CN1483833A - Optimizing method for biolgical transformation cloud point system of androst-1, 4 diene-3, 17-diketone - Google Patents

Optimizing method for biolgical transformation cloud point system of androst-1, 4 diene-3, 17-diketone Download PDF

Info

Publication number
CN1483833A
CN1483833A CNA031421156A CN03142115A CN1483833A CN 1483833 A CN1483833 A CN 1483833A CN A031421156 A CNA031421156 A CN A031421156A CN 03142115 A CN03142115 A CN 03142115A CN 1483833 A CN1483833 A CN 1483833A
Authority
CN
China
Prior art keywords
concentration
substrate
transformation
cloud point
cholesterol
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.)
Granted
Application number
CNA031421156A
Other languages
Chinese (zh)
Other versions
CN1223681C (en
Inventor
陈代杰
王志龙
戈梅
叶伟东
金一平
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.)
Shanghai Laiyi Biomedical Research And Development Center LLC
Original Assignee
Shanghai Laiyi Biomedical Research And Development Center LLC
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
Application filed by Shanghai Laiyi Biomedical Research And Development Center LLC filed Critical Shanghai Laiyi Biomedical Research And Development Center LLC
Priority to CN 03142115 priority Critical patent/CN1223681C/en
Publication of CN1483833A publication Critical patent/CN1483833A/en
Application granted granted Critical
Publication of CN1223681C publication Critical patent/CN1223681C/en
Anticipated expiration legal-status Critical
Expired - Fee Related legal-status Critical Current

Links

Images

Landscapes

  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The present invention belongs to the field of microbial technology. Said invention provides an optimization method of cloud point system is microbial transformation application, in particular, it discloses the optimization parameters of cloud point system from cholesterol to production of ADD and 4-AD, and when the concentration of substrate adopted by said invention is 0.5-2.0/100ml, the ADD and 4-AD yield obtained by microbial transformation can be up to 3.5-10g/L, and the correspondent conversion rate can be 80-93%.

