Chemistry: molecular biology and microbiology – Carrier-bound or immobilized enzyme or microbial cell;... – Enzyme or microbial cell is immobilized on or in an organic...
Reexamination Certificate
2000-04-03
2002-10-15
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Carrier-bound or immobilized enzyme or microbial cell;...
Enzyme or microbial cell is immobilized on or in an organic...
C435S182000, C435S174000, C435S177000
Reexamination Certificate
active
06465227
ABSTRACT:
This application is a 371 of PCT/EP98/05729, filed Aug. 9, 1998, which claims priority to Austrian applications Ser. No. 1505/97, filed Sep. 9, 1997, Ser. No. 1506/97, filed Sep. 9, 1997 and Ser. No. 1507/97, filed Sep. 9, 1997.
The present invention relates to a process for inhibition of esterase activity in enzyme preparations, e.g. useful in enzymatic camlysed production of 7-aminocephalosporanic acid (7-ACA); and to the production of enzyme preparations, e.g. useful in 7-ACA and/or 7-amino-3-hydroxymethyl-3-cephem-4-carboxylic acid production.
7-ACA is a key intermediate in the production of e.g. pharmaceutically, active cephalosporin antibiotics, 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 in the production of cephalosporin antibiotics or known as a valuable group in an intermediate for their production; e.g. to obtain a 7-arninoacylated cephalosporin having a methylaceroxy group in position 3 of the ring system, such as cefotaxime, cerpodoxime (proxetil), cephaloglycin, and in the production of other cephalosporin antibiotics or intermediates for their production which may be derived from 7-ACA; e.g. by further reacting the methylacetoxy group in position 3 of the ring system to obtain a 3-substituted cephalosporin substituted by a group which is different from the acetoxymethyl group; e.g. a group known as a valuable group in the production of cephalosporin antibiotics or known as a valuable group in an intermediate for their production; e.g. by nucleophilic substitution of the aceroxy group, or e.g. by deacerylation of the methylacetoxy group to obtain the hydroxymethyl group (e.g. HACA); and e.g. further reacting a hydroxymethyl group obtained in position 3 of the ring system, e.g. by nucleophilic substitution of the hydroxy group; or removal of the hydroxy function of a hydroxymethyl group or removal of the hydroxymethyl group in position 3 of the ring system: and, if desired, esterification of the carboxylic group in position 4 of the ring system, e.g. by a group known as a valuable group in the production of cephalosporin antibiotics or known as a valuable group in an intermediate for their production: and, if desired, salt and/or solvent formation of a cephalosporin compound obtained in such a reaction e.g. according to a conventional method. 7-ACA and HACA may e.g. be obtained from cephalosporin C (Ceph C) by deacylation of the amine group in position 7 of the ring system, and deacetylation of the methylaceroxy group in position 3 of the ring system, respectively, e.g. enzymatically. An enzyme useful in enzymatic Ceph C deacylation is e.g. D-amino acid oxidase (DAO) which catalyses the oxydative desamination of Ceph C to form giutaryl-7-aminocephalosporanic acid (G1-7-ACA) via the intermediate a-ketoadipoyl-7-aminocephalosporanic acid (KA-7-ACA). G1-7-ACA may be hydrolised to obtain glutaric acid and 7-ACA, e.g. by a glutarylacylase (GAC). It is known that DAO and GAC containing enzyme preparations, such as microorganism cells may contain esterase activity additionally. Undesired deacerylation of the side chain in position 3 of the ring structure of the cephalosporin may occur, e.g. due to the presence of esterase activity in enzymatically catalysed Ceph C deacylation, e.g. 3-hydroxymethyl derivatives of Ceph C and G1-7-ACA may be formed. This may result in a significant decrease of 7-ACA quality and yield. Conventional methods for decreasing esterase activity present in the presence of DAO activity by acetone or CuSO
4
treatment may decrease esterase activity incompletely.
It was now surprisingly found that the treatment of a mixture having esterase activity in the presence of DAO activity or having esterase activity in the presence of GAC activity with phenyimethyisulphonyl fluoride (PMSF) may decrease esterase activity 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 the present invention PMSF, known e.g. as an irreversible inhibitor of serine containing proteins by serine sulphonviation, may decrease esterase activity present in the presence of DAO activity or in the presence of GAC activity selectively without, e.g. substantial influence on DAO or GAC activity; and even more surprising is the selective and effective inhibition by PMSF of esterase activity present in the presence of GAC activity, e.g. because of similar function and structure of acylases and esterases.
In one aspect the present invention provides a process for decreasing, e.g. substantially removing 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 glutarylacylase activity, e.g. present in the form of microorganism cells or in the form of a cell-free extract thereof, e.g. in the form of a cell-free extract thereof, comprising treating a mixture having esterase activity and D-amino acid oxidase activity and/or glutarylacylase activity with phenyimethyisulphonyl fluoride, e.g. wherein D-amino acid oxidase activity or glutarylacylase activity remains, e.g. substantially the same after treatment with phenyimethyisulphonyl fluoride as before said treatment. Typically D-amino acid oxidase activity remains more than 91% of the original value and in the case of glutarylacylase activity even more than 97% of the original value.
A process of the present invention may e.g. be carried out as follows: Known and e.g. commercially available microorganisms producing DAO activity include e.g. Trigonopsis, Aspergillus, Penicillium, preferably
Trigonopsis variabilis
microorganisms. Known and e.g. commercially available microorganisms producing GAC activity include e.g Pseudomonas, Achromobacter,
Bacillus cereus
or e.g. transformants, e.g.
E. coli
transformants, e.g. transformed according to e.g. a conventional method. A mixture having DAO activity or GAC activity and esterase activity may e.g. be obtained commercially or e.g. according to a conventional method, and may be e.g. in the form of a cell-free extract, e.g. in immobilised form, or in the form of microorganism cells, such as in the form of e.g. partly purified or impurified cells, and/or permeabilised or non-permeabilised cells, and/or partly destroyed or intact cells, and/or immobilised or non-immobilised cells; such as obtainable from a fermentation broth, e.g. according to a conventional method, preferably in the form of a cell-free extract, e.g. immobilized. E.g. microorganism cells may be isolated, e.g. harvested from a fermentation broth and used as such, e.g. in moist form, e.g. after centrifugation of the fermentation broth, or the cells may be further treated before or after isolation from the fermentation broth, e.g. according to a conventional method, such as homogenising cells to obtain, e.g. partly, destroyed cells, and/or permeabilising cells or fragments thereof to obtain permeabilised cells or cell fragments and/or purifying cells or cell fragments to obtain e.g. partly purified cells and/or cell fragments and/or cell-free (e.g. by cell flocculation) 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. immobilisation may be carried out according to a conventional method; e.g. in the presence of acrylic (immobilisation) beads. such as Eupergite® or in the presence of an ion exchange resin, e.g. such as Relite Dianion® according to a method described in the examples below; or e.g. according to another aspect of the present invention which is described below.
The microorganism cells may be used in the form of an, e.g. buffered, aqueous cell suspension, obtainable e.g. by re-suspension of cells in water or in a buffer solution after isolation from a fermentation broth, or in case of a cell-free extract an aqueous solution of the cell-free extract may be used. The pH of a s
Knauseder Franz
Palma Norbert
Reichert Arno
Riethorst Waander
Biochemie Gesellschaft m.b.H.
Dohmann George R.
Lopez Gabriel
McNally Lydia
Weber Jon P.
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