JPS5918988B2 - Production method of biocatalyst - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a biocatalyst, and particularly to a method for producing a pearl-like biocatalyst incorporated in a polymer.

Biocatalysts are of great significance for the direct recovery of primary and secondary metabolic products.

Examples of technical interest include:

Recovery of fructose from glucose by glucose isomesase.

6- from penicillin G by penicillin acylase
Manufacture of APS.

Production of L-aspartic acid from ammonium fumarate by E. coli.

Brevibacterium
) Production of L-malic acid from acylase with ammoniagenic cells.

The term "biocatalyst" is visible to the naked eye in industrial microbiology and microbial industry.
) is understood as a biological system consisting of whole-cell microorganisms or enzymes immobilized by a carrier.

In the past, immobilized microorganisms have been frequently used as biocatalysts for reasons of production cost and flexibility in production methods, especially from the viewpoint of the wide variety of enzymatic reactions.

The production of biocatalysts is basically carried out by physically incorporating microorganisms into a polymer matrix.

To produce conventional matrices, the following materials were used according to individual methods:

They are polyacrylamide, polymethacrylamide, collagen, cellulose triacedate, carboxymethylcellulose, agar, maleic acid-styrene copolymer', and carrageen.

From a technical point of view, the conventional method for producing biocatalysts using the above materials has difficulties due to its technical method and drawbacks in the properties of the biocatalyst obtained according to this method.

Alginate-based, CMC-based and copolymer-based catalysts, which are easily obtained by gel formation with polyvalent ions, have the disadvantage, in particular, of not being resistant to the phosphate buffer solutions in which many reactions are actually carried out.

By using natural electrolytes, microbial attack and combined with this, IJ lux destruction begins.

Moreover, catalysts of the above type can hardly be used in fixed bed reactors due to their mediocre mechanical load-bearing properties.

The production of cellulose triacetate catalyst by wet spinning method requires the use of highly toxic solvents such as toluene and methylene chloride, and the immobilization method is meaningful only for a few microorganisms.

Depending on the fiber shape, the catalyst can be partially added to the fixed bed reactor beforehand.

The production of collagen-based catalysts is very wasteful.

＼.

Its addition is limited to spiral reactors by the membrane geometry.

Additionally, the curing process with toxic glutaric aldehydes cannot be avoided.

Polyacrylamide catalysts, like the products of block polymerization, are irregular particles with sharp edges.

When charged into a stirred reactor, they exhibit high frictional properties;
Preparation of microorganisms is limited.

These are proven by the following West German patent publication.

DE-O82252815: 4.8 fE, Collie 1
20ml catalyst/4% by volume DE-O82420102: In' cells-170rI
ll catalyst/10% by volume DE-082414128: 1;l cell=120ml
Catalyst/10% by volume However, despite much research, only a few methods have been put into practical use.

This is due to the following drawbacks.

The mechanical strength of many supports is too low to add them to larger scale reactors.

The immobilization method is too wasteful and the introduction of biocatalysts is not economical.

The loadings obtained are so low that only unfavorable spatial and hourly yields are obtained.

One known method utilizes physical entrapment within a polymer network.

Examples are polyacrylamide, polymethacrylamide, collagen, cellulose triacetate, ionotropic gels.

Using this method of immobilization, relatively little inactivation of enzymes, cell debris, and whole cells occurs.

In contrast to polyacrylamide gels or polymethacrylamide gels, it is known to encapsulate, for example, alginate Ca2+ salts into ionotropic gels treated with substantially non-toxic compounds.

M, Kielstein and C, Bouquet from Biotec
hn, &Bioeng, 19 (1977), 3
87 describes a method for encapsulating enzymes, cell debris, and whole cells in Ca alginate gel.

They propose to produce fibers by injecting a Na-alginate solution into a Ca2+ coagulation bath.

However, this fiber reportedly exhibits only moderate stability.

Another drawback is that the fibers can only be fed into fixed bed reactors as biocatalysts.

IJ, Harakel's paper (T ech, Universitate Braunschwein 19f 6r Polymer Einschürs phono microloganismen zu aufbau land reactivitate phono biocatalysatoren (Polymereinschluss von Mi
kroognanismenzu Aufbau u
ndReaktivitit von Bioka
J) describes the preparation of ionotropic spherical gels as biocatalysts by injecting polyelectrolytes into a crosslinking bath.

It also discusses the utility of various types of polyelectrolytes, including fully synthetic products such as styrene-maleic acid copolymers.

However, these proposals do not yield biocatalysts with sufficient strength and high content of wet biomass (BF"M).

Increasing strength by increasing the polymer content in the biocatalyst has been unsuccessful due to the difficulty of handling high viscosity solutions.

An object of the present invention is to provide a method for producing a biocatalyst that has high mechanical strength, high enzyme substance content, and porosity.

