JP2003284579A - Method of producing 2-ketobutyric acid - Google Patents
Method of producing 2-ketobutyric acidInfo
- Publication number
- JP2003284579A JP2003284579A JP2002094835A JP2002094835A JP2003284579A JP 2003284579 A JP2003284579 A JP 2003284579A JP 2002094835 A JP2002094835 A JP 2002094835A JP 2002094835 A JP2002094835 A JP 2002094835A JP 2003284579 A JP2003284579 A JP 2003284579A
- Authority
- JP
- Japan
- Prior art keywords
- ketobutyric acid
- producing
- threonine
- microorganism
- threonine deaminase
- 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.)
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【0001】[0001]
【発明の属する技術分野】本発明は、2−ケト酪酸およ
び2−ケト酪酸エステル、2−ケト酪酸塩の製造法に関
するものである。2−ケト酪酸を含むケト酸類は、医薬
中間体などの合成原料として使用され、需要が高まりつ
つある。TECHNICAL FIELD The present invention relates to a method for producing 2-ketobutyric acid, 2-ketobutyric acid ester, and 2-ketobutyric acid salt. Keto acids including 2-ketobutyric acid are used as synthetic raw materials for pharmaceutical intermediates and the demand for them is increasing.
【0002】[0002]
【従来の技術】従来、2−ケト酪酸を工業的に合成する
方法として、プロピオン酸ジエチルとシュウ酸ジエチル
を縮合させた後、濃硫酸で加水分解する方法が知られて
いる。また、微生物を用いた2−ケト酪酸の合成法とし
て、ロドコッカス(Rhodococcus)属微生物を用いた
1,2−ブタンジオールからの2−ケト酪酸の製造法
(特開平2−207783)、シュードモナス(Pseudo
monas)属微生物を用いた2,3−ブタンジオールから
の2−ケト酪酸の製造法(特開平3−183476)が
報告されている。また、大腸菌にスレオニンデアミナー
ゼと改変pheAプロモーターを組み込み、スレオニンデア
ミナーゼを発現させた報告(Journal of Molecular Cat
alysisB:199,(2000)、米国特許第6197558号公報)もあ
る。2. Description of the Related Art Conventionally, as a method for industrially synthesizing 2-ketobutyric acid, a method is known in which diethyl propionate and diethyl oxalate are condensed and then hydrolyzed with concentrated sulfuric acid. Further, as a method for synthesizing 2-ketobutyric acid using a microorganism, a method for producing 2-ketobutyric acid from 1,2-butanediol using a microorganism of the genus Rhodococcus (Japanese Patent Application Laid-Open No. 2-207783), Pseudomonas (Pseudo)
A method for producing 2-ketobutyric acid from 2,3-butanediol using a microorganism belonging to the genus monas (JP-A-3-183476) has been reported. In addition, it was reported that threonine deaminase was expressed by incorporating threonine deaminase and a modified pheA promoter into Escherichia coli (Journal of Molecular Cat.
alysisB: 199, (2000), US Pat. No. 6,1975,58).
【0003】[0003]
【発明が解決しようとする課題】しかしながら、1,2
−ブタンジオールおよび2,3−ブタンジオールは工業
的に安価であるとは言い難く、さらに生成した2−ケト
酪酸の濃度も満足できるものではなかった。さらに、上
記報告(Journal of Molecular CatalysisB:199,(200
0)、米国特許第6197558号公報)記載の方法では、2−
ケト酪酸の蓄積量が十分ではない。[Problems to be Solved by the Invention]
It is hard to say that -butanediol and 2,3-butanediol are industrially inexpensive, and the concentration of 2-ketobutyric acid produced was also unsatisfactory. Furthermore, the above report (Journal of Molecular Catalysis B: 199, (200
0) and US Pat. No. 6,1975,58), the method described in 2-
Not enough ketobutyric acid accumulated.
【0004】本発明の課題は、微生物による2−ケト酪
酸の高効率な工業的製造方法を提供することである。An object of the present invention is to provide a highly efficient industrial production method of 2-ketobutyric acid by a microorganism.
