IL39158A - Enzymatic scission and synthesis of penicillins and cephalosporins - Google Patents

Abstract

1348359 Enzymatic preparation of cepnalosporins and penicillins SNAM PROGETTI SpA 27 April 1972 [28 April 1971] 19726/72 Heading C2A [Also in Division C3] The invention comprises a process for the treatment of a substance which contains a ring structure characteristic of penicillins or cephalosporins to convert it to another substance containing the ring structure, which process comprises contacting a reaction medium containing the substance with a filament in which is distributed in finely divided form at least one enzyme capable of causing the desired conversion, the or each enzyme being encased in the filament in such a manner that the or each enzyme can effect conversion and that it or they are substantially retained in the filament during the contacting. The enzyme or enzymes may be incorporated in a filament formed from a nitrated, esterified or etherified cellulosic polymer, a polyolefine, a polymer or copolymer of acrylonitrile, an acrylate, methacrylate, vinyl ester, vinyl chloride, vinylidene chloride or styrene, a polyamide or polyvinylbutyral. Suitable polymers include ethyl cellulose, poly-γ- methylglutamate and cellulose triacetate. Hydrolysis of natural penicillins may be carried out by this process. Such penicillins include those of Formula I wherein R is alkyl of 1 to 10 carbon atoms, with from 3 to 10 carbon atoms, aralkyl, R<SP>1</SP>OCH 2 - or R<SP>1</SP>SCH 2 -, wherein R<SP>1</SP> is alkyl, alkenyl, phenyl or optionally substituted phenyl. The pH of the reaction medium may be buffered at 8. The filament may contain an acylase derived from bacterial cells such as E. coli, B. megaterium, Aerobacter aerogenes or Alcaligenes faecalis cells. The filament may contain an enzyme derived from Actinomyces or fungal cells, e.g. Aspergillus, Penicillum, Botrytis cinerea, Fusanium, Mucor, Alternaria or Streptomyces cells. Synthesis of some penicillins, e.g. ampicillin, may be effected by the process of the invention. The invention also comprises a filament in which is distributed in finely divided form at least one enzyme capable of causing conversion of a substance containing a ring structure characteristic of penicillins or cephalosporins to another substance containing the ring structure, the enzyme being present in the filament in a manner such that it is retained in the filament, when the latter is contacted by a reaction medium containing the substance and that it is capable of effecting the conversion whilst being so retained. [GB1348359A]

Description

Enzymatic scission and synthesis of penicilins and cephalosporins SS& moaBTTI S.R.4.

C. 37183 This invention relates to a method of enzymatically treating an antibiotic substance selected rom the classes of penicillins and cephalosporins.

Although natural penicillins have been known for a considerable number of years, their utility as a titabtic substances has been reduced by the increasing immunity of microorganisms to such antibiotic substances. Ά considerable amount o research has been carried out to obtain related substances having a broader spectrum of anti-bacterial activity. These substances are usually similar in structure to natural penicillins, differing therefrom in the amide group reacted with the 6-araino group of the basic 6-amino penfeillanic acid structure of penicillins. Since such related penicillins are generally derived from natural penicillins, they are generally termed semi-synthetic penicillins. By the term "natural" penicillins as used herein is meant a penicilli produced by fermentation of Penicillum chrysogenum in the presence of suitable precursors. Although total syntheses for both natural and semisynthetic penicillins have been devised, owing to the relatively cheap fermentation procedures available for production of natural penicillins, such methods are generally used for the production of natural penicillins and the semisynthetic penicillins are obtained from the natural penicillins so produced.

In the production of semisynthetic penicillins from natural penicillins, it is generall necessary to first form 6-amino-penicillanic acid (i-APA) and then to acylate the 6-amino group with the desired acyl group to produce the desired new semisynthetic penicillin stable to ^-lactamase and having a larger spectrum of activity than the natural penicillins.

