Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing heterocyclic carbon compound having only o – n – s,...
Patent
1995-12-05
1998-01-27
Rollins, John W.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing heterocyclic carbon compound having only o, n, s,...
435117, 435125, C12P 1702, C12P 1706, C12P 1700
Patent
active
057121303
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The invention belongs to the field of biotechnology and relates to a process for the isolation of the hypolipaemic active substance lovastatin from a fermentation broth. Lovastatin is produced as a secondary metabolite of the fungus Aspergillus terreus (U.S. Pat. No. 4,231,938) deposited in American Type Culture Collection under Nos. ATCC 20541, ATCC 20542, and Monascus ruber deposited in Fermentation Research Institute Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan (DE 30 06 216 A1) under No. Ferm 4822. Other kinds of microorganisms producing lovastatin are known as well, e.g. a mutant of the microorganism Aspergillus terreus and Aspergillus oryzae marked ATCC 74135.
Lovastatin is chemically 1',2',6',7',8a'-hexahydro-3,5-dihydroxy-2',6'-dimethyl-8'-2"-methyl-1"-oxo butoxy)-1-naphtalene heptanoic acid-5-lactone (Stubbs et al., 1986) of the formula. (EP 0 033 537 A1) ##STR1##
An active form of lovastatin is also an acid, which is chemically 1,2,6,8,8a-hexahydro-.beta.,.delta.-dihydroxy-1-naphtalene heptanoic acid (Alberts et al., 1980) of the formula (EP 0 022 478 A1) ##STR2##
The lactone form of lovastatin is used as an agent for reducing cholesterol level in blood (Scott M. G. and Vega G. L., 1985). It inhibits the biosynthesis of mevalonic acid by inhibition of 3-hydroxy-3-methylglutaryl A reductase coenzyme (HMG-CoA reductase, E.C. 1.1.1.34) (Zubay et al., 1984).
PRIOR ART
After the completed fermentation, lovastatin is present in the broth in the lactone form (compound I) and in the acid form (compound II). In the isolation process as disclosed in EP 0 033 536 A2, lovastatin is extracted from the broth with ethyl acetate. The extract is concentrated by vacuum distillation. Since lovastatin is present in the lactone form as well as in the acid form and only the lactone is of commercial interest, the acid form should be converted into the lactone. The lactonisation is carried out by the reflux of the concentrate in toluene at 106.degree. C. for 2 hours. After the lactonisation is complete, the solution is concentrated to a small volume. A pure substance is obtained by means of purifying the concentrate on columns packed with silica gel, in the presence of solvents such as ethyl acetate or n-hexane. The collected fractions are again concentrated in vacuo and then pure lovastatin crystallizes in the lactone form.
Due to the sophisticated multi-step procedure and vigorous conditions applied during the isolation, the yields of lovastatin are generally low. Different solvents, which in part exhibit toxicity, are used such as benzene, toluene, acetonitrile or ethyl acetate. Hence working with these solvents endangers the health of the persons involved and poses a problem with respect to the environment.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 represents the results of IR spectroscopy confirming the structure of the substance isolated according to the present invention;
FIG. 2 represents the results of mass spectroscopy confirming the structure of the substance isolated according to the present invention;
FIG. 3 represents the results of NMR confirming the structure of the substance isolated according to the present invention; and
FIG. 4 represents the results of UV spectroscopy confirming the structure of the substance isolated according to the present invention.
DESCRIPTION OF THE INVENTION
An object of the present invention was to overcome the above deficiencies of the state of the art and to provide a process for isolating lovastatin in an efficient and economic way. In addition, attention should be paid to health aspects of the workers involved in the isolation and to the environment.
As a result of extensive search leading to the present invention, the present inventors have found that lovastatin may be easily isolated by extracting lovastatin from a sample with butyl acetate, concentrating the solution and carrying out a crystallization.
In a preferred embodiment the concentration of the solution is carried out under reduced pre
REFERENCES:
patent: 4231938 (1980-11-01), Monaghan et al.
patent: 5156960 (1992-10-01), Jekkel nee Bokany et al.
patent: 5403728 (1995-04-01), Jekkel et al.
Hajko Pavica
Pokorny Miroslav
Radez Ivan
Vesel Tanja
KRKA tovarna zdravil, p.o
Rollins John W.
Tate Christopher R.
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