CN102433360B - Method for preparing alpha-ketobutyric acid by using L-threonine as substrate - Google Patents
Method for preparing alpha-ketobutyric acid by using L-threonine as substrate Download PDFInfo
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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 a kind of method of the α of production-one base butyric acid, specifically, relate to utilize using contain synthesis type L-threonine dehydratase the microorganism intact cell as biological catalyst, using L-threonine as substrate, produce the method for α-one base butyric acid.
Background technology
α-one base butyric acid is a kind of important intermediate, can be applicable to the fields such as chemistry and medicine.For example, α-one base butyric acid can be used for producing ILE
[1], the 1-propyl alcohol
[2] [3], the food spice component
[4]And D-alpha-hydroxybutyric acid
[5].Wherein, the D-alpha-hydroxybutyric acid can be used to prepare azinothricin family antitumor antibiotic and the poly-alpha-hydroxybutyric acid [P (2HB) of superpolymer
[6].In addition, α-one base butyric acid can change the L-butyrine into, and the latter is the chiral precurser of the antiepileptic drug such as synthetic Levetiracetam
[7] [8].
The production method of α-one base butyric 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-one base butyric acid
[9].And that the latter who reports in document comprises is following several: with 1, the 2-butyleneglycol is initial substrate, and the full cell of Rhodococcus equi IF03730 of take is catalyzer, and final product concentration is 153.8 mM/ls, molar yield is 68%, and production intensity is 4.8 mM/ls/hour
[10]The β-crotonic acid of take is initial substrate, and the full cell of osmium and pseudomonas putida of take is catalyzer, and final product concentration is 47 mM/ls, and production intensity is 9.4 mM/ls/hour
[9]The DL-2-hydroxybutyric acid of take is substrate, and the Pseudomonas stutzeri SDM of take is catalyzer, and final product concentration is 434.9 mM/ls, and molar yield is 91.5%, and production intensity is 18.1 mM/ls/hour
[11]The Threonine of take is substrate, and 78.4 mM/ls of α-one base butyric acid are produced in the ilv-3 mutant fermentation of neurospora crassa, molar yield>90%, and production intensity is 0.7 mM/l/hour
[4].1,2-butyleneglycol, β-crotonic acid and DL-2-hydroxybutyric acid can only obtain by chemical method, can not realize the green production of α-one base butyric acid.And Threonine can be by microorganism extensive acquisition of fermenting, the output of whole world L-threonine reaches 20,000 tons
[12], be suitable as very much the initial substrate of α-one base butyric acid.By L-threonine, transform and generate α-one base butyric acid, passed through the L-threonine enzymatic deamination of dehydration and dehydration
[13].
L-threonine dehydratase (L-threonine desaminase) is divided into two kinds of synthesis type and degraded types.Synthesis type L-threonine dehydratase constructive expression under aerobic conditions, be subject to the inhibition of ILE; Under degraded type L-threonine dehydratase anaerobic condition, be subjected to the L-threonine abduction delivering, be not subjected to the inhibition of ILE
[14] [15] [16].Through retrieval, find, utilize using contain synthesis type L-threonine dehydratase the microorganism intact cell as biological catalyst, using L-threonine as substrate, the method for producing α-one base butyric acid yet there are no report.
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
For the deficiencies in the prior art, the problem to be solved in the present invention be to provide a kind of using contain synthesis type L-threonine dehydratase the microorganism intact cell as biological catalyst, using L-threonine as substrate, produce the method for α-one base butyric acid.
Of the present inventionly using L-threonine and as substrate, produce the method for α-one base butyric acid, 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 dehydratase, with conventional shaking flask or fermentor tank mode aerobic, cultivate in the LB substratum; Separate and collect thalline, washing thalline 2～4 times with pH 7.2～7.5 potassium phosphate buffers, separating the microorganism intact cell obtained and be biological catalyst; Biological catalyst is resuspended in above-mentioned potassium phosphate buffer or deionized water, make the biocatalysis agent concentration reach 200 gram wet cells/liter, namely obtain containing the suspension of biological catalyst, 4 ℃ store for future use;
(2) transform
By the suspension that contains biological catalyst and the L-threonine aqueous solution made in step (1), and make the concentration of L-threonine in 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, with 50～250 rev/mins of oscillatory reactions 1～30 hour, obtain conversion fluid;
(3) produce the solution that contains α-one base butyric acid
By the conversion fluid of step (2) with 5,000～14,000 rev/min centrifugal 5～25 minutes, or with 200～400 order filter-cloth filterings, remove the biological catalyst added in step (2), the gained clear liquid is the solution that contains α-one base butyric acid;
(4) α-one base butyric acid and L-threonine detect
By the above-mentioned solution that contains α-one base butyric 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 rear gained supernatant is sample.
