HUT67012A - Method for separation of two solid components - 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

The present invention relates to a process for separating an undissolved catalyst and one or more other non-solution components from a reaction mixture.

A solution for precipitation from a solution is well known; for example, by filtration or centrifugation. If the reaction mixture contains two or more different, undissolved components, it is not easy to separate the undissolved components from one another. Filtration followed by mechanical separation is not possible for the various undissolved components. In some cases, it is possible, after filtration, to separate the undissolved components by chemical methods, which, however, are usually complicated.

There are many areas in the chemical literature where it is necessary to separate two undissolved components in a liquid. In the design of various chemical processes, this solution is generally ruled out and does not form a reaction mixture containing two or more different, undissolved components, since they must then be separated. An area in which two or more undissolved components are to be separated is the case of reactions in which an undissolved catalyst is employed. These may be enzyme preparations bound to a solid support.

The cost of the catalyst in processes employing a catalyst is generally a significant factor in the overall cost of the process, and it is therefore necessary to develop a process in which the catalyst can be reused without significant loss of catalyst activity. The recovery and reusability of the catalyst is often compromised if the catalyst is present in a reaction mixture that also contains other undissolved components that may be formed during the reaction or may be present throughout the process.

One particular area of interest to the process of the present invention is the preparation of peptides such as Aspartame ™. One of the basic steps in the preparation of Aspartame (L-alpha-aspartyl-L-phenylalanine methyl ester) involves the enzyme-catalyzed coupling between an aspartic acid derivative and phenylalanine methyl ester hydrochloride, e.g., N- (benzyloxy) carbonyl) -L-aspartic acid and L-phenylalanine methyl ester hydrochloride are coupled to give N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester (which is hereafter referred to as ZAPM). ZAPM forms an addition compound with unreacted L-phenylalanine methyl ester (or, if present in the reaction mixture, D-phenylalanine methyl ester), which has a very low solubility; it therefore precipitates during synthesis and reverses the reaction equilibrium towards condensation. In order to separate the catalyst (e.g., the Thermolysin ™ catalyst) from the reaction mixture and the reaction product, a partially purified soluble enzyme preparation is used. The reaction product is dissolved and the enzyme is precipitated with an organic solvent (e.g. acetone) and the enzyme is removed, for example, by filtration.

However, during the process, 14%, or at least 60%, of the enzyme may suffer from catalytic activity loss (see Nonaka et al., U.S. Patent No. 4,212,945 and

Meijer et al., Biocatalysis in Organic Synthesis (Eds., Tramper, van Dér Plas & Linko), 135-156 (1985). The greatest loss of activity occurs during precipitation as well as during enzyme isolation of the catalyst.

Another particularly important area in which an appropriate method for separating undissolved components from one or more other undissolved components is to be devised is the enzymatic or catalytic acylation of beta-lactam compounds during penicillin production or cephalosporin production. Therefore, the following is a more detailed description of this field in order to illustrate the scope of the present invention. More particularly, the process relates to the enzymatic preparation of beta-lactam derivatives, which may be, in part, 6-aminopenicillinic acid (6-APA), 7-aminodeacetoxycephalosporinic acid (hereinafter referred to as 7-ADCA), 7-aminocephalosporinic acid 7-ACA) or 3-chloro-7-amino-deacetoxycephalosporinic acid. On the other hand, D-phenylglycine or D-p-hydroxyphenylglycine can be used as the side chain used in the catalytic reaction for the corresponding beta-lactam molecule by reaction with a suitable D-phenylglycine or D-p-hydroxyphenylglycine derivative.

The above semi-synthetic beta-lactam compounds are generally commercially prepared by chemical processes, for example the 6-APA compound, which usually contains a carboxyl group in protected form, is reacted with a derivative of an activated side chain and subsequently deprotected by hydrolysis. For example, ampicillin can be prepared by reacting a 6-APA containing a properly protected carboxyl group with D-phenyl

-glycyl chloride and the product is hydrolyzed. These reactions are generally carried out using expensive reaction steps such as those below 0 ° C (in some cases below -25 ° C), the use of silylating reagents or the use of organic solvents such as dichloromethane which are hazardous to health, and therefore its management is environmentally important and may pose a threat to the environment.

