ESTERASE FREE ENZYMES

Abstract: A process for decreasing esterase activity present in the presence of D-amino acid oxidase activity or in the presence of glutarylacylase activity in a mixture having esterase activity and D-amino acid oxidase activity and/or having esterase activity and glutarylacylase activity by treatment with phenylmethylsulphonyl fluoride; immobilized biocatalysts in spherical particle form obtainable by treating microorganism cells having enzyme activity with a primary or secondary amine containing polymer, an organic solvent which is able to form a two-phase system with water and a bifunctional agent; and the use of that process and biocatalysts in the production of 7-aminocephalo-sporanic acid, 7-amino-3-hydroxymethyl-3-cephem-4-carboxylic acid and cephalosporin antibiotics.

Description: Esterase free enzymes The present invention relates to a process for inhibition of esterase activity in enzyme preparanons, e.g. useful in enzymaπc catalysed production of 7-amιnocephalosporanιc acid (7-ACA); and to the production of enzyme preparations, e.g. useful in 7-ACA and/or 7-ammo-3-hydroxymethyl-3- cephem-4-carboxvhc aαd production. 7-ACA is a key intermediate in the production of e.g. pharmaceutically, acπve cephaiosporm annbioncs, e.g. 7-ACA may be acylated at the amine group in position 7 of the ring system, e.g. a group known as a valuable group m the production of cephaiosporm antibiotics or known as a valuable group in an intermediate for their producπon; e.g. to obtain a 7-amιnoacylated cephaiosporm having a methylacetoxy group in position 3 of the ring system, such as cefotaxime, cefpodoxime (proxeul), cephaloglyαn; and in the producπon of other cephaiosporm annbioncs or intermediates for their production which may be derived from 7-ACA; e.g. by further reacung the methylacetoxy group m position 3 of die ring system to obtain a 3-subsπtuted cephaiosporm subsntuted by a group which is different from the acetoxymethyl group; e.g. a group known as a valuable group m the production of cephaiosporm annbioncs or known as a valuable group m an intermediate for their producπon; e.g. by nucieophihc subsπtuπon of the acetoxy group, or e.g. by deacetylanon of the methylacetoxy group to obtain the hydroxymethyl group (e.g. HACA); and e.g. further reacπng a hydroxymethyl group obtained in posiπon 3 of die ring system, e.g. by nucieophihc subsπtuπon of die hydroxy group; or removal of the hvdroxy funcπon of a hydroxvmethyl group or removal of the hydroxmediyl group in posiπon 3 of the ring system: and, if desired, esteπficauon of die carboxvhc group m posiπon 4 of d e ring system, e.g. by a group known as a valuable group in the producπon of cephaiosporm annbioncs or known as a valuable group in an intermediate for their producnon: and, if desired, salt and/or solvent formaπon of a cephaiosporm compound obtained such a reacπon e.g. according to a convenπonal method. 7- ACA and HACA may e.g. be obtained from cephaiosporm C (Ceph C) by deacylaπon of the amine group m posiπon 7 of the ring system, and deacetylanon of die mediylacetoxy group in posiπon 3 of die ring system, respecπvely, e.g. enzymancally. An enzyme useful in enzymaπc Ceph C deacylaπon is e.g. D-amino acid oxidase (DAO) which catalyses die oxydaπve desaminanon of Ceph C to form glutaryl-7-amιnocephalosporanιc acid (G1-7-ACA) via die intermediate α-ketoadιpoyl-7- aminocephalosporamc acid (KA-7-ACA). G1-7-ACA may be hydrohsed to obtain glutanc acid and 7-ACA, e.g. by a glutarylacylase (GAC). It is known that DAO and GAC containing enzyme preparanons, such as microorganism cells mav contain esterase acπviry addmonally. Undesired deacetylanon of d e side chain m posmon 3 of die ring structure of the cephaiosporm may occur, e.g. due to die presence of esterase acπvity m enzymarically catalysed Ceph C deacylaπon, e.g. 3- hydroxymethyl denvaπves of Ceph C and G1-7-ACA may be formed. This may result in a significant decrease of 7-ACA quality and yield. Convenπonal methods for decreasing esterase activity present in the presence of DAO acπvity by acetone or CuSO 4 treatment may decrease esterase activity incompletely. It was now surprisingly found diat the treatment of a mixture having esterase acπvity in die presence of DAO acπvity or having esterase activity in die presence of GAC activity widi phenyimeri ylsuiphonyl fluoride (PMSF) may decrease esterase acπvity considerably, e.g. substantially complete, whereas DAO, or GAC activity, respectively may remain high, e.g. substantially unchanged. This finding is surprising because according to die present mvenπon PMSF, known e.g. as an irreversible inhibitor of seπne containing proteins by seπne sulphonviaπon, may decrease esterase acnvity present m the presence of DAO acπvity or in the presence of GAC acπviry seiecnveiy without, e.g. substanπal influence on DAO or GAC acπvity; and even more surprising is the selecnve and effecnve mhibmon by PMSF of esterase acπvity present m the presence of GAC acπvity, e.g. because of similar funcnon and structure of acylases and esterases. In one aspect the present invenπon provides a process for decreasing, e.g. substantially removing esterase acnviry present in the presence of D-ammo acid oxidase acπvity or in the presence of glutarylacylase acπvity m a mixture having esterase acπvity and D-ammo acid oxidase activity and/or glutarylacylase activity, e.g. present the form of microorganism cells or in the form of a cell-free extract ti ereof, e.g. in die form of a cell-free extract thereof, comprising treaπng a mixture having esterase acnvity and D-amino acid oxidase acπviry and/or glutarylacylase acπvity with phenyimeύ vlsulphonyl fluoride, e.g. wherein D-ammo acid oxidase acπvity or glutarviacylase acπviry remains, e.g. substanπally the same after treatment with phenylmediylsulphonyl fluoride as before said treatment. Typically D-ammo acid oxidase acπvity remains more than 91% of die original value and in the case of glutarylacylase acπvity even more than 97% of the original value. A process of the present invenπon may e.g. be carried out as follows: Known and e.g. commercially available microorgansisms producing DAO acnvity include t.g.Trtgonopsis, Asperχιllus, Pemcilli m, preferably Trtgonopsts variabilis microorganisms. Known and e.g. commercially available microorgansisms producing GAC acnvity include e.g Pseuάomonas, Achromobacter, Bacillus cereus or e.g. transformants, e.g. £. colt transformants, e.g. transformed according to e.g. a convennonal mediod. A mixture having DAO acπvity or GAC acπvity and esterase acπvity may e.g. be obtained commercially or e.g. according to a conventional mediod, and may be e.g. in the form of a cell-free extract, e.g. in immobilised form, or m die form of microorganism cells, such as in die form of e.g. pardy purified or impurified cells, and/or permeabilised or non-permeabilised cells, and or pardy destroyed or intact cells, and/or immobilised or non-immobilised cells; such as obtainable from a fermentation broth, e.g. according to a convenπonal mediod, preferably in the form of a cell-free extract, e.g. immobilized. E.g. microorganism cells may be isolated, e.g. harvested from a fermentaπon broth and used as such, e.g. in moist form, e.g. after cenrrifugaπon of the fermentauon brodi, or the cells may be further treated before or after isolaπon from the fermentanon broth, e.g. according to a convennonal mediod, such as homogenising cells to obtain, e.g. partly, destroyed cells, and/or permeabilismg cells or fragments thereof to obtain permeabilised cells or cell fragments, and/Or purifying cells or cell fragments to obtain e.g. pardy purified cells and/or cell fragments and/or cell-free (e.g. by cell floccuiaπon) extracts, and/or immobilising cells or cell-free extracts to obtain immobilised cells and/or immobilised cell fragments and/or immobilised cell-free extracts. E.g. immobilisaπon mav be carried out according to a convennonal mediod; e.g. in die presence of acrylic (lmmobihsauon) beads, such as Eupergit * or in the presence of an ion exchange resin, e.g. such as Relite Dianion* according to a method described die examples below; or e.g. according to anodier aspect of die present invenπon which is described below. The microorganism cells may be used in die form of an, e.g. buffered, aqueous cell suspension, obtainable e.g. by