JPH0328188B2 - - Google Patents
Info
- Publication number
- JPH0328188B2 JPH0328188B2 JP57095293A JP9529382A JPH0328188B2 JP H0328188 B2 JPH0328188 B2 JP H0328188B2 JP 57095293 A JP57095293 A JP 57095293A JP 9529382 A JP9529382 A JP 9529382A JP H0328188 B2 JPH0328188 B2 JP H0328188B2
- Authority
- JP
- Japan
- Prior art keywords
- alanine
- alanine dehydrogenase
- action
- dehydrogenase
- pyruvic acid
- 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.)
- Expired - Lifetime
Links
- 108010031025 Alanine Dehydrogenase Proteins 0.000 claims description 31
- 229960003767 alanine Drugs 0.000 claims description 18
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 claims description 16
- QNAYBMKLOCPYGJ-UWTATZPHSA-N L-Alanine Natural products C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 claims description 16
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 16
- 230000000694 effects Effects 0.000 claims description 16
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 7
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims description 6
- 241000589499 Thermus thermophilus Species 0.000 claims description 6
- 238000002523 gelfiltration Methods 0.000 claims description 6
- 229940107700 pyruvic acid Drugs 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 239000000758 substrate Substances 0.000 claims description 4
- 241000894006 Bacteria Species 0.000 claims description 3
- 229940024606 amino acid Drugs 0.000 claims description 3
- 150000001413 amino acids Chemical class 0.000 claims description 3
- 125000003277 amino group Chemical group 0.000 claims description 3
- 238000007833 oxidative deamination reaction Methods 0.000 claims description 3
- 230000002441 reversible effect Effects 0.000 claims description 3
- 229960001153 serine Drugs 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 235000015097 nutrients Nutrition 0.000 claims description 2
- 230000009849 deactivation Effects 0.000 claims 2
- 108090000790 Enzymes Proteins 0.000 description 13
- 102000004190 Enzymes Human genes 0.000 description 13
- 239000000872 buffer Substances 0.000 description 12
- 239000000243 solution Substances 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 5
- 235000011130 ammonium sulphate Nutrition 0.000 description 5
- 239000012141 concentrate Substances 0.000 description 5
- 229920001223 polyethylene glycol Polymers 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 2
- TUENNOFVPLQEJC-UHFFFAOYSA-L [OH-].[Cl-].[K+].[K+].NCC(O)=O Chemical compound [OH-].[Cl-].[K+].[K+].NCC(O)=O TUENNOFVPLQEJC-UHFFFAOYSA-L 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229940076788 pyruvate Drugs 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 206010006500 Brucellosis Diseases 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 241000589596 Thermus Species 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229940093429 polyethylene glycol 6000 Drugs 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
Description
本発明はＬ−アラニンデヒドロゲナーゼYK−
１およびその製造法に関する。
Ｌ−アラニンデヒドロゲナーゼは以下の反応を
触媒する酵素として知られている。
Ｌ−Alanine＋H2O＋NADPyruvate＋NH3
＋NADHL−アラニンデヒドロゲナーゼは、ピ
ルビン酸からのＬ−アラニンの酵素的合成あるい
はＬ−アラニン、ピルビン酸等の定量等の応用面
が考えられるが、実際上余り実用化されていな
い。この理由としては、まず第１には従来のＬ−
アラニンデヒドロゲナーゼは工業的に利用するう
えで安定性に問題があつたからである。即ち工業
的にＬ−アラニンを生産させるためには、酵素を
固定化したり、反応温度を高めたりする必要があ
り、より安定な酵素が求められているが従来のも
のでは不十分であつた。つぎにKm値が大きすぎ
ることも問題であつた。Ｌ−アラニンを酵素的に
測定する場合、微量に存在するＬ−アラニンと反
応する必要があり、従つてKm値の小さいＬ−ア
ラニンデヒドロゲナーゼが要望されるからであ
る。
従来、比較的安定性の高いＬ−アラニンデヒド
ロゲナーゼとしては、サーマス・サーモフイラス
（Z.Vali.、Biochimica et Biophisica Acta615
巻、34頁、1980年）、バチルス・ズブチリス（A.
