.

11. A spherical biocatalyst according to claim 10, wherein the in-organic anions are tripolyphosphate.

12. A spherical biocatalyst according to claim 1, wherein the polyvalent anions are polyvalent organic anions.

13. A spherical biocatalyst according to claim 12, wherein the organic anions are polymeric organic carboxylates, sulfonates or hydroxy compounds.

14. A spherical biocatalyst according to claim 1, wherein the silicic acid is a precipitated silicic acid.

15. A spherical biocatalyst according to claim 14, wherein the pre-cipitated silicic acid has an average particle size in the range of from 5 to 50 µm and an average bulk density in the range of from 5 to 100 g/100 ml.

16. A process for the manufacture of spherical biocatalysts according to claim 1, which comprises mixing enzymatically active material with precipitated silicic acid in solid form, an aqueous buffer solution and an aqueous solution of a cationic polyelectrolyte to form a suspension, while avoiding the formation of foam, introducing the suspension dropwise into an aqueous crosslinker bath containing polyvalent anions and shrinking and solidifying the resulting biocatalyst beads in the cross-linker bath, and optionally separating them from the crosslinker bath.

17. A process according to claim 16, which comprises avoiding the forma-tion of foam by carrying out the mixing process in the presence of silicic acid.

18. A process according to claim 16, wherein chitosan in aqueous solution having a viscosity in the range of from 1,000 to 20,000 cP is used as cationic polyelectrolyte.

19. A process according to claim 16, wherein the ratio by weight of silicic acid to cationic polyelectrolyte is in a range of from 1.5 to 50 : 1.

20. A process according to claim 16, wherein the final concentration in the suspension consisting of enzymatically active material, silicic acid, buffer solution and cationic polyelectrolyte is in a range of from 2 to 15 % (w/w) for the silicic acid and in a range of from 0.3 to 1.5 % (w/w) for the cationic electrolyte.

21. A process according to claim 16, wherein the polyvalent anion in the crosslinker bath is tripolyphosphate.

22. A process according to claim 21, wherein tripolyphosphoric acid or a salt thereof is used for the crosslinker bath in a concentration in the range of from 0.5 to 10 % (w/v).

23. A process according to claim 21, wherein the volume of the cross-linker bath is at least about three times the volume of the suspension of enzymatically active material, silicic acid, buffer solution and cationic polyelectrolyte.

24. Spherical biocatalysts of high mechanical strength manufactured by a process according to claim 16.

25. A process for the conversion of organic substances, wherein a spherical biocatalyst according to claim 1 is used for the conversion.

26. A process for the manufacture of .alpha.-hydroxycarboxylic acids from .alpha.-ketocarboxylic acids, wherein a spherical biocatalyst according to claim 4 is used for the conversion.

27. A process according to claim 26 for the manufacture of 2-hydroxy-4-phenylbutyric acid from 2-oxo-4-phenylbutyric acid.

28. A process according to claim 26 for the manufacture of 2-(R)-hydroxy-4-phenylbutyric acid from 2-oxo-4-phenylbutyric acid, wherein the spherical biocatalysts are employed in a fixed-bed reactor through which there is continuously passed an aqueous solution of the substrate 2-oxo-4-phenylbutyric acid, formate and an electron mediator.

FO 7.4/AW/kg*