^ Hence 6-APA is a particularly valuable starting \^ material for the production of semisynthetic penicillins. is generally produced from natural penicillins rather than by direction fermentation with Penicillum ehrysogensi in the absence of an amide precursor because such a procedure is technically very complex and the yields are very low. Although chemical methods have been proposed for the conversio of \v natural penicillins to 6-APA, such methods are relatively \ expensive to operate, particularly in view of difficulties encountered in achieving the desired course of reaction in the presence of the hydrolysis mediuia. In generalv, too great a proportion of by-products is obtained to make the"process commercially feasible. More important are enzymatic procedures which employ cells of microorganisms which produce penicillin acylase. It is necessary to use these cells in relatively large amounts to shorten the hydrolysis time, and as these cells can be only used once, it is necessary to continually grow cells containing the enzymes. A further disadvantage of such a procedure is that the extraction of the hydrolysis products, 6-APA, om the reaction material obtained is particularly complex and the resulting product requires repeated crystallization and, even then, often contains proteinaceous material which is not removed, for example b enzymatic digestion with proteolytic enzymes, and is liable to cause such problems as allergy phenomena during administration of penicillins derived from the 6-APA.

An analogous situation arises with cephalosporin derivatives which are of similar structure to penicillins and find similar uses. In this case the valuable Intermediate for the production of semisynthetic cephalosporin is 7-amino a cephalosporo*nic acid. Other compounds which retain the basic 39158/2 One method for the preparation of 6-APA is known from Israel patent specification No, 31925» According to tha I patent specification benzylpenicillin or phenoxy methyl-penicillin or a non-toxic sal thereof Is contacted with a water-insoluble enzyme preparation comprising a deacylase enzyme which is bonded to a water-insoluble polymeric substrate.

Apart from the difficulty to chemically bind an enzyme to a substrat withou deactivating the enzyme, the direct contact of the enzyme with the reaction medium may also lead to deactivation of the enzyme.

According to the presen invention, there is provided a process for the enzymatic treatment of a substance which .? contains a ring structure characteristic of penicillins o cephalosporins, which comprises, contacting a reaction medium containing the substance wi h a filament throughout which is distributed in inely divided form at least one enzyme capable of causing the desired conversion, the enzyme(s) being encased in the filament in a manne such that the or each enzyme can effect said conversion, and such that the or each enzyme is substantially retained in the filament during the contacting* ~ The presen invention also provides the enzyme-containing ilaments per se.

The present invention makes it possible to avoid the release of enzyme and its dispersio into the reaction medium employed. - · The filamentary structures employed and the method for their production ca be, for example, of the type described in Israel patent specification Ho. 32406, according to which the filaments encasing the enzyme are produced from a polymeric solution capable of being produced in the form of a filament, wherein are dispersed very small drops of the enzymatic compaKnds so as to form a emulsion. The emulsions so obtained can be wet or dry spun to provide filaments having small cavities in which are disposed the enzymes which are separated from the exterior of the filaments by a very thin membran which prevents the loss of enzymes into the reaction medium in which the enzyme is employed, but allows the reaction medium to pass therethrough to contact the enzyme which is then able to exert its catalytic action.

Examples of polymers which may be used to encase the enzymes are nitrated, etherified and esterif ed cellulosic polymers polyolefins, polymers and copolymers obtained from acrylonitrile, acrylates, methacrylates, vinyl esters, vinyl chlorides, vinylidene chloride, styrene, polyamides and polyvinyl butyral. A particular advantage of the use of the enzyme-containing filaments is that the filaments have been show to retain their catalytic activity for a least one year when in continual use. This makes the use of the filaments particularly useful commercially. The use of the enzyme(s> in a manner which prevents its/thei entrainment in the product obviates the need for removal of the enzyme from the product; this allo s continuous working, thereby simplifying the plant employed.

The enzyme-containing laments are particularly suitable for use in the conversion of penicillins and cephalosporins to the corresponding 6-amino and 7-amino derivatives respectively. For this purpose, it is necessary for at least one of the enzymes to be an acylase. Where the basic 6-amino penieillanic acid or 7-amino cephalosporanic acid structure is substituted in the 6 or 7 position respectively by ah a-phenoxy methyl group as in the case for example of penicillin V (cs-phenoxy methyl penicillin) 39158/2 the or each enzyme employed will preferably be in a crude cell extract obtained from moulds or actinomycetes, for example Aspergillus, Peniclllium, Botrytis cinerea, Fusarium, Mucor, Alternaria, Streptomyces, When the substituent at the 6-amino or 7-amino position of the pasic penicillanic acid o cephalo-sporonic acid structure respectively is a benzyl grouppas in penicillin G (benzyl penicillin), the or each e zyme is preferably derived from bacteria such as E Coli, B Megaterium, Aerobacter Aerogenes and Alcaligenes faecalls.

