WO1992012782A1

WO1992012782A1 - Process for separation of two solid components

Info

Publication number: WO1992012782A1
Application number: PCT/DK1992/000024
Authority: WO
Inventors: Svend Kaasgaard; Lars Guldbaek Karlsen; Ib Schneider
Original assignee: Novo Nordisk A/S
Priority date: 1991-01-25
Filing date: 1992-01-24
Publication date: 1992-08-06
Also published as: NO932576D0; HU9302146D0; EP0569462A1; BG97981A; JPH06504947A; HUT67012A; CZ148593A3; CA2101256A1; SK78593A3; NO932576L; MX9200313A; AU1235992A

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

PROCESS FOR SEPARATION OF TWO SOLID COMPONENTS

TECHNICAL FIELD

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 re¬ action mixture.

BACKGROUND ART

Separation of a precipitate from a solution is a known process which, for example, can be carried out by fil- tration or centrifugation. If a reaction mixture contains two or more different non-dissolved components, there is no easy way of separating the non-dissolved components from each other. Filtration followed by mechanical separation of the different non-dissolved components is almost always out of the question. In some cases, it may be possible, after a filtration to separate the non-dissolved components from each other by chemical methods which, however, normally are complicated.

There are several areas within the chemical field where there is a need for separating two non-dissolved components, present in a liquid, from each other. When considering the development of new chemical processes, such developments are usually cancelled if a reaction mixture containing two or more different non-dissolved components, which it is necessary to separate from each other, is ob¬ tained. One area where there is a need for a process for separating two or more non-dissolved components from each other is reactions where a non-dissolved catalyst is used. Such catalysts may be immobilized enzyme preparations. In reactions involving a catalyst, the price of the catalyst is often an important element in the overall cost of production and the need then arises to design a process in which the catalyst can be reused without significant loss of catalytic activity. The isolation and reusability of the catalyst is often impared when the catalyst is present in a reaction mixture together with another non-dissolved compo¬ nent which may be formed during the reaction or may be present during the whole process. One specific area which is important in connection with this invention is the synthesis of peptides, for example, synthesis of Aspartame™. In the production of Aspartame (L-α-aspartyl-L-phenylalanine methyl ester) , one of the critical steps involves an enzyme catalyzed coupling of an aspartic acid derivative and phenylalanine methyl ester hydrochloride (for example coupling N-benzyloxy- carbonyl-L-aspartic acid and L-phenylalanine methyl ester hydrochloride forming N-benzyloxycarbonyl-L-aspartyl-L- phenylalanine methyl ester (herein after designated ZAPM) ) . The ZAPM forms an addition compound with unreacted L- phenylalanine methyl ester (or D-phenylalanine methyl ester if present) with a very poor solubility and thus precipi¬ tates during the synthesis shifting the equilibrium of the reaction towards condensation. In order to separate the catalyst (for example Thermolysin™) from the reaction product, a semi-purified soluble enzyme preparation is used. The reaction product is dissolved and the enzyme is precipi¬ tated by addition of an organic solvent (for example acetone) and the enzyme can be removed, for example, by fil- tration. However, from 14% up to at least 60% of the cataly¬ tic activity is lost during the process (Nonaka et al. , US patent specification No. 4,212,945, and Meijer et al. in "Biocatalysts in Organic Syntheses" (Eds.: Tramper, van der Plas and Linko) , pp. 135 - 156 (1985)). The major loss of activity occurs during the precipitation and isolation of the enzyme.

Another specific area where there is a need for developing a process for separating a non-dissolved compo¬ nent from one or more other non-dissolved components is the enzymatic or catalytic acylation of a jS-lactam nucleus preparing penicillins or cephalosporins. Hence, as an example, this field is further dealt with in the following in order to show, in more details, the problems with the present processes. More specifically, the following relates to a process for the enzymatic preparation of β-lactam derivatives consisting of, on one hand, 6-aminopenicillanic acid (hereinafter designated 6-APA) , 7-aminodesacetoxy- cephalosporanic acid (hereinafter designated 7-ADCA) , 7- aminocephalosporanic acid (hereinafter designated 7-ACA) or 3-chloro-7-aminodesacetoxycephalosporanic acid, and, on the other hand, D-phenylglycine or D-p-hydroxyphenylglycine as a side chain by catalytic reaction of the corresponding β-lac¬ tam nucleus with a derivative of D-phenylglycine or D-p- hydroxyphenylglycine.

