MXPA98000406A - An acilase of penicillin g immobilized, better - Google Patents

Abstract

The present invention relates to a new penicillin acylase G has been provided with surprisingly good performance. In preparing these new immobilized enzymes, the beta-lactam derivatives are prepared with high yield by the enzymatic reaction of a aminobeta-lactam ancestor and a corresponding acylating agent.

Description

AN ACILASE OF PENICILLIN G IMMOBILIZED, IMPROVED.

Technical field The invention relates to an improved immobilized penicillin G acylase. In addition, the invention relates to the preparation of β-lactam antibiotics by enzymatic acylation of the predecessor amino β-lactam nuclei with a corresponding acylating agent using said immobilized enzyme.

BACKGROUND AND FIELD OF THE INVENTION Enzymatic production of semi-synthetic ß-lactam antibiotics by acylation of the amino-β-lactam with an acid derived from the end of the chain, such as an amide or ester, is known from the German patent 158847, the applications of the international patents O92 / 01061 and WO93 / 12250, the North American patent 3816253, and the documents of the German patents Western 2163792 and 2621618. The enzymes used in the techniques are in most cases penicillin acylases obtained from Escherichia coli and are immobilized in various types of water-insoluble materials. REF: 26585 A disadvantage of the enzymatic methods known for the production of amoxicillin, ampicillin, cefadroxil, cephalexin, and cephradine is the high cost due to the selefivity of the immobilized enzyme. Said immobilized enzymes are able to condense the two activated sides of the chains such as D (-) - phenylglycine ida (PGA), methyl ester D (-) - phenylglycine (PGM), D (-) -4-hydroxyphenylglycinamide (HPGA), methyl ester D (-) -4-hydroxyphenylglycine (HPGM), D (-) -2, 5-dihydroxyphenylglicinamide (DPGA), and methyl ester D (-) -2, 5-dihydroxyphenylglycine (DPGM) with amino β-lactams 6-amino-penicillanic acid (6-APA), 7-aminocephalosporanic acid (7-ACA), 7-amino-3-cephem-4-carboxylic acid. On the other side, said immobilized enzymes may also hydrolyse the activated derivative at the end of the chain with the negligible acids at the end of the chain. Also the desired product is hydrolyzed to form the acid at the end of the chain and the amino-β-lactam ancestor. A high ratio between synthesis and hydrolysis can decrease the cost of the activated derivative at the end of the chain. From the application of the international patent O93 / 12250 it is known that the synthesis / hydrolysis relation for the synthesis of cefadroxil and cefalixin by acylase of penicillin G Escherichia coli immobilized in PCA is strongly dependent on the reaction conditions such as pH, concentration of the reagents and temperature. The influence of the nature of the carrier material in relation to the synthesis / hydrolysis however has not been taught. From European patent 222462 it is known that amino groups can be introduced into the carrier material by the addition of amino polymers such as alginamine, chitosan, pectin, or polyethylene imine to the constituent gellating base of the carrier. Surprisingly it has been found that the immobilization of penicillin G Escherichia coli acylase in a transporter consisting of a gelling agent and a polymer containing free amino groups that give an enzymatic catalyst with superior characteristics with respect to the synthesis / hydrolysis ratio in the reaction of condensation of the derivatives activated at the end of the chain with amino ß-lactams compared to the acylases of penicillin G immobilized in other transporters.

