【作者】 陈希杨；

【导师】 王普；

【作者基本信息】 浙江工业大学 ， 生物化工， 2002， 硕士

【摘要】 格氏物是合成倍他米松的重要中间体，倍他米松（Betamethasone），即16β-甲基-9α-氟氢化泼尼松（16β-Methy1-9α-fluoroprednisolone），是目前糖类皮质激素中作用最强的药物之一。其抗炎作用是氢化可的松（Hydrocortisone）的35倍，是地塞米松的2.5倍。 倍他米松的合成一般采用剑麻或薯蓣皂素为起始原料，在合成路线中C_1,2的脱氢采用简单节杆菌进行，其反应式如下： 本文首先对甾体药物工业现状、甾体药物生产新资源的开发、微生物转化在甾体药物转化中的应用、甾体激素的微生物脱氢研究进展及新技术的开发进行全面综述，并进行了展望。 对实验室保藏的简单节杆菌通过初筛后并进行菌种的分离纯化，获得一株转化率较高的菌株02-7-6-14，以此为出发株进行菌种选育。采用紫外诱变，并结合CS¹³⁷-γ射线辐照复合诱变处理，获得较佳诱变株Q2-2-2-1。应用于格氏物脱氢反应，转化率为86.54％，转化率比出发株02-7-6-14提高了5％左右，且遗传性状稳定。 本文考察了培养基组成及环境因子对该诱变株产脱氢酶的影响。通过单因素条件优化及正交试验确定最佳培养基组成为：葡萄糖0.4％，玉米浆1.2％，蛋白胨0.3％，KH₂PO₄0.2％。产酶最适条件为：接种量20％，发酵初始培养基pH7.0～8.0，摇瓶装液量为100ml/250ml，诱导物加量为5mg／100ml，在培养期初期加入格氏物（诱导物）对产酶有很大的促进作用。金属离子Cu²⁺和Fe³⁺对产酶有很大的的抑制作用，Zn²⁺和Fe²⁺对产酶也有一定的抑制作用，Co²⁺和Mg²⁺对产酶有浙江工业大学硕士论文 摘要一定的促进作用。表面活性剂泡敌和吐温80对产酶有一定的促进作用，而洗衣粉和SDS对菌体生长和产酶有很大的抑制作用。 本文对转化工艺条件进行了较深入的研究，确定了发酵工艺的基本条件：接种量为 20％，装量为 100 ml／250 ml三角瓶，培养基初始 pH为 7刀～7．5，诱导物加量为 10 mg／L发酵液，试验中选用乙醇D5％)作为格氏物的增溶介质，其加量为7％，投料浓度为0．7％。合适浓度的氯化钻、外源电子受体(辅酶I、辅酶＊、维生素K／和吐温80对转化都有一定的促进作用。首次将培养液稀释新工艺应用于格氏物简单节杆菌脱氢反应。试验中选用无菌水为稀释介质，当稀释比为1：1时，其转化率与原工艺相近，所以能大大降低生产成本。实验中还考察了超声处理菌体。投料后的发酵液及底物（稀释介质中析出后）对转化结果的影响。结合培养液稀释工艺，将底物在无菌水中析出并经超声处理后投料，转化率高达92．54％。发现超声处理能有效使底物颗粒细化，提高固体颗粒的比表面积，从而提高底物的溶解速率，起到强化传质的作用。 在 10 L罐中进行小试，发现转化过程中提高通气量，转化速率加快，转化周期可以缩短。在该 10 L罐小试中，采用该诱变株转化率达到 86．7％左右。 本文首次采用简单节杆菌游离细胞进行了格氏物脱氢反应动力学研究。其中包括酶稳定性试验，底物浓度对反应速度的影响，酶量对转化初速度的影响。由此建立了该反应的动力学模型及相应的动力学方程，并经线性回归得出反应动力学参数，模型计算结果与实验数据的比较表明提出的动力学模型能较合理的描述该转化过程。更多还原

【Abstract】 17 α-hydroxy-16 β-methyl-pregn-4,9(11)-diene-3,20-dione was one of the important intermediates used to manufacture beta-methasone (16 ?-methyl -9 a -fluoroprednisolone) which was one of the most effective anti-inflammatory drug. The anti-inflammatory activity of beta-methasone was 35 times than that of hydrocortisone and 2.5 times than dexamethasone.The commercial production of beta-methasone involves several steps from the precursors (Diosgenin and Tigogenin). One of the important step needed A l -dehydrogenation by Arthrobacter simplex. The reaction as follows:Firstly, the current status of steroids industry > development of the steroids resource > progress on the microbial dehydrogenation of steroids and applications of new technology used on the steroid bio-transformation were reviewed and the prospects were described as well in this paper.Strain Q2-2-2-1 was obtained after isolation and rejuvenescention by UV treatment and Cs137- Y irradiation. The production capability was about 86.54%, which was 5% higher than that of the original strain 02-7-6-14 and the strain had hereditary stability.In this article, the effects of the medium composition and the environmental factors on dehydrogenase production were investigated. The optimal medium composition, which was selected by employing single -factor and orthogonal optimization procedures, was as follows (g/L): 4 glucose, 12 corn steep liquor, 3 peptone, 2 KH2PO4. The optical fermentation conditions were: medium’s initial pH value 7.0 ~ 8.0, the inoculum amount 20%, loading amount 100 ml in 250 ml shaken flask, amount of inducer 5 mg/100 ml. Dehydrogenase production was promoted by Co2+ ion and Mg2+ ion, but enzyme production was markedly inhibited in the presence of Cu2+ ion and Fe3+ ion, and somewhat inhibited by Zn2+ ion and Fe2+ ion. The growth of Arthrobacter simplex and dehydrogenase production were inhibited strongly by detergent and SDS. However, Tween 80 and pao-di (triatomic alcohol poly ether) benefited to the enzyme production.Studies on bio-transformation conditions of beta-methasone intermediate 17 α -hydroxy-16 β-methyl-pregn-4,9(11)-diene-3,20-dione were investigated in depth and optimized as follows: inoculum size was 20% , loading amount was 100 ml in 250 ml shake vessel , initial pH was 7.0 ~ 7.5 , amount of inducer was 10 mg/L, amount of ethanol used to improve the solubility of the substrate was 7%, substrate concentration was 0.7%. Optimum concentration of cobalt chloride ^ external electron acceptor (NADP> NAD and menadione) and Tween 80 had considerable effect on the conversion. It was the first time to apply diluting cell culture brothes process to 17 α -hydroxy-16 β -methyl-pregn-4,9(11)-diene-3,20-dione dehydrogenation by Arthrobacter simplex. Aseptic water was selected as the diluent. When the ratio of dilution was 1:1, the conversion ratio was found to be comparable with that of the conventional process, so the production costs could be greatly decreased. The effects of ultrasonic irradiation on free cell and substrate were also investigated. Coupled with the process byKdiluting cell culture brothes, substrate which dissolved in ethanol and then precipitated in the sterile water was irradiated by ultrasound. The conversion rate was over 92.54%. It was found that ultrasound irradiation could induce dispersal of cluster of the substrate.Tests in 10 L fermentor were also carried out. It was found that the conversion cycle could be shortened by enhancing the aeration. In the test the conversion rate was about 86.7%,Studies on kinetic behaviors of transformation of 17 a -hydroxy-16 J3 -methyl-pregn-4,9(ll)-diene-3,20-dione by free cell of Arthrobacter simplex were also investigated, including: the effect of initial substrate and the enzyme concentration on initial rate, the stability of the enzyme, the time course of A ’-dehydrogenation of the transformation process. Based on the experiment results, a kinetic model for the dehydrogenation of 17 a -hydroxy-16 {3 -methyl-pregn-4,9(ll)-diene更多还原