格氏物简单节杆菌微生物脱氢研究
|
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CS~(137)-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％，KH_2PO_40.2％。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Cu~(2+)和Fe~(3+)对产酶有很大的的抑制作用，Zn~(2+)和Fe~(2+)对产酶也有一定的抑制作用，Co~(2+)和Mg~(2+)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。
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 by
K
diluting 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
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CS~(137)-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％，KH_2PO_40.2％。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Cu~(2+)和Fe~(3+)对产酶有很大的的抑制作用，Zn~(2+)和Fe~(2+)对产酶也有一定的抑制作用，Co~(2+)和Mg~(2+)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。
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 by
K
diluting 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
引文
[1] 俞俊棠，唐孝宣．生物工艺学(下册)．上海：华东化工学院出版社，1991：278～292
[2] 沈裕康．微生物转化在甾体激素生产中的应用．工业微生物，1987，17(1)：26～33
[3] 刘其明．我国甾体微生物工业的发展．工业微生物，1989，(3)：28～31
[4] 崔杨棣．油脂中甾醇资源开发及其在甾体药物工业中的应用．粮食与油脂，1998，(4)：25～31
[5] Morii S, Fujii C, Miyoshi T, et al. 3-Ketosteroid-Δ'-dehydrogenase of Rhodococcus rhodochrous: sequencing of the genomic DNA and hyperexpression, purification, and characterization of the recombinant enzyme. J Biochem, 1998, 124(5): 1026～1032
[6] 阳葵，王福东，冯霞，等．生长调节剂对甾体微生物催化转化过程的影响．分子催化，1999，13(2)：137～139
[7] 李晓静，施秀芳，冯霞，等．外加因子的生物效应及其在生物技术中的应用．安阳师范学院学报，2001，2：9-12
[8] Zabaneh M, Bar R. Ultrasound-enhanced bioprocess Ⅱ: Dehydrogenation of hydrocortisone by Arthrobacter simplex. Biotechnol Bioeng, 1991, 37: 998～1003.
[9] León R, Fernandes H M, Cabral J M S. Whole—cell biocatalysis in organic media. Enzyme Microb Technol, 1998, 23: 483～500.
[10] Zhang L Q, Zhang Y D, Li X L, et al. Lipase-catalyzed synthesis of RGD diamide in aqueous water miscible organic solvents. Enzyme Microb Technol, 2001, 29: 129～135
[11] Cruz A, Fernandes P, Cabral J M S, et al. Whole-cell bioconversion of β-sitosterol in aqueous-organic two-phase systems. J Mol Catalysis B: Enzymatic, 2001, 11: 579～585
[12] Manosroi J, Sripalakit P, Manosroi A. Bioconversion of hydrocortisone to prednisolone by immobilized bacterial cells in a two-liquid-phase system. Journal of Chemical Technology and Biotechnology. 1998, 73 (3): 203～210
[13] 韩广甸，韩卫亚．关于我国皮质激素的发展策略．中国医药工业杂志，1997，28(12)：559～562．
[14] 翁玲玲，楼荣良．国外正在研制的甾体药物．中国药学杂志，1994，29(3)：174～176
[15] 黄丕铂．甾体类药物的微生物转化研究现状．医学信息，1995，7：9～10
[16] 秦天才，张友德，张君芝．湖北黄姜生产中的问题与对策．长江流域资源与环境，1997，16(1)：35～38
[17] 徐诗伟，法幼华．A环饱和甾体的微生物转化作用Ⅲ．倍他美松中间体16β-甲基-5α-Δ~(9(11))-孕甾烯-3β，17α，21-三羟基-20-酮-21-醋酸酯的Δ~(1，4)作用．微生物学报，1984，24(1)：46～49
[18] Arnezeder C, Hampel W A. Influence of growth rate on the Accumulation of Ergosterol in yeast cells in a phosphate limited continuous culture. Biotechnol Lett, 1991, 13(2), 97～100
[19] 诸有义，生物合成药物学，化学工业出版社，2000，647-665
[20] Lizuka H, Natio A. Microbial conversion of steroids and alkaloids. Springer-Veriag.Berlin Heidelberg. New York. University of Tokyo Press, 1981,329-347
[21] 张丽青，微生物转化在甾体药物合成中的应用，医药工业,1985，16 (1) , 37-41