Yoshida.、Biochimica et Biophisica Acta96
巻、248頁、1965年）、バチルス属菌（I.Epstein.、
Biochimica et Biophisica Acta445巻、549頁、
1976年）等が知られていたが、本発明者は、さら
に安定性に優れたＬ−アラニンデヒドロゲナーゼ
について鋭意探索したところ、サーマス・フラバ
スに属する菌株サーマス・フラバスAT−62を培
養することによつて、培養物中に著量の熱安定性
に優れかつまたKm値の小さい新規なＬ−アラニ
ンデヒドロゲナーゼYK−１を生産蓄積すること
を見出し、本発明を完成したものである。サーマ
ス・フラバスAT−62の菌学的性質については
（T.Saikiら、Agr.Biol.Chem.、36巻、2357〜
2366頁、1972年）に記載されており、又本菌は工
業技術院微生物工業研究所に微工研菌寄第6529
（FERM−ＰNo.6529）として寄託されている。
本発明のＬ−アラニンデヒドロゲナーゼは次の
理化学的性質を有している。
(1) 作用：Ｌ−アラニンを酸化的脱アミノ反応に
よりピルビン酸にする作用およびピルビン酸に
アミノ基を付加してＬ−アラニンを生成する可
逆反応を触媒する作用を有する。
(2) 基質特異性：Ｌ−アラニンに特異的に作用
し、Ｌ−セリンには1.5％の相対力価を示した
が、他のアミノ酸にはほとんど作用しない。
(3) 至適PH：本酵素の至適PHは10〜11付近にある
（第１図に示す通り）。なお用いた緩衝液として
は、PH9.2〜10.3では50ｍＭグリシン−塩化カ
リウム−水酸化カリウム緩衝液、PH10.3〜
11.05では、50ｍＭ燐酸ニナトリウム緩衝液で
ある。
(4) 安定性：50℃、60分処理した場合、PH6.0〜
8.5付近で最も安定である（第２図に示す通
り）。
(5) 至適温度：60〜70℃付近にある（第３図に示
す通り）。
(6) 熱安定性：80℃、80分処理では全く失活せ
ず、90℃、80分処理で約30％の失活が認められ
る（第４図に示す通り）。
(7) 阻害剤に対する影響：
(a) 各種阻害剤1.0ｍＭの影響について表１に
示す。
The present invention provides L-alanine dehydrogenase YK-
1 and its manufacturing method. L-alanine dehydrogenase is known as an enzyme that catalyzes the following reactions. L−Alanine+ H2O +NADPyruvate+ NH3
+NADHL-alanine dehydrogenase can be considered for applications such as the enzymatic synthesis of L-alanine from pyruvate or the quantification of L-alanine, pyruvate, etc., but it has not been put into practical use much. The reason for this is, firstly, that the conventional L-
This is because alanine dehydrogenase has had stability problems when used industrially. That is, in order to industrially produce L-alanine, it is necessary to immobilize the enzyme or raise the reaction temperature, and a more stable enzyme is required, but conventional enzymes have not been sufficient. Another problem was that the Km value was too large. This is because when measuring L-alanine enzymatically, it is necessary to react with a trace amount of L-alanine, and therefore an L-alanine dehydrogenase with a small Km value is required. Conventionally, as L-alanine dehydrogenase with relatively high stability, Z. Vali., Biochimica et Biophisica Acta615
Vol. 34, 1980), Bacillus subtilis (A.
Yoshida., Biochimica et Biophisica Acta96
Vol. 248, 1965), Bacillus (I. Epstein.
Biochimica et Biophisica Acta volume 445, page 549,
1976), etc., but the present inventor conducted an intensive search for L-alanine dehydrogenase with even better stability, and discovered it by culturing Thermus flavus AT-62, a strain belonging to Thermus flavus. Therefore, the present invention was completed by discovering that a novel L-alanine dehydrogenase YK-1, which has excellent thermostability and a small Km value, can be produced and accumulated in a culture. Regarding the mycological properties of Thermus flavus AT-62 (T. Saiki et al., Agr. Biol. Chem., vol. 36, 2357-
2366, 1972), and this bacterium was submitted to the Institute of Microbiology, Agency of Industrial Science and Technology, Microbiological Research Institute No. 6529.