For example, the process for preparing Amoxicillin, Cephalexin and Ampicillin starting from pure 6-APA and a D-phenylglycine derivative (such as a lower alkyl ester) is described in the literature. Examples of such literature are German Patent Application 2,163,792, Australian Application 243,986, Dutch Application 70-09138, German Application 2,621,618, and European Patent Application 339,751. Generally, the D-phenylglycine derivative below 300 mmol and 6-APA at a concentration below 25 mmol is used in the methods described above.

The known above processes for the preparation of the above semi-synthetic beta-lactams (not yet used on an industrial scale) have the disadvantage that the initial concentration of 6-APA is very low (generally less than 25 mmol) and thus the semi-synthetic beta-lactam formed isolation is more difficult and costly. Therefore, the production described is low; is usually less than 85% and requires a recycle process for the unreacted beta-lactam parent compound, which consequently involves an additional cost.

One problem that arises with regard to increasing the concentration of substrate in the synthetic enzymatic process of semi-synthetic beta-lactams is that some of the substrates have very low solubility and also very low solubility of the products. Therefore, in order for the process to be carried out with sufficient economy, the concentration of the substrates and the products obtained must be above the solubility values, i.e. they may be in the form of a crystalline, amorphous or solid, present in the reaction mixture. They may be present in soluble form. In addition, it must be ensured that the enzyme is present in a reusable form in the reaction mixture, for example in the form of a solid support.

Therefore, it is necessary to remove the catalyst from the reaction mixture which, in addition to other products or unreacted products, which may be crystalline, amorphous or other solid forms or soluble forms after completion of the enzyme reaction, further purification of unreacted compounds and products. For example, it may be necessary to dissolve all substrates and products at a low pH (pH: 0.5 to 2.0) prior to purifying and adversely affecting enzyme activity.

If the reaction uses an undissolved catalyst, such as a catalyst supported on a solid support, which has a density lower than the density of the liquid used in the reaction, it is possible to separate the undissolved catalyst from one or more undissolved components present in the reaction mixture. the undissolved components have a higher density than the reaction liquid. One method for this purpose is to separate the catalyst from the reaction mixture or to separate the catalyst from the reaction mixture suspension after the reaction is complete. This is possible when, for example, the catalyst accumulates on the surface of the reaction mixture after a certain amount of time in the reaction mixture, from where it can be removed, optionally together with a portion of the reaction medium used. The reaction liquid may be removed from the catalyst by known methods, such as filtration, or, if necessary, rinsed with a conventional rinse aid. The catalyst has to be reused with very little or no loss of activity. Before or after removal of the insoluble catalyst, other undissolved components having a density greater than the density of the reaction mixture may be removed, for example, in crystalline form, for example by removal from the bottom of the reaction vessel; optionally together with the reaction liquid or a portion thereof, and inter alia with soluble substrates and products. The reaction products can then be further purified and the unreacted substrates can be further purified and recycled.

The process of the invention is illustrated in detail in the following accompanying drawings. The following symbols are used in the figures:

···· * ···

Figure 1:

The process of the invention is used in the reaction; as a result of mixing, the undissolved catalyst and other undissolved components are distributed in the reaction mixture (liquid).

Figure 2:

Presentation of the process of the invention after completion of the reaction; the undissolved catalyst and other undissolved components, such as a precipitate, appear at the top or bottom of the reaction liquid, respectively.

The present invention relates to a process for removing an undissolved catalyst from one or more other undissolved reaction components, each of which is present in the reaction mixture, wherein the catalyst has a lower density than the reaction liquid and the other undissolved components have a higher density. the liquid density of the reaction mixture, and separating one of the undissolved components, optionally containing a portion of the reaction medium, from the other reaction mixture containing the undissolved component. Generally, the separation of one undissolved component from the reaction mixture containing the other undissolved component is accomplished by physical methods.

Above and throughout the specification, the reaction mixture is defined as a liquid (or solute) that is part of the reaction mixture. The reaction mixture treated by the process of the present invention comprises at least two non-solubilized solvents.

