CN106349056A - Separation and purification method of alpha-ketoglutaric acid - Google Patents
Separation and purification method of alpha-ketoglutaric acid Download PDFInfo
- Publication number
- CN106349056A CN106349056A CN201610732998.3A CN201610732998A CN106349056A CN 106349056 A CN106349056 A CN 106349056A CN 201610732998 A CN201610732998 A CN 201610732998A CN 106349056 A CN106349056 A CN 106349056A
- Authority
- CN
- China
- Prior art keywords
- ketoglutaric acid
- fermentation
- liquid
- solution
- concentration
- 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
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
- C07C51/43—Separation; Purification; Stabilisation; Use of additives by change of the physical state, e.g. crystallisation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
- C07C51/47—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
-
- 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
- C12P7/44—Polycarboxylic acids
- C12P7/50—Polycarboxylic acids having keto groups, e.g. 2-ketoglutaric acid
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/10—Process efficiency
Abstract
The invention relates to a separation and purification method of alpha-ketoglutaric acid. The method is implemented by converting L-glutamic acid by L-dglutamic oxidase to prepare alpha-ketoglutaric acid and separating and purifying the alpha-ketoglutaric-acid-containing conversion solution. The method comprises the following steps: ion exchange, nanofiltration, activated carbon decolorization, reverse osmosis concentration, concentration crystallization and drying. The membrane separation, nanofiltration decolorization, membrane concentration and other advanced treatment techniques are utilized, thereby saving the energy consumption, saving the activated carbon consumption, reducing the environmental pollution, lowering the production cost and enhancing the product quality.
Description
Technical field
The present invention relates to a kind of isolation and purification method of a-ketoglutaric acid is and in particular to a kind of l- glutamic acid that is directed to is through l-
When a-ketoglutaric acid is prepared in the conversion of dglutamic oxidase, isolation and purification method to a-ketoglutaric acid, belong to microbial technique
Field.
Background technology
L- dglutamic oxidase (ec 1.4.3.11, l-glutamate oxidase, glod) is with flavin adenine two
Nucleotide (fad) is a kind of l- amino acid oxidase of coenzyme, is a kind of new enzyme starting the beginning of the eighties to find, earliest from Serpentiss
Isolate and purify in the organisms such as venom, the kidney of Mus, invertebratess and microorganism and obtain.L- dglutamic oxidase can not add
Plus aoxidize l- glutamic acid deamination under conditions of exogenous cofactor, generate α-ketoglutaric acid and hydrogen peroxide.Glod is to catalysis
Reaction substrate has a stereoisomerism selectivity of height, high catalytic efficiency, and reaction condition is gentle, have been widely used for food, light industry,
The every field such as chemical industry, medicine, environmental protection, the energy and scientific research.This enzyme from 20th century since the 80's were found, always work
One of focus of tool enzyme research.The main research of enzyme concentrates at this stage, and one is by modern observation and mensuration means research
The structure of enzyme and mechanism of action, two is how to serve people using enzyme using the technique study of enzyme viability and engineering
The production of class, life.
In recent years, domestic and international scientist is successively separated to streptomycete, the actinomycetes that can produce glod respectively from soil,
And establish more perfect liquid fermentation producing enzyme system, and it is prepared for glod crude product, by studying to its zymologic property, pure
Change this enzyme obtaining and all have characteristics that can be catalyzed l- glutamic acid in specific manner generates hydrogen peroxide, ammonia and a- ketone penta 2
Acid.At present, glod fermenting and producing, away from industrialized production also very big distance, which greatly limits its application.Therefore
Need to study its fermentation technology further, by process optimization, improve fermentation medium components and fermentation condition, create and be suitable for bacterium
Bulk-growth and the optimum condition of biological metabolism, give full play to the productive potentialities of strain, significantly improve fermentation yield.
L- dglutamic oxidase one side may apply to measure l- glutamic acid and its coupling reaction；On the other hand then may be used
For the preparation of a-ketoglutaric acid, and then prepare l- glutamic acid-a-ketoglutaric acid.Domestic also do not have producer so far to it
Produced, abroad also only have a production to be reconstituted in colibacillary l- dglutamic oxidase.The costliness of price, source
Single, seriously govern application and the development of l- dglutamic oxidase.
