微生物转化甾体药物关键中间体4AD的研究
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摘要
本研究以 4AD 为底物，筛选具有转化功能的微生物，得到了三个菌株：Beauveria
bassiana HCB-00059 菌株、Rhizopus stolonife HCB-00643 菌株和 Rhizopus stolonife
HCB-00644 菌株。并从它们的转化产物中分离到 10 个转化化合物，其中 6 个结构已经
确定。4 个分子量已经确定。
1. 从 Beauveria bassiana HCB-00059 菌株的转化液中分离到化合物 BB059-1、
BB059-2、BB059-3 和 BB059-4，其中化合物 BB059-2 和 BB059-4 的化学结构已被确
定，分别是 6，11-二羟基雄甾烯二酮和 6-羟基睾酮。化合物 BB059-1 和 BB059-3
的分子量已被确定，均为 318。
2. 从 Rhizopus stolonife HCB-00643 菌株的转化液中分离到化合物 RS643-1、RS643-2
和 RS643-3，其中化合物 RS643-2 和 RS643-3 的化学结构已被确定，分别是 6-羰基
睾酮和 6，15-二羟基雄甾烯二酮。化合物 RS643-1 的分子量已被确定，为 304。
3. 从 Rhizopus stolonife HCB-00644 菌株的转化液中分离到化合物 RS644-1、RS644-2
和 RS644-3，其中化合物 RS644-2 和 RS644-3 的化学结构已被确定，分别为 6-羟
基雄甾烯二酮和14-羟基雄甾烯二酮。化合物RS644-1的分子量为已被确定，为318。
4. 对影响三株菌株微生物转化过程的一些因素进行了考察，取得了较好的结果。
The transformation of androstenedione (4AD) was studied to obtain valuable derivatives.
A preliminary screening had showed that three strains were efficient for the production of
4AD derivatives: Beauveria bassiana HCB-00059, Rhizopus stolonife HCB-00643, and
Rhizopus stolonife HCB-00644. The metabolites of three strains were isolated and purified.
Ten pure compounds were obtained. The structures of six compounds had been identified,
the molecular weights of the other four had been known.
1.Four compounds, BB059-1, BB059-2, BB059-3 and BB059-4 were isolated and purified
from Beauveria bassiana HCB-00059 culture. The structures of BB059-2 and BB059-4 were
identified as 6,11-dihydroxyandrostenedione, 6-hydroxytestosterone. The molecular weights
of the other two compounds , BB059-2 and BB059-4, were 318.
2.Three compounds, RS643-1, RS643-2 and RS643-3, were isolated and purified from
Rhizopus stolonife HCB-00643 culture. The structures of RS643-2 and RS643-3 were
identified as 6-oxo-testosterone and 6,15-dihydroxyandrostenedione. The molecular weight
of RS643-1 was 304.
3. Three compounds, RS644-1, RS644-2 and RS644-3, were isolated and purified from
Rhizopus stolonife HCB-00644 culture. The structures of RS644-2 and RS644-3 were
identified as 6-Hydroxyandrostenedione and 14- hydroxyandrostenedione. The molecular
weight of RS644-1 was 318.
The parameters in the bioconversion process by three strains were optimized. The results
showed better progresses.
bassiana HCB-00059 菌株、Rhizopus stolonife HCB-00643 菌株和 Rhizopus stolonife
HCB-00644 菌株。并从它们的转化产物中分离到 10 个转化化合物，其中 6 个结构已经
确定。4 个分子量已经确定。
1. 从 Beauveria bassiana HCB-00059 菌株的转化液中分离到化合物 BB059-1、
BB059-2、BB059-3 和 BB059-4，其中化合物 BB059-2 和 BB059-4 的化学结构已被确
定，分别是 6，11-二羟基雄甾烯二酮和 6-羟基睾酮。化合物 BB059-1 和 BB059-3
的分子量已被确定，均为 318。
2. 从 Rhizopus stolonife HCB-00643 菌株的转化液中分离到化合物 RS643-1、RS643-2
和 RS643-3，其中化合物 RS643-2 和 RS643-3 的化学结构已被确定，分别是 6-羰基
睾酮和 6，15-二羟基雄甾烯二酮。化合物 RS643-1 的分子量已被确定，为 304。
3. 从 Rhizopus stolonife HCB-00644 菌株的转化液中分离到化合物 RS644-1、RS644-2
和 RS644-3，其中化合物 RS644-2 和 RS644-3 的化学结构已被确定，分别为 6-羟
基雄甾烯二酮和14-羟基雄甾烯二酮。化合物RS644-1的分子量为已被确定，为318。
4. 对影响三株菌株微生物转化过程的一些因素进行了考察，取得了较好的结果。
The transformation of androstenedione (4AD) was studied to obtain valuable derivatives.
A preliminary screening had showed that three strains were efficient for the production of
4AD derivatives: Beauveria bassiana HCB-00059, Rhizopus stolonife HCB-00643, and
Rhizopus stolonife HCB-00644. The metabolites of three strains were isolated and purified.
Ten pure compounds were obtained. The structures of six compounds had been identified,
the molecular weights of the other four had been known.
1.Four compounds, BB059-1, BB059-2, BB059-3 and BB059-4 were isolated and purified
from Beauveria bassiana HCB-00059 culture. The structures of BB059-2 and BB059-4 were
identified as 6,11-dihydroxyandrostenedione, 6-hydroxytestosterone. The molecular weights
of the other two compounds , BB059-2 and BB059-4, were 318.
2.Three compounds, RS643-1, RS643-2 and RS643-3, were isolated and purified from
Rhizopus stolonife HCB-00643 culture. The structures of RS643-2 and RS643-3 were
identified as 6-oxo-testosterone and 6,15-dihydroxyandrostenedione. The molecular weight
of RS643-1 was 304.
3. Three compounds, RS644-1, RS644-2 and RS644-3, were isolated and purified from
Rhizopus stolonife HCB-00644 culture. The structures of RS644-2 and RS644-3 were
identified as 6-Hydroxyandrostenedione and 14- hydroxyandrostenedione. The molecular
weight of RS644-1 was 318.
The parameters in the bioconversion process by three strains were optimized. The results
showed better progresses.
引文
[1] 俞俊棠, 唐孝宣.生物工艺学. 上海:华东化工学院出版社,1994.
[2] 杨纵, 吴庭生,马安良. 薯芋皂甙元的代用品制备皮质激素[J].国内外医药工业水平调研报
告 集. 1992
[3] 谭载友，廖清江. 甾体药物化学的新进展[J].药学进展，1998，22(1):1-3.
[4] 杨征宇, 夏弈, 郑筠青,等. 甾体药物作用类型的发展.国内外医药工业水平调研报告集.
1992.
[5] 廖清江.国外研究开发甾体药物进展.内部资料.1999.
[6] 翁玲玲,楼荣良. 国外正在研制的甾体药物[J].中国药学杂志，1994，29(3):174-175.
[7] Bondzynski ST, Humnicki V. History of the research (J). Drugs under Experimental and Clinical
Research, 1986, 12(l): 1-9.
[8] Hench P S, Kendall E C, Slocumb C H. Proc. Staff Meetings Mayo Clin, 1949, 24: 181-183.
[9] 锗志义.生物合成药物学.上海：上海医科大学出版社，1991,325-330
[10] 法幼华，朱慧天，张丽青,等. 全国微生物学 1963 年学术年会论文摘要集. 1963,45-48.
[11] Marshek WJ. Progress in industrial microbiol. Ed. by Hocken-Hull, DJD. Churchill Livingstone,
Edindurgh and London. 1971, 10: 49-54
[12] 张丽青.微生物转化在甾体药物合成中的应用[J].医药工业,1985,16(1):37-42.
[13] 叶丽，史济平.甾体微生物转化在制药工业中的应用[J].工业微生物，2001，31（4）:40－48.
[14] 法幼华.甾体药物合成中一步分离的各种发酵工艺[J].微生物学通报，1994,21(1):51-53.
[15] 李金峰，曹竹安. 丙烯酰胺反向悬浮共聚合包埋简单节杆菌的应用研究[J].微生物学报，
1991,31(5):410-412
[16] 夏鹏. 对我国甾体药物研究和生产的几点想法.国内外医药工业水平调研报告集. 1992
[17] Celso Perez, Leana Perez, Emilia Herve. Isolation and partials characterization of a new mutant for
sterol biotransformation in Mycobaterium sp. Biotechnology Letters, 1995, 17(11):1241-1246.
[18] 今田幸男,新田一诚.甾族化合物转化工业[J].发酵工业, 1980，28(12):1118-1121.
[19] Yukimatsu K, Kaji A. Preparation of androsta-3, 17-diones [P]. JP, 54067096, 1979-05-30.
[20] Sugano Susumu. Preparation of androsta-3, 17-diones [P]. JP, 54105294, 1979-08-18.
[21] Owen RW, Nason AN, Bilton RF. Biotransformation of chenodeoxycholic acid by Pseudomonas
species NCIB 10590 under anaerobic conditions [J]. Lipid Research, 1983, 24(9):1109-1112.
[22] 褚志义，史济平 .今年来微生物对甾体转化机制的研究及其应用[J].工业微生物，1983，1292：
25-44.
[23] Krasnova LA.Messinova OV. Baynova VI, et al. RP, 801517, 1987.
[24] Favero J, Winternitz. Microbiological transformation of 1, 2-diphenyl-3, 5 dioxo-4-n-butyl
pyrazolidine [J]. Drug Metabolism and Pharmacokinetics. 1980, 5(1): 1-7.
68
微生物转化甾体药物关键中间体 4AD 的研究
[25] Smith KE, Latif S, Kirk DN, et al.Microbial transformation of steroid－I.Rare transformation of
progesterone by Apiocrea chrysosperma [J]. Steroid Biochemistry and Molecular Biology, 1988,
31(1): 83-89.
[26] Yoshihama M, Nakakoshi M, Tamura K. 14 alpha-hydroxyandrost-4-ene-3, 6, 17-trione as a
mechanical-based irreversible inhibitor of estrogen biosynthesis [J]. Chemical and Pharmaceutical
bulletin (Tokyo). 1990, 38(10): 2834-2837.
[27] Masuda Y, Nishimura N, Tanaka K.JP 01171496.
[28] Nakakoshi M.Yoshihama M.Tamura K.et al. JP 0117149
[29] Gabinskaya KN, Bayunova VI, Malyutina OF.and Grinenko, G.S. (1991) Khim-Farm.Zh.25, 63
[30] Smith KE., Latif S, Kirk DN, et al. Microbial Transformation of Steroid－VI.Transformations of
testosterone and androstenedione by Botryosphaerica obtusa [J].Steroid Biochemistry and Molecular
Biology,1990,35(1): 115-120.
[31] Orabi KY, Li E.Clark AM, et al. Microbial transformation of sampangine [J]. Nature Product, 1999,
62(7): 988-992..
[32] Nakano H, Hara M, Aano H.et al. EP, 228700.
[33] 黄海华，钟大放，刘瑶，等.黑曲霉菌对 SFZ-47 的代谢转化.沈阳药科大学学报，1997，14（3）：
199-204.
[34] 王赓，王敏，杜连祥.甾体 1，4-脱氢和 11α-羟基化反应的两种不同微生物转化.生物工程学报，
2000，16（5）：651-653.
[35] 杜连祥，王敏，王赓，等.转化 RSA 为氢化可的松的新月弯孢霉筛选.微生物学报，2001，28
（1）：44-48.
[36] 徐庆丰.我国白僵菌研究的进展.全国生物防止学会讨论会论文集，北京：1991.
[37] Parshikov IA, Moody JD, Heinza TM.et al. Transformation of cinoxacin by Beauveria bassiana.
Microbiological Letters, 2002, 214(1): 133-6.
[38] Preisig CL, Laakso JA, Mocek UM.et al. Biotransformations of the cardiovascular drugs mexrenone
and canrenone [J] Nature Product, 2003, 66(3): 350-356.
[39] Sandhu SS, Kinghom JR, Rajak RC, et al. Transformation system of Beauveria bassiana and
Metarhizium anisopliae using nitrate reductase gene of Aspergillus nidulans [J]. Experimental
biology, 2001, 39(7): 650-653.
[40] Peczynska-Czoch W, Mordarski M, Kaczmarek L, et al. Microbial transformation of azacarbazoles.
IX. Preliminary studies on hydroxylation of 2, 3-benzo-1, 4-dimethyl-alpha-iso-carboline by
Paecilomyces flavinosus. Archivum immunologiae at therapiae experimentalis, 1987, 35(2): 143-146.
[41] Capek A, Hanc O, Tadra M, et al. Microbial transformations of steroids. XXV-The effect of
substituents on the direction of the transformation of steroids by Beauveria bassiana [J]. Folia
Microbiologica, 1966, 11(2): 159-162.
[42] Choudhary MI, Sulatan S, Yaqoob M, et al. Microbial transformation of cortisol and prolyl
endopeptidase inhibitory activity of its transformed products [J]. Nature Product Research, 2003,
17(6): 389-395.
69
福州大学硕士毕业论文
[43] Choudhary MI, Sultan S, Yaqoob M, et al. Microbial transformation of dehydroepiandrosterone [J].
Nature Product Research, 2003,17(3): 215-220.
[44] Pokorna J, Kasal A. Progesterone side-chain degradation beside hydroxylation with Rhizopus
nigricans depends on the presence of nutrients [J]. Steroid Biochemistry and Molecular Biology,
1990, 35(1): 155-156.
[45] Protiva J.Schwarz V.Martinkova J, et al. Steroid derivatives. LV. Microbial transformation of steroid
compounds of the pregnane type substituted in position 16 and 17[J]. Folia Microbiologia, 1968,
13(2): 146-152.
[46] Galal AM. Microbial transformation of pyrethrosin [J]. Nature Product, 2001, 64(8): 1098-9.
[47] Barrero AF, Oltra JE, Raslan DS, et al. Microbial transformation of sesquiterpene lactones by the
fungi cunninghamella echinulata and rhizopus oryzae [J]. Nature Product. 1999, 62(5):726-729.
[48] Silva EA.Takahashi JA.Boaventura MA.et al. The biotransformation of ent-kaur-16-en-19-oic acid
by Rhizopus stolonifer [J]. Phytochemistry, 1999, 52(3): 397-400.
[49] Yamamoto T.Fukuoka M.Fujimoto Y, et al. Inhibitory effect of a new androstenedione derivative, 14
alpha-hydroxy-4-androstene-3, 6,17-trione (14 alpha-OHAT) on aromatase activity of human uterine
tumors [J]. Steroid Biochemistry and Molecular Biology, 1990, 36(6): 517-521.
[50] Hu S, Genain G, Azerad R. Microbial transformation of steroids: contribution to 14
alpha-hydroxylations [J]. Steroids, 1995, 60(4): 337-352.
[51] Smith KE, Latif S, Kirk DN, et al.Microbial Transformation of Steroid－II.Transformations of
progesterone, testosterone and androstenedione by phycomyces blakesleeanus [J]. Steroid
Biochemistry and Molecular Biology, 1989, 32(3): 445-451.
[52] Smith KE, Latif S, Kirk DN, et al. Microbial Transformation of Steroid－IV.6,7-Dehydrogenation:a
new class of fungal steroid transformation product[J].Steroid Biochemistry and Molecular
Biology,1989,33(3): 271-276.
[53] Furuta T, Suzuki A, Matsuzawa M, et al, Synthesis of stable isotope-labeled 6 beta-hydroxycortisol,
6- beta-hydroxycortisone, and 6 beta-hydroxytestosterone [J]. Steroid, 68(7-8): 693-696
[54] Hamada Hiroki.Production of 6beta-hydroxytestosterone.JP, 4139193, 1992-5-13.
[2] 杨纵, 吴庭生,马安良. 薯芋皂甙元的代用品制备皮质激素[J].国内外医药工业水平调研报
告 集. 1992
[3] 谭载友，廖清江. 甾体药物化学的新进展[J].药学进展，1998，22(1):1-3.
[4] 杨征宇, 夏弈, 郑筠青,等. 甾体药物作用类型的发展.国内外医药工业水平调研报告集.
1992.
[5] 廖清江.国外研究开发甾体药物进展.内部资料.1999.
[6] 翁玲玲,楼荣良. 国外正在研制的甾体药物[J].中国药学杂志，1994，29(3):174-175.
[7] Bondzynski ST, Humnicki V. History of the research (J). Drugs under Experimental and Clinical
Research, 1986, 12(l): 1-9.
[8] Hench P S, Kendall E C, Slocumb C H. Proc. Staff Meetings Mayo Clin, 1949, 24: 181-183.
[9] 锗志义.生物合成药物学.上海：上海医科大学出版社，1991,325-330
[10] 法幼华，朱慧天，张丽青,等. 全国微生物学 1963 年学术年会论文摘要集. 1963,45-48.
[11] Marshek WJ. Progress in industrial microbiol. Ed. by Hocken-Hull, DJD. Churchill Livingstone,
Edindurgh and London. 1971, 10: 49-54
[12] 张丽青.微生物转化在甾体药物合成中的应用[J].医药工业,1985,16(1):37-42.
[13] 叶丽，史济平.甾体微生物转化在制药工业中的应用[J].工业微生物，2001，31（4）:40－48.
[14] 法幼华.甾体药物合成中一步分离的各种发酵工艺[J].微生物学通报，1994,21(1):51-53.
[15] 李金峰，曹竹安. 丙烯酰胺反向悬浮共聚合包埋简单节杆菌的应用研究[J].微生物学报，
1991,31(5):410-412
[16] 夏鹏. 对我国甾体药物研究和生产的几点想法.国内外医药工业水平调研报告集. 1992
[17] Celso Perez, Leana Perez, Emilia Herve. Isolation and partials characterization of a new mutant for
sterol biotransformation in Mycobaterium sp. Biotechnology Letters, 1995, 17(11):1241-1246.
[18] 今田幸男,新田一诚.甾族化合物转化工业[J].发酵工业, 1980，28(12):1118-1121.
[19] Yukimatsu K, Kaji A. Preparation of androsta-3, 17-diones [P]. JP, 54067096, 1979-05-30.
[20] Sugano Susumu. Preparation of androsta-3, 17-diones [P]. JP, 54105294, 1979-08-18.
[21] Owen RW, Nason AN, Bilton RF. Biotransformation of chenodeoxycholic acid by Pseudomonas
species NCIB 10590 under anaerobic conditions [J]. Lipid Research, 1983, 24(9):1109-1112.
[22] 褚志义，史济平 .今年来微生物对甾体转化机制的研究及其应用[J].工业微生物，1983，1292：
25-44.
[23] Krasnova LA.Messinova OV. Baynova VI, et al. RP, 801517, 1987.
[24] Favero J, Winternitz. Microbiological transformation of 1, 2-diphenyl-3, 5 dioxo-4-n-butyl
pyrazolidine [J]. Drug Metabolism and Pharmacokinetics. 1980, 5(1): 1-7.
68
微生物转化甾体药物关键中间体 4AD 的研究
[25] Smith KE, Latif S, Kirk DN, et al.Microbial transformation of steroid－I.Rare transformation of
progesterone by Apiocrea chrysosperma [J]. Steroid Biochemistry and Molecular Biology, 1988,
31(1): 83-89.
[26] Yoshihama M, Nakakoshi M, Tamura K. 14 alpha-hydroxyandrost-4-ene-3, 6, 17-trione as a
mechanical-based irreversible inhibitor of estrogen biosynthesis [J]. Chemical and Pharmaceutical
bulletin (Tokyo). 1990, 38(10): 2834-2837.
[27] Masuda Y, Nishimura N, Tanaka K.JP 01171496.
[28] Nakakoshi M.Yoshihama M.Tamura K.et al. JP 0117149
[29] Gabinskaya KN, Bayunova VI, Malyutina OF.and Grinenko, G.S. (1991) Khim-Farm.Zh.25, 63
[30] Smith KE., Latif S, Kirk DN, et al. Microbial Transformation of Steroid－VI.Transformations of
testosterone and androstenedione by Botryosphaerica obtusa [J].Steroid Biochemistry and Molecular
Biology,1990,35(1): 115-120.
[31] Orabi KY, Li E.Clark AM, et al. Microbial transformation of sampangine [J]. Nature Product, 1999,
62(7): 988-992..
[32] Nakano H, Hara M, Aano H.et al. EP, 228700.
[33] 黄海华，钟大放，刘瑶，等.黑曲霉菌对 SFZ-47 的代谢转化.沈阳药科大学学报，1997，14（3）：
199-204.
[34] 王赓，王敏，杜连祥.甾体 1，4-脱氢和 11α-羟基化反应的两种不同微生物转化.生物工程学报，
2000，16（5）：651-653.
[35] 杜连祥，王敏，王赓，等.转化 RSA 为氢化可的松的新月弯孢霉筛选.微生物学报，2001，28
（1）：44-48.
[36] 徐庆丰.我国白僵菌研究的进展.全国生物防止学会讨论会论文集，北京：1991.
[37] Parshikov IA, Moody JD, Heinza TM.et al. Transformation of cinoxacin by Beauveria bassiana.
Microbiological Letters, 2002, 214(1): 133-6.
[38] Preisig CL, Laakso JA, Mocek UM.et al. Biotransformations of the cardiovascular drugs mexrenone
and canrenone [J] Nature Product, 2003, 66(3): 350-356.
[39] Sandhu SS, Kinghom JR, Rajak RC, et al. Transformation system of Beauveria bassiana and
Metarhizium anisopliae using nitrate reductase gene of Aspergillus nidulans [J]. Experimental
biology, 2001, 39(7): 650-653.
[40] Peczynska-Czoch W, Mordarski M, Kaczmarek L, et al. Microbial transformation of azacarbazoles.
IX. Preliminary studies on hydroxylation of 2, 3-benzo-1, 4-dimethyl-alpha-iso-carboline by
Paecilomyces flavinosus. Archivum immunologiae at therapiae experimentalis, 1987, 35(2): 143-146.
[41] Capek A, Hanc O, Tadra M, et al. Microbial transformations of steroids. XXV-The effect of
substituents on the direction of the transformation of steroids by Beauveria bassiana [J]. Folia
Microbiologica, 1966, 11(2): 159-162.
[42] Choudhary MI, Sulatan S, Yaqoob M, et al. Microbial transformation of cortisol and prolyl
endopeptidase inhibitory activity of its transformed products [J]. Nature Product Research, 2003,
17(6): 389-395.
69
福州大学硕士毕业论文
[43] Choudhary MI, Sultan S, Yaqoob M, et al. Microbial transformation of dehydroepiandrosterone [J].
Nature Product Research, 2003,17(3): 215-220.
[44] Pokorna J, Kasal A. Progesterone side-chain degradation beside hydroxylation with Rhizopus
nigricans depends on the presence of nutrients [J]. Steroid Biochemistry and Molecular Biology,
1990, 35(1): 155-156.
[45] Protiva J.Schwarz V.Martinkova J, et al. Steroid derivatives. LV. Microbial transformation of steroid
compounds of the pregnane type substituted in position 16 and 17[J]. Folia Microbiologia, 1968,
13(2): 146-152.
[46] Galal AM. Microbial transformation of pyrethrosin [J]. Nature Product, 2001, 64(8): 1098-9.
[47] Barrero AF, Oltra JE, Raslan DS, et al. Microbial transformation of sesquiterpene lactones by the
fungi cunninghamella echinulata and rhizopus oryzae [J]. Nature Product. 1999, 62(5):726-729.
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