- Contains 9 components in addition to the above reaction medium. The solubility of the above undissolved components depends on the reaction mixture. One of the undissolved components may be an undissolved catalyst, which may be an enzyme composition. Another undissolved component may be a desired reaction product which may be partially dissolved in the reaction medium, or else the other undissolved component may be a contaminant, a by-product, or an unreacted starting material, each of which has some solubility in the reaction medium. The undissolved component may also be a precipitate formed during the reaction. The reaction medium contains dissolved components and, if desired, one or more solvents. Generally, one or more solvents are optionally desired. The reaction medium is usually a single-phase system. In most cases, the reaction medium contains water; however, other organic solvents may be added. Alternatively, the reaction medium may be a biphasic or multiphase system. The reactants may be partially or completely dissolved in the reaction medium.

The undissolved catalyst used in the process may be a solid support composition having a density less than the density of the reaction mixture. In such a solid support composition, the enzyme may be in the form of an absorbed, adsorbed, covalently bonded, encapsulated or ionic bonded support. Methods for binding to a solid support are known in the art. In the preferred process of the invention, the undissolved catalyst comprises an enzyme. In general, any enzyme may be used. Enzymes used include proteases, metalloproteases, serine proteases, thermolysins, amidases, esterases and peptidases. In addition, enzymes useful for the hydrolysis of penicillin G, penicillin V, ampicillin or cephalexin are also useful.

To produce an enzyme with a density less than the density of the reaction mixture, the enzyme may be used either partially or wholly in purified form or in the form of an entire cell preparation and directly bound to substances or particles having a density lower than the density of the reaction medium.

In addition, the undissolved catalyst, which may be wholly or partially purified, may be coupled to a catalyst binder using materials known in the art (such as glass, cellular compositions or polymers such as agarose, polyacrylamide or styrene). Further, they consist of a substance having a density lower than the density of the liquid portion of the reaction mixture, which is present as a conglomerate incorporated therein, partially bound, or otherwise incorporated. It is a further object of the present invention that the undissolved enzyme-containing catalyst may be a whole cell or cell homogenate composition bound together with a low density material to a solid support.

In order to provide a lower density of the catalyst than the liquid portion of the reaction mixture, any known low density material may be used. Examples of such materials are plastics or polymers (e.g. polystyrene, polyurethane or polyacrylamide) or natural materials such as cork. In addition, they can be used in hollow or porous forms, for example, hollow or porous fibers or hollow or porous solid particles which have a "·· * ··" "

Materials may be filled with gases or other low density materials or may be partially evacuated under vacuum. Alternatively, the material which provides the undissolved enzyme composition with a lower density than the liquid phase of the reaction mixture may be a higher density material which has been rendered hollow or porous (e.g., hollow glass beds) or filled with gases or other low density materials.

The process of the invention is particularly advantageous in the following areas:

1) enzymatic processes carried out directly in a fermentation broth;

2) in the synthesis of peptides;

3) hydrolysis of amides and esters; and

4) in the manufacture of semi-synthetic penicillins and cephalosporins.

6-APA is generally prepared by enzymatic hydrolysis of penicillin G or penicillin V, which is obtained by fermentation. In general, the fermented penicillin G compound is purified (for example, by filtration of the medium by extraction in an organic solvent such as butyl acetate and subsequently by extraction with water) and then the hydrolysis step performed. In order to reduce the number of purification processes carried out and to increase the yield of 6-APA, it is possible to add a catalyst having a density less than the liquid portion of the reaction mixture to the hydrolysis of penicillin G immediately after the fermentation. PH value ··;

···; , ··, - · ► ··· ·· «.

. ·. * · * ·· »· *

·., · · .. · ·

For example, the product is usually adjusted to about 7.5, and the hydrolysis is allowed to proceed, keeping the pH constant and the temperature about 30 ° C. The catalyst may be removed from the top of the reaction mixture by decantation, and after thorough washing, the catalyst may be reused. The resulting 6-APA compound can be purified by known methods (e.g. adsorption and crystallization).

The use of thermolysin on a bound support having a density less than the density of the reaction mixture in the process described above simplifies the separation step and reduces the degree of activity loss in the separation step, which is less than about 1%.

For a more detailed description of the process of the invention, the following is the use of the process of the invention in the enzymatic acylation of beta-lactam compounds. Examples of the beta-lactam derivatives of the invention include Ampicillin, Amoxicillin, Cefaclor, Cephalexin, Cefadroxil and Cephaloglycin.

The reactant D-phenylglycine or Dp-hydroxyphenylglycine derivative to be used in the process may be a lower alkyl ester (methyl ester, ethyl ester, n-propyl ester or isopropyl ester) or a first order, second order or third order amid. Preferred derivatives include methyl ester, ethyl ester and amide. The derivative may be used in free or salt form, for example in the form of its hydrochloric or sulfuric acid salt.