Content of the invention
It is an object of the invention to provide a kind of isolation and purification method of a-ketoglutaric acid is and in particular to a kind of be directed to l- paddy
Propylhomoserin prepares a-ketoglutaric acid through the conversion of l- dglutamic oxidase, to the side of isolating and purifying containing a-ketoglutaric acid conversional solution
Method.The present invention saves energy consumption using advanced handling process such as membrance separation, nanofiltration decolouring, membrance concentration, saves the use of activated carbon
Amount, decreases the pollution to environment, reduces production cost, improve product quality.Technical scheme is as follows:
A kind of isolation and purification method of a-ketoglutaric acid, specifically comprises the following steps that
Ion exchange: conversional solution is limpid to effluent with water backwash resin after the absorption of d301 macroreticular weakly base negative resin, use
0.25n hydrochloric acid solution eluting, eluting yield reaches 95%；
Nanofiltration: a-ketoglutaric acid nanofiltration is obtained using 600-800 molecular weight nanofiltration membrane a-ketoglutaric acid eluent liquid
Clear liquid, after 3 cleaning concentrates of deionization moisture of 3 times of volume of concentrate, discards concentrated solution, collects containing citrulline product
Nanofiltration dislysate, this step yield reaches 97%；
Activated carbon decolorizing: ph 3.0, is decoloured with activated carbon, and the consumption of activated carbon is the 1% of a-ketoglutaric acid nanofiltration clear liquid
(mass fraction), bleaching temperature is 50 DEG C, and bleaching time is 30min, obtains destaining solution, destaining solution printing opacity with 0.22 μm of membrane filtration
Rate >=99%, decolouring yield reaches 99%；
Reverse osmosis concentration: a-ketoglutaric acid destaining solution is concentrated into original volume half through reverse osmosis membrane, and to obtain a-ketoglutaric acid pre- dense
Contracting liquid, reverse osmosiss yield reaches 99%；
Condensing crystallizing: pre-concentration liquid is concentrated into the dense of content >=80% under the conditions of vacuum >=0.095,50 DEG C of evaporating temperature
Contracting liquid, concentrated solution stirring is cooled to 20 DEG C of crystallization 5h.
It is dried: after terminating, sucking filtration obtains crystal, 50 DEG C of vacuum drying of crystallographic obtain a-ketoglutaric acid
Product.
The present invention produces a-ketoglutaric acid by microbial enzyme method, and specially l- glutamic acid is through l- dglutamic oxidase
A-ketoglutaric acid is prepared in conversion.The present invention adopts a step enzymatic reaction, adds catalase, it is to avoid transformed in conversional solution
The inhibitory action to l- dglutamic oxidase for the journey hydrogen peroxide, thus affecting the Synthesis of a-ketoglutaric acid, makes a- ketone penta 2
Acid can run up to higher concentration.
Specifically comprise the following steps that
(1) preparation of crude enzyme liquid: the fermentation liquid of l- dglutamic oxidase is first passed through ceramic membrane filter, removes thalline, supernatant
Concentrate 50 times of crude enzyme liquids being the l- dglutamic oxidase that enzymatic conversion uses through reverse osmosis membrane again.
(2) conversion culture: ph be 8.5 phosphate buffer in, add the crude enzyme liquid of l- dglutamic oxidase, h2o2Enzyme
And mncl2And substrate l- sodium glutamate, 37 DEG C, 200r/min converts 24 h, obtains final product the conversional solution containing a-ketoglutaric acid；Phosphorus
The final concentration of 50mmol of acid buffer, the final concentration of 15u/ml, h of l- dglutamic oxidase2o2The final concentration of 20u/ of enzyme
ml、mncl2Final concentration of 5mmol, the final concentration of 10%(mass fraction of l- sodium glutamate).
The method preparing l- dglutamic oxidase using bacterial strain mqo-160, step is as follows:
(1) slant culture: bacterial strain mqo-160 is aseptically inoculated on slant medium and carries out being inverted culture, training
Foster temperature is 28 DEG C, and incubation time is 48h；
Slant medium: soluble starch 20g, kno31g, k2hpo40.5g, mgso4·7h2O 0.5g, nacl 0.5g,
feso4·7h2O 0.01g, agar 20g, plus pure water to 1l, adjusts ph to 7.4-7.6, sterilising temp 115 with naoh
DEG C, 15min.
