IL31925A - Preparation of 6-aminopenicillanic acid by use of insoluble amidase - Google Patents

Description

Preparation of 6-aminopenicillanic acid by use of insoluble amidase BEEGHAM GROUP LIMITED♦ C: 30263 The present invention relates to an enzyme preparation, and particularly one prepared from deacylase enzymes known to split the amido bond in penicillins.

It is an object of the present invention to provide water-insoluble enzyme preparations that can be recovered from the reaction for re-use and are a convenient form for storing and transporting the enzyme. Also the resulting 6-aminopenicillanic acid is purer than when water soluble penicillin amidase is used, in its preparation from penicillins, since traces of the enzyme itself are not present in the product.

Accordingly the present invention provides water-insoluble enzyme preparation comprising a deacylase enzyme known to split the amido bond in penicillins, and bonded to a water-insoluble inert polymeric material.

Preferably the deacylase enzyme is obtained from bacteria, such as strains of Escherichia coli, when used for the splitting of benzylpenicillin into 6-aminopenicillanic acid: or for example from fungi and actinomycetes when used for the spllflting of phenoxymethylpenicillin.

Preferred polymeric materials include bromoacetylcellulose. and the azido derivative of carboxymethylcellulose. The water-insoluble ■'ψ derivatives may, for example, be prepared by the methods described in British Patent Specifications Nos. 916, 931 and 1 , 062, 596. The enzyme may be reacted for example with bromoacetylcellulose or coupled with CM-Sephadex ("Sephadex" is a registered trade mark) in presence of a suitable amide-forming reagent, such as a carbodii ide .

Other suitable polymeric materials include certain activated ion-exchange resins, such as the acid chloride of Zeokarb type 226, and ethylene-maleic anhydride resins cross-linked with hydrazine.

The water-insoluble derivatives retain the activity of the parent enzyme, but in use remain in the vrater-insoluble form, and so may be used in continuous processes in columns and other equipmen .

The invention also provides a process for preparing 6-aminopenicillanic acid, which process comprises contacting benzylpenicillin or phenoxy-methylpenicillin with a water-insoluble enzyme preparation as defined above, in presence of water, separating the resulting solution from the solid material, and recovering the 6-aminopenicillanic acid therefrom.

With the deacylase enzyme produced by E. coli, the water-insoluble preparation may be obtained by first isolating and purifying the free enzyme and then coupling it with the polymeric material. ψ φ Isolation of the enzyme may be effected by separating cells of E. coli from a fermented medium, killing the cells with aqueous formaldehyde, and harvesting them by filtration or centrifugation. The cells are then disrupted and the enzyme is separated from cell debris by centrifugation, and purified by ammonium sulphate precipitation and subsequent chromatography. Enzyme thus obtained is found to be pure by electrophoresis and it has high specific activity in splitting benzylpenicillin.

The following Examples illustrate the invention. In these Examples an amidase unit is numerically equivalent to the quantity of 6- aminopenicillanic acid (expressed as iig/ml.) produced in the conditions of the assay which were, the enzymation of -3 benzylpenicillin for 1 and 1/2 hours at pH 7.9 and 37°C.

EXAMPLE 1 The enzyme obtained from E. coli was agitated in aqueous solution with bromoacetylcellulose at room temperature for 30 hours and at pH 5.8 to give preparations having activities of 30,000 to 250,000 deacylase units/g. These materials were then kept at 4°C and pH 8.9 for 12 hours to give preparations having activities of 13,000 - 100,000 deacylase units/g. The enzyme preparations were freed from unreacted bromine by treatment with 0.05 M ethanolamine in water at °C for 24 hours, then they were washed twice with 0.15 aqueous sodium chloride, followed by water.

With one preparation obtained in this way having an activity of 40,000 units/g. dry bromoacetylcellulose, four successive enzymations of 2% aqueous benzylpenicillin were carried out under standard conditions. Each enzymation efficiency was satisfactory and the final preparation which retained activity was satisfactory for re-use; over 98$ pure 6-aminopenicillanic acid was isolated from the reaction mixtures, & -dji ifi'nj The azido derivative of carboxymethylcellulose was prepared by the known way.

Then 30g. of azido-carboxymethylcellulose was stirred in 3,000 ml. 0.05 M phosphate buffer at pH 8.7 and to this was added 5 X 10^ units of pure amidase enzyme (from E. coli). The mixture was stirred at 4°C for 24 hours. The active preparation was centrifuged and the solid was washed successively with 1 M NaCl, 0,5 M NaHCO^, 1 M NaCl and finally with water. The solid after final centrifugation was found to have an activity of 100,000 units/g. dry azido-carboxymethylcellulose.

A further preparation using the above-described method and which had an activity of 3 ,300 units/g. dry azido-carboxymethylcellulose was used for two consecutive enzymations of 2% benzylpenicillin on a 400 ml. scale at pH 7-8, 37°C. The active preparation was recovered at the end of the first enzymation for re-use in the secondj comparable enzymation efficiencies were obtained from the two experiments (67 and 70$, respectively).

