CN107083406A - (R) production method of 3 hydroxybutyric acids - Google Patents
(R) production method of 3 hydroxybutyric acids Download PDFInfo
- Publication number
- CN107083406A CN107083406A CN201710396141.3A CN201710396141A CN107083406A CN 107083406 A CN107083406 A CN 107083406A CN 201710396141 A CN201710396141 A CN 201710396141A CN 107083406 A CN107083406 A CN 107083406A
- Authority
- CN
- China
- Prior art keywords
- hydroxybutyrate
- microorganism
- salt
- acetoacetyl
- fermentation
- 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
Links
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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/42—Hydroxy-carboxylic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/15—Corynebacterium
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses the method that one kind produces (R) 3 hydroxybutyric acid by microorganism one-step fermentation, it uses the microorganism of no pathogenicity, carbon source and nitrogen source are directly converted into by (R) 3 hydroxybutyric acid by fermentation, salt such as sodium salt, sylvite, magnesium salts, calcium salt etc. can be further made in (R) 3 hydroxybutyric acid produced, as active constituents of medicine or nutritious supplementary pharmaceutical, with wide prospects for commercial application.The microorganism used in the above method includes a kind of Corynebacterium glutamicum built by genetic engineering, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.13957.
Description
Technical field
The invention belongs to bioengineering field, specifically, it is related to one kind and (R) -3- hydroxyls is produced by microbial fermentation
The method of butyric acid and its salt.
Background technology
(R) -3-hydroxybutyrate ((R) -3-Hydroxybutyrate, 3HB) is a kind of with optically active chiral compound
Thing, No. CAS is 625-72-9.(R) -3-hydroxybutyrate is the long-chain fat acid metabolic in mammal body in liver and produced
Compound, be present in as main ketoboidies in blood plasma and peripheral tissues, may be used as the energy in body most tissues
Source.(R) -3-hydroxybutyrate is in addition to trophic function, also with the effect for treating many diseases, including：1st, can be with
Treatment many has benefited from the disease of ketone body levels raising (as included the neurological disorders disease of epilepsy and myoclonia and including A Er
Hereby write from memory disease and dementia etc. neurodegenerative disorders)；2nd, free radical injury (such as ischemic disease) is reduced by aoxidizing ubiquinone；3、
Strengthen metabolic efficiency (improving training effectiveness and sports achievement, deficiency, angina pectoris, miocardial infarction etc. are supported in treatment)；4th, treat
Such as related disease of the cancer especially cancer of the brain (such as astrocytoma)；5th, for carbohydrate metabolism disturbance (such as type i diabetes, II types sugar
Urinate disease, the low ketoboidies disease of hypoglycemia etc.) there is good curative effect；6th, it can be used for preventing and treating sclerotin and reduce (osteopenia), sclerotin
Osteoporosis, serious osteoporosis and related fracture etc..Based on these functions, (R) -3-hydroxybutyrate and its salt may be used as
Food additives and medicine, with huge health care and medical value.
(R) preparation of -3-hydroxybutyrate is mainly chemical method, including direct chemical synthesis (R) -3-hydroxybutyrate, is used
Poly- 3-hydroxybutyrate depolymerase produces (R) -3-hydroxybutyrate by enzymic degradation poly 3-hydroxy butyrate.Chemical synthesis (R) -3-
The method of hydroxybutyric acid needs chiral metal catalyst of high temperature, the reaction condition of high pressure and costliness etc.；And enzymic degradation is poly-
3-hydroxybutyrate production (R) -3-hydroxybutyrate needs to expend substantial amounts of organic solvent, and the reaction time is longer, and needs high-purity
The poly 3-hydroxy butyrate of degree easily produces racemization as starting material, but this method is at present also in laboratory level, such as
Fruit enters the problems such as industrialized production still has high cost, low yield.
At present, most of 3-hydroxybutyrate on the market is raceme, i.e. (R) -3-hydroxybutyrate and (S) -3- hydroxyls
The equal amount of mixture of base butyric acid.Although research shows that (S) -3-hydroxybutyrate does not have physiologically active, the 3-hydroxybutyrate of racemization
And its salt especially sodium salt is still at present main commercial form and received by consumers in general.It is contemplated that single optics
(the R) -3-hydroxybutyrate and its salt of activity should can be increasingly becoming consumption market main flow from now on, replace without optically active 3- hydroxyls
Base butyric acid and its salt.