Today this group of semisynthetic /9-lactams are prepared in industry by chemical methods, for example, by reacting 6-APA, usually having its carboxyl group protected, with an activated side chain derivative, followed by the removal of the protecting groups by hydrolysis. For example, Ampicillin can be prepared by reacting 6-APA, having a suit¬ ably protected carboxylic group, with D-phenylglycyl chlor- ide, followed by hydrolysis. These reactions typically in¬ volve costly steps such as the use of temperatures below 0°C (in certain cases even below -25°C) , silylation reagents and organic solvents like methylene chloride, which is injurious to health and must be handled with care, and which is har - full to the environment.

Enzymatic production of, for example, .Amoxicillin, Cephalexin and Ampicillin from pure 6-APA and a D-phenylgly¬ cine derivative (such as a lower alkyl ester) is known from German patent application No. 2,163,792, Austrial patent No. 243,986, Dutch patent application No. 70-09138, German patent application No. 2,621,618 and European patent appli¬ cation having publication No. 339,751. Processes described in the prior art have typically used below 300 mM of the D- phenylglycine derivative and below 25 mM of 6-APA. The potential drawbacks of the known enzymatic methods for the production of these semisynthetic /3-lactams (none have yet been used in an industrial scale) are that the starting concentrations of 6-APA are very low (typically less than 25 mM) , thus making the isolation of the formed semisynthetic J-lactam more difficult and thus more costly. Furthermore, the reported yields are low, typically less than 85%, and a process for recycling the unreacted j8-lactam nucleus is required, which leads to more and costly unit operations.

One of the problems encountered by increasing the concentration of the substrates in the enzymatic synthesis of the semisynthetic 3-lactams is the rather low solubility of some of the substrates involved and of the products formed during the reaction. Hence, in order to run the pro¬ cess at economically feasible conditions, a concentration level of both^' substrates and products may be above their respective solubility, i.e., they may be present during all or part of the enzyme reaction both in a crystalline, amorphous or other solid form and in a soluble form, and the enzyme used should be in a reusable form, for example, immo¬ bilized. The need then arises to separate the catalyst from the reaction mixture containing among others products and unreacted substrates which may be present in both a crystal¬ line, amorphous or other solid form and in soluble form after the enzyme reaction is stopped, before the unreacted substrates and products are further purified as the purifi¬ cation steps may involve conditions harmful to the enzyme activity, for example, dissolution of all substrates and products at a low pH value (pH value: 0.5 - 2.0).

SUMMARY OF THIS INVENTION By using a non-dissolved catalyst, for example an immobilized enzymes, having a density lower than the density of the reaction liquid, it is possible to separate the non- dissolved catalyst from one or more other non-dissolved com¬ ponents present in the reaction mixture, provided that the last-mentioned non-dissolved components have a density higher than the density of the reaction liquid. One way of doing this is to separate the catalyst from the reaction mixture or slurry after termination of the reaction. This is possible, as the catalyst, for example after standing of the reaction mixture for a certain period of time, will accumu- late at the top of the reaction vessel from where it can be taken out, optionally together with a part of the reaction liquid. The reaction liquid can be removed from the catalyst in any known manner, for example, by filtration and, if desired, it may be flushed, for example, with an usual rinsing agent. The catalyst may be reused with little or no loss of activity. Before or after the removal of the non- dissolved catalyst from the reaction mixture, the other non- dissolved components having a density higher than the densi¬ ty of the reaction liquid, for example crystals, can be separated, for example by being taken out from the bottom of the reaction vessel, optionally together with the reaction liquid or a part thereof containing, among other, soluble substrates and products. The reaction products may be further purified and unreacted substrates may be further purified and recycled.

BRIEF DESCRIPTION OF DRAWINGS

The present invention is further illustrated by reference to the accompanying drawings, in which:

Fig. 1 shows a representation of the process of this inven- tion during the reaction; due to the agitation, the non-dis¬ solved catalyst and the other non-dissolved components are distributed throughout the reaction liquid.

