SK78593A3 - Method of separation of an insoluble catalyst from a reaction mixture - Google Patents

Abstract

The present invention relates to a process for separating, from each other, one non-dissolved catalyst from one or more other non-dissolved components present in a reaction mixture. The separation is obtained by using a catalyst having a density less than the density of the reaction liquid and in that the other non-dissolved components have a density which is above the density of the reaction liquid.

Description

Technical field

The invention relates to a process for separating the undissolved catalyst from one or more undissolved components in the reaction mixture.

BACKGROUND OF THE INVENTION

The separation of the precipitate from the solution is a known method and is carried out, for example, by filtration or centrifugation. If the reaction mixture contains two or more different undissolved components, it is not an easy task to separate such undissolved components from each other. Filtration followed by mechanical separation of the various undissolved components is almost always problematic. In some cases, it may be possible to separate undissolved ingredients by chemical methods after filtration, but this is generally complicated.

There are various fields of chemistry in which two undissolved components contained in a liquid need to be separated from one another. Considering the development of new chemical processes, such development is generally voided if the reaction mixture contains two or more undissolved components that need to be separated from each other. One of the fields where two or more undissolved components need to be separated are reactions using an undissolved catalyst. Such catalysts may be immobilized enzymatic agents.

In reactions where a catalyst is used, the cost of the catalyst is often a significant factor in the overall cost of production, and there is a need to develop processes in which the catalyst can be reused without significant loss of catalytic efficiency. Isolation and reuse of the catalyst are often difficult when the catalyst is contained in the reaction mixture together with another insoluble component that may be formed in the reaction or may be present in the reaction mixture throughout the process.

A specific area that is important in this sense is the synthesis of peptides, for example, the synthesis of Aspartame ™ (TM = trademark). In the preparation of Aspartame (La-aspartyl-L-phenylalanine methyl ester), one of the decisive steps is the enzyme-catalyzed coupling of an aspartic acid derivative and phenylalanine methyl ester hydrochloride (e.g. coupling of N-benzyloxycarbonyl-L-aspartic acid to L-phenylalanine methyl ester L-phenylalanine methyl ester) phenylalanine methyl ester

- referred to herein as ZAPM). N-Benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester (or with any present

D-phenylalanine methyl ester) with very poor solubility and thus precipitates shift the reaction equilibrium towards condensation during synthesis. A semi-pure dissolved enzyme reagent is used to separate the catalyst (e.g., Thermolyser) from the reaction product. The reaction product is dissolved and the enzyme is precipitated by the addition of an organic solvent (e.g. acetone) and the enzyme can be obtained, for example, by filtration. However, 14 to 60% of catalytic activity is lost during this operation (Nonaka et al., U.S. Patent 4,212,945 and Meijer et al., Biocatalysts in Organic Syntheses, Tramper, van der Plas and Linco). , pp. 135-156. A major loss of catalyst activity occurs upon enzyme clotting and isolation.

Another specific field where undissolved components need to be separated is enzymatic or catalytic acylation of the β-lactam nucleus in the preparation of penicillins and cephalosporins. More attention is given to this problem to demonstrate the importance of separating insoluble components. The method of enzymatic preparation of β-lactam derivatives relates to both

6-aminopenicilenic acid (hereinafter referred to as 7-APA), an acid

7-aminodesacetoxycephalosporane (referred to herein as

7-ADCA), 7-aminocephalosporanoic acid (hereinafter referred to as 7-ACA) or 3-chloro-7-aminodesacetoxycephalosporanoic acid, and D-phenylglycine or Dp-hydroxyphenylglycine as a side chain in the catalytic reaction of the corresponding β-lactam nucleus with the D- phenylglycine or Dp-hydroxyphenylglycine.

Currently, this class of semisynthetic β-lactams is prepared by industrial chemical methods, for example by reaction

6-aminopenicillanic acid (hereinafter referred to as 6-APA), having its carboxyl group generally protected, with an activated side chain derivative and removal of the protecting groups by hydrolysis. For example, Ampicillin can be prepared by reacting 6-aminopenicillanic acid having a carboxyl group suitably protected with D-phenylglycyl chloride and subsequent hydrolysis.

