Chemistry: molecular biology and microbiology – Process of utilizing an enzyme or micro-organism to destroy... – Treating animal or plant material or micro-organism
Patent
1997-12-03
1999-12-21
Lilling, Herbert J.
Chemistry: molecular biology and microbiology
Process of utilizing an enzyme or micro-organism to destroy...
Treating animal or plant material or micro-organism
435119, 435800, C12P 1718
Patent
active
060048018
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a purification process of K-252a.
BACKGROUND ART
K-252a is a physiologically active substance produced by microorganisms (Japanese Patent Laid-Open Application No. 41489/1985). It has an inhibition activity for protein kinase C and exhibits various pharmacological effects.
A conventional purification process of K-252a comprises the steps of: a) collecting microorganisms by filtering a culture solution; b) extracting the microorganisms by adding thereto a hydrous or anhydrous organic solvent such as methanol or acetone; c) removing the microorganisms by filtration; d) concentrating the resulting extracted solution; e) further extracting it with an anhydrous organic solvent in high yields; f) separating it by using a column filled with adsorbents such as active carbon or Diaion HP-10 and carriers such as silica gel, silanized silica gel, aluminum oxide or dextran; and g) concentrating it to obtain a crude K-252a crystal. However, the crystal thus obtained will not have sufficient purity. This process therefore further needs a recrystallization step in order to achieve higher purity. Furthermore, this process is not suitable for filtering a large amount of the culture solution because it is considerably difficult to filter the culture solution in this process.
Alternatively, it is also possible to collect an extracted solution by directly extracting a culture solution with an organic solvent and then filtering it. However, this process further needs a column treatment because the organic solvent used for extraction should be added in the same volume as the culture solution or more due to a lower solubility of K-252a in various solvents (5 to 10 g/l, for example 5 g/l in acetone), a liquid volume to be treated is extremely increased and the resulting extracted solution has lower purity.
DISCLOSURE OF THE INVENTION
The present invention provides a purification process of K-252a, which comprises: ##STR3## with an alkaline solution to convert K-252a into K-252b represented by formula (II): ##STR4## or alkali salts thereof, which are then released out of the cells, methylating K-252b or alkali salts thereof to convert them into K-252a again, and
The microorganism cells containing K-252a used in the present invention may be obtained by culturing microorganisms capable of producing K-252a in a nutrient medium.
The microorganisms capable of producing K-252a include Nocardiopsis sp. K-252 deposited at the Agricultural Research Service Culture Collection in the United States under accession number NRRL15532 (Japanese Patent Laid-Open Application No. 41489/1985).
As the nutrient medium any conventional medium may be used which is generally used for culturing microorganisms capable of producing K-252a. Examples include a nutrient medium used for culturing normal antinomycetes. The microorganisms may be suitably cultured in a liquid medium, particularly under a submerged culture condition. They may be preferably cultured at a temperature of 25 to 40.degree. C. and at a neutral pH of 5 to 9, more preferably 6 to 8.
After K-252a has been accumulated inside the microorganism cells, the culture of the cells is stopped to obtain microorganisms used for the purification of K-252a. K-252a may be preferably accumulated inside the cells at a concentration of at least 1.0 g/liter of cell volume, more preferably at least 5.0 g/liter of cell volume.
The microorganism cells can be collected, for example, by centrifugation. Preferably, an acid such as hydrochloric acid, sulfuric acid, nitric acid and acetic acid, preferably sulfuric acid, may be added to the culture solution to adjust its pH to 2-4 before collecting the cells.
Any centrifugal separator may be used for centrifugation. Particularly, Westfalia separator and .alpha.-Labal separator are preferred because they can continuously separate a large amount of the culture solution while washing the microorganism cells with washing water.
The collected cells maybe suspended into an alkaline solution at a concentration of 10 to
REFERENCES:
patent: 4555402 (1985-11-01), Matsuda et al.
Ross et al. "Differential Biological Effects of K252 Kinase Inhibitors are related to Membrane Solubility but not to Permeability" J. Neurocehm 65 No. 6 (1995) pp. 2748-2756.
Wood et al., "Design and Implementation of an Efficient Synthetic Approach to Furanosylated Indolocarbazoles: Total Synthesis of (+)-and(-)-K252A," J. Am. Chem. Soc. 119: 9641-9651 (1997).
Kase et al., "K-252a, a potent inhibitor of protein kinase C from microbial orgin," J. Antibiot. 39: 1059-1065 (1986).
Yasuzawa et al., "The structures of the novel protein kinase C inhibitors K-252a, b, c and d," J. Antibiot. 39: 1072-1078 (1986).
Nakanishi et al., "K-252 b, c, and d, potent inhibitors of protein kinase C from microbial orgin," J. Antibiot. 39: 1066-1071 (1986).
Lazarovici et al., "K-252a inhibits the increase in c-fos transcription and the increase in intracellular calcium produced by nerve growth factor in PC12 cells," J. Neurosci. Res. 23: 1-8 (1989).
Nakayama et al., "K252a inhibits the phosphorylation of pRb without changing the levels of G1 cyclins and Cdk2 protein in human hepatoma cells," Biochem. Biophys. Res. Commun. 224: 180-183 (1996).
Isono et al., "Epidermal growth factor induces PC12 cell differentiation in the presence of the protein kinase inhibitor K-252a," J. Neurochem. 4: 1235-1245 (1994).
Gschwendt et al., "Differentiative action of K252a on protein kinase C and a calcium-unresponsive, phorbol ester/phospholipid-activated protein kinase," Biochem. Res. Commun. 164: 974-982 (1989).
Kino Mitsutaka
Kinoshita Tadashi
Nagamura Satoru
Kyowa Hakko Kogyo Co. Ltd.
Lilling Herbert J.
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