(2) seed amplification culture: picking colony from the slant medium of step (1), dilute obtains phage solution,
Phage solution is inoculated on seed culture medium and carries out amplification culture, inoculum concentration is 10%(v/v), the temperature of amplification culture is
28 DEG C, shaking speed 120r/min, incubation time is 12h；
Seed culture medium: sucrose 30g, yeast extract 6g, (nh4)2so46g、caco330g、cacl23g、mgcl2·6h2o
1g, kcl 1g, plus pure water to 1l, adjusts ph to 7.0,115 DEG C of sterilising temp, 15min with naoh.
(3) fermentation culture: the thalline after will be enlarged by cultivating is inoculated in fermentation broth, inoculum concentration is 10%(v/
V), 30 DEG C of amplification culture temperature, shaking speed 150r/min, fermentation period 36h.
Fermentation medium: glucose 30g, Dried Corn Steep Liquor Powder 20g, sucrose 30g, yeast extract 1g, (nh4)2so410g, paddy
Propylhomoserin sodium 10g, mgcl20.5g, kcl 0.5g, nah2po40.5g, is made into 1l solution with tap water, adjusts ph with naoh
To 7.0,121 DEG C of sterilising temp, sterilization time 20min.
(4) 30l fermentor cultivation: the fermentation liquid after fermentation culture in step (3) is inoculated into 30l
In the fermentation medium of fermentation tank, inoculum concentration is 10%(v/v), control tank pressure to be 0.05mpa by force, train under the conditions of 28 DEG C
Support 48 hours, speed of agitator is 400rpm, air quantity is 10 l/min, obtains the fermentation containing l- dglutamic oxidase after fermentation
Liquid.
The present invention is through separating to streptomyces sp.206(soil) carry out ultraviolet mutagenesis and chemomorphosises, screening
To the higher bacterial strain of one plant of glutamic acid enzymatic activity, separated acquisition bacterial strain mqo-160 after purification, its deposit number is: cgmcc
no.12892；Depositary institution is: China Committee for Culture Collection of Microorganisms's common micro-organisms center；Preservation date is:
On August 22nd, 2016；Preservation address is: BeiChen West Road, Chaoyang District, BeiJing City 1 institute 3.
The biological nature of bacterial strain mqo-160 is: is milky with aerial hyphae in this bacterial strain base, mycelia has more
Barrier film, aerial hyphae has more dendron branch, and part mycelia is in vigorous trophophase, has bud shape structure, and this bacterial strain produces
Golden yellow or aubergine soluble pigment.This bacterial strain spore oval, spherical or cylindricality, smooth surface, fibrillae of spores is in the shape of a spiral.
Bacterial strain mqo-160 Classification And Nomenclature is streptomycete streptomyces sp..
The present invention compared with prior art, has the advantages that
(1) screen one plant of mqo-160, solve enzymatic translation technics enzyme source and the restriction of enzyme high cost；
(2) adopt l- glutamic acid fermentation refined solution as conversion raw material, overcome l- glucose oxidation enzyme transforming process and produce a- ketone
The bottleneck of 1,3-propanedicarboxylic acid, cheap be easy to get, process is simple, product purity is high；
(3) energy consumption is saved using advanced handling process such as membrance separation, nanofiltration decolouring, membrance concentration, saves the consumption of activated carbon,
Decrease the pollution to environment, reduce production cost, improve product quality.
(4) l- glucose oxidation enzyme transforming process of the present invention produces substrate conversion efficiency 87% in a-ketoglutaric acid, produces in conversional solution
Thing concentration is up to 105g/l, the disposable extract yield 50% of a-ketoglutaric acid.
Specific embodiment
To further describe the present invention with reference to specific embodiment, advantages of the present invention and feature will be with description and
Apparent.But embodiment is only exemplary, any restriction is not constituted to the scope of the present invention.Those skilled in the art should
It should be appreciated that, can be to repair to the details of technical solution of the present invention and form under without departing from the spirit and scope of the present invention
Change or replace, but these modifications and replacement each fall within protection scope of the present invention.