α-one base butyric acid assay adopts high performance liquid chromatography Agilent1100 system, chromatography column is Aminex HPX-87H (U.S.), analysis condition is moving phase for take 10 mM/ls of sulfuric acid, 55 ℃ of column temperatures, flow velocity is 0.4 ml/min, sample size 5 microlitres, adopt differential refraction detector.Mass spectrometric detection is adopted in the evaluation of α-one base butyric acid.
Measure L-threonine concentration 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-one base butyric acid of generation) * 100%.
Above-mentionedly take L-threonine and produce in the method for α-one base butyric acid as substrate:
The described 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 dehydratase of step (1).
The concentration of the described L-threonine of step (2) is 20～60 grams per liters preferably; Described biocatalysis agent concentration preferably 60～120 gram wet cells/liter; Preferably 30～60 ℃ of described temperature; Described pH scope preferably 8.0～10.0; The described reaction times is preferably 2～24 hours.
The present invention selects the microorganism intact cell that contains synthesis type L-threonine dehydratase as biological catalyst, usings L-threonine as substrate, has successfully realized the High-efficient Production of α-one base butyric acid.The inventive method has following characteristics:
(1) yeast culture and reaction time are all shorter.
(2) transformation efficiency of substrate L-threonine generation α-one base butyric acid is high, can reach more than 99.6%.
(3) product α-one base butyric acid can run up to higher concentration.
(4) bacterial strain uses therefor does not need fragmentation, can directly with intact cell, transform, easy to operate.
(5) utilize intact cell catalysis, without adding expensive cofactor.
(6) biological catalyst can pass through to filter or centrifugal removal, and product component is simple, is conducive to later separation and extracts.
The accompanying drawing explanation
Fig. 1: α-one base butyric acid high performance liquid chromatography detected result.Wherein a is standard substance, and b is response sample.
Fig. 2: the α-one base butyric acid mass spectrometric detection result of generation.
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, with conventional shaking flask or fermentor tank mode aerobic, cultivate in the LB substratum; Separate and collect thalline, washing thalline 3 times with pH 7.4 potassium phosphate buffers, separating the microorganism intact cell obtained and be biological catalyst.Biological catalyst is resuspended in potassium phosphate buffer or deionized water, make the biocatalysis agent concentration reach 200 gram wet cells/liter, namely obtain containing the suspension of biological catalyst, 4 ℃ store for future use;
(2) transform
By the suspension that contains biological catalyst and the L-threonine aqueous solution made in step (1), and make the concentration of L-threonine in mixture be 80 grams per liters, biocatalysis agent concentration be 150 gram wet cells/liter; At 20 ℃, under pH 11.0 conditions, with 200 rev/mins of oscillatory reactions 20 hours, obtain conversion fluid;
(3) produce the solution that contains α-one base butyric acid
By above-mentioned conversion fluid, with 12,000 rev/mins centrifugal 20 minutes, remove the biological catalyst added in step (2), the gained stillness of night is the solution that contains α-one base butyric acid.
(4) α-one base butyric acid and L-threonine detect
By the above-mentioned solution that contains α-one base butyric 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 rear gained supernatant is sample.
α-one base butyric acid assay adopts high performance liquid chromatography Agilent1100 system, chromatography column is Aminex HPX-87H (U.S.), analysis condition is moving phase for take 10 mM/ls of sulfuric acid, 55 ℃ of column temperatures, flow velocity is 0.4 ml/min, sample size 5 microlitres, adopt differential refraction detector.Mass spectrometric detection is adopted in the evaluation of α-one base butyric acid.
Measure L-threonine concentration 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-one base butyric acid of generation) * 100%.
After testing, in conversion fluid, the concentration of α-one base butyric acid is 34.2 grams per liters, and transformation efficiency is 49.9%.As shown in Figure 1, the mass spectrometric detection result as shown in Figure 2, confirms that product has the generation of α-one base butyric acid to the high performance liquid chromatography detected result of α-one base butyric acid.The generation of ammonia is arranged, as shown in Figure 3 in the amino acidanalyser working sample.
Product is α-one base butyric acid and ammonia, proves that of the present invention to using the process that L-threonine produces α-one base butyric acid as substrate be by the enzymatic dehydration deamination process of L-threonine dehydration.In addition, because microorganism cells is to cultivate under aerobic conditions, and culturing process do not need to add inductor, so the L-threonine dehydratase that catalytic process relates to is synthesis type.
Embodiment 2:
(1) preparation contains the suspension of biological catalyst
Choose subtilis ATCC23857, with conventional shaking flask or fermentor tank mode aerobic, cultivate in the LB substratum; Separate and collect thalline, washing thalline 3 times with pH 7.4 potassium phosphate buffers, separating the microorganism intact cell obtained and be biological catalyst.Biological catalyst is resuspended in potassium phosphate buffer or deionized water, make the biocatalysis agent concentration reach 200 gram wet cells/liter, namely obtain containing the suspension of biological catalyst, 4 ℃ store for future use;
(2) transform
By the suspension that contains biological catalyst and the L-threonine aqueous solution made in step (1), and make the concentration of L-threonine in mixture be 10 grams per liters, biocatalysis agent concentration be 60 gram wet cells/liter; At 30 ℃, under pH 8.0 conditions, with 200 rev/mins of oscillatory reactions 24 hours, obtain conversion fluid;
(3) produce the solution that contains α-one base butyric acid
By above-mentioned conversion fluid, with 12,000 rev/mins centrifugal 20 minutes, remove the biological catalyst added in step (2), the gained stillness of night is the solution that contains α-one base butyric acid.
(4) α-one base butyric acid and L-threonine detect
After testing, in conversion fluid, the concentration of α-one base butyric acid is 0.64 grams per liter, 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, with conventional shaking flask or fermentor tank mode aerobic, cultivate in the LB substratum; Separate and collect thalline, washing thalline 3 times with pH 7.4 potassium phosphate buffers, separating the microorganism intact cell obtained and be biological catalyst.Biological catalyst is resuspended in potassium phosphate buffer or deionized water, make the biocatalysis agent concentration reach 200 gram wet cells/liter, namely obtain containing the suspension of biological catalyst, 4 ℃ store for future use;
(2) transform
By the suspension that contains biological catalyst and the L-threonine aqueous solution made in step (1), and make the concentration of L-threonine in mixture be 60 grams per liters, biocatalysis agent concentration be 120 gram wet cells/liter; At 65 ℃, under pH 7.0 conditions, with 200 rev/mins of oscillatory reactions 24 hours, obtain conversion fluid;
(3) produce the solution that contains α-one base butyric acid
By above-mentioned conversion fluid, with 12,000 rev/mins centrifugal 20 minutes, remove the biological catalyst added in step (2), the gained stillness of night is the solution that contains α-one base butyric acid.
(4) α-one base butyric acid and L-threonine detect
After testing, in conversion fluid, the concentration of α-one base butyric acid is 19.6 grams per liters, and transformation efficiency is 38.1%.