Description

Androstane-1,4 diene-3, the optimization method of 17-diketone bio-transformation cloud point system
Technical field
The invention belongs to microbial technology field.Be specifically related to androstane-1,4 diene-3, the optimization method of 17-diketone bio-transformation cloud point system
Background technology
The special property of biological catalyst makes conversion technology be subjected to the attention of business circles and researcher again.Particularly highly selective and reaction conditions gentleness are particularly suitable for synthetic and the modification natural compounds.Even a certain bioconversion reaction has been developed simple chemosynthesis approach, the high chemo-selective of bio-transformation and spatial selectivity also can be got rid of protection necessary in the chemosynthesis approach and go to protect step.Yet although people have keen interest to bio-transformation, industrial application often is subjected to the restriction of practical difficulty.Low as the solubleness of hydrophobic substrate in water, limited its bioavailability; Substrate, product may suppress or poison microorganism.Non-aqueous media plays an important role in bio-transformation because of its unique function, has made significant headway in recent years.The inventor has developed a kind of new system, and this system is a nonionogenic tenside micellar solution, is higher than its cloud point or has under the condition that inductor exists in temperature, can automatic phase-splitting forms tensio-active agent dilute phase and the agglutinant layer that is rich in tensio-active agent mutually.Promptly adopt one or more nonionogenic tensides form aqueous solution system that cloud points are lower than the microbial transformation culture temperature be cloud point system (cloud point system, CPS), as the medium of microbial transformation.In CPS, water-in-oil and oil-in-water microcosmic emulsion have been formed.The tensio-active agent drop has solubilising, is serving as the storage of substrate and the inhibitor of product.Strengthen the availability of substrate, got rid of the inhibition of product.There is bubble in the successive tensio-active agent in mutually, is equivalent to a microreactor, can make microorganism avoid the toxic action of tensio-active agent.The biocompatibility of cloud point system and the strengthening effect of substrate bioavailability is proved.
The approach that ADD (androstane-1,4 diene-3,17-diketone) and 4-AD (androstane-4 alkene-3,17-diketone) are produced in the side chain excision that utilizes Mycrobacterium sp.NRRL B-3683 to carry out cholesterol as shown in Equation 1.In this microbial transformation, the mol ratio of ADD and 4-AD is 10: 1.
The approach of ADD and 4-AD is produced in the bio-transformation of formula 1 cholesterol:
Although it is very limited to introduce new steroid drugs, the productive rate of the side chain excision of the productive rate, particularly sterol that the raising selected microorganism transforms is the direction that people make great efforts so that utilize cheap natural matter always.People have attempted the whole bag of tricks with solubleness that increases substrate or the restraining effect of getting rid of product, as aqueous two-phase system, organic solvent biphasic system, organic medium, liposome system etc.
Summary of the invention
Technical problem to be solved by this invention is to design the optimization method that improves the bio-transformation productive rate in the cloud point system application, particularly provide the excision of cholesterol side chain to produce ADD (androstane-1,4 dienes-3, the 17-diketone) and the cloud point system of 4-AD (androstane-4 alkene-3,17-diketone) transform the optimization production method.
The present invention experiment showed, that through a large amount of surfactant concentration and concentration of substrate have bigger influence to conversion yield in cloud point system.The present invention has set up the optimal conditions of producing the CPS of ADD and 4-AD by optimizing substrate, surfactant concentration, and concentration of substrate is compared all with other conversion system with transformation efficiency and is significantly improved.
The cloud point system parameter of optimization production ADD disclosed by the invention and 4-AD is: nonionogenic tenside adopts Triton X-100 and Triton X-114, and its mass ratio is 1: 0.5-1.5; The concentration of mixed surfactant is 5-10g/100ml; The concentration of substrate cholesterol is 0.5-2.0g/100ml; Microbial strains is adopted Mycrobacterium sp.NRRL B-3683, and the microbial transformation time is 5-9 days.Under this optimal conditions, the ADD+4-AD productive rate is up to 3.5-10g/L, and corresponding transformation efficiency is 80-93%.
Is that the optimal conditions of the CPS of raw material production ADD and 4-AD is further described below by concrete experimentation with the cholesterol to the present invention:
1, microorganism and cultivation
Microorganism
Microbial strains Mycobacterium sp.NRRL B 3683.It can realize the side chain excision of cholesterol, generates product A DD and 4-AD, and its ratio is 10: 1.
Substratum
Slant medium (100ml): the 0.5g yeast leaches cream, 1.2g agar powder, 1g glycerine, 0.05g K 2HPO 4, 0.1g (NH 4) 2SO 4, 0.05g MgSO 47H 2O.
Seed culture medium (100ml): 0.5g (NH 4) 2SO 4, 0.45g Na 2HPO 4, 0.34gKH 2PO 4, 0.05g MgSO 47H 2O, 1.0g glycerine, 0.2g cholesterol, 0.2g TritonX-100.