re-suspension of cells in water or in a buffer solunon after isolation from a fermentanon broth, or in case of a cell-free extract an aqueous solution of the cell-free extract may be used. The pH of a suspension or solunon may be, e.g. approximately neutral, e.g. a pH of 6.5 to 8.5, such as 7.0 or around 7.0 may be appropriate. In a preferred embodiment of die present invenπon an esterase and DAO acπvity having mixture may be e.g. substanπally, free of e.g. nanve catalase acπvity, e.g. obtainable by treatment of an aqueous cell mixture having DAO, esterase and catalase acπvity in basic medium, e.g. m die presence of a causπc soda solunon, e.g. at pH 9 to 11.5, such as 10 to 11. An esterase acnvity and DAO acπvity having mixture or an esterase activity and GAC acπvity having mixture, may be treated, e.g. incubated at an appropriate temperature, e.g. at room temperature for an appropriate πme, e.g. for several hours, such as for 1 to 5, e.g. 2.5 to 4 hours or longer with PMSF, e.g. by addinon of a PMSF solution, e.g. of an appropriate concentranon range, e.g. of 0.5% to 25%, such as 1% to 10% PMSF m an appropriate solvent, e.g. m alcohol, such as (C, alkanol, e.g. e hanol. Per 100 liters of a cell suspension an amount of e.g. 1000 ml of PMSF solunon, e.g. in a concentranon range as indicated above, may be sufficient to remove pracπcally completely and irreversibly undesired esterase acπvity, e.g. without, e.g. substanπally decreasing the DAO or GAC acπvity present m the starπng mixture. Removal of esterase activity may be determined by determinanon of deacetylanon products obtained in G1-7-ACA or 7-ACA reacπon from Ceph C using a PMSF-treated DAO or GAC containing mixture. For comparison the reacπon may be carried out using a non-PMSF treated DAO or GAC containing mixture under comparable reacπon conditions. A mixture may be obtained wherein DAO or GAC activity is high, e.g. substantially die same as before PMSF treatment and wherein esterase activity is, e.g. substantially, completely removed. A mixture obtainable according to the present invention is useful, e.g. in d e producnon of 7-ACA from e.g. Ceph C. Yields and purity of 7-ACA obtained may be improved if PMSF treated DAO and/or GAC acnvity is used in comparison with die use of non-PMSF treated DAO and/or GAC acnvity under comparable reacnon condinons. Thus, e.g. die removal of esterase acπviry according to the present mvennon, results in less, e.g. substantially no, 3 -deacetylanon by-products which are due to esterase acnvity during DAO and or GAC reacπon. The undesired amount of 3- deacervlaπon by-products in GI-7-ACA or 7-ACA obtained by a process according to d e present invenπon may be, e.g. substantially (typically within 1% in a process giving 3% starπng acπvity whereas when PMSF is not used it may increase to 6%), the same as in Ceph C used as starπng material. In anod er aspect the present invenπon provides a process for the producπon of glutaryI-7-amιnocephalosporaπιc acid comprising the steps treanng a mixture having esterase acnvity and D-ammo acid oxidase activity widi phenylmed vlsulphonyl fluoride, e.g. to obtain a mixture which is substantially free of esterase acnvity, and e.g. having substannallv the same D-amino acid oxidase activity as before PMSF treatment, n) reacung cephaiosporm C with a mixture obtained in step I) to obtain glutaryl-7- aminocephalosporanic acid, and isolanng gluraryl-7-amιnocephalosporanιc acid, if desired. In anorher aspect d e present mvennon provides a process for the producπon of 7- aminocephalosporanic acid comprising the steps 0 treanng a mixture having esterase acnvity and D-amino aαd oxidase acπvity wid phenylmedivlsulphonyl fluoride, e.g. to obtain a mixture which is substantially free of esterase acnvity, and e.g. having substannally die same D-ammo acid oxidase activity as before PMSF treatment, n) reacπng cephalosporin C wi h a mixture obtained m step l) to obtain glutaryl-7-aπuno- cephalosporanic acid, iii) converting giutaryl-7-aminocephalosporanic acid into 7-aminocephalosporanic acid, and isolating 7-aminocephalosporanic acid obtained, if desired. Conversion of glutaryl-7-aminocephalosporanic acid into 7-aminocephalosporanic acid may be cariied out according to a conventional mediod, e.g. chemically or enzymatically, or e.g. according to another aspect of die present invention, e.g. as described herein. In another aspect the present invention provides a process for the production of 7-amino cephalosporanic acid comprising the steps i) treating a mixture having esterase activity and D-amino acid oxidase activity widi phenylmediylsulphonyl fluoride, e.g. to obtain a mixture which is substantially free of esterase activity, and e.g. having substantially d e same D-amino acid oxidase activity as before PMSF treatment, ii) reacting cephalosporin C with a mixture obtained in step i) to obtain glutaryl-7-amiπo- cephalosporanic acid, iii) treating a mixture having esterase activity and glutarylacylase activity with phenylmediylsulphonyl fluoride, e.g. to obtain a mixture which is substantially free of esterase activity, and e.g. having substantially d e same glutarylacylase activity as before PMSF treatment, iv) reacting glutaryl-7-aminocephalosporanic acid obtained in step ii) widi a mixture obtained in step iii) to obtain 7-aminocephalosporanic acid; and isolating 7-aminocephalosporanic acid obtained in step ii), if desired. A process of the present invention to obtain 7-ACA from Ceph C may e.g. be carried out as follows: The starting material Ceph C may be e.g. in the form of an aqueous suspension or solution of Ceph C in free-form or in salt form; e.g. Ceph C may be e.g. in the form of a Ceph C containing fermentation broth, e.g. wherein cells and solid have been removed, e.g. according to a conventional mediod, or in the form of a salt of Ceph C, e.g. in the form of a sodium salt. The suspension or solution of Ceph C may be treated with a mixture having DAO activity which is pre-treated with PMSF according to die present invention, e.g. in a form as described above, e.g. an aqueous microorganism cell suspension or a cell-free extract may be added to an aqueous suspension or solution of Ceph C (or vice versa); under introduction of oxygen into the suspension or solution, e.g. in the form of oxygen gas or in the form of air, or mixtures thereof, e.g. if desired under pressure. The reaction may be carried out at appropriate pH, e.g. at around neutral pH, e.g. at a pH of between 6.5 to 8.0, such as 7.0 to 7.5, at appropriate temperatures such as 10°C to 30°C, e.g. at room temperature. The amount of DAO activity, e.g. d e amount of a cell suspension or of a cell- free extract having DAO activity in respect with the amount of Ceph C is depending on the DAO activity present in the cell suspension used and on die amount of Ceph C to be reacted and is not critical; an amount e.g. necessary for a short reaction time may be easily determined e.g. by determination of the amount of G1-7-ACA formed in die mixture during, e.g. regular rime intervalls by a conventional mediod, e.g. by HPLC determination. G1-7-ACA obtained may be isolated and purified, e.g. by a conventional mediod or may be used as such for further processing, e.g. in the production of 7-ACA. Oxidase activity may be determined as follows: 0.2 to 5 g of a mixture having DAO activity in die form of permeabilised cells is suspended under stirring in an aqueous 20 mM Ceph C solution at pH 8.0, at 25°C under introduction of an of oxygen stream into the mixture to oxygen saturation. The increase of G1-7-ACA and KA-7-ACA is determined by HPLC. The activity of "1 U oxidase" corresponds to he formation of 1 μmol/min G1-7-ACA and KA-7-ACA under the above conditions. An aqueous solution or suspension of G1-7-ACA, e.g. obtainable or obtained according to the present invention may be brought into contact wid a mixture having GAC activity which is pretreated wid PMSF according to d e present invention, e.g. an aqueous, e.g. buffered cell suspension or a cell-free extract may be added to an aqueous suspension or solution of G1-7-ACA (or vice versa). The reaction may be carried out at appropriate pH, e.g. at slighdy basic pH, e.g. including an pH of between 7.5 to 8.5, such as around 8.2, at appropriate temperatures including 10°C to 30°C, e.g. room temperature. The amount of GAC activity in respect widi die amount of G1-7-ACA is depending on die GAC activity present in the cell suspension or in the cell-free extract used and on the amount of G1-7-ACA to be reacted and is not critical; an amount e.g. necessary for a short reaction time may be easily determined e.g. by determination of die amount of 7-ACA formed in die mixture during, e.g. regular time intervalls by a conventional mediod, e.g. by HPLC determination. GAC acπvity may be determined as follows: 0.5 g to 2.0 g of a mixture having GAC activity are suspended under stirring in a 2% aqueous G1-7-ACA solution containing 5 mM phosphate and of a pH of 8.0 at 37°C. The pH is kept at 8.0 by addition of an aqueous 100 mM sodium hydroxide solution. The amount of sodium hydroxide used is determined and corresponds to the amount of glutaric acid or 7-ACA, respectively formed from G1-7-ACA. The activity of "1 U acylase" corresponds to die formation of 1 μmol/min glutanc acid or 7-ACA, respectively under the above conditions. 