WO2004069845A1 - Solid state fermentation and fed batch for the production of an immunosuppressant - Google Patents

Solid state fermentation and fed batch for the production of an immunosuppressant Download PDF

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Publication number
WO2004069845A1
WO2004069845A1 PCT/IN2003/000023 IN0300023W WO2004069845A1 WO 2004069845 A1 WO2004069845 A1 WO 2004069845A1 IN 0300023 W IN0300023 W IN 0300023W WO 2004069845 A1 WO2004069845 A1 WO 2004069845A1
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Prior art keywords
fermentation
rice
bran
wheat
solid substrate
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PCT/IN2003/000023
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French (fr)
Inventor
Ainn Nazhath Ul
Sowmyalakshmi Khandige
Nitin Sopanrao Patil
Anand Prakash Khedkar
Ramakrishnan Melarkode
Ramavana Gururaja
Shrikumar Suryanarayan
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Biocon Limited
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Priority to PCT/IN2003/000023 priority Critical patent/WO2004069845A1/en
Priority to AU2003209664A priority patent/AU2003209664A1/en
Publication of WO2004069845A1 publication Critical patent/WO2004069845A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
    • C07D498/18—Bridged systems
    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms

Definitions

  • the invention provides a fermentation process in which the compound of Formula I or any of it's salt form, is produced on solid nutritious matrix.
  • the fermentation is also carried out in fed-batch mode to increase the productivity/yields of the final product.
  • the fermentation is also carried out in a contained bio-reactor.
  • Feed-batch fermentation or “fed-batch technique”: The term fed-batch fermentation as used herein, means a fermentation process carried out where substrate or nutrients are added in small increments as the fermentation progresses.
  • the substrate or nutrient is added in small increment that would encourage the production of secondary metabolites, because some secondary metabolite production is inhibited by high concentrations of substrate or substrates, so this method would encourage the production of such metabolites.
  • Supplement of nutrients at a time when the initially fed nutrient are consumed by the microorganisms or culture also help in providing more energy to the microorganism which in turn increases the overall production of the secondary metabolites.
  • bioreactor means a device capable of holding fermentation media inoculated with microorganism and carrying out the process of solid state fermentation in a contained manner.
  • a bioreactor can be used to grow any microorganism capable of growing, under specified conditions in a contained environment.
  • Some examples of microorganisms capable of growing in a bioreactor are fungi, yeast and bacteria.
  • the nitrogen source for feeding is selected from ammonium sulphate, dried yeast, ammonium nitrate, sodium nitrate, bacteriological peptone, yeast extract, casein hydrolyzate, soy peptone, soy flour, cotton seed flour, corn steep liquor or a mixture of two or more of these.
  • the solid substrate fermentation is also carried out in a fed- batch mode.

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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)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Nitrogen And Oxygen Or Sulfur-Condensed Heterocyclic Ring Systems (AREA)

Abstract

The present invention provides a novel method for producing compound of formula (I) or any of its salt form, by solid state fermentation, optionally using fed-batch technique by culturing microorganisms capable of producing the compound of formula (I).