It has been recognized that the compound of natural or biological source is safer, come for medicine, food, cosmetic composition
Increasingly pursue " naturalization " in source.For marketing purpose, medicine, food, Cosmetic Manufacture commercial city are more gladly using biology
The product in source substitutes the same substance for being chemically synthesized out.(R) -3-hydroxybutyrate is used as widely used naturalization
Compound, finds a kind of biological method and substitutes chemical method to produce as a kind of study hotspot.
The content of the invention
(the R) -3-hydroxybutyrate of " safety non-toxic " is produced to realize biological method, inventor is for the step of microorganism one
Fermentation method is explored, and is selected the microorganism of no pathogenicity as host, is transformed using technique for gene engineering, by increasing
The expression of the gene related to the biosynthesis of (R) -3-hydroxybutyrate by force, weakens branched metabolic pathway, filters out high yield (R) -3-
The production bacterial strain of hydroxybutyric acid.This several (R) -3-hydroxybutyrate production bacterium can effectively accumulate (R) -3- hydroxyls during the fermentation
Base butyric acid, with wide industrial applications prospect.
Therefore, first purpose of the invention is to provide a kind of method of production (R) -3-hydroxybutyrate.
Second object of the present invention is (the R) -3-hydroxybutyrate and its salt for providing a kind of food-grade.
Third object of the present invention is the raceme 3-hydroxybutyrate and its salt for providing food-grade, to meet market
Need.
Fourth object of the present invention is to provide a kind of (R) -3-hydroxybutyrate production bacterium.
The present invention includes following technical scheme：
The method of one kind production (R) -3-hydroxybutyrate, using the microorganism of no pathogenicity, by fermentation directly by carbon source
(the R) -3-hydroxybutyrate produced in (R) -3-hydroxybutyrate, the microorganism is converted into nitrogen source to be secreted into zymotic fluid, from
Extraction purification (R) -3-hydroxybutyrate in zymotic fluid,
The microorganism is selected from Corynebacterium glutamicum, bacillus subtilis, brevibacterium lactofermentum, scattered branch brevibacterium, Huang
Color brevibacterium and brevibacterium ammoniagene, and the microorganism has following bioconversion functions of carrying out successively：Make carbon source with it is auxiliary
Enzyme A is converted into acetyl coenzyme A；Acetyl coenzyme A is converted into acetoacetyl-CoA；Acetoacetyl-CoA is converted into (R) -3-
Hydroxybutyryl A；(R) -3- hydroxybutyryls A is converted into (R) -3-hydroxybutyrate.
Preferably, the microorganism is overexpressed the enzyme that more than one are selected from the group：Succinyl-coenzyme A transferase
(Succinyl-CoA transferase), acetoacetyl-CoA synzyme (acetoacetyl-CoA synthetase), second
Acyl acetyl coenzyme A reductase (acetoacetyl-CoA reductase, thioesterase (thioesterase).
More preferably described microorganism is overexpressed acetoacetyl-CoA synzyme, Acetoacetyl-CoA reductase.
Preferably, the expression of the Antimicrobial or downward beta-Ketothiolase (β-ketothiolase).
In a preferred embodiment, the microorganism is Corynebacterium glutamicum, is preserved in Chinese microorganism strain preservation
Administration committee's common micro-organisms center, deposit number is CGMCC No.13957.
In fermentation process, for different microorganisms, different culture mediums can be used, wherein the carbon source can be selected from Portugal
Grape sugar, sucrose, maltose, molasses, starch and glycerine.Nitrogen source in fermentation medium can be selected from organic nitrogen source and inorganic nitrogen
Source, the organic nitrogen source is selected from corn steep liquor, wheat bran hydrolyzate, soya-bean cake hydrolyzate, yeast extract, dusty yeast, peptone and urea；
It is described inorganic nitrogen-sourced selected from ammonium sulfate, ammonium nitrate or ammoniacal liquor.
According to a preferred embodiment of the invention, when microorganism uses Corynebacterium glutamicum, fermentation medium composition is as follows：
Glucose 75g/L, corn steep liquor 25-30g/L, (NH4)2SO4 20g/L、KH2PO4 1.5g/L、MgSO4·7H2O0.5g/L, urea
1.0g/L, histidine 30mg/L, molasses 25g/L, biotin 100 μ g/L, defoamer 0.2g/L.