Fig. 2 shows a representation of the process of this inven¬ tion after termination of the reaction; the non-dissolved catalyst and the other non-dissolved components, for example, a precipitate, appear at the top and the bottom, respectively, of the reaction liquid. DETAILED DISCLOSURE OF THIS INVENTION

This invention provides a process for separating a non-dissolved catalyst from one or more other non-dissolved components, all of which are present in a reaction mixture whereby the catalyst has a density less than the density of the reaction liquid and that the other non-dissolved compo¬ nents have a density which is above the density of the reac¬ tion liquid and that one of the non-dissolved components, optionally containing a part of the reaction liquid, is separated from the reaction mixture containing the other non-dissolved components. Usually, the separation of one of the non-dissolved components the reaction mixture containing the other non-dissolved component is performed by a physical process. Herein, the reaction liquid is defined as the liquid (or dissolved) part of the reaction mixture. The re¬ action mixture dealt with in this invention consists of at least two non-dissolved components and the so-called reac¬ tion liquid. The solubility of these non-dissolved compo- nents referes to the reaction mixture in question. One of the non-dissolved components is a non-dissolved catalyst which may be an enzyme preparation. Another non-dissolved component may be a desired reaction product which, partly, may be dissolved in the reaction liquid. Alternatively, the other non-dissolved components may be an impurity, a by-pro¬ duct or unreacted starting material, all of which may have a certain solubility in the reaction liquid. The non-dissolved component may be a precipitate formed during the reaction. The reaction liquid contains dissolved components and, op- tionally, one or more solvents. Preferably, the reaction liquid is a one phase system. In most cases, the reaction liquid contains water, although organic solvents may be added. Alternatively, the reaction liquid may be a two or multi phase system. The reactants may be fully or partly dissolved in the reaction liquid.

The non-dissolved catalyst to be used in this pro¬ cess may exist in the form of an immobilized enzyme prepara- tion having a density less than the density of the reaction liquid. In this preparation, the enzyme may be adsorbed, absorbed, covalently bound, entrapped or bound by ionic forces. Immobilization methods are known in the art. In a preferred embodiment of this invention, the non-dissolved catalyst contains an enzyme. In general, any enzyme can be used. Examples of enzymes are proteases, metalloproteases, serine proteases, thermolysine, amidases, esterases and peptidases. Also, an enzyme which is able to hydrolyze peni- cillin G, penicillin V, .Ampicillin or Cephalexin can be used.

To confer the catalyst a density lower than the density of the reaction liquid, the enzyme, being fully or partly purified or being a whole cell preparation, can be immobilized directly onto a material or particles with a density lower than the density of the reaction liquid.

Also, the non-dissolved catalyst, being fully or partly purified, may be immobilized on any material known in the art (for example glass, cell preparations or polymers, for example, agarose, polyacrylamide or styrene) and having material with a density lower than the density of the reac¬ tion liquid incorporated, conglomerated, coimmobilized or by other means entrapped into the preparation.

In another particular embodiment, the non-dissolved catalyst containing enzyme may be a whole cell or cell homo- genate preparation co-immobilized with a low density mate¬ rial.

As a material conferring the catalyst a density lower than the density of the reaction liquid, particles in any form of any known low density material may be used. Examples of such low density materials are plastic materials or polymers (for example polystyrene, polyurethane or poly¬ acrylamide) or natural products such as cork. Also, these materials could be used as hollow or porous materials, for example, hollow or porous fibres or hollow or porous beads, which materials may be filled with gasses or other low den¬ sity materials, possibly partly evacuated. Alternatively, a material conferring the non-dissolved enzyme preparation a density lower than the reaction liquid could be a more heavy material made hollow or porous (for example hollow glass beads) or it may be filled with gasses or other low density materials.

Specific areas wherein this invention has an important use is: 1) enzymatic processes performed directly in a ferma- tion broth, 2) the synthesis of peptides, 3) the hydrolysis of amides and esters and 4) the preparation of semisynthetic penicillins and cephalosporins.

6-APA is usually produced by an enzymatic hydrolysis of penicillin G or penicillin V which is produced by fermentation. Usually, the fermented penicillin G is puri¬ fied (for example by filtration of the culture broth, ex- traction 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 increasing the overall yield, 6-APA can be produced by adding, directly to the fermentation broth at the end of the fermentation, a catalyst having a density below the density of the reaction liquid, thereby hydrolyzing penicillin G. The pH value is adjusted, for example, to about 7.5, and the hydrolyzis is allowed to proceed by keeping the pH value constant at a temperature of about 30°C. The catalyst can be recovered from the top of the reaction mixture by decanta- tion and after a thorough wash, the catalyst can be reused. Formed 6-APA is purified by methods known per se (for example adsorption and crystallization) .