Description

TITLE OF INVENTION
SOLID STATE FERMENTATION AND FED BATCH FOR THE PRODUCTION OF AN IMMUNOSUPPRESSANT FIELD OF THE INVENTION
The present invention provides a novel method for producing compound of Formula I or its any salt form by solid state fermentation, optionally with fed-batch technique by fermentation of microorganisms of Streptomyces sp. capable of producing the compound of Formula I. BACKGROUND OF THE INWEϋϊIOPi The compound of Formula I or its any salt form produced by the inventive method is used as an immunosuppressant therapeutically. Compound of Formula I is also referred as FK520 This agent inhibits the proliferative response of lymphocytes to alloantigen stimulation, and a variety of T cell associated immune reaction. The compound suppresses immune responses in vivo as well as in vitro. The compound can be further chemically modified to another form which is useful in the treatment of psoriasis and does not have immunosuppressive activity.
Figure imgf000004_0001
Formula I
The prior art literature does not disclose production of compound of Formula I or its any salt form, by solid substrate fermentation or solid state fermentation with fed-batch technique.
SUMMARY OF THE INVENTION The present invention provides a novel method for production of compound of Formula I or its salts.
In preferred embodiments, the invention provides a fermentation process in which the compound of Formula I or any of it's salt form, is produced on solid nutritious matrix. The fermentation is also carried out in fed-batch mode to increase the productivity/yields of the final product. The fermentation is also carried out in a contained bio-reactor.
©ETM EE) _ ES«H_PTIE(α« @F IMEFΞβE-D EMIBOIDMEOTS
_D@ffD_π]flfcD®ιπ)$ "§®M sKe ff©™©G i£a'£-©_fl-,, ©B* "s©lid sffcafoe cultivation": The term "solid state fermentation" or "solid state cultivation", sometimes referred to as "semi-solid state fermentation" as used herein, means the process of fermenting microorganisms on a solid medium that provides anchorage points for the microorganisms in the absence of any freely flowing substance. The amount of water in the solid medium can be any amount of water. For example, the solid medium could be almost dry, or it could be slushy. A person skilled in the art knows that the terms "solid state fermentation" and "semi-solid state fermentation" are interchangeable.
"Fed-batch fermentation" or "fed-batch technique": The term fed-batch fermentation as used herein, means a fermentation process carried out where substrate or nutrients are added in small increments as the fermentation progresses. The substrate or nutrient is added in small increment that would encourage the production of secondary metabolites, because some secondary metabolite production is inhibited by high concentrations of substrate or substrates, so this method would encourage the production of such metabolites. Supplement of nutrients at a time when the initially fed nutrient are consumed by the microorganisms or culture also help in providing more energy to the microorganism which in turn increases the overall production of the secondary metabolites.
,,-5D®D-es} -:©- The term "bioreactor" as used herein, means a device capable of holding fermentation media inoculated with microorganism and carrying out the process of solid state fermentation in a contained manner. A bioreactor can be used to grow any microorganism capable of growing, under specified conditions in a contained environment. Some examples of microorganisms capable of growing in a bioreactor are fungi, yeast and bacteria.
The present invention discloses a process for production of compound of Formula I or its any salt form, by culturing a microorganism capable of producing such compound on solid nutrient matrix wherein, optionally the nutrients are fed in adequate quantities during the growth of the culture so that the production of the product increases significantly.
The instant invention discloses a process for the manufacture of compound of Formula ϊ and its salts by solid substrate fermentation.
The solid substrate fermentation is carried out using Streptomyces hygroscopicus.
The solid substrate for fermentation is selected from wheat bran, wheat rava, oat meal, broken wheat, boiled rice, rice bran, rice rava, beaten rice, maize bran, maize grits, oat bran, bagasse, tapioca residue, soy grits, soy flakes, rice flakes, ceramic beads, glass beads, sponge or a mixture of two or more of these. The carbon source for feeding is selected from glucose, sucrose, starch (maize, wheat, tapioca, potato), modified starch, maltose, malto-dextrin, soybean oil, acetate or a mixture of two or more of these.
The nitrogen source for feeding is selected from ammonium sulphate, dried yeast, ammonium nitrate, sodium nitrate, bacteriological peptone, yeast extract, casein hydrolyzate, soy peptone, soy flour, cotton seed flour, corn steep liquor or a mixture of two or more of these.