【0005】[0005]
【課題を解決するための手段】上記問題点を解決するた
めに、本発明者らは更に生産性の高い2−ケト酪酸の製
造方法について鋭意研究を行った結果、微生物にスレオ
ニンデアミナーゼ遺伝子を組み込み、その微生物を用い
たスレオニンの酵素的変換を行うことにより、2−ケト
酪酸の蓄積濃度、生成収率が向上することを見出し、本
発明に到達した。In order to solve the above-mentioned problems, the inventors of the present invention have conducted diligent research on a method for producing 2-ketobutyric acid, which has higher productivity, and as a result, have incorporated the threonine deaminase gene into microorganisms. The present inventors have found that the concentration of 2-ketobutyric acid accumulated and the production yield are improved by enzymatically converting threonine using the microorganism, and arrived at the present invention.
【0006】すなわち、本発明は「スレオニンデアミナ
ーゼをコードする遺伝子をlacプロモーターまたはT7プ
ロモーターの制御により発現させるよう該遺伝子を組み
込んだ微生物またはその微生物の抽出物を用いて、スレ
オニンから2−ケト酪酸を生産することを特徴とする2
−ケト酪酸の製造方法。」であり、スレオニンデアミナ
ーゼをコードする遺伝子を組み込んだ微生物を培養して
回収し、スレオニンを含む反応液中に該微生物またはそ
の抽出液を添加し、反応液中に2−ケト酪酸を蓄積せし
め、前記反応液より2−ケト酪酸を回収することを特徴
とする2−ケト酪酸の製造方法である。[0006] That is, the present invention "uses a microorganism or an extract of the microorganism in which a gene encoding threonine deaminase is inserted so that the gene is expressed under the control of the lac promoter or T7 promoter, and 2-ketobutyric acid is extracted from threonine. 2 characterized by producing
-Method for producing ketobutyric acid. , Culturing and recovering a microorganism incorporating a gene encoding threonine deaminase, adding the microorganism or an extract thereof to a reaction solution containing threonine, and accumulating 2-ketobutyric acid in the reaction solution, A method for producing 2-ketobutyric acid, which comprises recovering 2-ketobutyric acid from the reaction solution.
【0007】[0007]
【発明の実施の形態】スレオニンはスレオニンデアミナ
ーゼの作用により脱アミノ化され、2−ケト酪酸が生成
する。BEST MODE FOR CARRYING OUT THE INVENTION Threonine is deaminated by the action of threonine deaminase to produce 2-ketobutyric acid.
【0008】本発明に使用する宿主微生物は、微生物で
あるならば特に制限はないが、細菌が好ましく用いら
れ、特に好ましくはエシェリヒア・コリ(Esherichia c
oli)である。The host microorganism used in the present invention is not particularly limited as long as it is a microorganism, but bacteria are preferably used, and Escherichia coli (Esherichia c) is particularly preferable.
oli).
【0009】また、微生物であれば、他に薬剤に対する
耐性、栄養要求性などの性質があってもよい。In addition, microorganisms may have other properties such as drug resistance and auxotrophy.
【0010】宿主に組み込むべきスレオニンデアミナー
ゼをコードする遺伝子は、宿主によって発現され、スレ
オニンデアミナーゼとしての活性が保持されるならば特
に制限はないが、エシェリヒア・コリ(Esherichia col
i)、サルモネラ・ティフィムリウム(Salmonella typh
imurium)、シュードモナス・プチダ(Pseudomonasputi
da)、コリネバクテリウム・グルタミカム(Corynebact
erium glutamicum)、サッカロマイセス・セレビシエ
(Saccharomyces cerevisiae)、キャンディダ・ユティ
リス(Candida utilis)などの遺伝子が知られており、
これらが好ましく用いられる。The gene encoding threonine deaminase to be incorporated into the host is not particularly limited as long as it is expressed by the host and retains the activity as a threonine deaminase, but is not limited to Escherichia coli.
i), Salmonella typh
imurium), Pseudomonasputi
da), Corynebacterium glutamicum (Corynebact
erium glutamicum), Saccharomyces cerevisiae, Candida utilis, and other genes are known.
These are preferably used.
【0011】スレオニンデアミナーゼ遺伝子の取得方法
としては特に制限はなく、PCR法、ゲノムライブラリ
ーやcDNAライブラリーからのスクリーニング法など
が用いられる。The method for obtaining the threonine deaminase gene is not particularly limited, and a PCR method, a screening method from a genomic library or a cDNA library and the like are used.