The use of an immobilized thermolysin with a density lower than the density of the reaction mixture in the above mentioned process for preparing Aspartame simplifies the separation step and reduces the loss of activity in the separation step to less than about 1%. In order to explain this invention in more details, the fol¬ lowing comments relates to the use of this invention within the field of enzymatic acylation of a 3-lactam nucleus. Examples of 3-lactam derivatives that may be produced by the process of this invention are Ampicillin, Amoxicillin, Cefaclor, Cephalexin, Cefadroxil and Cephaloglycin.

The derivative of D-phenylglycine or D-p-hydroxy¬ phenylglycine to be used as reactant in the process may be a lower alkyl (methyl, ethyl, n-propyl or isopropyl) ester or primary, secondary or tertiary amide. The methyl ester, ethyl ester, and the amide are preferred. The derivative may be used in the free form or in the form of a salt, for example, the HC1 salt or the H₂S0₄ salt.

The enzyme to be used may be any enzyme catalyzing the reaction in question. Such enzymes are usually termed penicillin amidases, penicillin acylases, or ampicillin hydrolases. A number of microbial enzymes are known to have this activity, derived from, for example, Acetobacter. Xan- thomonas. Mycoplana. Protaminobacter. Aeromonas (German patent application No. 2,163,792) Pseudomonas (Austrian patent No. 243,986), Flavobacterium (Dutch patent applica¬ tion No. 70-09138) , Aphanocladium. Cephalosporium (German patent application No. 2,621,618), Acetobacter pasteurianus (German patent application No. 2,163,792 A), Acetobacter turbidans (Takahashi et al. , Biochem.J. 137 (1974), 497 - 503) , Pseudomonas melano enum (Kim & Byun, Biochim.Biophys. Acta, 1040 (1990) , 12 - 18) , Xanthomonas citrii (European patent application having publication No. 339,751), Kluyvera citrophila (Okachi et a_l. , A r.Biol.Che . , 37 (1973), 2797 - 2804) , Escherichia coli (German patent application No. 2930794) , and Bacillus meqaterium (Chiang & Bennett, J.Bac- teriol.. 93 (1967), 302).

After separation of reaction liquid and crystals from the catalyst, the catalyst may be flushed with a rinsing agent and may be reused. Products may be further purified and unreacted substrates may be further purified and recycled. The substrates, inert compounds and the products may be present as precipitates during the reaction.

Generally, the reaction temperature may vary between 0°C and 40°C, especially 10 - 35°C. 20 - 35°C may be preferred for convenient operation. The suitable pH value depends on the type and purity of enzyme. With E.coli enzyme, it is typically in the range 5.5 - 8.0. The pH value should be controlled. Suitable reaction times are from several minutes to several hours, in particular from about 0.5 hours to about 24 hours. Suitable enzyme concentrations are from about 1 U/ml to about 200 U/ml (1 U = one unit of enzyme activity, see below) .

Recovery and purification of the desired product can be achieved by methods known per se, for example, by crystallisation.

The following, non-limiting examples further illustrate the present invention.

Example 1

Preparation of a catalyst with a density lower than the reaction liquid

Penicillin G acylase from E.coli was fermented

(vide, for example, Cole et al. , Met.Enzvmol. , 4_3 (1975) ,

698) and semipurified by methods known per se (precipitation with ammonium sulfate, adsorption, ultrafiltration etc.) to contain approximately 20 mg of protein/ml and 340 U/ml activity (units as defined below) .

Agarose beads (6% agarose) containing hollow glass beads (50 - 75 μ.m) were prepared by the procedure described by Pertoft and Hallen, J.Chro . 128 (1976) , 112 - 131, replacing the silica with hollow glass beads (50 - 75 μm) from, for example, Schott (Schott Glaswerke, D-6500 Mainz) . Then, the agarose beads were cross-linked using epichlor- hydrin (Porath and Axen, Meth.Enzymol. 44 (1976) , 23) . Agarose beads having the desired density properties were obtained by classifying the particles in a fluid bed.

The agarose beads were divinylsulfone activated by the method described by Lihme et al. , J.Chrgm. 376 (1986), 299 - 305. Hereafter, the enzyme was added and immobilised to the activated agarose beads.

The resulting catalyst had a particle size of 350 -

500 μm and an enzyme activity of 500 U/g moist catalyst.

However, the particle size and activity of the catalyst may vary considerably depending on the reaction conditions chosen or desired.

