JPS6348514B2 - - Google Patents
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- Publication number
- JPS6348514B2 JPS6348514B2 JP59274299A JP27429984A JPS6348514B2 JP S6348514 B2 JPS6348514 B2 JP S6348514B2 JP 59274299 A JP59274299 A JP 59274299A JP 27429984 A JP27429984 A JP 27429984A JP S6348514 B2 JPS6348514 B2 JP S6348514B2
- Authority
- JP
- Japan
- Prior art keywords
- sod
- molecular weight
- superoxide dismutase
- range
- gluconobacter
- 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
Links
- 230000000694 effects Effects 0.000 claims description 16
- 102000019197 Superoxide Dismutase Human genes 0.000 claims description 15
- 108010012715 Superoxide dismutase Proteins 0.000 claims description 15
- 241000589236 Gluconobacter Species 0.000 claims description 11
- 241000894007 species Species 0.000 claims description 10
- 230000001580 bacterial effect Effects 0.000 claims description 9
- 229910052751 metal Inorganic materials 0.000 claims description 5
- 239000002184 metal Substances 0.000 claims description 5
- 238000002211 ultraviolet spectrum Methods 0.000 claims description 4
- 238000001429 visible spectrum Methods 0.000 claims description 4
- 238000010521 absorption reaction Methods 0.000 claims description 3
- 238000007323 disproportionation reaction Methods 0.000 claims description 3
- 239000000758 substrate Substances 0.000 claims description 3
- 239000000539 dimer Substances 0.000 claims description 2
- 150000002739 metals Chemical class 0.000 claims 1
- 239000000126 substance Substances 0.000 claims 1
- 239000008363 phosphate buffer Substances 0.000 description 13
- 102000004190 Enzymes Human genes 0.000 description 10
- 108090000790 Enzymes Proteins 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 239000002244 precipitate Substances 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 8
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 8
- 102100030497 Cytochrome c Human genes 0.000 description 6
- 108010075031 Cytochromes c Proteins 0.000 description 6
- 150000002978 peroxides Chemical class 0.000 description 6
- -1 superoxide ion Chemical class 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 5
- 235000011130 ammonium sulphate Nutrition 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- LRFVTYWOQMYALW-UHFFFAOYSA-N 9H-xanthine Chemical compound O=C1NC(=O)NC2=C1NC=N2 LRFVTYWOQMYALW-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 241000607720 Serratia Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000006866 deterioration Effects 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- NJYFRQQXXXRJHK-UHFFFAOYSA-N (4-aminophenyl) thiocyanate Chemical compound NC1=CC=C(SC#N)C=C1 NJYFRQQXXXRJHK-UHFFFAOYSA-N 0.000 description 1