(FERM-P No. 6529). The L-alanine dehydrogenase of the present invention has the following physical and chemical properties. (1) Action: It has the action of converting L-alanine into pyruvic acid by oxidative deamination reaction and the action of catalyzing the reversible reaction of adding an amino group to pyruvic acid to produce L-alanine. (2) Substrate specificity: Acts specifically on L-alanine, showing a relative potency of 1.5% on L-serine, but has almost no effect on other amino acids. (3) Optimal PH: The optimal PH of this enzyme is around 10 to 11 (as shown in Figure 1). The buffers used were 50mM glycine-potassium chloride-potassium hydroxide buffer for pH9.2-10.3, and 50mM glycine-potassium chloride-potassium hydroxide buffer for pH10.3-10.
11.05, 50mM disodium phosphate buffer. (4) Stability: PH6.0~ when treated at 50℃ for 60 minutes
It is most stable around 8.5 (as shown in Figure 2). (5) Optimum temperature: around 60-70℃ (as shown in Figure 3). (6) Thermal stability: There is no inactivation at all when treated at 80°C for 80 minutes, and approximately 30% inactivation is observed when treated at 90°C for 80 minutes (as shown in Figure 4). (7) Effects on inhibitors: (a) Table 1 shows the effects of 1.0mM of various inhibitors.
【表】
(b) 金属イオン（1.0ｍＭ）等の影響について
表２に示す。[Table] (b) Table 2 shows the effects of metal ions (1.0mM), etc.
【表】【table】
【表】
ある。
(8) 分子量：約290000〜300000（ゲル過法によ
る）
(9) 等電点：7.56（焦点電気泳動法による）
(10) Km値：約３×10-4M
上記理化学的性質を持つたＬ−アラニンデヒド
ロゲナーゼYK−１は他の微生物起源のＬ−アラ
ニンデヒドロゲナーゼとは明らかに異なつた新規
酵素である。
表３に各種微生物起源の耐熱性Ｌ−アラニンデ
ヒドロゲナーゼと本酵素との比較を示す。[Table] Yes.
(8) Molecular weight: approximately 290,000 to 300,000 (by gel filtration method) (9) Isoelectric point: 7.56 (by focal electrophoresis method) (10) Km value: approximately 3×10 -4 M L-alanine dehydrogenase YK-1 is a novel enzyme that is clearly different from L-alanine dehydrogenases derived from other microorganisms. Table 3 shows a comparison between heat-stable L-alanine dehydrogenases derived from various microorganisms and this enzyme.