Bacterial strain mqo-160, its deposit number is: cgmcc no.12892；Depositary institution is: Chinese microorganism strain preservation
Administration committee's common micro-organisms center；Preservation date is: on August 22nd, 2016；Preservation address is: Chaoyang District, Beijing City north
Occasion West Road 1 institute 3.
The biological nature of embodiment 1 bacterial strain mqo-160
It is milky with aerial hyphae, mycelia has more barrier film, and aerial hyphae has more in the base of bacterial strain mqo-160
Dendron branch, part mycelia is in vigorous trophophase, has bud shape structure, and this bacterial strain produces golden yellow or aubergine solubility color
Element.This bacterial strain spore oval, spherical or cylindricality, smooth surface, fibrillae of spores is in the shape of a spiral.Bacterial strain mqo-160 Classification And Nomenclature is
Streptomycete streptomyces sp..
A kind of isolation and purification method of a-ketoglutaric acid of embodiment 2, specifically comprises the following steps that
Ion exchange: conversional solution is limpid to effluent with water backwash resin after the absorption of d301 macroreticular weakly base negative resin, use
0.25n hydrochloric acid l eluant solution, eluting yield reaches 95%；
Nanofiltration: a-ketoglutaric acid nanofiltration is obtained using 600-800 molecular weight nanofiltration membrane a-ketoglutaric acid eluent liquid
Clear liquid, after 3 cleaning concentrates of deionization moisture of 3 times of volume of concentrate, discards concentrated solution, collects containing citrulline product
Nanofiltration dislysate, this step yield reaches 97%；
Activated carbon decolorizing: ph 3.0, is decoloured with activated carbon, and the consumption of activated carbon is the 1% of a-ketoglutaric acid nanofiltration clear liquid
(mass fraction), bleaching temperature is 50 DEG C, and bleaching time is 30min, obtains destaining solution, destaining solution printing opacity with 0.22 μm of membrane filtration
Rate >=99%, decolouring yield reaches 99%；
Reverse osmosis concentration: a-ketoglutaric acid destaining solution is concentrated into original volume half through reverse osmosis membrane, and to obtain a-ketoglutaric acid pre- dense
Contracting liquid, reverse osmosiss yield reaches 99%；
Condensing crystallizing: pre-concentration liquid is concentrated into the dense of content >=80% under the conditions of vacuum >=0.095,50 DEG C of evaporating temperature
Contracting liquid, concentrated solution stirring is cooled to 20 DEG C of crystallization 5h.
It is dried: after terminating, sucking filtration obtains crystal, 50 DEG C of vacuum drying of crystallographic obtain a-ketoglutaric acid
Product.
The method that embodiment 3 prepares l- dglutamic oxidase using bacterial strain mqo-160, step is as follows:
(1) slant culture: bacterial strain mqo-160 is aseptically inoculated on slant medium and carries out being inverted culture, training
Foster temperature is 28 DEG C, and incubation time is 48h；
Slant medium: soluble starch 20g, kno31g, k2hpo40.5g, mgso4·7h2O 0.5g, nacl 0.5g,
feso4·7h2O 0.01g, agar 20g, plus pure water to 1l, adjusts ph to 7.4-7.6, sterilising temp 115 with naoh
DEG C, 15min.
(2) seed amplification culture: picking colony from the slant medium of step (1), dilute obtains phage solution,
Phage solution is inoculated on seed culture medium and carries out amplification culture, inoculum concentration is 10%(v/v), the temperature of amplification culture is
28 DEG C, shaking speed 120r/min, incubation time is 12h.
Seed culture medium: sucrose 30g, yeast extract 6g, (nh4)2so46g、caco330g、cacl23g、mgcl2·
6h2O 1g, kcl 1g, plus pure water to 1l, adjusts ph to 7.0,115 DEG C of sterilising temp, 15min with naoh.
(3) fermentation culture: the thalline after will be enlarged by cultivating is inoculated in fermentation broth, inoculum concentration is 10%(v/
V), 30 DEG C of amplification culture temperature, shaking speed 150r/min, fermentation period 36h.