The enzyme to be used can be any enzyme that is

- Catalyze 13 specific reactions. Such enzymes are generally penicillin amidases, penicillin acylases or ampicillin hydrolases. Many microbiological enzymes known to possess this activity are known, for example, Acetobacter, Xanthomonas, Mycoplana, Protaminobacter, Aeromonas (German Patent Application 2,163,792), Pseudomonas (Austrian Application 243,986), Flavobacterium (70-09138), Aphanocladium, Chan German Patent Application 2,621,618), Acetobacter pasteurianus (German Patent Application 2,163,792 A), Acetobacter turbidans (Takahashi et al., Biochem. J., 137, 497-503 (1974)), Pseudomonas melanogenum (Kim and Byun, Biochim. Biophys. Acta 1040: 12-18 (1990), Xanthomonas citrii (European Patent Application 339,751), Kluyvera citrophila (Okachi et al., Agr. Bioi. Chem. 37 (1973) 2797-2804), Escherichia coli (German Patent Application No. 2,930,794) and Bacillus megaterium (Chiang and Bennett, J. Bacteriol., 93, 302 (1967)).

After the liquid portion of the reaction mixture and the crystals are separated from the catalyst, the catalyst can be washed, for example, with a rinse agent and reused. The products can be further purified, or unreacted substrates can be further purified and recycled. Substrates, inorganic compounds and products may be present as precipitates during the reaction.

The reaction temperature is usually 0 to 40 ° C, in particular 10 to 35 ° C. The preferred temperature for proper operation is shown in FIG

35 ° C. The occasion

- 14 glazed pH depends on the purity and type of enzyme. For E. coli, the pH is generally between 5.5 and 8.0. The pH should be controlled. Suitable reaction times are from several minutes to several hours, in particular from about 0.5 to about 24 hours.

The enzyme concentration used is from about 1 U / ml to about 200 U / ml (1 U = 1 unit of enzyme activity, see below).

Recovery and purification of the desired product can be accomplished by known methods, including, for example, crystallization.

The following examples illustrate the invention. The examples are not to be construed as limiting the scope of the invention.

Example 1

Preparation of a catalyst with a density less than the liquid portion of the reaction medium

Penicillin G acylase from E. coli is fermented (see, e.g., Colé et al., Met. Enzymol., 435 (1975) 698) and then semi-purified by known methods (ammonium sulfate precipitation, adsorption, ultrafiltration, etc.). This gives 20 mg protein / ml and 340 U / ml (unit definitions are given below).

An agarose bed (6% agarose) was prepared containing hollow glass particles (50-75 mm). The preparation is carried out according to the procedure described in Pertoft and Hallen, J. Chrom., 128, 112-1133 (1976) by replacing silica gel with hollow glass particles, 50-75 μπι, obtained, for example, from Schott. Schott Glaswerke, D-6500 Mainz). The agarose particles are then crosslinked using epichlorohydrin / Porath and Axen, Meth.

Enzymol., 44, 23 (1976).

Agarose particles of sufficient density are obtained by sorting them in a fluidized bed.

Agarose Particles / Lihme et al., J.

Chrom., 376, 299-305 (1986), is activated with divinylsulfone. The enzyme is then added to the agarose activated particles and bound thereto.

The resulting catalyst has a particle size of 350 to 500 μπι and an enzyme activity of 500 U / g wet catalyst. However, the particle size and activity of the catalyst may vary considerably depending on the reaction conditions chosen or used.

enzyme Activity

The following are used to define enzyme activity:

One unit (U) activity corresponds to the amount of enzyme which hydrolyzes 1 μπ penicillin G per minute under standard conditions (5% penicillin G, 0.2 molar sodium phosphate buffer / pH 8.0 / 28 ° C) ).

• · «

Example 2

Ampicillin synthesis

D-Phenylglycine Methyl Ester (hereinafter D-PGM) and 6-APA were dissolved in 50 mM phosphate buffer and adjusted to pH 6.0 for 10 minutes at 35 ° C. equilibrate (final concentration is 450 mmol and 100 mmol, respectively). To the mixture was added 0.75 g of wet catalyst (as in Example 1). The total volume is 20 ml and the synthesis is allowed to proceed, keeping the pH constant (6.0). The reaction is stirred vigorously.

