# Patent

## Title
Method of producing a peptide mixture

## Abstract

Penicillin G acylase is immobilized by covalent bonding to a crosslinked mixture of a gelled gelling agent such as gelatin and a polymer containing free amino groups such as alginate amine, chitosan or polyethylene imine. The immobilized penicillin G acylase provides a higher synthesis/hydrolysis ratio as compared to immobilizing with other carriers when producing .beta.-lactam derivatives by a condensing reaction of an amino .beta.-lactam with an acylating agent. The acylating agent may be a derivative of D-phenylglycine, a derivative of D-p-hydroxyphenylglycine or a derivative of D-2,5-dihydro-phenylglycine. Examples of .beta.-lactam derivatives that can be produced are amoxycillin, ampicillin, cephaclor, cephadroxil, cephprozil, cephalexin and cephradine.

## Background
The present invention relates to an improved immobilized Penicillin G acylase. Furthermore, the invention relates to the preparation of .beta.-lactam-antibiotics by enzymatic acylation of the parent amino .beta.-lactam nucleus with the corresponding acylating agent using said immobilized enzyme.

Enzymatic production of semisynthetic .beta.-lactam antibiotics by acylation of the parent amino .beta.-lactam moiety with an activated side chain acid derivative, such as an amide or an ester, is known from Dutch patent 158847, European patent applications 339751 and 473008, international patent applications WO 92/01061 and WO 93/12250, U.S. Pat. No. 3,816,253, and West German patent documents 2163792 and 2621618. The enzymes used in the art are in most cases penicillin acylases obtained from Escherichia coli and are immobilized on various types of water-insoluble materials.

A drawback of the known enzymatic methods for the production of amoxycillin, ampicillin, cephadroxil, cephalexin, and cephradine is the high cost due to the selectivity of the immobilized enzyme. Said immobilized enzymes are capable of condensing activated side chain derivatives such as D(-)-phenylglycine amide (PGA), D(-)-phenylglycine methyl ester (PGM), D(-)-4-hydroxyphenylglycine amide (HPGA), D(-)-4-hydroxyphenylglycine methyl ester (HPGM), D(-)-2,5-dihydro-phenylglycine amide (DPGA), and D(-)-2,5-dihydrophenylglycine methyl ester (DPGM) with amino .beta.-lactams such as 6-amino-penicillanic acid (6-APA), 7-aminocephalosporanic acid (7-ACA), 7-amino-3-chloro-3-cephem-4-carboxylic acid (7-ACCA), 7-aminodesacetoxycephalosporanic acid (7-ADCA) and 7-amino-3-[(Z)-1-propenyl]-3-cephem-4-carboxylic acid. On the other hand, said immobilized enzymes will also hydrolyse the activated side chain derivatives to worthless side chain acids. Also, the desired product hydrolyses to form side chain acid and the parent amino .beta.-lactam. A high ratio between synthesis and hydrolysis will lower the cost of activated side chain derivative.

From international patent application Wo 93/12250 it is known that the ratio synthesis/hydrolysis for cephadroxil and cephalexin synthesis by Escherichia coli penicillin G acylase immobilized on Eupergit PCA is strongly dependent on the reaction conditions such as pH, concentration of reactants and temperature. The influence of the nature of the carrier material on the ratio synthesis/hydrolysis however, has not been taught.

From European Patent No. 222,462 and corresponding U.S. Pat. No. 5,137,818. It is known that amino groups can be introduced onto the carrier material by adding aminopolymers such as alginate amine, chitosan, pectin, or poly-ethylene imine to the base gelling constituent of the carrier.

Surprisingly, it has been found that immobilization of Escherichia coli penicillin G acylase on a carrier consisting of a gelling agent and a polymer containing free amino groups gives an enzymatic catalyst with superior characteristics regarding the ratio synthesis/hydrolysis in the condensation reaction of activated side chain derivatives with amino .beta.-lactams as compared to penicillin G acylases immobilized on other carriers.

The present invention provides Penicillin G acylase immobilized on a carrier comprising a gelling agent and a polymer containing free amino groups. Preferably the polymer is selected from the group consisting of alginate amine, chitosan, pectin, or polyethylene imine, and more preferably, the gelling agent is gelatin. Furthermore, by applying such an immobilized enzyme, an improved process for the preparation of a .beta.-lactam derivative by an enzymatic reaction of the parent amino .beta.-lactam with the corresponding acylating agent has been provided for.