【表】
表３より明らかの如く、本発明のＬ−アラニン
デヒドロゲナーゼYK−１（生産菌：サーマス・
フラバスAT−62）は他のいずれの酵素よりKm
値が小さく、かつ安定性に優れていることがわか
る。本発明の新規Ｌ−アラニンデヒドロゲナーゼ
YK−１はサーマス・フラバスAT−62（FERM−
ＰNo.6529）を培養することによつて得られるが、
使用する培地としては、炭素源、窒素源、無機物
その他栄養素を程よく含有する培地ならば、合成
培地または天然培地のいずれも使用可能であり、
液状でも固状でもよいが、通常は液体培地を使用
する。
培養条件としては、培養開始時のPHは６〜８の
範囲で、培養温度は60〜80℃の範囲で行われる。
このような条件下で、12〜50時間培養することに
よつて培養物中にＬ−アラニンデヒドロゲナーゼ
YK−１が著量生成する。
こうして培養物中に生産蓄積されたＬ−アラニ
ンデヒドロゲナーゼYK−１は次の如き方法で採
取される。Ｌ−アラニンデヒドロゲナーゼYK−
１は主に菌体中に存在するので、培養終了後、菌
体は遠心分離、過等の方法で集められ、水また
は緩衝液で洗浄し、次いでPH５〜８の適当な緩衝
液に懸濁し、ダイノーミル等で菌体を磨砕し、菌
体内Ｌ−アラニンデヒドロゲナーゼYK−１を抽
出する。
こうして菌体抽出物より得られる粗Ｌ−アラニ
ンデヒドロゲナーゼYK−１をさらに精製するに
は、硫安塩析、透析、DEAE−セルロースカラム
クロマトグラフイー、ブルーセフアロースカラム
クロマトグラフイー、濃縮、セフアデツクスＧ−
200ゲル過等の通常の精製手段が用いられる。
次に本発明において用いたＬ−アラニンデヒド
ロゲナーゼYK−１の活性測定法を示す。
44ｍML−アラニン水溶液0.1ml、50ｍＭグリシ
ン緩衝液（PH10.0）2.56ml、および14ｍMNAD水
溶液0.1mlをキユベツト（径1.0cm）にとり、37
℃、５分間予備加温する。次いで、酵素液を加え
て37℃で340nmの吸光度を２〜３分間記録し、直
線部分から１分間当りの吸光度の変化を求める
（ΔODt）。一方、コントロールとして基質の代り
に水を用いたもので上記と同様の操作を行い吸光
度の変化を求める（ΔODb）。
Ｌ−アラニンデヒドロゲナーゼの単位は37℃で
１分間に1μmoleのNADを還元する酵素量と定義
する。一方、１ｍＭのNADの吸光係数は6.22と
報告されているから、求める酵素溶液１ml当りの
力価は次式より求められる。
ｕ／ml＝（ΔODt−ΔODb）×2.95／6.22×0.1
以下実施例について述べる。
実施例 １
ポリペプトン2.0％、酵母エキス1.0％からなる
培地（PH7.2、120℃、20分間滅菌）300ml有する
坂口フラスコにサーマス・フラバスAT−62
（FERM−ＰNo.6529）を接種し68℃、24時間培養
して種菌を調製した。次いでこの種菌を上記と同
一組成からなる培地20を有する30容ジヤーフ
アーメンターに接種し、72℃、18時間通気撹拌培
養し、培養終了後、培養物を6000rpm、10分間遠
心分離して集菌し、この菌体を0.85％の食塩水で
洗浄後、0.2M燐酸緩衝液（PH7.0）２に懸濁せ
しめ、ダイノーミル（Dyno−Mill：WillyA.
Bachofen社製スイス）にかけ菌体を磨砕した。
この磨砕液を10000rpm、20分間遠心分離し上清
液を得る。この上清液に硫安を加え、硫安45％飽
和で沈澱する区分を採取する。この沈殿を0.2M
燐酸緩衝液（PH7.0）200mlに再び溶解した後、
40000rpm、30分間遠心分離して不溶物を除去し、
上清液に硫安を加え、硫安30〜45％飽和で沈澱す
る区分を集め、10ｍＭトリス−塩酸緩衝液（PH
7.65）100mlに溶解後、同緩衝液10で24時間透
析した。この透析して得られた液の酵素活性は
435単位であつた。
次いで透析液を同緩衝液で平衡化したDEAE−
セルロースカラム（2.6×32cm）に通過させ、食
塩濃度0.1〜0.2Mにて吸着された蛋白質を溶出せ
しめ、溶出液を集め、カーボワツクス（別名ポリ
エチレングリコール6000）にて濃縮した。こうし
て得られた濃縮液のＬ−アラニンデヒドロゲナー
ゼ活性は284単位であつた。濃縮液47mlを10ｍＭ
トリス−塩酸緩衝液（PH7.65）に対して透析した
後、80℃、20分間の加熱処理を行い、微量の沈澱
物を10000rpm、10分間の遠心分離により除去し
た。加熱処理後の液のＬ−アラニンデヒドロゲナ
ーゼの活性は273単位であつた。
次に、同緩衝液で平衡化したDEAE−セルロー
スカラム（2.6×22cm）に上記酵素液を通過させ、
食塩濃度が０〜0.3Mである直線勾配にて溶出せ
しめ、食塩濃度0.15M付近で溶出されるＬ−アラ
ニンデヒドロゲナーゼ活性区分を集め、カーボワ
ツクスにて濃縮した。この濃縮液38ml（Ｌ−アラ
ニンデヒドロゲナーゼ活性は216単位であつた。）
を50ｍＭトリス−塩酸緩衝液（PH7.65）に対して
透析した後、同緩衝液で緩衝化したブルーセフア
ロースカラム（２×12cm）に通過させ、NADH
濃度が０〜0.1ｍＭである直線勾配にて溶出せし
めた活性区分を集めカーボワツクスにて濃縮し
た。この濃縮液５ml（Ｌ−アラニンデヒドロゲナ
ーゼ活性は168単位である。）を50ｍＭ燐酸緩衝液
（PH6.5）で予め緩衝化したセフアデツクスＧ−
200カラム（２×75cm）チヤージしてゲル過を
行い、活性区分を集めた後、再度カーボワツクス
濃縮し、同カラムにてゲル過を行い、活性区分
を集め、コロジオンバツグにより濃縮し、最終的
にＬ−アラニンデヒドロゲナーゼ活性136u／ml、
OD280nｍ＝9.7なるＬ−アラニンデセドロゲナー
ゼYK−１の精製溶液を1.0mlを得た。[Table] As is clear from Table 3, L-alanine dehydrogenase YK-1 of the present invention (producing bacteria: Thermus
flavus AT-62) is more Km than any other enzyme.
It can be seen that the value is small and the stability is excellent. Novel L-alanine dehydrogenase of the present invention