The solid substrate fermentation is also carried out in a fed- batch mode.
The feeding for fed-batch fermentation is done at the beginning of the fermentation or at intervals, throughout the fermentation.
The first aspect of the invention is production of compound of Formula I or its any salt form. The compound is afforded by culturing Strepto yces sp. on solid nutrient matrix e.g. wheat bran, wheat rawa, broken wheat, boiled rice, rice bran, rice rawa, beaten rice, rice flakes, maize bran, maize grits, oat bran, bagasse, tapioca residue, soy grits, soy flakes, ceramic beads, glass beads, sponge or a mixture of one or more of these. The product is then purified by conventional techniques comprising filtration, centrifugation, chromatography, extraction, distillation, concentration, precipitation, crystallization and drying.
The second aspect of invention is production of compound of Formula I or its any salt form. The compound is afforded by culturing Streptomyces sp. on solid nutrient matrix e.g. wheat bran, oatmeal, soybean meal, wheat flour, soybean flakes, maize bran etc The culture is then fed with nutrients to increase production of the final product. The product is purified by conventional techniques comprising filtration, centrifugation, chromatography, extraction, distillation, concentration, precipitation, crystallization and drying. The advantages of the present invention over the other reported methods are:
(i) cost effective process
(ii) higher productivity of compound of Formula I with feeding (iii) self-contained bioreactor decreases hazardous exposure and risk of contamination. The following Examples further illustrate the invention, it being understood that .the invention is not intended to be limited by the details disclosed therein.
EXAMPLES EXAMPLE 1
A grown slant of Streptomyces hygroscopicus was taken and 5ml of distilled water was added. It was shaken thoroughly and 4ml of this spore suspension was used fqr the inoculation of 400ml seed medium taken in 2000 ml conical flask. The composition of seed medium is as follows:
Dextrose = 20g/L
Yeast extract = 15g/L
Malt extract = lOg/L Agar = 5g/L
pH of this medium is adjusted to 6.5 after making up the volume with water. The seed flasks were grown at 28°C for 4 days and used as an inoculum for solid state fermentation. Solid state fermentation: lOgm of different raw materials as given below were taken in separate petri plates. Adequate amount of water was added and sterilized at 121 deg C for 30 minutes. 10 ml inoculum from 4 day old seed medium was added. The entire substrate was mixed properly with the inoculum and incubated at 28 deg C for 7 days. Following results were obtained.
Figure imgf000009_0001
Figure imgf000010_0001
E1CMPELE 2
Solid state fermentation was conducted as in Example 1 using 75 g ceramic beads as the solid support in a petri-plate, 15 mL of Streptomyces hygroscopicus Inoculum grown in seed medium was added. The result obtained is given in the table below.
Figure imgf000010_0002
PIE S
Solid state fermentation was conducted as in Example 1 using different solid supports in combination. lOgm of the combination substrates were taken in petri plate and 10ml of Streptomyces hygroscopicus inoculum grown in seed medium was added. The results obtained are given in the table below.
Figure imgf000011_0001
EXAMPLE 4 Solid state fermentation was conducted as in Example 3 using a mixture of wheat bran, broken wheat and beaten rice. lOgm of this substrate is taken in petri plate and 10ml of Streptomyces hygroscop/cus inoculum grown in seed medium was added along with a liquid nutrient feed consisting of glucose and yeast extract. The feed was added every alternate day up to 4th day.
Figure imgf000011_0002
E AMPLE 5 Seed inoculum of Streptomyces hygroscop/cus is obtained as explained in Example 1. 3.5L of this inoculum was used for inoculating 35 L of the same seed medium taken in a 50L fermenter. This is grown for 48 hr at 28°C. This is used as an inoculum for solid state fermentation. 15 kg of substrate mixture consisting of wheat bran, oatmeal and rice flake was loaded into a bioreactor having 22600 cm2 surface area. The bioreactor was sterilised at 121 deg C for 1 to 2 hours using steam. After the sterilization the temperature of the solid substrate was brought down to 28 deg C. 15 L of the above inoculum was added to the solid substrate along with 2 L of glucose and dried yeast feed and mixed. This was incubated at 27 to 29 deg C. On 2nd and 4th day 2 L of the above feed was added to the solid substrate matrix and mixed well. The entire biomass along with the solid substrate was harvested on 7th day and processed to get pharmaceutically acceptable grade of FK520.