Examples of .beta.-lactam derivatives that may be produced by the process of this invention are amoxycillin, ampicillin, cephaclor, cephadroxil, cephprozil, cephalexin, and cephradine. The acylase activity is independent of the substituents at the 3-position of the cephem compounds, e.g. hydrogen, halogen, (lower) alkoxy, methyl or methyl substituted with, for instance, (lower) alkoxy, (lower) alkanoyloxy, halogen, S-R.sub.5 (where R.sub.5 is (lower) alkyl, (lower) alkanoyl or an optionally substituted heterocyclic ring), N.sub.+ -R.sub.6 (where R.sub.6 is (lower) alkyl or an optionally substituted heterocyclic ring). By lower is meant 1-6 carbon atoms. A heterocyclic ring is defined as an unsaturated ring structure comprising at least one nitrogen, sulphur or oxygen atom.

The acylating agent may be a derivative of D(-)-phenylglycine, D(-)-4-hydroxyphenylglycine or D(-)-2,5-dihydro-phenylglycine such as a lower alkyl (methyl, ethyl, n-propyl or isopropyl) ester or an amide which is unsubstituted in the --CONH.sub.2 group.

The corresponding amino .beta.-lactam contains the same .beta.-lactam nucleus as the .beta.-lactam derivative prepared.

Generally, the reaction temperature of the process of this invention may vary between 0.degree. C. and 35.degree. C. The optimal temperature depends on the substrates as has been mentioned in European patent application 473008 and has not been optimized in the comparative examples given. The suitable pH value depends on the nature and concentration of the substrates and is typically in the range of 5 to 9. For convenient operation control of pH is used. Suitable reaction times are from several minutes to several hours, in particular from 30 minutes to three hours.

In commercial processes involving the use of a catalyst e.g. an enzyme, the price of the catalyst is often an important parameter in the overall economy of the process. In such cases it is an advantage if the catalyst can be reused without loss of catalytic activity. To this end, it is advantageous to have the enzyme in a reusable form, for example, in immobilized or entrapped form. The following immobilized Escherichia coli penicillin acylases were investigated: international patent application Wo 92/12782. Immobilization was carried out as described in European Patent No. 222,462 and corresponding U.S. Pat. No. 5,137,818. acylase from Recordati, Italy, as described in European patent application No. 473008. acylase from Boehringer Mannheim GmbH, Germany, known as Enzygel.RTM..

Suitable enzyme concentrations may be from 0.1 U.ml.sup.-1 to 100 U.ml.sup.-1 (1 U=one unit of enzyme activity, see below). Using the process according to this invention, extraordinary high synthesis/hydrolysis ratio's can be obtained.

Enzyme Activity

As definition of penicillin G acylase activity the following is used: one unit (U) corresponds to the amount of enzyme that hydrolyses per minute 1 .mu.mole penicillin G under standard conditions (100 g.l.sup.-1 penicillin G potassium salt, 0.05 M potassium phosphate buffer, pH 8.0, 28.degree. C.).

HPLC Analysis

Procedure A (amoxycillin) buffer, pH 5 0.2% sodium dodecyl sulphate, pH 2.6 min); HPGM (7.3 min)

Procedure B (cephalexin) buffer, pH 5 0.2% sodium dodecyl sulphate, pH 3.1 min); PGM (7.8 min)

Procedure C (cephradine) pH 3.0 eluent A, 50% acetonitrile Gradient: 0-5 min: 100% A; 5-10 min: from 100% A to 70% A; 10-18 min: 70% A; 18-18.1 min: from 70% A to 100% A. min); cephradine (18.5 min)

Procedure D (cephaclor) pH 3.0 eluent A, 50% acetonitrile Gradient: 0-5 min: 100% A; 5-10 min: from 100% A to 70% A; 10-18 min: 70% A; 18-18.1 min: from 70% A to 100% A.

cephaclor (14.9 min)

Procedure E (ampicillin) buffer, pH 6.9 0.1% sodium dodecyl sulphate, pH 3.0 min); PGM (5.8 min) Coli Penicillin G Acylase