YK-1 is Thermus Flavas AT-62 (FERM-
P No. 6529), but
As the medium to be used, either a synthetic medium or a natural medium can be used as long as it contains a suitable amount of carbon sources, nitrogen sources, inorganic substances, and other nutrients.
Although it may be in liquid or solid form, a liquid medium is usually used. As for the culture conditions, the pH at the start of culture is in the range of 6 to 8, and the culture temperature is in the range of 60 to 80°C.
By culturing for 12 to 50 hours under these conditions, L-alanine dehydrogenase is produced in the culture.
YK-1 produces a significant amount. L-alanine dehydrogenase YK-1 thus produced and accumulated in the culture is collected by the following method. L-alanine dehydrogenase YK-
1 is mainly present in the bacterial cells, so after the completion of the culture, the bacterial cells are collected by centrifugation, sieving, etc., washed with water or a buffer solution, and then suspended in an appropriate buffer solution with a pH of 5 to 8. The bacterial cells are ground using a dyno mill or the like, and intracellular L-alanine dehydrogenase YK-1 is extracted. In order to further purify the crude L-alanine dehydrogenase YK-1 obtained from the bacterial cell extract, the steps are ammonium sulfate salting out, dialysis, DEAE-cellulose column chromatography, Brucellose column chromatography, concentration, and Sephadex G-
Conventional purification procedures such as 200 gel filtration are used. Next, a method for measuring the activity of L-alanine dehydrogenase YK-1 used in the present invention will be described. Place 0.1 ml of 44mM-alanine aqueous solution, 2.56ml of 50mM glycine buffer (PH10.0), and 0.1ml of 14mM NAD aqueous solution in a cuvette (diameter 1.0cm), and add 37
Prewarm at ℃ for 5 minutes. Next, the enzyme solution is added and the absorbance at 340 nm is recorded at 37° C. for 2 to 3 minutes, and the change in absorbance per minute is determined from the linear portion (ΔODt). On the other hand, as a control, water was used instead of the substrate and the same procedure as above was performed to determine the change in absorbance (ΔODb). A unit of L-alanine dehydrogenase is defined as the amount of enzyme that reduces 1 μmole of NAD per minute at 37°C. On the other hand, since the extinction coefficient of 1mM NAD is reported to be 6.22, the desired titer per ml of enzyme solution can be calculated from the following formula. u/ml=(ΔODt−ΔODb)×2.95/6.22×0.1 Examples will be described below. Example 1 Thermus flavus AT-62 was placed in a Sakaguchi flask containing 300 ml of a medium consisting of 2.0% polypeptone and 1.0% yeast extract (PH7.2, sterilized at 120°C for 20 minutes).