[22] Lee M K , Bae M Enzymatic characteristics of steroid △'-dehydrogenase from Arthrobacter simplex. J Microbiol Biotechnol, 1994,4(2) : 119-25
[23] Choi K P, Istvan M , Mitsuo Y, et al. Purification and characterization of the 3-ketosteroid-△'-dehydrogenase of Arthrobacter simplex produced in Streptomyces lividans. J Biochem, 1995, 117(5) , 1043-1049
[24] Chen K C, Chang C C, Chiu C F, et al. Mathematical simulation of pseudo-crystallofermentation of hydrocrotione by Arthrobacter simplex. Biotechnol Bioeng, 1985,27:253-259
[25] Maxon W D, Chen J W, Hanson F R. Simulation of a steroid bioconversion with a matermatical model.Ind Eng Chem Process Design Rev, 1966, 5: 285-289
[26] Constantinidis A. Steroid transformation at high substrate concentrations using immobilized Corynebacterium simplex cells. Biotechnol Bioeng. 1980, 22: 119-136
[27] Kondo E, Eitaro M. Pseudo-crystallofermentation of steroids: A new process for preparing prednisolone by a microorganism. J Gen Appl Microbiol, 1961, 7: 113-117
[28] Goetschel R, Bar, R. Formation of mixed crystals in microbial conversion of sterols and steroids. Enzyme Microb Technol, 1992,14(6) : 462-469
[29] Sonomoto K, Jin I N, Tanaka S F. Application of urethane prepolymers to immobilization of biocatalysts: A '-dehydrogenation of hydrocortisone by Arthrobacter simplex cells entrapped with urethane prepolymers. Agric Biol Chem, 1980,44(5) : 1119-1126
[30] Montes M C, Magana P I. △'-Dehydrogenation of steroids by Arthrobacter simplex immobilized in calcium polygalacturonate beads. J Ind Microbiol, 1991, 8(4) : 259-64
[31] Pinheiro H M, Carbral J M S. Activity and stability of an entrapped-cell system for the △'-dehydrogenation of steroids in organic media. Biotechnol Bioeng, 1992, 40: 1123-1127
[32] Zhang Y, Yao C Y, Wang M Y, et al. Transformation of cortisone acetate using immobilized Arthrobacter simplex cell. Shengwu Gongcheng Xuebao, 1998, 14(2) : 176-180
[33] Fukui S, Ahmed S A, Omata T, et al.Bioconversion of lipophilic compounds in nonaqueous solvent. Effect of gel hydrophobicity on diverse conversions of testosterone by gel-entrapped Nocardia rhodocrous cells. Eur J Appl Microbiol Biotechnol, 1980,10: 289-301
[34] Krysteva M A, Grigorova P M. Transformation of cortisol to prednisolone by viable cells of Arthrobacter simplex covalently immobilized on cellulose granules. Enzyme Microb Technol, 1987, 9: 534-541
[35] 张瑛，王明耀，姚传义．固定化简单节杆菌转化醋酸可的松动力学．天津大学学报，1999，32(1)：41～44
[36] Kaul R, Adlercreutz P, Mattiasson B. Coimmobilization of substrate and biocatalyst: A method for bioconversion of poorly soluble substances in water milieu. Biotechnol Bioeng, 1991, 37: 998～1003.
[37] 孙黎，法幼华．原生质体在甾体转化研究中的发展和应用．微生物学通报，1991，18(5)：299～301
[38] Wilmanska D, Milcazarek K, Rumijiowska A, et al. Elimination of by-products in 11β-hydroxylation of substrance S using Curvularia lunata clones regenerated from NTG-treated protoplasts. Appl Microbio Biotechnol, 1992, 37(5): 626～630.
[39] Flygare S, Larsson P O. Steroid transformation on aqueous two-phase systems: side-chain degration of cholestrol by Mycobacterium sp. Enzyme Microb Technol, 1989, 11: 752～759.
[40] Kaul R, Mattiasson B. Appl Microbiol Biotechnol. 1986, 24:259～265
[41] 张丽青，王敬一，5α-Δ~(9(11))-16α-甲基-3β，17α，21-三羟基-孕甾烯-3β，21-双醋酸酯-20-酮的微生物转化．药学学报，1986，21(9)：674～679
[42] 张丽青．5α-Δ~(9(11))-16β-甲基-3β，17α，21-三羟基-孕甾烯-3β，21-双醋酸酯-20-酮和5α，17α-甲基-17β-羟基雄甾-3-酮的微生物转化．药学学报，1981，16：356～358
[43] 陈家任，蒲自莲，曾本秀，等．微生物学方法制备16α-甲基-11α，17α，21-三羟基孕甾-1，4-二烯-3，20-二酮。微生物学报，1991，31(4)：308～314
[44] 徐诗伟，徐清，曹桂芳，等．地塞美松中间体的C_(14)脱氢和11α-羟基化．生物工程学报，2000，16(6)：763～765．
[46] 徐诗伟，徐清，法幼化．梨头霉11α-羟基化制备16β-甲基-11α，17α，21-三羟基孕甾-1，4-二烯-3,20-二酮．生物工程学报，2000，16(4)：482～484．
[45] Lee C Y, Chen C D, Liu W H. Production ofandrosta-1,4-diene-3,17-dione from cholesterol using two-step microbial transformation. Appl Microbiol Biotechnol. 1993, 38(4): 447～452
[47] Buckland B C, Dunill P, Lilly M D. The enzymatic transformation of waterinsoluble reactions in nonaqueous solvents conversion of cholesterol to cholest-4-ene-3-one by a Nocardia sp. Biotechnol Bioeng, 1975, 17: 815～826.
[48] Laane C, Boeren S, Vos K, Veeger C. Rules for the optimization of biocatalysis in organic solvents. Biotechnol Bioeng, 1987, 30:81～87
[49] Hocknull M D, Lilly M D. Stability of the steroid Δ'-dehydrogenation system of Arthrobacter simplex in organic solvent-water two-liquid phase enviroments. Enzyme Microb Technol. 1998, 10:669～674
[50] Pinheiro H M, Cabral J M S, Adlercreutz P. Quinones as external electron acceptors in steroid Δ'-dehydrogenation with entrapped cells in organic medium. Biocatalysis, 1993, 7:83～96
[51] Boeren S, Laane C. Steroid conversions by Flavobactriurn dehyrogenans in two-liquid-phase systems. Biotechnol Bioeng, 1987, 29:305～309
[52] Liu W H, Houng W C, Tsai M S. Bioconversion of cholesterol to cholestenone in aqueous/organic two-phase reactors. Enzyme Microb Technol, 1996, 18: 184～189
[53] Ceen E G, Dunnill P, Herrman J P R. Two-liquid phase reactor studies of 11α-hydroxylation of pregestrone by Aspergillus ochraceus. Biotechnol Bioeng, 1988, 31: 743～746
[54] Luisi P L, Steinmann-Hofman B. Activity and conformation of enzymes in reverse micellar solutions. Methods Enzymol, 1987, 136(1): 188～216
[55] Smolder A J J, Pinheiro H M, Noromha P, et al. Steroid bioconversion in a microemul sion system. Biotechnol Bioeng, 1991, 38:1210～1217
[56] Hedstrom G, Stotte J P, Molunder O, et al. Enzyme-catalyzed oxidation of cholesterol in physically characterized water-in-oil microemulsions. Biotechnol Bioeng, 1992, 39:218～224
[57] Houng J Y, Chiang W P, Chen K G, et al. 11α-hydroxylation of progesterone in biphasic media using alginate-entropped Asperigillus ochraceus gel beads covered with polyurea. Enzyme Microb Technol, 1994, 16:485～491
[58] Dias A C P. Carbral J M S, Pinheiro H M. Sterol side-chain cleavage with immobilized Mycobacterium cells in water-immisible organic solvents. Enzyme Microb Technol, 1994, 16:708～714
[59] Pinheiro H M, Cabral J M S. Screening of whole-cell immobilization procedures for the Δ'-dehydrogenation of steriods in organic medium. Enzyme Microb Technol, 1992, 14:619～624
[60] Steinert H J, Vorlop K D, Klein J.Steroid side chain cleavage with immobilized cells in organic medium. In: Biocatalysis in organic media (Laane C, Lilly MD). Elsevier Science, Amsterdam, 1987, 51～63
[61] 阳葵，王福东，冯霞，等．磁化水处理菌种在甾体微生物转化过程中的效应．微生物学通报，1999，2(5)：336～338
[62] Marazban S, Barbara L K, Chen ching-shih. Enzymatic catalysis in cosolvent modified pressurized organic solvents. Biotechnol Bioeng, 1999, 65(3): 258～264
[63] 马海乐．超临界CO_2萃取技术及其在生物资源开发利用中应用的最新进展(Ⅱ)．包装与食品机械，2001，19(3)：7～10(17)
[64] 冯若，赵逸云，李化茂，等．超声在生物技术中的应用进展[J]．生物化学与生物物理进展，1994(6)：500～504
[65] Bar R. Ultrasound enhanced bioprocesses: cholesterol oxidation by Rhodoccus erythopolis. Biotechnol Bioeng. 1988, 32:655～663
[66] Yang K, Wang F D, Feng X, et al. The effects of chemical and physical factors on biocatalytic transformation of steroid. Chinese Journal of Catalysis, 1998, 19(5): 391～392
[67] Yang H S, Studebaker J K. Continuous dehydrogenation of a steroid with immobilized microbial cells: effect of an exogenous electron acceptor. Biotechnol Bioeng. 1978, 20:17～25
[68] Silbiger E, Freeman A. Continuous Δ'-hydrocortisone dehydrogenation with in situ product recovery. Enzyme Microb Technol, 1991, 13:869～872
[69] 贾淑颖，穆国平，任树林．国果蔬中还原糖、蔗糖和淀粉的快速测定-3，5-二硝基水杨酸分光光度法．食品与发酵工业，1983，(2)：19～21
[70] 金青萍，郑自龙，刘维达，等．醋酸可的松微生物转化工艺条件的研究．微生物学通报，1985，12(1)：19～21
[71] Pinheiro H M, Carbral J M S. Effect of solvent molecular toxicity and microenvironment composition on the Δ'-dehydrogenation activity of Arthrobacter simplex cells. Biotechnol Bioeng, 1991, 37:97～102
[72] 四川省生物所，上海第十二制药厂．培他美松中间体-17α-羟基-16β-甲基孕甾-4,9(11)-二烯-3，20-二酮．微生物脱氢的研究．微生物学报，1977，17(1)：41～46
[73] 施巧琴，吴松刚．工业微生物育种学．福建科学技术出版社，1991，44～95
[74] 陈代杰，朱宝泉．工业微生物菌种选育与发酵控制技术．上海科学技术文献出版社，1995，176～178
[75] 鲁梅芳，路福平，王昌禄，等．醋酸泼尼松生产菌株的选育及转化条件的研究．微生物学通报，1992，21(3)：84～89
[76] 姚汝华．华南理工大学出版社，1995，20～21
[77] 俞俊棠，唐孝宣．生物工艺学(下册)．上海：华东化工学院出版社，1992：99～104
[78] Ohlson S,Larsson P O,Mosbach K. Steroid transformation by activated living immobilized Arthrobacter simplex cells. Biotechnol Bioeng, 1978, 20:1267～1284
[79] 黄淑惠，徐诗伟，法幼华．用新月弯孢霉11β-羟基化-16α-甲基化合物RS-21醋酸酯．微生物学报，1989，29(1)：68～71
[80] Freeman A, Lilly M D. The effect of water miscible solvents on the Δ'-dehydrogenase activity of free and PAAH-entrapped Arthrobacter simplex. Appl Microbiol Biotechnol. 1987, 25:495～501
[81] Pinheiro H M, Carbral J M S, Adlercreutz P. Quinones as external electron acceptors in steroid Δ'-dehydrogenation with entrapped cells in organic medium. Biocatalysis, 1993, 7(2): 83～96
[82] SonomotoR, Matsumo R, Tanoka A. Kinetic study on Δ'-dehydrogenation of hydrocortisone by gei-entrapped Arthrobacter simplex cell. J Ferment Technol, 1984, 62(2): 157～163
[83] 钟丽婵，曹竹安，李强，等．简单节杆菌转化氢化可的松的Δ'-脱氢反应动力学Ⅰ．简单节杆菌BY-2-13自由细胞转化氢化可的松的Δ'-脱氢反应动力学．微生物学报，1987，27(1)：45～51
[84] 曹竹安，李强，施燕，等．简单节杆菌转化氢化可的松的Δ'-脱氢反应动力学Ⅱ．固定化简单节杆菌细胞转化氢化可的松的Δ'-脱氢反应动力学．微生物学报，1987，27(2)：151～155
[2] 沈裕康．微生物转化在甾体激素生产中的应用．工业微生物，1987，17(1)：26～33
[3] 刘其明．我国甾体微生物工业的发展．工业微生物，1989，(3)：28～31
[4] 崔杨棣．油脂中甾醇资源开发及其在甾体药物工业中的应用．粮食与油脂，1998，(4)：25～31
[5] Morii S, Fujii C, Miyoshi T, et al. 3-Ketosteroid-Δ'-dehydrogenase of Rhodococcus rhodochrous: sequencing of the genomic DNA and hyperexpression, purification, and characterization of the recombinant enzyme. J Biochem, 1998, 124(5): 1026～1032
[6] 阳葵，王福东，冯霞，等．生长调节剂对甾体微生物催化转化过程的影响．分子催化，1999，13(2)：137～139
[7] 李晓静，施秀芳，冯霞，等．外加因子的生物效应及其在生物技术中的应用．安阳师范学院学报，2001，2：9-12
[8] Zabaneh M, Bar R. Ultrasound-enhanced bioprocess Ⅱ: Dehydrogenation of hydrocortisone by Arthrobacter simplex. Biotechnol Bioeng, 1991, 37: 998～1003.
[9] León R, Fernandes H M, Cabral J M S. Whole—cell biocatalysis in organic media. Enzyme Microb Technol, 1998, 23: 483～500.
[10] Zhang L Q, Zhang Y D, Li X L, et al. Lipase-catalyzed synthesis of RGD diamide in aqueous water miscible organic solvents. Enzyme Microb Technol, 2001, 29: 129～135
[11] Cruz A, Fernandes P, Cabral J M S, et al. Whole-cell bioconversion of β-sitosterol in aqueous-organic two-phase systems. J Mol Catalysis B: Enzymatic, 2001, 11: 579～585
[12] Manosroi J, Sripalakit P, Manosroi A. Bioconversion of hydrocortisone to prednisolone by immobilized bacterial cells in a two-liquid-phase system. Journal of Chemical Technology and Biotechnology. 1998, 73 (3): 203～210
[13] 韩广甸，韩卫亚．关于我国皮质激素的发展策略．中国医药工业杂志，1997，28(12)：559～562．
[14] 翁玲玲，楼荣良．国外正在研制的甾体药物．中国药学杂志，1994，29(3)：174～176
[15] 黄丕铂．甾体类药物的微生物转化研究现状．医学信息，1995，7：9～10
[16] 秦天才，张友德，张君芝．湖北黄姜生产中的问题与对策．长江流域资源与环境，1997，16(1)：35～38
[17] 徐诗伟，法幼华．A环饱和甾体的微生物转化作用Ⅲ．倍他美松中间体16β-甲基-5α-Δ~(9(11))-孕甾烯-3β，17α，21-三羟基-20-酮-21-醋酸酯的Δ~(1，4)作用．微生物学报，1984，24(1)：46～49
[18] Arnezeder C, Hampel W A. Influence of growth rate on the Accumulation of Ergosterol in yeast cells in a phosphate limited continuous culture. Biotechnol Lett, 1991, 13(2), 97～100
[19] 诸有义，生物合成药物学，化学工业出版社，2000，647-665
[20] Lizuka H, Natio A. Microbial conversion of steroids and alkaloids. Springer-Veriag.Berlin Heidelberg. New York. University of Tokyo Press, 1981,329-347
[21] 张丽青，微生物转化在甾体药物合成中的应用，医药工业,1985，16 (1) , 37-41
[22] Lee M K , Bae M Enzymatic characteristics of steroid △'-dehydrogenase from Arthrobacter simplex. J Microbiol Biotechnol, 1994,4(2) : 119-25
[23] Choi K P, Istvan M , Mitsuo Y, et al. Purification and characterization of the 3-ketosteroid-△'-dehydrogenase of Arthrobacter simplex produced in Streptomyces lividans. J Biochem, 1995, 117(5) , 1043-1049
[24] Chen K C, Chang C C, Chiu C F, et al. Mathematical simulation of pseudo-crystallofermentation of hydrocrotione by Arthrobacter simplex. Biotechnol Bioeng, 1985,27:253-259
[25] Maxon W D, Chen J W, Hanson F R. Simulation of a steroid bioconversion with a matermatical model.Ind Eng Chem Process Design Rev, 1966, 5: 285-289
[26] Constantinidis A. Steroid transformation at high substrate concentrations using immobilized Corynebacterium simplex cells. Biotechnol Bioeng. 1980, 22: 119-136
[27] Kondo E, Eitaro M. Pseudo-crystallofermentation of steroids: A new process for preparing prednisolone by a microorganism. J Gen Appl Microbiol, 1961, 7: 113-117
[28] Goetschel R, Bar, R. Formation of mixed crystals in microbial conversion of sterols and steroids. Enzyme Microb Technol, 1992,14(6) : 462-469
[29] Sonomoto K, Jin I N, Tanaka S F. Application of urethane prepolymers to immobilization of biocatalysts: A '-dehydrogenation of hydrocortisone by Arthrobacter simplex cells entrapped with urethane prepolymers. Agric Biol Chem, 1980,44(5) : 1119-1126
[30] Montes M C, Magana P I. △'-Dehydrogenation of steroids by Arthrobacter simplex immobilized in calcium polygalacturonate beads. J Ind Microbiol, 1991, 8(4) : 259-64
[31] Pinheiro H M, Carbral J M S. Activity and stability of an entrapped-cell system for the △'-dehydrogenation of steroids in organic media. Biotechnol Bioeng, 1992, 40: 1123-1127
[32] Zhang Y, Yao C Y, Wang M Y, et al. Transformation of cortisone acetate using immobilized Arthrobacter simplex cell. Shengwu Gongcheng Xuebao, 1998, 14(2) : 176-180
[33] Fukui S, Ahmed S A, Omata T, et al.Bioconversion of lipophilic compounds in nonaqueous solvent. Effect of gel hydrophobicity on diverse conversions of testosterone by gel-entrapped Nocardia rhodocrous cells. Eur J Appl Microbiol Biotechnol, 1980,10: 289-301
[34] Krysteva M A, Grigorova P M. Transformation of cortisol to prednisolone by viable cells of Arthrobacter simplex covalently immobilized on cellulose granules. Enzyme Microb Technol, 1987, 9: 534-541
[35] 张瑛，王明耀，姚传义．固定化简单节杆菌转化醋酸可的松动力学．天津大学学报，1999，32(1)：41～44
[36] Kaul R, Adlercreutz P, Mattiasson B. Coimmobilization of substrate and biocatalyst: A method for bioconversion of poorly soluble substances in water milieu. Biotechnol Bioeng, 1991, 37: 998～1003.
[37] 孙黎，法幼华．原生质体在甾体转化研究中的发展和应用．微生物学通报，1991，18(5)：299～301
[38] Wilmanska D, Milcazarek K, Rumijiowska A, et al. Elimination of by-products in 11β-hydroxylation of substrance S using Curvularia lunata clones regenerated from NTG-treated protoplasts. Appl Microbio Biotechnol, 1992, 37(5): 626～630.
[39] Flygare S, Larsson P O. Steroid transformation on aqueous two-phase systems: side-chain degration of cholestrol by Mycobacterium sp. Enzyme Microb Technol, 1989, 11: 752～759.
[40] Kaul R, Mattiasson B. Appl Microbiol Biotechnol. 1986, 24:259～265
[41] 张丽青，王敬一，5α-Δ~(9(11))-16α-甲基-3β，17α，21-三羟基-孕甾烯-3β，21-双醋酸酯-20-酮的微生物转化．药学学报，1986，21(9)：674～679
[42] 张丽青．5α-Δ~(9(11))-16β-甲基-3β，17α，21-三羟基-孕甾烯-3β，21-双醋酸酯-20-酮和5α，17α-甲基-17β-羟基雄甾-3-酮的微生物转化．药学学报，1981，16：356～358
[43] 陈家任，蒲自莲，曾本秀，等．微生物学方法制备16α-甲基-11α，17α，21-三羟基孕甾-1，4-二烯-3，20-二酮。微生物学报，1991，31(4)：308～314
[44] 徐诗伟，徐清，曹桂芳，等．地塞美松中间体的C_(14)脱氢和11α-羟基化．生物工程学报，2000，16(6)：763～765．
[46] 徐诗伟，徐清，法幼化．梨头霉11α-羟基化制备16β-甲基-11α，17α，21-三羟基孕甾-1，4-二烯-3,20-二酮．生物工程学报，2000，16(4)：482～484．
[45] Lee C Y, Chen C D, Liu W H. Production ofandrosta-1,4-diene-3,17-dione from cholesterol using two-step microbial transformation. Appl Microbiol Biotechnol. 1993, 38(4): 447～452
[47] Buckland B C, Dunill P, Lilly M D. The enzymatic transformation of waterinsoluble reactions in nonaqueous solvents conversion of cholesterol to cholest-4-ene-3-one by a Nocardia sp. Biotechnol Bioeng, 1975, 17: 815～826.
[48] Laane C, Boeren S, Vos K, Veeger C. Rules for the optimization of biocatalysis in organic solvents. Biotechnol Bioeng, 1987, 30:81～87
[49] Hocknull M D, Lilly M D. Stability of the steroid Δ'-dehydrogenation system of Arthrobacter simplex in organic solvent-water two-liquid phase enviroments. Enzyme Microb Technol. 1998, 10:669～674
[50] Pinheiro H M, Cabral J M S, Adlercreutz P. Quinones as external electron acceptors in steroid Δ'-dehydrogenation with entrapped cells in organic medium. Biocatalysis, 1993, 7:83～96
[51] Boeren S, Laane C. Steroid conversions by Flavobactriurn dehyrogenans in two-liquid-phase systems. Biotechnol Bioeng, 1987, 29:305～309
[52] Liu W H, Houng W C, Tsai M S. Bioconversion of cholesterol to cholestenone in aqueous/organic two-phase reactors. Enzyme Microb Technol, 1996, 18: 184～189
[53] Ceen E G, Dunnill P, Herrman J P R. Two-liquid phase reactor studies of 11α-hydroxylation of pregestrone by Aspergillus ochraceus. Biotechnol Bioeng, 1988, 31: 743～746
[54] Luisi P L, Steinmann-Hofman B. Activity and conformation of enzymes in reverse micellar solutions. Methods Enzymol, 1987, 136(1): 188～216
[55] Smolder A J J, Pinheiro H M, Noromha P, et al. Steroid bioconversion in a microemul sion system. Biotechnol Bioeng, 1991, 38:1210～1217
[56] Hedstrom G, Stotte J P, Molunder O, et al. Enzyme-catalyzed oxidation of cholesterol in physically characterized water-in-oil microemulsions. Biotechnol Bioeng, 1992, 39:218～224
[57] Houng J Y, Chiang W P, Chen K G, et al. 11α-hydroxylation of progesterone in biphasic media using alginate-entropped Asperigillus ochraceus gel beads covered with polyurea. Enzyme Microb Technol, 1994, 16:485～491
[58] Dias A C P. Carbral J M S, Pinheiro H M. Sterol side-chain cleavage with immobilized Mycobacterium cells in water-immisible organic solvents. Enzyme Microb Technol, 1994, 16:708～714
[59] Pinheiro H M, Cabral J M S. Screening of whole-cell immobilization procedures for the Δ'-dehydrogenation of steriods in organic medium. Enzyme Microb Technol, 1992, 14:619～624