7-ACA obtained may be isolated and purified, e.g. by a conventional method, or, if desired may be used as such, e.g. for further processing, e.g. in a 7-ACA reaction, e.g. according to a convennonal mediod. 7-ACA obtained according to the present invention may be of high purity, e.g. 7-ACA obtained may contain a low amount of 3-deacetylation products and may be obtained in high yields; e.g. in vields which are higher than in a process wherein a non pretreated DAO activity and/or a non-pre-σeated GAC acnvity is used under comparable reacπon conditions. It is known tiiat e.g. enzyme containing microorganism cells may be immobilised. Immobilised enzyme containing microorganism cells may be useful e.g. because of d e possibility of easy recovering and re-use. Known processes for the producnon of immobilized microorgansim celis may have disadvantages, e.g. immobilised cells having poor enzyme stability and/or low specific enzyme acnvity may be obtained, and/or die immobilisation process may be complicated. Surprisingly a simple immobilisaπon process for microorganism cells having an enzyme activity, such as DAO or GAC activity, was now found wherein immobilised cells may be obtained in die form of solid spherical particles which may contain stable and highly specific enzyme activity. In anod er aspect the present invenπon provides a process for the producπon of spherical parπcles, e.g. in solid form, from microogranism cells having an enzyme activity, comprising the steps I) treaung microorganism cells wi h a primary or secondary amine containing polymer, in contacπng an organic solvenr which is abie to form a two-phase system with water with a mixture of microorganism cells, e.g. adding a mixture of microorganism cells to a solvent which is able to form a two phase system with water, iiι) treaπng a mixture obtained in step ii) witii a bifuncnonal agent; and isolaπng die spherical particles obtained; e.g. further comprising pre-treaung the microorganism cells with a bifuncnonal reagent before carrying out steps i) or ii); and/or adding a water-miscible solvent to a mixture obtained in step iii) before isolation of the spherical particles: and/or adding a solid to a mixture obtained in step i) or to the microorganism cells before carrying out step i). The process of the present mvennon is useful for immobilising microorganism cells having an enzyme activity which are commonly designated as biocatalysts. Microorganisms which may be used according to the present mvennon include all types of microorganisms, such as bacteπas, yeasts, funghi and acπnomycetes, e.g. Trtgonopsts, such as Trtgonopsts variabihs, Agrobactertum, such as Agrobactertum radtobacter, Rhodotorula, such as Rhodotorula glutmts, Pleurotus, such as Pleurotus ostreatus, Aspergillus, Penictlltum, Pseudomonas, Achromobacter, Bacillus, such as Bacillus cereus, Schtzosaccharomyces, such as Schtzosaccharomyces potnbe; or e.g. transformants such as e.g. Escherichia colt transformants having an enzyme acnvity. Enzyme acnvity includes all types of enzyme acnvity, such as hydrolase, isomerase, lyase, decarboxylase, oxireductase acnvity. Examples of microorganism cells having an enzyme acnvity includes e.g. Pleurotus, e.g. Pleurotus ostreatus cells having e.g. penicillin-V acylase acnvity, Agrobactertum, e.g. Agrobactertum radtobacter cells having e.g. hydantomase and/or N-decarbamoylase acnvity, Rhodotorula, e.g. Rhodotorula glutmts cells having e.g. esterase and/or oxidase acnvity, Trtgonopsts, e.g. Trtgonopsts variabths having e.g. DAO acnvity, Schtzosaccharomyces, such as Schtzosaccharomyces pombe having e.g. GAC acnvity; and cells of transformants such as Escherichia colt having e.g. GAC and/or esterase acπvity. The process of the present mvennon may be e.g. of parπcular interest in the producnon of immobilised microorganism cells which are useful in 7-ACA QΓ HACA producπon, e.g. starπng from Ceph C, such as cells having DAO acnvity, e.g. in the presence of esterase (and/or deacetylase) acnvity, and/or cells having GAC acnvity, e.g. m die presence of esterase (and/or deacetylase) acnvity. The process according to the present mvennon may be carried out as follows: Microorganism cells having enzyme acπvitv may e.g. be obtained from a fermentanon broth, e.g. according to a convennonal method. Microorganism cells include cells in any form e.g. m the form of, e.g. pardy purified or impunfied cells, and/or permeabilised or non-permeabihsed cells, and/or, e.g. pardy, destroyed or intact cells. E.g. microorganism cells may be isolated, e.g. harvested from a fermentanon broth and used as such, e.g. m moist form, e.g. after cenmfugaπon of die fermentaπon broth, or the cells may be further treated before or after isolaπon from the fermentanon broth, e.g. according to a convennonal mediod, such as homogenising cells to obtain, e.g. pardy, destroyed cells and/or permεabihsmg cells or fragments thereof to obtain permeabilised cells or fragments thereof, and/or purifying cells to obtain e.g. pardy purified cells and/or cell fragments. Undesired enzvme acnvity in microogrganism cells may be removed before immobilisaπon of the cells, e.g. according to a convennonal mediod. E.g. catalase acnvity in Trtgonopsts, e.g. Trtgonopsts vartabύs cells may e.g. be removed by treatment under a basic pH, e.g. a pH of 9 to 12, e.g. by addinon of a causπc soda solunon, e.g. in a mixture of microorganism cells alone, or in a mixture of microorganism cells in the presence of a primary and/or secondary amme containing polymer, opnonally in the presence of additives, such as β-mercaptoethanol, eriιylenedιamme-N,N,N',N'- tetraacenc acid (EDTA) for some time, e.g. ca. 15 minutes or longer. According to die present mvennon the microorganism cells may be pre-treated with a bifuncnonal agent. A bifuncnonal agent as used herein is to be understood as a compound having at least two reacnve groups which are able to react with primary and/or secondary amine groups, including e.g. glutaraldehyde, dimeriiyl pimehmidate, epichlorhydnn, N,N'-carbonyldiimidazole, 1,4-butanediol- diglyαdyleriier, maleic acid anhydride, dicyclohexylcarbodiimide, hexamediylenediisocyanate, preferably glutaraldehyde. Pre-treatment of die cells may be carried out in d e fermentation broth, or after isolaπon of microorganism cells, preferably in die fermentaπon broth. E.g. the bifuncnonal agent may be added to a microorganism cell suspension, e.g. in dissolved form, e.g. glutaraldehyde may be used in aqueous solunon, e.g. in 10% to 30% aqueous solunon and the mixture obtained after addinon may be snrred for some πme, e.g. 30 minutes to some hours, such as 30 minutes to 5 hours. The amount of the bifuncnonal agent in respect with the amount of cells is not criπcal; e.g. per 100 g of dry cell weight e.g. 0.1 g to 100 g, e.g. 5 g to 50 g of a bifuncnonal agent may be used conveniendy; e.g. if glutaraldehyde is used as a bifuncnonal agent 0.5 g to 200 g, such as 1 g to 100 g of a 25% aqueous solunon may be appropriate. After pre-treatment with a bifuncnonal agent the cells may be harvested and isolated, e.g. according to a conventional method, such as concentrated by microfiltranon and/or cenmfuganon and stored in a cool place, e.g. in frozen state. For further use frozen cells may be thawed and washed witii spring water, and, if desired, concentrated, e.g. according