When the enzyme-containing filamentary structures are employed in the hydrolysis of penicillins to produce 6-APA, an aqueous solution of the salt of the penicillin is generally used, the solution containing the salt in a concentration of from 1 to 6% by weight, preferably rom 2 to 3% by weight. The reaction solution J.s kept slightly alkaline, generally at a pH value of 8.0 by addition of alkali. In a preferred operating procedure, the filamentar structure which, before use is washed to remove adsorbed impurities , is preferably packed in a column through which the reaction solution is continuously passed in a cycle. To avoid difficulties in the control of pH a differen heights of the column, the penicillin or cephalosporin solution employed is preferably buffered at pH 8. Hydrolysis can be carried out at temperature of from 50°C to 60eC, preferably from 37eC to 40°C. _ Acylases which yield 6-amino penicillanic acid and 7-amino cephalosporanic acid structures from penicillin and cephalosporin precursors respectively under alkaline conditions are known to catalyse the reverse reaction under acid conditions. This affords a method of readily producing semisynthetic penicillins and cephalosporin from reaction mixtures containing 6-amino penicillanic acid or 7-aminocephalosporanic acid and a suitable carboxylic acid or deri at weethereof. The same apparatus as used for the hydrolysis procedure described above can be employed in the synthesis of semisynthetic penicillins or cephalosporins to yield the desired amide or n-acyl derivative or glycine. For the synthesis reaction, the pH of the reaction medium should be kept at about 6.0. As with the hydrolysis reaction, synthesis can be carried out at a temperature of from 50 to G0eC, preferably from 37 to 40°C. The reaction solutio preferably has a concentration of from 1 to 3% by weight. In addition to carrying out the hydrolysis or synthesis procedure described above on a continuous basis using a column packed with the filamentary structures containing the enzyme, it is possible to carry out the procedure on a discontinuous basis with the filamentary structures present in a reaction vessel. When hydrolysis is complete, the reaction mixture is removed therefrom and replaced by a fresh mixture and the reaction is repeated.

Penicillins which will be deacylated by the hydrolysis procedure described above will generally be natural penicillins having the following general formula:- wherein R is alkyl with from 1 to 10 carbon atoms, alkenyl with from 3 to 10 carbon atoms, aralkyl, R'0CH2— or R'SCH2 wherein R* is alkyl, alkenyl, phenyl or phenyl substituted for example with halogen, nitro, alkyl, alkenyl or alkoxy groups.

Penicillins which can be synthesised from 6-APA produced by the hydrolysis procedure and which are of substantial therapeutic use include 6- (a-phenoxypropionamido) -penicillanic acid, 6- (a-phenoxybutyramido) -penicillanic acid, 6- (2,6-diraethoxy-benzoaraido) -penicillanic acid, 6- (5-methyl~3-phenyl-4-isoxazol- ,- * acetamido) -penicillanic acid.

In an analogous manner, it is possible to employ the method of the present invention to enzymatically deacylate 7- acyl amino cephalosporin derivatives to produce the corresponding 7-amino cephalosporih derivatives. Thus, for example, 7-(2- phenyl acetamido) cephalosporanic acid and 7-(2-phenoxy acetamido) cephalosporanic acid can be deacylated to produce 7-amino oxy cephalosporanic acid and 7-(2-phenyl acetamido desacet-y-4- cephalosporanic acid can be deacylated to produce 7—amino desa- oxy cety-l- cephalosporanic acid. An example of a synthesis product which can be obtained from such 7-amino cephalosporanic acid compound is 7- (2-D-a-aminophenylacetamido) cephalosporanic acid.

The following Examples illustrate the invention: Example 1 (a) Production of supported enzyme Cells of E. Coli (60 gram moist weight) obtained by fermentation in a suitable nutrient in the presence of phenyl- acetitic acid while being subjected to ultrasonic waves were *— placed in a phosphate buffer (0.01 Μ,ρΗ 7.0). The raw fermentation product was then heated for three minutes at 50eC and ammonium sulphate solution at pH 5.5 was added as precipitant. The fractions at 25 to 75% saturation were retained and were dissolved in 30 ml of a phosphate buffer (0.01 M, pH 8.0 containing 30% by volume of glycerol) .