These reactions generally include costly steps such as the use of temperatures below 0 ° C (in some cases even below -25 ° C), the use of silylating agents and organic solvents such as methylene chloride, which are harmful and must be handled with care and which are negative for the environment.

Enzymatic production for example

Ampicillin, Cephalexin and Amoxidiline from pure 6-APA and (for example, the lower alkyl ester) of the D-phenylglycine derivative are known from German patent 2 163792, Austrian patent 243986, Dutch patent 70-09138, German patent No. 2,626,118 and European Patent Specification No. 339,751. Methods known in the art generally employ below 300 mM D-phenylglycine derivative and below 25 mM 6-APA.

A possible drawback of the known enzymatic methods of producing these semisynthetic β-lactams (none of which have been utilized on an industrial scale so far) is the fact that the starting concentration of 6-APA is very low (generally less than 25 mM), which is isolation of the semisynthetic β- lactam is more difficult and expensive. In addition, the reported yields are low, generally less than 85% of the theory, and the unreacted β-lactam nucleus needs to be recycled, resulting in more expensive operations.

One of the problems with increasing substrate concentration in the enzymatic synthesis of semisynthetic β-lactams is rather the low solubility of some substrates and formed products during the reaction. Therefore, in order to operate the process under economically feasible conditions, the concentration of substrates and products may be above their respective solubility, that is, they may be present during all or part of the enzymatic reaction stages in crystalline, amorphous or other solid form and in soluble form; the enzyme should be in a re-usable form, for example in an immobilized form.

It is an important requirement to separate the catalyst from the reaction mixture containing, among other products and unreacted substrates, which may be contained in both crystalline, amorphous or other solid form and in dissolved form after completion of the enzymatic reaction before further purification of unreacted substrates and products under harmless efficiency conditions. enzymes, for example, by dissolving all substrates and products at a low pH (pH value of 0.5 to 2.0).

SUMMARY OF THE INVENTION

The method of separating the undissolved catalyst from one or more other undissolved constituents contained in the same reaction mixture is to use a catalyst having a lower density than the reaction liquid and other undissolved components having a higher density than the reaction liquid. wherein one of the reactants, optionally containing a portion of the reaction liquid, is separated from the reaction mixture containing other insoluble components preferably by physical methods.

By using undissolved catalysts, for example, immobilized enzymes having a specific gravity lower than that of the reaction liquid, it is possible to separate the undissolved catalyst from one or more other undissolved components contained in the reaction mixture, provided that these insoluble components of the mixture have a higher specific gravity. than the specific gravity of the reaction liquid. One method of such a solution is to separate the catalyst from the reaction mixture or suspension after the reaction is complete. This is possible if, for example, after the reaction mixture has been standing for some time, the catalyst has accumulated upwards in the reaction vessel and from there can optionally be removed with a little reaction liquid. The reaction liquid was.

it is separated from the catalyst by known methods, for example by filtration, and can optionally be rinsed with known rinsing agents. The catalyst can then be used with little loss of activity or no loss of activity. Before the undissolved catalyst from the reactant having a specific gravity higher, or after removal of the mixture, other insoluble than the specific gravity of the reaction liquid, for example crystals, may be separated, for example, by removing from the bottom of the reaction vessel or with the reaction liquid or substrates or products. The reaction products can then be further purified and unreacted substrates can be further purified and recycled.

The method according to the invention is illustrated in more detail in the accompanying drawings: FIG. 1 shows the process of the invention during the reaction;

as a result of stirring, the undissolved catalyst and the undissolved components are separated in the reaction liquid; 2 shows the process of the invention after completion of the reaction; the undissolved catalyst and other undissolved components, for example a precipitate, are at the top and bottom of the reaction vessel.

Thus, the invention is a method of separating the undissolved catalyst from one or more other undissolved components, all of which are contained in the reaction mixture, wherein the catalyst has a lower specific gravity than the reaction liquid and the other undissolved components have a higher specific gravity than the reaction liquid. optionally containing a portion of the reaction liquid is separated from the reaction mixture containing other undissolved components. Typically, the separation of one undissolved component from the reaction mixture containing the other undissolved component is carried out in a physical manner.