Fermentation medium: glucose 30g, Dried Corn Steep Liquor Powder 20g, sucrose 30g, yeast extract 1g, (nh4)2so410g, paddy
Propylhomoserin sodium 10g, mgcl20.5g, kcl 0.5g, nah2po40.5g, is made into 1l solution with tap water, adjusts ph with naoh
To 7.0,121 DEG C of sterilising temp, sterilization time 20min.
(4) 30l fermentor cultivation: the fermentation liquid after fermentation culture in step (3) is inoculated into 30l
In the fermentation medium of fermentation tank, inoculum concentration is 10%(v/v), control tank pressure to be 0.05mpa by force, train under the conditions of 28 DEG C
Support 48 hours, speed of agitator is 400rpm, air quantity is 10 l/min, obtains the fermentation containing l- dglutamic oxidase after fermentation
Liquid.
Embodiment 4 l- glutamic acid is through the conversion method of preparing a-ketoglutaric acid of l- dglutamic oxidase
Specifically comprise the following steps that
(1) preparation of crude enzyme liquid: the fermentation liquid of l- dglutamic oxidase is first passed through ceramic membrane filter, removes thalline, supernatant
Concentrate 50 times of crude enzyme liquids being the l- dglutamic oxidase that enzymatic conversion uses through reverse osmosis membrane again.
(2) conversion culture: ph be 8.5 phosphate buffer in, add the crude enzyme liquid of l- dglutamic oxidase, h2o2Enzyme
And mncl2And substrate l- sodium glutamate, 37 DEG C, 200r/min converts 24 h, obtains final product the conversional solution containing a-ketoglutaric acid；Phosphorus
The final concentration of 50mmol of acid buffer, the final concentration of 15u/ml, h of l- dglutamic oxidase2o2The final concentration of 20u/ of enzyme
ml、mncl2Final concentration of 5mmol, the final concentration of 10%(mass fraction of l- sodium glutamate).
The determination of the conversion condition of test example 1 a-ketoglutaric acid
(1) impact to a-ketoglutaric acid yield for the determination reaction temperature of reaction temperature, the difference in the range of 25 DEG C -42 DEG C
At a temperature of, the situation of conversion reaction l0h, temperature higher a-ketoglutaric acid yield is higher, and 37 DEG C reach highest, then produce more than 37 DEG C
Rate declines.
(2) impact to a-ketoglutaric acid yield for the determination ph of ph, prepares the substrate solution under the conditions of different ph
(ph6.0-10.0), at 37 DEG C, react 10h, investigate the impact to enzyme activity (product amount) for the ph, ph is purpose product concentration when 8.5
Highest, that is, relative to enzyme activity highest.
(3) impact to a-ketoglutaric acid yield for the determination rotating speed of rotating speed, the a-ketoglutaric acid when rotating speed is for 200r/min
Yield is maximum.
(4) impact to a-ketoglutaric acid yield for the determination concentration of substrate and response time in concentration of substrate and response time,
With the increase of l- aminoglutaric acid concentration, the time that a-ketoglutaric acid concentration reaches required for maximum gradually extends.When l- glutamic acid
When concentration is 10%, conversion 24h a-ketoglutaric acid concentration reaches maximum, extends time production concentration and is not further added by and tends to steady
Fixed；When l- aminoglutaric acid concentration continues to increase, the yield of product a-ketoglutaric acid is not further added by and tends towards stability, and now increases bottom
Thing concentration is nonsensical, so selecting substrate l- aminoglutaric acid concentration to be 10%, transformation time is 24h.
The mensure of test example 2 a-ketoglutaric acid molar yield
Thalline is inoculated in 50ml fluid medium and cultivates 48 h in 28 DEG C.Cultured sending out containing dglutamic oxidase
At 4 DEG C of zymotic fluid, 4000 r/min centrifugation 30min, removes thalline, supernatant is enzyme through 50 times of despining evaporation and concentration again
The crude enzyme liquid of the l- dglutamic oxidase that conversion uses.Ph be 8.5 phosphate buffer in, add l- dglutamic oxidase
Crude enzyme liquid, h2o2Enzyme and mncl2And substrate l- sodium glutamate, 37 DEG C, 200r/min converts 24 h, obtains final product ketone containing a- penta 2
The conversional solution of acid；The final concentration of 50mmol of phosphate buffer, the final concentration of 15u/ml, h of l- dglutamic oxidase2o2Enzyme
Final concentration of 20u/ml, mncl2Final concentration of 5mmol, the final concentration of 10%(mass fraction of l- sodium glutamate).6000 r/
Min is centrifuged 10 min, the a-ketoglutaric acid concentration in mensure supernatant, and calculates the molar yield y of substrate glutamic acid,
The vigor of dglutamic oxidase is represented with this.It is 87% that the present invention records glutamic acid molar yield.
y=na/nb
Wherein na is the amount (mol) of the material of a-ketoglutaric acid in conversional solution；Nb is the material of original glutamic acid in conversional solution
Amount (mol).