EXAMPLE 3 200 mg. ethylene-maleic anhydride polymer resin (EMA 21) were stirred in 25 ml. phosphate buffer at pH 7.6 at 5°C. The cross linking agent, 2 ml. of 1% solution of hydrazine hydrate was added, followed after 1 - 2 minutes by 10 mg. of pure amidase enzyme (from E. coli and having an activity of 5-7 107 units/g. ) dissolved in a little phosphate buffer. The mixture was stirred in the ice bath for 2 hours and then kept overnight at 5°C. The active preparation was recovered and washed as described in Example 2 and found to have an activity of 360,000 units/g. dry resin.

Further experiments using the same method gave activities in the range of 100,000 to 60Q000 units/g. dry resin.

EXAMPLE 4 Zeokarb - 226 was converted to the acid chloride by refluxing with thionyl chloride and dimethylformamide in chloroform. The chlorine assay was l4#. To 30 ml. of phosphate buffer at pH 7.6 containing 250,000 units of pure amidase enzyme (from E. coli) was added 1.0 g. of the Zeokarb -22β aoid chloride and the reaction mixture was stirred for 5 hours at 5°C. The active preparation was recovered and washed as described in Example 2 and found to have an activity 119 , 000 units/g. dry Zeokarb -226 acid chloride.

EXAMPLE 5 Zeokarb -225 was converted to the sulphonyl chloride y refluxing with phosphorus oxychloride.

The chlorine assay was 12. 5$. To >0 ml of phosphate buffer at pH 8. 5 containing 270, 000 units of pure amidase enzyme (from E. coli) was added 1 . 0 g. of the Zeokarb -225 sulphonyl chloride and the reaction mixture was stirred for 5 hours at 5°C. The active preparation was recovered and washed as described in Example 2 and found to have an activity of 4, 800 units/g. dry Zeokarb -225 sulphonyl chloride.

EXAMPLE 6 Carboxymethylcellulose was converted to the acid chloride by reaction with thionyl chloride and dimethylformamide in pyridine. The chlorine assay was 9.9%. To 30 ml. of phosphate buffer at pH 8. 5 containing 270, 000 units of pure amidase enzyme (from E. coli) was added 1 . 0 g. of the carboxymethylcellulose acid chloride and the reaction mixture was stirred for 2 and 1/2 hours at 5°C. The active preparation was recovered and washed as described in Example 2 and found to have an activity of II.90O units/g. dry carboxymethylcellulose acid chloride. 2-amino-4 ,6-dichloro-193·5-triazine was prepared and attached to diethylaminoethylcellulose in known manner. 125 g« of one such preparation was resuspended in 1500 ml. of 0.05M 8 borate buffer pH 8.75 containing 1.2 x 10 units of partially-purified penicillin amidase. The slurry was stirred at room temperature overnight and the pH was controlled by the addition of dilute NaOH. The insoluble amidase preparation was recovered by filtration and washed with 0.02M phosphate buffer pH 7.8 containing 1 KaCl (twice) and with buffer alone (twice). The final product (125 g) was stored in buffer at 4°C. The activity of the insoluble enzyme was 2.6 x 10 6 units per gramme dry weight. 6 85.2 g of damp enzyme (total activity 59.0 x 10 units) were used to dephenylacetylate 1 1. of l> (w/v) potassium benzylpenicillin in 12 successive experiments at pH 7·8 and 37°C, The average yield of 6-amino-penicillanic acid was 90$.

EXAMPLE 8 1 g of hydrophilic copolymer based on acrylamide and having an acid hydrazlde as the functional site for coupling was added to 50 ml. 2M HC1 cooled to below 5°C 40 ml of 2 M02 cooled to below 5°C as added slowly with stirring. Stirring was continued for 1 minutes and the solid was recovered by filtration and washed 4 times with 0·05Μ phosphate buffer con-taining 2 x 10 units of partially purified penicillin amidase and stirred for 24 hours. After filtering, the solid was treated with 20 ml. of 0.01 phenol in 10$ sodium acetate for 15 minutes with stirring. The insoluble enzyme was then washed with 0.05 phosphate buffer pH 7.5 containing 0.5M NaCl and with buffer without salt. The damp solid weighed 5«81 g and had an activity of 4.8 x 10 units per gram.

The method described in Example 8 was repeatedaw-ith ^φ hydrophilic copolymer based on acr lamide and having an aromatic amino acid as the functional site for coupling. The product weighed 04 g and had an activity of 1.6 x 10^ units per gram* EXAMPLE 10 1 g (dry wt) of carboxymethylcellulose was added to 4 ml of a partially-purified amidase solution. To this was added a solution of dicyclohexylcarbodi-imide (400 mg) in tetrahydro-furan (1 ml) and water (2 ml) with stirring. The whole was stirred for a further 4 days at 4°C. The insoluble enzyme was then recovered by filtration and washed 3 times for 5 hours with water. The product weighed 636 mg and had an activity of 2.8 x 6 0 unife per gram dry weight.

EXAMPLE 11 The method in Example 10 was repeated using amino-ethyl-cellulose. The product weighed 410 mg and had an activity of 1.2 x 10 units per gram dry weight.