Transform substratum (100ml): 1.0g (NH 4) 2SO 4, 0.45g Na 2HPO 4, 0.34gKH 2PO 4, 0.2g MgSO 47H 2O, a certain amount of cholesterol and mixed surfactant Triton X-100: Triton X-114=1: 1).
Microorganism culturing
Microorganism activation culture 3 days under 28 ℃, 220rpm, aerobic, 20/250ml loading amount condition, getting activated seed is to transform under the 22/250ml condition to cultivate 7 days in 1: 10 ratio in loading amount.Get and transform the nutrient solution analysis.
2, analytical procedure
Get the 1ml sample and soak 2hr with 4ml methyl alcohol.Get the 0.8ml supernatant after centrifugal and carry out the HPLC analysis.With Hypersil C18 post, moving phase is methyl alcohol: water (4: 1), flow velocity are 0.7ml/min, and the detection wavelength is 254nm.
3, result and discussion
1) optimization of microbial transformation time
The mass ratio of mixed surfactant is Triton X-100 and the ratio of Triton X-114 is 1: 1.With the concentration of substrate is that 1.5g/100ml, surfactant concentration are that 4g/100ml carries out microbial transformation, and the influence of time as shown in Figure 1.Fig. 1 is the time curve of microbial transformation.Production concentration when the result shows 7 days is the highest.Prolonging transformation time again reduces on the contrary.This shows that product can further be degraded by microorganism.Make the further key enzyme of degraded of ADD and 4-AD during possible induction mutation of bacterium, the 9a-hydroxylase does not have complete deactivation.
2) influence of mixed surfactant concentration
Mixed surfactant concentration is 0.5～4.0g/100ml, and the result of microbial transformation as shown in Figure 2 when concentration of substrate was 0.5g/100ml.Fig. 2 is the variation of final product concentration with surfactant concentration.When surfactant concentration was low, productive rate increased with the increase of surfactant concentration, and this is attributable to the solubilising of tensio-active agent.Surfactant micelle serves as the storage vault of substrate and the extraction agent of product, and with the increase of surfactant concentration, the bioavailability of substrate increases and the restraining effect of product is disengaged.But when surfactant concentration was higher, productive rate reduced with the increase of surfactant concentration, showed the restraining effect of high surface agent concentration.Final product concentration shows with the curve that surfactant concentration changes, and there is optimum surfactant concentration in certain concentration of substrate.
3) influence of concentration of substrate
Concentration of substrate is 0.025～3g/100ml, and the microbial transformation result as shown in Figure 3 when mixed surfactant concentration was 2g/100ml.Fig. 3 is the variation of product with concentration of substrate.Less at concentration of substrate, productive rate stably increases with the increase of concentration of substrate and shows better linearity relation.When concentration of substrate was higher, productive rate was stable at a constant.This is attributable to the solubilising of tensio-active agent.The result shows, obtain higher production concentration and transformation efficiency, the critical concentration of substrate (as surfactant concentration is 2g/100ml, and maximum end product concentration is 6200mg/L, and corresponding critical concentration of substrate is 0.85g/100ml) that certain surfactant concentration is existed a correspondence.
4) optimization of concentration of substrate
High concentration of substrate is an important factor that influences the microbial transformation economy.Substrate and the surfactant concentration final product concentration that influencing each other.So adopt concentration of substrate 1～2.2g/100ml, different mixed surfactant concentration are carried out microbial transformation, the result as shown in Figure 4.Fig. 4 is the optimization of concentration of substrate.Can estimate the critical concentration of substrate of maximum final product concentration correspondence when the certain surface surfactant concentration from Fig. 4.The result as shown in Figure 5.Fig. 5 is the mutual relationship of critical concentration of substrate and surfactant concentration.When surfactant concentration was 2～8g/100ml, the maximum production concentration during corresponding certain surface surfactant concentration increased with the increase of surfactant concentration.When surfactant concentration reached 10g/100ml, its maximum production concentration no longer obviously increased, and stagnation behavior appears in maximum production concentration during the high surface agent concentration.With the increase of surfactant concentration, may cause material transfer, particularly the oxygen transmission is suppressed.
The basic parameter of table 1 microbial transformation system
Substrate product medium microbial substrates concentration transformation efficiency
Cholesterol ADD, 4-AD aqueous two-phase system mycobacterium 1g/L 80%
Sitosterol 4-AD organic medium immobilization mycobacterium 12mM 89%
Plant sterol 4-AD PPG solvent mycobacterium 5-30g/L 90%
Organic diphase system
Sitosterol 4-AD system immobilization mycobacterium 5g/L 70%
Cholesterol ADD, 4-AD cloud point system mycobacterium 1.45g/100ml 93%