Preferably, when carrying out feed supplement in fermentation process, that is, when taking Fed batch fementation mode, used feed-batch culture
Base composition is as follows：Ammonium sulfate 500g/L, glucose 650g/L.
(R) -3-hydroxybutyrate purity prepared by the inventive method reaches more than 95%, preferably more than 96%, more than 97%,
More than 98%, more preferably more than 99%.
(the R) -3-hydroxybutyrate prepared by the above method is free of bacterial endotoxin, and without chemical peculiar smell such as bitter taste
And dissolvent residual.
(R) -3-hydroxybutyrate is a kind of acid, can be with alkali forming salt.Alternatively, (R) -3-hydroxybutyrate of the invention is in
The form of salt, such as sodium salt, sylvite, magnesium salts, calcium salt；Particular certain cancers.These salt also can be optically active compound.
Traditionally by food and pharmaceutical production manufacturer and disappear in view of raceme 3-hydroxybutyrate and its sodium salt
Expense person is received, (R) -3-hydroxybutyrate and its salt can be prepared through racemization processing racemic 3-hydroxybutyrate and its
Salt.For example, by the way that (R) -3-hydroxybutyrate to be heated to simultaneously held for some time in alkaline solution such as sodium hydroxide solution, can
To realize racemization.
Raceme 3-hydroxybutyrate salt can be 3-hydroxybutyrate sodium, 3-hydroxybutyrate potassium, 3-hydroxybutyrate magnesium, 3- hydroxyls
Calcium butyrate or above-mentioned salt-mixture.
(the R) -3-hydroxybutyrate and its salt prepared by the method for the present invention, without bacterial endotoxin, also not comprising having
The chemical substance of toxic, therefore its foodsafety can be ensured.Further, since without chemical residue or chemically reacting miscellaneous
Matter, (R) -3-hydroxybutyrate of the invention is free of chemical peculiar smell such as bitter taste, is used directly for making medicine and health products.
A kind of Corynebacterium glutamicum, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, protects
It is CGMCC No.13957 to hide numbering.
(R) -3-hydroxybutyrate production bacterium constructed by the present invention can realize in zymotic fluid (R) -3- during the fermentation
Effective accumulation of hydroxybutyric acid, obtains (R) -3-hydroxybutyrate of food-grade, thus with wide prospects for commercial application.
The Classification system for (R) -3-hydroxybutyrate high-yield genetic engineering bacterium that the present invention is built is Corynebacterium
Glutamicum, Chinese is Corynebacterium glutamicum or corynebacterium glutamicum, has been preserved in Chinese microorganism strain preservation
Administration committee's common micro-organisms center, preservation date is on March 30th, 2017, and preservation address is Chaoyang District, Beijing City North Star west
No. 3 Institute of Microorganism, Academia Sinica of institute of road 1, deposit number is CGMCC No.13957.
Embodiment
The present invention is described in further details below in conjunction with specific embodiment.It should be understood that following examples are only used for
The bright present invention is not for restriction the scope of the present invention.
Addition, content and the concentration of many kinds of substance is referred to herein, wherein described percentage composition, except special instruction
Outside, weight/mass percentage composition is all referred to.
In embodiments of the invention, illustrated if do not made for operation temperature, the temperature is often referred to room temperature
(15-30℃)。
Herein, term " Corynebacterium glutamicum ", " corynebacterium glutamicum ", " bacterial strain CGMCC No.13957 " expressions
Identical meaning, the bacterial strain for fermenting and producing (R) -3-hydroxybutyrate built all referring to inventor.
In order to meet consumer and medicine and food production business for (R) -3-hydroxybutyrate and its salt, 3-hydroxybutyrate
And its pursuit in salt " naturalization " or " bioid " source, inventor have studied with microbial fermentation to produce (R) -3- hydroxyl fourths
The method of acid.Inventor is screened for the microorganism kind for building engineering strain, although abandoned it is conventional,
But there are potential pathogenic microorganism such as colibacillus, select the Corynebacterium glutamicum, bacillus subtilis, breast of no pathogenicity
Sugar fermentation brevibacterium, scattered branch brevibacterium, brevibacterium flavum and brevibacterium ammoniagene etc. construct genetic engineering bacterium, led to as host
Screening is crossed, obtains that the several bacterial strains of (R) -3-hydroxybutyrate can be produced by one-step fermentation.These bacterial strains are during the fermentation not
The endotoxin that may be caused harm for majority of populations can be produced, meets the design philosophy of " nontoxic ".