Embodiment 4:
(1) preparation contains the suspension of biological catalyst
Choose Corynebacterium glutamicum ATCC13032, with conventional shaking flask or fermentor tank mode aerobic, cultivate in the LB substratum; Separate and collect thalline, washing thalline 3 times with pH 7.4 potassium phosphate buffers, separating the microorganism intact cell obtained and be biological catalyst.Biological catalyst is resuspended in potassium phosphate buffer or deionized water, make the biocatalysis agent concentration reach 200 gram wet cells/liter, namely obtain containing the suspension of biological catalyst, 4 ℃ store for future use;
(2) transform
By the suspension that contains biological catalyst and the L-threonine aqueous solution made in step (1), and make the concentration of L-threonine in mixture be 20 grams per liters, biocatalysis agent concentration be 40 gram wet cells/liter; At 60 ℃, under pH 10.0 conditions, with 200 rev/mins of oscillatory reactions 20 hours, obtain conversion fluid;
(3) produce the solution that contains α-one base butyric acid
By above-mentioned conversion fluid, with 12,000 rev/mins centrifugal 20 minutes, remove the biological catalyst added in step (2), the gained stillness of night is the solution that contains α-one base butyric acid.
(4) α-one base butyric acid and L-threonine detect
After testing, in conversion fluid, the concentration of α-one base butyric acid is 12.1 grams per liters, 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, with conventional shaking flask or fermentor tank mode aerobic, cultivate in the LB substratum; Separate and collect thalline, washing thalline 3 times with pH 7.4 potassium phosphate buffers, separating the microorganism intact cell obtained and be biological catalyst.Biological catalyst is resuspended in potassium phosphate buffer or deionized water, make the biocatalysis agent concentration reach 200 gram wet cells/liter, namely obtain containing the suspension of biological catalyst, 4 ℃ store for future use;
(2) transform
By the suspension that contains biological catalyst and the L-threonine aqueous solution made in step (1), and make the concentration of L-threonine in mixture be 30 grams per liters, biocatalysis agent concentration be 60 gram wet cells/liter; At 50 ℃, under pH 8.0 conditions, with 200 rev/mins of oscillatory reactions 6 hours, obtain conversion fluid;
(3) produce the solution that contains α-one base butyric acid
By above-mentioned conversion fluid, with 12,000 rev/mins centrifugal 20 minutes, remove the biological catalyst added in step (2), the gained stillness of night is the solution that contains α-one base butyric acid.
(4) α-one base butyric acid and L-threonine detect
After testing, in conversion fluid, the concentration of α-one base butyric acid is 25.6 grams per liters, and transformation efficiency is 99.6%.
Bacterial strain Pseudomonas stutzeri (P.stutzeri) the SDMCCTCC NO:M206010 that contains synthesis type L-threonine dehydratase related in the present invention is applicant laboratory preservation strain, and this bacterial strain had before carried out patent protection and preservation; Corynebacterium glutamicum ATCC13032 and subtilis ATCC23857 are purchased from US mode culture collection warehousing (American type culture collection).
Claims (1)
1. take the method that L-threonine produces α-one base butyric acid as substrate for one kind, step is:
(1) preparation contains the suspension of biological catalyst
Choose the bacterial strain that Rhodopseudomonas, Corynebacterium or bacillus contain synthesis type L-threonine dehydratase, with conventional shaking flask or fermentor tank mode aerobic, cultivate in the LB substratum; Separate and collect thalline, with pH7.2～7.5 potassium phosphate buffer washing thalline 2～4 times, separating the microorganism intact cell obtained and be biological catalyst; Biological catalyst is resuspended in above-mentioned potassium phosphate buffer or deionized water, make the biocatalysis agent concentration reach 200 gram wet cells/liter, namely obtain containing the suspension of biological catalyst, 4 ° of C store for future use;
(2) transform
By the suspension that contains biological catalyst and the L-threonine aqueous solution made in step (1), and make the concentration of L-threonine in mixture be 10～80 grams per liters, biocatalysis agent concentration be 40～150 gram wet cells/liter; At 20～65 ° of C, under pH7.0～11.0 conditions, with 50～250 rev/mins of oscillatory reactions 1～30 hour, obtain conversion fluid;
(3) produce the solution that contains α-one base butyric acid
By the conversion fluid of step (2) with 5,000～14,000 rev/min centrifugal 5～25 minutes, or with 200～400 order filter-cloth filterings, remove the biological catalyst added in step (2), the gained clear liquid is the solution that contains α-one base butyric acid;
It is characterized in that: the described bacterial strain that contains synthesis type L-threonine dehydratase of step (1) is Pseudomonas stutzeri (P.stutzeri) SDM CCTCC NO:M206010.
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