- PQMFVUNERGGBPG-UHFFFAOYSA-N (6-bromopyridin-2-yl)hydrazine Chemical compound NNC1=CC=CC(Br)=N1 PQMFVUNERGGBPG-UHFFFAOYSA-N 0.000 description 1
- XNCSCQSQSGDGES-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]propyl-(carboxymethyl)amino]acetic acid Chemical compound OC(=O)CN(CC(O)=O)C(C)CN(CC(O)=O)CC(O)=O XNCSCQSQSGDGES-UHFFFAOYSA-N 0.000 description 1
- KJHJAABRDASKAF-UHFFFAOYSA-N 3,7-dihydropurine-2,6-dione Chemical compound OC1=NC(O)=C2N=CNC2=N1.O=C1NC(=O)NC2=C1NC=N2 KJHJAABRDASKAF-UHFFFAOYSA-N 0.000 description 1
- 101000950981 Bacillus subtilis (strain 168) Catabolic NAD-specific glutamate dehydrogenase RocG Proteins 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N EtOH Substances CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 102000016901 Glutamate dehydrogenase Human genes 0.000 description 1
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 1
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 1
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000012288 Phosphopyruvate Hydratase Human genes 0.000 description 1
- 108010022181 Phosphopyruvate Hydratase Proteins 0.000 description 1
- 108091000080 Phosphotransferase Proteins 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
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- 108010093894 Xanthine oxidase Proteins 0.000 description 1
- 102100033220 Xanthine oxidase Human genes 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N adenyl group Chemical group N1=CN=C2N=CNC2=C1N GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000001479 atomic absorption spectroscopy Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 239000000385 dialysis solution Substances 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 229960002089 ferrous chloride Drugs 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- NMCUIPGRVMDVDB-UHFFFAOYSA-L iron dichloride Chemical compound Cl[Fe]Cl NMCUIPGRVMDVDB-UHFFFAOYSA-L 0.000 description 1
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 1
- 235000020778 linoleic acid Nutrition 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 235000014593 oils and fats Nutrition 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 239000008057 potassium phosphate buffer Substances 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 235000021419 vinegar Nutrition 0.000 description 1
- 239000000052 vinegar Substances 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 229940075420 xanthine Drugs 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
Description
(産業上の利用分野)
本発明は新規な酵素としてのスーパーオキシド
ジスムターゼに関する。
(従来の技術)
一般に、スーパーオキシドジスムターゼ(以下
単にSODという)は、下記の反応
2O2 -+2H+−→H2O2+O2
によつて、基質であるO2 -の不均化を起こさせる
ことのできる酵素として知られている。
そして、このようなSODとしては、従来では