To an aqueous solution (50 ml) containing 10 mM HPGA and 30 mM 6-APA is added 50 U of immobilized Escherichia coli penicillin G acylase at 21C. The pH is adjusted to 6.0 and the reaction is allowed to proceed under a nitrogen atmosphere with pH control using a 0.05 M solution of H.sub.2 SO.sub.4 in water. At different time intervals (see tables below) samples are analyzed according to procedure A as described above. The molar ratio synthesis/hydrolysis (S/H) is calculated from the results thus obtained. ______________________________________ Synthesis of amoxycillin using type A enzyme Time (min) 5      10  15    20  25    30  60    90  120 ______________________________________ S/H-ratio 1.1    1.3   1.3   1.4 1.2   1.2 1.2   1.1 1.1 ______________________________________ ______________________________________ Synthesis of amoxycillin using type B enzyme Time (min) 18  60       90    110    150  180 ______________________________________ S/H-ratio 0.6   0.7      0.7 0.7    0.6  0.5 ______________________________________ ______________________________________ Synthesis of amoxycillin using type C enzyme Time (min) 18        30  60       90  120 ______________________________________ S/H-ratio 0.7       0.7   0.6      0.6 0.5 ______________________________________ Coli Penicillin G Acylase

To an aqueous solution (50 ml) containing 10 mM HPGM and 30 mM 6-APA is added 50 U of immobilized Escherichia coli penicillin G acylase at 21.degree. C. The pH is adjusted to 6.0 and the reaction is allowed to proceed under a nitrogen atmosphere with pH control using a 0.05 M solution of H.sub.2 SO.sub.4 in water. At different time intervals (see tables below) samples are analyzed according to procedure A as described above. The molar ratio synthesis/hydrolysis (S/H) is calculated from the results thus obtained. ______________________________________ Synthesis of amoxycillin using type A enzyme Time (min) 10  20          40    60 ______________________________________ S/H-ratio    1.6   1.4         1.3 1.2 ______________________________________ Coli Penicillin G Acylase

To an aqueous solution (50 ml) containing 10 mM PGA and 30 mM 7-ADCA is added 50 U of immobilized Escherichia coli penicillin G acylase at 21.degree. C. The pH is adjusted to 7.0 and the reaction is allowed to proceed under a nitrogen atmosphere with pH control using a 0.05 M solution of H.sub.2 SO.sub.4 in water. At different time intervals (see tables below) samples are analyzed according to procedure B as described above. The molar ratio synthesis/hydrolysis (S/H) is calculated from the results thus obtained. ______________________________________ Synthesis of cephalexin using type A enzyme Time (min) 5   10          20    30 ______________________________________ S/H-ratio    6.5   4.2         3.4 2.4 ______________________________________ ______________________________________ Synthesis of cephalexin using type B enzyme Time (min) 5   10          20    30 ______________________________________ S/H-ratio    1.0   0.9         0.8 0.7 ______________________________________ Escherichia Coli Penicillin G Acylase

To an aqueous solution (120 ml) containing 300 mM DPGM.HCl and 300 mM 7-ADCA is added immobilized Escherichia coli penicillin G acylase (units as given in tables). The pH is adjusted to the value given in the tables below and the reaction is allowed to proceed under a nitrogen atmosphere. At different time intervals samples are analyzed according to procedure C as described above. The molar ratio synthesis/hydrolysis (S/H) is calculated from the results thus obtained. ______________________________________ Synthesis of Cephradine at pH 7.5 using type A enzyme (12 U .multidot. ml.sup.-1) Time (min) 26      62    75      106   120 ______________________________________ Conversion (%) 40      63      63      58    54 S/H-ratio 12   4.0   2.9   2.0   1.9 ______________________________________ ______________________________________ Synthesis of Cephradine at pH 7.0 using type B enzyme (33 U .multidot. ml.sup.-1) Time (min) 45    110        170     255 ______________________________________ Conversion (%) 33      49         51    68 S/H-ratio   2.4   1.7   1.4   1.4 ______________________________________ Coli Penicillin G Acylase

To an aqueous solution (120 ml) containing PGA and 7-ACCA (concentrations and enzyme units as given in tables below) is added immobilized Escherichia coli penicillin G acylase. The pH is adjusted to 7.7 and the reaction proceeds with pH control using a 2.0 M solution of H.sub.2 SO.sub.4 in water. At different time intervals (see tables below) samples are analyzed according to procedure D as described above. The molar ratio synthesis/hydrolysis (S/H) is calculated from the results thus obtained. ______________________________________ Synthesis of cephaclor from PGA (0.5 M) and 7-ACCA (0.6 M) using type A enzyme (9 U .multidot. ml.sup.-1) Time (min) 2           62    90 ______________________________________ Conversion (%) 3           58      66 S/H-ratio   2.0   6.2   4.0 ______________________________________ ______________________________________ Synthesis of cephaclor from PGA (0.6 M) and 7-ACCA (0.6 M) using type B enzyme (47 U .multidot. ml.sup.-1) Time (min) 26      62      124   161     266 ______________________________________ Conversion (%) 25      40        50    55      58 S/H-ratio   5.3   4.4   3.4   3.2   2.6 ______________________________________ Coli Penicillin G Acylase

