WO1997021830A1 - MICROBIAL 11α-HYDROXYLATION OF STEROIDS - Google Patents

MICROBIAL 11α-HYDROXYLATION OF STEROIDS Download PDF

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Publication number
WO1997021830A1
WO1997021830A1 PCT/EP1996/005729 EP9605729W WO9721830A1 WO 1997021830 A1 WO1997021830 A1 WO 1997021830A1 EP 9605729 W EP9605729 W EP 9605729W WO 9721830 A1 WO9721830 A1 WO 9721830A1
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WO
WIPO (PCT)
Prior art keywords
steroid
microbial
rhizopus
purity
steroids
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP1996/005729
Other languages
French (fr)
Inventor
Marten Wiersma
Pieter Van Der Meijden
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Akzo Nobel NV
Original Assignee
Akzo Nobel NV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to EP96944023A priority Critical patent/EP0900283B1/en
Priority to NZ325276A priority patent/NZ325276A/en
Priority to JP9521752A priority patent/JP2000501940A/en
Priority to DK96944023T priority patent/DK0900283T3/en
Priority to PL96327439A priority patent/PL185223B1/en
Priority to DE69620474T priority patent/DE69620474T2/en
Priority to US09/077,982 priority patent/US6046023A/en
Priority to AU13758/97A priority patent/AU713685B2/en
Application filed by Akzo Nobel NV filed Critical Akzo Nobel NV
Priority to AT96944023T priority patent/ATE215606T1/en
Priority to KR1019980704372A priority patent/KR19990072066A/en
Publication of WO1997021830A1 publication Critical patent/WO1997021830A1/en
Priority to NO982700A priority patent/NO982700L/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P33/00—Preparation of steroids
    • C12P33/06—Hydroxylating
    • C12P33/08—Hydroxylating at 11 position
    • C12P33/10—Hydroxylating at 11 position at 11 alpha-position

Definitions

  • the present invention relates to a microbial method of 1 l ⁇ -hydroxyiation of steroids
  • Microbial 1 l -hydroxylation of steroids is a well known process, in vivo as well as in vitro
  • 1 l ⁇ -hydroxylation of progesterone by cell-free preparations of Aspergillus ochraceus has been reported by Shibahara et al Biochim Biophys Acta, 202 ( 1970), 172-179
  • microbial 1 l ⁇ -hydroxylation reactions of steroids are unpredictable, and invariably lead to incomplete transformations
  • Typicallv conversion degrees of 80-85% are obtained
  • Mathematical models in the optimization of such fermentation processes are discussed by Deshayes et al , Bull Soc Chim Fr , ( 1980), II 24-34
  • the invention therefore relates to a microbial method of in vitro transformation of a steroid into its corresponding 1 l -hydroxy analogue using oxygen and a micro-organism selected from A speigiJlus oclvaceus Aspergillus niger Rhizopus stolonifer, Rhizopus nigricans, Rhizopus art hizits, and strains of ' Pestelolia, characterized in that a steroid having a purity of less than 97% is used
  • the micro-organism is Aspergillus ochraceus
  • the purity of the steroid is preferably more than 90% More preferably the purity of the steroid is between
  • the present microbial method can be used with steroids having an unsubstituted 1 1- position.
  • steroids having an unsubstituted 1 1- position.
  • Preferred examples are estr-4-ene-3, 17-dione and canrenone
  • a shake flask containing a mineral growth medium with glucose was inoculated with spores of A. ochraceus and placed on a reciprocal shaker at 28 °C for 15 h
  • a stirred fermentor containing 5 1 of medium was subsequently inoculated with 250 ml of germinated spore suspension.
  • the used medium contained a glucose/yeast extract medium (glucose 40 g/1, yeast extract 10 g/1, pH 5 0)
  • the culture conditions were as follows stirrer speed 750 rpm, airflow 0 2 1/1/m, temp 28 °C Foaming was measured with an antifoam electrode and controlled by automatic addition of a silicon based antifoam agent
  • the culture had a biomass concentration of at least 2 g/1 a small portion of estr-4-ene-3, 17-dione ( 1 g/1) was added to induce the synthesis of the hydroxylation enzymes
  • Three hours later higher concentrations of steroid were added to reach the desired concentrations as given in Table I The steroid trans ⁇ formation was stopped when repeatedly no increase in conversion was observed
  • Example 2 In a shake flask the same culture as described in Example 1 was prepared When the culture had a biomass concentration of at least 4 g 1 the canrenone was added at once to reach the desired concentrations as given in Table II The steroid transformation was stopped when repeatedly no increase in conversion was observed