Enzyme activity

As definition of enzyme activity, the following is used: one unit (U) corresponds to the amount of enzyme that hydrolyses per minute 1 μmole penicillin G under standard conditions (5% penicillin G, 0.2 M sodium phosphate buffer

(pH value: 8.0) , 28°C) .

Example 2

Synthesis of Ampicillin D-phenylglycine methyl ester (hereinafter desig¬ nated D-PGM) and 6-APA were dissolved in 50 mM phosphate buffer adjusted to a pH value of 6.0 and equilibrated at 35°C for 10 minutes (final concentrations being 450 mM and 100 mM, respectively) . 0.75 g of moist catalyst (from Example 1) was added, the total volume being 20 ml, and the synthesis was allowed to proceed keeping a constant pH value (6.0) and maintaining an efficient stirring.

After approximately 0.5 hours, D-phenylglycine (hereinafter designated D-PG) started to precipitate from the reaction mixture and after about 3 hours, the Ampicillin concentration reached a maximum. At this point, the stirring was stopped, the catalyst accumulated at the top of the reaction vessel and the formed precipitate (a mixture of D- phenylglycine and Ampicillin) was drained from the bottom of the reaction vessel together with the reaction liquid (con¬ taining, among other, dissolved, unreacted substrates and products) .

Ampicillin can be further purified by known chroma- tographic and/or crystallization methods.

The catalyst in the reaction vessel was washed with 50 mM phosphate buffer (pH value: 6.0) and can be reused without a significant loss of activity (Table 1) .

Table 1

Penicillin G acylase activity before and after synthesis and following separation of catalyst from reaction mixture. The activity assayed by the p-dimethylaminobenzaldehyde method as described by Balasingham et al. , Biochi .Biophys.Acta, 276 (1972), 250 - 256.

Amount of Activity catalyst, l≤ϋ fu/q)

Before synthesis 0.75 498.5 After separation 0.74 497.2

HPLC analysis of reaction components

Column: RP LC-18, (250 x 4.6 mm; 5 μm) , Eluent A: 25 mM Phosphate buffer, pH value 6.5, Eluent B: acetonitrile, Gradient: Time B

Flow: 1 ml/minute Detection: 215 nm Retention times in minutes: 4.1 (D-PG) , 8.1 (6-APA), 13.9 (Ampicillin) , 18 (D-PGM) .

SUBSTITUTE SHEET Example 3

D-phenylglycine methyl ester, HCl-salt, (1.6526 g) and 7-aminodesacetoxycephaloranic acid (0.4278 g) (7-ADCA) were dissolved in 50 mM phosphate buffer (pH value: 6.5) and equilibrated to 35°C. 100 mg of catalyst (from example 1) was added (total volume 20 ml) and the reaction was allowed to proceed under efficient mixing, keeping the pH value and temperature constant.

After approximately 35 minutes of reaction, a precipitate was formed and after further 10 minutes of reaction, the stirring was stopped. After a short period of time, approximately 1 minut, the catalyst was situated in the top layer. The precipitate and reaction liquid con¬ taining, among other, Cephalexin, D-phenylglycine, D-phenyl- glycine methylester and 7-aminodesacetoxycephaloranic acid, was easily removed from the catalyst by draining from the bottom of the reaction vessel.

The Cephalexin may be further purified by the known methods and the catalyst may be reused with out any signifi- cant loss of activity. A rinsing step may be introduced before the catalyst is reused. More than 99% of the cataly¬ tic activity was retained after the separation step.

The reaction was followed by HPLC using the same conditions as described in Example 2 where 7-ADCA and Cephalexin elutes after 6.3 and 13.4 minutes, respectively.

Example 4

A slurry of 2.247 g of D-p-hydroxyphenylglycine amide (hereinafter designated HPGA) and 0.715 g of 6-amino- penicillanic acid was prepared in a 50 mM phosphate buffer

(pH value: 6.5) and equilibrated to 25 °C. The reaction was started by adding 2 g of catalyst (from Example 1) (final volume: 20 ml) , and the pH value was kept constant during the reaction by titration with 2 molar H₂S0₄. After 10 hours, the concentration of Amoxicillin reached a maximum and the reaction mixture contained, among other, crystals of Amoxicillin and unreacted HPGA. The stirring was stopped allowing the catalyst to accumulate at the top of the reaction liquid and the crystals to precipitate. Crystals and liquid were drained from the bottom of the reaction vessel. The Amoxicillin may be further purified by methods known in the art.