It is also an object of the present invention to produce a biocatalyst having high pressure resistance (P/Pearl) and high content (2 enzyme active substances/Guparl-like catalyst) and exhibiting a relative shrinkage of 415 parts to 115 parts after careful drying. It is to provide.

The biocatalyst according to the invention is preferably produced by the method of the invention.

However, this biocatalyst is not limited to the method of this invention.

As such biocatalysts are known in the art, other methods of production may be developed.

In order to achieve the object of this invention, according to this invention:
Enzyme active substance (A1) was added to an aqueous solution of polyelectrolyte at 0.0%.
Mixture (A) prepared by suspending or dissolving the mixture (A) in a concentration range of 5 to 15% by weight is mixed with 0.5 to 3% of a compound containing polyvalent ions having opposite polarity to the polyelectrolyte in water.
A mixture as a coagulation bath formed by dissolving it at a concentration of 5% by weight (
B) Particle size of about 05 to 5 mm by injection into
(2) filtration of the thus obtained pearl-like catalyst containing an enzyme active substance; Wash with physiological saline solution and (/-)8
Shrink and solidify the pearl catalyst by drying by passing air through it at a temperature of up to 0° C. for a period of up to about 48 hours (→ the pearl catalyst is transferred to the coagulation bath (B) for post-curing). Provided is a method for producing a pearl-like biocatalyst, characterized in that the pearl-like catalyst is charged, re-swelled, solidified by post-crosslinking or re-crosslinking, subjected to a washing step, and taken out in a wet state.

The method of this invention includes the following aspects.

(1) In an aqueous solution of a polymer electrolyte with a concentration of 0.5 to 15% by weight, A mixture ('A) is prepared by suspending the enzyme at a content of up to /TLl, which is mixed in step (a) into a mixture (B) consisting of an aqueous solution of polyelectrolytes with a concentration of 0.5 to 35% by weight. The size of the injection is 0.
．． 5 to 5 mm pearls are obtained, which are solidified by uniformly stirring for 0.25 to 5 hours, and the pearl-shaped catalyst thus obtained is washed with physiological saline solution in step (b), and step ()→ This pearl-shaped catalyst is then dried by passing air through it at a temperature of up to 80°.
Coagulation bath (B) or concentration 0 for post-curing in
．． A method characterized in that the pearl-shaped catalyst is poured into a coagulating solution consisting of a solution of 5 to 5% by weight of a polyelectrolyte, and then subjected to a subsequent washing step and recovered in a wet state.

(2) Enzyme active substance is proliferative whole cells, non-proliferative whole cells,
A method comprising cell fragments, isolated enzymes, or enzyme complexes, which is prepared as a suspension or solution.

(3) As the polyelectrolyte in the mixture (A), a natural polyelectrolyte, a chemically modified natural polymer, or a molecular weight 20
000 to 200,000 alginate or pectate at a concentration of 0.5 to about 15% by weight, or carboxymethyl cellulose as a chemically modified natural polymer at a concentration of 0.5 to about 15% by weight. Method.

(4) As the ionic compound in the mixture (,B), a low molecular weight salt having multivalent ions with opposite polarity to the polymer electrolyte, or CaCl2, A12(SO4)3, Fe(N03)3 or these A method characterized in that the mixture is introduced into the mixture (A) at a concentration of 0.05 to 1 mol/l.

(5) Convert whole cells or cell debris into sodium alginate solution.
A mixture (A) is prepared by suspending it in an aqueous solution of 0.05 to 10% by weight, a 0.05 to 1 molar CaCl2 solution is used as a coagulation bath (B) in step (a), and step (b) and ( /→ After step (→, 0.05 to 1 molar concentration of AI2 is added as coagulation bath (C).
(SO4) using s solution or 2 to 1
A mixture (A) is prepared by suspending or dissolving the isolated enzyme or enzyme complex in a 0% by weight sodium alginate solution, and is added to a coagulation bath (B) in a shield (4).
Fe(No3)3 at a concentration of 0.05 to 1 molar as
Using a solution, after steps (b) and (′),
A method characterized in that a 0.05 to 1 molar Fe (NO3) s solution is used as the coagulation bath (B).

(6) A method characterized by shrinking the pearl-shaped catalyst to 415 to 115 before drying by drying step (/→).