Claims (9)
1. a kind of isolation and purification method of a-ketoglutaric acid, specifically comprises the following steps that
Ion exchange: water backwash resin will be used extremely containing a-ketoglutaric acid conversional solution after the absorption of d301 macroreticular weakly base negative resin
Effluent is limpid, with 0.25n hydrochloric acid solution eluting；
Nanofiltration: a-ketoglutaric acid nanofiltration is obtained using 600-800 molecular weight nanofiltration membrane a-ketoglutaric acid eluent liquid
Clear liquid, after 3 cleaning concentrates of deionization moisture of 3 times of volume of concentrate, discards concentrated solution, collects containing citrulline product
Nanofiltration dislysate；
Activated carbon decolorizing: ph 3.0, is decoloured with activated carbon, and the consumption of activated carbon is the 1% of a-ketoglutaric acid nanofiltration clear liquid
(mass fraction), bleaching temperature is 50 DEG C, and bleaching time is 30min, obtains destaining solution with 0.22 μm of membrane filtration；
Reverse osmosis concentration: a-ketoglutaric acid destaining solution is concentrated into original volume half through reverse osmosis membrane, and to obtain a-ketoglutaric acid pre- dense
Contracting liquid；
Condensing crystallizing: pre-concentration liquid is concentrated into the dense of content >=80% under the conditions of vacuum >=0.095,50 DEG C of evaporating temperature
Contracting liquid, concentrated solution stirring is cooled to 20 DEG C of crystallization 5h；
It is dried: after terminating, sucking filtration obtains crystal, 50 DEG C of vacuum drying of crystallographic obtain a-ketoglutaric acid finished product.
2. the method isolating and purifying according to claim 1 it is characterised in that described containing a-ketoglutaric acid conversional solution
Preparation method, specifically comprises the following steps that
(1) preparation of crude enzyme liquid: the fermentation liquid of l- dglutamic oxidase is first passed through ceramic membrane filter, removes thalline, supernatant
Concentrate 50 times of crude enzyme liquids being the l- dglutamic oxidase that enzymatic conversion uses through reverse osmosis membrane again；
(2) conversion culture: ph be 8.5 phosphate buffer in, add the crude enzyme liquid of l- dglutamic oxidase, h2o2Enzyme and
mncl2And substrate l- sodium glutamate, 37 DEG C, 200r/min converts 24 h, obtains final product the conversional solution containing a-ketoglutaric acid；Phosphoric acid
The final concentration of 50mmol of buffer, the final concentration of 15u/ml, h of l- dglutamic oxidase2o2The final concentration of 20u/ml of enzyme,
mncl2Final concentration of 5mmol, final concentration of the 10% of l- sodium glutamate；
The method preparing l- dglutamic oxidase using bacterial strain mqo-160, step is as follows:
(1) slant culture: bacterial strain mqo-160 is aseptically inoculated on slant medium and carries out being inverted culture, training
Foster temperature is 28 DEG C, and incubation time is 48h；
(2) seed amplification culture: picking colony from the slant medium of step (1), dilute obtains phage solution, by bacterium
Liquid solution is inoculated on seed culture medium and carries out amplification culture, and inoculum concentration is 10%(v/v), the temperature of amplification culture is 28 DEG C,
Shaking speed 120r/min, incubation time is 12h；
(3) fermentation culture: the thalline after will be enlarged by cultivating is inoculated in fermentation broth, inoculum concentration is 10%(v/v), expand
Big 30 DEG C of cultivation temperature, shaking speed 150r/min, fermentation period 36h；
(4) 30l fermentor cultivation: the fermentation liquid after fermentation culture in step (3) is inoculated into 30l
In the fermentation medium of fermentation tank, inoculum concentration is 10%(v/v), control tank pressure to be 0.05mpa by force, train under the conditions of 28 DEG C
Support 48 hours, speed of agitator is 400rpm, air quantity is 10 l/min, obtains the fermentation containing l- dglutamic oxidase after fermentation
Liquid；
The deposit number of described bacterial strain mqo-160 is: cgmcc no.12892；Depositary institution is: Chinese microorganism strain preservation
Administration committee's common micro-organisms center；Preservation date is: on August 22nd, 2016；Preservation address is: Chaoyang District, Beijing City north
Occasion West Road 1 institute 3.