(FERM-P No. 6529) was inoculated and cultured at 68°C for 24 hours to prepare an inoculum. Next, this inoculum was inoculated into a 30-volume jar fermenter containing medium 20 with the same composition as above, and cultured at 72°C for 18 hours with aeration. After the culture was completed, the culture was collected by centrifugation at 6000 rpm for 10 minutes. After washing the bacterial cells with 0.85% saline, they were suspended in 0.2M phosphate buffer (PH7.0) 2, and Dyno-Mill (WillyA.
Bachofen (Switzerland)) to grind the bacterial cells.
This homogenate is centrifuged at 10,000 rpm for 20 minutes to obtain a supernatant. Add ammonium sulfate to this supernatant, and collect the fraction that precipitates at 45% ammonium sulfate saturation. 0.2M of this precipitate
After redissolving in 200ml of phosphate buffer (PH7.0),
Centrifuge at 40,000 rpm for 30 minutes to remove insoluble matter.
Add ammonium sulfate to the supernatant, collect the fraction that precipitates at 30-45% saturation with ammonium sulfate, and add 10mM Tris-HCl buffer (PH
7.65) After dissolving in 100 ml, it was dialyzed against the same buffer 10 for 24 hours. The enzyme activity of the solution obtained by this dialysis is
It was 435 units. Then, the dialysate was equilibrated with the same buffer and DEAE-
The mixture was passed through a cellulose column (2.6 x 32 cm), and the adsorbed protein was eluted at a salt concentration of 0.1 to 0.2 M. The eluate was collected and concentrated with Carbowax (also known as polyethylene glycol 6000). The L-alanine dehydrogenase activity of the concentrate thus obtained was 284 units. 47ml of concentrated solution to 10mM
After dialysis against Tris-HCl buffer (PH7.65), heat treatment was performed at 80°C for 20 minutes, and a trace amount of precipitate was removed by centrifugation at 10,000 rpm for 10 minutes. The L-alanine dehydrogenase activity of the solution after heat treatment was 273 units. Next, the enzyme solution was passed through a DEAE-cellulose column (2.6 x 22 cm) equilibrated with the same buffer.
Elution was performed using a linear gradient with a salt concentration of 0 to 0.3M, and the L-alanine dehydrogenase activity fraction eluted at a salt concentration of around 0.15M was collected and concentrated using carbo wax. 38 ml of this concentrate (L-alanine dehydrogenase activity was 216 units)
After dialyzing against 50mM Tris-HCl buffer (PH7.65), it was passed through a BlueSepharose column (2 x 12cm) buffered with the same buffer, and NADH
The active fraction eluted with a linear gradient having a concentration of 0 to 0.1 mM was collected and concentrated with carbo wax. 5ml of this concentrate (L-alanine dehydrogenase activity is 168 units) was pre-buffered with 50mM phosphate buffer (PH6.5).
Charge a 200 column (2 x 75 cm) and perform gel filtration, collect the active fraction, concentrate it again with Carbowax, perform gel filtration on the same column, collect the active fraction, concentrate with a collodion bag, and finally L-alanine dehydrogenase activity 136u/ml,
1.0 ml of a purified solution of L-alanine decedrogenase YK-1 having an OD280nm of 9.7 was obtained.
第１図、第２図、第３図および第４図は本発明
によるＬ−アラニンデヒドロゲナーゼYK−１の
至適PH、安定PH、至適温度および熱安定性をそれ
ぞれ示す図である。なお第４図において白丸は、
80℃で処理した場合であり、黒丸は、90℃で処理
した場合である。
FIG. 1, FIG. 2, FIG. 3, and FIG. 4 are diagrams showing the optimal pH, stable pH, optimal temperature, and thermostability of L-alanine dehydrogenase YK-1 according to the present invention, respectively. In addition, in Figure 4, the white circles are
This is the case when the treatment was performed at 80°C, and the black circle is the case when the treatment was performed at 90°C.
