CN102433360A - Method for preparing alpha-ketobutyric acid by using L-threonine as substrate - Google Patents
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Abstract
The invention discloses a method for preparing alpha-ketobutyric acid by using L-threonine as a substrate, wherein a product, alpha-ketobutyric acid is prepared by using microbial complete cells containing synthetic L-threonine dehydratase as a biological catalyst, using the L-threonine as the substrate, and performing oscillating transformation at a rate of 50 to 250 turns/minute under the conditions that the temperature is between 20 and 65 DEG C and the pH is between 7.0 and 11.0. The method has the following characteristics that (1) a biological catalysis method is used, a reaction system is simple, reaction conditions are mild, steps are easy, and the operation is simple; and the biological catalyst can be easily removed, and the separation and purification of subsequent products are facilitated; (2) the transformation rate of the alpha-ketobutyric acid generated by the substrate, L-threonine can reach more than 99.6 percent, and the product, alpha-ketobutyric acid can be accumulated to reach a higher concentration; and (3) the substrate, L-threonine has a low price and is easy to obtain. According to the method, a basis of realizing high-efficient production of the alpha-ketobutyric acid is established.
Description
Technical field
The present invention relates to the method for a kind of α of production-alpha-ketobutyric acid, specifically, relate to utilization with the mikrobe intact cell that contains synthesis type L-threonine dehydra(ta)se as biological catalyst, as substrate, produce the method for α-alpha-ketobutyric acid with the L-Threonine.
Background technology
α-alpha-ketobutyric acid is a kind of important midbody, can be applicable to fields such as chemistry and medicine.For example, α-alpha-ketobutyric acid can be used for producing the L-Isoleucine
[1], the 1-propyl alcohol
[2] [3], the food spice component
[4]And D-Alpha-hydroxy butyric acid
[5]Wherein, D-Alpha-hydroxy butyric acid can be used to prepare azinothricin family antitumor antibiotic and superpolymer gathers Alpha-hydroxy butyric acid [P (2HB)
[6]In addition, α-alpha-ketobutyric acid can change the L-butyrine into, and the latter is the chiral precurser of antiepileptic drug such as synthetic UCB-L 059
[7] [8]
The working method of α-alpha-ketobutyric acid is divided into traditional chemical method and microorganism catalysis method.The former refers to by oxalic acid diethyl ester and ethyl propionate mixed hydrolysis and forms α-alpha-ketobutyric acid
[9]And the latter who reports in the document comprises following several kinds: with 1; The 2-butyleneglycol is initial substrate, is catalyzer with the full cell of Rhodococcus equi IF03730, final product concentration be 153.8 mmoles/liter; Molar yield is 68%, and production intensity is 4.8 mmoles/liter/hour
[10]With Ba Dousuan is initial substrate, is catalyzer with osmium and the full cell of pseudomonas putida, final product concentration be 47 mmoles/liter, production intensity is 9.4 mmoles/liter/hour
[9]With the DL-2-hydroxybutyric acid is substrate, is catalyzer with Pseudomonas stutzeri SDM, final product concentration be 434.9 mmoles/liter, molar yield is 91.5%, production intensity is 18.1 mmoles/liter/hour
[11]With the Threonine is substrate, the fermentation of the ilv-3 two mutants of coarse arteries and veins born of the same parents bacterium produce α-alpha-ketobutyric acid 78.4 mmoles/liter, molar yield＞90%, production intensity is 0.7 mmole/liter/hour
[4]1,2-butyleneglycol, Ba Dousuan and DL-2-hydroxybutyric acid can only obtain through chemical method, can not realize the green production of α-alpha-ketobutyric acid.And Threonine can obtain through microbial fermentation on a large scale, and the output of whole world L-Threonine reaches 20,000 tons
[12], be suitable as very much the initial substrate of α-alpha-ketobutyric acid.Transform generation α-alpha-ketobutyric acid by the L-Threonine, passed through catalytic deamination of L-threonine dehydra(ta)se and dehydration
[13]
L-threonine dehydra(ta)se (L-threonine deaminase) is divided into two kinds of synthesis type and degraded types.Synthesis type L-threonine dehydra(ta)se constructive expression under aerobic conditions receives the inhibition of L-Isoleucine; Receive L-Threonine abduction delivering under the degraded type L-threonine dehydra(ta)se anaerobic condition, do not receive the inhibition of L-Isoleucine
[14] [15] [16]Find through retrieval, utilize with the mikrobe intact cell that contains synthesis type L-threonine dehydra(ta)se, as substrate, produce the method for α-alpha-ketobutyric acid and also do not appear in the newspapers with the L-Threonine as biological catalyst.