to a convennonal mediod such as microfiltranon. The microorganism cells, e.g. pre-treated as described above, may be used in die form of an, e.g. buffered, aqueous cell suspension e.g. by re-suspenion of cells in water after isolaπon from a fermentaπon broth and opπonal further treatment, e.g. at a pH of 6.0 to 10.0, such as 8.0 or around 8.0. If desired, an additive, e.g. as convennonal, such as β-mercaptoethanol, ethylenedιamιne-N,N,N',N'-tetraaceuc acid (EDTA) may be added to die suspension, e.g. in a convennonal amount. To the suspension obtained a primary and/or secondary amine containing polymer including e.g. a polyethylene lmme and a polyvmylamme, which is preferably water- soluble, e.g. in aqueous solunon, such as polyediylene imme in e.g. 10% to 50% aqueous solunon, e.g. having a molecular weight of 600,000 to 1,000,000 is added. Primary and/or secondary amme containing polymers are e.g. commercially available or e.g. may be produced according to a convennonal mediod. The amount of a primary and/or secondary amme containing polymer in respect with die dry cell mass is not cnncal and includes e.g. an amount of 1 g to 100 g, such as 5 g to 70 g, such as 10 g to 50 g per 100 g of dry microorganism cell weight. The cell/polymer mixture is contacted widi an organic solvent which is able to form a two-phase system with water, e.g. the cell/polymer mixture is introduced into an organic solvent which is able to form a two-phase system with water. Appropriate organic solvents include e.g. tπalkyl phosphates, such as tπbutyl phosphates, e.g. n-tributyl phosphate, alkanes, e.g. n-hexane, n-heptane, aromaπc hydrocarbons, e.g. toluene, natural or syndieπc oils, e.g. soya oil, silicone oil, diesel oil, aromaπc or aiiphauc e.g. (C 3 ,)alkyl, such as (C, alkyl, e.g. mono- or di- carboxy c acid alkyl, e.g. (C, Jalkyl, such as (C, Jalkyl, including e.g. glutaπc, sucαnic, adipic acid dimeriiyl- or diethylester, benzoic or phthalic acid eriiyl-, butyl-, dibutyl- or diisobutylester and anise alcohol, preferably tπalkyl phosphate. The amount of organic solvent may be such that the microorganism cell supension may be dispersed in the organic solvent, e.g. by, e.g. laminar, sπrnng into die solvent; per g of drv cell weight e.g. 1 ml to 100 ml, such as 3 ml to 50 ml of solvent may conveniendv be used. The mixture containing die cells, die polymer and die organic solvent is snrred for some πme, e.g. unπl a homogeneous supension is obtained and treated widi a bifuncnonal agent, including e.g. glutaraldehyde, epichlorohydnn, N,N'-carbonyldiimιdazole, 1,4-butanediol-dιglycιdvledιer. maleic acid anhydride, dicyclohexylcarbodiimide, hexamediylened socyanate, preferably glutaraldehyde. If a pre-treatment of the microorganism cells with a bifuncnonal agent was carried out, die same bifuncnonal agent may convemendy be used as in die pre-treatment. The amount of the bifuncnonal agent is not cnncal and includes an amount of e.g. 0.5 g to 100 g of a bifuncnonal agent per 100 g of dry cell weight, e.g. in case of using glutaraldehyde as a bifuncnonal agent e.g. 1 g to 200 g of a 25% aqueous solunon mav convemendy be used. Upon addinon of die bifuncπonai agent to the mixture containing the cells, d e polymer and the organic solvent crosslinking may start ana may be terminated e.g. within a few minutes and a suspension of, e.g. solid spherical parncles of the microorganism cells in die solvent mixture used may be obtained. If desired, die suspension obtained may be contacted with an organic solvent which is water-miscible and which may be harmless in respect with enzyme acnvity, e.g. which may be non-damagmg in respect with enzyme acnvity, including e.g. alcohols, ketones, polyediviene glycols, glycerol, preferably glycerol, e.g. the cell suspension obtained may be introduced into die organic solvent or the solvent may be introduced into d e cell suspension obtained. The amount of die water-miscible organic solvent is not cnncal and includes an amount which is sufficient to form a liquid-liquid two phase system in the reacnon mixture; e.g. per 100 g of dry cell weight convemendy e.g. 100 g to 5000 g and more, e.g. 500 g to 5000 g of a water-miscible organic solvent may be used. Phase separaπon, e.g. immediate, may occur upon contact of the solvent with the cell suspension. Spherical cell parncles, e.g. solid, formed may be in the lower aqueous phase obtained. The upper phase, e.g. containing mainly die organic solvent which is able to form a two phase system witii water may be separated easily and, e.g. substantially completely from the lower phase and may be re-used, e.g. in a further immobilisation process. The spherical cell particles obtained may be in solid form and may be isolated form the organic-miscible-solvent phase, e.g. by a conventional mediod, e.g. by pouring the suspension through a vessel with perforated bottom, centrifugation, filtration. The isolated spherical particles may be freed from solvent residues, e.g. washed, e.g. with spring water. Prior to crosslinking, i.e. prior to addition of die bifunctional agent, e.g. prior to addition of a solvent which is able to form a two-phase system with water, a solid, including e.g. aluminium oxide, acnvated carbon, bentonite may be added to the mixture of cells. The amount of a solid is not critical; conveniendy an amount of 5 g to 200 g, such as 50 g to 150 g per 100 g dry cell weight may be used. After suspension in die solvent which is able to form a two phase system with water and crosslinking by addition of the bifunctional agent into such a mixture, spherical cell parncles may be obtained which may particularly easy be separated and which may have a favourable narrow size-distribution-range and excellent mechanical stability. The biocatalyst, e.g. spherical microorganism cell particles, e.g. in solid form, obtained according to die present invention may be stored for later use e.g. in a cool place, e.g. in an appropriate buffer or e.g. in frozen form or lyophilised form. Spherical particles containing microorganism cells which contain enzyme activity obtainable, e.g. obtained, according to the present invention are novel. In anorher aspect die present invention provides spherical particles containing microorganism cells, e.g. in solid form, which contain enzyme acnvity obtainable, e.g. obtained, from a process comprising the steps i) treaπng microorganism cells having enzyme activity with a primary or secondary amine containing polymer, ii) contacting an organic solvent which is able to form a two-phase system with water with a mixture of microorganism cells, e.g. after carrying out step i), e.g. by addition of a mixture of microorganism cells to a solvent which is able to to form a two-phase system with water, iii) treaπng a mixture obtained in step ii) widi a bifunctional agent and isolating d e spherical particles obtained; e.g. furrher comprising pre-rreaπng die microorganism cells with a bifunctional reagent before carrying out steps i) or ii); and/or adding a water-miscible solvent to a mixture obtained in step iii) before isolation of die spherical particles; and/or adding a solid to a mixture obtained in step i) or to microorganism cells before carrying out step i) Spherical particles, e.g. solid, having an enzyme activity obtained according to a process of the present invention may be useful in enzymatic reactions, e.g. spherical particles, e.g. solid, having DAO activity, e.g. (pre-)rreated with PMSF and spherical particles, e.g. solid having GAC activity, e.g. (pre-)treated widi PMSF may be useful in the producπon of 7-ACA and/or G1-7-ACA and/or HACA. In anod er aspect the present invention provides a process for the production of glutaryl-7- aminocephalosporanic acid comprising die steps i) treating microorganism cells having D-amino acid oxidase activity widi a primary or secondary amine containing polymer, ii) contacting an organic solvent which is able to form a two-phase system with water with a mixture of microorganism cells, , e.g. after carrying out step i), e.g. by addition of a mixture of microorganism cells to a solvent which is able to to form a two-phase system with water, iii) treating a mixture obtained in step ii) widi a bifunctional agent; and isolating d e spherical particles obtained, and if desired, treating a mixture having esterase activity