Claims

We claim:
1. A process for the manufacture of compound of Formula I and its salts by solid substrate fermentation with feeding comprising the steps of
Figure imgf000013_0001
Formula I preparing an inoculum of the microorganism of the genus
Streptomyces^ inoculating a solid substrate matrix with the inoculum prepared - incubating the inoculated solid substrate matrix for 4-7. days at 25-30 deg.C extracting the incubated solid substrate matrix to obtain the said product.
2. A process as claimed in claim 1 further comprising the step of purifying the said extract.
3. A process as claimed in claim 1, wherein said microorganism is Streptomyces hygroscopicus.
4. A process as claimed in claim 1, wherein the solid substrate for fermentation is selected from wheat bran, wheat rava, oat meal, broken wheat, boiled rice, rice bran, rice rava, beaten rice, maize bran, maize grits, oat bran, barley bran, bagasse, tapioca residue, soy grits, soy flakes, rice flakes, ceramic beads, glass beads, sponge or a mixture of two or more of these.
5. A process as claimed in claim 1, wherein the feeding for the solid state fermentation is done at the beginning of the fermentation or at intervals throughout the fermentation.
6. A process as claimed in claims 6, wherein the carbon source for feeding is selected from glucose, sucrose, starch (maize, wheat, tapioca, potato), modified starch, maltose, malto-dextrin, soybean oil, acetate or a mixture of two or more of these.
7. A process as claimed in claim 6, wherein the nitrogen source for feeding is selected from ammonium sulphate, dried yeast, ammonium nitrate, sodium nitrate, bacteriological peptone, yeast extract, casein hydrolyzate, soy peptone, soy flour, cotton seed flour, corn steep liquor or a mixture of two or more of these..
8. A process as claimed in claim 1 wherein in the step of purification is carried by filtration, centrifugation, chromatography, extraction, distillation, concentration, precipitation, crystallization and drying.
PCT/IN2003/000023 2003-02-10 2003-02-10 Solid state fermentation and fed batch for the production of an immunosuppressant WO2004069845A1 (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104694423A (en) * 2015-02-12 2015-06-10 青岛青泉港生物工程有限公司 Method for preparing biological desulfurization deodorant by solid-state fermentation
CN105671102A (en) * 2015-06-24 2016-06-15 重庆乾泰生物医药有限公司 Method of preparing ascomycin through fermentation
CN107245460A (en) * 2017-05-18 2017-10-13 福建省微生物研究所 A kind of streptomyces hygroscopicus mutagenic strain of high yield ascosin and its application

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3244592A (en) * 1962-06-09 1966-04-05 Arai Tadashi Ascomycin and process for its production
EP0184162B1 (en) * 1984-12-03 1994-04-27 Fujisawa Pharmaceutical Co., Ltd. Tricyclo compounds, a process for their production and a pharmaceutical composition containing the same

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3244592A (en) * 1962-06-09 1966-04-05 Arai Tadashi Ascomycin and process for its production
EP0184162B1 (en) * 1984-12-03 1994-04-27 Fujisawa Pharmaceutical Co., Ltd. Tricyclo compounds, a process for their production and a pharmaceutical composition containing the same

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
"Production of a novel FK520 analog in Streptomycs hygroscopicus: Improving titer while minimizing impurities", J. OF INDUSTRIAL MICROBIOLOGY&BIOTECHNOLOGY, vol. 28, 2002, pages 12 - 16 *
JUNKER B. ET AL.: "Secondary metabolite scale-up to minimize homolog impurity levels", BIOTECHNOLOGY AND BIOENGINEERING, vol. 59, no. 5, 1998, pages 595 - 604, XP002175770, DOI: doi:10.1002/(SICI)1097-0290(19980905)59:5<595::AID-BIT10>3.0.CO;2-9 *
JUNKER B. ET AL.: "Use of soybean oil and ammonium sulfate additions to optimize secondary metabolite production", BIOTECHNOLOGY AND BIOENGINEERING, vol. 60, no. 5, 1998, pages 580 - 588, XP002287283, DOI: doi:10.1002/(SICI)1097-0290(19981205)60:5<580::AID-BIT8>3.0.CO;2-D *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104694423A (en) * 2015-02-12 2015-06-10 青岛青泉港生物工程有限公司 Method for preparing biological desulfurization deodorant by solid-state fermentation
CN105671102A (en) * 2015-06-24 2016-06-15 重庆乾泰生物医药有限公司 Method of preparing ascomycin through fermentation
CN107245460A (en) * 2017-05-18 2017-10-13 福建省微生物研究所 A kind of streptomyces hygroscopicus mutagenic strain of high yield ascosin and its application

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