During the fermentation, can be to bars such as dissolved oxygen, temperature, pH in order to obtain higher (R) -3-hydroxybutyrate yield
Part carries out necessary regulation and control.
It is preferred that when being fermented, constant dO2Control is 15%~25%.For example fermentation can be carried out under the following conditions：
Air mass flow is about 1vvm, wherein vvm (ventilation ratio) be throughput per minute with the ratio of the actual material liquid volume of tank body (for example,
For a fermentation tank containing 30 liters of zymotic fluids, 1vvm is equal to 30L/min；And for a fermentation containing 5 liters of zymotic fluids
Tank, 1vvm is equal to 5L/min).
It is preferred that when being fermented, temperature control is at 30~32 DEG C；After fermentation the phase, temperature can be brought up to 34~37 DEG C,
In order to (R) -3-hydroxybutyrate synthesis and be discharged in zymotic fluid.
It is preferred that when being fermented, pH is controlled in pH6.0-8.0, preferably pH6.5~7.0；Phase after fermentation, can be by pH controls
6.8~7.0, in order to (R) -3-hydroxybutyrate synthesis and be discharged in zymotic fluid.
Above-mentioned term " fermentation later stage " refers to the microorganism growth platform phase to decline phase.Such as use OD600nmValue monitoring is micro-
During biological concentration, OD600nmValue no longer rises and tends to decline.
Whole fermentation process residual sugar control is more preferably controlled 1.5%~2.5% 1%~3.0% or so.
, it is necessary to be reclaimed to zymotic fluid after fermentation ends, (R) -3-hydroxybutyrate is therefrom extracted.For example, passing through centrifugation
Method is removed the supernatant of thalline, if necessary concentrated supernatant；By the post processing means such as drying, purifying, isolate
(R) -3-hydroxybutyrate.Or thalline and macromolecular substances are removed by filtering (including ultrafiltration, nanofiltration etc.) method, filtered
Liquid, concentrates filtrate if necessary；Again by the post processing means such as drying, purifying, (R) -3-hydroxybutyrate is isolated.Or by from
Heart method is removed the supernatant of thalline, then removes macromolecular substances by methods such as ultrafiltration or nanofiltrations, obtains filtrate, necessary
When concentrate filtrate；Again by the post processing means such as drying, purifying, (R) -3-hydroxybutyrate is isolated.
When preparing (R) -3-hydroxybutyrate salt such as sodium salt, sylvite, magnesium salts, calcium salt, (R) -3- hydroxyls of equivalent are answered
The reaction temperature of butyric acid and corresponding alkali or metal oxide such as sodium hydroxide is controlled below 30 DEG C, preferably less than 25 DEG C,
It is more preferably following less than 20 DEG C, racemization is avoided as far as possible.
Because, without using organic solvent, resulting (R) -3-hydroxybutyrate and its product salt are without chemistry in preparation process
Peculiar smell such as bitter taste, can be directly used for the manufacture of medicine, health products.
Embodiment
Embodiment 1：Preculture and fermentation
Bacterial strain CGMCC No.13957 preservative fluids in glycerol tube are thawed, seed culture medium (glucose is inoculated in
75g/L, corn steep liquor 25-30g/L, (NH4)2SO4 20g/L、KH2PO4 1.5g/L、MgSO4·7H2O 0.5g/L, urea 1.0g/
L, histidine 30mg/L, molasses 25g/L, biotin 100 μ g/L, pH 7.0) in, 5000mL triangular flask liquid amounts 500mL, 30 DEG C
Culture 18 hours, absorbance Δ OD=0.4-0.5 shaking tables at present, obtains seed culture fluid.After testing, no miscellaneous bacteria.