By reaction liquid is meant herein the liquid (or dissolved portion) of the reaction mixture. The reaction mixture according to the invention consists of at least two undissolved components and a so-called reaction liquid. The solubility of these undissolved components refers to the respective reaction mixture. One of the undissolved components is an insoluble catalyst, which may be an enzymatic agent. Another undissolved component may be the desired reaction product, which may be partially dissolved in the reaction liquid. Alternatively, other undissolved components may be impurities, by-product, unreacted starting material, all having some solubility in the reaction liquid. The insoluble component may be a precipitate formed during the reaction. The reaction liquid comprises dissolved components and optionally one or more solvents. Preferably, the reaction liquid is a single phase system. In most cases, the reaction liquid is water, although organic solvents may be added. Alternatively, the reaction liquid may be a two-phase or multi-phase system. The reactants may be completely or partially dissolved in the reaction liquid.

The undissolved catalyst used in the process of the invention may be in the form of an immobilized enzyme reagent having a lower weight than the specific gravity of the reaction liquid. In the case of such an agent, the enzyme may be absorbed, adsorbed, covalently bound, entrained, or bound by ionic forces. Immobilization procedures are known in the art. According to a preferred embodiment of the process according to the invention, the undissolved catalyst comprises an enzyme. In general, any enzyme can be used. Exemplary enzymes useful include proteases, metalloproteases, serine proteases, thermolysin, amidases, esterases, and peptidases. An enzyme capable of hydrolyzing penicillin G, penicillin V, Ampicillin or Cephalexin may also be used.

In order to have a specific gravity of less than the specific gravity of the liquid, the enzyme may be fully or partially purified or completely prepared immobilized directly on the material or on particles with a lower specific gravity than the specific gravity of the reaction liquid.

Thus, the undissolved catalyst, wholly or partially purified, can be immobilized on any known material in the art (e.g., glass, foamed polymers, e.g., agarose, polyacrylonitrile or styrene) having a specific gravity lower than that of the unreacted liquid by introduction, conglomeration, by co-immobilization or other incorporation into the reagent.

Particles of any shape of known low density material may be used as the material providing the catalyst with a lower specific gravity than that of the reaction liquid. Examples of such low density particles are plastics or polymers (e.g. polystyrene, polyurethane or polycarlyamine) or natural products such as cork. It is also possible to use these materials in hollow or porous form, such as hollow or porous fibers or hollow or porous beads, these materials being filled with gas or other low density media or partially evacuated. Alternatively, the material supplying the undissolved enzyme agent a lower specific gravity than the reaction liquid may be a heavier material that is hollow or porous (e.g., hollow glass beads) or may be filled with gases or other low density materials.

The specific surface area of the process of the invention is important:

(1) for enzymatic processes carried out directly in the fermentation broth,

2) for peptide synthesis,

(3) for the hydrolysis of amides and esters; and

4) for the preparation of semisynthetic penicillins and cephalosporins.

6-APA is generally prepared by enzymatic hydrolysis of penicillin G or penicillin V, which are prepared by fermentation. Typically, the fermented penicillin G is purified (e.g., by filtration from the soil, extraction into an organic solvent such as butyl acetate from which it is extracted into water) before the hydrolysis step. In order to limit the number of unit operations and thus increase the overall yield, 6-APA can be produced by adding directly to the fermentation broth at the end of fermentation of a catalyst having a specific gravity lower than the reaction liquid, thereby hydrolyzing penicillin G. 7.5 and hydrolysis is performed by maintaining a constant pH at a temperature of about 30 ° C. The catalyst is then removed from the surface of the reaction mixture by decantation and, after thorough washing, the catalyst can be reused. The 6-APA formed is purified by known methods (e.g., adsorption and crystallization).

The use of immobilized thermolysin with a lower specific gravity than the reaction mass of the above process simplifies the separation method of Aspartame and reduces the catalyst activity loss in the separation step by less than about 1%.

To further elucidate the invention, its use in the field of enzymatic acylation of the β-lactam nucleus is described. Examples of β-lactam derivatives which can be prepared by the process according to the invention are Ampicillin, Amoxicillin, Cafalor, Cefalexin, Cefadroxil, Cephaloglycine.

The D-phenylglycine or D-p-hydroxyphenylglycine derivatives which may be used as reactants in the process of the invention may be lower alkyl esters (methyl, ethyl, n-propyl or isopropyl ester) or a primary, secondary or tertiary amide. Preferred derivatives are methyl ester, ethyl ester and amide. The derivatives may be used in free form or in the form of salts, for example in the form of a salt with hydrochloric acid or with sulfuric acid. The enzyme used can be any enzyme catalysing the reaction. Such enzymes are generally referred to as penicillin amidases, penicillin acylases or ampicillin hydroxylases. Many microbial enzymes are known which are effective, derived, for example, from Acetobacter, Xanthomonas, Mycoplana, Protaminobacter, Aeromonas (German Patent No. 2163792), Psudomonas (Austrian Patent No. 243986), Flavobacterium (Dutch Patent No. 70-09138), Aphanocladium, Cephalosporium (German Patent No. 2 621618), Acetobacter pasteurianus (German Patent No. 2 163792 A), Acetobacter turbidans (Takahashi et al., Biochem. J. 137, 1974, pp. 497-503),

Pseudomonas melanogenum (Kim & Byun, Biochem. Biophys, Acta, 1040, pp. 12-18, 1990), Xanthomonas citrii (European Patent Specification 339 751), Kluyvera citrophila (Okachi et al., Arg. Biol. Chem. 37 2797-1804 (1973), Escherichia coli (German Patent No. 2,930,794) and Bacillus megaterium (Chiang & Bennett, J. Bactteriol., 93, 302 (1967)).

After separation of the reaction liquid and crystals from the catalyst, the catalyst may be washed with a washing agent and reused. The products may be further purified and unreacted substrates may be further purified and reused.

Substrates, inert compounds and products may be present as precipitates during the reaction.

In general, the reaction temperature is 0 to 40 ° C, preferably 10 to 35 ° C. For conventional operations, a temperature of 20 to 35 ° C may be preferred.

The appropriate pH value depends on the type and purity of the enzyme. In the case of the E. coli enzyme, a pH of 5.5 to 8.0 is generally chosen. The pH should be controlled. A suitable reaction time is several minutes to several hours, especially about 30 minutes to about 24 hours. A suitable enzyme concentration is about 1 U / ml to about 200 U / ml (1 U = unit of enzyme activity, as explained below).

The isolation and purification of the desired product can be carried out by known methods, for example by crystallization.

The invention is illustrated in more detail by the following examples.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Preparation of a catalyst with a lower specific gravity than the reaction liquid

Penicillin G acylase from E. coli is fermented (see, e.g., Cole et al., Met. Enzymol., 43, 698, 1975) and purified by methods known in the art (e.g. ammonium sulfate precipitation, adsorption, ultrafiltration) to a protein content of approximately 20 mg / ml and an activity of 340 U / ml (unit to be explained below).

Agarose beads (6% agarose) containing hollow glass beads were prepared as described by Pertoft and Hallen, J. Chromium. 128, p. 112-131, 1976, replacing silica with hollow glass beads (50-75 microns), for example, Schott (Schott Glaswerke, D-6500, Mainz). The agarose beads are then cross-linked using epichlorohydride (Porath and Axen, Meth. Enzymol. 44, 23, 1976). Agarose beads having desirable specific gravity characteristics are prepared by screening particles in a fluidized bed.

The agarose beads are activated by divinylsulfone as described by Lihme et al., J. Chromium. 376, p. 299 to 305, 1986. The enzyme is then added and immobilized to yield activated agarose beads.

The catalyst obtained has a particle size of 350-500 microns and an enzymatic efficiency of 500 U / g wet catalyst. However, the particle size and activity can vary widely depending upon the reaction conditions selected or desired.

Enzyme activity

The enzyme activity is defined as follows: one unit (U = unit) corresponds to the amount of enzyme which hydrolyzes per minute 1 μιποί of penicillin G under normalized conditions (5% penicillin G, 0.2 M sodium phosphate buffer, pH 8.0 and temperature 28) "C).

Example 2

Synthesis of Ampicillin

D-Phenylglycine methyl ester (referred to herein as D-PGM) and 6-APA are dissolved in 50 mM phosphate buffer adjusted to pH 6 and allowed to equilibrate at 35 ° C for 10 minutes (final concentration is 450 mM and optionally 100 mM). 0.75 g of wet catalyst (according to Example 1) is added, the total volume is 20 ml, and the synthesis is allowed to proceed at a constant pH (6.0) and with efficient stirring.

After about 30 minutes, D-phenylglycine (referred to herein as D-PG) begins to precipitate from the reaction mixture and after about three hours the Ampicillin concentration reaches its maximum. At this point, stirring is stopped, the catalyst is collected at the surface and the precipitate formed (a mixture of D-phenylglycine and Ampicillin) is withdrawn from the bottom of the reaction vessel along with the reaction liquid (containing inter alia dissolved, unreacted substrates and products).

The ampicillin may be further purified by known methods by chromatography and / or crystallization.

The catalyst is washed in the reaction vessel with 50 mM phosphate buffer (pH 6.0) and can be reused without significant loss of efficacy (Table I).

Table I

Efficacy of penicillin G acylase before and after synthesis and after separation of the catalyst from the reaction mixture. Assay for p-dimethylaminobenzaldehyde activity by Blasingham et al., Biochim. Biophys. Acty, 276, p. 250-256, 1972.

Amount of catalyst

activity

U / g

Before synthesis after separation

0.75

0.74

498.5

497.2

HPLC analysis of reagents column: RP LC-18 (250 x 4.6 mm; 5μτη), eluent A: 25 mM phosphate buffer, pH 6.5 eluent B: acetonitrile

Gradient Time% A% B

0 99 1 10 80 20 20 80 20 21 99 1 35 99 1

flow: 1 ml / min detection: 215 nm minutes retention time: 4.1 (D-PG), 8.1 (6-APA), 13.9 (Ampicillin), 18 (D-PGM)

Example 3

D-Phenylglycine methyl ester hydrochloride salt (1.6526 g) and 7-aminodesacetoxycephalosporanoic acid (0.4278 g) (7-ADCA) are dissolved in 50 mM phosphate buffer adjusted to pH 6.5 and allowed to equilibrate at 35 " C. 100 g of catalyst (according to Example 1) are added, the total volume being 20 ml, and the reaction is allowed to proceed with constant pH, constant temperature and efficient stirring.

After about 35 minutes, a precipitate formed and after a further 10 minutes the reaction was stopped. After a short time, about 1 minute, the catalyst was collected on the surface. The precipitate formed and the reaction liquid containing, among other ingredients, Cephalexin, D-phenylglycine, D-phenylglycine methyl ester and 7-aminodesacetoxycephalosporanoic acid are readily separated from the catalyst by drawing them from the bottom of the reaction vessel.

Cephalexin can be further purified by known methods and the catalyst can be reused without any significant loss of activity. The catalyst may be purged prior to re-use. After the separation process, the catalyst retains more than 99% of its activity.

The reaction was monitored by HPLC under the same conditions as in Example 2, with 7-ADCA and Cefalexin eluting after 6.3 and 13.4 minutes, respectively.

Example 4

A suspension of 2.247 g of Dp-hydroxyphenylglycine (referred to herein as HPGA) and 0.715 g of 6-aminopenicillanic acid is prepared in 50 mM phosphate buffer adjusted to pH 6.5 and allowed to equilibrate at 25 C. The reaction is started by adding 2.0. g of catalyst (according to Example 1), the total volume is 20 ml, and the reaction is allowed to proceed by maintaining the pH constant by titration with 2 molar sulfuric acid.

After 10 hours, the concentration of Amoxicillin reaches its maximum and the reaction mixture contains, among other components, Amoxicillin and HPGA crystals.

Stirring is stopped and the catalyst is allowed to collect at the surface of the reaction liquid and the crystals precipitate. The formed crystals and reaction liquid are readily separated from the catalyst by drawing them from the bottom of the reaction vessel. The amoxicillin can be further purified by known methods.

The catalyst can be reused without any significant loss of activity (retaining more than 99% of its activity), directly or after rinsing with water or phosphate buffer.

The reaction is monitored by HPLC under the same conditions as in Example 2, using 5% acetonitrile in 95% 25 mM phosphate buffer (pH 6.0) for isocratic elution (1 mL / min) of the compounds. Under these conditions, the retention time (in minutes: 2.5 (D-p-hydroxyphenylglycine), 3.3 (D-HPGA), 6.0 (6-APA) and 15.0 (Amoxicillin)).

Example 5

A mixture of 8.35 g HPGA in water adjusted to pH 6.0 was charged with 0.75 g wet catalyst (according to Example 1), the total volume was 100 ml, and the hydrolysis was allowed to proceed at 35 ° C while maintaining a constant value. pH 6.0 by titration with 2 molar sulfuric acid and with stirring.

After three hours, HPGA is completely hydrolyzed to the free acid, D-p-hydroxyphenylglycine (referred to herein as HPG), and the reaction mixture appears to be a suspension of catalyst and partially precipitated HPG.

When stirring is stopped, the catalyst is collected at the level of the reaction liquid and the precipitated HPG is separated easily from the catalyst by drawing it from the bottom of the reaction vessel along with the reaction liquid containing, among other components, salt and dissolved HPG.

The HPG can then be further purified by known methods by chromatography or crystallization.

The catalyst is washed in the reaction vessel with 50 mM phosphate buffer (pH 6.0) and can be reused without any significant loss of activity (losing less than 1% of its activity in synthesis and separation).

The reaction was monitored by HPLC under the same conditions as described in Example 4.

Example 6

Preparation of a thermolysis grain catalyst having a specific gravity lower than that of the reaction liquid

Dissolve 8 g of Termolysin (Sigma P-1512) in 25 mM phosphate buffer (pH 7) to obtain a solution of about 20 mg protein per ml and about 1500 U / ml. Agarose beads were prepared and activated as in Example 1, protease was added and immobilized to obtain activated agarose beads. Catalyst activity is approximately 3000 U / g. Activity is measured by a casein digestion method (1 unit hydrolyses casein to give a color equivalent of 1.0 μιηοΐ tyrosine per minute at pH 7.5 at 35 ° C (Folin-Ciocalteu color reagent).

Synthesis of N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester (an intermediate for the synthesis of Aspartame)

0.1 mol of the acid is dissolved

Of N-benzyloxycarbonyl-L-aspartic acid and 0.25 mol

The pH of the L-phenylalanine methyl ester hydrochloride in water was adjusted to 6.0, with a final volume of 350 ml and allowed to equilibrate at 35 ° C for 10 minutes. 15 g of catalyst are added. The reaction was allowed to proceed at 40 ° C for 10 minutes and an additional 15 g of catalyst was added under stirring. The reaction is allowed to proceed at 40 ° C while maintaining a constant pH and efficient shear mixing. The reaction produces a condensation product,

The N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester (referred to herein as ZAPM), which precipitates almost quantitatively as the product, forms a dipeptide addition compound with unreacted L-phenylalanine methyl ester with very poor water solubility. After 20 hours, stirring was stopped and the reaction mixture was cooled to about 5 ° C. The catalyst was collected on the surface. The precipitate formed and the reaction liquid are readily separated from the catalyst by drawing them from the bottom of the reaction vessel.

The catalyst is reused in the reaction vessel after washing, first with one volume of 25 mM phosphate buffer (pH 6.5) and then with one volume of water. More than 99% of the original activity remains.

ZAPM can be further processed to Aspartame in a known manner (for example, by removing nitrogen protecting groups from the aspartic fraction, by crystallization).

The preparation of ZAPM is monitored by HPLC under the conditions described by Oyama et al., J.C. Perkin II, p. 356, 1981.