Claims (1)
を特徴とするＬ−アラニンデヒドロゲナーゼYK
−１。 (1) 作用：Ｌ−アラニンを酸化的脱アミノ反応に
よりピルビン酸にする作用およびピルビン酸に
アミノ基を付加してＬ−アラニンを生成する可
逆反応を触媒する。 (2) 基質特異性：Ｌ−アラニンに特異性を有し、
Ｌ−セリンに僅かに作用するが、他のアミノ酸
には実質的に作用しない。 (3) 至適PH：10〜11付近 (4) 至適温度：60〜70℃ (5) 熱安定性：80℃、80分処理では実質的に失活
しない。 (6) Km値：３×10-4M (7) 分子量：約290000〜300000（ゲルろ過法）。 ２ サーマス・フラバスに属するＬ−アラニンデ
ヒドロゲナーゼYK−１生産菌を栄養培地に培養
し、得られた培養物から少なくとも下記の理化学
的性質を有することを特徴とするＬ−アラニンデ
ヒドロゲナーゼYK−１を採取することを特徴と
するＬ−アラニンデヒドロゲナーゼYK−１の製
造法。 (1) 作用：Ｌ−アラニンを酸化的脱アミノ反応に
よりピルビン酸にする作用およびピルビン酸に
アミノ基を付加してＬ−アラニンを生成する可
逆反応を触媒する。 (2) 基質特異性：Ｌ−アラニンに特異性を有し、
Ｌ−セリンに僅かに作用するが、他のアミノ酸
には実質的に作用しない。 (3) 至適PH：10〜11付近 (4) 至適温度：60〜70℃ (5) 熱安定性：80℃、80分処理では実質的に失活
しない。 (6) Km値：３×10-4M (7) 分子量：約290000〜300000（ゲルろ過法）。[Claims] 1. L-alanine dehydrogenase YK characterized by having at least the following physicochemical properties:
-1. (1) Action: Catalyzes the action of converting L-alanine into pyruvic acid through oxidative deamination reaction and the reversible reaction of adding an amino group to pyruvic acid to produce L-alanine. (2) Substrate specificity: has specificity for L-alanine,
It has a slight effect on L-serine, but has no substantial effect on other amino acids. (3) Optimum pH: Around 10-11 (4) Optimum temperature: 60-70°C (5) Thermal stability: Substantially no deactivation after treatment at 80°C for 80 minutes. (6) Km value: 3×10 -4 M (7) Molecular weight: approximately 290,000 to 300,000 (gel filtration method). 2. Culture L-alanine dehydrogenase YK-1-producing bacteria belonging to Thermus flavus in a nutrient medium, and collect L-alanine dehydrogenase YK-1 characterized by having at least the following physicochemical properties from the obtained culture. A method for producing L-alanine dehydrogenase YK-1, characterized in that: (1) Action: Catalyzes the action of converting L-alanine into pyruvic acid through oxidative deamination reaction and the reversible reaction of adding an amino group to pyruvic acid to produce L-alanine. (2) Substrate specificity: has specificity for L-alanine,
It has a slight effect on L-serine, but has no substantial effect on other amino acids. (3) Optimum pH: Around 10-11 (4) Optimum temperature: 60-70°C (5) Thermal stability: Substantially no deactivation after treatment at 80°C for 80 minutes. (6) Km value: 3×10 -4 M (7) Molecular weight: approximately 290,000 to 300,000 (gel filtration method).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP57095293A JPS58212782A (en) | 1982-06-02 | 1982-06-02 | L-alanine dehydrogenase yk-1 and preparation thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP57095293A JPS58212782A (en) | 1982-06-02 | 1982-06-02 | L-alanine dehydrogenase yk-1 and preparation thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS58212782A JPS58212782A (en) | 1983-12-10 |
| JPH0328188B2 true JPH0328188B2 (en) | 1991-04-18 |
Family
ID=14133724
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP57095293A Granted JPS58212782A (en) | 1982-06-02 | 1982-06-02 | L-alanine dehydrogenase yk-1 and preparation thereof |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS58212782A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022074772A1 (en) | 2020-10-07 | 2022-04-14 | 日本電信電話株式会社 | Identifier change management device, identifier change management method and identifier change management program |
