The Streptomyces tsukubaensis was found in the early 1980s near Mt. Tsukuba, Ibraki, Japan. In 1985 S. tsukubaensis was found to produce a 23-membered macrolide antibiotic - Tacrolimus (also FK-506, Fujimycin, Prograf®), a potent immunosuppressive agent. It was introduced into the clinic in the late 1980s for use as both primary and rescue therapy in patients receiving solid organ transplants. Its main function is to reduce the activity of the patient's immune system after allogenic organ transplant and therefore the risk of organ rejection. Tacrolimus hydrate is isolated from the whole fermentation broth of a bacterium Streptomyces tsukubaensis cultivated in submerged culture.

Our research project has been aimed at developing an original, novel method of biosynthesis of the macrolide by means of submerged cultivation using liquid media containing degradation products of food industry.

In our research the strain of Streptomyces tsukubaensis (FERM BP-927) has been used. Several media have been tested to find the relationship between the composition of the cultivation medium and the mycelial growth and tacrolimus productivity by the microorganism.

The method of determination of FK 506 by High-Performance Liquid Chromatography has also been studied in this research. Main problems in HPLC determination of Tacrolimus are caused by two kinds of conformational heterogenity of this compound. First kind is a cis-trans conformational isomerization involving restricted rotation of the amide bond in a pipecolic moiety, second is reported for aqueous solution epimerization of FK-506 to an intermediate tautomer I (cis) which is converted into tautomer II (cis-trans). For quantitative chromatographic method it creates a problem when there is a difference in water content between a sample and a standard solution. To avoid problems caused by the epimerization and isomerization during inprocess monitoring of bulk drug substance during the Tacrolimus biosynthesis, combined methods of Akashi [1] and Nishikawa [2] were used in our research.

The best strain growth was recorded for media containing starch as a carbon source and soybean extract as a nitrogen source. For cheap media, containing beat molasses and grain worth as carbon and nitrogen source, growth of cultivated strain was two times lower, however productivity of tacrolimus biosynthesis observed for these cultivations was comparably good.

The highest productivity recorded during our preliminary tests was equal 2,8 mcg/ml of medium. Obtained productivity is two times lower than that declared in Okuhara et al. US patent [3]. We hope that optimization of cultivation conditions and culture media will positively affect the macrolide productivity.

[1] T. Akashi, T. Nefuji, M.Yoshida, J. Hosoda; J. Pharm. Biomed. Anal. 14 (1996) 339-346

[2] T. Nishikawa, H.Hasumi, S.Suzuki, H.Kubo, H.Ohtani; Pharm. Res.10 (1993) 12, 1785-1789

[3] Okuhara et al., US Patent 4,894,366 (1990)

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1. ACCUMULATION OF ZINC BY THE LENTINUS EDODES (BERK.) MYCELIUM CULTIVATED IN SUBMERGED CULTURE.