【0012】取得した遺伝子はColE1タイプの複製起点
をもつpBR322、pACYCタイプの複製起点をもつpSTV28お
よびそれらの派生ベクターに組み込む。特に好ましくは
pUC18/19、pET16bベクター(Novagen社製)が用いられ
る。またスレオニンデアミナーゼの発現を制御するプロ
モーターとしては、lacプロモーターまたはT7プロモー
ターを使用する。The obtained gene is incorporated into pBR322 having a ColE1 type origin of replication, pSTV28 having a pACYC type origin of replication and their derivative vectors. Particularly preferably
pUC18 / 19 and pET16b vectors (Novagen) are used. As a promoter that controls the expression of threonine deaminase, the lac promoter or T7 promoter is used.
【0013】導入方法としては特に制限はないが、例え
ば大腸菌の場合は塩化カルシウム法、エレクトロポレー
ション法などが用いられる。The introduction method is not particularly limited, but for example, in the case of Escherichia coli, the calcium chloride method, electroporation method or the like is used.
【0014】以上のような方法でスレオニンデアミナー
ゼ遺伝子を大量発現する菌体が得られる。By the method as described above, bacterial cells expressing a large amount of the threonine deaminase gene can be obtained.
【0015】本発明における2−ケト酪酸蓄積評価方法
について説明する。2−ケト酪酸は常法に従い、高速液
体クロマトグラフによって評価される。 本発明におけ
る培養方法について説明する。スレオニンデアミナーゼ
発現用培地は炭素源、窒素源、無機イオンおよび必要に
応じてその他の有機微量成分を含有する通常の培地であ
るが、スレオニンデアミナーゼが発現する限り特に制限
はない。The method for evaluating 2-ketobutyric acid accumulation in the present invention will be described. 2-Ketobutyric acid is evaluated by high performance liquid chromatography according to a conventional method. The culture method in the present invention will be described. The medium for expressing threonine deaminase is an ordinary medium containing a carbon source, a nitrogen source, inorganic ions and, if necessary, other organic trace components, but is not particularly limited as long as threonine deaminase is expressed.
【0016】培養は通常、好気的条件下で行うが、スレ
オニンデアミナーゼの発現に応じて適宜条件を変更する
ことが可能である。培養温度は15℃〜45℃、好まし
くは20℃〜35℃が用いられる。The culture is usually carried out under aerobic conditions, but the conditions can be appropriately changed depending on the expression of threonine deaminase. The culture temperature is 15 ° C to 45 ° C, preferably 20 ° C to 35 ° C.
【0017】これらの条件の下に24〜120時間振盪
または攪拌培養することで好ましい結果が得られる。誘
導性プロモーターを用いた場合には、培養中に適宜誘導
物質を添加する方が望ましい。[0024] Under these conditions, preferable results can be obtained by shaking or stirring culture for 24-120 hours. When an inducible promoter is used, it is desirable to add an inducer appropriately during culture.
【0018】スレオニンデアミナーゼを発現させた菌体
は、集菌後酵素反応に用いる。この際そのまま使用して
もよいし、菌体破砕後の抽出液を使用することもでき
る。The bacterial cells expressing threonine deaminase are used in an enzymatic reaction after collecting the bacteria. At this time, it may be used as it is, or an extract obtained by crushing the cells may be used.
【0019】スレオニンを適当量含む緩衝液中に菌体ま
たは抽出液を適宜添加し、振盪または攪拌することで2
−ケト酪酸の生成がみられる。緩衝液はpH緩衝作用を
もつものであればよいが、リン酸カリウム緩衝液、リン
酸ナトリウム緩衝液、酢酸ナトリウム緩衝液などが用い
られる。The cells or the extract are appropriately added to a buffer containing an appropriate amount of threonine, and the mixture is shaken or stirred to
-The formation of ketobutyric acid is seen. Any buffer may be used as long as it has a pH buffering action, and potassium phosphate buffer, sodium phosphate buffer, sodium acetate buffer and the like are used.
【0020】スレオニンの添加量に関して特に制限はな
いが、10mMから1Mが好ましい。The amount of threonine added is not particularly limited, but is preferably 10 mM to 1M.
