Improving Productivity of Pravastatin, HMG-CoA Reductase Inhibitor

HMG-CoA Reductase Inhibitor인 Pravastatin의 생산성 향상

  • 전동수 (단국대학교 식품공학과) ;
  • 배동훈 (단국대학교 식품공학과)
  • Received : 2009.09.01
  • Accepted : 2009.10.13
  • Published : 2009.11.30

Abstract

Pravastatin sodium, competitive inhibitors of HMG-CoA(3-hydroxy-3-methylglutaryl coenzyme A) reductase, is produced from the culture broth of Streptomyces carbophilus KCCM 10370, The production of Pravastatin sodium was increased about 45 fold compared to wild type by UV mutation. Production of Pravastatin was also improved by continuous feeding of Compactin sodium to 24% and bioconversion ratio was also increased to 4.3% by intermittent addition. In main culture, concentration of Compactin sodium was kept less than 0.1%(w/v) under continuous feeding of Compactin sodium then product was 0.49% and bioconversion was 70%. After finishing the fermentation, Pravastatin was purified by various chromatographies such as Diaion HP20 resin column, Partition, and ODS(Octa-Decylsilyl Silicagel) resin column with a final yield of 70~72% and over 99.7% purity. The IR, UV, and NMR study of the purified Pravastatin sodium showed the same pattern as that of EP(European Pharmacopoeia).

프라바스타틴 나트륨은 HMG-CoA(3-hydroxy-3-methylglutaryl coenzyme A) 환원효소 저해제로서, 콜레스테롤 생합성의 초기 율속단계(rate-limiting step)에 작용하여 메 발로네이트(mevalonate)의 합성을 저해하는 광범위한 고지혈증 치료제이다. 본 연구에서는 효율적 발효와 고품질의 정제를 통한 생산성 향상을 위해 수행하였다. 프라바스타틴 나트륨의 경쟁력있는 생산성 향상을 위하여 Streptomyces carbophilus KCCM 10370을 UV 돌연변이 처리하여 프라바스타틴 나트륨의 생산량을 약 45배 향상시키는 S. carbophilus K-212(KCCM 10370) 선별하였다. 생산성을 극대화시키기 위하여 콤팩틴 나트륨의 연속식 투입이 회분식 투입보다 생산량이 24%, 전환율 4.3%로 향상 되었으며, 본배양에서 콤팩틴 나트륨의 농도를 연속적으로 투입하여 0.1%(w/v) 이하로 유지시켜 프라바스타틴 나트륨의 생산량이 0.49%, 전환율 70%로 확인하였다. 발효가 종료된 후 배양액을 Diaion HP20 수지 컬럼, 분배(Partition), 그리고 ODS(Octa-Decylsilyl Silicagel) 수지 컬럼 크로마토 그래피를 하여 유럽약전기준(EP)에 적합한 순도 99.7% 이상, 70~72%의 수율이었다. 프라바스타틴 나트륨을 적외부스펙트럼측정법, 자외부스펙트럼측정법, 수소핵자기공명스펙트럼측정법 및 탄소핵자기공명스펙트럼측정법으로 분석한 결과 유럽약전기준(EP)과 일치하였다.

