CA1143680A - Method for producing bio-catalysts - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
The invention refers to a process for the manufacture of mechanically and chemically stable, porous biocatalysts with high enzymatic activity by including whole cells, cell fragments, or enzymes in polymers, whereby mixture (A) is in-serted into mixture (B) as a precipitation bath. Mixture (A) is composed of an enzymatically active substance in an aqueous solution or suspension and a water-soluble polyelectrolyte, or of an enzymatically active substance in an aqueous solution or suspension of an epoxyprepolymer component, a multifunctional harden-ing component and a water-soluble polyelectrolyte, and mixture (B) is composed of the aqueous solution of a compound containing polyvalent ions charged oppositely to the polyelectrolytes. When stirred, the bead-like particles that have formed are solidified, washed, and following filtering off are carefully dried to contraction and solidification, and hardened, washed and discharged in the moist condition in mixture (B), or washed and discharged in the moist condition follow-ing solidification of the epoxy matrix in a phosphate buffer solution for dis-solving out the polyelectrolytes.

Description

Biocatalysts are becoming increasingly important for the direct recovery of primary and secondary metabolic products. The following are mentioned as examples of practices for which technological interest exists:
- obtaining fructose from glucose by means of glucose isomerase;
- producing 6-APS from pencillin G by means of penicillin-acylase;
- producing L-asparaginic acid from ammonium fumarate by means of E. coli;
- producing L-malic acid from fumarase with brevibacterium ammonia-genic cells.
In technical microbiology and microbial engineering, the term biocatalysts is understood to mean a biological system, fixed by a macroscopic carrier, con-sisting of whole-cell micro-organisms or enzymes.
Preference was originally given to the use of fixed micro-organisms as biocatalysts for reasons of production costs and flexibility, especially from the point of view of multiple enzyme reaction.
Biocatalysts are produced mainly by physically enclosing the micro-organism in a poly~er matrix.
Depending upon the method used, these matrices were produced from the following substances:
` polyacrylamide/polymethylacrylamide/collagen/cellulose triacetate/
20 carboxy ethyl cellulose/agar/co- poly-(maleic-acid sty ol~/carrageenan.

~ . !

" .
' According to the state of the art, known methods for producing biocatalysts with these substances present teehnieal production problems, and biocatalysts produced by such methods have disadvantageous properties.
In the alginate/CNC/eopoly-eatalyst group whieh is produeed in a simple manner by forming gels with polyvalent eations, there exists the partieular disadvantage of inresistanee to the phosphate buffer~solutions in whieh many of the reactions are earried out. If a natural eleetrolyte is used, mierobial attaek and, assoeiated therewith, destruction of the matrix may occur.
In addition to this, because of its not very high load-earrying capacity, this type of catalyst has the dis-advantage that it can scarcely be used in a fixed-bed reaetor.
The produetion of the eellulose-triaeetate eatalys-t by a wet-spinning proeess, using highly toxic solvents such as toluene or methylene chloride, is a fixation method which can be used only for a few micro-organisms. This fibre-form assumes one-sided use of the eatalyst in fixed-bed reaetors.
The produetion of eollagen eatalysts is extremely eostly. The diaphragm shape restricts its use to the spiral reaetor. It is impossible to dispense with the toxie step of hardening with glutarie dialdehyde.
~olyacrylamide catalysts, as products of block-polymerization, are described as sharp-edged, irregular granules.
When used in agitator-reactors, these granules show high abrasion and loading with miero-organisms is restricted. This is con-'' .