3. the method isolating and purifying according to claim 2 is it is characterised in that described bacterial strain mqo-160's is biological special
Property is:
It is milky with aerial hyphae, mycelia has more barrier film, and aerial hyphae has more branch in the base of bacterial strain mqo-160
Shape branch, part mycelia is in vigorous trophophase, has bud shape structure, and bacterial strain produces golden yellow or aubergine soluble pigment,
The spore oval of bacterial strain mqo-160, spherical or cylindricality, smooth surface, fibrillae of spores is in the shape of a spiral.
4. the method that isolates and purifies according to claim 2 is it is characterised in that the preparation of described slant medium: solubility
Starch 20g, kno31g, k2hpo40.5g, mgso4·7h2O 0.5g, nacl 0.5g, feso4·7h2O 0.01g, agar
20g, plus pure water is to 1l.
5. the method isolating and purifying according to claim 4 is it is characterised in that described slant medium sterilising temp is 115
DEG C, sterilization time is 15min.
6. the method isolating and purifying according to claim 2 is it is characterised in that the preparation of described seed culture medium: sucrose
30g, yeast extract 6g, (nh4)2so46g、caco330g、cacl23g、mgcl2·6h2O 1g, kcl 1g, plus pure water is extremely
1l.
7. the method isolating and purifying according to claim 6 is it is characterised in that the sterilising temp of described seed culture medium is
115 DEG C, sterilization time is 15min.
8. the method isolating and purifying according to claim 2 is it is characterised in that the preparation of described fermentation medium: Fructus Vitis viniferae
Sugared 30g, Dried Corn Steep Liquor Powder 20g, sucrose 30g, yeast extract 1g, (nh4)2so410g, sodium glutamate 10g, mgcl20.5g,
Kcl 0.5g, nah2po40.5g, is made into 1l solution with tap water.
9. the method isolating and purifying according to claim 8 is it is characterised in that the sterilising temp of described fermentation medium is
121 DEG C, sterilization time is 20min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610732998.3A CN106349056B (en) | 2016-08-27 | 2016-08-27 | A kind of isolation and purification method of a-ketoglutaric acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610732998.3A CN106349056B (en) | 2016-08-27 | 2016-08-27 | A kind of isolation and purification method of a-ketoglutaric acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106349056A true CN106349056A (en) | 2017-01-25 |
| CN106349056B CN106349056B (en) | 2018-11-23 |
Family
ID=57854295
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610732998.3A Expired - Fee Related CN106349056B (en) | 2016-08-27 | 2016-08-27 | A kind of isolation and purification method of a-ketoglutaric acid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106349056B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108315364A (en) * | 2018-01-31 | 2018-07-24 | 山东民强生物科技股份有限公司 | Bioconversion prepares the separation purifying technique of α-ketoglutaric acid |
| CN108486173A (en) * | 2018-03-27 | 2018-09-04 | 河南巨龙生物工程股份有限公司 | A kind of preparation method of α-ketoglutaric acid |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103352058A (en) * | 2013-07-23 | 2013-10-16 | 江南大学 | Biocatalysis method for preparing Alpha ketoglutarate from L-soda glutamate |
| CN105017086A (en) * | 2015-07-01 | 2015-11-04 | 滨州市生物技术研究院有限责任公司 | Separation and purification method for L-citrulline |
| CN105175275A (en) * | 2015-07-01 | 2015-12-23 | 山东民强生物科技股份有限公司 | Separation and purification method of L-ornithine |
-
2016
- 2016-08-27 CN CN201610732998.3A patent/CN106349056B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103352058A (en) * | 2013-07-23 | 2013-10-16 | 江南大学 | Biocatalysis method for preparing Alpha ketoglutarate from L-soda glutamate |
| CN105017086A (en) * | 2015-07-01 | 2015-11-04 | 滨州市生物技术研究院有限责任公司 | Separation and purification method for L-citrulline |