Keywords

References

  1. Dietschy JM, Wilson JD. 1970. Regulation of cholesterol metabolism.N. Engl. J. Med., 282: 1179-1183 https://doi.org/10.1056/NEJM197005212822105
  2. Endo A, and Kuroda M. 1976. Citrinin, an inhibitor of cholesterolsynthesis. J. Antibiotics. 29: 841-843 https://doi.org/10.7164/antibiotics.29.841
  3. Endo A, Kuroda M, Tsujita Y. 1976. ML-236A, ML-236B andML-236C, new inhibitors of cholestero genesis produced byPenicillium citrinum. J. Antibiot. 29: 1346-1348 https://doi.org/10.7164/antibiotics.29.1346
  4. Cleeman JI. 2001. Executive Summary of The Third Report ofThe National Cholesterol Education Program(NCEP) ExpertPanel on Detection, Evaluation, And Treatment of High BloodCholesterol In Adults JAMA. 285(19): 2486-2497 https://doi.org/10.1001/jama.285.19.2486
  5. Freeman DJ, Norrie J, Sttar N, Neely DG, Cobbe SM, Ford I,Isles C, Lorimer AR, Macfarlane PW, McKillop JH, PackardCJ, Shepherd J, Gaw A. 2001. Pravastatin and the developmentof diabetes mellitus: Evidence for a protective treatment effectin the West of Scotland Coronary Prevention Study. Circulation103: 357-362 https://doi.org/10.1161/01.CIR.103.3.357
  6. Hideyuki H, Harumitsu K, Takeshi K, Akira T, Takashi N. 1986.Structure elucidation of bioactive metabolites of ML-236B(Mevastatin)isolated from dog urine. Chem. Pharm. Bull. 34: 1459-1467 https://doi.org/10.1248/cpb.34.1459
  7. Hosobuchi M, Testsuya S, Masatoshi A, Jo O, Seigo I, Hiroji Y.1992. Production of ML-236B, and inhibitor of 3-hydroxy-3-methylglutaryl Co A reductase, by Penicillium citrinum :Improvements of strain and culture conditions. Biosci. Biotech.Biochem. 57: 1414-1419 https://doi.org/10.1271/bbb.57.1414
  8. Kiyoshima K, Takada K, Yamamoto M, Kubo K, Okamoto R,Fukagaway, Ishikura T, Naganawa H, Sawa T, Takeuchi, UmexawaH. 1987. New tylosin anologs produced by mutants ofStreptomyces fradiae. J. Antibiot. 40: 1123-1130 https://doi.org/10.7164/antibiotics.40.1123
  9. Masahiko H, Ogawa K, Yoshikawa H. 1993. Morphology study inproduction of ML-236B, a precursor of pravastatin sodium, byPenicillium citrinum. J. Ferment. Bioeng. 76: 470-475 https://doi.org/10.1016/0922-338X(93)90243-2
  10. Miller GL. 1959. Use of dinitrosalicylic acid regent for determinationof reducing Suar. Anal. Chem. 31: 426-428 https://doi.org/10.1021/ac60147a030
  11. Ni W, Egashira K, Kataoka C, Kitamoto S, Koyanagi M, Inoue S,Takeshima A. 2001. Antiinflammatory and antiarterioscleroticactions of HMG-CoA reductase inhibitor in a rat model ofchronic inhibition of nitric oxide synthesis. Circ. Res. 89: 415-421 https://doi.org/10.1161/hh1701.096614
  12. Nobufusa S, Hosobuchi N, and Yoshikawa H. 1996. Biochemicaland molecular-approaches for production of Pravastatin, a potentcholesterol-lowering drug. Biotech. Ann. Rev. 2; 373-389 https://doi.org/10.1016/S1387-2656(08)70017-6
  13. Ongini E, Impagnatiello F, Bonazzi A, Guzzetta M, Govoni M,Monopoli A, Soldato PD, Ignarro LJ. 2004. Nitric oxide (NO)-releasing statin derivatives, a class of drugs showing enhancedantiproliferative and antiinflammatory properties. Proc. Natl.Acad. Sci. 101: 8497-8502 https://doi.org/10.1073/pnas.0401996101
  14. Serizawa N, Nakagawa K, Hamano K, Tsujita Y, Terahara A,Kuwano H. 1983. Microbial hydroxylation of ML-236B(compactin)and monacolin K. J. Antibiot. 36: 604-607 https://doi.org/10.7164/antibiotics.36.604
  15. Stryer L.1995. Biochemistry (4th ed). W.H. Freeman and CompanyVirginia, USA, pp. 685-712
  16. Tobert JA. 1987. New developments in lipid-lowering therapy :the role of inhibiters of hydroxymethyl-glutaryl-coenzyme Areductase, Circulation, 76(3), 534-538 https://doi.org/10.1161/01.CIR.76.3.534
  17. Tsujita Y, Kuroda M, Shimada T, Tanzawa K, Arai M, Kaneko I,Tanaka M, Masuda H, Tarumi C, Watanabe Y, Fujii S. 1986.CS-514, a competitive inhibitor of 3-hydroxy-3-methylglutarylcoenzyme A reductase : tissue-selective inhibition of sterol synthesisand hypolipidemic effect on various animal species. Biochim.Biophys. Acta. 877: 50-60 https://doi.org/10.1016/0005-2760(86)90117-7
  18. Wassmann S, Laufs U, Muller K, Konkol C, Ahlbory K, BaumerAT, Linz W, Bohm M, Nickenig G. 2002. Cellular antioxidanteffects of atrovastatin in vitro and in vivo. Arteriolscler ThrombVasc Biol. 22: 300-305 https://doi.org/10.1161/hq0202.104081
  19. Witkin EM. 1976. Ultraviolet mutagenesis and inducible DNArepair in Escherichia coli. Bacteriol. Rev. 40: 869-907