【0021】反応液中に生成した2−ケト酪酸は、その
まま単離精製することなく利用することもできるし、常
法により単離精製し利用することもできる。The 2-ketobutyric acid formed in the reaction solution can be used as it is without being isolated and purified, or can be isolated and purified by a conventional method.
【0022】また、こうして得られた2−ケト酪酸は公
知の方法によりエステルまたは塩にできる。The 2-ketobutyric acid thus obtained can be converted into an ester or salt by a known method.
【0023】[0023]
【実施例】以下、実施例により本発明を具体的に説明す
る。EXAMPLES The present invention will be specifically described below with reference to examples.
【0024】実施例1
(1)スレオニンデアミナーゼ遺伝子のクローニング
スレオニンから2−ケト酪酸を産生させるために、スレ
オニンデアミナーゼ遺伝子のクローニングを行った。Example 1 (1) Cloning of the threonine deaminase gene In order to produce 2-ketobutyric acid from threonine, the threonine deaminase gene was cloned.
【0025】公知となっているスレオニンデアミナーゼ
遺伝子の塩基配列はデータベース(GenBank)の配列を元
に、エシェリヒア・コリ(Esherichia coli)用のPC
Rプライマーを設計した。PCRに用いたプライマーの
配列は配列表1、配列表2に示した。The known nucleotide sequence of the threonine deaminase gene is a PC for Esherichia coli based on the sequence in the database (GenBank).
The R primer was designed. The sequences of the primers used for PCR are shown in Sequence Listing 1 and Sequence Listing 2.
【0026】これらのプライマーを用い、Esherichia c
oli K12株のゲノムDNAを鋳型としてPCRを行い、1578
塩基対の増幅断片を得た。この増幅断片は大腸菌のベク
ターであるpUC18のHincII切断部位にサブクローニング
し、塩基配列を確認した結果、この増幅断片が実際にEs
herichia coliのスレオニンデアミナーゼ遺伝子をコー
ドしていることを確認した。Using these primers, Esherichia c
PCR was performed using the genomic DNA of the oli K12 strain as a template, and 1578
A base-pair amplified fragment was obtained. This amplified fragment was subcloned into the HincII cleavage site of pUC18, which is an E. coli vector, and the base sequence was confirmed.
It was confirmed that it encodes the threonine deaminase gene of herichia coli.
【0027】(2)発現ベクターの作製
(1)で増幅したDNA断片をNdeIとBamHIで消化した後、
T7プロモーターをもつpET16bベクター(Novagen社製)
のNdeI-BamHIサイトに挿入し、pETTDAプラスミドを構築
した(図)。(2) Preparation of expression vector After digesting the DNA fragment amplified in (1) with NdeI and BamHI,
PET16b vector with T7 promoter (Novagen)
Was inserted into the NdeI-BamHI site of pETTDA to construct a pETTDA plasmid (Fig.).
【0028】(3)宿主への発現ベクターの導入
(2)で作製した発現ベクターpETTDAをEsherichia col
i BL21(DE3)株(Novagen社)に塩化カルシウム法を用い
て導入した。(3) Introduction of the expression vector into the host The expression vector pETTDA prepared in (2) was transformed into Esherichia col.
It was introduced into the iBL21 (DE3) strain (Novagen) using the calcium chloride method.
【0029】発現ベクターを導入後、組換え株の選択は
抗生物質であるアンピシリン耐性を指標に行い、形質転
換体を得た。この形質転換株をEsherichia coli TDA株
と命名した。After the introduction of the expression vector, the recombinant strain was selected with the resistance to the antibiotic ampicillin as an index to obtain a transformant. This transformant was named Esherichia coli TDA strain.
【0030】実施例2(形質転換株でのスレオニンデア
ミナーゼ発現)
Esherichia coli BL21(DE3)株、Esherichia coli TDA株
を培養した。すなわち、これらの菌株を各々LB培地5
mlに1白金耳植菌し、37℃で16時間振盪して前培
養した。Example 2 (Expression of threonine deaminase in transformant) Esherichia coli BL21 (DE3) strain and Esherichia coli TDA strain were cultured. That is, each of these strains was added to LB medium 5
1 platinum loop was inoculated in ml and precultured by shaking at 37 ° C. for 16 hours.