Reference
【1】Eggeling，I.，Codes，C.，Eggeling，L.，Sahm，H.，1987.Regulation?of?acetohydroxy?acid?synthase?in?Corynebacterium?glutamicum?during?fermentation?of?α-ketobutyrate?to?L-isoleucine.Appl.Microbial.Biotechnol.25，346-351.
【2】Atsumi，S.，Hanai，T.，Liao，J.C.，2008.Non-fermentative?pathways?for?synthesis?of?branched-chain?higher?alcohols?as?biofuels.Nature?451，86-89.
【3】Shen，C.R.，Liao，J.C.，2008.Metabolic?engineering?of?Escherichia?coli?for?1-butanol?and?1-propanol?production?via?the?keto-acid?pathways.Metab.Eng.10，312-320.
【4】Zurbriggen，B.D.，Rekhif，N.，Mehlmann-De-Campos，M.，Lerch，K.，2006.Production?of?α-ketobutyrate.U.S.patent?7144715.
【5】Simon，E.S.，Plante，R.，Whitesides，G.M.，1989.D-lactate?dehydrogenase.Substrate?specificity?and?use?as?a?catalyst?in?the?synthesis?of?homochiral?2-hydroxy?acids.Appl.Biochem.Biotechnol.22，169-179.
【6】Gao，C.，Zhang，W.，Ma，C.，Liu，P.，Xu，P.，2011.Kinetic?resolution?of?2-hydroxybutanoate?racemic?mixtures?by?NAD-independent?L-lactate?dehydrogenase.Bioresour.Technol.102，4595-4599.
【7】Zhang，K.，Li，H.，Cho，K.M.，Liao，J.C.，2010.Expanding?metabolism?for?total?biosynthesis?of?the?nonnatural?amino?acid?L-homoalanine.Proc.Natl.Acad.Sci.USA?107，6234-6239.
【8】Park，E.，Kim，M.，Shin，J.S.，2010.One-pot?conversion?of?L-threonine?mto?L-homoalanine：biocatalytic?production?of?an?unnatural?amino?acid?from?a?natural?one.Adv.Synth.Catal.352，3391-3398.
【9】Furuyoshi，S.，Nawa，Y.，Kawabata，N.，1991.Microbial?production?of?2-oxobutyric?acid?from?crotonic?acid.Agric.Biol.Chem.55，123-128.
【10】Nakahara，T.，Nakajima-kambe，T.，and?Sato，S.，1994.Production?of?2-ketobutyric?acid?from?1，2-butanediol?by?resting?cells?ofRhodococcus?equi?IFO?3730.Biotechnol.Lett.16，263-268.
【11】Gao，C.，Zhang，W.，Lv，C.，Li，L.，Ma，C.，Hu，C.，Xu，P.，2010.Efficient?production?of?2-oxobutyrate?from?2-hydroxybutyrate?by?using?whole?cells?of?Pseudomonas?stutzeri?stram?SDM.Appl.Envrion.Microbiol.76，1679-1682.
【12】Debabov，V.G.，2003.The?threonine?story.Adv.Biochem.Eng.Biotechnol.79，113-136.
【13】Husain，A.，Jeelani，G.，Sato，D.，Ali，V.，Nozaki，T.，2010.Characterization?of?two?isotypes?of?L-threonine?dehydratase?from?Entamoeba?histolytica.Mol.Biochem.Parasitol.170，100-104.
【14】Lam，V.M.S.，Chan，I.P.R.，Yeung，Y.G.，1980.Role?of?L-threonine?deaminase?and?L-threonine?3-dehydrogenase?in?the?utilization?of?L-threonine?by?Pseudomonas?aerugmosa.J.Gen.Microbiol.117，539-542.