(Wet enzyme active substance mass (A1) is mixed with a resin component (A2) consisting of a substantially pure polyfunctional epoxy prepolymer component, and the weight ratio of (A2) and (A1) is 0.5:
1 to 5:1, and then in step (a) the polyfunctional curing component (B1) is added to the mixture (A2) + (AI
), (B1) and ((A2) + (At)) in a weight ratio of 0.2:1 to 0.8:1 and substantially homogeneous by stirring to cause polycondensation. This system was mixed with an aqueous solution of the polymer electrolyte (D) at 1:0.
6 to 1:2.8 weight ratio, and this mixture is
The low molecular weight electrolyte (E) containing polyvalent ions is injected into an excessively present aqueous solution to form pearls with a size of 0.5 to about 5 mm, and this is stirred for about 5 to 50 minutes to remove the outer layer. Cured to a solidified pearl, subjected to a washing step in step (B), and carefully cured (dry and removed) by contact with air at a temperature of up to 80°C for up to about 30 hours in step (2). In step (e), the polymer electrolyte is eluted from the pearl-like material by washing with an ionic solution competing with the above (E) at a concentration of 0.05 to 5 M/l, and separated and recovered in a wet state. Features.

(8) A method characterized by using an emulsifying low-viscosity epoxy resin or a modified bisphenol A/epichlorohydrin-epoxy compound as the resin component (A2).

(9) A method characterized in that a viscous polyaminoamide is used as the curing component (B1) in the form of an approximately 20 to 50% by weight aqueous solution.

00) A process characterized in that a natural polyelectrolyte or a chemically modified natural polyelectrolyte is used as the polyelectrolyte solution (D), or in that sodium alginate is used in a concentration of 5 to 10% by weight.

aυ As a low molecular electrolyte (E), a high molecular electrolyte (E)
or when using an anionic polyelectrolyte (D) in the mixture (A), a 0.05 to 1 molar concentration of CaCl
The manufacturing method according to any one of claims 8 to 11, characterized in that two solutions are used.

(12) Mix the resin component (A2), curing component (B1), biological mass (A1), and polymer electrolyte (D), and periodically inject this mixture into an aqueous solution of a low molecular electrolyte consisting of an aqueous CaCl2 solution. A method characterized by:

(13) Concentration ratio (D): (A2)+ in process →
A method characterized in that the porosity of the pearl-like catalyst is adjusted by changing (Bl)+(AI).

I In step (a), the mixture (A) or (A2+
A method characterized in that A1) is periodically injected into the mixture (B) or (B1).

The invention takes advantage of the effect that, by careful drying, the pearl-like catalyst undergoes a certain shrinkage, whereby higher strength, higher content of enzyme-active substances are achieved at higher porosity.

It is a surprising effect that no limited reswelling occurs due to new equilibration with the coagulation baths (B) or (C).

This effect leads to solving the problem/objective of this invention.

Such an effect has not been known in the past, and this is also the usefulness of the present invention.

The biocatalyst of this invention is made by encapsulating an enzyme active substance selected from whole cells, cell fragments, or enzymes in a pearl shape in a polymer, and has a pressure resistance (P/pearl) of 800 to 1000 and a pressure resistance of 0.9. It has an enzyme active substance content (enzyme active substance/gupal-like catalyst) of up to .

The method of this invention produces such a biocatalyst.

The effectiveness of the method of this invention is clear from the following experiment.

Gel pearls were produced by crosslinking an 8% alginate solution with a 2% CaCl2 solution in a conventional manner.

Careful drying consisting of passing air through for 5 hours at room temperature caused the pearl to shrink in diameter from 3.5 mm to 1.511L11L.

This corresponds to approximately 215 mm of the initial diameter.

2 m diameter by fresh equilibration with 2% CaCl2 solution.
A slight re-expansion to rn occurred.

Even when this was left in a 30% glucose solution at 65° C. for 6 weeks, no further swelling occurred.

The relationship between catalyst pearl shrinkage and the resulting polymer solids content in the biocatalyst is shown in the accompanying figure.

This figure graphically shows the diameter (w) and solids content (%) of biocatalyst pearls in relation to drying time.

According to this example, the diameter of the biocatalyst pearl decreases sharply when drying up to 21i, and then gradually decreases along a flat curve for up to 5 hours (solid line).

On the other hand, the increase in solids content shows a turning point after 1 and 4 hours, during which time it progresses quickly (progresses) with a uniform slope.
(dotted line), the increase in solids content can be considered fairly regular.

This invention will be described in detail below based on examples.

Example 1 First, 150g of compressed yeast was made up to 150ml with water (enzyme active substance), and an 8% by weight solution of Na alginate (
A mixture (A) was prepared by suspending it in 45 omi (aqueous solution of polyelectrolyte).

A 0.2 molar CaCl2 solution was used as the coagulation bath (B) containing a compound having multivalent ions of opposite polarity to the polyelectrolyte of the mixture (A).

Next, in step (a), the mixture (A) is transferred from a capillary having a diameter of 0.4 mru to a coagulation bath (B) under stirring.
dripped inside.

In this way, pearls with a diameter of 4 mw were obtained.

These were allowed to solidify by leaving under stirring for 15 minutes.