The enzyme-containing solution obtained was then added to a solution obtained by dissolving 20 gram of cellulose triacetate -nitrate in 280 gram of methylene dichloride, with agitation.

A binary emulsion was obtained by vigorous agitation of the mixture so produced at 0°C for 30 minutes. The emulsion was then fed to a spinneret maintained at -6*C and was spun in an atmosphere of nitrogen into toluene having the ambient emulsion in the toluene were wound around a spindle. The ^ continuous filaments were dried in vacuo for several hours to remove the toluene. 37 gram of the filaments were then placed in a jacketed glass distillation column 77 centimetres high and 3 centimetres in diameter and were washed with glycerol until the wash liquid obtained from the column was free from proteinaceous material. (b) Deacylation of penicillin 600 ml of an aqueous 2% by weight solution of potassium penicillin G were continuously cycled through the column triacetate containing the washed enzyme-containing cellulose -niter-afeo-filaments. The pH of the penicillin solution was monitored by means of a pH meter and 0.5 N.NaOH was continuously added to maintain the pH of the solution at 8.0. The column was kept at 37eC throughout. After about 5 hours, the rate at which the NaOH needed to be added to maintai the pH of the reaction solution at 8.0 had dropped to 5% of the initial rate. The penicillin solution was then collected from the reaction system, was cooled to 3eC, acidifed with 2N.HC1 until its pH dropped to 3.0, 33.5 ml of the 2N.HC1 being required for this purpose.

The acidifed reaction solution was then twice extracted with 200 ml portions of butyl acetate. The presence of unreacted penicillin G in the organic extracts was determined by the hydroxylamine text yielding a hydroxamic acid derivative which gave a colour reaction with a nickel salt. By comparison of the intensity of the colouration obtained with a standard, it was possible to determine that the first extract contained 298 mg. of unchanged penicillin G and that the second extract contained 2 mg of unchanged penicillin G. Since the total quantity of penicillin G used was 12 gram, 97% thereof had undergone conversion. Chromatographic analysis of the organic phase did The pH of the aqueous phase (800 ml) which was free from unconverted penicillin and from phenyl acetic acid was adjusted to 7.0 by addition of 33 ml of 2N.NaOH and was concentrated in vacuo at 37°C until its volume was reduced to 110 ml. The concentrated solution so obtained was cooled to 0eC and its pH was adjusted to 4.2 by addition of 3N.HC1. 6-APA precipitated and was separated by filtration and dried, in vacuo, at 45eC until constant weight was reached. 6.6 gram of 6-APA were obtained. The concentration of 6-APA in the acidifed mother liquor (130 ml) was 0.4% by volume.

Evidence of the retention of the penicillin structure in the crystalline product was obtained by the 97.5% conversion of the 6-APA to a hydroxyamic acid derivative by reaction with hydrox lasiine. The IR spectrum, chromatographic analysis and optical rotary power of the crystalline salt showed it to be a practically pure product.

The above deacylation of penicillin G was repeated 100 times over a six month period using the same sample of enzyme-containing filaments. 6-APA was produced in each case without any loss of yield over the six month period.

Example 2 (a) Production of supported enzyme triacetat Example 1 (a) was repeated to produce cellulose -nitrate filaments containing an enzymatic preparation obtained from cells of E. Coli, which cells had been purified fifteen times in relation to the purification of the cells of B. Coli used in Example 1. The enzyme-containing filaments obtained possessed an activity ten times the activity of an equal weight of filaments produced in Example 1(a). Before use, 37 gram of the fibres packed in a column of the type used in Example 1 and having a capacity of 480 ml, were washed with water and glycerol (b) Deacylation of penicillin 3 Litres of a 2% by weight aqueous solution of potassium penicillin G were placed in a 5 litre container and a cycle was set up between the container and the column and back to the container, employing a peristaltic pump to transfer the aqueous penicillin solution to the column. It was thus possible to achieve continuous transfer of reaction solution to the column. In order to reduce the time required for penicillin hydrolysis, it was necessary to ensure homogeneity of the reaction medium. The container therefore was vigorously agitated and the rate of circulation of the reaction medium through the column was kept at 50 ml/min. The pH of the reaction medium was constantly monitored and kept at a value of 8.0 by addition of aqueous 0.5N NaOH solution. The temperature of the column was kept constant at 37°C.