After about 0.5 hours, D-phenylglycine (hereinafter D-PG) begins to precipitate from the reaction mixture and after about 3 hours the concentration of ampicillin reaches its maximum. At this point, stirring is stopped, the catalyst is collected at the top of the reaction vessel, and the precipitate formed (which is a mixture of D-phenylglycine and ampicillin) is drained from the bottom of the reaction vessel along with the liquid portion of the reaction mixture. contain).

The ampicillin can be further purified by known chromatography and / or crystallization procedures.

The catalyst remaining in the reaction vessel was washed with 50 mM phosphate buffer, pH 6.0, and then reused without loss of activity (Table I).

• · • · • "···· ···· • • · · · · · · · ····· ·· ·· ·· · · ·

- 17 TABLE I

Penicillin G acylase activity before and after synthesis and after catalyst separation from the reaction mixture. Activity is determined by the p-dimethylaminobenzaldehyde method as described by Balasingham et al., Biochim. Biophys. Acta, 276: 250-256 (1972).

Quantity of catalyst (g)

Activity (U / g) before synthesis

0.75

498.5 after weaning

0.74

497.2

HPLC (high performance liquid chromatography) analysis of reaction components

Column: RP LC-18 (250 x 4.6 mm; 5 μτη), Elution A: 25 mM phosphate buffer, pH 6.5, B eluent: acetonitrile.

• · ···· ···· · · · · 4 · · · · ·

Gradient: Time % THE % B 0 99 1 10 80 20 20 80 20 21 99 1 35 99 1

Flow rate: 1 ml / min

Detection: 215 nm

Retention times per minute: 4.1 (D-PG), 8.1 (6-APA), 13.9 (ampicillin), 18 (D-PGM).

Example 3

1.6526 g of D-phenylglycine methyl ester hydrochloride salt and

7-Amino-deacetoxycephaloric acid (7-ADCA) (0.4278 g) was dissolved in 50 mM phosphate buffer (pH 6.5) and equilibrated at 35 ° C. Then, 100 mg of catalyst (Example 1) was added (20 mL total volume), and the reaction was allowed to proceed with stirring, keeping the pH and temperature constant. After about 35 minutes, a precipitate formed and after a further 10 minutes stirring was stopped. Within a short time - about 1 minute - the catalyst is located in the top layer. The precipitate and the liquid portion of the reaction mixture, including Cephalexin, D-phenylglycine, D-phenylglycine methyl ester and 7-amino-deacetoxycephaloric acid ············· ··· • · · · · · ······ · ·

- 19, can be easily separated from the catalyst by lowering it at the bottom of the reaction mixture.

Cephalexin is further purified by known methods and can be reused without significant loss of activity. When using the catalyst, a washing step is introduced before this. After the separation step, more than 99% of the catalyst activity remains.

The progress of the reaction is monitored by HPLC

Using the conditions described in Example 2, wherein the elution times of 7-ADCA and Cephalexin are, respectively, 6.3 and 13.4 minutes.

Example 4

A suspension of 2.247 g of Dp-hydroxyphenylglycine amide (hereinafter "HPGA") and 0.715 g of 6-aminopenicillanic acid is prepared in 50 mM phosphate buffer, pH 6.5, and the solution is heated to 25 ° C. balance. The reaction was started by adding 2 g of catalyst (Example 1, final volume: 20 mL) and maintaining the pH constant by titration with 2 M sulfuric acid.

After 1 hour, the concentration of Amoxicillin reaches its maximum and the reaction mixture contains, among other things, crystalline Amoxicillin and unreacted HPGA.

Stirring is then stopped and the catalyst is allowed to collect at the top of the liquid portion of the reaction mixture and the crystals settle. The crystals and the liquid in the reaction mixture are drained from the bottom of the reaction mixture. Amoxicillin can be further purified by methods known in the art.

···· ···················································································································································································································· ·

The catalyst in the reaction vessel can be reused without losing its catalytic activity (more than 99% of the activity is retained), directly or preferably after washing with water or phosphate buffer.