in the presence of D-amino acid oxidase activity with phenylmediylsulphonyl fluoride, e.g. before step iii), e.g. before step i); or treating spherical particles obtained in step iii) having esterase activity in the presence of D-amino acid oxidase activity widi phenylmediylsulphonyl fluoride, iv) reacting cephalosporin C with spherical particles obtained in step iii) to obtain glutaryl-7- aminocephalosporanic acid, and isolating glutaryl-7-aminocephalosporanic acid, if desired; e.g. and furrher converting glutaryl-7-aminocephalosporanic acid obtained in step iv) into 7- aminocephalosporanic acid. In anodier aspect the present invention provides a process for the production of glutaryl-7- aminocephalosporanic acid which comprises reacting cephalosporin C with spherical particles obtainable by step iii) to obtain glutaryl-7-aminocephalosporanic acid, and isolating glutaryl-7- aminocephalosporanic acid, if desired. Conversion of glutaryl-7-aminocephalosporanic acid obtained in step iv) into 7-aminocephalo- sporanic acid may e.g. be carried out according to a conventional method, e.g. chemically or enzymatically, or according to another aspect of the present invention, e.g. using spherical particles having GAC activity obtainable or obtained as described above according to the present invention. If according to d e present invention spherical particles are used having DAO enzyme activity in die presence of deacetylase activity; and/or having GAC activity in the presence of deacetylase activity, HACA may be obtained in a reaction with Ceph C or G1-7-ACA. In another aspect the present invention provides die use of a process for the production of spherical particles according to die present invention or the use of spherical particles produced according to die present invention in die production of 7-amino-3-hydroxymethyl-3-cephem-4-carboxylic acid from cephalosporin C or from glutaryI-7-aminocephalosporanic acid. 7-ACA or HACA produced according to the present invention may be useful as an intermediate in the production of a cephalosporin, e.g. a cephalosporin antibiotic or anodier intermediate in the production thereof, e.g. by - acylating d e amine group in position 7 of the ring system - further reacting the acetoxymediyl group in position 3 of the ring system - esterification of the carboxy group in position 4 of the ring system - salt and/or solvent formation, e.g. as described above. In anodier aspect the present invention provides a process for the production of 7- aminocephalosporanic acid comprising the steps i) treating microorganism cells having glutarylacylase activity with a primary or secondary amine containing poiymer, ii) contacting an organic solvent which is able to form a two-phase system with water with a mixture of microorganism cells, iii) treating a mixture obtained in step ii) widi a bifunctional agent; and isolating d e spherical particles obtained, iv) if desired, treating a mixture having esterase activity in the presence of glutarylacylase activity with phenylmediylsulphonyl fluoride, e.g. before step iii), e.g. before step i) if desired; or treating spherical particles obtained in step iii) having esterase activity in the presence of glutarylacylase acylase with phenylmediylsulphonyl fluoride, if desired, v) reacting glutaryl-7-aminocephalosporanic acid widi spherical particles obtained in step iii) or obtained in step iv) to obtain 7-aminocephalosporanic acid; and isolating 7- aminocephaiosporanic acid obtained, if desired. In anodier aspect the present vennon provides a process for the producπon of a cephalosporin, e.g. a cephaiosporm antibiotic or an intermediate in its production, wherein 7-ammocephalo- sporamc acid or 7-amιno-3-hydroxymethyl-3-cephem-4-carboxylιc acid, e.g. 7-ammocephalo- sporanic acid, produced according to die present invention is used as an intermediate. "Substannally" as used herein means e.g. greater riian 95%, e.g. 96%, 97% of the original value or less than 5%, e.g. 4%, 3% of die original value as appropriate. In the following examples all temperatures are given in degrees Celsius. The following abbreviaπons are used: 7-ACA: 7-amιnocephalosporanιc acid Ceph C. Cephalosporin C DAO: D-ammo acid oxidase GAC: glutarylacylase GDA: glutaraldehyde GI-7-ACA: Glutaryl-7-amιnocephalosporanιc acid KA-7-ACA:α-ketoadtpoyl-7-amιnocephaiosporanιc acid PEI Polyediylene imine PMSF: Phenylmethylsulphonyl fluoride TBP: n-Tributyl phosphate The acnvity of "1 U oxidase" corresponds to the formaπon of 1 μmol/min G1-7-ACA and KA-7- ACA in an oxygen saturated 20 mM aqueous Ceph C solunon at pH 8 and 25 β C. The acnvity of " 1 U acyiase" corresponds to die formaπon of 1 μmol/min glutanc acid or 7-ACA in a 2% aqueous G1-7-ACA solunon at pH 8 and 37 β C. Example 1 General Procedure to obtain a mixture having DAO activity which is substantially free of esterase activity From an aqueous fermentation broth having esterase activity and DAO activity in water, a cell pellet is obtained from harvested cells according to a conventional mediod (see e.g. Kubicek-Pranz, E.M. et al., Can. J. Microbiol. 1985, 31, pp 624-628), 10 g of the cell pellet obtained are re-suspended in 50 ml of water and treated for ca. 90 minutes at a pH of ca. 11 with a caustic soda solution at room temperature to remove catalase activity. The pH of die mixture is adjusted to ca. 7 by addition of phosphoric acid. To die mixture obtained 500 μl of an edianolic solution containing 10 mg of PMSF/ml edianol are added and d e mixture obtained is incubated at room temperature for ca. 3 hours. The cell suspension obtained may be used as such in a reaction which requires DAO activity. DAO activity of cell pellet: 530 U oxidase DAO activity after caustic soda treatment: 490 U oxidase DAO activity after PMSF treatment: 450 U oxidase (= 92% of activity before PMSF treatment). Example 2 A PMSF-treated cell suspension having DAO activity obtained as described example 1 is added to a solution of 10 g Ceph C in 1000 ml of water and die pH of die solution obtained is adjusted to 7.5. The mixture obtained is stirred for ca. 180 minutes at room temperature under introduction of oxygen into die mixture. Yield of Gl-7-ACA: 91%. Sum of 3-deacetylation products in d e starting Ceph C solution: 3% relative to Ceph C Sum of 3-deacetylation products in reacπon solution obtained: 3% relative to starπng Ceph C Example 2 is repeated but using a DAO activity widiout PMSF treatment Yield of Gl-7-ACA: 88%. Sum of 3-deacetylation products in die starnng Ceph C solution: 3% relative to Ceph C Sum of 3-deacetyiation products in reaction solution obtained: 6% relative to starting Ceph C Example 3 A cell suspension obtained as described in example 1 is homogenised before PMSF treatment (DAO activity in homogenised cells: 385 U oxidase) and die homogenised cells obtained are treated with PMSF as described in example 1 (DAO activity in homogenised and PMSF treated cells: 372 U oxidase corresponding to 96% of activity before PMSF treatment). The cell suspension obtained is reacted with Ceph C as described in example 1. Yield of G1-7-ACA: 93% Sum of 3-deacerylarion products in starting Ceph C solution: 3% relative to Ceph C Sum of 3-deacetylation products in reaction solution obtained: 3% relative to starting Ceph C Example 3 is repeated but using a DAO activity widiout PMSF treatment. Yield of G1-7-ACA: 89%. Sum of 3-deacetylarion products in starting Ceph C solution: 3% relative to Ceph C Sum of 3-deacetylation products in reaction solution obtained: 7% relative to starting Ceph C Example 4 10 g of a cell pellet obtained as described in example 1 before caustic soda treatment are suspended in 50 ml of water, 2 g of polyethylene imine (molecular weight 600,000 - 1,000,000) are added to die supension obtained and die mixture obtained is incubated for 90 minutes at pH 11 (adjusted by caustic soda solution addition) at room temperature. The pH of a mixture obtained is adjusted to 8.5 by addition of phosphoric acid, and die suspension obtained is stirred widi 50 ml of toluene. An emulsion of fine droplet-like cell particles in solvent is obtained to which 8 ml of 25% GDA solution are added. The fine droplet-like cell particles solidify to form solid spherical particles. The solid spherical particles are isolated and washed wid spring water. DAO activity in spherical