【15】Umbarger，H.E.，1956.Evidence?for?a?negative-feedback?mechanism?in?the?biosynthesis?of?isoleucine.Science?123，848.
【16】Luginbuhl，G.H.，Hofler，J.G.，Decedue，C.J.，Burns，R.O.，1974.Biodegradative?L-threonine?deaminase?of?Salmonella?typhimurium.J.Bacteriol.120，559-561.
Summary of the invention
To the deficiency of prior art, the problem that the present invention will solve provide a kind of with the mikrobe intact cell that contains synthesis type L-threonine dehydra(ta)se as biological catalyst, as substrate, produce the method for α-alpha-ketobutyric acid with the L-Threonine.
Method of producing α-alpha-ketobutyric acid as substrate of the present invention with the L-Threonine, step is:
(1) preparation contains the suspension of biological catalyst
Choose the bacterial strain that Rhodopseudomonas, Corynebacterium or bacillus etc. contain synthesis type L-threonine dehydra(ta)se, in the LB substratum, shake bottle or the cultivation of fermentor tank mode aerobic with routine; Separate and the collection thalline, wash thalline 2～4 times, separate the mikrobe intact cell that obtains and be biological catalyst with pH 7.2～7.5 potassium phosphate buffers; Biological catalyst is resuspended in above-mentioned potassium phosphate buffer or the deionized water, make the biocatalysis agent concentration reach 200 gram wet cells/liter, promptly obtain containing the suspension of biological catalyst, 4 ℃ store for future use;
(2) transform
With suspension that contains biological catalyst that makes in the step (1) and L-Threonine aqueous solution, and make the concentration of L-Threonine in the mixture be 10～80 grams per liters, biocatalysis agent concentration be 40～150 gram wet cells/liter; At 20～65 ℃, under pH7.0～11.0 conditions,, get conversion fluid with 50～250 rev/mins of oscillatory reactions 1～30 hour;
(3) produce the solution that contains α-alpha-ketobutyric acid
With the conversion fluid of step (2) with 5,000～14,000 rev/min centrifugal 5～25 minutes, perhaps filter with 200～400 order filter clothes, remove the biological catalyst that adds in the step (2), the gained clear liquid is the solution that contains α-alpha-ketobutyric acid;
(4) α-alpha-ketobutyric acid and L-Threonine detect
With the above-mentioned solution that contains α-alpha-ketobutyric acid in 100 ℃ of heating 10 minutes, with 5,000～14,000 rev/min centrifugal 1～10 minute, the gained stillness of night is diluted with 4% sulphosalicylic acid, 4 ℃ of hold over night, centrifugal gained supernatant afterwards is sample.
α-alpha-ketobutyric acid assay adopts performance liquid chromatography Agilent1100 system, and chromatography column is Aminex HPX-87H (U.S.), and analysis condition is for being moving phase with 10 mmoles/rise sulfuric acid; 55 ℃ of column temperatures; Flow velocity is 0.4 ml/min, and sample size 5 microlitres adopt differential refraction detector.Mass spectrometric detection is adopted in the evaluation of α-alpha-ketobutyric acid.
Measure L-Threonine concentration with amino acidanalyser (Hitachi, L-8900, Japan).Transformation efficiency=(the amount of substance concentration of the former L-Threonine of amount of substance concentration ÷ of the α-alpha-ketobutyric acid of generation) * 100%.
Above-mentioned is that substrate is produced in the method for α-alpha-ketobutyric acid with the L-Threonine:
The said preferred Pseudomonas stutzeri of bacterial strain (Pseudomonas stutzeri) SDM CCTCC NO:M206010, Corynebacterium glutamicum ATCC13032 or the subtilis ATCC23857 that contains synthesis type L-threonine dehydra(ta)se of step (1).
Preferred 20～60 grams per liters of concentration of the said L-Threonine of step (2); Preferred 60～120 gram wet cells of said biocatalysis agent concentration/liter; Preferred 30～60 ℃ of said temperature; Said pH scope preferred 8.0～10.0; The said reaction times is preferably 2～24 hours.