The progress of the reaction was monitored by HPLC analysis using the conditions of Example 2 but using isocratic elution (1 mL / min) eluting with 5% acetonitrile in 95% 25 mM phosphate buffer (pH (6.0). Under these conditions, the retention times per minute for compounds of interest to us are: 2.5 (Dp-hydroxyphenylglycine), 3.3 (D-HPGA), 6.0 (6-APA), and 15.0 ( amoxicillin).

Example 5

To a mixture of 8.35 g of HPGA in water adjusted to pH 6.0, 0.75 g of wet catalyst (material of Example 1) was added to make a total volume of 100 ml. The hydrolysis was allowed to proceed at 35 ° C by keeping the pH constant at 6.0 by titration with 2 M sulfuric acid and vigorous stirring.

After 1 h, HPGA was completely hydrolyzed to the free acid, D-p-hydroxyphenylglycine (hereinafter HPG), and the reaction mixture was a suspension of catalyst and partially precipitated HPG.

When the stirring is stopped, the catalyst is collected at the top of the reaction mixture, the HPG precipitate can be removed from the bottom of the reaction vessel along with the liquid in the reaction mixture, which is · · ·

- 21 liquids including salts and dissolved HPG.

The HPG can be further purified by chromatography and / or crystallization techniques known in the art.

The catalyst in the reaction vessel (pH:

6.0), washed with 50 mM phosphate buffer and reused without significant loss of catalytic activity (less than 1% of catalytic activity is lost during synthesis and subsequent separation).

The reaction was monitored by HPLC as in Example 4.

Example 6

Preparation of a Thermolysin Catalyst with Density Less Than the Liquid Part of the Reaction Dissolve g Thermolysin (Sigma, P-1512) in 25 mM phosphate buffer (pH 7) to give a concentration of about 20 mg protein / ml and an activity of about 1500 U / ml. a. Agarose beads were prepared and activated as in Example 1, and the protease was added and bound to the activated agarose beads. The activity of the catalyst is about 3000 U / g. Activity is measured by a casein digestion (1 unit of casein is hydrolyzed to give a color corresponding to 1.0 μπιόΐ tyrosine activity over 1 minute at pH 7.5 at 35 ° C) / the color reaction is adjusted with Folin-Ciocalteu reagent live/.

···· · «· · · · · · · · · · · · · · · · · · ···

- Preparation of 22 N- (Benzyloxycarbonyl) -L-aspartyl-L-phenylalanine methyl ester (intermediate for the synthesis of aspartame)

Dissolve 0.1 mol of N-benzyloxycarbonyl-L-aspartic acid and 0.25 mol of L-phenylalanine methyl ester hydrochloride in water and bring the pH of the solution to 6.5 and make up a final volume of 350 ml. The substrates were equilibrated at 40 ° C for 10 minutes and then 15 g of catalyst was added. The reaction was allowed to proceed at 40 ° C, while maintaining the pH constant at 6.5 and under vigorous stirring (low shear). In the reaction, N- (benzyloxycarbonyl) -L-aspartyl-L-phenylalanine methyl ester (hereinafter ZAPM) is formed as a condensation product and precipitates almost quantitatively as the product forms a dipeptide addition compound. unreacted with L-phenylalanine methyl ester, which has very low water solubility. After 20 hours, stirring was stopped and the reaction mixture was cooled to about 5 ° C. The catalyst is collected at the top of the reaction vessel and the precipitate as well as the liquid in the reaction mixture is drained from the bottom of the reaction mixture. The catalyst in the reaction vessel can be reused by first washing with 1 volume of 25 mM phosphate buffer (pH 6.5) followed by 1 volume of water. More than 99% of the total catalytic activity remains after the separation step. The ZAPM compound can be further processed into aspartame by methods known in the art (Removal of N-protecting group from aspartic acid unit, crystallization, etc.). Progress of ZAPM Synthesis · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

23 HPLC followed by Oyama et al., J.C.

S. Perkin II, 356 (1981).

Example 7

Penicillium culture medium, direct preparation of 6-APA

5.5 L in a fermentor such as Likidis and Schügerl, Biotechnol. Letters, 9, 229-232 (1987) / Penicillin G is fermented for 7 days. The contents of the fermentor were transferred to a reaction vessel using a low shear mixing propeller. The culture was adjusted to pH 8.0 with 4N ammonium hydroxide solution at 30 ° C and 50 g of the catalyst of Example 1 was added. The hydrolysis is carried out at a constant pH, the temperature is kept constant and effective stirring is used. After 5 hours, 98% of the fermented penicillin G is hydrolyzed and stirring is stopped. After a few minutes, the catalyst is decanted from the top of the reaction mixture. Hydrolysis products, which are 6-APA and phenylacetic acid, can be purified from cells or media by known methods (e.g., extraction, adsorption and crystallization). The catalyst was thoroughly washed with 50 mM phosphate buffer (pH 8.0) and used as a new wood. 95% of the total activity of the recovered catalyst is retained.