particles: 290 U oxidase The solid spherical particles obtained are suspended in 50 ml of 20 mM phosphate buffer pH 7.0, mixed widi 500 ui of PMSF solution analogously as described in example 1 and die mixture obtained is incubated for 180 minutes. DAO acnvity in PMSF treated solid spherical particles: 305 U oxidase. The solid PMSF treated spherical particles obtained are used in a Ceph C reaction analogously as described in example 2. Yield of G1-7-ACA: 92% Sum of 3-deacetylarion products in starring Ceph C solution: 3% relative to Ceph C Sum of 3-deacetylarion products in reaction solution obtained: 3% relative to starting Ceph C Example 4 is repeated but using solid spherical particles widiout PMSF treatment. Yield of G1-7-ACA: 89%. Sum of 3-deacetylation products in starπng Ceph C solution: 3% relative to Ceph C Sum of 3-deacetyiarion products in reacπon solution obtained: 7% relative to starting Ceph C Example 5 10 g of a moist cell pellet of the recombinant E. colt strain, e.g. CCM 4229 (GAC acnvity: 720 U acyiase) isolated from the fermentation broth are washed and re-suspended in 50 ml of 50 mM phosphate buffer pH 7.0. To die suspension obtained 0.5 ml of an etiianolic solution containing 10 mg PMSF/ml, are added and die mixture obtained is stirred for ca. 3 hours at room temperature. GAC acnvity: 708 U acyiase (98 % of activity before PMSF treatment). 10 g of G1-7-ACA are dissolved in 1000 ml of water and the pH is adjusted to 8.2. The PMSF- treated E. colt suspension obtained as described above is added and die mixture obtained is snrred for ca. 180 minutes at 12° whilst maintaining die pH at ca. 8.0 - 8.2. Yield of 7-ACA: 93% Sum of 3-deacetylanon products in starπng G1-7-ACA solution: 3% relative to G1-7-ACA Sum of 3-deacetylaπon products m reacnon solution obtained: 3% relative to starπng G1-7-ACA Example 5 is repeated but using GAC acnvity without PMSF treatment. Yield of G1-7-ACA: 90%. Sum of 3 -deacetylanon products in starnng G1-7-ACA solution: 3% relative to G1-7-ACA Sum of 3-deacetylarion products m reacπon solution obtained: 7% relative to starnng G1-7-ACA Example 6 10 g of a moist washed cell pellet of die recombinant E. coli strain, e.g. CCM 4229 (GAC acnvity: 720 U acyiase) are suspended in 50 ml of 50 mM phosphate buffer pH 7.0 and homogenised under a pressure of 700 bar. The homogenised cells (GAC acnvity: 752 U acyiase) are mixed with edianohc PMSF soiunon as described in example 5. GAC acπviry in PMSF treated cells: 768 U acyiase. The suspension obtained is added to a G1-7-ACA solunon and d e reacπon is carried out as described in example 5. Yield of 7-ACA: 89% Sum of 3 -deacetylanon products in starnng G1-7-ACA solunon: 3% relanve to G1-7-ACA Sum of 3 -deacetylanon products in reacπon solunon obtained: 3% relative to starnng GI-7-ACA Example 6 is repeated but using GAC activity without PMSF treatment Yield of G1-7-ACA: 83%. Sum of 3-deacetylauon products in starnng G1-7-ACA solution: 3% relanve to G1-7-ACA Sum of 3-deacetylation products in reacπon solution obtained: 6% relative to starπng G1-7-ACA Example 7 Homogenised cells from 10 g of a moist washed cell pellet of recombinant E. coli strain, e.g. CCM 4229 cells (GAC activity 7350 U acyiase) are suspended in 50 ml of 50 mM phosphate buffer pH 7.0 and mixed with a flocculation agent (1 ml of Sedifloc R CL 900-18/40). The pH of the mixture obtained is adjusted to 5.2 by addition of acetic acid and d e mixture obtained is incubated for ca 1 hour at ca. 40° under stirring and stirred ca. for a further hour at 10°. The mixture obtained is cleared by centrifugation and die GAC-containing cell-free supernatant (GAC activity: 6150 U acyiase) is adjusted to pH 7.5, mixed widi PMSF solution and incubated analogously as described in example 5. GAC activity of die cell-free extract obtained after PMSF treatment: 6220 U acyiase. The mixture obtained is mixed widi 10 g of acrylic (immobilisation) beads (Eupergit" C), treated widi 50 ml of an aqueous 1 mol sodium sulphate solution and incubated for 64 hours at room temperature under shaking. The enzyme-charged carrier material obtained is separated from the immobilisaπon solution and used as catalyst in a reaction with a G1-7-ACA solution analogously as described in example 5. Yield of 7-ACA: 90% Sum of 3-deacetylation products in starting G1-7-ACA solution: 3% relative to G1-7-ACA Sum of 3-deacetylation products in reaction solution obtained: 3% relative to starting G1-7-ACA Example 7 is repeated but using immobilised GAC activity widiout PMSF treatment. Yield o Gl-7-ACA: 83%. Sum of 3-deacetylation products in starting G1-7-ACA solution: 3% relative to GI-7-ACA Sum of 3-deacetylation products in reaction solution obtained: 6% relative to starting G1-7-ACA Example 8 GAC activity is immobilised analogously as described in example 7, but PMSF treatment is not carried out after immobilisation but during immobilisation by addition of 1 ml of PMSF solution analogously as described in example 5 to a mixture containing die GAC-containing cell-free supernatant, Eupergit and sodium sulphate. The enzyme-charged PMSF treated carrier material obtained is separated from the immobilisation solution and used as a catalyst in a reaction with a G1-7-ACA solution analogously as described in example 5. Yield of 7-ACA and deacetylation products are substantially as described in example 7. Example 9 GAC-containing supernatant is produced analogously as described in example 7, but widiout PMSF treatment (GAC activity: 5830 U acyiase). The solution obtained is mixed with 40 g of an ion exchange resin (Relite R Diaion-HPA25L), the mixture obtained is adjusted to pH 7.5 and incubated for ca. 300 minutes at room temperature under shaking. The ion exchange resin obtained is isolated and washed (GAC activity: 1865 U acyiase). The exchange resin obtained is suspended in 200 ml of 50 mM phosphate buffer, 2 ml of the PMSF solution as used in example 5 are added and die mixture obtained is incubated for ca. 180 minutes under stirring. GAC activity in die PMSF treated ion exchange resin: 1810 U acyiase corresponding to 97% of the activity in die catalyst before PMSF treatment. The enzyme-charged PMSF treated ion exchange resin obtained is used as a catalyst in a reaction with a G1-7-ACA solution analogously as described in example 5. Sum of 3 -deacetylation products in starting G1-7-ACA solution: 3% relative to G1-7-ACA Sum of 3 -deacetylation products in reaction solution obtained: 3% relative to starting GI-7-ACA Example 10 a. Pre-treatment Cells of Trtgonopsts variabilis, e.g. ATCC 58536 (535 g moist weight, DAO activity: 25,970 U oxidase) are pre-treated in 2.7 kg of a fermentation broth by stirring 21 g of an aqueous 25% GDA solution into the fermentation broth during ca. 1 hour at room temperature and at pH 7.5. The cells are harvested from the cell suspension obtained by centrifugation and stored in a frozen state. The frozen cell mass is thawed, washed widi spring water by means of micro-filtration and concentrated to a volume of ca. 735 ml. DAO activity in d e concentrated cell mass: 26,350 U. b. Polvmer treatment 735 ml of the cell mass obtained as described in step a. (100 g cell dry weight) are suspended widi β- mercaptoethanol (5 mM), EDTA (2 mM) and 40 g of an aqueous 50% PEI solution, molecular weight 600,000 - 1,000,000 in water (ca. 800 ml total volume) and stirred slowly for ca. 90 minutes at pH 11 which is adjusted by addition of a caustic soda solution at room temperature. The pH of the mixture obtained is adjusted to pH 9 by addition of phosphoric acid. DAO activity: 24,360 U (94 % of pre-treated DAO activity obtained as described in example 10 a.. c. Crosslinking To 3000 ml of TBP in a stirring vessel the cell/polymer mixture obtained in step b. is added at room temperature under stirring. A homogeneous dispersion of die cell/polymer droplets in TBP is obtained widiin ca. 30 minutes to which 40 g of an aqueous 25% GDA solution are added. Cosslinking starts immediately and is terminated after some minutes and die cells are obtained in the form of solid spherical particles. 