著者

添田 愼介 明石 健志 前田 清 川北 毅

出版者

日本生物工学会

雑誌

生物工学会誌 : seibutsu-kogaku kaishi (ISSN:09193758)

巻号頁・発行日

vol.76, no.9, pp.389-397, 1998-09-25

参考文献数

11

被引用文献数

2

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Tacrolimus is an immunosuppressant macrolide isolated from Streptomyces tsukubaensis. It is used clinically to prevent the rejection of tissue transplants. To achieve the industrial production of tacrolimus, development research was aimed at breeding strains that efficiently produce tacrolimus, optimizing the cultivation conditions, determining an effective purification method, and establishing a means of rapid quantitative analysis. The wild-type S. tsukubaensis was sequentially treated with ultra violet light to furnish various types of morphologically altered mutants, from which a desired strain was selected and bred. For the fermentation of the new strain, a cultivation medium was formulated with a low viscosity and resistant to thermo-denaturation on sterilization. In a scale-up study, in which the fermentor size was increased from 30l to 25kl, the productivity of tacrolimus was found to be well reproduced by keeping both the dissolved oxygen and the agitation at low levels during the growth phase of the producing strain. As a result of these procedures, the concentration of tacrolimus in the fermentation broth was increased 300-fold over that obtaind in the early stages of the research with the wild strain. S. tsukubaensis produces many kinds of proteins and oligosacchalides as well as various types of tacrolimus related compounds. To remove these impurities effectively, the cultivation broth was directly extracted with acetone. The extract was successively purified with a high porosity absorbance resin, and acidic and natural silica gel column chromatography, followed by recrystalization in aqueous acetonitrile, to obtain tacrolimus monohydrate. Tacrolimus itself is readily converted to optical and steric isomers in an aqueous solution. When tacrolimus was analyzed by HPLC at lower temperatures, the peaks corresponding to the macrolide were complex because of cis-trans isomerization in the column. The problem was overcome by heating the column to 50℃, when the isomerization rate was so high that the peaks were fused into a single, sharp one. The epimerization ratio was found to depend on the concentration of water in the solution, but the ratio remained constant when a Brij-35 solution used as a diluent. By these procedures, a simple, rapid and reliable analytical method was established. The industrial production of tacrolimus was thus achieved by a combination of fermentation, purification, and analytical investigations.