| CN105175275A (en) * | 2015-07-01 | 2015-12-23 | 山东民强生物科技股份有限公司 | Separation and purification method of L-ornithine |
Non-Patent Citations (1)
| Title |
|---|
| 占宏德 等: ""离子交换法纯化α-酮戊二酸"", 《食品与生物技术学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108315364A (en) * | 2018-01-31 | 2018-07-24 | 山东民强生物科技股份有限公司 | Bioconversion prepares the separation purifying technique of α-ketoglutaric acid |
| CN108486173A (en) * | 2018-03-27 | 2018-09-04 | 河南巨龙生物工程股份有限公司 | A kind of preparation method of α-ketoglutaric acid |
| CN108486173B (en) * | 2018-03-27 | 2022-04-01 | 河南巨龙生物工程股份有限公司 | Preparation method of alpha-ketoglutaric acid |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106349056B (en) | 2018-11-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103320362B (en) | One strain is produced the bacterial strain of L-Glutamic decarboxylase and is produced the method for γ-aminobutyric acid with it | |
| CN105017086B (en) | Separation and purification method for L-citrulline | |
| CN107955791B (en) | Strain capable of obtaining nicotinamide adenine dinucleotide through one-step catalysis, screening method and application thereof | |
| CN107267422B (en) | Comamonas testosteroni HHALA-001 and method for producing L-alanine by using same | |
| CN104788522A (en) | Method for extracting cyclic adenosine monophosphate from fermentation liquid | |
| CN102719378B (en) | Method for preparing (-) gamma-lactam by catalysis asymmetry of microorganism | |
| CN108299244A (en) | Bioconversion prepares the separation purifying technique of L-citrulline | |
| CN106349056B (en) | A kind of isolation and purification method of a-ketoglutaric acid | |
| CN105886573B (en) | Method for preparing trehalose by continuous extracellular enzyme biological method | |
| CN106337039A (en) | Method for preparing L-glutamate oxidase from strains MQO-160 | |
| CN106148240A (en) | The fermentation medium of bacterial strain MQO 160 | |
| CN102719502A (en) | Method for producing L-alanine by mutating lactate-production bacteria | |
| CN103602609A (en) | High-yield strain for producing L-alanine by fermentation and preparation method thereof | |
| CN106148434A (en) | A kind of microbial enzyme method produces the method for a ketoglutaric acid | |
| CN106318888A (en) | Strain MQO-160 for generating L-glutamate oxidase and application of strain | |
| CN106047713A (en) | Talaromyces pinophilum strain Li-93 and application thereof | |
| CN105176859A (en) | Strain MQO-153 for production of arginine deiminase | |
| CN104630195A (en) | Marine microorganism fermentation production method for low temperature gamma-lactamase | |
| CN105175275B (en) | A kind of isolation and purification method of L ornithine | |
| CN110511968A (en) | The method of one-step fermentation separation coupling generation diamine | |
| CN105586289B (en) | A kind of (+/-) gamma-lactam that can split obtains pseudomonad and its screening and application of (-) gamma-lactam | |
| CN105177075A (en) | Method for preparation of L-citrulline with arginine as raw material | |
| CN105567584A (en) | Bacillus capable of resolving (+/-)gamma-lactam to obtain (+)gamma-lactam and screening and application of bacillus | |
| CN108410749A (en) | The method that marine low temperature (+) gamma-lactam enzyme asymmetric hydrolysis prepares (-) gamma-lactam | |
| CN104789489B (en) | The bacillus cereus of one plant of High-yield arginine deiminase bacterial and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| PP01 | Preservation of patent right | ||
| PP01 | Preservation of patent right |
Effective date of registration: 20181206 Granted publication date: 20181123 |
|
| PD01 | Discharge of preservation of patent | ||
| PD01 | Discharge of preservation of patent |
Date of cancellation: 20211206 Granted publication date: 20181123 |
|
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20181123 Termination date: 20190827 |