The present invention selects for use the mikrobe intact cell that contains synthesis type L-threonine dehydra(ta)se as biological catalyst, as substrate, has successfully realized the High-efficient Production of α-alpha-ketobutyric acid with the L-Threonine.The inventive method has following characteristics:
(1) yeast culture and reaction time are all shorter.
(2) substrate L-Threonine generates the transformation efficiency height of α-alpha-ketobutyric acid, can reach more than 99.6%.
(3) product α-alpha-ketobutyric acid can run up to higher concentration.
(4) bacterial strain uses therefor does not need fragmentation, can directly transform with intact cell, and is easy to operate.
(5) utilize intact cell catalysis, need not to add expensive cofactor.
(6) biological catalyst can pass through to filter or centrifugal removal, and product component is simple, helps later separation and extracts.
Description of drawings
Fig. 1: α-alpha-ketobutyric acid performance liquid chromatography detected result.Wherein a is standard substance, and b is a response sample.
Fig. 2: the α of generation-alpha-ketobutyric acid mass spectrometric detection result.
Fig. 3: amino acidanalyser detects the generation of ammonia.
Embodiment
Buy and execute example 1:
(1) preparation contains the suspension of biological catalyst
Choose Pseudomonas stutzeri (P.stutzeri) SDM CCTCC NO:M206010, in the LB substratum, shake bottle or the cultivation of fermentor tank mode aerobic with routine; Separate and the collection thalline, wash thalline 3 times, separate the mikrobe intact cell that obtains and be biological catalyst with pH 7.4 potassium phosphate buffers.Biological catalyst is resuspended in potassium phosphate buffer or the deionized water, make the biocatalysis agent concentration reach 200 gram wet cells/liter, promptly obtain containing the suspension of biological catalyst, 4 ℃ store for future use;
(2) transform
With suspension that contains biological catalyst that makes in the step (1) and L-Threonine aqueous solution, and make the concentration of L-Threonine in the mixture be 80 grams per liters, biocatalysis agent concentration be 150 gram wet cells/liter; At 20 ℃, under pH 11.0 conditions,, get conversion fluid with 200 rev/mins of oscillatory reactions 20 hours;
(3) produce the solution that contains α-alpha-ketobutyric acid
With above-mentioned conversion fluid, with 12,000 rev/mins centrifugal 20 minutes, remove the biological catalyst that adds in the step (2), the gained stillness of night is the solution that contains α-alpha-ketobutyric acid.
(4) α-alpha-ketobutyric acid and L-Threonine detect
With the above-mentioned solution that contains α-alpha-ketobutyric acid in 100 ℃ of heating 10 minutes, with 10,000 rev/mins centrifugal 10 minutes, the gained stillness of night is diluted with 4% sulphosalicylic acid, 4 ℃ of hold over night, centrifugal gained supernatant afterwards is sample.
α-alpha-ketobutyric acid assay adopts performance liquid chromatography Agilent1100 system, and chromatography column is Aminex HPX-87H (U.S.), and analysis condition is for being moving phase with 10 mmoles/rise sulfuric acid; 55 ℃ of column temperatures; Flow velocity is 0.4 ml/min, and sample size 5 microlitres adopt differential refraction detector.Mass spectrometric detection is adopted in the evaluation of α-alpha-ketobutyric acid.
Measure L-Threonine concentration with amino acidanalyser (Hitachi, L-8900, Japan).Transformation efficiency=(the amount of substance concentration of the former L-Threonine of amount of substance concentration ÷ of the α-alpha-ketobutyric acid of generation) * 100%.
Through detecting, the concentration of α-alpha-ketobutyric acid is 34.2 grams per liters in the conversion fluid, and transformation efficiency is 49.9%.The performance liquid chromatography detected result of α-alpha-ketobutyric acid is as shown in Figure 1, and mass spectrometric detection result is as shown in Figure 2, confirms that product has the generation of α-alpha-ketobutyric acid.The generation of ammonia is arranged in the amino acidanalyser working sample, as shown in Figure 3.
Product is α-alpha-ketobutyric acid and ammonia, proves that process of producing α-alpha-ketobutyric acid as substrate with the L-Threonine of the present invention is by the catalytic dehydration deamination of L-threonine dehydra(ta)se process.In addition, because microorganism cells is cultivation under aerobic conditions, and culturing process need not add inductor, so the L-threonine dehydra(ta)se that catalytic process relates to is a synthesis type.