, ' 3~30 firmed in the following publications;
DE-OS 2 252 815: 4,8 g E. coli = 120 ml ca-talyst/4 Vol. %
DE-OS 2 420 102: 17 g cells = 170 ml catalyst/10 Vol. ~
DE-OS 2 414 128: 12 g cells = 120 ml catalyst/10 Vol. %
In spite of intensive research work, however, only a few methods have achieved practical application, apparently because of the following disadvantages:
- the mechanical stability of many carriers is too low for their use in large reactors;
- fixation methods are too costly, and the use of such biocatalysts is therefore uneconomical;
- achievable loadings are too low, and the space-~ime yie7d i~ ~g ~sa~is~ac~o~y.
one of the best~known methods uses physical encl(~u~e in a polymer network. Examples are: polyacrylamid/polymetha-crylamide, collagen, cellulose triacetate, ionotropic gels.
These fixation-methods are used, since this relatively slight inactivation of enzymes produces cell-fragments, whole cells.
It is known that this enclosure in ionotropic gels represents a method which, in contrast to polyacrylamide or polymethylacrylamide gels, makes it possible to operate with practically non-toxic substances, e.g. alginates, Ca2+ . .
In Biotech. and ~ioeng. l9 (1977) 387, M. Kierstein and C. Bucke discuss the enclosure of enzymes, cell-fragments and whole cells in Ca- alginate gels. These authors propose to produce fibres by injecting an Na- alginate solution into a Ca precipitation bath.
According to these authors, however, these Eibres have only moderate stability. Another disadvantage is that such fibres can be used as biocatalysts only in fixed bed reactors.
In his dissertation "Tech. University Braunschweig 1976 - Polymer inclusion of micro-organisms - formation an reactivity of biocatalysts", U. Hackel proposes to produce spherical ionotropic gels for biocatalysts by injecting the polyelectrolyte solution into a cross-linking bath. This author also mentions the use of different types of poly-electrolytes, including fully synthetic products such as styrene/maleic acid copolymer.
However, none of these proposals has produced bio-catalysts exhibiting high strength and the ahility to be heavily loaded with moist biomass.
Increasing the strength by increasing the polymer content in the hiocatalysts is unsuccessful since the highly viscous solutions cannot be processed.
It is the purpose of the invention to discover a method for producing biocatalysts which exhibit high mechanical strength and the ability to be heavily loaded with an enzymatic sub-stance, and which, to this end, are highly porous.
It is also the purpose of the invention to discover o biocatalysts whlch have high compressive strength (P/bead) and heavy loading (g enzymatically active substance/g catalyst beads) and which, after careful drying, exhibit a relative contraction of between ~/5ths and 1/5th.
According to the invention, such catalysts are preferably made by the method according to the invention. The said catalysts are not, however, bound to the method of the invention. Now that biocatalysts of this kind are known to the world of experts, other methods may be used in producing them.
Thus, this invention provides a method for producing biocatalysts having high mechanical strength and heavily loaded with enzymatically active substances by including whole cells, cell-fragments, or enzymes in polymers , in the form of catalyst beads having a high compressive strength (P/beads) of above 800 to 1000 and a loading of up to 0.9 g of enzymatic substance per g of catalytic beads, characterized in that mixtures produced by:
(A) suspending or dissolving an enzymatically active substance (Al) in an aqueous solution of polyelectrolytes in a concentration range between 0.5 and 15% by weight;
(B) as a precipitation bath, by dissolving a compound containing poly-valent ions charged oppositely to the polyelectrolytes in the mixture ~A), in a concentration of the compound between 0.5 and 35% by weight in water; there-after, in stage 1, the mixture (A) being converted by injection into the mixture (B), into bead-like particles of about 0.5 to 5 mm, the said particles, when stirred, adhering to their surfaces wi~hin a period of about 5 mins. to 4 h; thereafter, in stage 2, the catalyst beads thus formed being filtered out, with the enzymatic substances, being washed with a physiological salt solution and, in stage 3, being subjected to careful drying by passing thereover air at a temperature of up to 80C for up to about 48 h, so that the said catalyst beads are subject to contraction and solidification;

,:.

~3 t;~3~

thereafter, in stage 4, tlle said catalyst beads being introduced, for reharden-ing, into the precipitation bath (B) in stage 1, thus solidifying the said beads, by re-cross-linking and expansion, as compared with their original condition;
and thereafter, in stage 5, subjecting the said catalyst beads to a washing process and discharging them in tlle moist condition.
In a second aspect this invention provides catalyst beads having a high compressive strength (P/beads) of above 800 to 1000 and a loading of up to 0.9 grams of enzymatic substance per gram of catalytic beads.
In achieving the purpose of the invention, use is made for the first time of the technical effect of obtaining, by careful drying, catalysts beads having a specific contraction, leading to increased strength and increased loading withenzymatically active substances. In this connection, a surprising effect essential to the invention is that renewed equilibration with precipitation bathB or C produces only limited re-expansion. It is this surprising effect that made it possible to achieve the purpose of the invention. This technical effect was by no means obvious, otherwise it would have long ago been stated by the experts, since there is a considerable need for the biocatalysts obtained by themethod of the invention, and for biocatalysts having the specific properties of those thus obtained.
The method according to the invention, and the biocatalysts thereby obtain-ed, are defined in the claims.
The sub-claims relate to the preferred configuration of the method accord-ing to the invention.

- 5 a -, J', -3~B~3 The technical effect of the method of the invention may be obtained by the following experiment:
gel-beads are produced in known fashion by cross-linking an 8% alginate solution with a 2% CaC12 solution. Careful air-drying at room-temperature for a period of 5 h causes a particle to contract from 3.5 to 1.5 mm is diameter, i.e. to about 2/5ths of the original diameter.
According to the surprising technical effect of the method, renewed equilibration with a 2% CaC12 solution produces re-expansion only to a diameter of 2 mm. No further expansion was observed after technological use for 6 weeks at 65C in a 30% glucose solution.
The relationship between particle-contraction and the resulting proportion of polymer-solid in the biocatalyst is shown in the figure which shows the relationship between the diameter in mm of the biocatalyst beads, and the solid content in %, as a function of the drying time in h.
According to this example, the diameter of the biocatalyst beads decreases rapidly during the first two hours of drying, after which the curve flattens up to 5 h, during which the diameter decreases more slowly. After 1 and 4 hours, the increase in solid content shows flat changes in direction, with ~he slope therebetween almost uniform. The increase in solid content may be described as fairly regular.
The method according to the invention is described in the following examples.