The definition transformation efficiency is ADD and 4-AD and the ratio of the mole number of cholesterol.When transforming fully, the 1g cholesterol can arrive 0.74g ADD and 4-AD (ADD: 4-AD=10: 1).Can obtain from Fig. 6 that the critical concentration of substrate of correspondence is 1.45g/100ml when surfactant concentration is 8g/100ml.As shown in table 1, transformation efficiency, particularly concentration of substrate (corresponding final product concentration) all improve a lot.
Conclusion
Surfactant concentration and concentration of substrate influence each other to conversion yield.The present invention has set up the optimal conditions of CPS by optimizing substrate, surfactant concentration.The concentration of substrate is compared all with other conversion system with transformation efficiency and is significantly improved, and shows that CPS is good conversion system.
Description of drawings
The time curve of Fig. 1 microbial transformation
Fig. 2 final product concentration is with the variation of surfactant concentration
Fig. 3 productive rate is with the variation of concentration of substrate
The optimization of Fig. 4 concentration of substrate
Surfactant concentration (g/100ml): * (2); ◇ (4); △ (6); Zero (8);
●(10)
The mutual relationship of critical concentration of substrate of Fig. 5 and surfactant concentration
Embodiment
The side chain excision of embodiment cholesterol, the system parameter of optimization CPS
Embodiment 1.
1. microorganism and cultivation
Microorganism
Microbial strains Mycobacterium sp.NRRL B 3683.It can realize the side chain excision of cholesterol, generates product A DD and 4-AD, and its ratio is 10: 1.
Substratum
Slant medium (100ml): the 0.5g yeast leaches cream, 1.2g agar powder, 1g glycerine, 0.05g K 2HPO 4, 0.1g (NH 4) 2SO 4, 0.05g MgSO 47H 2O.
Seed culture medium (100ml): 0.5g (NH 4) 2SO 4, 0.45g Na 2HPO 4, 0.34gKH 2PO 4, 0.05g MgSO 47H 2O, 1.0g glycerine, 0.2g cholesterol, 0.2g TritonX-100.
Transform substratum (100ml): 1.0g (NH 4) 2SO 4, 0.45g Na 2HPO 4, 0.34gKH 2PO 4, 0.2g MgSO 47H 2O, 1.5g cholesterol, 10g tensio-active agent (TritonX-100: Triton X-114=1: 1).
Microorganism culturing
Microorganism activation culture 3 days under 28 ℃, 220rpm, aerobic, 20/250ml loading amount condition, getting activated seed is to transform under the 22/250ml condition to cultivate 7 days in 1: 10 ratio in loading amount.Get and transform the nutrient solution analysis.
Analytical procedure
Get the 1ml sample and soak 2hr with 4ml methyl alcohol.Get the 0.8ml supernatant after centrifugal and carry out the HPLC analysis.With Hypersil C18 post, moving phase is methyl alcohol: water (4: 1), flow velocity are 0.7ml/min, and the detection wavelength is 254nm.
The result
Production concentration (ADD: 4-AD=10: 1) be 10g/L.
Embodiment 2.
1. microorganism and cultivation
Microorganism
Microbial strains Mycobacterium sp.NRRL B 3683.It can realize the side chain excision of cholesterol, generates product A DD and 4-AD, and its ratio is 10: 1.
Substratum
Slant medium (100ml): the 0.5g yeast leaches cream, 1.2g agar powder, 1g glycerine, 0.05g K 2HPO 4, 0.1g (NH 4) 2SO 4, 0.05g MgSO 47H 2O.
Seed culture medium (100ml): 0.5g (NH 4) 2SO 4, 0.45g Na 2HPO 4, 0.34gKH 2PO 4, 0.05g MgSO 47H 2O, 1.0g glycerine, 0.2g cholesterol, 0.2g TritonX-100.
Transform substratum (100ml): 1.0g (NH 4) 2SO 4, 0.45g Na 2HPO 4, 0.34gKH 2PO 4, 0.2g MgSO 47H 2O, 1.5g cholesterol, 6g tensio-active agent (TritonX-100: Triton X-114=1: 1.2).
Microorganism culturing
Microorganism activation culture 3 days under 28 ℃, 220rpm, aerobic, 20/250ml loading amount condition, getting activated seed is to transform under the 22/250ml condition to cultivate 7 days in 1: 10 ratio in loading amount.Get and transform the nutrient solution analysis.
Analytical procedure
Get the 1ml sample and soak 2hr with 4ml methyl alcohol.Get the 0.8ml supernatant after centrifugal and carry out the HPLC analysis.With Hypersil C18 post, moving phase is methyl alcohol: water (4: 1), flow velocity are 0.7ml/min, and the detection wavelength is 254nm.
The result
Production concentration (ADD: 4-AD=10: 1) be 8g/L.
Embodiment 3.
1. microorganism and cultivation
Microorganism
Microbial strains Mycobacterium sp.NRRL B 3683.It can realize the side chain excision of cholesterol, generates product A DD and 4-AD, and its ratio is 10: 1.
Substratum
Slant medium (100ml): the 0.5g yeast leaches cream, 1.2g agar powder, 1g glycerine, 0.05g K 2HPO 4, 0.1g (NH 4) 2SO 4, 0.05g MgSO 47H 2O.
Seed culture medium (100ml): 0.5g (NH 4) 2SO 4, 0.45g Na 2HPO 4, 0.34gKH 2PO 4, 0.05g MgSO 47H 2O, 1.0g glycerine, 0.2g cholesterol, 0.2g TritonX-100.
Transform substratum (100ml): 1.0g (NH 4) 2SO 4, 0.45g Na 2HPO 4, 0.34gKH 2PO 4, 0.2g MgSO 47H 2O, 1.0g cholesterol, 10g tensio-active agent (TritonX-100: Triton X-114=1: 0.8).
Microorganism culturing
Microorganism activation culture 3 days under 28 ℃, 220rpm, aerobic, 20/250ml loading amount condition, getting activated seed is to transform under the 22/250ml condition to cultivate 7 days in 1: 10 ratio in loading amount.Get and transform the nutrient solution analysis.
Analytical procedure
Get the 1ml sample and soak 2hr with 4ml methyl alcohol.Get the 0.8ml supernatant after centrifugal and carry out the HPLC analysis.With Hypersil C18 post, moving phase is methyl alcohol: water (4: 1), flow velocity are 0.7ml/min, and the detection wavelength is 254nm.
The result
Production concentration (ADD: 4-AD=10: 1) be 7g/L.