The rate of consumption of NaOH solution was plotted and allowed the course of hydrolysis to be followed. When the period of two hours, ten minutes had elapsed, the rate of consumption of sodium hydroxide had dropped to less than 5% of the initial rate. At this point, reaction was stopped and the circuit was drained. 310 ml of 0.5N NaOH had been used.

About 3300 ml of an ajueous solution containing 6-APA and phenylacetic acid formed by hydrolysis of the penicillin together with some residual .'penicillin, were obtained* Phenylacetic acid and penicillin were removed from the reaction medium by extraction with butyl acetate. Two extractions were carried out, each with 1100 ml of fresh solvent.

For the extractions, the reaction medium was collected in a 6 litre container fitted with an agitator device, was cooled to 5eC and was adjusted to pH 3 by addition of aqueous normal HC1. The first 1100 ml sample of butyl acetate was 39158/2 ^ ~? added and the container was agitated to obtain good dispersid!? of the aqueous and organic phases. The agitation was continued for 15 rainutes and further HC1 was continuously added to maintain the pH of the aqueous phase at 3. While keeping the contents of the container at 5°C, they were decanted into a separating funnel over a period of about 20 minutes. The emulsion was allowed to separate out and in the bottom of the funnel was obtained an aqueous phase slightly turbid because of the presence of traces of solvent. A clear organic phase was obtained in the top of the funnel. The aqueous phase was separated off and subjected to centrifugation to remove th© small amount of butyl acetate dispersed therein. The aqueous phase was then treated with a further 1100 ml sample of fresh solvent and the above procedure was repeated to obtain an aqueous phase. A total of 180 ml of the normal aqueous hydrochloric acid were used in the two extractions.

The two butyl acetate fractions were subjected to the aforementioned hydroxylamine test to detect the presence of penicillin. The first butyl acetate fraction was found to contain 2 m /ml and the second fraction did not contain any penicillin.

The aqueous phases were adjusted to pH 6.9 by addition of 165 ml of a normal aqueous solution of sodium hydroxide and were evaporated in vacuo using a rotating evaporator. The solution being evaporated was maintained at 45eC by passage of liquid through a jacket. The vacuum pump was employed to ensure the pressure over the liquid was maintained at a 1 millimetre of mercury. After five hours, the volume of the aqueous phase had been reduced to l/8th of its initial volume.

The residu -solution was collected in a 1 litre container cooled to 2°C and was adjusted to pH 4.2 by addition of 6 N.HC1. During the addition of the hydrochloric acid, 6-APA present formed a crystalline precipitate. The precipitate iae filtered off from the aqueous medium in vacuo and was dried in vacuo (0.5 mH.Hg/45°C) until constant weight was achieved. 32.4 gram of the dried product were obtained. The mother liquor {450 ml) when estimated for penicillin-nucleus content by the above mentioned hydroxylamine test, was found to contain 180 mM/ litre of 6-ΆΡΑ, that is a concentration of 6-APA of 0.39% by weight. The crystalline product obtained was redissolved in water and the solution obtained was also tested by the hydroxyl-amine test. Colorometric tests with a standard solution of very pure 6-APA which had been obtained by consecutive crystallisations and dissolutions showed that the crystalline product obtained contained 98.5% by weight of 6-APA. The molar amount of 6-APA recovered was therefore 32.4 X 0.985 ________________ r 216 that is 147 mM. Since 161 mM of penicillin had been originally used, the yield of 6-APA was 91.4% of theoretical.

In a continuously operating plant, the mother liquor would be recycled to the evaporation stage. In this way, the total product recovery could be increased to a yield of 95% of theoretical.

Example 3 The procedure of Example 1(a) was repeated to produce a cellulose »2rfci?afce filament containing encased therein enzymatic material obtained from 100 gram of a moist mycelium of Penicillium chrysogenum. 30 gram of the enzyme-containing filaments so obtained were placed in the column used in Example 1 which was thermostatically kept at 37°C. 600 ml of an aqueous solution of the potassium salt of penicillin V was constantly recyied through the column. The pH of this aqueous solution was maintained at 3.0 in the same way as set out in Example 1. In different tests it was found that within five to seven hours, about 90% of the penicillin V had been hydrolysed. Numerous tests were carried out using the same filaments without any detectable loss of activity of the filaments being shown during the period of the tests.

Example 4 The column used in Example 1 containing cellulose triacetate filaments containing encased therein enzymes cerived from E._JJoli, was used to prepare ampicillin [6-(D( } -a*?arainophenyl-acetamido) -penicillin] . For this purpose, an aqueous solution obtained from the enzymatic hydrolysis of penicillin G, as carried out in Example 1, and freed from residual penicillin G and from phenylacetic acid, containing about 8.5 mg/ml of 6-APA was recycled through the column. The pH of the aqueous solution was maintained at 6.0. D-a-aminophenylacetamidehydrobromide was added to the aqueous solution in a final concentration of 18 iag/ ml. The aqueous solution was kept at a temperature of 37eC.

After circulation of the reaction solution through the column for from 6 to 7 hours, 90S of the 6-APA was found to have been converted to ampicillin.

Example 5 Example 4 v;as repeated, but. using DL-mandelamide in place of the D-a-aminophenyl ac amide bromohydrate . The DL-mandel amide was used in a concentration of .12 mg/ml and the aqueous Eeaction solution was kept at a pH of about 5.5. After 6 hours, the AFA was found to have been converted to a-hydroxybensyl penicillin in a yield of §0%.

Example 6 16 gram of ethyl cellulose were dissolved in 184 gram moist cells of E. Coli obtained by fermentation of E. C'oli in a suitable nutrient medium were cen ridfuged and the enzyme was precipitated from the aqueous fraction obtained by addition of ammonium sulphate and the precipitate was dissolved in 30 ml of a phosphate buffer containing glycerol, The enzymatic solution thus obtained was added to the polymer solution and a homogeneous two phase emulsion was obtained by vigorous agitation at 0°C for 30 minutes. The emulsion was then fed to a spinneret maintained at -6eC and filaments were spun therefrom under an atmosphere of nitrogen into petroleum ether maintained at 2Q°C in ¾rf ich the filament coagulated* The procedure of Example 1(b) was repeated using 30 gram of the enzyme-containing filaments in the column. After about 5 hours, when it was clear that the reaction was almost complete, 5.15 g of 6-APA had been obtained.

Example 7 The procedure of Example 6 was repeated to obtain filaments of poly - γ - methyl - glutamate (PLG-30 of Kyowa Hakko Kogyo C. Ltd containing enzymes derived from E. Coli.

The filaments were spun into toluene kept at the ambient temperature. 30 gram of the enzyme-containing filaments so produced were used in the procedure of Example Kb). After abou 5 hours, 6 gram of 6-ΔΡΑ had been produced.

Example 8 A column containing 30 gram of enzyiae-containing filaments, as used in Example 1(b) was employed in ths deacylation of a cephalosporin derivative. For this ourpose, 600 ml of a 2% by weight aqueous solution of the potassiui salt of 7- (2- through the column. The pH of this solution was kept at 8.0 by addition of aqueous sodium hydroxide and the column was kept at 37eC. After 5 hours, it was found that 98% hydrolysis had taken place and 7-amino cephalosporanic acid was recovered from the reaction mixture.

Example 9 A column of the type described injExample 1 was packed with 30 gram of the enzyme-containing filaments obtained in Example 3. 600 ml of a 2% by weight aqueous solution of the potassium salt of 7- (2-phenoxy-acetamido) cephalosporanic acid were passed through the column. After* 6 hours, 85% hydrolysis was found to have taken place and 7-araino cephalosporanic acid was recovered from the reaction mixture.

Example 10 600 ml of a 2% by weight aqueous solution of the oxy potassium salt of 7-(2-phenyl-acetamido) desacefcyi- cephalosporanic acid was constantly cycled through the reactor used in Example 1(b) containing 37 grams of enzyme-containing filaments. The pH of the aqueous solution was kept at 8.0 and the temperature thereof was maintained at 37°C. After 5 hours, no further hydrolysis of the desacetyl cephalosporanic acid derivative oxy employed took place and 7-amino desacetyd cephalosporanic acid was recovered from the reaction solution in a yield of 85%.

Example 11 500 ml of an aqueous solution containing 5 gram of the potassium salt of 7-amino cephalosporanic acid and 13.1 gram of D-a-phenylglycinemethyl ester hydrochloride were constantly cycled through the column used in Example 1(b) containing 37 grams of 39158/2 the enzyme-containing filaments. The pH of the aqueous solution was kept at 6.5, being monitored thsroughout by means of a pH meter. The temperature of the aqueous reaction solution was kept at 37°C. After 1.5 hours,, analysis of the aqueous solution showed that 40% of the 7-amino cephalosporanic acid had been converted to 7- (2-D- -aminophenylacetamido) cephalosporanic acid. 39158/2

Claims (47)

1. A process for the enzymatic treatment of a substance which contains a ring structure characteristic of penicillins or cephalosporins to convert it to another substance containing the .ring structure, which comprises contacting a Λ reaction medium containing the substance with a filament throughout which is distributed in finely divided form at least one enzyme capable of causing the desired conversion, the enzyme (s) being encased in the filament in a manner such that the or each enzyme can effect conversion and that the or each enzyme is substantially retained in the filament during the contacting.

2. A process as claimed in Claim 1, wherein the contacting is effected in a continuous operation.

3. A process according to Claim 1 or 2, wherein the enzyme (s) is/are incorporated in a filament formed from a ^wLtrafeed, esterified or etherified cellulosic polymer, a poly-olefin, a polymer or copolymer of acr lonitrile, an aerylate, methacrylate, vinyl ester, vinyl chloride, vinylidene chloride or styrene, a polyamide or pdyvinylbutyral .

4. A process according to Claim 3, wherein the filament is formed from ethyl cellulose.

5. A process as claimed in Claim 3, wherein the filament is formed from poly - γ - methyl glutamate.

6. A process as claimed in Claim 3. wherein the filament is formed from cellulose uiLiuiL .

7. A process as claimed in any one of the preceding claims, wherein the contacting of the reactio medium with the filaments results in hydrolysis of said substanc contained in the reaction medium.

8. A process as claimed wherein said substance which is hydrolysed is a natura pen cillin. 39158/2

9. , A process as claimed in Claim 8, in which said substance is a natural penicillin of formula: wherein R is alk with from 1 to 10 carbon atoms, alkenyi tfith from 3 to 10 carbon a oms aralkyl, R'—0CH5— or '—SCH„—, wherein R' is alkyl, dkenyl, phenyl, or optionally substituted phenyl , which penicillin is thus hydroiysed to 6-aminopenicillanic acid.

10. A process as claimed in Claim 9, wherein R* is phenyl substituted by halogen, nitre, alkyl, alkenyi or alkoxy.

11. A process as claimed in Claim 9 orjlO, wherein the reaction medium is an aqueous solution containing the potassium salt of said substance.

12. A process as claimed in any one of Claims 9 to 11 v/herein the reaction medium contains from 1 to 6% by weight of said substance.

13. A process as claimed in Claim 12, wherein the reaction medium contains from 2 to 3% by weight of said substance.

14. A process as claimed in any one of Claims S to 13, wherein the pH of the reaction medium is maintained at 8.

15. A process as claimed in Claim 14, wherein the reaction medium is buffered at pH 8.

16. A process as claimed in Claim 7, wherein said substance which is hydroiysed contains a cephalosporin ring structure.

17. A process as claimed in Claim 16, wherein said substance is a derivative of cephalosporanic acid.

18. A process as claimed in Claim 17, wherein said r 39158/2

19. A process as claimed in Claim 17, wherein said substance is 7- (2-phenoxyacetamido) cephalosporanic acid which is hydrolysed to 7-aminocephalosporanic acid.

20. A process as claimed in Claim 17, wherein said oxy substance is 7- (2-phenylacetaaiido) desacet-^4 cephalosporanic acid.

21. A process as claimed in any one of Claims 17 to ID, wherein the reaction medium is an aqueous solution containing said substance in the form of its potassium salt.

22. A process as claimed in any one of Claims 7 to 9, 11 to 18, 20 and 21, wherein when a phenylacetamido group is h/drol&tically cleaved from said substance} said filament contains an acylase derived from bacterial cells.

23. A process as claimed in Claim 22, wherein the acylase is derived from S. oli, B. Megateriuia, Aerobacter aeroge css or Alcaligenes faecalis cells.

24. A process as claimed in any one of Claims 7 to 17, 19 and 21, wherein when a phenoxyacetamido group is hydrox tically cleared from said substance, said filament contains an acylase derived from fungal or ac inomyces cells.

25. A process as claimed in Claim 24, whereintthe acylase is derived from Aspergillus , Penicillium, Botrytis cinerea , Fusartum, Mucor, Alternaria or Streptomyces cells.

26. A process as claimed in any one of the preceding claims, wherein the filament contains a purified enzyme- containing cell extract.

27. A process as claimed in any one of Claims 1 to 6, wherein the contacting of the reaction medium with the filament results in a synthetic reaction taking place between said substance and another substance present in the reaction medium. 39158/2

28. A process as claimed in Claim 27, wherein said substance is the product of hydrolysis of a precursor thereof which has been hydrolysed by contacting a reacting medium containing the same with said filament under different reaction conditions.

29. A process as claimed in Claim 27 or 28, in which said substance is e-aminopenicillanic acid.

30. A process as claimed in Claim 29 f wherein -hydroxybeiieylpeniciilin is synthesised.

31. A process as claimed in Claim 29, wherein ^ ampicillin is synthesised.

32. A process as claimed in Claim 30 to 31, wherein the filament contains enzymes derived from E. Coli cells.

33. A process as claimed in any one of Claims 29 to 32, wherein the reaction medium is an aqueous solution containing the potassium salt of 6-aminopenicillanic acid.

34. A process as claimed in any one of Claims 29 to 33, wherein the reaction medium is maintained at pH 6.

35. A process as claimed in any one of Claims 29 to 34 , wherein the reaction medium contains om 1 to 3% by weight of 6-aminopenicillanic acid.

36. A process as claimed in Claim 27 or 28, in which said substance is 7-aminocephalosporanic acid and the synthesis product obtained therefroiu is 7- (2-D-a-aminophenylacetamido) cephalosporanic acid.

37. A process as claimed in Claim 26, wherein the reaction medium is an aqueous solution containing the potassium salt of 7~aminocephalosporanic acid. 39158/2 0

38. A process as claimed in any one of Claims 7 to 37, which is carried out at a temperature of from 15 to 60°C.

39. A process as claimed in Claim 38, which is carried out at a temperature of from 37 to 40°C.

40. A process as claimed in Claim 1 for the enzymatic treatment of a substance which contains a ring structure characteristic of penicillins and cephalosporins, as described in any one of the foregoing Examples.

41. A substance containing a ring structure characteristic of penicillins and cephalosporins, whenever produced by the process claimed in any one of the foregoing claims.

42. A filament in which is present at least one enzyme capable of causing conversion of a substance containing a ring structure characteristic of penicillins and cephalosporins to another substance containing the ring structure, the or each enzyme being present throughout a filament in finely divided form and being encased in the filament in a manner such that the or each enzyme is retained in the filament when the latter is contacted by a reaction medium containing said substance and such that the or each enzyme is capable of effecting the conversion whilst being so retained.

43. A filament as claimed in Claim 42, which contains a penicillin acylase obtained from fungal or actinomycetes cells.

44. A filament as claimed in Claim 43, wherein the acylase is obtained from cells of Aspergillus , Penicillium, Botrytic cinerea, Fusarium, Mucor , Alternari or Strep omyces.

45. A filament as claimed in Claim 42, which contains a penicillin acylase obtained from bacterial cells. 39158/2 ^

46. A filament as claimed in Claim 45, wherein the acylate is obtained from cells of E. Coli, B. Magaterium, Aerobacte Aerogenes and Alcaligenes f ecal s.

47. A filament as claimed in Claim 42, substantially as described in any one of the foregoing Examples 1 to 3, 6 and 7. KEsmr