500mL seed culture fluids are inoculated in 7 liters of fermentation tanks, 5 liters of liquid amount.Medium component and seed culture medium phase
Together, pH=6.4~6.7 (after sterilizing).Supplemented medium：Ammonium sulfate 500g/L, glucose 650g/L.30 DEG C of cultivation temperature, tank
Press 0.05Mpa, initial ventilation ratio 1vvm.Stir 600rpm.PH controls are in pH6.5 or so during fermentation；After fermentation the phase, pH is controlled
Temperature is brought up to 35 DEG C or so by system 6.7 or so.The regulation of ventilation and speed of agitator is flexibly controlled according to dissolved oxygen,
Dissolved oxygen constant dO2Control is 15~25%.Residual sugar is controlled：When sugar is down to 3.0% or so originally, start flow feeding, whole hair
Ferment process residual sugar is controlled 1.5%~2.0% or so.Fermentation total time is 72 hours, product (R) -3-hydroxybutyrate concentration
11.5g/L。
Embodiment 2：The separation of zymotic fluid and the extraction purification of (R) -3-hydroxybutyrate
5.2 liters of zymotic fluids of gained in embodiment 1 are centrifuged under 4500rpm, thalline is discarded.Add 1% diatom in supernatant
Filtered after soil.Then 1% activated carbon is added, stirring is filtered after decolourizing 30 minutes, obtains limpid filtrate.
Nanofiltration processing is carried out to filtrate with NF membrane, gained filtrate crosses 732 cationic ion-exchange resins, and collection penetrates liquid.It is dense
1000g/L is reduced to, in grease, is poured out while hot, 55.9g (R) -3-hydroxybutyrate, yield 91% is obtained.Using high-efficient liquid phase color
Spectrum determines (R) -3-hydroxybutyrate purity.Chromatographic column is Shim-pack Vp-ODSC18 posts (150L × 4.6), and mobile phase is second
Nitrile：Water (v/v)=15：85, ultraviolet detection wavelength is 210nm, sample size 20 μ L, flow velocity 1mL/min, 10 DEG C of column temperature.After testing,
(R) purity of -3-hydroxybutyrate is 99.2%, specific rotatory power：[α]D 20=-25.1 ° of (C=6%, H2O)。
Embodiment 3：(R) preparation of -3-hydroxybutyrate sodium
(the R) -3-hydroxybutyrate obtained in 5g embodiments 2 is slowly added into the 2N sodium hydroxide solutions of equivalent
Row neutralization reaction, reaction temperature is controlled below 25 DEG C.Concentrated by rotary evaporation stands 2 hours, collected by suction is solid to there is solid precipitation
Body, 60 DEG C of forced air dryings obtain 4.8 grams of (R) -3-hydroxybutyrate sodium powder ends, yield 79%.Fusing point is 152 DEG C, specific rotatory power：
[α]D 20=-14.2 ° of (C=10%, H2O)。
Embodiment 4：(R) racemization of -3-hydroxybutyrate
10g (R) -3-hydroxybutyrate is slowly added into 100mL 2N sodium hydroxide solutions, 60 DEG C of reactions are heated to
4 hours.Room temperature is down to, it is 0 ° that polarimeter, which determines optical activity,.Be neutralized with hydrochloric acid to pH 7.0, concentrated by rotary evaporation to there is solid precipitation,
2 hours are stood, collected by suction solid obtains 3-hydroxybutyrate sodium powder end 10.8g, yield 89%, and polarimeter determines optical activity and is
0 ° of (C=10%, H2O).As a result show to obtain is racemization 3-hydroxybutyrate sodium.
In summary, (R) -3-hydroxybutyrate genetic engineering bacterium constructed by the present invention can realize hair during the fermentation
Effective accumulation of (R) -3-hydroxybutyrate in zymotic fluid, produces food-grade (R) -3-hydroxybutyrate of safety non-toxic, with wide
Prospects for commercial application.
Claims (10)
1. the method for one kind production (R) -3-hydroxybutyrate, using the microorganism of no pathogenicity, by fermentation directly by carbon source and
Nitrogen source is converted into (the R) -3-hydroxybutyrate produced in (R) -3-hydroxybutyrate, the microorganism and is secreted into zymotic fluid, from hair
Extraction purification (R) -3-hydroxybutyrate in zymotic fluid,
It is short that the microorganism is selected from Corynebacterium glutamicum, bacillus subtilis, brevibacterium lactofermentum, scattered branch brevibacterium, yellow
Bacillus and brevibacterium ammoniagene, and the microorganism has following bioconversion functions of carrying out successively：Carbon source is set to turn with coacetylase
Turn to acetyl coenzyme A；Acetyl coenzyme A is converted into acetoacetyl-CoA；Acetoacetyl-CoA is converted into (R) -3- hydroxyls
Butyryl coenzyme A；(R) -3- hydroxybutyryls A is converted into (R) -3-hydroxybutyrate.