Then, in step (b), the obtained pearl-like catalyst (
(containing enzyme active substances) was filtered and washed with physiological NaCl solution.

The catalyst pearls were then dried in a drying chamber by careful drying with air at 30° C. for 20 hours.

These treatments caused shrinkage and solidification of the pearl-like catalyst.

The shrinkage rate for this state before drying was about 215.

Next, in the step (2), A1 with a concentration of 0.1 mol/l
2 (SO4) in a coagulation bath (C) consisting of a solution of 30
Post-curing of the pearl-shaped catalyst was performed by soaking for a minute.

Then, in step ((e)), the pearl-like catalyst is exposed to physiological N
It was subjected to a washing step with aC1 solution and collected in a wet state.

The pearl-shaped catalyst of this example exhibited the following high mechanical strength and enzyme active substance content.

Since the volume of mixture (A) was reduced from 600 ml to 170 ml during the manufacturing process, and this catalyst volume includes the initially added 1,507 ml of compressed yeast as a wet biological mass, 0. 88 f compressed yeast/g biocatalyst content.

According to the method of this invention, the pressure resistance was 750 p/pearl at failure load.

Example 2 First, E, Co IJ (ATCC111
05) 8 wt% aqueous solution of Na alginate (polymer electrolyte) with 100f adjusted to 1507711 with water 450
Mixture (A) was prepared by suspending in m1.

The rest was treated in the same manner as in Example 1.

A total of 165 TLl of pearl-shaped biocatalyst was obtained from the mixture (A).

Moreover, the content was 0.6 PBFw-encapsulated biocatalyst.

The pressure resistance according to the method described above was 800 p/pearl.

Example 3 First, a mixture (A) was prepared by suspending 100 mg of amyloglucosidase in 10 ml of an 8% aqueous solution of alginic acid.

As the coagulation bath (B), a 0.5-E, Roux-CaC1z solution was used.

In step (a), the mixture (A) was dropped into the coagulation bath (B) under stirring from a capillary with a diameter of 0.4 mrn, and pearls with a diameter of 4 mrn were produced.

These were further allowed to solidify by standing under stirring for 15 minutes.

Then, in step (b), the obtained pearl-like catalyst (containing the enzyme active substance) was filtered and washed with physiological NaCl solution.

Next, the pearl-shaped catalyst was heated to 30°C and air was blown into it at step 0 →.
It was dried by standing for 0 hours.

This treatment caused shrinkage and solidification of the pearl-like catalyst.

■, The relative shrinkage rate was about 215 at a diameter of 5 mm.

Then, in the step), 0.1 mol A12 (SO4)
The pearl-shaped catalyst was post-cured by immersing it in a coagulation bath (C) consisting of three solutions for 30 minutes.

Next, in step (e), the pearl-shaped catalyst was subjected to a washing step with a physiological NaCl solution and recovered in a wet state.

This pearl-shaped catalyst had a diameter of 1.8 mm in the final state, from which a content of 32 to 32 enzyme/ml biocatalyst was obtained by the ratio of mass balance.

According to the pressure resistance according to the invention, a strength of 780 p/pearl was obtained.

The biocatalyst produced according to the method of the present invention has significantly increased pressure resistance (p/pearl) and the content of enzyme active substance (p/pearl) and the content of enzyme active substance (p/pearl-like catalyst) compared to those according to the conventional method.

For example, the pressure resistance of uncured alginate pearls is comparable to biocatalyst pearls according to the present invention.

The measurement method for determining pressure resistance will be described below.

The mechanical components of the test device are a fixed test stand, a stamp placed on it and fixedly connected by a force-forming device (suction device Ul/IK pound manufactured by Hotsteiner-Baldwin Messtechnik), and a drive consisting of a motor and gears. It is a device.

The signal of the above-mentioned force generating device is measured using a measuring amplifier type KWS3072.
(manufactured by HBM) and input into the recorder.

To measure the load carrying capacity of the catalyst particles, each particle is placed on the test stand.

The stamp is then pressed onto the catalyst particles from above at a feed rate of 1.45 mm/min.

The force acting on the catalyst particles passes through the stamp, the force forming device and the amplifier and is recorded as a direct measured quantity by a recorder on a time chart.

The results regarding the fracture resistance are clear from the above diagram since the fracture operation is clearly indicated by the tooth-like discontinuities in the above graph.

The allowable error in force measurement is 1% or less, and although it naturally fluctuates due to measuring a large number of pearls obtained in one batch, it does not exceed ±5%.

By the above measurement method, the pressure resistance of dimension p/pearl is defined as the thickness constrained by the shape of each pearl.

The above measurement method simply expresses force in units of force, more precisely in pounds/pearl.

"p/pearl" refers to this definition.

The force unit "pound" is converted in the SI force unit "newton" as follows.