EXAMPLE 12 Polystyrene beads (approximate molecular weight 100,000) were ground in a colloid mill and then converted to polynitropolystyrene and finally to polyaminopolystyrene in known manner. g of this product was suspended in 1 1. of 2N hydrochloric acid cooled to between 0° and 5°C. The diazo derivative was prepared by the dropwise addition of 150 ml of 14 sodium nitrite solution (pre-cooled) with stirring. After a further 20 minutes excess HNO,, was destroyed by the addition . solution with vigorous agitation. 120 ml of a solution %f partially-purified amidase in \% sodium chloride was added and the pH was quickly adjusted to 6.0 by the addition of sodium acetate. Stirring was continued overnight at 0oC. The pH was adjusted to 8.0 with dilute sodium carbonate solution and stirring was resumed for a further 24 hours at 0°C. The product was harvested by filtration, washed with 0.9 sodium chloride and stored in 0.05 phosphate buffer pH 7.6. The insoluble enzyme (20 g) had an activity of 4.2 x 10^ units per gram.

EXAMPLE 13 A polymeric carrier with carboxyl anhydride groups was prepared by disseizing acrylamide (45 g) , Η,Ν'-methylene-bisacrylamide (3 g) and maleic acid (15 g) in 0.05 M phosphate buffer of pH 7.6 (350 ml) and mixing under nitrogen with a 5 ammonium peroxide disulphate solution (1 ml). The reaction mixture was heated briefly to 80°Gi After 1 hours the gellike polymerisate was pressed through a sieve having a mesh width of 0.5 mm, and after careful washing with water was freeze-dried. It was then heated in a vacuum for 2 hours at 180¾ Purified penicilli acylase (1 mg) which had been obtained from E, .coll and had a specified enzymatic activity of 1.5 x 10 deacylase units/mg were dissolved in water (20 ml), at a pH of 5.8 and 4°Q, 1.5 g of mixed polymerisate made as described above was added while stirring. The pH value of the reaction mixture was held constant at 5·8 with diluted aodium hydroxide. After 24: ours it was filtered and the insoluble enzyme successively washed with water (100 ml), 0.5 M sodium chloride solution- (300 ml) and water (100 ml). The moist, carrier-bonded enzyme (30< g wet) had a specific activity of By means of the enzyme preparation produced ±rr this-way, ten consecutive enzymatic fissions are carried out nder™ standard conditions with a 6$ solution of aqueous benzyl penicillin potassium salt. The enzyme preparation separated after the last enzjasaitic fission still shows sufficient activity for further repeated usages.

EXAMPLE 14 There were placed in an Erlenmeyer flask with a magneti stirrer at room temperature a 0.05 phosphate buffer of pH 4.5 (20 ml), a bead-like copolymer of 19 acrylic acid with 81 tetra ethylene glycol dimethacrylate (1 g), an aqueous solution of penicillin acylase from E. coli with an activity of 2·95 x 10 units/ml (20 ml) and isobutyraldehyde (50 mg) as a 5% aqueous solution. After agitation for 1 hour at room temperature, a newly made 10$ solution of 3-morpholinopropyl-isonitrile (0.5 ml) in water was added. The formulation was agitated for 22 hours at room temperature, the resin sucked off and washed with 0.05M phosphate buffer of pH 7.5 to which 1 mol of sodium chloride per litre has been added, and then with the same buffer without any addition. Residue and washing solutions contained 8.2 x 10 units ( 4$) and the washed enzyme resin 3.5 x 0 units, that is 9 of the original penicillinacylase activity.

EXAMPLE 1 A copolymer from 1 S glycidyl methacrylate and 8 $ tetra-ethylene glycol dimethacrylate (1 g), penicillin acylase solution in a 0.025 M phosphate buffer of pH 8.0 with an enzyme activity of 5-5 x 10^ units (100 ml) and 1 ml of toluene as a bacterio tat were placed in a 250 ml flask. The flask was closed and slowly shaken for 3 days at 34°C. The resin was. then filtered off and successively washed with 0.5 M sodium chloride 31925/2

Claims (1)

1. CLAIMS A enzyme preparation comprising a deacylase enzyme known to split the amido bond in penicillins and bonded to a inert polymeric An enzyme preparation as claimed in Claim wherein the deacylase enzyme is derived from An enzyme preparation as claimed in Claim wherein the deacylase enzyme is derived from Escherichia An enzyme preparation claimed in Claim wherein the deacylase enzyme is derived from fungi or An enzyme preparation as claimed in Claims 1 to wherein the polymeric material is anhydrid polymer resin with hydrazine or lose acid an A process for preparation as claimed in Claim 1 substantially as described with reference to any one of the specific Examples hereinbefore set A process for preparing acid which process comprises contacting benzylpenicillin or phenoxy m thylpenicillin or a salt thereof with an enzyme preparation as claimed in any one of Claims 1 to 6 in presence of water separating the resulting solution from the solid material and recovering the acid A process for preparing acid substantially as described with reference to any one of the specific Examples hereinbefore set when prepared by a process claimed in Claim 7 or insufficientOCRQuality