[60] Steinert H J, Vorlop K D, Klein J.Steroid side chain cleavage with immobilized cells in organic medium. In: Biocatalysis in organic media (Laane C, Lilly MD). Elsevier Science, Amsterdam, 1987, 51～63
[61] 阳葵，王福东，冯霞，等．磁化水处理菌种在甾体微生物转化过程中的效应．微生物学通报，1999，2(5)：336～338
[62] Marazban S, Barbara L K, Chen ching-shih. Enzymatic catalysis in cosolvent modified pressurized organic solvents. Biotechnol Bioeng, 1999, 65(3): 258～264
[63] 马海乐．超临界CO_2萃取技术及其在生物资源开发利用中应用的最新进展(Ⅱ)．包装与食品机械，2001，19(3)：7～10(17)
[64] 冯若，赵逸云，李化茂，等．超声在生物技术中的应用进展[J]．生物化学与生物物理进展，1994(6)：500～504
[65] Bar R. Ultrasound enhanced bioprocesses: cholesterol oxidation by Rhodoccus erythopolis. Biotechnol Bioeng. 1988, 32:655～663
[66] Yang K, Wang F D, Feng X, et al. The effects of chemical and physical factors on biocatalytic transformation of steroid. Chinese Journal of Catalysis, 1998, 19(5): 391～392
[67] Yang H S, Studebaker J K. Continuous dehydrogenation of a steroid with immobilized microbial cells: effect of an exogenous electron acceptor. Biotechnol Bioeng. 1978, 20:17～25
[68] Silbiger E, Freeman A. Continuous Δ'-hydrocortisone dehydrogenation with in situ product recovery. Enzyme Microb Technol, 1991, 13:869～872
[69] 贾淑颖，穆国平，任树林．国果蔬中还原糖、蔗糖和淀粉的快速测定-3，5-二硝基水杨酸分光光度法．食品与发酵工业，1983，(2)：19～21
[70] 金青萍，郑自龙，刘维达，等．醋酸可的松微生物转化工艺条件的研究．微生物学通报，1985，12(1)：19～21
[71] Pinheiro H M, Carbral J M S. Effect of solvent molecular toxicity and microenvironment composition on the Δ'-dehydrogenation activity of Arthrobacter simplex cells. Biotechnol Bioeng, 1991, 37:97～102
[72] 四川省生物所，上海第十二制药厂．培他美松中间体-17α-羟基-16β-甲基孕甾-4,9(11)-二烯-3，20-二酮．微生物脱氢的研究．微生物学报，1977，17(1)：41～46
[73] 施巧琴，吴松刚．工业微生物育种学．福建科学技术出版社，1991，44～95
[74] 陈代杰，朱宝泉．工业微生物菌种选育与发酵控制技术．上海科学技术文献出版社，1995，176～178
[75] 鲁梅芳，路福平，王昌禄，等．醋酸泼尼松生产菌株的选育及转化条件的研究．微生物学通报，1992，21(3)：84～89
[76] 姚汝华．华南理工大学出版社，1995，20～21
[77] 俞俊棠，唐孝宣．生物工艺学(下册)．上海：华东化工学院出版社，1992：99～104
[78] Ohlson S,Larsson P O,Mosbach K. Steroid transformation by activated living immobilized Arthrobacter simplex cells. Biotechnol Bioeng, 1978, 20:1267～1284
[79] 黄淑惠，徐诗伟，法幼华．用新月弯孢霉11β-羟基化-16α-甲基化合物RS-21醋酸酯．微生物学报，1989，29(1)：68～71
[80] Freeman A, Lilly M D. The effect of water miscible solvents on the Δ'-dehydrogenase activity of free and PAAH-entrapped Arthrobacter simplex. Appl Microbiol Biotechnol. 1987, 25:495～501
[81] Pinheiro H M, Carbral J M S, Adlercreutz P. Quinones as external electron acceptors in steroid Δ'-dehydrogenation with entrapped cells in organic medium. Biocatalysis, 1993, 7(2): 83～96
[82] SonomotoR, Matsumo R, Tanoka A. Kinetic study on Δ'-dehydrogenation of hydrocortisone by gei-entrapped Arthrobacter simplex cell. J Ferment Technol, 1984, 62(2): 157～163
[83] 钟丽婵，曹竹安，李强，等．简单节杆菌转化氢化可的松的Δ'-脱氢反应动力学Ⅰ．简单节杆菌BY-2-13自由细胞转化氢化可的松的Δ'-脱氢反应动力学．微生物学报，1987，27(1)：45～51
[84] 曹竹安，李强，施燕，等．简单节杆菌转化氢化可的松的Δ'-脱氢反应动力学Ⅱ．固定化简单节杆菌细胞转化氢化可的松的Δ'-脱氢反应动力学．微生物学报，1987，27(2)：151～155