Claims (1)

1. androstane-1,4 dienes-3, the optimization method of 17-diketone bio-transformation cloud point system, this method is raw material with the cholesterol, with microorganism Mgcobacterium sp.NRRL B 3683. is that bacterial classification carries out microbial transformation, it is characterized in that the used medium of this method is a cloud point system, each parameter is: it is 1 that nonionogenic tenside adopts mass ratio: Triton X-100 and the TritonX-114 of 0.5-1.5, the concentration of mixed surfactant is 5-10g/100ml, the concentration of substrate cholesterol is 0.5-2.0g/100ml, and the microbial transformation time is 5-9 days.
CN 03142115 2003-08-07 2003-08-07 Optimizing method for biolgical transformation cloud point system of androst-1, 4 diene-3, 17-diketone Expired - Fee Related CN1223681C (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN 03142115 CN1223681C (en) 2003-08-07 2003-08-07 Optimizing method for biolgical transformation cloud point system of androst-1, 4 diene-3, 17-diketone

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN 03142115 CN1223681C (en) 2003-08-07 2003-08-07 Optimizing method for biolgical transformation cloud point system of androst-1, 4 diene-3, 17-diketone

Publications (2)

Publication Number Publication Date
CN1483833A true CN1483833A (en) 2004-03-24
CN1223681C CN1223681C (en) 2005-10-19

Family

ID=34155612

Family Applications (1)

Application Number Title Priority Date Filing Date
CN 03142115 Expired - Fee Related CN1223681C (en) 2003-08-07 2003-08-07 Optimizing method for biolgical transformation cloud point system of androst-1, 4 diene-3, 17-diketone