2. the method as described in claim 1, it is characterised in that the microorganism is overexpressed the enzyme that more than one are selected from the group：
Succinyl-coenzyme A transferase, acetoacetyl-CoA synzyme, Acetoacetyl-CoA reductase, thioesterase.
3. the method as described in claim 1, it is characterised in that the microorganism is overexpressed acetoacetyl-CoA synzyme, second
Acyl acetyl coenzyme A reductase.
4. method as claimed in claim 3, it is characterised in that the microorganism is Corynebacterium glutamicum, is preserved in China micro-
Biological inoculum preservation administration committee common micro-organisms center, deposit number is CGMCC No.13957.
5. the method as described in claim 1, it is characterised in that the carbon source is selected from glucose, sucrose, maltose, molasses, shallow lake
Powder and glycerine；The nitrogen source is selected from organic nitrogen source and inorganic nitrogen-sourced, and the organic nitrogen source is selected from corn steep liquor, wheat bran hydrolyzate, beans
Cake hydrolyzate, yeast extract, dusty yeast, peptone and urea；It is described inorganic nitrogen-sourced selected from ammonium sulfate, ammonium nitrate or ammoniacal liquor.
6. (the R) -3-hydroxybutyrate prepared by method any one of claim 1-5, it is free of bacterial endotoxin, pure
Degree reaches more than 95%.
7. (R) -3-hydroxybutyrate as claimed in claim 6, it is characterised in that it is rendered as the shape of (R) -3-hydroxybutyrate salt
Formula, the salt is selected from the group：Sodium salt, sylvite, magnesium salts, calcium salt.
8. raceme 3-hydroxybutyrate, it is by by (R) -3-hydroxybutyrate described in claim 6 or claim 7 institute
(the R) -3-hydroxybutyrate salt stated is handled through racemization and prepared.
9. 3-hydroxybutyrate as claimed in claim 8, it is characterised in that it is rendered as the form of 3-hydroxybutyrate salt, described
Salt is selected from the group：Sodium salt, sylvite, magnesium salts, calcium salt.
10. a kind of Corynebacterium glutamicum, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation
Numbering is CGMCC No.13957.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710396141.3A CN107083406B (en) | 2017-05-27 | 2017-05-27 | Method for producing (R) -3-hydroxybutyric acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710396141.3A CN107083406B (en) | 2017-05-27 | 2017-05-27 | Method for producing (R) -3-hydroxybutyric acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107083406A true CN107083406A (en) | 2017-08-22 |
| CN107083406B CN107083406B (en) | 2021-02-02 |
Family
ID=59608670
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710396141.3A Active CN107083406B (en) | 2017-05-27 | 2017-05-27 | Method for producing (R) -3-hydroxybutyric acid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107083406B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018187324A1 (en) * | 2017-04-04 | 2018-10-11 | NNB Nutrition USA, LLC | Preparation of (r)-3-hydroxybutyric acid or its salts by one-step fermentation |
| CN110862316A (en) * | 2018-08-27 | 2020-03-06 | 浙江华睿生物技术有限公司 | Crystal form of (R) -3-hydroxybutyric acid and application thereof |
| CN110904161A (en) * | 2019-12-27 | 2020-03-24 | 浙江英玛特生物科技有限公司 | Method for producing high-purity (R) - (-) -3-hydroxybutyric acid by adopting enzyme method |
| CN111304140A (en) * | 2020-03-05 | 2020-06-19 | 清华大学 | Recombinant intestinal bacterium for producing (R) -3-hydroxybutyric acid and construction method thereof |
| CN111534561A (en) * | 2020-05-19 | 2020-08-14 | 南京纽邦生物科技有限公司 | Preparation method of (R) -3-hydroxybutyric acid |
