甾体药物中间体HMPDD的C_（1,2）位微生物脱氢工艺和动力学研究
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摘要
抗炎甾体激素药物的母核在C_(1,2)位置导入双键后，能成倍地提高其抗炎活性。与化学方法相比，采用微生物法脱氢，具有反应条件温和、专一性强(包括结构专一性和立体专一性)以及转化率高等优点，已成为甾体激素药物生产中的重要反应之一。
倍他米松(Betamethasone)，即16β-甲基-9α-氟氢化泼尼松(16β-methyl-9 α-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，比强的松高10倍，是地塞米松的2.5倍，且副作用小。倍他米松的生产，一般采用剑麻或薯蓣皂素为合成的起始原料。在合成路线中，17α—羟基—16β—甲基—孕甾—4，9(11)—二烯—3，20—二酮(简称HMPDD)，经微生物脱氢生成17α—羟基—16β—甲基—孕甾—1，4，9(11)—三烯—3，20—二酮(简称HMPTD)，是关键反应之一。该反应可由简单节杆菌催化实现。反应式如下：
论文在对我国甾体药物工业的现状，微生物转化技术在甾体药物生产中的应用，甾体激素的微生物脱氢研究进展，以及新技术的开发等方面加以全面综述的基础上，针对HMPDD生物转化脱氢的研究报道很少，以及在实际生产中存在的转化率偏低等问题，就HMPDD生物转化脱氢的菌种选育，甾体脱氢酶产酶条件，生物转化新工艺以及生物转化的动力学等方面进行了深入的研究。
首先对适用于HMPDD生物转化脱氢的微生物菌种进行了选育。从实验室收集、保藏的15株简单节杆菌中，通过摇瓶转化的方法，进行菌种的初筛，并经过菌种的分离、纯化，得到转化率较高的2-76菌株。以该菌株为出发菌株，采用紫外线诱变处理，并结合Cs~(137)-γ射线辐照的复合诱变处理方法，选育得到了较佳正变株Q4-2-51，其摇瓶发酵的转化率达86.63％，较原始菌株提高了8％。
浙江大学博士学位论文
该诱变株经群体连续传代方式考察，遗传性能比较稳定。
研究中优化了发酵培养基的组成，考察了Q4一2一51菌株产幽体脱氢酶的较佳
工艺条件。通过单因素实验和正交试验优化，确定的较佳培养基组成为:葡萄糖
0.4%，玉米浆1.2%，蛋白陈0.3%，KHZPO40.2%。简单节杆菌产幽体脱氢酶
的最适条件为:接种量20%，培养基初始pH 7.0，摇瓶装量100 ml/250 ml。
HMPDD可诱导菌体产酶，其较适加量为0.05 g/L，且诱导物以在菌体生长初期
加入为好。金属离子cuZ十和Fe3+明显抑制菌体产酶，zn2+和Fe2+对产酶的抑制
作用较弱，c犷+和Mg2+则对产酶有一定的促进作用。培养基中加入适量的表面
活性剂，如泡敌或吐温一80，对产酶有一定的促进作用。
论文还就HMPDD的微生物转化工艺条件进行了研究，得到较佳的转化工艺
条件:接种量巧%，摇瓶装量为100 ml/250 ml，培养基初始pH7.0一7.5，诱导物
加量O.05g/L。试验中选用乙醇作为HMPDD的增溶介质，其最适加量为7%(v/v)。
研究中还探讨了水析投料法促进微生物脱氢反应的机理。较适的底物投料浓度为
0.7%，并且HMPDD以一次性投料为好。转化过程中加入适量的氯化钻、外源
电子受体(辅酶I、辅酶11、维生素K3)或表面活性剂吐温一80，对转化率的提
高均有一定的促进作用。论文首次将培养液稀释转化新工艺，应用于简单节杆菌
催化的HMPDD脱氢过程。研究开发的稀释转化新工艺中选用无菌水为稀释介
质，当稀释比为1:1(v/v)时，HMPDD的转化率与不稀释的原工艺(对照)相近，
故能大大减少转化反应中作为生物催化剂的菌体用量，提高设备利用率，降低生
产成本。此外，还考察了采用超声波分别处理微生物菌体、投料后的菌悬液以及
HMPDD底物(水析料)对转化结果的影响。结合底物超声波处理的培养液稀释
转化新工艺，可使摇瓶发酵转化率高达92.54%。研究发现，超声波处理能有效
地使底物颗粒细化，提高固体颗粒的比表面积，进而提高底物的溶解速率，起到
强化传质的作用。
论文分别采用10L罐、3OOL罐和3T罐，对HMPDD的微生物脱氢工艺进
行了逐级放大试验。10L罐小试中发现，提高转化阶段的通气量，有利于提高转
化速率，缩短转化周期，提高转化得率。转化30h的HMPDD脱氢转化率可达
86.54%。采用300L罐进行了三个批次的转化试验，控制菌体生长和转化阶段的
通气量分别为0.4 VVm和0.3 VVm，搅拌转速为200 r/min，24h时的平均转化
率达到80.32%。3T罐发酵时，控制菌体生长和转化阶段的通气量分别为0.25
VVm和0.2 VVm，搅拌转速为200:/min，六个批次的平均转化率为840%。以
上研究结果表明，HMPDD生物转化反应的放大过程中虽存在一定的放大效应，
但并不严重。
摘要
本文首次研究了简单节杆菌游离细胞催化HMPDD脱氢的反应动力学。考察
了不同投料方式下HMPDD晶体的粒径大小和分布，幽体脱氢酶的稳定性，以及
底物浓度和酶量等对转化反应初速度的影响，建立了相应的动力学模型。并从实
验数据回归分析，得到了有关的动力学参数。模型计算结果与实验数据比较表明，
提出的动力学模型能较好地描述HMPDD的微生物脱氢过程。动力学研究结果为
调控转化条件，提高脱氢酶的催化活性提供了理论依据。
街体的生物转化过程不同于常规的发酵过程
After dehydrogenation and double bond formation in C1,2 of steroid ring system, the anti-inflammatory activity of hormone-type medicines will be many times higher than that of the original one. At mild conditions, (such as low temperature, low pressure, etc.), the dehydrogenation reaction of steroid can be catalyzed by certain microorganisms with higher selectivity and productivity comparing with that of the chemical methods, so that the biodehydrogenation has become an important reaction in the production of hormone-type medicines.
Beta-methasone, 16p-methyl -9α-fluoroprednisolone, was one of the most effective anti-inflammatory drug. The anti-inflammatory activity of beta-methasone is 35, 10 and 2.5 times higher than that of hydrocortisone, prednisone and dexamethasone, respectively. 17α-hydroxy-16β-methyl-pregn-l, 4, 9(ll)-triene-3, 20-dione (HMPTD) is an important intermediate in the production of beta-methasone, which is one of the key reactions in beta-methasone production and is a dehydrogenation product of 17 α-hydroxy-16 β-methyl-pregn-4, 9(ll)-diene-3, 20-dione (HMPDD) , which was catalyzed by Arthrobacter simplex. The reaction is as follows:
The current status of steroids industry, the progress in microbial dehydrogenation of steroids and the applications of new technology used in the steroid biotransformation are reviewed, and the prospects of steroid medicines are discussed as well. Based on the fact that only a few reports on the bio-dehydrogenation of HMPDD, and low conversion ratio of HMPDD currently, the purposes of this work are: the strain screening, better fermentation conditions for the formation of 1-dehydrogenase, the improvement for efficient bio-dehydrogenation of HMPDD, the research and development of new technology for the bioconversion of HMPDD, and the kinetics of biotransformation processes.
A strain 2-76 with higher bioconversion ratio of HMPDD was screened out by
bioconversion test in shake bottle after strain separation and purification from 15 strains of Arthrobacter simplex. The strain 2-76 was further mutated by UV treatment arid Cs137-γ irradiation, and a better strain Q4-2-51 was obtained by isolating and rejuvenescention. The conversion ratio of HMPDD catalyzed by Q4-2-51 reached 86.63%, which was 8% higher than that of the original strain 2-76. The hereditary stability of this positive mutant was satisfactory.
The effects of medium composition and culture conditions on the dehydrogenase activity of strain Q4-2-51 were evaluated experimentally. The optimized medium composition was as follows (g/L): glucose 4, corn steep liquor 12, peptone 3, KH2PO4 2. The optimized fermentation conditions for cell growth and formation of 1-dehydrogenase were as follows: initial pH value, pH7.0; inoculum volume, 20%; amount of inducer, 0.05g/L; and 100 ml medium in 250 ml shaken flask. Dehydrogenase production was enhanced by Co2+ and Mg2+, and slightly inhibited by Zn2+and Fe2+, but was markedly inhibited in the presence of Cu 2+ and Fe3+. The growth of Arthrobacter simplex and the production of dehydrogenase were also inhibited strongly by detergent and SDS. However, the addition of Tween 80 and a kind of defoamer, triatomic alcohol polyether, was beneficial for the enzyme production.
The biotransformation conditions for the dehydrogenation of HMPDD were also investigated in depth, and the optimal conditions were obtained as follows: inoculum volume, 15%; loading amount, 100 ml in 250 ml shake vessel; initial pH value of medium, pH7.0~7.5, amount of inducer, 0.05g/L; amount of ethanol used to improve the solubility of steroid substrate, 7% (v/v); and the concentration of HMPDD should be kept at 0.7%. It was found that the addition of appropriate amount of cobalt chloride, external electron acceptor (NADP NAD and menadione), or Tween 80, had a considerable positive effect on the bioconversion ratio. A new strategy was proposed to perform the biotransformation: the fermentation broth was diluted with aseptic water in the ratio of l:l(v/v), and the conversion ratio was m
倍他米松(Betamethasone)，即16β-甲基-9α-氟氢化泼尼松(16β-methyl-9 α-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，比强的松高10倍，是地塞米松的2.5倍，且副作用小。倍他米松的生产，一般采用剑麻或薯蓣皂素为合成的起始原料。在合成路线中，17α—羟基—16β—甲基—孕甾—4，9(11)—二烯—3，20—二酮(简称HMPDD)，经微生物脱氢生成17α—羟基—16β—甲基—孕甾—1，4，9(11)—三烯—3，20—二酮(简称HMPTD)，是关键反应之一。该反应可由简单节杆菌催化实现。反应式如下：
论文在对我国甾体药物工业的现状，微生物转化技术在甾体药物生产中的应用，甾体激素的微生物脱氢研究进展，以及新技术的开发等方面加以全面综述的基础上，针对HMPDD生物转化脱氢的研究报道很少，以及在实际生产中存在的转化率偏低等问题，就HMPDD生物转化脱氢的菌种选育，甾体脱氢酶产酶条件，生物转化新工艺以及生物转化的动力学等方面进行了深入的研究。
首先对适用于HMPDD生物转化脱氢的微生物菌种进行了选育。从实验室收集、保藏的15株简单节杆菌中，通过摇瓶转化的方法，进行菌种的初筛，并经过菌种的分离、纯化，得到转化率较高的2-76菌株。以该菌株为出发菌株，采用紫外线诱变处理，并结合Cs~(137)-γ射线辐照的复合诱变处理方法，选育得到了较佳正变株Q4-2-51，其摇瓶发酵的转化率达86.63％，较原始菌株提高了8％。
浙江大学博士学位论文
该诱变株经群体连续传代方式考察，遗传性能比较稳定。
研究中优化了发酵培养基的组成，考察了Q4一2一51菌株产幽体脱氢酶的较佳
工艺条件。通过单因素实验和正交试验优化，确定的较佳培养基组成为:葡萄糖
0.4%，玉米浆1.2%，蛋白陈0.3%，KHZPO40.2%。简单节杆菌产幽体脱氢酶
的最适条件为:接种量20%，培养基初始pH 7.0，摇瓶装量100 ml/250 ml。
HMPDD可诱导菌体产酶，其较适加量为0.05 g/L，且诱导物以在菌体生长初期
加入为好。金属离子cuZ十和Fe3+明显抑制菌体产酶，zn2+和Fe2+对产酶的抑制
作用较弱，c犷+和Mg2+则对产酶有一定的促进作用。培养基中加入适量的表面
活性剂，如泡敌或吐温一80，对产酶有一定的促进作用。
论文还就HMPDD的微生物转化工艺条件进行了研究，得到较佳的转化工艺
条件:接种量巧%，摇瓶装量为100 ml/250 ml，培养基初始pH7.0一7.5，诱导物
加量O.05g/L。试验中选用乙醇作为HMPDD的增溶介质，其最适加量为7%(v/v)。
研究中还探讨了水析投料法促进微生物脱氢反应的机理。较适的底物投料浓度为
0.7%，并且HMPDD以一次性投料为好。转化过程中加入适量的氯化钻、外源
电子受体(辅酶I、辅酶11、维生素K3)或表面活性剂吐温一80，对转化率的提
高均有一定的促进作用。论文首次将培养液稀释转化新工艺，应用于简单节杆菌
催化的HMPDD脱氢过程。研究开发的稀释转化新工艺中选用无菌水为稀释介
质，当稀释比为1:1(v/v)时，HMPDD的转化率与不稀释的原工艺(对照)相近，
故能大大减少转化反应中作为生物催化剂的菌体用量，提高设备利用率，降低生
产成本。此外，还考察了采用超声波分别处理微生物菌体、投料后的菌悬液以及
HMPDD底物(水析料)对转化结果的影响。结合底物超声波处理的培养液稀释
转化新工艺，可使摇瓶发酵转化率高达92.54%。研究发现，超声波处理能有效
地使底物颗粒细化，提高固体颗粒的比表面积，进而提高底物的溶解速率，起到
强化传质的作用。
论文分别采用10L罐、3OOL罐和3T罐，对HMPDD的微生物脱氢工艺进
行了逐级放大试验。10L罐小试中发现，提高转化阶段的通气量，有利于提高转
化速率，缩短转化周期，提高转化得率。转化30h的HMPDD脱氢转化率可达
86.54%。采用300L罐进行了三个批次的转化试验，控制菌体生长和转化阶段的
通气量分别为0.4 VVm和0.3 VVm，搅拌转速为200 r/min，24h时的平均转化
率达到80.32%。3T罐发酵时，控制菌体生长和转化阶段的通气量分别为0.25
VVm和0.2 VVm，搅拌转速为200:/min，六个批次的平均转化率为840%。以
上研究结果表明，HMPDD生物转化反应的放大过程中虽存在一定的放大效应，
但并不严重。
摘要
本文首次研究了简单节杆菌游离细胞催化HMPDD脱氢的反应动力学。考察
了不同投料方式下HMPDD晶体的粒径大小和分布，幽体脱氢酶的稳定性，以及
底物浓度和酶量等对转化反应初速度的影响，建立了相应的动力学模型。并从实
验数据回归分析，得到了有关的动力学参数。模型计算结果与实验数据比较表明，
提出的动力学模型能较好地描述HMPDD的微生物脱氢过程。动力学研究结果为
调控转化条件，提高脱氢酶的催化活性提供了理论依据。
街体的生物转化过程不同于常规的发酵过程
After dehydrogenation and double bond formation in C1,2 of steroid ring system, the anti-inflammatory activity of hormone-type medicines will be many times higher than that of the original one. At mild conditions, (such as low temperature, low pressure, etc.), the dehydrogenation reaction of steroid can be catalyzed by certain microorganisms with higher selectivity and productivity comparing with that of the chemical methods, so that the biodehydrogenation has become an important reaction in the production of hormone-type medicines.
Beta-methasone, 16p-methyl -9α-fluoroprednisolone, was one of the most effective anti-inflammatory drug. The anti-inflammatory activity of beta-methasone is 35, 10 and 2.5 times higher than that of hydrocortisone, prednisone and dexamethasone, respectively. 17α-hydroxy-16β-methyl-pregn-l, 4, 9(ll)-triene-3, 20-dione (HMPTD) is an important intermediate in the production of beta-methasone, which is one of the key reactions in beta-methasone production and is a dehydrogenation product of 17 α-hydroxy-16 β-methyl-pregn-4, 9(ll)-diene-3, 20-dione (HMPDD) , which was catalyzed by Arthrobacter simplex. The reaction is as follows:
The current status of steroids industry, the progress in microbial dehydrogenation of steroids and the applications of new technology used in the steroid biotransformation are reviewed, and the prospects of steroid medicines are discussed as well. Based on the fact that only a few reports on the bio-dehydrogenation of HMPDD, and low conversion ratio of HMPDD currently, the purposes of this work are: the strain screening, better fermentation conditions for the formation of 1-dehydrogenase, the improvement for efficient bio-dehydrogenation of HMPDD, the research and development of new technology for the bioconversion of HMPDD, and the kinetics of biotransformation processes.
A strain 2-76 with higher bioconversion ratio of HMPDD was screened out by
bioconversion test in shake bottle after strain separation and purification from 15 strains of Arthrobacter simplex. The strain 2-76 was further mutated by UV treatment arid Cs137-γ irradiation, and a better strain Q4-2-51 was obtained by isolating and rejuvenescention. The conversion ratio of HMPDD catalyzed by Q4-2-51 reached 86.63%, which was 8% higher than that of the original strain 2-76. The hereditary stability of this positive mutant was satisfactory.
The effects of medium composition and culture conditions on the dehydrogenase activity of strain Q4-2-51 were evaluated experimentally. The optimized medium composition was as follows (g/L): glucose 4, corn steep liquor 12, peptone 3, KH2PO4 2. The optimized fermentation conditions for cell growth and formation of 1-dehydrogenase were as follows: initial pH value, pH7.0; inoculum volume, 20%; amount of inducer, 0.05g/L; and 100 ml medium in 250 ml shaken flask. Dehydrogenase production was enhanced by Co2+ and Mg2+, and slightly inhibited by Zn2+and Fe2+, but was markedly inhibited in the presence of Cu 2+ and Fe3+. The growth of Arthrobacter simplex and the production of dehydrogenase were also inhibited strongly by detergent and SDS. However, the addition of Tween 80 and a kind of defoamer, triatomic alcohol polyether, was beneficial for the enzyme production.
The biotransformation conditions for the dehydrogenation of HMPDD were also investigated in depth, and the optimal conditions were obtained as follows: inoculum volume, 15%; loading amount, 100 ml in 250 ml shake vessel; initial pH value of medium, pH7.0~7.5, amount of inducer, 0.05g/L; amount of ethanol used to improve the solubility of steroid substrate, 7% (v/v); and the concentration of HMPDD should be kept at 0.7%. It was found that the addition of appropriate amount of cobalt chloride, external electron acceptor (NADP NAD and menadione), or Tween 80, had a considerable positive effect on the bioconversion ratio. A new strategy was proposed to perform the biotransformation: the fermentation broth was diluted with aseptic water in the ratio of l:l(v/v), and the conversion ratio was m
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