| WO2022074773A1 (en) | 2020-10-07 | 2022-04-14 | 日本電信電話株式会社 | Identifier change management device, identifier change management method, and identifier change management program |
| WO2022074771A1 (en) | 2020-10-07 | 2022-04-14 | 日本電信電話株式会社 | Identifier change management device, identifier change management method, and identifier change management program |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3376340D1 (en) * | 1982-08-24 | 1988-05-26 | Unitika Ltd | Leucine dehydrogenase and a process for production thereof |
| JP2672845B2 (en) * | 1988-09-27 | 1997-11-05 | 旭化成工業株式会社 | Method for producing L-alanine dehydrogenase |
-
1982
- 1982-06-02 JP JP57095293A patent/JPS58212782A/en active Granted
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022074772A1 (en) | 2020-10-07 | 2022-04-14 | 日本電信電話株式会社 | Identifier change management device, identifier change management method and identifier change management program |
| WO2022074773A1 (en) | 2020-10-07 | 2022-04-14 | 日本電信電話株式会社 | Identifier change management device, identifier change management method, and identifier change management program |
| WO2022074771A1 (en) | 2020-10-07 | 2022-04-14 | 日本電信電話株式会社 | Identifier change management device, identifier change management method, and identifier change management program |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS58212782A (en) | 1983-12-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2850515B2 (en) | Glucose dehydrogenase and method for producing the same | |
| JPS59198972A (en) | Microbiologically produced l-phenylalanine dehydrogenase, obtaining method thereof and production of l-alpha-amino- carboxylic acid | |
| JPH0328188B2 (en) | ||
| EP0229219B1 (en) | Urease and process for preparation thereof | |
| EP0365158B1 (en) | Process for production of l-alanine dehydrogenase and a microorganism strain of the genus sporolactobacillus | |
| JP3029915B2 (en) | Thermostable adenosine-5'-phosphosulfate kinase and method for producing the same | |
| JPS5915625B2 (en) | Novel acyl coenzyme A oxidase and its production method | |
| SUGIURA et al. | Purification and some properties of glycerol dehydrogenase from Erwinia aroideae | |
| JPH0783712B2 (en) | Novel proline acylase and method for producing the same | |
| JPH0364107B2 (en) | ||
| JPS6139035B2 (en) | ||
| Nakamura et al. | Purification and some properties of β-phosphoglucomutase from Lactococcus lactis subsp. cremoris IFO 3427 | |
| JP2801694B2 (en) | New enzyme | |
| JPH0655137B2 (en) | L-phenylalanine dehydrogenase and method for producing the same | |
| JPS5934884A (en) | Leucine dehydrogenase and its preparation | |
| JPS5817590B2 (en) | Method for producing L-malic acid dehydrogenated oxygen | |
| JPH0797986B2 (en) | Heat-resistant acetic acid kinase and method for producing the same | |
| JPH0634714B2 (en) | Process for producing pyrroline-5-carboxylate dehydrogenase | |
| Dagley | [44] 4-Hydroxy-2-ketopimelate aldolase | |
| JPH01148184A (en) | Heat-resistant mannitol dehydrogenase and production thereof | |
| JPH0829079B2 (en) | L-phenylalanine dehydrogenase and method for producing the same | |
| JPS5840471B2 (en) | Method for producing glutamate dehydrogenase | |
| JPS6022914B2 (en) | Method for producing 3α-hydroxysteroid dehydrogenase | |
| JPS6012977A (en) | Aminoacylase | |
| GAOXIANG et al. | Application of Microbial Enzymes in Diagnostic Analysis |