To an aqueous solution (100 ml) containing 500 mM PGA and 300 mM 6-APA is added 100 U of immobilized Escherichia coli penicillin G acylase. The pH is adjusted to 7.5 and the reaction is allowed to proceed with pH control using a 6.0 M solution of HCl in water. At different time intervals samples are analyzed according to procedure E as described above. The conversion and the molar ratio synthesis/hydrolysis (S/H) are calculated from the results thus obtained and given in the tables below. ______________________________________ Synthesis of Ampicillin using type A enzyme (as polymer alginate amine has been used) Alginate amine (%) 0         1.0        2.0    3.0 ______________________________________ Conversion (%) 5      5        10   5      5    10 Time (min) 115 54 116 151 68 135 S/H-ratio 2.4 4.6 3.5 3.9 3.9 2.9 ______________________________________ __________________________________________________________________________ Synthesis of Ampicillin using type A enzyme (as polymer chitosan amine has been used) Chitosan (%) 0  1.0   1.5   2.0   2.5   3.0 __________________________________________________________________________ Conversion (%) 5  5  10 5  10 5  10 5  10 5  10 Time (min) 115 34 73 22 51 26 62 30 57 26 52 S/H-ratio 2.4 2.5 2.6 2.4 2.4 2.1 2.1 2.5 2.0 3.4 3.4 __________________________________________________________________________ ______________________________________ Synthesis of Ampicillin using type A enzyme (as polymer pectin has been used) Pectin (%) 0        2.0          3.0 ______________________________________ Conversion (%) 5        5      10      5    10 Time (min) 115 65 133 45 94 S/H-ratio 2.4 2.4 1.9 3.5 2.7 ______________________________________ ______________________________________ Synthesis of Ampicillin using type A enzyme (as polymer polyethylene imine has been used) Polyethylene imine (%) 0      1.0      2.0      3.0 ______________________________________ Conversion (%) 5      5       10   5     10   5     10 Time (min) 115 64 132 49 100 43 93 S/H-ratio 2.4 2.5 2.4 2.4 2.8 2.7 2.5 ______________________________________ ______________________________________ Synthesis of Ampicillin using type B enzyme Conversion (%) 5     10 ______________________________________ Time (min)      43      92 S/H-ratio   2.3   2.4 ______________________________________ ______________________________________ Synthesis of Ampicillin using type C enzyme Conversion (%) 5     10 ______________________________________ Time (min)      33      69 S/H-ratio   3.3   2.8 ______________________________________

## Claims

1. Penicillin G acylase immobilized by covalent bonding to a carrier comprising a crosslinked mixture of gelled gelatin and a polymer containing free amino groups.

2. Penicillin G acylase according to claim 1, wherein the penicillin G acylase is from Escherichia coli, Actetobacter pasteurianum, Xanthomonas citrii, kluyvera citrophila, Bacillus megaterium or Alcaligenes faecalis.

3. A process for the preparation of a .beta.-lactam derivative comprising reacting an amino .beta.-lactam with an acylating agent in the presence of the immobilized Penicillin G acylase, as defined in claim 1.

4. The process according to claim 3, wherein the acylating agent is selected from the group consisting of a derivative of D-phenylglycine, a derivative of D-p-hydroxyphenylglycine, and a derivative of D-2,5-dihydro-phenylglycine.

5. The process according to claim 3, wherein the .beta.-lactam derivative is selected from the group consisting of ampicillin, amoxycillin, cephaclor, cephalexin, cephadroxil, cephradine and cephprozil.

6. The process according to claim 3, wherein reacting is performed at a temperature in the range from about 0 to about 35.degree. C.

7. The process according to claim 3, wherein reacting is performed at a pH value in the range from above about 5 through about 9.

8. The process of claim 6 wherein the temperature is above about 10.degree. C.