Brief summary of the invention. The present invention provides a penicillin G acylase immobilized on a carrier comprising a gelling agent and a carrier polymer containing free amino groups. It is preferred that the polymer be selected from the group consisting of alginatamine, chitosan, pectin, or polyethyleneimine, and it is even more preferred that the gelling agent be gelatin. In addition, by the similar application of an immobilized enzyme, an improved process for the preparation of a β-lactam derivative has been provided by an enzymatic reaction of the amino-lactam precursor with the corresponding acylating agent. Specific realizations. Examples of the β-lactam derivatives that can be produced by the process of this invention are amoxicillin, ampicillin, cefaclor, cefadroxil, cefprozil, cephalexin, and cephradine. The activity of acylase is independent for substituents at the 3-position of the cephem compounds, for example hydrogen, halogen, (light) alkoxy, methyl or substituted with methyl, for example (light) alkoxy, (light) alkanoyloxy, halogen , SR (where R is a (light) alkyl, an alkanoi (light), or an optionally substituted heterocyclic ring, N + -R6 (where R6 is a (light) alkyl or an optionally substituted heterocyclic ring). 1-6 carbon atoms A heterocyclic ring is defined as an unsaturated ring structure comprising at least one nitrogen, sulfur, or oxygen atom.The acylating agent may be a derivative of D (-) - phenylglycine, D (-) -4-hydroxyphenylglycine or D (-) - 2,5-dihydroxyphenylglycine with a light alkyl (methyl, ethyl, n-propyl, or isopropyl) ester or an amide which is unsubstituted in the -CONH2 group The corresponding amino-lactam contains the same nucleus as the ß-lact derivative In general, the reaction temperature of the process of this invention can vary between 0 ° C and 35 ° C. The optimum temperature depends on the substrates as mentioned in the European patent application 473008 and has not been optimized in the given comparative examples. The pH value conveniently depends on the nature and concentration of the substrate and is typically within the range of 5 to 9. It is used for convenient control of the operation. Suitable reaction times are from several minutes to several hours, in particular from 30 minutes to three hours. In the commercial process involving the use of a catalyst such as an enzyme, the price of the catalyst is often an important parameter in the overall economy of the process. In similar cases this is an advantage if the catalyst can be rejected without loss of catalyst activity. In the end, it is advantageous to provide enzymes in a reusable form, for example, a trapped or immobilized form. The following penicillin acylases Escherichia coli were investigated: Type A: Penicillin acylase Escherichia coli isolated as described in the application of the international patent W092 / 12782. Immobilization was performed as described in European Patent Application No. 222462. Type B: Commercially immobilized penicillin G Escherichia coli Acylase available from Recordati, Italy, as described in European Patent Application No. 473008. Type C: Commercially immobilized penicillin G Escherichia coli acylase available from Boehringer Mannheim, GmbH, Germany, known as Enzygel® The concentrations of available enzymes can range from 0.1 U / ml to 100 U / ml (1U = one unit of activity) of the enzyme, see below). Using the process according to this invention, extraordinarily high ratios of synthesis / hydrolysis can be obtained.

Definitions and methods of analysis. Enzyme activity As definition of penicillin G acylase activity the following are used: One unit (U) corresponds to the amount of enzyme that hydrolyzes per minute lμmol of penicillin G under standard conditions (100g / l of penicillin salt G potassium 0.05M potassium phosphate buffer, pH 8.0, 28 ° C.

HPLC Analysis Procedure A (Amoxicillin) Sample: Dilution 1:10 using 25% acetonitrile in 2mM potassium phosphate buffer, pH 5. Column: Cromosphere C18, 5μm (100x3 .Omm). Solvent: 25% acetonitrile in 12mM potassium phosphate buffer containing 0.2% sodium dodecyl sulfate, pH 2.6. Flow: lml / min. Detection: 214nm. Retention: HPG (1.9min); HPGA (3 lmin); 6- APA (3.4min); Amoxicillin (4.8min); HPGM (7.3min).

Procedure B (Cephalexin) sample: Dilution 1:10 using 25% acetonitrile in 2mM potassium phosphate buffer, pH 5.

Column: Cromosphere C18, 5μm (100x3 .Omm). Solvent: 29% acetonitrile in 5mM potassium phosphate buffer containing 0.2% sodium dodecylsulfate, pH 3.1. Flow: lml / min. Detection: 214nm. Retention: PG (0.8min); 7-ADCA (l3min); PGA (3.7min) cephalexin (6.2min); PGM (7.8min).

Procedure C (Cephradine). Sample: Dilution 1: 150 using 3% 1-propanol in 50mM phosphoric acid buffer, pH 3.0. Column: Nucleosil 120 3 C18 (250x4.Omm) Solvent: Eluent A: 50mM phosphoric acid buffer, pH 3.0 Eluent B: 50% eluent A, 50% cetonitrile. Gradient: 0-5min: 100% A; 5-10min: from 100% A to 70% A; 10-18min: 70% A; 18-18. lmin: from 70% A to 100% A. Flow: lml / min Detection: 220nm Retention: 7-ADCA (5.3min); DPG (6. Omin); DPGA (9.1); DPGM (15.9min); cephradine (18.5min).