The catalyst in the reaction vessel may be reused, without loss of catalytic activity (more than 99% of the activity was retained) , directly or preferably after being flushed with water or phosphate buffer. The reaction was followed by the same HPLC system as described in Example 2 but using 5% acetonitrile in 95% 25 mM phosphate buffer (pH value: 6.0) for an isocratic elu- tion (1 ml/min) of the compounds. These conditions gives the following retention times (in minutes) for the compounds of interest: 2.5 (D-p-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 a pH value of 6.0, 0.75 g of moist catalyst (from Example 1) was added, the total volume being 100 ml. The hydrolysis was allowed to proceed at 35°C keeping the pH value constant at 6.0 by titration with 2 M sulfuric acid, while main¬ taining an efficient stirring. After 3 hours, the HPGA was fully hydrolyzed to the free acid, D-p-hydroxyphenylglycine (hereinafter designated HPG) , and the reaction mixture appeared as a slurry of cata¬ lyst and partly precipitated HPG.

When stirring was stopped, the catalyst accumulated at the top of the reaction mixture and the HPG precipitate was drained from the butto of the reaction vessel together with the reaction liquid containing, among other, salts and dissolved HPG.

The HPG can be further purified by known chromatographic and/or crystallization methods.

The catalyst in the reaction vessel was washed in 50 mM phosphate buffer (pH value: 6.0) and was reused with- out a significant loss of catalytic activity (less than 1% of the total activity were lost during the synthesis and following separation) .

The HPLC method described in Example 4 was used for following the reaction.

Example 6

Preparation of a Thermolvsin catalyst with a density lower thanthe reaction liquid.

8 g of Thermolysin (Sigma P-1512) were dissolved in 25 mM of phosphate buffer (pH value: 7) to approximately 20 mg protein per ml and approximately 1500 U per ml. Agarose beads were prepared and activated as described in Example 1 and the protease was added and immobilized to the activated agarose beads. The activity of the catalyst was approximate- ly 3000 U/g. The activity was measured by the casein digestion method (1 unit will hydrolyze casein to produce color equivalent to 1.0 μmole of tyrosine per minute at a pH value of 7.5 at 35°C (color by Folin-Ciocalteu reagent)).

Synthesis of N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester fan intermediate in the synthesis of Aspar¬ tame) .

0.1 moles of N-benzyloxycarbonyl-L-aspartic acid and 0.25 moles of L-phenylalanine methyl ester hydrochloride were dissolved in water and adjusted to a pH value of 6.5 and a final volume of 350 ml. The substrates were eqili- brated to 40°C for 10 minutes after which 15 g of catalyst was added. The reaction was allowed to proceed at 40°C keeping the pH value constant at 6.5 maintaining an effi¬ cient low shear stirring. As the condesation product, N- benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester (hereinafter designated ZAPM) , is formed during the reac¬ tion, it precipitates almost quantitatively as the product dipeptide forms an addition compound with unreacted L- phenylalanine methyl ester with a very poor solubility in water. After 20 hours, the stirring was stopped and the reaction mixture was cooled to approximately 5°C. The cata¬ lyst accumulated at the top of the reaction vessel and the precipitate plus reaction liquid was drained from the bottom of the reaction vessel. The catalyst in the reaction vessel can be reused after being washed with first 1 volume of 25 mM phosphate buffer (pH value: 6.5) followed by 1 volume of water. More than 99% of the total catalytic activity were retained after the separation step. The ZAPM can be further processed to Aspartame by methods known per se (removal of the N-protection group of the aspartic moiety, crystalliza¬ tion etc.). The HPLC method of Oyama et al. , J.C.S. Perkin II, 356 (1981) , was used to follow the synthesis of ZAPM.

Example 7

Production of 6-APA directly in a Penicillium culture broth. Penicillin G was fermented in a 5.5 1 fermentor (vide, for example, Likidis and Schύgerl, Biotechnol.Let¬ ters, 9. (1987), 229 - 232), for 7 days. The content of the fermentor was transferred to a reaction vessel with a low shear stirring propeller. The pH value in the culture broth was adjusted to 8.0 with 4 N NH₄0H at 30°C and 50 g of the catalyst from Example 1 was added. The hydrolysis was carried out at a constant pH value and temperature main¬ taining an efficient stirring. After 5 hours, 98% of the fermented penicillin G was hydrolyzed and the stirring was stopped. After a few minutes, the catalyst was decanted from the top of the reaction mixture. The hydrolysis products, 6- APA and phenylacetic acid, can be purified from the the cells and the medium by methods known per se (for example extraction, adsorption and crystallisation) . The catalyst can be reused after a thorough wash with 50 mil phosphate buffer (pH value: 8.0). 95% of the total activity was found in the recovered catalyst.