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Steroid Compounds (AREA)

Abstract

The invention relates to a microbial method of in vitro transformation of a steroid into its corresponding 11α-hydroxy analogue using oxygen and a microorganism selected from Aspergillus ochraceus, Aspergillus niger, Rhizopus stolonifer, Rhizopus nigricans, Rhizopus arrhizus, and strains of Pestelotia, characterized in that a steroid having a purity of less than 97 % is used.

Description

MICROBIAL llα-HYDRO\YLΛTION OF STEROIDS
The present invention relates to a microbial method of 1 lα-hydroxyiation of steroids
Microbial 1 l -hydroxylation of steroids is a well known process, in vivo as well as in vitro For instance 1 lα-hydroxylation of progesterone by cell-free preparations of Aspergillus ochraceus has been reported by Shibahara et al Biochim Biophys Acta, 202 ( 1970), 172-179 It has also been known that microbial 1 lα-hydroxylation reactions of steroids are unpredictable, and invariably lead to incomplete transformations Typicallv conversion degrees of 80-85% are obtained For industrial applications it is however of importance to obtain high predictable conversion rates, which preferably lead to higher than 95% yields of l lα- hydroxylated steroids Mathematical models in the optimization of such fermentation processes are discussed by Deshayes et al , Bull Soc Chim Fr , ( 1980), II 24-34 For example, in the l lα-hydroxylation of canrenone, Deshayes disclosed that under optimum conditions better than 95% yields could be attained when Aspergillus ochraceus was used in a medium containing I a 10 g/1 of glucose m a matπx of malt-extract and trypticase Under these conditions up to 1 5 g/1 of canrenone could be transformed, which was considered to be an improvement in the art, for instance as disclosed by Blunt et al , J Chem Soc , 6 ( 1971 ), 1 136 who were not able to obtain more than 90% yield of 1 lα-hvdroxylated canrenone using at the most 0 5 g 1 of substrate
Since it can be reasoned that contaminations will have a detrimental influence on the microbial process, it can be expected that further optimization could be obtained by increasing the purity of the starting materials Surprisingly however, it has now been found that a further improvement is obtained by using less than pure substrate, in particular by using a substrate having a purity of less than 97% The invention therefore relates to a microbial method of in vitro transformation of a steroid into its corresponding 1 l -hydroxy analogue using oxygen and a micro-organism selected from A speigiJlus oclvaceus Aspergillus niger Rhizopus stolonifer, Rhizopus nigricans, Rhizopus art hizits, and strains of ' Pestelolia, characterized in that a steroid having a purity of less than 97% is used Preferably the micro-organism is Aspergillus ochraceus The purity of the steroid is preferably more than 90% More preferably the purity of the steroid is between 90 and 95 % The present method using impure substrates affords microbial transformations at substrate concentrations, which are substantially greater than the maximum concentrations as disclosed by Blunt or Deshayes
The present microbial method can be used with steroids having an unsubstituted 1 1- position. Preferred examples are estr-4-ene-3, 17-dione and canrenone
The surprising effect of impurities on the conversion degree is illustrated in the following tables:
Table I
Conversion of estr-4-ene-3, 17-dione by A. ochraceus purity substrate concentration conversion degree
(%) (g/i) (%)
99 15 78
99 10 83
98 25 85
94* 15 91
15 91
94*
15 94
94* 25 97
93 10 98
92
various natural impurities added to pure substrate Table II
Conversion of canrenone by A. ochraceus purity substrate concentration conversion degree
(%) (g i) (%)
100 5 74
100 10 78
100 20 73
100 35 72
96 10 98
94 15 96
96 22 96
95 22 95
The invention is further illustrated by the following examples.
EXAMPLES
Example 1
A shake flask containing a mineral growth medium with glucose was inoculated with spores of A. ochraceus and placed on a reciprocal shaker at 28 °C for 15 h A stirred fermentor containing 5 1 of medium was subsequently inoculated with 250 ml of germinated spore suspension. The used medium contained a glucose/yeast extract medium (glucose 40 g/1, yeast extract 10 g/1, pH 5 0) The culture conditions were as follows stirrer speed 750 rpm, airflow 0 2 1/1/m, temp 28 °C Foaming was measured with an antifoam electrode and controlled by automatic addition of a silicon based antifoam agent When the culture had a biomass concentration of at least 2 g/1 a small portion of estr-4-ene-3, 17-dione ( 1 g/1) was added to induce the synthesis of the hydroxylation enzymes Three hours later higher concentrations of steroid were added to reach the desired concentrations as given in Table I The steroid trans¬ formation was stopped when repeatedly no increase in conversion was observed
Example 2
In a shake flask the same culture as described in Example 1 was prepared When the culture had a biomass concentration of at least 4 g 1 the canrenone was added at once to reach the desired concentrations as given in Table II The steroid transformation was stopped when repeatedly no increase in conversion was observed

Claims

5
1 A microbial method of in vitro transformation of a steroid into its corresponding 1 lα- hydroxy analogue using oxygen and a micro-organism selected from Aspergillus ochraceus, Aspergillus niger, Rhizopus stolonifer, Rhizopus nigricans, Rhizopus airhizus, and strains of Pestelotia, characterized in that a steroid having a purity of less than 97% is used
2 The method according to claim 1 , wherein the micro-organism is Aspergillus ochraceus
3 The method according to claim 1 or 2, wherein the purity of the steroid is between 90 and 95%
4 The method according to any one of claims 1 -3, wherein a steroid concentration greater than 0 5 g/1 is used
5 The method according to any one of claims 1-4, wherein the steroid is selected from estr-4-ene-3, 17-dione and canrenone
PCT/EP1996/005729 1995-12-12 1996-12-10 MICROBIAL 11α-HYDROXYLATION OF STEROIDS Ceased WO1997021830A1 (en)

Priority Applications (11)

Application Number Priority Date Filing Date Title
US09/077,982 US6046023A (en) 1995-12-12 1996-12-10 Microbial 11α-hydroxylation of steroids
JP9521752A JP2000501940A (en) 1995-12-12 1996-12-10 11α-Hydroxylation of steroids by microorganisms
DK96944023T DK0900283T3 (en) 1995-12-12 1996-12-10 Microbial 11alpha-hydroxylation of steroids
PL96327439A PL185223B1 (en) 1995-12-12 1996-12-10 Microbiological method of 11alpha- hydroxylating the steroids
DE69620474T DE69620474T2 (en) 1995-12-12 1996-12-10 MICROBIAL 11ALPHA HYDROXYLATION OF STEROIDS
EP96944023A EP0900283B1 (en) 1995-12-12 1996-12-10 MICROBIAL 11alpha-HYDROXYLATION OF STEROIDS
KR1019980704372A KR19990072066A (en) 1995-12-12 1996-12-10 11α-hydroxylation of Steroids by Microorganisms
AU13758/97A AU713685B2 (en) 1995-12-12 1996-12-10 Microbial 11alpha-hydroxylation of steroids
AT96944023T ATE215606T1 (en) 1995-12-12 1996-12-10 MICROBIAL 11ALPHA HYDROXYLATION OF STEROIDS
NZ325276A NZ325276A (en) 1995-12-12 1996-12-10 Microbial method of in vitro transformation of a steroid into the 11a-hydroxy analogue using oxygen and a micro-organism
NO982700A NO982700L (en) 1995-12-12 1998-06-11 Microbial 11 <alpha> hydroxylation of steroids

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP95203451.0 1995-12-12
EP95203451 1995-12-12

Publications (1)

Publication Number Publication Date
WO1997021830A1 true WO1997021830A1 (en) 1997-06-19

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PCT/EP1996/005729 Ceased WO1997021830A1 (en) 1995-12-12 1996-12-10 MICROBIAL 11α-HYDROXYLATION OF STEROIDS

Country Status (21)

Country Link
US (1) US6046023A (en)
EP (1) EP0900283B1 (en)
JP (1) JP2000501940A (en)
KR (1) KR19990072066A (en)
CN (1) CN1138861C (en)
AR (1) AR005043A1 (en)
AT (1) ATE215606T1 (en)
AU (1) AU713685B2 (en)
CA (1) CA2240315A1 (en)
CZ (1) CZ289764B6 (en)
DE (1) DE69620474T2 (en)
DK (1) DK0900283T3 (en)
ES (1) ES2175175T3 (en)
IL (1) IL119818A (en)
NO (1) NO982700L (en)
NZ (1) NZ325276A (en)
PL (1) PL185223B1 (en)
PT (1) PT900283E (en)
RU (1) RU2213143C2 (en)
WO (1) WO1997021830A1 (en)
ZA (1) ZA9610485B (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1352054A2 (en) * 2000-10-30 2003-10-15 Pharmacia Corporation Aspergillus ochraceus 11 alpha hydrolase and oxidoreductase
WO2004011663A3 (en) * 2002-07-24 2004-07-15 Schering Ag Microbiological method for the production of 7 alpha-substituted 11 alpha-hydroxysteroids
US7262023B2 (en) 2002-07-24 2007-08-28 Bayer Schering Pharma Ag Microbiological process for the production of 7-substituted 11-hydroxy steroids, 7,17-substituted 11-Halogen steroids and uses thereof

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MXPA03004112A (en) * 2000-11-09 2003-08-19 Astrazeneca Ab Oral pharmaceutical composition containing a block copolymer.
FR2817284B1 (en) 2000-11-27 2003-02-14 Alstom Power Nv PIECE, IN PARTICULAR HYDRAULIC COMPONENT, IN COMPOSITE MATERIAL AND METHOD FOR MANUFACTURING SUCH A PART
US20070066579A1 (en) * 2002-08-16 2007-03-22 White Michael J 5-androsten-3beta-ol steroid intermediates and processs for their preparation
US20040034215A1 (en) * 2002-08-16 2004-02-19 White Michael J. 5-Androsten-3beta-ol steroid intermediates and processes for their preparation
CN100338226C (en) * 2005-07-29 2007-09-19 天津科技大学 Method for preparing 11 alpha bydroxy canrenone through conversion method of batch fermentation of microbe
US20070212751A1 (en) * 2006-01-18 2007-09-13 Solvay Pharmaceuticals Gmbh Microbial method for the 11beta hydroxylation of 9beta, 10alpha-steriods
RU2731712C2 (en) * 2018-12-07 2020-09-08 Федеральное государственное бюджетное образовательное учреждение высшего образования "Московский государственный университет имени М.В. Ломоносова" (МГУ) Microbiological method of producing 11α-acetoxyprogesterone
CN111593086B (en) * 2020-05-27 2023-05-05 湖北葛店人福药业有限责任公司 Method for reducing impurities in ethyldiketone 11a hydroxylation process by using mixed solvent

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FR1080205A (en) * 1950-08-19 1954-12-07 Upjohn Co Steroid Treatment Method
FR1091743A (en) * 1952-09-24 1955-04-14 Pfizer & Co C Improvements in manufacturing processes for steroid compounds
FR1261181A (en) * 1958-02-21 1961-05-19 Merck & Co Inc Production by fermentation of 11 alpha-hydroxyl derivatives of steroids of the 16 alpha-alkyl-4-pregnene-3, 20-dione series
FR1406102A (en) * 1963-06-17 1965-07-16 Upjohn Co Microbiological progesterone oxygenation process
NL6409039A (en) * 1964-08-06 1966-02-07
US3294646A (en) * 1963-10-23 1966-12-27 American Home Prod Microbiological hydroxylation of norsteroids using aspergillus ochraceus
FR1555991A (en) * 1967-02-20 1969-01-31

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US2602769A (en) * 1952-02-23 1952-07-08 Upjohn Co Oxygenation of steroids by mucorales fungi

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR1080205A (en) * 1950-08-19 1954-12-07 Upjohn Co Steroid Treatment Method
FR1091743A (en) * 1952-09-24 1955-04-14 Pfizer & Co C Improvements in manufacturing processes for steroid compounds
FR1261181A (en) * 1958-02-21 1961-05-19 Merck & Co Inc Production by fermentation of 11 alpha-hydroxyl derivatives of steroids of the 16 alpha-alkyl-4-pregnene-3, 20-dione series
FR1406102A (en) * 1963-06-17 1965-07-16 Upjohn Co Microbiological progesterone oxygenation process
US3294646A (en) * 1963-10-23 1966-12-27 American Home Prod Microbiological hydroxylation of norsteroids using aspergillus ochraceus
NL6409039A (en) * 1964-08-06 1966-02-07
FR1555991A (en) * 1967-02-20 1969-01-31

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DESHAYES, CHRISTIAN M. P.: "Use of mathematical models for optimization in fermentation. Applications to transformations by microorganisms", BULL. SOC. CHIM. FR. (1980), (1-2, PT. 2), 24-34 CODEN: BSCFAS;ISSN: 0037-8968, 1980, XP002029426 *
TAN, LIAT ET AL: "Interactions of steroids and fungi. III. 11.alpha.- Hydroxylation and degradation of progesterone-4-14C by a cell-free preparation from Aspergillus ochraceus", J. STEROID BIOCHEM. (1970), 1(3), 221-7 CODEN: JSTBBK, 1970, XP000671122 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1352054A2 (en) * 2000-10-30 2003-10-15 Pharmacia Corporation Aspergillus ochraceus 11 alpha hydrolase and oxidoreductase
WO2004011663A3 (en) * 2002-07-24 2004-07-15 Schering Ag Microbiological method for the production of 7 alpha-substituted 11 alpha-hydroxysteroids
EA008147B1 (en) * 2002-07-24 2007-04-27 Шеринг Акциенгезельшафт Microbiological method for the production of 7 alpha-substituted 11 alpha-hydroxysteroids
US7262023B2 (en) 2002-07-24 2007-08-28 Bayer Schering Pharma Ag Microbiological process for the production of 7-substituted 11-hydroxy steroids, 7,17-substituted 11-Halogen steroids and uses thereof
CN100339486C (en) * 2002-07-24 2007-09-26 舍林股份公司 Microbial process for the preparation of 7α-substituted 11α-hydroxy steroids
EA010572B1 (en) * 2002-07-24 2008-10-30 Шеринг Акциенгезельшафт 7 alpha, 17 alpha substituted 11 beta halogen steroids, processes for production thereof, use thereof and pharmaceutical preparation based thereon
KR101041328B1 (en) * 2002-07-24 2011-06-14 바이엘 쉐링 파마 악티엔게젤샤프트 7 Alpha-Substituted 11 Microbiological Methods for the Production of Alpha-hydroxy Steroids

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Publication number Publication date
PL327439A1 (en) 1998-12-07
AU713685B2 (en) 1999-12-09
DK0900283T3 (en) 2002-07-15
RU2213143C2 (en) 2003-09-27
ZA9610485B (en) 1997-06-24
EP0900283A1 (en) 1999-03-10
JP2000501940A (en) 2000-02-22
PL185223B1 (en) 2003-04-30
US6046023A (en) 2000-04-04
CN1207136A (en) 1999-02-03
CN1138861C (en) 2004-02-18
PT900283E (en) 2002-09-30
DE69620474T2 (en) 2002-11-14
NZ325276A (en) 1999-11-29
NO982700D0 (en) 1998-06-11
NO982700L (en) 1998-06-11
CA2240315A1 (en) 1997-06-19
ATE215606T1 (en) 2002-04-15
CZ289764B6 (en) 2002-04-17
AR005043A1 (en) 1999-04-07
EP0900283B1 (en) 2002-04-03
CZ183498A3 (en) 1998-10-14
ES2175175T3 (en) 2002-11-16
DE69620474D1 (en) 2002-05-08
IL119818A0 (en) 1997-03-18
KR19990072066A (en) 1999-09-27
IL119818A (en) 2000-02-29
AU1375897A (en) 1997-07-03

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