Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing heterocyclic carbon compound having only o – n – s,...
Patent
1997-08-26
1999-11-16
Lankford, Jr., Leon B.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing heterocyclic carbon compound having only o, n, s,...
435136, 435886, 514210, 540349, C12P 740, C07D48708, C12N 120
Patent
active
059856246
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a process for increasing the production of clavulanic acid and other clavams including those with a strong beta-lactamase inhibitory activity from organisms having the appropriate biosynthetic pathways.
Micro-organisms, in particular Streptomyces sp. produce a number of antibiotics including clavulanic acid and other clavams, cephalosporins and penicillins.
Clavulanic acid is an important beta-lactamase inhibitor which is a key ingredient of the antibiotic sold under the name AUGMENTIN (Trade Mark of SmithKline Beecham plc). The commercial method by which clavulanic acid is produced is via fermentation of Streptomyces clavuligerus. A suitable fermentation medium for producing clavulanic acid is described in UK Patent Specification No. 1,508,977.
Whilst clavulanic acid and other clavams can be prepared in acceptable amounts by existing methods there remains a need for improving the titre of clavulanic acid in the fermentation broth so that the product can be produced more economically. One way in which the problem can be addressed is to seek mutant strains of Streptomyces clavuligerus ATCC 27064 or other clavulanic acid-producing microorganisms which give rise to higher titre. To date relatively little has been published regarding process improvements by which a higher titre of clavulanic acid can be achieved by varying the reaction conditions in the fermentor.
It is customary to include ammonia as a source of nitrogen as manipulation of the nitrogen input to the fermentation is critical to clavulanic acid yield. However we have found that fermentation methods for producing clavulanic acid are particularly sensitive to the concentration of ammonia in the system.
According to the present invention there is provided a method for preparing clavams by fermentation of a clavam--producing organism in a suitable medium, characterised in that the amount of ammonia in the fermentation is kept at a low level during the fermentation in order to avoid repression of one or more key enzymes by ammonia.
By a low level we mean less than about 50 .mu.g/ml. The level of ammonia should be maintained below 50 .mu.g/ml for sufficient time, suitably between 1 and 10 hours, to allow derepression and biosynthesis of the key ammonia-repressible enzymes. Under customary fermentation conditions (with ammonia levels exceeding 100 .mu.g/ml) it has been found that the ammonia acts to repress a number of enzymes involved in the metabolism of nitrogen including urease which catalyses the conversion of urea to ammonia and carbon dioxide (See V. Bascaran et al. J. Gen Microbiol. 1989 vol 135 pp 2465 to 2474 ). However the latter studies were not linked to antibiotic titre and made no suggestion for any link with improvement in clavam or clavulanic acid production in particular.
A surprising finding in the work leading up to the present invention was that urea accumulates in clavulanic acid fermentation broths. Urea is produced from the clavulanic biosynthetic process but there may well be other sources of this urea.
It has been found in accordance with a further aspect of the present invention that if the urea which can build up during clavulanic acid production is caused to react with a urease enzyme titres of clavulanic acid are considerably improved, for example in the order of 10%.
Accordingly in a further aspect of the invention there is provided a method for preparing clavams by fermentation of a clavam-producing micro-organism in a suitable medium characterised in that urea produced during the fermentation process is caused to react with a urease enzyme.
The urease can be either intrinsic (endogenous) or extrinsic (exogenous), that is to say can be caused to be produced by the clavulanic acid producing micro-organism (intrinsic urease) or added to the system (extrinsic urease).
If it is desired to use extrinsic urease the enzyme may be obtained from any suitable source and added to the fermentation reaction. If the urease is intrinsic it can, in one aspect of the invention, be liberated by derepressing expre
REFERENCES:
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patent: 5185139 (1993-02-01), Krishnamurthy et al.
Romero et al. Appl. Microbiol. Biotechnol. vol. 20, pp. 318-325, 1984.
Zhang et al. Can. J. Microbiol. vol. 35, pp. 399-402, 1989.
Zhang et al. Biotechnol. Adv. vol. 9 (4), pp. 623-642, 1991.
Aharonowitz et al. Can. J. Microbiol. vol. 25, pp. 61-67, 1979.
Bascaran et al., "Regulationof Nitrogen Catabolic Enzymes in Streptomyces clavuligerus", Journal of General Microbiology, 135, 2465-2474 (1989).
Holms William Henry
Jeffkins Paul Alan
Mousdale David Michael
Valentine Brian Peter
Kerekas Zoltan
Kinzig Charles M.
Lankford , Jr. Leon B.
SmithKline Beecham p.l.c.
Tate Christopher R.
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