1000 pounds = 9.806 newtons 10 newtons.

According to the above method, conventional uncured Ca alginate pearls with a diameter of 3-5 mm break at a pressure of 10 to 150 p.

On the other hand, the pearl-shaped biocatalyst obtained by the method of the present invention has a diameter of 2 mw and breaks only at a pressure of 600 to 1000 p/pearl.

The pearl-like biocatalyst produced by the method of the present invention exhibits a previously unattainable content due to the concentration of biological (enzyme) mass associated with a reduction in capacity by a factor of 3-5.

The aforementioned M, Kielstein and C, Bouquet are 0, 2
Achieved 58 F M/i catalyst.

In German Patent Applications 2252815, 2420102 and 2414128, catalyst contents of only 0.04 to 0.11 BFM/ffl are obtained by immobilization in polyacrylamide gels.

On the other hand, the biocatalyst pearl obtained by the method of this invention is 0.88? BFM/? The content of pearly catalyst is shown.

The biocatalyst pearls obtained by the method of this invention further exhibit porosity as observed from Raster Electron Microscopy (REM) photographs.

The high content of enzymatically active substances is demonstrated, for example, by reacting penicillin G with immobilized E. coli cells (pearl-like Biocatalyst: 2.5m in diameter
- Content rate 0.51BFM/crit: Temperature: 37°C; C
8-5%; penicillin GNa salt).

Example 4 E-coli cells in the form of centrifuged fluid BFM (ATCC
111os)3oP (as component (A1)) was mixed with epoxy resin "Epi-Coat DX255" (manufactured by Shell, Germany) 11' (as resin component (A2)).

Next, [Casamide CA360J] was used as the polyamine amide curing agent component (B1).
A 25% aqueous solution 201 (manufactured by Akzo-Heamy Co., Ltd.) was sufficiently dispersed by stirring to perform polycondensation of the epoxy resin.

The system of (A2) + (B1) + (A1) was thoroughly mixed with 20 ml of an 8% by weight aqueous solution of Na alginate (Manukol LD, manufactured by Alpinate India Ltd.) as solution (D), and then this was added to the step ( a) diameter of 0.4 mm
2 wt% Ca as a coagulation bath under pressure from a capillary of
Injected into C12 solution.

Pearls with a diameter of 3 to 4 mvt were thus obtained.

After stirring for 20 minutes, the pearls were recovered as stable particles from the CaCl2 solution and washed in step (b).

Careful drying was then carried out in step ('→) by bubbling air at 28 DEG C. for 24 hours.

This caused the pearl to shrink to about 215 mm of its initial diameter.

The dried pearls hardened with respect to components (A2) and (B1) are washed in step (→) for 40 minutes with stirring in a 0.1 molar phosphate buffer, with the addition of the polyelectrolyte component (D) The alginate was eluted from the particles and the pearls shrunk by 3 mm in diameter.

The porous biocatalyst thus obtained exhibits a high cell mass content and good mechanical stability, as shown in the following table.

Relative activity 28% (fixed cells/same number of free cells) and absolute activity (penicillin G-acylase, 37°C, pH
=7.8) is 38μ catalyst/l catalyst.

The pressure resistance according to the method of this invention was 651 p/pearl.

Example 5 35 g of compressed yeast as component (A1) was mixed with resin component (A2).
) as epoxy resin (Epicoat DX-255) 1
The mixture was stirred and mixed into 0V.

Add "Casamide CA36" as component (B1) to this mixture.
A 25% by weight aqueous solution of 0J was well dispersed by stirring.

The system of (A2) + (Bl) + (AI) is Manukol L
35 ml of an 8% by weight aqueous solution of D
The sample was injected into a 1% by weight CaC2 solution as component (E) through a 2 mm capillary under pressure.

In this way, pearls with a diameter of 3 to 4 mm were obtained.

Thereafter, the same operations as in Example 4 were performed.

The pearl-shaped biocatalyst had a final diameter of 3 mm.

The content was 1.188 F'M/P biocatalyst and the absolute activity measured during the decomposition of glucose to hetanol was 0.18 fglucose/ml catalyst·hr.

The pressure resistance according to this invention was 6501)/pearl.

Since this pearl-shaped biocatalyst exhibits substantially the same shape,
A constant amount of material is obtained for pressure resistance.

According to the second method of the invention, the biocatalyst polymer matrix)
Substantive synthesis of IJ Lux is accomplished by polycondensation of a multifunctional, water-emulsifiable epoxy prepolymer component (A2) with a curing component (B1) such as a polyaminoamide.

The technical advantage of this general reaction type is that the synthesis of the polymer can be carried out at room temperature without the formation of by-products by proper selection of the reaction components.

The polymer networks obtained according to the method of the invention have high mechanical and chemical stability.