Claims (10)

PATENT CLAIMS A process for separating an undissolved catalyst from one or more other undissolved components, all of which are present in the same reaction mixture, characterized in that the catalyst has a lower density than the density of the liquid in the reaction mixture and that the other undissolved components higher density than the density of the liquid in the reaction mixture and separating an undissolved component which may optionally contain a portion of the liquid in the reaction mixture from the reaction mixture containing other undissolved components, preferably by physical processes. 2. A process according to claim 1 wherein the undissolved catalyst is an enzyme composition. 3. The process according to claim 1 or 2, wherein the undissolved catalyst is a fully or partially purified enzyme which is directly bound to particles having a density lower than the density of the liquid in the reaction mixture or combined. particles having a density lower than the density of the liquid in the reaction mixture. 4. A process according to claim 1 or 2, wherein the undissolved catalyst is a whole cell or ····· Ϊ ·· · «* 25 cell homogenate formulations are used which are bound together with particles which have a density lower than the density of the liquid in the reaction mixture and which may be hollow glass particles. The process according to any one of claims 1 to 4, wherein the catalyst comprises an enzyme which is co-bound with hollow glass particles in agarose or wherein the catalyst comprises whole cells or cell homogenates, optionally agarose bound with hollow glass particles. . Process according to any one of claims 1 to 5, characterized in that the reaction mixture comprises at least one component present in particulate form at the end of the reaction in addition to the undissolved catalyst, and optionally the particulate form in the reaction mixture during the reaction. result of precipitation. 7. A process according to any one of claims 1 to 6, wherein the beta-lactam derivative is one of 6-aminopenicillanic acid, 7-amino-deacetoxycephalosporinic acid, 7-aminocephalosporinic acid or 3-chloro- 7-Amino-deacetoxycephalosporinic acid and on the other hand side chain containing D-phenylglycine or Dp-hydroxyphenylglycine is prepared by catalytic reaction, from the corresponding beta-lactam core with a D-phenylglycine or Dp-hydroxyphenyl- using glycine, wherein the catalyst used is a non-dissolved catalyst having a density are lower ···· »* ···· ·· · · · • *« «* • ₀ · ♦ ♦ ··· · · 4 · · »· 9 9 ·· ·· ·» ·· · 4 · 26 is thinner than the density of the liquid in the reaction mixture, wherein the reaction mixture contains an additional undissolved component other than the catalyst, which component has a density greater than the density of the liquid in the reaction mixture. A process according to any one of claims 1 to 6, wherein an amino acid ester or amino acid amide is hydrolyzed to the free amino acid by catalytic reaction starting from the corresponding amino acid ester or amino acid amide, wherein the amino acid ester or amino acid is preferably -amide D-phenylglycine or Dp-hydroxyphenylglycine derivative, D-valine, L-valine, L-phenylalanine or L-tyrosine. A process according to any one of claims 1 to 6, wherein the peptide is prepared by catalytic reaction of an amino acid derivative with an amino acid amide or a lower alkyl ester derivative of an amino acid, preferably an N-protected protecting group. aspartyl L-phenylalanine methyl ester is prepared from a N-protected L-aspartic acid and L-phenylalanine methyl ester or D, L-phenylalanine methyl ester by catalytic reaction. The process according to any one of claims 1 to 9, wherein the insoluble enzyme composition comprises an enzyme such as a protease, a metallo-protease, a serine protease, a thermolysin enzyme, an amidase or an esterase, and preferably the insoluble the enzyme used in the enzyme preparation is obtained from microorganisms of Escherichia coli, Acetobacter pasteurianus, Acetobacter turbi27 dans, Xanthomonas citrii, Kluyvera citrophila or Bacillus megaterium, Pseudomonas melanogenum or penacillus thermoproteolyticus, hydrolysis of ampicillin or cephalexin.