Procedure D (Cefaclor). Sample: Dilution 1: 150 using 3% 1-propanol in 50mM phosphoric acid buffer, pH 3.0. Column: Nucleosil 120 3 C18 (250x4.Omm) Solvent: Eluent A: 50mM phosphoric acid buffer, pH 3.0 Eluent B: 50% eluent A, 50% cetonitrile. Gradient: 0-5min: 100% A; 5-10min: from 100% A to 70% A; 10-18min: 70% A; 18-18. lmin: from 70% A to 100% A. Flow: lml / min Detection: 22Onm Retention: 7-ACC (3.2min); PG (3.8min); PGA (5.6), • cefaclor (14.9min).

Procedure E (ampicillin) Sample: Dilution 1: 200 using 335 acetonitrile in 3.4mM potassium phosphate buffer, pH 6.9. Column: Chromosphere C18, 5μm (100x3.Omm) Solvent: 30% acetonitrile in 5mM potassium phosphate buffer containing 0.1% sodium dodecyl sulfate, pH 3.0. Flow: lml / min Detection: 214nm Retention: PG (1. Omin); 6-APA (l3min); PGA (2.6min); ampicillin (4.5min); PGM (5.8min).

Example 1 Synthesis of amoxicillin from 6-APA and HPGA using penicillin acylase G immobilized Escherichia col.

To an aqueous solution (50ml) containing lOmM of HPGA and 30mM of 6-APA was added 50U of penicillin acylase G Escherichia coli at 21 ° C. The pH was adjusted to 6.0 and the reaction was allowed to proceed under a nitrogen atmosphere with the pH control using a 0.05M solution of H2SO4 in water. Samples according to procedure A as described above. The molar ratio synthesis / hydrolysis (S / H) was calculated from the results thus obtained.

Table 1. 1 Synthesis of amoxicillin using enzyme A Table 1 .2 Synthesis of amoxicillin using enzyme B Example 2 Synthesis of amoxycillin from 6-APA and HPGA using immobilized penicillin G Eschßrichia coli acylase. To an aqueous solution (50ml) containing lOmM of HPGM and 30mM of 6-APA was added 50U of penicillin acylase G Escherichia coli at 21 ° C. The pH was adjusted to 6.0 and the reaction was allowed to proceed under a nitrogen atmosphere with the pH control using a solution of 0.05M H2S0 in water. Samples according to procedure A as described above. The molar ratio synthesis / hydrolysis (S / H) was calculated from the results thus obtained.

Table 2. 1 Amoxicillin synthesis using enzyme A.

Ex «ampio 3 Synthesis of cephalexin from 7-ADCA and PGA using penicillin acylase G immobilized Escherichia coli. 50U of penicillin G Escherichia coli acylase at 21 ° C was added to an aqueous solution (50ml) containing 10 mg of PGA and 30mM of 7-ADCA. The pH was adjusted to 7.0 and the reaction was allowed to proceed under a nitrogen atmosphere with pH control using a 0.05M solution of H2SO4 in water. Samples at different time intervals were analyzed according to procedure B as described above. The molar ratio synthesis / hydrolysis (S / H) was calculated from the results thus obtained.

Table 3. 1 Synthesis of cephalexin using enzyme A Table 3.2 Synthesis of cephalexin using enzyme B Example 4 Synthesis of cefradine 7-ADCA and DPGM.HCL using penicillin acylase G immobilized Escherichia coli. To an aqueous solution (120ml) containing 300mM of DPGM.HCL and 300mM of 7-ADCA was added immobilized penicillin G Escherichia coli acylase (the units are given in the tables). The pH was adjusted to the values given in the tables below and the reaction was allowed to proceed under a nitrogen atmosphere. The samples were analyzed at different time intervals according to procedure C described above. The molar ratio synthesis / hydrolysis (S / H) was calculated from the results thus obtained.

Table 4. 1 Synthesis of cephradine at pH 7. 5 using enzyme A (12U / ml).

Table 4.2 Synthesis of cephradine at pH 7. 0 using the enzyme.

Example 5 Synthesis of cefaclor from 7-ACCA and PGA using penicillin acylase G immobilized Escherichia coli. To an aqueous solution (120ml) containing PGA and 7-ACCA (concentrations and units of enzymes are given in the tables below) was added penicillin acylase G Escherichia coli. The pH was adjusted to 7.7 and the reaction was allowed to proceed under pH control using a 2.0M solution of H2SO4 in water. The samples were analyzed according to procedure D at different time intervals as described above. The molar ratio synthesis / hydrolysis (S / H) was calculated from the results thus obtained.