Example 1 First of all, mixture A is produced by suspension of 150 g of compressed yeast, extended to 150 ml with water, as an enzymatically active substance, in 450 ml of an 8% by weight Na- alginate solution, as an aqueous solution of a poly-electrolyte.
A 0.2 molecular CaC12 solution is used as a pre-cipitation bath B with a compound containing polyvalent ions charged oppositely to the polyelectrolytes in mixture A.
In stage 1, mixture A is then dripped into mixture B, with stirring, from a 0.4 mm diameter capillary tube. This produces bead-like particles 4 mm in diameter. These are solidified by further stirring over a period of 15 min. There-after, in stage 2, the catalyst beads thus formed are filtered out, with the enclosed enzymatically active substance, and are washed with a physiological NaCl solution. In stage 3, the catalyst beads are dried carefully in a drying cabinet by pass-ing air thereover for 20 h at a temperature of 30C. This step produces contraction and solidification of the catalyst beads.
As compared with the condition prior to drying, the relative contraction amounts to about 2/5ths. Thereafter, in stage 4, the catalyst beads are rehardened in precipitation bath C, consisting of an AL2(SO4)3 solution having a concentration of 0.1 mole/l, for 30 mins.
Thereafter, in stage 5, the catalyst beads are sub-jected to a washing process with a physiological NaCl solution, and are discharged in the moist condition.

~3~

The catalyst beads of this examples show the following values for mechanical strength and loading with enzymatieally active subs-tanee.
Since the volume of mixture A is redueed by the pro-duction process from 600 ml to 170 ml, and since the original 150 g of compressed yeast are contained in this catalyst-volume in the form of a moist biomass, the loading obtained is 0.88 g of eompressed yeast/g of bioeatalyst.
The eompressive-strength test - by the method aceord-ing to the invention - gives a loadability at the rupture point of 750 p/beads.
Example 2 Mixture A is first produeed by suspending 100 g of E. eoli ATCC 11105, in the form of a moist eentrifuged mass extended to 150 ml with water, in 450 ml of an 8% by weight of an aqueous Na- alginate solution as the polyeleetrolyte.
Otherwise, the procedure is as in Example 1.
From the original 600 ml solution A, a total of 165 ml of biocatalyst beads are obtained. This is the equivalent of a loading of 0.6 g of BFM (moist biomass)/ml of biocatalyst.
The compressive strength, aceording to the test described, is 800 p/beads.
Example 3 Mixture A is first produeed by dissolving 100 mg of amyloglueosidase in 10 ml of an 8% by weight Na- alginate solution.
A 0.5 molar CaC12 solution is used as precipitation -~ i~3~0 bath s, with a compound which contains polyvalent ions charyed oppositely to the polyelectrolytes in mixture A.
In stage l, mixture A is then dripped into mixture B, with s-tirring, from a 0.4 mm diameter capillary tube, thus forming bead-like particles ~ mm in diameter. These are solidified by further stirring Eor 15 mins.
Thereafter, in stage 2, the catalyst beads thus formed are filtered out, with the enlosed en~ymatically active sub-stance, and are washed with a physiological NaCl solution.
In stage 3, the catalyst beads are then subjected to careful drying by passing air at a temperature of 30C there-over for 20 h. This step produces contraction and solidi-fic-ation of the catalyst beads. At 1.5 mm, the relative contrac-tion amounts to about 2/5ths.
Thereafter, in stage 4, the catalyst beads are re-hardened in a precipitation bath C consisting of a 0.1 molar A12(SO4)3 solution, for a period of 30 minutes.
Thereafter, in stage 5, the catalyst beads are sub-jected to a washing process with a physiological NaCl solution, and are discharged in the moist condition.
In their final condition, the biocatalyst beads are 1.8 mm in diameter which, according to the quantitive balance, is the equivalent of a loading of 32 mg of enzyme/ml of bio-catalyst.
The compressive-strength test - by the method accord-ing to the invention - gives a strength of 780 p/beads.
As compared with the prior art, the biocatalysts pro-_ g _ ~3~30 duced by the method according to the invention show a substan-tial increase in compressive strength (p/beads) and loading with enzymatic substance (g of enzymatically active substance/g of catalyst beads).
By way of example, the compressive strength of unhardened alginate beads was compared with that of biocatalyst beads produced by the method according to the invention.
The method for determining compressive strength will be described hereinafter.
The mechanical portion of the test arrangement con-sists of a sample table clamped firmly in position, a ram located thereabove and connected rigidly to a power pick-up (Messrs. Hottinger Baldwin Messtechnik, Darmstadt: power pick-up U 1/1 kpond), and a drive unit consisting of a motor and transmission.
The signal from the power pick-up is amplified in a type KWS 3072 measuring amplifier (Messrs. HBM, Darmstadt) and is passed to a recorder.
For the purpose of measuring the loadability (load-carrying capacity) of catalyst spheres, a sphere is placed uponthe sample-table. The ram then applies pressure from above to the sample, at a feed velocity of 1.45 mm/min.
The force acting upon the sphere is recorded, through the ram, force pick-up, and amplifier, as a direct measurement, by the recorder, in the form of a power-time diagram.
This diagram makes it possible to predict the breaking strength, in that the breaking process is clearly marked by a 3 ~ ~368(3 serrated discontinuity in the power-time diagram.
The margin of error in the force-measuremen-t itself is less than 1~. In measuring a plurality of beads in one pro-duction batch, major fluctuations naturally occur, but these do not exceed ~5~.
This procedure simultaneously defines the compressive strength in the dimension "P/beads" as a factor linked to the geometry of the respective beads.
This method indicates the Eorce only in one unit of power, namely in "pond/beads", written hereinafter as "P/beads".
The unit of power "pond" may be converted to the SI
power unit "Newton" as follows:
1000 ponds = 9.806 Newtons ~ 10 Newtons.
This method shows that unhardened Ca- alginate beads according to the prior art, 3.5 mm in diameter, are destroyed by a compressive force of 10 to 150 _. In contrast to this, biocatalyst beads produced by the method of the invention, 2 mm in diameter, are destroyed only by a compressive force of above 600 to 1000 ~ beads.
The biocatalyst beads produced by the method of the invention exhibit, by enrichment of -the biomass associated with a reduction in volume by a factor of 3 to 5, loadings which are unobtainable according to the prior art.
M. Kierstein and C. Bucke (loc. cit.) obtain a value of 0.25 BFM/cm of catalyst. According to Disclosure texts