【0031】次に、前培養液を新たなLB培地10ml
に1%植菌し、37℃で6時間振盪培養した。培養液に
イソプロピルチオ−β−D−ガラクトピラノシド(IPT
G)を終濃度1mMになるように添加し、さらに37℃
で16時間振盪した。続いて培養液を遠心分離し、菌体
を得た。Next, the preculture liquid was added to 10 ml of a fresh LB medium.
Was inoculated with 1% and cultured at 37 ° C. for 6 hours with shaking. Isopropylthio-β-D-galactopyranoside (IPT
G) was added to a final concentration of 1 mM, and then 37 ° C.
It was shaken for 16 hours. Subsequently, the culture solution was centrifuged to obtain bacterial cells.
【0032】実施例3(形質転換株を用いた2−ケト酪
酸生成)
50mMのリン酸カリウム緩衝液10mlに0.3gのスレオニンを
加えた溶液に、得られた菌体10ml培養液相当を添加し3
0℃で24時間酵素反応を行った。Example 3 (Production of 2-ketobutyric acid using transformant) To a solution obtained by adding 0.3 g of threonine to 10 ml of 50 mM potassium phosphate buffer solution, 10 ml of the obtained bacterial cell culture solution was added. Three
The enzymatic reaction was carried out at 0 ° C. for 24 hours.
【0033】反応終了後、高速液体クロマトグラフィー
を用いて定量した。カラムはSCR-101H(島津製作所製)
を使用し、緩衝液は0.1%リン酸、210nmの吸光を測定し
た。その結果を以下表1に示した。After completion of the reaction, quantification was performed using high performance liquid chromatography. Column is SCR-101H (Shimadzu)
The buffer solution was 0.1% phosphoric acid, and the absorbance at 210 nm was measured. The results are shown in Table 1 below.
【0034】[0034]
【表1】 [Table 1]
【0035】コントロールであるEsherichia coli BL21
(DE3)株に比較して組換え株であるEsherichia coli TDA
株では2−ケト酪酸の著量蓄積が見られた。Control Esherichia coli BL21
Escherichia coli TDA, which is a recombinant strain compared to the (DE3) strain
The strain showed a significant accumulation of 2-ketobutyric acid.
【0036】実施例4(形質転換株抽出液を用いた2−
ケト酪酸生成)
50mMのリン酸カリウム緩衝液10mlに0.6gのスレオニンを
加えた溶液に、得られた菌体1ml培養液相当を超音波破
砕した上清を添加し30℃で24時間酵素反応を行っ
た。Example 4 (2-using a transformant extract)
Ketobutyric acid production) To a solution obtained by adding 0.6 g of threonine to 10 ml of 50 mM potassium phosphate buffer solution, the supernatant obtained by ultrasonically crushing 1 ml of the obtained bacterial cell culture solution was added, and the enzyme reaction was performed at 30 ° C for 24 hours. went.
【0037】反応終了後、高速液体クロマトグラフィー
を用いて定量した。カラムはSCR-101H(島津製作所製)
を使用し、緩衝液は0.1%リン酸、210nmの吸光を測定し
た。その結果を以下表2に示した。After the reaction was completed, the amount was quantified by high performance liquid chromatography. Column is SCR-101H (Shimadzu)
The buffer solution was 0.1% phosphoric acid, and the absorbance at 210 nm was measured. The results are shown in Table 2 below.
【0038】[0038]
【表2】 [Table 2]
【0039】コントロールであるEsherichia coli BL21
(DE3)株に比較して組換え株であるEsherichia coli TDA
株では2−ケト酪酸の著量蓄積が見られた。Control Esherichia coli BL21
Escherichia coli TDA, which is a recombinant strain compared to the (DE3) strain
The strain showed a significant accumulation of 2-ketobutyric acid.
【0040】[0040]
【発明の効果】本発明の微生物を用い、酵素法により2
−ケト酪酸を反応液中に生産すると、既存の方法に比較
してより経済的な2−ケト酪酸の生産が可能となる。EFFECT OF THE INVENTION Using the microorganism of the present invention, the enzyme method 2
-Production of ketobutyric acid in the reaction liquid enables more economical production of 2-ketobutyric acid as compared to existing methods.