Embodiment 2:
(1) preparation contains the suspension of biological catalyst
Choose subtilis ATCC23857, in the LB substratum, shake bottle or the cultivation of fermentor tank mode aerobic with routine; Separate and the collection thalline, wash thalline 3 times, separate the mikrobe intact cell that obtains and be biological catalyst with pH 7.4 potassium phosphate buffers.Biological catalyst is resuspended in potassium phosphate buffer or the deionized water, make the biocatalysis agent concentration reach 200 gram wet cells/liter, promptly obtain containing the suspension of biological catalyst, 4 ℃ store for future use;
(2) transform
With suspension that contains biological catalyst that makes in the step (1) and L-Threonine aqueous solution, and make the concentration of L-Threonine in the mixture be 10 grams per liters, biocatalysis agent concentration be 60 gram wet cells/liter; At 30 ℃, under pH 8.0 conditions,, get conversion fluid with 200 rev/mins of oscillatory reactions 24 hours;
(3) produce the solution that contains α-alpha-ketobutyric acid
With above-mentioned conversion fluid, with 12,000 rev/mins centrifugal 20 minutes, remove the biological catalyst that adds in the step (2), the gained stillness of night is the solution that contains α-alpha-ketobutyric acid.
(4) α-alpha-ketobutyric acid and L-Threonine detect
Through detecting, the concentration of α-alpha-ketobutyric acid is 0.64 grams per liter in the conversion fluid, and transformation efficiency is 7.5%.
Embodiment 3:
(1) preparation contains the suspension of biological catalyst
Choose Pseudomonas stutzeri (P.stutzeri) SDM CCTCC NO:M206010, in the LB substratum, shake bottle or the cultivation of fermentor tank mode aerobic with routine; Separate and the collection thalline, wash thalline 3 times, separate the mikrobe intact cell that obtains and be biological catalyst with pH 7.4 potassium phosphate buffers.Biological catalyst is resuspended in potassium phosphate buffer or the deionized water, make the biocatalysis agent concentration reach 200 gram wet cells/liter, promptly obtain containing the suspension of biological catalyst, 4 ℃ store for future use;
(2) transform
With suspension that contains biological catalyst that makes in the step (1) and L-Threonine aqueous solution, and make the concentration of L-Threonine in the mixture be 60 grams per liters, biocatalysis agent concentration be 120 gram wet cells/liter; At 65 ℃, under pH 7.0 conditions,, get conversion fluid with 200 rev/mins of oscillatory reactions 24 hours;
(3) produce the solution that contains α-alpha-ketobutyric acid
With above-mentioned conversion fluid, with 12,000 rev/mins centrifugal 20 minutes, remove the biological catalyst that adds in the step (2), the gained stillness of night is the solution that contains α-alpha-ketobutyric acid.
(4) α-alpha-ketobutyric acid and L-Threonine detect
Through detecting, the concentration of α-alpha-ketobutyric acid is 19.6 grams per liters in the conversion fluid, and transformation efficiency is 38.1%.
Embodiment 4:
(1) preparation contains the suspension of biological catalyst
Choose Corynebacterium glutamicum ATCC13032, in the LB substratum, shake bottle or the cultivation of fermentor tank mode aerobic with routine; Separate and the collection thalline, wash thalline 3 times, separate the mikrobe intact cell that obtains and be biological catalyst with pH 7.4 potassium phosphate buffers.Biological catalyst is resuspended in potassium phosphate buffer or the deionized water, make the biocatalysis agent concentration reach 200 gram wet cells/liter, promptly obtain containing the suspension of biological catalyst, 4 ℃ store for future use;
(2) transform
With suspension that contains biological catalyst that makes in the step (1) and L-Threonine aqueous solution, and make the concentration of L-Threonine in the mixture be 20 grams per liters, biocatalysis agent concentration be 40 gram wet cells/liter; At 60 ℃, under pH 10.0 conditions,, get conversion fluid with 200 rev/mins of oscillatory reactions 20 hours;
(3) produce the solution that contains α-alpha-ketobutyric acid
With above-mentioned conversion fluid, with 12,000 rev/mins centrifugal 20 minutes, remove the biological catalyst that adds in the step (2), the gained stillness of night is the solution that contains α-alpha-ketobutyric acid.
(4) α-alpha-ketobutyric acid and L-Threonine detect
Through detecting, the concentration of α-alpha-ketobutyric acid is 12.1 grams per liters in the conversion fluid, and transformation efficiency is 70.6%.
Embodiment 5:
(1) preparation contains the suspension of biological catalyst
Choose Pseudomonas stutzeri (P.stutzeri) SDM CCTCC NO:M206010, in the LB substratum, shake bottle or the cultivation of fermentor tank mode aerobic with routine; Separate and the collection thalline, wash thalline 3 times, separate the mikrobe intact cell that obtains and be biological catalyst with pH 7.4 potassium phosphate buffers.Biological catalyst is resuspended in potassium phosphate buffer or the deionized water, make the biocatalysis agent concentration reach 200 gram wet cells/liter, promptly obtain containing the suspension of biological catalyst, 4 ℃ store for future use;
(2) transform
With suspension that contains biological catalyst that makes in the step (1) and L-Threonine aqueous solution, and make the concentration of L-Threonine in the mixture be 30 grams per liters, biocatalysis agent concentration be 60 gram wet cells/liter; At 50 ℃, under pH 8.0 conditions,, get conversion fluid with 200 rev/mins of oscillatory reactions 6 hours;
(3) produce the solution that contains α-alpha-ketobutyric acid
With above-mentioned conversion fluid, with 12,000 rev/mins centrifugal 20 minutes, remove the biological catalyst that adds in the step (2), the gained stillness of night is the solution that contains α-alpha-ketobutyric acid.
(4) α-alpha-ketobutyric acid and L-Threonine detect
Through detecting, the concentration of α-alpha-ketobutyric acid is 25.6 grams per liters in the conversion fluid, and transformation efficiency is 99.6%.
Bacterial strain Pseudomonas stutzeri (P.stutzeri) the SDMCCTCC NO:M206010 that contains synthesis type L-threonine dehydra(ta)se that relates among the present invention is an applicant laboratory preservation strain, and this bacterial strain had before carried out patent protection and preservation; Corynebacterium glutamicum ATCC13032 and subtilis ATCC23857 are available from US mode culture collection warehousing (American type culture collection).
Claims (7)
1. one kind is the method that substrate is produced α-alpha-ketobutyric acid with the L-Threonine, and step is:
(1) preparation contains the suspension of biological catalyst
Choose the bacterial strain that Rhodopseudomonas, Corynebacterium or bacillus contain synthesis type L-threonine dehydra(ta)se, in the LB substratum, shake bottle or the cultivation of fermentor tank mode aerobic with routine; Separate and the collection thalline, wash thalline 2～4 times, separate the mikrobe intact cell that obtains and be biological catalyst with pH 7.2～7.5 potassium phosphate buffers; Biological catalyst is resuspended in above-mentioned potassium phosphate buffer or the deionized water, make the biocatalysis agent concentration reach 200 gram wet cells/liter, promptly obtain containing the suspension of biological catalyst, 4 ℃ store for future use;
(2) transform
With suspension that contains biological catalyst that makes in the step (1) and L-Threonine aqueous solution, and make the concentration of L-Threonine in the mixture be 10～80 grams per liters, biocatalysis agent concentration be 40～150 gram wet cells/liter; At 20～65 ℃, under pH7.0～11.0 conditions,, get conversion fluid with 50～250 rev/mins of oscillatory reactions 1～30 hour;
(3) produce the solution that contains α-alpha-ketobutyric acid
With the conversion fluid of step (2) with 5,000～14,000 rev/min centrifugal 5～25 minutes, perhaps filter with 200～400 order filter clothes, remove the biological catalyst that adds in the step (2), the gained clear liquid is the solution that contains α-alpha-ketobutyric acid.
2. be the method that substrate is produced α-alpha-ketobutyric acid with the L-Threonine according to claim 1; It is characterized in that the said bacterial strain that contains synthesis type L-threonine dehydra(ta)se of step (1) selects Pseudomonas stutzeri (P.stutzeri) SDM CCTCC NO:M206010, Corynebacterium glutamicum ATCC13032 or subtilis ATCC23857.
3. be the method that substrate is produced α-alpha-ketobutyric acid with the L-Threonine according to claim 1, it is characterized in that the concentration of the described L-Threonine of step (2) is 20～60 grams per liters.
4. be the method that substrate is produced α-alpha-ketobutyric acid with the L-Threonine according to claim 1, it is characterized in that, the described biocatalysis agent concentration of step (2) be 60～120 gram wet cells/liter.
5. be the method that substrate is produced α-alpha-ketobutyric acid with the L-Threonine according to claim 1, it is characterized in that the described temperature of step (2) is selected 30～60 ℃.
6. be the method that substrate is produced α-alpha-ketobutyric acid with the L-Threonine according to claim 1, it is characterized in that the described pH scope of step (2) is 8.0～10.0.
7. be the method that substrate is produced α-alpha-ketobutyric acid with the L-Threonine according to claim 1, it is characterized in that the described reaction times of step (2) is 2～24 hours.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103865869A (en) * | 2014-04-03 | 2014-06-18 | 天津科技大学 | Genetically engineered bacterium for producing alpha-ketobutyric acid and application of genetically engineered bacterium |
| CN105296455A (en) * | 2015-11-23 | 2016-02-03 | 江南大学 | Threonine dehydratase active inclusion body preparation and application |
| CN105695519A (en) * | 2016-04-22 | 2016-06-22 | 中国科学院天津工业生物技术研究所 | Novel biological preparation method of optically pure 2-hydroxy butyric acid |
| CN106148432A (en) * | 2015-09-11 | 2016-11-23 | 天津科技大学 | A kind of fermentation manufacturing technique of α alpha-ketobutyric acid |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003284579A (en) * | 2002-03-29 | 2003-10-07 | Toray Ind Inc | Method of producing 2-ketobutyric acid |
| CN1592788A (en) * | 2001-11-29 | 2005-03-09 | 雀巢产品有限公司 | Production of alpha-keto butyrate |
| CN101333554A (en) * | 2008-08-05 | 2008-12-31 | 山东大学 | Method for producing D-lactic acid by enzyme resolution of D,L-lactic Acid |
| CN101928734A (en) * | 2009-11-13 | 2010-12-29 | 山东大学 | Method for preparing alpha-ketobutyric acid |
-
2011
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1592788A (en) * | 2001-11-29 | 2005-03-09 | 雀巢产品有限公司 | Production of alpha-keto butyrate |
| JP2003284579A (en) * | 2002-03-29 | 2003-10-07 | Toray Ind Inc | Method of producing 2-ketobutyric acid |
| CN101333554A (en) * | 2008-08-05 | 2008-12-31 | 山东大学 | Method for producing D-lactic acid by enzyme resolution of D,L-lactic Acid |
| CN101928734A (en) * | 2009-11-13 | 2010-12-29 | 山东大学 | Method for preparing alpha-ketobutyric acid |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103865869A (en) * | 2014-04-03 | 2014-06-18 | 天津科技大学 | Genetically engineered bacterium for producing alpha-ketobutyric acid and application of genetically engineered bacterium |
| CN106148432A (en) * | 2015-09-11 | 2016-11-23 | 天津科技大学 | A kind of fermentation manufacturing technique of α alpha-ketobutyric acid |
| CN106148432B (en) * | 2015-09-11 | 2020-08-18 | 天津科技大学 | Fermentation production process of alpha-ketobutyric acid |
| CN105296455A (en) * | 2015-11-23 | 2016-02-03 | 江南大学 | Threonine dehydratase active inclusion body preparation and application |
| CN105296455B (en) * | 2015-11-23 | 2020-02-14 | 安徽华恒生物科技股份有限公司 | Preparation and application of threonine deaminase active inclusion body |
| CN105695519A (en) * | 2016-04-22 | 2016-06-22 | 中国科学院天津工业生物技术研究所 | Novel biological preparation method of optically pure 2-hydroxy butyric acid |
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