For example, treatment with salt solutions and extreme pH values in the acidic and alkaline ranges has no effect on the physical properties of the network.

The epoxy prepolymer used according to the method of this invention provides a pearl-like biocatalyst that is substantially non-toxic in an aqueous medium and upon exclusion of toxic solvents.

Wet biological mass (A1) is mixed with a viscous epoxy resin component (A2)
The cells are coated with component (A2) by direct introduction into the cell, with this non-toxic resin acting as a fixed mortuary protective colloid.

The special suitability of the epoxide for the inclusion of whole cells or cell fragments results from the block condensation of (A2) + (A'l) with (B1).

Due to the high proportion of biomass (A1) in proportion to the added amount of resin and curing component (A2) + (Bl), the epoxy polymer synthesized by block polymerization has a high porosity as confirmed by REM. Having is achieved.

The content of biomass (A1) with a higher enzymatic activity is increased by the shrinkage of the carrier material of the substances (A2) and (B1) used according to the method of the invention during block polymerization in the presence of water. It gets expensive.

Furthermore, approximately 3 to 4 mm, which is necessary for technical methods.
The method of operation using the nucleus yields a modest enzyme activity of about 115 times the initial activity.

This results in proliferation prevention.

Due to the high content of biomass (A1), the substrate molecules cannot obtain all the cells.

These confirmations can be verified by the following tests.

When the epoxy blocks containing cells are crushed into irregular 50-100 micron particles with sharp edges in a polymer mill, these particles exhibit high enzymatic activity.

This is a relatively mild immobilization technique.

This also confirms the REM results for the porosity of the material.

However, as the core size of this type of crushed catalyst particles increases, the activity decreases.

Products of this type are therefore not suitable for use in conventional methods.

In contrast, the pearl-like catalyst obtained by the method of the present invention exhibits high enzyme activity with a technically necessary core size.

Drying preferably at room temperature according to the present invention, the long time required for curing the pearlized catalyst, up to about 30 hours (which means a long pot life for the raw material), is a desirable time for the solidification process. Provides the benefit of retention.

In the conventional method, the consumption of crushing energy is low.

There is also a drawback.

In addition to this, considerable abrasion of irregular, dry, brittle particles occurs, and problems of reaction inhibition due to diffusion restriction also occur.

These disadvantages are overcome by the process of the invention and the pearly catalyst according to the invention.

That is, according to the method of this invention, polycondensation (A2) +
(Bl) and ionotropic gel formation (D) + (A1)
A pearl-like porous biocatalyst is obtained, which exhibits high mechanical and chemical stability and a high content of enzymatically active substances compared to conventional biocatalysts, and is technically not polymerizable. It exhibits high enzymatic activity with a nucleus size of about 2 to 5, and can be incorporated into known reaction systems due to its pearl-like morphology and strength.

A preferred method is to use a polyelectrolyte such as Na alginate (
D) as epoxy-hardener-biomass ((A2)+(Bl
)+(AI)) is periodically injected into a coagulation bath (E) consisting of an aqueous CaCl2 solution, which immediately causes ionic cross-linking of the alginate on the outer surface of the droplet, A pearl-like catalyst is thereby produced.

At this time, polycondensation of the resin-curing agent system (A2)+(Bl) starts simultaneously inside the Ca alginate film.

After a short time, the droplets solidify into pearls, which can be collected, washed, and subjected to careful drying by introducing air.

After a drying process of about 20 hours at room temperature, the alginate uniformly distributed in the pearl-shaped catalyst is washed out with a phosphate buffer.

This procedure creates a penetrating capillary channel, resulting in a porous pearl-like biocatalyst consisting of epoxide (A2) + (Bl) and biomass (A1).

According to REM, the porosity obtained by enclosing alginate in step (a) and elution in step (/→) shows that the swollen catalyst pearls obtained by washing out the alginate are 30% larger than the pearls containing dry alginate. It can be proven optically.

The advantage of the pearl-like catalyst according to the invention is also that it has a significantly higher enzyme activity than conventional pearl-like catalysts of the same size.

(A2) in polyelectrolyte matrix (D) + (E)
Due to the structural change of the epoxy network obtained by carrying out the polycondensation of Bl, the macroscopic material behavior of the support Ma) IJ lux shifts from a brittle behavior in block condensation particles to an elastic behavior in the pearl-like catalyst according to the invention.

This technical advantage is of great significance for the versatile application of catalyst pearls according to the invention.

The following differences show that the pearl-shaped catalyst according to the invention is superior to the conventional one in terms of pressure resistance (p/par).

No destruction of catalyst pearls according to the invention was observed even under extreme conditions in batch-type stirred kettle operation.

The following table shows a comparison of pressure resistance (p/perle) for commercially available ion exchange resins according to a similar measurement method.