【0041】[0041]
【配列表】 SEQUENCE LISTING <110> TORAY <120> 2−ケト酪酸の製造方法 <130> 51E16560 <160> 2 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> PCR primer to obtain Threonine deaminase from Esherichia coli <400> actggggggt tacatatggc tgactcgcaa <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> PCR primer to obtain Threonine deaminase from Esherichia coli <400> gctatcaggc atggatccct aacccgccaa[Sequence list] SEQUENCE LISTING <110> TORAY <120> Method for producing 2-ketobutyric acid <130> 51E16560 <160> 2 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> PCR primer to obtain Threonine deaminase from Esherichia coli <400> actggggggt tacatatggc tgactcgcaa <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> PCR primer to obtain Threonine deaminase from Esherichia coli <400> gctatcaggc atggatccct aacccgccaa
【図1】 スレオニンデアミナーゼ発現ベクターpETTDA
のフィジカルマップを示す図である。Figure 1: Threonine deaminase expression vector pETTDA
It is a figure which shows the physical map of.
Claims (6)
伝子をlacプロモーターまたはT7プロモーターの制御に
より発現させるよう該遺伝子を組み込んだ微生物または
その微生物の抽出物を用いて、スレオニンから2−ケト
酪酸を生産することを特徴とする2−ケト酪酸の製造方
法。1. A method of producing 2-ketobutyric acid from threonine using a microorganism or an extract of the microorganism in which a gene encoding threonine deaminase is expressed so as to be expressed under the control of lac promoter or T7 promoter. A method for producing 2-ketobutyric acid, which is characterized.
伝子がエシェリヒア(Esherichia)属、サルモネラ(Sa
lmonella)属、シュードモナス(Pseudomonas)属、コ
リネバクテリウム(Corynebacterium)属、サッカロマ
イセス(Saccharomyces)属、キャンディダ(Candida)
属のいずれか由来であることを特徴とする請求項1に記
載の2−ケト酪酸の製造方法。2. A gene encoding threonine deaminase is a genus of Esherichia, Salmonella (Sa).
Lmonella), Pseudomonas, Corynebacterium, Saccharomyces, Candida
The method for producing 2-ketobutyric acid according to claim 1, which is derived from any of the genera.
求項1または2に記載の2−ケト酪酸の製造方法。3. The method for producing 2-ketobutyric acid according to claim 1 or 2, wherein the microorganism is a bacterium.
請求項3に記載の2−ケト酪酸の製造方法。4. The method for producing 2-ketobutyric acid according to claim 3, wherein the microorganism is Escherichia coli.
−ケト酪酸をエステル化することを特徴とする2−ケト
酪酸エステルの製造方法。5. The obtained in any one of claims 1 to 4.
-A method for producing a 2-ketobutyric acid ester, which comprises esterifying ketobutyric acid.
−ケト酪酸を中和し塩にすることを特徴とする2−ケト
酪酸塩の製造方法。6. The obtained in any one of claims 1 to 4.
-A method for producing 2-ketobutyric acid salt, which comprises neutralizing ketobutyric acid to form a salt.
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2005333907A (en) * | 2004-05-28 | 2005-12-08 | Kyowa Hakko Kogyo Co Ltd | Method for producing 5-aminolevulinic acid |
CN102433360A (en) * | 2011-11-23 | 2012-05-02 | 山东大学 | Method for preparing alpha-ketobutyric acid by using L-threonine as substrate |
CN105461544A (en) * | 2016-01-28 | 2016-04-06 | 天津科技大学 | Method for extracting 2-ketobutyric acid from enzymatic conversion solution |
-
2002
- 2002-03-29 JP JP2002094835A patent/JP2003284579A/en active Pending
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2005333907A (en) * | 2004-05-28 | 2005-12-08 | Kyowa Hakko Kogyo Co Ltd | Method for producing 5-aminolevulinic acid |
JP4520219B2 (en) * | 2004-05-28 | 2010-08-04 | 協和発酵バイオ株式会社 | Process for producing 5-aminolevulinic acid |
CN102433360A (en) * | 2011-11-23 | 2012-05-02 | 山东大学 | Method for preparing alpha-ketobutyric acid by using L-threonine as substrate |
CN105461544A (en) * | 2016-01-28 | 2016-04-06 | 天津科技大学 | Method for extracting 2-ketobutyric acid from enzymatic conversion solution |
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