2,252,815, 2,420,102, 2,414,128, loadings of only 0.04 to 0.1 g BFM/cm3 of catalyst are obtained by fixation in polyacrylamide ~3~80 gels. In contrast to this, the biocatalyst beads produced by the method of the invention show a loading of 0.88 g BFM/g of catalyst beads.
Ilowever, the catalyst beads produced by the method of the invention also e~hibit high porosity, as shown by photographs with the scanning electron mlcroscope (S~M).
Th0 high loading with enzymatic activity ln confirmed, for example, by converslon of penicillin G with immobilized E. coli cells to a relative activity of 61% and an absolute activity of 4 400 U (units)/l Kat (per litre of catalyst).
(Biocatalysts beads 2.5 mm in diameter; loading 0.5 g BFM/cm3; temperature 37C; c5 - 5%; penicillin G Na- salt).
Exam~
30 g of E. Coli cells ATCC 1105 are mixed, in the form of centrifuged-out wet biomass (BFM), as componen* Al, with 10 g of the epoxy resin "Epikote Dx-255* (Deutsche Shell Ag, ~rankfurt), as resin component A2.
20 g of a 25% aqueous solution of "Casamide CA 360"* (Akzo Chemie, DUren), as the polyaminoamide-hardener component B1J are then thoroughly distributed by stirring, which initiates polycondensation oE the epoxy resin.
This (A2)+(Bl)~(Al) system is now thoroughly mixed with 20 ml of an 8% by weight aqueous Na- alginate solution ("Mannucol LD"* by Messrs. Alginate Ind., Hamburg) as solution D and this is injected thereafter, in stage 1, into a 2%
by weight CaC12 solution, as a precipitation bath, from a 0.4 mm diameter capillary, by the application of pressure. This produces bead-like particles of between 3 and 4 mm in diameter. After stirring for 20 minutes, the par-ticles, now stabilized, are removed from the CaC12 solution, and are washed in stage 2. This is followed, in stage 3, by careful drying by passing air there-over at 28C for a period of 24 h~ This causes the beads to contract to about *Trade Mark c j - 12 -' ':7' J

3t~

2/5ths of their original diameter. The dried beads, with components A2 and B
hardened out, are washed, in stage 4, for 40 mins. in a 0.1 molar phosphate buffer-solution, with stirring, the alginate being dissolved as the Poly-electrolyte component D from the spheres and the beads expanding back to a diameter of 3 mm. The porous biocatalyst thus obtained exhibits a high loading in cell masses combined with good mechanical stability, as confirmed by the following data:

Type of E. Coli LoadingRelative Absolute Catalyst LoadingVol % Activity Activity g BFM per % ~ cat/l of g Catalyst Catalyst . . .

Irregular --partciles from block-condensation 100 ~m 1.24 70 40 80

3-4 mm 1.24 70 11 16.4 Spherical beads according to the method of the invention ~ 3 mm 1.13 67 28 38 . .
tActivity of the fixated E. coli cells as compared with the activity of the same amount of cells in free suspension.
At a relative activity of 28% ~fixated cells/similar number of free cells), the absolute activity (pencillin G-acylase~ 37C, pH = 7.8) is 38 ~ cat/l catalyst.