"Levateit" is a product of Bayer AG.

Material: Styrene-divinylbenzene (DVB).

The DVB content indicates the degree of crosslinking.

The above comparison proves the high pressure resistance of the pearl-shaped catalyst according to the invention.

The long enzymatic stability of biocatalysts is an important contribution for formulation under industrial conditions.

The pearl-like catalyst obtained according to the present invention with E. coli cells immobilized in epoxide pearls has 0.9% NaC
When stored in a single solution at 9°C, the activity remains about 21% of the initial activity even after 120 days.

Free cells stored under similar conditions are unusable for 3-4 days.

Kinetic long-term tests were carried out in a spiral bed reactor with stirred pot characteristics.

For this purpose, a pearl-shaped catalyst newly produced according to the method of the present invention was used.

A series of batch reaction operations were repeated.

A 0.5% penicillin G solution 11 was continuously pumped through a spiral bed reactor containing E. coli cells as the enzyme active and a pearlized catalyst.

The temperature was 37°C and the pH was 7.8. The reaction solution is 24
It was completely replaced with fresh 0.5% penicillin G solution in an hour.

The enzymatic activity of the biocatalyst remained essentially constant for 30 days.

A comparative test conducted on a polyacrylamide-based biocatalyst having immobilized E. coli cells showed only about half the stability at 40° C. for 17 days.

In addition to this ([Continuous Production of 6-Amizpenicillin Antamide Chromium Penicillin Pi Immobilized Microvial Cells] by Yoshito, Tosa, and Shibata (European Applied Microbiology) See Academic Journal, 2153160 (1976)).

A feature of the biocatalyst according to the invention is that it has higher mechanical and chemical stability and porosity as well as higher enzymatic activity due to higher cell content.

The pearl shape of the biocatalyst according to the invention allows it to be routinely loaded into various reactor types with very good space-time yields and long-term stability.

This proves the superiority of the biocatalyst of the present invention over conventional ones.

Additionally, the invention is economically advantageous because it is easier to implement than the prior art.

This is also due to the fact that cost-effective starting materials are used.

The method of the invention also has the advantage of allowing a wide selection of polymer components and crosslinking agents.

This allows the ionotropic gel to optimally modulate the physiological properties of the biological mass.

The wide range of options means that it is easy to encapsulate whole cells, cell fragments, and enzymes as a biomass.

This increases the pot life of the biocatalyst of the invention.

For example, by incorporating aminoglucosidase into alginate Fe catalyst pearls, the residual activity after 6 days was 90% or more.

Another advantage of this invention is that the crosslinking reaction to produce the ionotropic gel is reversible.

This allows the polymer component of the biocatalyst to be recovered by dissolving it after use.

[Brief explanation of the drawing]

The attached figure is a characteristic diagram of the diameter and solid content of the biocatalyst pearls of the present invention as a function of drying time.

Claims (1)

[Scope of Claims] 1 (a) A mixture (A) obtained by suspending or dissolving the enzyme active substance (A1) in an aqueous solution of a polymer electrolyte at a concentration range of 0.5 to 15% by weight, A compound containing multivalent ions of opposite polarity to the molecular electrolyte was added to water at 0.0%.
By injecting into the mixture (B) as a coagulation bath, particles with a particle size of about 0.5
to 5 mm in size, the pearls are left under stirring for about 5 minutes to 4 hours to solidify the surface, and (b) the thus obtained pearl-shaped catalyst containing the enzyme active substance is filtered. the catalyst beads are washed with a physiological saline solution and dried by passing air through them for up to about 48 hours at a temperature up to 0.980° C. to shrink and solidify the catalyst beads (→ post-cure the catalyst beads). (b) The pearl-shaped catalyst is subjected to a washing step and taken out in a wet state. 2. Producing the mixture (A) by suspending the enzyme at a content of up to 0.5?/ml in an aqueous solution of a polyelectrolyte with a concentration of 0.5 to 15% by weight, In step (a), this is injected into a mixture (B) consisting of an aqueous solution of a polyelectrolyte with a concentration of 0.5 to 35% by weight to obtain pearl-like particles with a size of 0.5 to 5 mm. It is solidified by stirring uniformly for 25 to 5 hours, and the pearl-shaped catalyst thus obtained is washed with physiological saline in step (b) and dried by passing air through it at a temperature of up to 80°C in step (b). In order to post-cure the pearl-shaped catalyst in the step (→), it is put into a coagulation bath (B) or a coagulation bath consisting of a solution of a polyelectrolyte with a concentration of 0.5 to 5% by weight, and then in the subsequent step ((1) The production method according to claim 1, characterized in that the pearl-shaped catalyst is subjected to a washing step and recovered in a wet state. 3. The enzyme active substance is a proliferating whole cell, a non-proliferating whole cell, or a cell. The production method according to claim 1, characterized in that the mixture (A) consists of an isolated enzyme, an isolated enzyme, or an enzyme complex, and is prepared as a suspension or a solution.4. Polyelectrolytes, chemically modified natural polymers, or molecular weight 200
00 to 200,000 alginate or pectate at a concentration of 0.5 to about 15% by weight, or carboxymethyl cellulose as a chemically modified natural polymer at a concentration of 0.5 to about 15% by weight. A manufacturing method according to any one of claims 1 to 3. 5 As the ionic compound in the mixture (B), a low molecular weight salt having multivalent ions of opposite polarity to the polyelectrolyte, or CaCl2, A12 (5O4)3, F
e (NOs) s or a mixture of these to 0
．． 5. The method according to any one of claims 1 to 4, characterized in that it is introduced into the mixture (A) at a concentration of 0.05 to 1 mol/l. 6 Prepare the mixture (A) by suspending whole cells or cell debris in a 2 to 10% by weight aqueous solution of sodium alginate, and add 0 as the coagulation bath (B) in step →.
．． 0.05 to 1 molar Ca C12 solution is used, and after step (b) and (-→, 0.05 to 1 molar Al2(S) is used as a coagulation bath (C) in step (ii).
The mixture (
A) is prepared, and a 0.05 to 1 molar Fe (NO3) s solution is used as the coagulation bath (B) in step (a), and coagulation is carried out in step (b) and (after -→ to step). The manufacturing method according to any one of claims 1 to 5, characterized in that a 0.05 to 1 molar Fe (NOs) s solution is used as the bath ('B). 7. Step (-) The manufacturing method according to any one of claims 1 to 6, characterized in that the pearl-like catalyst is shrunk to 415 to 115 before drying by surface drying. 8. Step (I) ), the manufacturing method according to any one of claims 1 to 1, characterized in that the mixture (A) is periodically injected into the mixture (B).9 (a) In a wet state. The enzyme-active biomass (A1) is mixed with a resin component (A2) consisting of a substantially pure polyfunctional epoxy prepolymer component, and the weight ratio of (A2) and (80) is 0.
5:1 to 5:1, and then the polyfunctional curing component (B1) is mixed with the mixture ((A2)+(AI)) and (
B1) and ((A2+(AI)) are mixed in a weight ratio of 0.2:1 to 0.8:1 and dispersed substantially homogeneously by stirring to cause polycondensation, and the system is Mix with an aqueous solution of a polymer electrolyte (D) at a weight ratio of 1:0.6 to 1:2.8, and add this mixture to an aqueous solution containing an excess of a polyelectrolyte (E) having polyvalent ions. Inject to produce pearls with a size of 0.5 to about 5r/Lr/L, stir for about 5 to 50 minutes to harden into an outer solidified pearl, and (b) Filtered, washed with physiological saline solution (approximately 3
Dry by contact for up to 0 hours)
A pearl-like living body characterized in that the polymer electrolyte is eluted from the pearl-like material by washing it with an ionic solution that competes with the above (E) at a concentration of 0.05 to 5 M/l, and then separated and collected in a wet state. Catalyst manufacturing method. 10. The manufacturing method according to claim 9, characterized in that an emulsifying low-viscosity epoxy resin or a modified bisphenol A/epichlorohydrin-epoxy compound is used as the resin component (A2). 11. The manufacturing method according to claim 9 or 10, characterized in that a viscous polyaminoamide is used as the curing component (B1) in the form of an approximately 20 to 50% by weight aqueous solution. 12 Claims 9 to 12, characterized in that a natural polyelectrolyte or a chemically modified natural polyelectrolyte is used as the polyelectrolyte solution (D), or sodium alginate is used at a concentration of 5 to 10% by weight. The manufacturing method according to any one of Item 11. 13 As a low molecular electrolyte (E), a high molecular electrolyte (D
), or when using an anionic polyelectrolyte (D) in the mixture (A), a 0.05 to 1 molar concentration of Ca
The manufacturing method according to any one of claims 9 to 12, characterized in that a Cl2 solution is used. 14 Mixing the resin component (A2), curing component (B1), biological mass (A1), and polymer electrolyte (D), and periodically injecting this mixture into an aqueous solution of a low molecular electrolyte consisting of an aqueous CaCl2 solution. A manufacturing method according to any one of claims 9 to 13. 15. Claims 9 to 10 are characterized in that the porosity of the pearl-like catalyst is adjusted by changing the concentration ratio (D): (A2)+(Bl)+(AI) in the step (/→) The manufacturing method according to any one of Item 13. 16 In step (a), the mixture ((A2)+(A
16. A manufacturing method according to any one of claims 9 to 15, characterized in that I)) is periodically injected into the mixture (B1).