| CN111944853A (en) * | 2019-05-17 | 2020-11-17 | 南京纽邦生物科技有限公司 | Method for producing (R) -3-hydroxybutyric acid by microbial fermentation |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1429912A (en) * | 2002-01-04 | 2003-07-16 | 清华大学 | Method of producing D-(-)-3-hydroxy butanoic acid |
| CN101969769A (en) * | 2008-01-04 | 2011-02-09 | 伊希斯创新有限公司 | Ketone bodies and ketone body esters as blood lipid lowering agents |
| CN103890187A (en) * | 2011-10-18 | 2014-06-25 | 昭和电工株式会社 | Production method for organic acid using coa-transferase |
| CN105050594A (en) * | 2013-03-19 | 2015-11-11 | 南佛罗里达大学 | Compositions and methods for producing elevated and sustained ketosis |
| CN105950517A (en) * | 2016-06-30 | 2016-09-21 | 江南大学 | Corynebacterium glutamicum and application thereof |
| CN106148440A (en) * | 2016-08-10 | 2016-11-23 | 洛阳华荣生物技术有限公司 | Fermentative Production gamatine |
| WO2017016902A1 (en) * | 2015-07-29 | 2017-02-02 | Evonik Degussa Gmbh | Production of 3-hydroxybutyrate |
| WO2017066498A1 (en) * | 2015-10-13 | 2017-04-20 | Lanzatech New Zealand Limited | Genetically engineered bacterium comprising energy-generating fermentation pathway |
-
2017
- 2017-05-27 CN CN201710396141.3A patent/CN107083406B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1429912A (en) * | 2002-01-04 | 2003-07-16 | 清华大学 | Method of producing D-(-)-3-hydroxy butanoic acid |
| CN101969769A (en) * | 2008-01-04 | 2011-02-09 | 伊希斯创新有限公司 | Ketone bodies and ketone body esters as blood lipid lowering agents |
| CN103890187A (en) * | 2011-10-18 | 2014-06-25 | 昭和电工株式会社 | Production method for organic acid using coa-transferase |
| CN105050594A (en) * | 2013-03-19 | 2015-11-11 | 南佛罗里达大学 | Compositions and methods for producing elevated and sustained ketosis |
| WO2017016902A1 (en) * | 2015-07-29 | 2017-02-02 | Evonik Degussa Gmbh | Production of 3-hydroxybutyrate |
| WO2017066498A1 (en) * | 2015-10-13 | 2017-04-20 | Lanzatech New Zealand Limited | Genetically engineered bacterium comprising energy-generating fermentation pathway |
| CN105950517A (en) * | 2016-06-30 | 2016-09-21 | 江南大学 | Corynebacterium glutamicum and application thereof |
| CN106148440A (en) * | 2016-08-10 | 2016-11-23 | 洛阳华荣生物技术有限公司 | Fermentative Production gamatine |
Non-Patent Citations (8)
| Title |
|---|
| KEN’ICHIRO MATSUMOTO: "Efficient (R)-3-hydroxybutyrate production using acetyl CoA-regenerating pathway catalyzed by coenzyme A transferase", 《APPL MICROBIOL BIOTECHNOL》 * |
| QIAN LIU等: "Microbial production of R-3-hydroxybutyric acid by recombinant E. coli harboring genes of phbA，phbB，and tesB", 《APPL MICROBIOL BIOTECHNOL》 * |
| 孙勇民等: "《微生物技术及应用（第二版）》", 31 January 2012, 华中科技大学出版社 * |
| 张宽厚著: "《细菌生理学》", 31 July 1964, 人民卫生出版社 * |
| 李季伦等: "《微生物生理学》", 31 December 1993, 北京农业大学出版社 * |
| 王德山等著: "《全国中医院校硕士研究生入学考试辅导丛书 生理学 生物化学》", 31 October 2001, 上海中医药大学出版社 * |
| 路福平: "《微生物学》", 31 July 2005, 中国轻工业出版社 * |
| 韩北忠: "《发酵工程》", 31 January 2013, 中国轻工业出版社 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018187324A1 (en) * | 2017-04-04 | 2018-10-11 | NNB Nutrition USA, LLC | Preparation of (r)-3-hydroxybutyric acid or its salts by one-step fermentation |
| US10428357B2 (en) | 2017-04-04 | 2019-10-01 | NNB Nutrition USA, LLC | Preparation of (R)-3-hydroxybutyric acid or its salts by one-step fermentation |
| US11198890B2 (en) | 2017-04-04 | 2021-12-14 | NNB Nutrition USA, LLC | Preparation of (R)-3-hydroxybutyric acid or its salts by one-step fermentation |
| CN110862316A (en) * | 2018-08-27 | 2020-03-06 | 浙江华睿生物技术有限公司 | Crystal form of (R) -3-hydroxybutyric acid and application thereof |
| CN111944853A (en) * | 2019-05-17 | 2020-11-17 | 南京纽邦生物科技有限公司 | Method for producing (R) -3-hydroxybutyric acid by microbial fermentation |