O

Compressive strength, by the method of the invention, amounts to 651 p/beads.
Example 5 35 g of compressed yeas-t, as component Al, are stirred into 10 g of epoxy-resin ~Epikote Dx-255 - Deutsche Shell AG, Frankfurt) as the resin component A2.
In this, 20 g of a 25~ by weight aqueous solution of "Casamide CA 360"
~Akzo Chemie, DUren), as the polyaminoamide-hardener component Bl, are well distributed by stirring, which -.3~

initiates polycondensation of the epoxy-resin.
This (A2)+(sl)+(Al) system is now thoroughly mixed with 35 ml of an 8% by weight Na alginate solution ("Mannucol LD" by Messrs. Algina-te Ind., Hamburg) as solution D, and this is injected into a 1% by weight CaC12 solution, as component E, from a 0.4 mm capillary, by the application of pressure. This produces bead-shaped particles between three and our millimetres in diameter.
Otherwise, the procedure is as in Example 1.
The final diameter of the biocatalyst beads is 3.
The loading amounts to 1.18 g of moist biomass per g of bio-catalyst; the absolute activity, measured by the breakdown of glucose into ethanol, amounts to 0.18 g of glucose/ml of catalyst . h.
Compressive strength, according to the method of the invention, amounts to 650 p/beads biocatalyst.
"Measuring the loading" of catalyst beads in the biocatalyst according to the invention produces the expression "compressive strength" in p/beads as a factor linked with the geometry of individual beads. Since the catalyst beads are nearly all of the same shape, this provides a definite numerical value for "compressive strength".
According to the method of the invention, the material build-up of the polymer matrix of a blocatalyst is effected by polycondensation of a multi-functional, water-emulsifiable, epoxy-prepolymer component A2 with a multi-functional hardener component Bl such as polyaminoamide. The technical advantage 3~

of this general type of reaction is that the polymer build-up takes place with suitably selected reaction-components, at room temperature, without the formation of by-products. The polymer network produced according to the method of the invention has high mechanical and chemical stability. Treatment with a salt-solution, and extreme pH values in the acid and alkaline range, have no effect upon the material network properties.
The epoxy prepolymers used, according to the method of the invention, in an aqueous medium, with all detrimental solvents excluded, lead to practically non-toxic biocatalyst beads.
Direct introduction of the moist biomass Al into the viscous epoxy-resin component A2 causes the cells to be coated with component A2, the non-toxic resin also performing the function of a protective colloid during the fixation.
The special suitability of epoxy for enclosing whole cells, or cell fragments, already appears in the simple block-condensation of A2 + Al with B. The high proportion of biomass Al, in relation to the amount of resin- and hardening-components Al and A2, ensures, as confirmed by tests with the scanning electron microscope, porosity by block-polymerization of a synthesized epoxy-polymer networkO
Contraction of the carrier material of substances A2 and Bl, used according to the method of the invention, appearing during block polymerization with the discharge of water, leads to high loadings of biomass Al with high enzymatic activity. With this method, however, and the approximately 3 to 4 mm grain-size required for the technical processes, B~

the enzymatic activity achieved is only about l/5th of the original activity. This result is attributable to diffusion-inhibition. As a result of the high loading with biomass Al, the substrate molecules cannot reach all of the cells.
This fact is confirmed by the following investigation.
If a block of epoxy containing cells is broken down, in a polymer mill, into sharp-edged, irregularly shaped catalyst-particles of between 50 and 100 microns, these particles have high enzymatic activity, indicating a relatively mild kind of fixation. This also confirms the scanning electron microscope (SEM) results relating to porosity of the material~
As the grain-size of such ground catalyst particles increases, however, there is a reduction in activity. Thus products of this kind cannot be used in the technology.
In contrast to this, the catalyst beads produced according to the method of the invention display high enzymatic activity, even in the technically necessary grain-sizes.
The long period of up to about 30 h for drying and hardening out the catalyst beads, preferably at room temperature, according to the method of the invention ~ which may also be called the long "pot time" of the initial materials - offers the advantage of time reserves so desirable in technical fixation processes. However, the method according to the prior art has the disadvantage of requiring large amounts of grinding energy.
In addition to this, there is considerable abrasion of the irregular, dry, brittle particles, as well as inhibition of the reaction by limitation of diffusion. These disadvantages are - .

3~

overcome by the method of the invention and the catalyst beads according to the invention.
The method of the invention produces, by polycon-densation of A2 and Bl and ionotropic gel-formation of D and Al, a bead-shaped, porous biocatalyst which, as compared with biocatalysts according to prior art, has high mechanical and chemical stability and high loading of enzymatic biomass, has high enzymatic activity in the technically important grain size of about 2 to 5, and which, because of its shape and strength, may be used in known reactor systems.
A preferred example of embodiment consists in mixing a polyelectrolyte D, such as Na- alginate, with the epoxy-hardener biomass A2 + Bl + Al by periodic injection into a precipitation bath consisting of an aqueous CaC12 solution, which permits direct ionic cross-linking of the alginate with the surface of the drops, and thus the forming of the bead-shaped catalyst.
Polycondensation of the resin-hardener system A2 + B
within the Ca- alginate sheath begins to take place similtan-eously with this. After a few minutes, the drops havesolidified into beads to such an extent that the latter may be separated and washed and then subjec-ted to careful drying and hardening-out by passing air thereover, which takes about 20 h at room temperature. Thereafter, the alginate uniformly dis-tributed in the catalyst beads is washed out with a phosphate buffer-solution. This procedure brings about the formation of continuous capillary passages, thus producing the porous, 3~

bead-shaped biocatalyst from epoxy A2 + Bl and biomass A1.
The Forosity achieved by inclusion of the alginate in stage 3 and the dissolution in stage 6, optically recognizable with the SEM, is revealed by expansion of the catalyst beads, when the alginate is washed out, by 30% as compared with the beads containing dried alginate.
This considerable advantage of the catalyst beads according to the invention, and produced according to the method of the invention, is the result of substantially higher enzymatic activity as compared with catalysts in the same grain-size range produced in accordance with the prior art.
The table overleaf shows this technical advantage of the activity of the epoxy biocatalyst according to the invention, as compared with catalysts produced according to the prior art, for penicillin G-acylase activity by means of fixated E.
coli.
The structural change in the epoxy network, produced by poly-condensation of A + B in a polyelectrolyte matrix D + E, converts the macroscopic behaviour of the substance of the carrier matrix from brittle, in the case of block-condensation particles, to resilient, in the case of catalyst beads according to the invention. This technical advantage is of considerable significance in the universal application of the catalyst beads according to the invention.
The following table shows the technical superiority of the catalyst beads according to the invention, as compared with those according to the prior art, in the matter of com-3ti~0 pressive s-trength expressecl in p/beads.

Carrier System Compressive (1) to (4) see strength foo-tnote pond/beads Polyacrylamide (1) less -than 10 Polymethacrylamide (2) 30 to 80 Copoly-maleic acid- -styrene (3) 200 to 400 Epoxy block (4) more than 1000 Epoxy beads accord-ing to the invention 650 to 895 (1) P. Schara, Disertation Techn. University sraunschweig 1977.
(2) as (1).
(3) U. Hackel, Disertation Techn. Unlversity Braunschweig 1976.

(4) Unpublished investigation with epoxy block according to the prior art.

There is no abrasion of the catalyst beads according to the invention, even under extreme conditions in batch-- operated agitator-kettle operation.
The following table gives by way of comparison com-pressive strength in p/beads by the measuring method used for commercially available ion-exchange resins.
Product: "Lewatit"*, a commercial product by Bayer AG, Leverkusen.
Material: styrene-divinyl benzene (DVB). The DVB
content indicates the degree of cross-linkage.

* Trade Mark ,~, .,~

o Macroporous Type Designation DVB % Compressive Strength Lewatit SPC 108/1l 8 380 Lewatit SP 112 12 710 In gel form I.ewatit SC 104/~1 4 50 . .
This comparison confirms the high compressive strength of the catalyst beads according to the invention.
A description will now be given of the method used to determine the compressive strength, expressed in p/beads.
The enzymatic long-term stability of a biocatalyst is an important requirement for its technical use under economic conditions.
A bead-shaped biocatalyst produced according to the method of the invention~
with E. coli cells fixed in epoxy beads, and stored at 9C in a 0.9% NaCl solution, still retains, after 120 days, about 21% of its original activity.
Free cells stored under the same conditions are unusable after 3 to 4 days.
A reaction-kinetics long-term test was carried out in a fluidized-bed reactor having the characteristics of an agitator kettle~ using freshly produced catalyst beads according to the method of the invention. A series of "batch-reaction-runs" was carried out. One litre of a 0.5% penicillin-G
solution was circulated continuously through the fluidized-bed reactor contain-ing the catalyst beads carrying E. coli -.

~3i~30 cells as the enzymatically active substance. Temperature 37C;
pH value 7.~. The reaction solution was completely replaced every 24 hours with a fresh 0.5~ penicillin-G solution.
The enzymatic activity of the biocatalyst remained practically constant within 30 days.
The long-term test, carried out by way of comparison with a polyacrylamide biocatalyst with fixed E. coli cells, has a half-value time of 17 days at 40C. Reference is made in this connection to "Continuous production of 6-aminopenicillin-anic acid from penicillin by immobilized microbial cells",Sato, Tosa, Chibata, in European J. Appl. Microbiol., 2, 153 160 (1976).
The special properties of the biocatalyst according to the invention, preferably produced according to the method of the invention, meet the requirements of high mechanical and chemical stability, porosity, and high enzymatic activity at high cell-loading.
The beaded shape of the biocatalyst according to the invention allows it to be used universally in the widest variety of reactor types, with very good space-time yield and long-term stability. This demonstrates the superiori-ty of the biocatalyst according to the invention as compared with those of the prior art. This surprising number of technical advantages is the result of considerable inventive effortO In addition to this, however, is the simple production process of the method of the invention, as compared with the prior art, which has the advantage of being economical. Another factor is ~3~

the inexpensive initial materials used.
The method of the invention also has the advantage of a wide choice of polymer components and cross-linking agents.
This makes possible optimal adaptation of the ionotropic gel to the physiological properties of the biomass.
The said wide variety also applies to the use of whole cells, cell fragments, and enzymes as the biomass. This means that the biocatalyst according to the invention has a long storage life.
For example, the inclusion of amyl glucosidase in Fe-alginate catalyst beads resulted in a residual activity of more than 90% after 6 days.
Another technical and economic advantage oE the method according to the invention, and the biocatalyst according to the invention, is that the cross-linking reaction used to build ionotropic gels is reversible. After being used in technical processes, the polymer components of the biocatalyst may be recovered by dissolving the network.
The accompanying drawings shows the behaviour of the diameter of biocatalyst beads in mm and the solids content in as a function of drying time in h.
The invention refers to a process for the manu-facture of mechanically and chemically stable, porous bio-catalysts with high enzymatic activity by including whole cells, cell fragments, or enzymes in polymers, whereby mixture (A) is inserted into mixture (B) as a precipitation bath. Mixture (A) is composed of an enzymatically active substance in an aqueous solution or suspension and a water-soluble polyelectrolyte, or of an enzymatically ac-tive substance in an aqueous solution or suspension of an epoxyprepolymer component, a multifunctional hardening component and a water-soluble polyelectrolyte, and mixture (B) is composed of the a~ueous solution of a compound containing polyvalent ions charged oppositely to the poly-electrolytes. When stirred, the bead-like particles that have formed are solidified, washed, and following filtering off are carefully dried to contraction and solidiEication, and rehardened, washed and discharged in the moist condition in mixture (B), or washed and discharged in the moist condition following solidi-fiction of the epoxy matrix in a phosphate buffer solution for dissolving out the polyelectrolytes.

Claims (16)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method for producing biocatalysts having high mechanical strength and heavily loaded with enzymatically active substances by including whole cells, cell-fragments, or enzymes in polymers, in the form of catalyst beads having a high compressive strength (P/beads) of above 800 to 1000 and a loading of up to 0.9 g of enzymatic substance per g of catalytic beads, characterized in that mixtures produced by:
(A) suspending or dissolving an enzymatically active substance (Al) in an aqueous solution of polyelectrolytes in a concentration range between 0.5 and 15% by weight;
(B) as a precipitation bath, by dissolving a compound containing poly-valent ions charged oppositely to the polyelectrolytes in the mixture (A), in a concentration of the compound between 0.5 and 35% by weight in water;
thereafter, in stage 1, the mixture (A) being converted by injection into the mixture (B), into bead-like particles of about 0.5 to 5 mm, the said particles, when stirred, adhering to their surfaces within a period of about 5 mins. to 4 h; thereafter, in stage 2, the catalyst beads thus formed being filtered out, with the enzymatic substances, being washed with a physiological salt solution and, in stage 3, being subjected to careful drying by passing thereover air at a temperature of up to 80°C for up to about 48 h, so that the said catalyst beads are subjected to contraction and solidification; thereafter, in stage 4, the said catalyst beads being introduced, for rehardening, into the precipita-tion bath (B) in stage 1, thus solidifying the said beads, by re-cross-linking and expansion, as compared with their original condition; and thereafter, in stage 5, subjecting the said catalyst beads to a washing process and discharg-ing them in the moist condition.

2. A method for producing biocatalysts having high mechanical strength and heavily loaded with enzymatically active substances according to claim 1, characterized in that the mixture (A) is made by suspending an enzyme, with a content of, up to 0.5 g/ml, in an aqueous solution of a polyelectrolyte having a concentration of 0.5 to 15% by weight, the said mixture being injected into the mixture (B), consisting of an aqueous solution of a polyvalent electrolyte in a concentration range of 0.5 to 35% by weight, in stage 1, thus producing bead-like particles in a grain-size range of 0.5 to 5 mm, the said particles being solidified by slow, uniform stirring for between 0.25 to 5 h; thereafter, in stage 2, the catalyst beads thus formed being filtered out with the enclosed enzymatically active substances, being washed with a physiological salt solution and, in stage 3, being subjected to careful drying by passing thereover air at a temperature of up to 80°C; thereafter, in stage 4, the catalyst beads being introduced, for rehardening, into a precipitation bath (B) or (C) consisting of a solution of polyvalent electrolyte in a concentration of 0.5 to 15% by weight; and thereafter, in stage 5, the said beads being subjected to a washing process and being discharged in the moist condition.

3. A method according to claim 1, characterized in that the enzymatically active substance consists of whole cells capable of reproduction, whole cells incapable of reproduction, cell fragments, an isolated enzyme, or an enzyme-complex, and used in the form of a suspension or solution.

4. A method according to claim 1, 2 or 3, characterized in that the poly-electrolyte in the mixture (A) is a natural polyelectrolyte; a natural polymer after chemical modification; or alginate or pectinate in a molecular-weight range of between 20,000 and 200,000 and in a concentration range of 0.5 up to about 15% by weight; or carboxymethyl-cellulose as a natural, chemically modified polymer in a concentration range of 0.5 up to about 15% by weight.

5. A method according to claim 1, 2 or 3, characterized in that in the mixture (B) the ionic compound is a low-molecular-weight salt with polyvalent ions charged oppositely to the polyelectrolyte; or CaCl2, Al2(SO4)3, Fe(NO3)3 or mixtures thereof, using anionic polyelectrolytes in the mixture (A) in a concentration range of 0.05 to 1 mole/l (litre).

6. A method according to claim 1, 2 or 3, characterized in that the mixture (A) is made by suspending whole cells of cell-fragments in an aquous 2 to 10%
by weight Na-alginate solution; thereafter, in stage 1, a 0.05 to 1 molar CaCl2 solution being used as the precipitation bath (B) and, after stages 2 and 3, in stage 4, a 0.05 to 1 molar Al2(SO4)3 solution being used as the precipitation bath (C); or by suspending or dissolving an isolated enzyme or an enzyme-complex in a 2 to 10% by weight Na-alginate solution; thereafter, in stage 1, a 0.05 to 1 molar fe(NO3)3 solution being used as the precipitation bath (B) and, after stages 2 and 3, in stage 4, a 0.05 to 1 molar Fe(NO3)3 solution being used as the precipitation bath (B).

7. A method according to claim 1, 2 or 3, characterized in that careful drying by passing air over at a temperature of up to 80°C for about 48 h produces a relative contraction of 4/5ths to 1/5th as compared with the con-dition prior to drying.

8. A method according to claim 1, characterized in that a moist enzymatically active biomass (A1) is mixed with a resin component (A2) consisting of a pra-tically pure, multifunctional epoxy-prepolymer component in a weight ratio (A2):
(A1) of 0.5:1 to 5:1; thereafter, an aqueous solution of a multifunctional hard-ening component (B1) is mixed with the mass in a weight ratio (B1):(M) - wherein (M) = (A2 + B1) - of 0.2:1 to 0.8:1 and is distributed practically homogeneously, with stirring, in order to produce poly-condensation, this system being mixed with an aqueous solution of a polyelectrolyte (D) in a weight ratio of 1:0.6 to 1:2.8, the mixture being thereafter injected into an excess aqueous solution of a lower-molecular-weight electrolyte with polyvalent ions (E), thus pro-ducing bead-like particles of a specific grain-size of 0.5 to about 5 mm, the said particles being hardened, by stirring for about 5 to 50 mins., into externally solidified particles; being subjected, in stage 2, to a washing process and thereafter, in stage 3, being dried carefully in contact with air at a temperature of up to 80°C for about 30 h; thereafter, in stage 4, being hardened out and, in stage 5, the polyelectrolyte being dissolved out of the bead-like particles by washing with an ionic solution concurrently with (E), thus producing bead-like particles of a specific grain-size of 0.5 to about 5mm, the said beads being hardened by stirring, in about 5 to 50 mins., into externally solidified particles and, in stage 2, being subjected to a washing process; thereafter, in stage 3, being carefully dried in contact with air at a temperature of up to 80°C for up to about 30 h and in stage 4, being hardened out; thereafter, in stage 5, the polyelectrolyte being dissolved out of the bead-like particles by washing with an ionic solution, concurrently with (E), having a concentration of 0.05 to 5 M/l, and being separated and discharged in the moist condition.

9. A method according to claim 8, characterized in that the resin-component (A2) is a low, viscous epoxy-resin emulsifiable in water; or a modified bis-phenol A/epichlorohydrin-epoxy compound; or an epoxy-resin obtainable on the date of the application under the name Epikote DX-255 and having the following characteristics:
- equivalent epoxy weight (EEW) 182 to 212;
- specific weight 1.05 at 20°C.

10. A method according to claim 8, characterized in that the hardening com-ponent (B1) is a viscous polyaminoamide in an approximately 20 to 50% by weight aqueous solution; or a viscous polyaminoamide obtainable on the date of the application under the name Casamide CA 360 and having the following characteristics:
- amino value : 130 to 160 mg of KOH/g;
- viscosity : 300 to 500 poises at 25°C;
- solids content : 50 -/1 1%.

11. A method according to claim 8, characterized in that the polyelectrolyte solution (D) is a natural polyelectrolyte or a chemically modified natural polymer; or the polysaccharide Na-alginate in a concentration of 5 to 10% by weight.

12. A method according to claim 8, characterized in that the lower-molecular-weight electrolyte (E) is a salt having polyvalent ions charged oppositely to the polyelectrolytes (D); or, if anionic polyelectrolytes (D) are used in the mixture (A), a 0.05 to 1 molar CaCl2 solution.

13. A method according to claim 8, characterized in that the polyelectrolyte (D) is mixed with the resin-component (A2), the hardening component (B1) and the biomass (A1), the mixture being introduced, by periodic injection, into the aqueous solution of the lower-molecular-weight electrolyte consisting of an aqueous CaCl2 solution.

14. A method according to claim 8, characterized in that the porosity of the catalyst beads is controlled by altering the concentration ratio (D) :

(A2) + (B) (A1) in stage 3.

15. A method according to claim 8, characterized in that, in stage 1, the muxture (A) or (A2 + A1) is periodically injected into the mixture (B) or (B1).

16. Catalyst beads having a compressive strength (P/beads) of above 800 to 1000 and a loading of up to 0.9 g of enzymatic substance per gram of cat-alytic beads.