2000 g of glycerol are added to the mixture obtained at 15° to 20° and phase separation between the TBP phase and d e aqueous phase is achieved. The lower phase containing mainly glycerol and solid spherical cell particles is separated from the upper phase containing mainly TBP. The upper phase obtained (2900 ml) may be re-used e.g. in another crosslinking batch. The solid spherical particles are isolated from rhe lower phase after ca. 90 minutes and washed widi spring water in order to remove TBP and glycerol residues. 580 g of moist solid spherical particles having oxidase activity are obtained, corresponding to 121 g of dry weight. Specific DAO activity in the solid spherical particles is 32 U/g moist weight (153 U/g dry weight), i.e. total DAO activity is 18,560 U (71% of the pre-treated DAO activity obtained as described in example 10 a.. Example 11 580 g of the solid spherical particles (moist weight) obtained in example 10 c. are suspended in 2000 ml of spring water and stirred at room temperature. 20 mg of PMSF are dissolved in 20 ml of absolute edianol and die solution obtained is added to die oxidase suspension witiiin ca. 2 minutes. The mixture is incubated for ca. 3 hours, the solid spherical particles obtained are isolated and washed widi aqueous edianol (1000 ml, 10%) and widi spring water. 563 g of solid spherical particles (moist weight; corresponding to 118 g of dry weight) are obtained. Specific DAO activity in die PMSF treated solid spherical particles is 31 U/g moist weight (148 U/g dry weight), i.e. total DAO activity is 17,450 U (67% of d e pre-treated DAO activity obtained as described in example 10 a. and 94% of the activity before PMSF treatment). The esterase acπvity in the spherical particles obtained is substantially removed. Example 12 The pH of a cell/polymer mixture produced as described in examples 10 a. and b. is adjusted to 9.0 by addition of phosphoric acid and 80 g of aluminium oxide are added. Crosslinking is carried out widi die mixture obtained as described in example 10 c. 630 g of solid spherical particles (moist weight, corresponding to 193 g of dry weight) are obtained. Specific DAO activity in the solid spherical particles is 26 U/g moist weight (85 U/g dry weight), i.e. total DAO activity is 16,440 U (63% of the pre-treated DAO activity obtained as described in example 10 a.). A narrow size-distribution-range of the particles of 240-500 μm is obtained. Example 13 Is carried out analogously as described in example 10 but without addition of glycerol. After crosslinking the suspension is stirred for ca. 60 minutes. The solid spherical particles obtained in TBP suspension are isolated by running the suspension obtained riirough a vessel having a perforated bottom, wherein the perforation holes are smaller than d e size of die solid spherical particles obtained. 2680 ml of TBP are obtained. TBP residues are removed from the solid spherical particles obtained by washing wid spring water. 610 g of solid spherical particles (moist weight, corresponding to 126 g of dry weight) are obtained. Specific DAO activity in die solid spherical particles is 36 U/g moist weight (174 U/g dry weight), i.e. total activity is 21,960 U (85 % of die pre-treated DAO activity obtained as described in example 10 a.. Example 14 PMSF treated solid spherical particles obtained analogously as described in examples 10 and 11 (81 g moist weight, DAO activity: 2,500 U) are mixed widi 1 litre of a 75 mM aqueous Ceph C solution of pH 7.2. Air under 5 bar pressure is introduced into die mixture obtained for ca. 80 minutes. The solution is separated off from the solid spherical particles and is analysed by HPLC. 0.6 mM Ceph C, 70.7 mM Gl-ACA and 3 mM of KA-7-ACA are determined in die separated solution. Example 14 is repeated 142 times each rime using a fresh Ceph C-solution and each rime using die same PMSF treated solid spherical particles described above in example 14. The solution is separated off from the solid particles in batch 142 after ca. 160 minutes of contact with Ceph C and is analysed by HPLC. 0.5 mM Ceph C, 69.8 mM Gl-ACA and 0.4 mM of KA-7- ACA are determined in die separated solution. After batch 142 the solid spherical particles had been used for 308 hours. 93 g of solid spherical particles (moist weight, corresponding to 24 g of dry weight) are isolated. Specific DAO activity in die solid spherical particles obtained is 12 U/g moist weight corresponding to 47 U/g dry weight, i.e. total activity is 1,120 U (45 % of d e original activity). Example 15 Cells of Schtzosaccharomyces po be, e.g. ATCC 38399 and e.g. ATCC 38436 (82 g and 90 g moist weight, respectively) having DAO activity are each pre-treated (separately) analogously as described in example 10 a. widi 3.3 g of an aqueous 25% GDA solution and harvested and stored in a frozen state. After thawing die mixture is washed widi spring water and concentrated to obtain 20.4 g of dry substance in 164 ml from e.g. ATCC 38399 and 24.1 g of dry substance in 180 ml from e.g. ATCC 38436. 7 g of cell dry weight of each of e.g. ATCC 38399 and e.g. ATCC 38436 are treated (separately) with 2.8 g of an aqueous 50% PEI solution as described in example 10 b.. Crosslinking is carried out witii borii suspensions obtained (separately) analogously as described in example 10 c. in 230 ml TBP under addition of 3.5 g of an aqueous 25% GDA solution and under addition of 400 g of glycerol. The solid spherical particles obtained are washed widi spring water over a sieve in order to remove glycerol and TBP residues. 59.5 g moist weight of solid spherical particles having DAO activity (14.0 g dry weight) are obtained from e.g. ATCC 38436 and 55.6 g moist weight of solid spherical particles having DAO activity (16.1 g dry weight) are obtained from e.g. ATCC 38399. The solid spherical particles obtained show similar DAO reaction characteristics and similar stability characteristics as the spherical particles obtained as described in example 10 c. Schtzosaccharomyces pombe is suitable for the cloning and expression of various enzymes. Other biocatalysts, e.g. solid spherical particles having other enzyme activity may be obtained in a simple manner analogously as described in the present example. Example 16 Cells of Pleurotus ostreatus (90 g moist weight, 18,970 U penicillin V acyiase) are pretreated with 3.3 g of an aqueous 25% GDA solution analogously as described in example 10 a.. The cells from the cell suspension obtained are harvested, stored in a frozen state, and after thawing are washed widi spring water and concentrated by microfilrration. 18.6 g of cells obtained (dry weight) are rreated with 7.4 g of an aqueous 50% PEI solution. Cell crosslinking is carried out analogously as described in example 10 c. in 560 ml TBP under addition of 7.4 g of an aqueous 25% GDA solution and under addition of 370 g of glycerol. The solid spherical particles obtained are washed widi spring water through a sieve in order to remove glycerol and TBP residues. 223 g of solid spherical particles containing penicillin V acyiase (moist weight corresponding to 29.4 g dry weight) are obtained. Specific penicillin V acyiase is 50.4 U/g moist weight, i.e. total penicillin V acyiase is 11,240 U (59% die original activity). 1 U penicillin V acyiase corresponds to the formation of 1 μmol per minute of 6-aminopenicillanic acid in an aqueous phosphate-buffered K-penicillin-V solution (50 g/1) at pH 7.5 and 28°. Example 17 Cells of Trtgonopsts variabilis, e.g. ATCC 58536 (54 g moist weight, DAO activity: 2,600 U oxidase) are produced analogously as described in examples 10 a. and 10 b.. Crosslinking is effected analogously as described in example 10 c, but a cell/polymer mixture containing Trigonopsis variabilis cells (ca. 10 g dry weight) is added under stirring into either 300 ml of glutaric dimethylester or 300 ml of adipic dimed ylester (instead of 3000 ml of TBP to 100 g cell dry weight). Crosslinking in each of die mixtures obtained (separately) is carried out under addition of 5 g of an aqueous 25% GDA solution to each of die mixtures and under addition of 400 g of glycerol to each of the mixture at a temperature of 15° to 20° analogously as described in example 10c. 93.4 g and 86.9 g, respectively of solid spherical particles having DAO activity are obtained (moist weight, corresponding to 20.6 g and 20.2 g of dry weight, respectively). Specific DAO activity is 14.8 U/g moist weight or 12.5 U/g moist weight, respectively, i.e. total activity is 1382 U (53% of die original activity) or 1086 U (42% of the original activity), respectively.