Claims

1. A process for separating a non-dissolved cata¬ lyst from one or more other non-dissolved components, all of which are present in the same reaction mixture, character- ized in that the catalyst has a density less than the densi¬ ty of the reaction liquid and that the other non-dissolved components have a density which is above the density of the reaction liquid and that one of the non-dissolved compo¬ nents, optionally containing a part of the reaction liquid, is separated from the reaction mixture containing the other non-dissolved components, preferably, using a physical pro¬ cess.

2. The process, according to Claim l, in which the non-dissolved catalyst is an enzyme preparation.

3. The process, according to Claim 1 or 2, in which the non-dissolved catalyst comprises an enzyme, fully or partly purified, immobilized directly with particles with a density lower than the density of the reaction liquid, or co-immobilized with particles with a density lower than the density of the reaction liquid.

4. The process, according to Claim 1 or 2, in which the non-dissolved catalyst is a whole cell or cell homoge- nate preparation co-immobilized with particles, which par¬ ticles have a density lower than the density of the reaction liquid, and which particles may be hollowed glass beads.

5. The process, according to any one of the pre¬ ceding claims, in which the catalyst contains an enzyme co- immobilized with hollow glass beads in agarose or in which the catalyst contains whole cells or a cell homogenate immo- bilized with hollow glass beads, optionally, in agarose.

6. The process, according to any one of the pre¬ ceding claims, in which at least one of the components in the reaction mixture, besides the non-dissolved catalyst, is present in a particulate form at the end of the reaction, and, optionally, the particulate form is a result of a pre¬ cipitation in the reaction mixture during the reaction.

7. A process, according to any one of the preceding claims wherein a β-lactam derivative consisting, on one hand, of 6-aminopenicillanic acid, 7-aminodesacetoxycephalo- sporanic acid, 7-aminocephalosporanic acid or 3-chloro-7- aminodesacetoxycephalosporanic acid and, on the other hand, a side chain of D-phenylglycine or D-p-hydroxyphenylglycine is prepared by a catalytic reaction of the corresponding β- lactam nucleus with a derivative of D-phenylglycine or D-p- hydroxyphenylglycine wherein the catalyst used is a non-dis¬ solved catalyst having a density which is lower that the density of the reaction liquid and which reaction mixture in addition to the catalyst contains a further non-dissolved component having a density which is higher that the density of the reaction liquid.

8. A process, according to any one of the Claims 1 through 6, characterized in that an amino acid ester or an amino acid amide is hydrolyzed to the free amino acid by a catalytic reaction of the corresponding amino acid ester or amino acid amide and, preferably, the amino acid ester or amino acid amide is a D-phenylglycine or a D-p-hydroxyphen- ylglycine derivative, D-valine, L-valine, L-phenylalanine or L-tyrosine.

9. A process, according to Claim 1 through 6, char¬ acterized in that a peptide is prepared by a catalytic reac¬ tion of an amino acid derivative and a derivative of an amino acid amide or lower alkyl ester and, preferably, an N- protected L-aspartyl-L-phenylalanine methyl ester is formed by a catalytic reaction of an N-protected L-aspartic acid and L-phenylalanine methyl ester or D,L-phenylalanine methyl ester.

10. The process, according to any one of the pre¬ ceding claims, in which the enzyme in the non-dissolved en¬ zyme preparation is a protease, a metalloprotease, a serine protease, a thermolysin, an amidase or an esterase and, pre- ferably, the enzyme in the non-dissolved enzyme preparation is obtainable from members of Escherichia coli. Acetobacter pasteurianus. Acetobacter turbidans. Xanthomonas citrii. Kluyvera citrophila or Bacillus meqaterium. Pseudomonas melanoqenum or Bacillus thermoproteolyticus. or, preferably, the enzyme in the non-dissolved enzyme preparation is able to hydrolyse penicillin G, penicillin V, Ampicillin or Cephalexin.