Example 7

Production of 6-APA directly in penicillin culture broth

Penicillin G is fermented in a 5.5 liter fermenter (see, e.g., Likidis and Schugerl, Biotechnol. Letters, 9: 229-232, 1987) for seven days. The fermenter contents are transferred to a reaction vessel with a low shear stirrer. The pH of the culture broth is adjusted to 8.0 with a 4N ammonium hydroxide solution at 30 ° C and 50 g of the catalyst of Example 1 are introduced. The hydrolysis is carried out at a constant pH and at a constant temperature with efficient stirring. After 5 hours, 98% of the fermented penicillin G is hydrolyzed and stirring is stopped. After 5 minutes, the catalyst was decanted from the reaction mixture. Hydrolytic products, 6-APA and phenylacetic acid, can be purified by removal of cells and culture medium by known methods (e.g., extraction, adsorption and crystallization). The catalyst may be reused after thorough washing with 50 mM phosphate buffer (pH 8.0). The catalyst obtained had 95% of the original activity.

Industrial usability

A process for separating the undissolved catalyst from one or more other undissolved components always contained in the same reaction mixture using a lower density catalyst and other undissolved components having a higher density than the reaction liquid.

Claims (10)

A process for separating an undissolved catalyst from one or more other undissolved components contained in the same reaction mixture, characterized in that a catalyst having a lower density than the reaction liquid and other undissolved components having a higher density than the reaction liquid is used, wherein one of the reactants, optionally containing a portion of the reaction liquid, is separated from the reaction mixture containing the other undissolved components preferably using physical methods. Process according to claim 1, characterized in that the undissolved catalyst is an immobilized enzyme. The process according to claim 1 or 2, characterized in that the undissolved catalyst is an enzyme, fully or partially purified, immobilized directly with particles having a lower density than the reaction liquid or co-immobilized particles having a lower density than the reaction liquid. The method of claim 1 or 2, wherein the undissolved catalyst is an entire cell or a cell homogenizer co-immobilized with particles of lower density than the reaction liquid, wherein the particles are hollow glass beads. The method of claims 1 to 4, wherein the undissolved catalyst comprises an enzyme co-immobilized with hollow glass beads in agarose or the catalyst comprises whole cells or cell homogenate immobilized with hollow glass beads or optionally in agarose. The process according to claims 1 to 5, characterized in that at least one component in the reaction mixture in addition to the undissolved catalyst is present in particulate form at the end of the reaction, and optionally the particulate form is the result of precipitation of the reaction mixture during the reaction. The process according to claim 1, preparing β-lactam 6-aminopenicillanic acid, up to 6, characterized in that the derivatives are, on the one hand, 7-aminodesacetoxycephalosporanic acid, 7-aminocephalosporanic acid or 3-chloro-7-aminodesacetoxycephalosporanic acid, or Dp-hydroxyphenylglycine as D-phenylglycine side chain corresponding to the catalytic reaction a β-lactam nucleus with a D-phenylglycine or Dp-hydroxyphenylglycine derivative using an undissolved catalyst with a specific gravity lower than that of the reaction liquid, wherein the reaction mixture in addition to the undissolved catalyst contains further undissolved constituents with a specific gravity greater than the specific gravity weight of reaction liquid The method according to claims 1 to 6, characterized in that the amino acid ester or amino acid amide is hydrolyzed to the free amino acid by catalytic reaction of the corresponding amino acid ester or amide, preferably the ester or amino acid amide being a N-phenylglycine or Dp-hydroxyphenylglycine derivative, D -valine, L-valine, L-phenylalanine or L-tyrosine. The method according to claims 1 to 6, characterized in that the peptide is prepared by catalytic reaction of an amino acid derivative and an amide derivative or a lower alkyl amino acid ester, and preferably the nitrogen-protected L-aspartyl-L-phenylalanine methyl ester is prepared by catalytic reaction on nitrogen-protected L-aspartic acid. acid and L-phenylalanine methyl ester or D, L-phenylalanine methyl ester. V V 0 ^ g5 · -9 N Method according to claims 1 to 9, characterized in that the enzyme in the undissolved enzymatic agent is a protease, metalloprotease, serine protease, thermolysin, amidase, esterase or peptidase, preferably the enzyme in the undissolved enzymatic agent is obtainable from the group comprising Escherichia coli, Acetobacter pasteurianus, Acetobacter turbidans, Xanthomonas citrii, Kluyvera citrophila or Bacillus megaterium, Pseudomonas melanogenum or Bacillus thermoproteolyticus or preferably an enzyme in the undissolved enzyme reagent is capable of hydrolyzing penicillin G, penicillin C, penicillin C, penicillin.