Claims: Patent Claims 1. A process for decreasing esterase activity present in the presence of D-ammo acid oxidase activity or in the presence of glutarylacylase activity in a mixture having esterase activity and D- ammo acid oxidase activity and or glutarylacylase activity comprising treating a mixture having esterase acnvity and D-aπuno acid oxidase activity and/or glutarylacylase activity widi phenylmediylsulphonyl fluoride. 2. A process for the production of glutaryl-7-amιnocephalosporanιc acid comprising the steps I) σeanng a mixture having esterase activity and D-ammo acid oxidase activity witii phenylmediylsulphonyl fluoride, ii) reacnng cephalosporin C with a mixture obtained in step i) to obtain glutaryl-7- ammocephalosporanic acid; and isolanng glutaryl-7-amιnocephalosporanιc acid obtained, if desired. 3. A process for the producnon of 7-ammocephalosporanic acid comprising the steps I) treanng a mixture having esterase activity and glutarylacylase activity widi phenylmediylsulphonyl fluoride, ii) reacnng glutaryl-7-amιnocephalosporanic acid widi a mixture obtained in step i) to obtain 7-amιnocephalosporanic acid; and isolanng 7-amιnocephalosporanιc acid obtained in step ii), if desired. 4 A process for the producnon of spherical parncles from microogranism cells having an enzvme acnvity, comprising the steps I) treaπng microorganism cells with a primary or secondary amine containing polymer, ii) contacπng an organic solvent which is able to form a two-phase system with water with a mixture of microorganism cells, iiι)πeaπng a mixture obtained in step ii) widi a bifuncnonal agent; and isolanng die spherical parncles obtained. 5. Spherical parncles containing microorganism cells which contain enzyme activity obtainable from a process comprising the steps I) treaπng microorganism cells having enzyme acnvity with a primary or secondary amme containing polymer ii) contacting an organic solvent which is able to form a two-phase system with water with a mixture of microorganism cells, iii) treating a mixture obtained in step ii) wid a bifunctional agent; and isolating die spherical particles obtained. 6. Spherical particles obtained by a process comprising the steps as defined in claim 5. 7. A process for the production of glutaryl-7-aminocephalosporanic acid comprising the steps i) treating microorganism cells having D-amino acid oxidase activity witii a primary or secondary amine containing polymer, ii) contacting an organic solvent which is able to form a two-phase system with water with a mixture of microorganism cells, iii) treating a mixture obtained in step ii) widi a bifunctional agent; and isolating die spherical particles obtained, and if desired, treating a mixture having esterase activity in die presence of D-amino acid oxidase activity widi phenylmediylsulphonyl fluoride, or treating spherical particles obtained in step iii) having esterase activity in d e presence of D-amino acid oxidase activity wid phenylmediylsulphonyl fluoride, iv) reacting cephalosporin C with spherical particles obtained in step iii) to obtain glutaryl-7- aminocephalosporanic acid, and isolating glutaryl-7- aminocephalosporanic acid, if desired. 8. A process for the production of glutaryl-7-aminocephalosporanic acid which comprises reacting cephalosporin C with spherical particles obtainable by step iii) as defined in claim 7 to obtain glutaryl-7-amιnocephalosporanic acid, and isolating glutaryl-7-aminocephalosporanic acid, if desired. 9. A process for the production of 7-aminocephalosporanic acid wherein glutaryl-7- aminocephalosporanic acid obtained according to claim 2, step ii) or claim 7, step iv) is converted into 7-aminocephalosporanic acid. 10. Use of a process as claimed in claim 4 or use of spherical particles as claimed in claim 5 in die production of 7-amino-3-hydroxymethyl-3-cephem-4-carboxylic acid from cephalosporin C or from glutaryl-7-aminocephalosporanic acid. 11. A process for die production of 7- aminocephalosporanic acid comprising the steps i) treating microorganism cells having glutarylacylase activity widi a primary or secondary amine containing polymer, ii) contacting an organic solvent which is able to form a two-phase system with water with a mixture of microorganism cells, iii) treating a mixture obtained in step ii) with a bifunctional agent; and isolating die spherical particles obtained, iv) if desired, treating a mixture having esterase activity in die presence of glutarylacylase activity widi phenylmediylsulphonyl fluoride, or treating spherical particles obtained in step iii) having esterase activity in the presence of glutarylacylase acyiase widi phenylmediylsulphonyl fluoride, if desired, v) reacting glutaryl-7-aminocephalosporanic acid widi spherical particles obtained in step iii) or obtained in step iv) to obtain 7-aminocephalosporanic acid; and isolating 7- aminocephalosporanic acid obtained, if desired. 12. A process for the production of a cephalosporin wherein 7-aminocephalo-sporanic acid or 7-amino-3-hydroxymethyl-3-cephem-4-carboxylic acid produced according to die present invention according to any one of claims 3 or 9 is used as an intermediate. 13. Product produced by a process according to any of claims 1 to 4, 7 to 9 or 11 to 12.

Inventors: KNAUSEDER FRANZ, PALMA NORBERT, REICHERT ARNO, RIETHORST WAANDER, KNAUSEDER, FRANZ, PALMA, NORBERT, REICHERT, ARNO, RIETHORST, WAANDER
Applicants: BIOCHEMIE GMBH, KNAUSEDER FRANZ, PALMA NORBERT, REICHERT ARNO, RIETHORST WAANDER, BIOCHEMIE GESELLSCHAFT MBH, KNAUSEDER, FRANZ, PALMA, NORBERT, REICHERT, ARNO, RIETHORST, WAANDER
Application year: 1998
Main classification: C12N9
IPC codes: C12N9, C12N11, C12P35