たとえば牛の赤血球から抽出されたSOD(Mark
owitz、J.Biol.Chem234巻第40頁、1959年)等が
知られており、又、このSODが、活性に関係す
る金属原子としてCuやZuを含むことも公知であ
る。
(本発明が解決しようとする問題点)
ところで、上記SODに限らず、一般に酵素は
熱に下安定であり、それ故、酵素の工業的な応用
も制限されることとなつていた。
本発明は、このような問題点を解決せんとし
て、新規な酵素としてのスーパーオキシドジスム
ターゼの開発を主目的とし、併せて熱に安定な酵
素を提供することを目的とする。
(問題点を解決するための手段)
本発明は、このような目的を達成するために開
発された新規なスーパーオキシドジスムターゼで
あり、分子量が48000±3000でサブユニツトの分
子量が約24000の二量体構造を有し、且つ活性に
関係する金属としてMnを含有し、250〜800nm
の範囲の可視紫外スペクトルで280nm付近及び
470nm付近で極大吸収値を有し、しかもグルコ
ノバクター属に属する菌種から得られることを特
徴とするものである。
(実施例)
以下、本発明の実施例について説明する。
実施例 1
本実施例は、一例としてのスーパーオキシドジ
スムターゼの精製法いついて説明するものであ
る。
すなわち、この実施例においては、先ず、グル
コノバクター・セリナス(IF03268)の細菌を
あたり、下記のg数の下記成分を含む培地上で培
養する。
酵母エキス粉末 5g
グリセロール 10g
K2HPO4 1g
この培地を110℃において20分間滅菌した。こ
れに予め同じ培地で予備培養しておいた細菌を入
れ好気的に24時間培養した。
その後、遠心分離機により細菌を収集し、さら
に0.85%(W/W)の食塩水により2回洗浄し、
その細菌よりSODを抽出した。
次に、上記のように洗浄した細菌を0.1Mリン
酸緩衝液(PH7.8)に懸濁させ、ダイノミルを用
いて細菌を破砕した。尚、この細菌の破砕及びこ
れ以後の操作はすべて4℃の条件下で行つた。
そして、この溶液に硫酸アンモニウムを45%飽
和になるまで加えた。これにより生じた沈澱物を
遠心分離機を用いて除き、得られた溶液に硫酸ア
ンモニウムを100%飽和になるまで加えた。これ
によつて得られた沈澱物には殆どのSODが存在
した。
次に、この沈澱物を少量の0.1Mリン酸緩衝液
(PH7.8)に溶解し、約10倍量の0.1Mリン酸緩衝
液(PH7.8)に一夜透析した。この間、透析外液
を3度交換した。透析を行つた溶液に60%飽和に
なるまで硫酸アンモニウムを加え、遠心分離によ
り沈澱物を除き、その上澄液を60%飽和硫酸アン
モニウムを含む0.1Mリン酸緩衝液で平衡化した
DEAE−トーヨーパール(DEAE−Toy opeal)
カラムに流し、SODをカラムに吸着させた。
次に、このSODの吸着したカラムを60%飽和
硫酸アンモニウムを含む0.1Mリン酸緩衝液で洗
い、50%飽和硫酸アンモニウムを含む0.1Mリン
酸緩衝液でSODを溶出させ、フラクシヨンコレ
クターで分画分取した。
そして、SODを含む分画成分を集め、この溶
液に硫酸アンモニウムを約100%飽和になるまで
加え、SODを沈澱させる。この溶液を一夜静置
し、沈澱物を遠心分離機により集めた。SODは
この沈澱物に大多数存在した。
次に、この沈澱物に0.01Mリン酸緩衝液を少量
加え、沈澱物を溶解させ、この溶液を0.01Mリン
酸緩衝液に一夜透析した。この間、透折外液を3
回交換した。
この溶液を、0.01Mリン酸カリウム緩衝液で平
衡化したDEAE−トーヨーパールカラムに流し、
このカラムに非吸着であるSODの分画成分を集
めた。このようにして集めた分画成分に100%飽
和になるまで硫酸アンモニウムを加え、SODを
沈澱させた。この沈澱物を遠心分離により集め、
これを少量の0.02Mリン酸緩衝液に溶解させ、
0.02Mリン酸緩衝液(PH7.2)に一夜透析した。
次に、この溶液を0.02Mリン酸緩衝液(PH7.2)
で平衡化したセフアデツクスG−100カラムに流
した。この溶出液をフラクシヨンコレクターで分
画分取し、SOD活性分画成分を得た。
以上の操作によつて得られたSODは、ポリア
クリルアミドゲル電気泳動で単一のバンドを示
し、又、超遠心分析によつて均一性が認められ
た。
そして、このSODの分子量の測定は、公知の
カラムとしてFinepak SIL AF−102(日本分光
製)を用いる高速液体クロマトグラフイーで行つ
た。
尚、この測定には、分子量が既知の下記のマー
カーを用いた。
分子量
glutamate dehydrogenase 290000
lactate dehydrogenase 142000
enolase 67000
adenyl kinase 32000
cytocrome c 12400
測定の結果、上記SODの分子量は約50000であ
つた。
次に、上記SODの蛋白構造のサブユニツトを
定めるため、ドデシル硫酸ナトリウムを加えたポ
リアクリルアミドゲル電気泳動を行つた結果、分
子量約24000のサブユニツトの単一型を観察した。
それ故、得られたSODの分子量は約24000×2す
なわち約48000と推定される。従つて、このサブ
ユニツトの観察と、上記高速液体クロマトグラフ
イーの結果との双方から考察すると、本発明にお
けるSODの分子量は約48000±3000の範囲と推定
できる。
また、これにより、上記SODはサブユニツト
の分子量が約24000の二量体構造を持つと推定さ
れる。
次に、本実施例におけるSODの溶液の可視紫
外スペクトルを測定したところ、第1図に示すよ
うな結果が得られた。すなわち、紫外領域におい
ては約280nm付近に、又、可視領域においては
約470nm付近に極大吸収値が認められた。
又、原子吸光分析による金属分析の結果、
Mn、Cuが検出されたが、上記SODは、5mMの
KCNで失活しないことから、このSODは、活性
に関係する金属としてMnを含有するMn−SOD
であると推定できる。
尚、SODの活性測定は、Arg.Biol.Chem.、38
(2)471〜473(1974)に、K.Asadaらが記載したの
と類似の方法で測定した。
すなわち、光学セル(1ml用)に50mMリン酸
カリウム(PH7.8)、0.1mMEDTA、0.1mMキサ
ンチン、10μMチトクロムc(ウマ心臓由来)
SODを入れ、全容を0.99mlとする。
これにキサンチン酸化酵素を、0.01ml加え、反
応を開始し、チトクロムc還元を550nmの吸光
度増加の初速度から求め、この値をvとする。
SODを加えないときのチトクロムc還元速度を
Vとすると、SODの酵素量はV/vに比例する。
この条件下で、チトクロムc還元を50%阻害する
(すなわち、V/v=2)SOD酵素量を1酵素単
位とした。
本実施例のSODは、スーパーオキシドイオン
(O2 -)を基質とし、このO2 -を不均化を起こさせ
る作用を有する。
従つて、一般に光、熱、その他の原因により過
酸化物を生成せしめ、品質を著しく変質、劣下さ
せることがある油脂等の酸化性成分を含む製品に
本実施例のSODを配合することにより、O2 -の不
均化を起こさせ、製品の過酸化物による変質、劣
下等を防止することができる。
又、上記SODの安定PHについて試験したとこ
ろ、次のとおりであつた。
先づ0.98g/のリン酸と、0.6g/の酢酸
と、0.62g/のホウ酸とを0.2Nの水酸化ナトリ
ウムでPHを調整して得られた1/100MのBritton
−Robinson緩衝液にてPHを調整しながら、上記
SODを37℃、30分の条件下で、種々のPHの範囲
で放置し、SODの相対活性を測定した。この結
果、第2図に示すように、上記SODはPH6.0〜
11.0の範囲で安定であることが認められた。
次に、上記SODの至適PHを決定するために、
25℃でキサンチン−キサンチン、オキシダーゼ−
チトクロムcの系で上記組成の1/20Mの
Britton−Robinsonの緩衝液にて種々のPHの範囲
でSODの相対活性を測定した。この結果、第2
図に示すように、PHの塩基性領域において8.5〜
10.5の範囲で相対活性が高いことが認められた。
これによつて、上記SODの至適PH範囲が8.5〜
10.5であることが認められる。
実施例 2
本実施例は、SODの過酸化物生成抑制効果を
試験するための実施例である。
先ず、過酸化物生成の系として次のようなモデ
ルを設け、抗酸化効果を調べた。
0.55Mリン酸緩衝液(PH6.9) 25ml
ポリオキシエチレンノニルフエニルエーテル
1.25g
リノール酸 0.28g
上記組成の混合物を50℃恒温器中に放置すると
過酸化物が生成する。
上記の系に上記実施例1のSODを0.1mg/mlに
なるように加え50℃の恒温器中に放置し、3日後
の過酸化脂質抑制効果を調べた。
過酸化脂質量の測定は、満田らのロダン鉄法
(栄養と食糧19巻第3号、210頁1966年)を用い
た。
すなわち、試料溶液0.1mlに75%−エタノール
4.7ml、35%−ロダン化アンモニウム水溶液0.1
ml、0.02M−塩化第一鉄の1N−塩酸溶液0.1mlを
加え、正確に3分経過後に500nmにおける吸光
度を測定した。
参考例として、上記SODのかわりに牛血清ア
ルブミンを同量加えたもの、及びSODと牛血清
アルブミンの両者とも加えないものを調整し、比
較のために同様の試験を行つたところ、次の表1
のような結果が得られた。
尚、表中の数値はすべて吸光度で示している。
(Industrial Application Field) The present invention relates to superoxide dismutase as a novel enzyme. (Prior art) Generally, superoxide dismutase (hereinafter simply referred to as SOD) causes dismutation of the substrate O 2 - by the following reaction 2O 2 - +2H + -→H 2 O 2 + O 2 . It is known as an enzyme that can Conventionally, such SOD is, for example, SOD (Mark) extracted from cow red blood cells.
owitz, J. Biol. Chem vol. 234, p. 40, 1959), and it is also known that this SOD contains Cu and Zu as metal atoms related to activity. (Problems to be Solved by the Present Invention) Incidentally, not only the above-mentioned SOD, but enzymes in general are stable under heat, and therefore, the industrial application of enzymes has been limited. In order to solve these problems, the main purpose of the present invention is to develop superoxide dismutase as a new enzyme, and also to provide a heat-stable enzyme. (Means for Solving the Problems) The present invention is a novel superoxide dismutase developed to achieve the above object, and is a dimer with a molecular weight of 48,000±3,000 and a subunit molecular weight of approximately 24,000. It has a structure and contains Mn as a metal related to activity, and has a diameter of 250 to 800 nm.
Visible and ultraviolet spectrum in the range of around 280nm and
It is characterized by having a maximum absorption value around 470 nm and being obtained from a bacterial species belonging to the genus Gluconobacter. (Example) Examples of the present invention will be described below. Example 1 This example describes an exemplary method for purifying superoxide dismutase. That is, in this example, first, bacteria of Gluconobacter selinus (IF03268) are tested and cultured on a medium containing the following components in the following grams. Yeast extract powder 5g Glycerol 10g K 2 HPO 4 1g This medium was sterilized at 110°C for 20 minutes. Bacteria that had been precultured in the same medium were added to this and cultured aerobically for 24 hours. After that, the bacteria were collected using a centrifuge and further washed twice with 0.85% (W/W) saline solution.
SOD was extracted from the bacteria. Next, the bacteria washed as described above were suspended in 0.1M phosphate buffer (PH7.8), and the bacteria were disrupted using a Dynomill. The disruption of the bacteria and subsequent operations were all carried out at 4°C. Ammonium sulfate was then added to this solution until it reached 45% saturation. The resulting precipitate was removed using a centrifuge, and ammonium sulfate was added to the resulting solution until it reached 100% saturation. Most of the SOD was present in the resulting precipitate. Next, this precipitate was dissolved in a small amount of 0.1M phosphate buffer (PH7.8) and dialyzed against about 10 times the amount of 0.1M phosphate buffer (PH7.8) overnight. During this period, the dialysis fluid was exchanged three times. Ammonium sulfate was added to the dialyzed solution until it was 60% saturated, the precipitate was removed by centrifugation, and the supernatant was equilibrated with 0.1M phosphate buffer containing 60% saturated ammonium sulfate.
DEAE-Toy opeal
It was applied to a column and SOD was adsorbed onto the column. Next, the column with this SOD adsorbed was washed with 0.1M phosphate buffer containing 60% saturated ammonium sulfate, SOD was eluted with 0.1M phosphate buffer containing 50% saturated ammonium sulfate, and fractionated using a fraction collector. I took it. Then, the fractionated components containing SOD are collected, and ammonium sulfate is added to this solution until it reaches approximately 100% saturation to precipitate SOD. This solution was allowed to stand overnight, and the precipitate was collected using a centrifuge. SOD was present in the majority in this precipitate. Next, a small amount of 0.01M phosphate buffer was added to this precipitate to dissolve the precipitate, and this solution was dialyzed against 0.01M phosphate buffer overnight. During this time, add 3
Exchanged twice. This solution was applied to a DEAE-Toyo Pearl column equilibrated with 0.01M potassium phosphate buffer.
Fractionated components of SOD that were not adsorbed were collected on this column. Ammonium sulfate was added to the fractionated components thus collected until 100% saturation was achieved to precipitate SOD. This precipitate was collected by centrifugation,
Dissolve this in a small amount of 0.02M phosphate buffer,
Dialysis was performed overnight against 0.02M phosphate buffer (PH7.2). Next, add this solution to 0.02M phosphate buffer (PH7.2)
The mixture was applied to a Sephadex G-100 column equilibrated with This eluate was fractionated using a fraction collector to obtain SOD active fraction components. The SOD obtained by the above procedure showed a single band in polyacrylamide gel electrophoresis, and homogeneity was confirmed by ultracentrifugation analysis. The molecular weight of this SOD was measured by high performance liquid chromatography using Finepak SIL AF-102 (manufactured by JASCO Corporation) as a known column. In this measurement, the following markers with known molecular weights were used. Molecular weight glutamate dehydrogenase 290000 lactate dehydrogenase 142000 enolase 67000 adenyl kinase 32000 cytochrome c 12400 As a result of measurement, the molecular weight of the above SOD was approximately 50000. Next, in order to determine the subunits of the protein structure of SOD, polyacrylamide gel electrophoresis with sodium dodecyl sulfate was performed, and as a result, a single type of subunit with a molecular weight of about 24,000 was observed.
Therefore, the molecular weight of the SOD obtained is estimated to be about 24,000 x 2, or about 48,000. Therefore, considering both the observation of this subunit and the results of the high performance liquid chromatography described above, the molecular weight of SOD in the present invention can be estimated to be in the range of about 48,000±3,000. Furthermore, from this, it is estimated that the above SOD has a dimeric structure in which the molecular weight of the subunits is approximately 24,000. Next, when the visible and ultraviolet spectra of the SOD solution in this example were measured, the results shown in FIG. 1 were obtained. That is, the maximum absorption value was observed in the vicinity of about 280 nm in the ultraviolet region and in the vicinity of about 470 nm in the visible region. In addition, the results of metal analysis by atomic absorption spectrometry,
Mn and Cu were detected, but the above SOD was 5mM.
Since it is not inactivated by KCN, this SOD is a Mn-SOD containing Mn as a metal related to activity.
It can be estimated that In addition, the activity measurement of SOD is carried out by Arg.Biol.Chem., 38
(2) 471-473 (1974), by a method similar to that described by K. Asada et al. That is, 50mM potassium phosphate (PH7.8), 0.1mM MEDTA, 0.1mM xanthine, 10μM cytochrome c (derived from horse heart) were added to the optical cell (for 1ml).
Add SOD to make the total volume 0.99ml. Add 0.01 ml of xanthine oxidase to this to start the reaction, determine cytochrome c reduction from the initial rate of increase in absorbance at 550 nm, and let this value be v.
If the cytochrome c reduction rate when SOD is not added is V, the amount of SOD enzyme is proportional to V/v.
Under these conditions, the amount of SOD enzyme that inhibits cytochrome c reduction by 50% (ie, V/v = 2) was defined as one enzyme unit. The SOD of this example uses superoxide ion (O 2 - ) as a substrate and has the effect of causing disproportionation of this O 2 - . Therefore, by blending the SOD of this example into products containing oxidizing components such as oils and fats, which generally produce peroxides due to light, heat, and other causes, and which can significantly alter or deteriorate quality. , O 2 - can be caused to disproportionate, thereby preventing product deterioration and deterioration due to peroxides. In addition, when the stable PH of the above SOD was tested, it was as follows. First, 1/100M Britton was obtained by adjusting the pH of 0.98g/phosphoric acid, 0.6g/acetic acid, and 0.62g/boric acid with 0.2N sodium hydroxide.
−While adjusting the pH with Robinson buffer,
SOD was left at 37°C for 30 minutes at various pH ranges, and the relative activity of SOD was measured. As a result, as shown in Figure 2, the above SOD is PH6.0 ~
It was found to be stable within the range of 11.0. Next, in order to determine the optimal pH of the above SOD,
Xanthine - xanthine, oxidase - at 25℃
Cytochrome c system with 1/20M of the above composition
The relative activity of SOD was measured at various pH ranges in Britton-Robinson's buffer. As a result, the second
As shown in the figure, in the basic region of PH 8.5~
It was observed that the relative activity was high in the range of 10.5.
As a result, the optimal PH range for the above SOD is 8.5 ~
10.5. Example 2 This example is an example for testing the peroxide production suppressing effect of SOD. First, the following model was set up as a peroxide production system, and the antioxidant effect was investigated. 0.55M phosphate buffer (PH6.9) 25ml polyoxyethylene nonyl phenyl ether
1.25g Linoleic acid 0.28g When a mixture with the above composition is left in a thermostat at 50°C, peroxide is generated. The SOD of Example 1 was added to the above system at a concentration of 0.1 mg/ml, and the system was left in a thermostat at 50°C, and the lipid peroxide suppressing effect was examined after 3 days. The amount of lipid peroxide was measured using the Rodan iron method of Mitsuda et al. (Nutrition and Shokuryo Vol. 19, No. 3, p. 210, 1966). That is, 75%-ethanol to 0.1 ml of sample solution.
4.7ml, 35% - ammonium rhodanide aqueous solution 0.1
ml, 0.1 ml of a 1N hydrochloric acid solution of 0.02M ferrous chloride was added, and the absorbance at 500 nm was measured after exactly 3 minutes. As a reference example, the same amount of bovine serum albumin was added instead of the above SOD, and the same amount of bovine serum albumin was added instead of SOD, and a similar test was conducted for comparison, and the following table is shown. 1
The following results were obtained. All numerical values in the table are expressed in absorbance.
【表】
0日目から3日後の吸光度の増加が過酸化脂質
量の増加を表している。又、過酸化脂質量と吸光
度とは比例する。このことと、上記試験結果よ
り、本実施例のSODには極めて優れた過酸化物
生成抑制効果が認められた。
実施例 3
本実施例は、上記SODの耐熱性を試験するた
めの実施例である。
上記実施例におけるSODの耐熱性を、グルコ
ノバクターと同じく常温付近で生育するセラチ
ア・マルセツセンスからのSODと比較した。
それぞれのSODは、0.01Mリン酸緩衝液(PH
7.2)に溶かし、60℃でそれぞれ5分間加温し、
氷中で冷却して活性測定を行つた。
それぞれ60℃加温前にも活性測定を行い、60℃
加温による失活を観察した。結果を下記の表2に
示す。[Table] An increase in absorbance after 3 days from day 0 represents an increase in the amount of lipid peroxide. Moreover, the amount of lipid peroxide and the absorbance are proportional. From this and the above test results, the SOD of this example was found to have an extremely excellent peroxide production suppressing effect. Example 3 This example is an example for testing the heat resistance of the above SOD. The heat resistance of SOD in the above example was compared with SOD from Serratia marsetuscens, which grows near room temperature like Gluconobacter. Each SOD was prepared in 0.01M phosphate buffer (PH
7.2) and heated at 60℃ for 5 minutes each.
The activity was measured after cooling in ice. The activity was also measured before heating to 60℃, and
Inactivation due to heating was observed. The results are shown in Table 2 below.
【表】
上記のように、本実施例におけるSODは、常
温付近で生育する細菌由来のSODに比べ、耐熱
性が著しく優れていることが認められた。
尚、上記実施例では、グルコノバクター・セリ
ナスにより抽出精製されたSODを用いたが、菌
種はこれに限らず、要は本発明のSODは、グル
コノバクター属に属する菌種から得られたもので
あればよい。
又、SODの精製方法も上記実施例に限定され
ない。
(発明の効果)
(イ) 叙上のように、本発明によつて、グルコノバ
クター属の菌種からの全く新規なスーパーオキ
シドジスムターゼが得られるに至つた。
(ロ) 又、一般に常温で生育する菌種から得られる
スーパーオキシドジスムターゼは、耐熱性が弱
いものであるが、本発明のスーパーオキシドジ
スムターゼは、常温で生育するグルコノバクタ
ー属の菌種から得られるものであるにもかかわ
らず、常温で生育する菌種由来の他のスーパー
オキシドジスムターゼ、たとえばセラチア・マ
ルセツセンス等から得られるものに比べると、
耐熱性が非常に優れたものであつた。
従つて、酵素の工業的な応用範囲が広がると
いう実益がある。
(ハ) さらに、グルコノバクター属の菌種は、たと
えば食酢やビタミンCの原料の製造等、従来か
ら食品工業に使用されていてその安全性も確認
されているため、この菌種から得られる本発明
のスーパーオキシドジスムターゼも、安全性が
高いものであると認められる。[Table] As described above, the SOD in this example was found to have significantly better heat resistance than the SOD derived from bacteria that grows near room temperature. In the above example, SOD extracted and purified by Gluconobacter selinus was used, but the bacterial species is not limited to this.In short, the SOD of the present invention can be obtained from a bacterial species belonging to the genus Gluconobacter. It is fine as long as it is. Furthermore, the method for purifying SOD is not limited to the above example. (Effects of the Invention) (a) As described above, the present invention has led to the production of a completely new superoxide dismutase from a species of the genus Gluconobacter. (b) In addition, superoxide dismutase obtained from bacterial species that grow at room temperature generally has weak heat resistance, but the superoxide dismutase of the present invention is obtained from a bacterial species of the genus Gluconobacter that grows at room temperature. However, compared to other superoxide dismutases derived from bacterial species that grow at room temperature, such as those obtained from Serratia marsetuscens,
It had very good heat resistance. Therefore, there is a practical benefit of expanding the range of industrial applications of enzymes. (c) Furthermore, the bacterial species of the genus Gluconobacter has been used in the food industry for a long time, for example in the production of raw materials for vinegar and vitamin C, and their safety has been confirmed. The superoxide dismutase of the present invention is also recognized to be highly safe.