Table 5. 1 Synthesis of cefaclor from PGA (0. 5M) and 7 -ACCA (0. 6M) using enzyme type A (9U / ml).

Table 5.2 Synthesis of cefaclor from PGA (0. 5M) and 7-ACCA (0. 6M) using the type B enzyme (47U / ml).

Ex amp 'ampicillin 6 synthesis from 6-APA and PGA using penicillin acylase G immobilized Escherichia coli.

To an aqueous solution (100ml) containing 500mM of PGA and 300mM of 6-APA was added 100U of penicillin acylase G Escherichia coli. The pH was adjusted to 7.5 and the reaction was allowed to proceed under the control of pH using a solution of 6. OM of HCL in water. Samples at different time intervals were analyzed according to procedure E as described above. The molar ratio synthesis / hydrolysis (S / H) was calculated from the results thus obtained.

Table 6. 1. 1 Synthesis of Ampicillin using type A enzyme (as algin has been used as a polymer).

Table 6.1.2 Synthesis of Ampicillin using type A enzyme (as a polymer chitosan has been used).

Table 6.1.3 Synthesis of Ampicillin using the type A enzyme (polyethyleneimine has been used as a polymer).

Table 6.1.4 Synthesis of Ampicillin using the type A enzyme (polyethyleneimine has been used as a polymer).

Conversion (%) 5 10 Table 6.2 Synthesis of Ampicillin using enzyme B Table 6. 3 Ampicillin synthesis using enzyme C.

Claims (7)

Claims I. Penicillin G acylase immobilized on a carrier comprises a gelling agent and a polymer containing free amino groups. 2. The penicillin acylase G according to claim 1, characterized in that the polymer is selected from the group consisting of alginatamine, chitosan, pectin, or polyethyimine. 3. The penicillin acylase G according to claim 1 or 2, characterized in that the gelling agent is gelatin. 4. The penicillin acylase G according to any of the methods of the claims, characterized in that the enzyme used is Escherichia coli, Acetobacter pasteurianum, Xanthomonas citrii, Kluyvera citrophila, Bacillus megaterium or Alcaligenes faecalis. 5. The process for the preparation of a β-lactam derivative by an enzymatic reaction of the original amino-lactam with the corresponding acylating agent by applying an immobilized enzyme, characterized by the 5. The process for the preparation of a β-lactam derivative by an enzymatic reaction of the original amino-lactam with the corresponding acylating agent by applying an immobilized enzyme, characterized by the application of an enzyme defined in any of claims 1-4. 6. A process according to claim 5, characterized in that 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-dihydrophenylglycine. 7. A process according to claim 5 or 6, characterized in that the resulting β-lactam derivative is selected from the group consisting of ampicillin, amoxicillin, cefaclor, cephalexin, cefadroxil, cephradine, and cefprozil. 8. A process according to any of claims 5-7, characterized in that the reaction is developed at a temperature within the range of about 0 to about 35 ° C, preferably above and about 10 ° C. 9. A process according to any of claims 5-8, characterized in that the reaction is developed at a pH value within the range near and above 5 to about 9. AMENDED VERSION. CLAIMS.
1. I. Penicillin acylase G selected from the group consisting of Escherichia coli, Acetobacter pasteurianum, Xanthomonas citrii, Kluyvera citrophila, Bacillus megaterium or Alcaligenes faecalis, immobilized on a carrier comprising a gelling agent and a polymer selected from the group consisting of alginatamine, chitosan, pectin or polyethyleneamide.
2. 2. The penicillin acylase G according to claim 1, characterized in that the gelling agent is gelatin.
3. 3. The process for the preparation of a β-lactam derivative by an enzymatic reaction of the amino-lactam ancestor with the corresponding acylation agent that applies an immobilized enzyme, characterized by the application of an enzyme defined in any of claims 1-2.
4. 4. A process according to claim 3, characterized in that 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-dihydrophenylglycine.
5. 5. A process according to claim 3 or 4, characterized in that the resulting β-lactam derivative is selected from the group consisting of ampicillin, amoxicillin, cefaclor, cephalexin, cefadroxil, cephradine and cefprozil.
6. 6. A process according to any of claims 3-5, characterized in that the reaction is developed at a temperature within the range close to -20 ° C and close to 35 ° C, preferably near and above 10 ° C.
7. 7. A process according to any of claims 3-6, characterized in that the reaction is developed to a pH value within the above and close range of 3 to about 7.