Country Status (1)

Country Link
CN (1) CN1223681C (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005014845A1 (en) * 2003-08-07 2005-02-17 Shanghai Health Creation Center For Biopharmaceutical R & D Co., Ltd. Application of cloud point system in biological transforming
WO2005052178A1 (en) * 2003-11-28 2005-06-09 Shanghai Health Creation Center For Biopharmaceutical R & D Co., Ltd. A method of being biological transformed using resting cells in cloud point system
CN102827913A (en) * 2012-08-31 2012-12-19 湖南诺凯生物医药有限公司 Method for preparing 11 alpha-OH-ADD by mixed fermentation of microbes

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005014845A1 (en) * 2003-08-07 2005-02-17 Shanghai Health Creation Center For Biopharmaceutical R & D Co., Ltd. Application of cloud point system in biological transforming
WO2005052178A1 (en) * 2003-11-28 2005-06-09 Shanghai Health Creation Center For Biopharmaceutical R & D Co., Ltd. A method of being biological transformed using resting cells in cloud point system
CN102827913A (en) * 2012-08-31 2012-12-19 湖南诺凯生物医药有限公司 Method for preparing 11 alpha-OH-ADD by mixed fermentation of microbes
CN102827913B (en) * 2012-08-31 2014-01-15 湖南成大生物科技有限公司 Method for preparing 11 alpha-OH-ADD by mixed fermentation of microbes

Also Published As

Publication number Publication date
CN1223681C (en) 2005-10-19

Similar Documents

Publication Publication Date Title
CN101346470B (en) Method for preparing long chain dicarboxylic acid
Kachlishvili et al. Modulation of Cerrena unicolor laccase and manganese peroxidase production
CN100347312C (en) Method for producing aurantin by liquid-state fermentation of monacolin
CN1946855A (en) The method of making optically active ester derivatives and their acids from racemic esters
CN1246156A (en) Novel bacterial strains and use thereof in fermentation processes for 2-keto-L-gulonic acid production
CN101078005A (en) Bacillus pumilus and application of the same in producing natural vanillin by biologically converting iso-eugenol
CN102321678A (en) The method of 1 alpha-hydroxy vitamin D is made in a kind of microbial transformation
CN110724654B (en) Pseudomonas aeruginosa for producing 5-hydroxymethyl-2-furancarboxylic acid and application thereof
CN1223681C (en) Optimizing method for biolgical transformation cloud point system of androst-1, 4 diene-3, 17-diketone
JP2002540760A (en) Improved production method of vitamin B12
CN1354798A (en) Process for preparation of pseudomonic acid A antibiotic by microbiological method
CN1920046A (en) Method of preparing lycopene from trispore Bruce mould
CN1876808A (en) Streptomyces virginiae and its application in transformation and degradation of steroid
CN1854302A (en) Production of 6-hydroxy-3-succinyl-pyridine with nicotine as substrate and biological conversion
CN1212404C (en) Fermentation process for raising ebomycin A yield
CN1188527C (en) Process for biotransformation of colchicone compounds into the correspoonding 3-o-glycosyl derivatives
CN1420935A (en) Process for production of optically active beta-amino alcohols
CN1483815A (en) Application of cloud point system in biotransformation
CN101054561A (en) Microorganism capable of hydrolyzing glycosyl to hydroxyl and application thereof
CN1255530C (en) Biotransformation method for resting cell in cloud point system
CN1840671A (en) Process for preparing (R)-mandelic acid by microbial asymmetric resolution
NL2027994B1 (en) Process for remediation of heavy metal-containing soil by using pig manure and trichoderma viride jx993849
CN1175101C (en) Process for culturing mycelial fungus in suspension of solid nutrient matrix
CN1614007A (en) Gluconobacter and its use
KR20070096099A (en) Manufacturing method of germanium

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20051019

Termination date: 20140807

EXPY Termination of patent right or utility model