第1図は一実施例としてのスーパーオキシドジ
スムターゼの可視、紫外スペクトルのチヤート
図。第2図は酸素活性とPHの相関関係を示す図。
FIG. 1 is a chart of visible and ultraviolet spectra of superoxide dismutase as an example. Figure 2 is a diagram showing the correlation between oxygen activity and PH.
Claims (1)
せる作用を有し、且つ安定PH範囲が6.0〜11.0で
あるとともに至適PH範囲が8.5〜10.5であるスー
パーオキシドジスムターゼにおいて、分子量が
48000±3000でサブユニツトの分子量が約24000の
二量体構造を有し、且つ活性に関係する金属とて
Mnを含有し、250〜800nmの範囲の可視紫外ス
ペクトルで280nm付近及び470nm付近に極大吸
収値を有し、しかもグルコノバクター属に属する
菌種から得られることを特徴とする新規スーパー
オキシドジスムターゼ。 2 前記グルコノバクター属に属する菌種がグル
コノバクター・セリナスである特許請求の範囲第
1項記載の新規スーパーオキシドジスムターゼ。[Claims] 1. A substance that uses O 2 - as a substrate, has an action of causing disproportionation of O 2 - , and has a stable PH range of 6.0 to 11.0 and an optimal PH range of 8.5 to 10.5. In some superoxide dismutases, the molecular weight is
It has a dimer structure with a subunit molecular weight of 48,000±3,000 and approximately 24,000, and metals related to activity are
A novel superoxide dismutase containing Mn, having maximum absorption values around 280 nm and around 470 nm in the visible and ultraviolet spectrum in the range of 250 to 800 nm, and being obtained from a bacterial species belonging to the genus Gluconobacter. 2. The novel superoxide dismutase according to claim 1, wherein the bacterial species belonging to the genus Gluconobacter is Gluconobacter selinus.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP59274299A JPS61185184A (en) | 1984-12-28 | 1984-12-28 | Novel superoxide dismutase and preparation thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP59274299A JPS61185184A (en) | 1984-12-28 | 1984-12-28 | Novel superoxide dismutase and preparation thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS61185184A JPS61185184A (en) | 1986-08-18 |
JPS6348514B2 true JPS6348514B2 (en) | 1988-09-29 |
Family
ID=17539708
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JP59274299A Granted JPS61185184A (en) | 1984-12-28 | 1984-12-28 | Novel superoxide dismutase and preparation thereof |
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JP (1) | JPS61185184A (en) |
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CN104726422A (en) * | 2015-02-10 | 2015-06-24 | 江南大学 | Method for producing heat-resistant superoxide dismutase (SOD) by utilizing Stilbella thermophila |
CN105624126B (en) * | 2016-02-23 | 2017-02-01 | 杭州睿道医药科技有限公司 | Novel recombinant high-stability superoxide dismutase and application thereof |
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1984
- 1984-12-28 JP JP59274299A patent/JPS61185184A/en active Granted
Also Published As
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