| CN110904161A (en) * | 2019-12-27 | 2020-03-24 | 浙江英玛特生物科技有限公司 | Method for producing high-purity (R) - (-) -3-hydroxybutyric acid by adopting enzyme method |
| CN111304140A (en) * | 2020-03-05 | 2020-06-19 | 清华大学 | Recombinant intestinal bacterium for producing (R) -3-hydroxybutyric acid and construction method thereof |
| CN111534561A (en) * | 2020-05-19 | 2020-08-14 | 南京纽邦生物科技有限公司 | Preparation method of (R) -3-hydroxybutyric acid |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107083406B (en) | 2021-02-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11198890B2 (en) | Preparation of (R)-3-hydroxybutyric acid or its salts by one-step fermentation | |
| CN107083406A (en) | (R) production method of 3 hydroxybutyric acids | |
| CA2741621C (en) | Process for preparing nutritional, therapeutic or organoleptic products from crude glycerol | |
| CN112481336B (en) | Method for biosynthesis of compounds using lignocellulose derivatives | |
| TWI558722B (en) | Use of extraction residue of yeast extract | |
| CN107418995B (en) | A kind of ellagic acid and preparation method thereof of granatanine liquid state fermentation preparation | |
| CN102250981B (en) | Method for preparing ellagic acid by solid fermentation with granatum as raw material | |
| CN104804183B (en) | Method for purifying and separating gamma-polyglutamic acid from fermentation liquor | |
| WO2017025339A1 (en) | Improved production of vanillin by fermentation | |
| CN106148440B (en) | Production of agmatine by fermentation method | |
| CN107201384A (en) | A kind of method of separation and Extraction D-ALPHA-Hydroxypropionic acid in sodium zymotic fluid from D-ALPHA-Hydroxypropionic acid | |
| EP2850213A1 (en) | Strain producing turanose and uses thereof | |
| CN104694614B (en) | A kind of extraction process of L-Trp | |
| TWI502070B (en) | Production method of γ-butyric acid | |
| US20110201054A1 (en) | Process for improved recovery of fermentation products from intracellular and extracellular presence | |
| CN109234328B (en) | Method for producing gamma-polyglutamic acid | |
| CN101857886A (en) | Method for preparing xylitol and co-producing L-arabinose | |
| CN109182407A (en) | A kind of tryptophan preparation method and its fermentation medium and tryptophan that use fermentation special nutritional member | |
| CN109206310A (en) | A method of extracting D-ALPHA-Hydroxypropionic acid from D-ALPHA-Hydroxypropionic acid calcium fermentation liquid | |
| CN107400686A (en) | Ellagic acid prepared by a kind of solid state fermentation granatum and preparation method thereof | |
| CN109206312A (en) | A method of D-ALPHA-Hydroxypropionic acid is isolated and purified from D-ALPHA-Hydroxypropionic acid ammonium fermentation liquid | |
| CN105018540A (en) | Method for producing GABA (Gamma-Amino-Butyric Acid) by liquid state and solid state fermentation of acetic acid bacteria | |
| KR0180986B1 (en) | Preparation process of xylitol by microbial fermentation from hydrolysate | |
| CN115927496A (en) | Method for producing gamma-aminobutyric acid by using saccharomyces cerevisiae | |
| CN110684810A (en) | Method for preparing powdery gamma-aminobutyric acid from fermentation liquor rich in gamma-aminobutyric acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20180102 Address after: 313000 Huzhou, Huzhou City, Zhejiang, No. 6, Hongfeng Road, No. 1366, floor 502, west side Applicant after: Zhejiang Huari Biotechnology Co., Ltd. Address before: 313000 Hongfeng Road, Huzhou, Huzhou, Zhejiang, 3, 2 floors and 205 rooms Applicant before: Huzhou Heng Rui Nutrition Health Technology Co., Ltd. |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |