JP4423283B2 - Hydrogen peroxide-degrading enzyme - Google Patents
Hydrogen peroxide-degrading enzyme Download PDFInfo
- Publication number
- JP4423283B2 JP4423283B2 JP2006288522A JP2006288522A JP4423283B2 JP 4423283 B2 JP4423283 B2 JP 4423283B2 JP 2006288522 A JP2006288522 A JP 2006288522A JP 2006288522 A JP2006288522 A JP 2006288522A JP 4423283 B2 JP4423283 B2 JP 4423283B2
- Authority
- JP
- Japan
- Prior art keywords
- hydrogen peroxide
- medium
- enzyme
- lactic acid
- pediococcus
- 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 - Fee Related
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Description
本発明は、乳酸菌が産生する過酸化水素分解酵素に関する。 The present invention relates to a hydrogen peroxide decomposing enzyme produced by lactic acid bacteria.
生体内外の過酸化水素を除去するために、生物は様々な過酸化水素分解酵素を有しており、代表的な過酸化水素分解酵素として、例えば、カタラーゼ、アスコルビン酸ペルオキシダーゼ、グルタチオンペルオキシダーゼ、ヘムペルオキシダーゼ等が挙げられる。 In order to remove hydrogen peroxide inside and outside the living body, organisms have various hydrogen peroxide-degrading enzymes, and examples of typical hydrogen peroxide-degrading enzymes include catalase, ascorbate peroxidase, glutathione peroxidase, and heme peroxidase. Etc.
カタラーゼは、好気的な代謝を営む生物全般に広く存在し、下記化学式（１）によって過酸化水素を水に変換する（非特許文献１〜２、４参照）。 Catalase is widely present in all organisms that perform aerobic metabolism, and converts hydrogen peroxide into water according to the following chemical formula (1) (see Non-Patent Documents 1 to 2, and 4).
アスコルビン酸ペルオキシダーゼは、植物をはじめとする生物全般に広く存在し、下記化学式（２）によって過酸化水素を水に変換する（非特許文献１〜４参照）。 Ascorbic acid peroxidase is widely present in all living organisms including plants, and converts hydrogen peroxide into water by the following chemical formula (2) (see Non-Patent Documents 1 to 4).
グルタチオンペルオキシダーゼは、動物をはじめとする生物全般に広く存在し、複数段階の反応によって過酸化水素を水に変換する（非特許文献１〜４参照）。 Glutathione peroxidase is widely present in all living organisms including animals, and converts hydrogen peroxide into water by a multi-step reaction (see Non-Patent Documents 1 to 4).
ヘムペルオキシダーゼは、生物全般に広く存在し、下記化学式（３）によって過酸化水素を水に変換する（非特許文献１〜４参照）。 Heme peroxidase is widely present in all living organisms, and converts hydrogen peroxide into water according to the following chemical formula (3) (see Non-Patent Documents 1 to 4).
このように、過酸化水素分解酵素は動植物をはじめとする生物に広く存在し、強酸、強アルカリ、加熱等による変性失活のおそれがない限り、抗酸化能力を有する。 Thus, hydrogen peroxide-degrading enzymes are widely present in organisms including animals and plants, and have an antioxidant ability as long as there is no fear of denaturation and deactivation due to strong acids, strong alkalis, heating, or the like.
ところで、ヨーグルトなど広く発酵食品に利用されている乳酸菌の一部は抗酸化酵素を有していると言われているが、その諸性質については不明な点が多く、そのため前記酵素の諸性質を明らかにすることが求められている。 By the way, some lactic acid bacteria widely used in fermented foods such as yogurt are said to have antioxidant enzymes, but there are many unclear points about their properties, so the properties of the enzymes are There is a need to clarify.
そこで、本発明者らは、種々の発酵食品から乳酸菌を分離し、該乳酸菌の培養菌体の諸性質について鋭意検討したところ、ペディオコッカス属(Pediococcus)に属する乳酸菌が産生する酵素が過酸化水素に対して分解特性を示すことを見出し、本発明を完成した。 Therefore, the present inventors have isolated lactic acid bacteria from various fermented foods and conducted extensive studies on the properties of the cultured cells of the lactic acid bacteria. As a result, enzymes produced by lactic acid bacteria belonging to the genus Pediococcus are peroxidized. The present invention was completed by finding that it exhibits decomposition characteristics against hydrogen.
すなわち、本発明の要旨は以下のとおりである。
〔１〕 ペディオコッカス・ペントサス(Pediococcus pentosaceus) TY-1573株（NITE P-91）由来で、下記の理化学的性質を有する過酸化水素分解酵素、
（１）基質特異性
ＮＡＤ（Ｐ）Ｈの存在下、過酸化水素に高い反応性を示し、かつ過酸化物には実質的に作用しない
（２）分子量
５０ｋＤａ（ＳＤＳポリアクリルアミドゲル電気泳動による測定）
〔２〕 配列番号１に記載のアミノ酸配列をＮ末端に有する、前記〔１〕記載の過酸化水素分解酵素、
〔３〕 前記〔２〕において、配列番号１に記載のアミノ酸配列が、１もしくは複数のアミノ酸が付加、欠失若しくは置換されたアミノ酸配列からなり、かつ過酸化水素分解活性を維持している、過酸化水素分解酵素、
〔４〕 前記〔１〕〜〔３〕のいずれか記載の過酸化水素分解酵素を含有する飲食品、化粧品または医薬品。
That is, the gist of the present invention is as follows.
[1] Hydrogen peroxide-degrading enzyme derived from Pediococcus pentosaceus TY-1573 strain (NITE P-91) and having the following physicochemical properties:
(1) Substrate specificity High reactivity with hydrogen peroxide in the presence of NAD (P) H and substantially no action on peroxides (2) Molecular weight 50 kDa (measured by SDS polyacrylamide gel electrophoresis) )
[2] The hydrogen peroxide-degrading enzyme according to [1], which has the amino acid sequence of SEQ ID NO: 1 at the N-terminus,
[3] In the above [2], the amino acid sequence set forth in SEQ ID NO: 1 consists of an amino acid sequence in which one or more amino acids are added, deleted or substituted, and maintains hydrogen peroxide decomposing activity. Hydrogen peroxide-degrading enzyme ,
[4] A food, beverage, cosmetic or pharmaceutical product containing the hydrogen peroxide decomposing enzyme according to any one of [1] to [3].
本発明によれば、乳酸菌に由来する新規な過酸化水素分解酵素を提供することができる。また、前記酵素を飲食品、化粧品または医薬品に含有させることにより、該酵素が有する過酸化水素除去能力を活用することが期待できる。 According to the present invention, a novel hydrogen peroxide decomposing enzyme derived from lactic acid bacteria can be provided. In addition, by incorporating the enzyme into foods, beverages, cosmetics or pharmaceuticals, it can be expected to utilize the hydrogen peroxide removing ability of the enzyme.
本発明の過酸化水素分解酵素の製造に用いられる乳酸菌は、例えば、ペディオコッカス属(Pediococcus)に属する乳酸菌のうち、ＮＡＤ（Ｐ）Ｈの存在下、過酸化水素に対して分解特性を示すものであって、ＳＤＳポリアクリルアミドゲル電気泳動（ＳＤＳ−ＰＡＧＥ）による測定で、分子量が約５０ｋＤａを示すものであれば特に限定されない。ペディオコッカス属(Pediococcus)に属する乳酸菌の菌種として、例えば、ペディオコッカス・ペントサス(Pediococcus pentosaceus)、ペディオコッカス・アシディラクティシ（Pediococcus acidilactici）、ペディオコッカス・セレビシェ（Pediococcus cerevisiae）、ペディオコッカス・セレビシェ・バー・デキストリニカス（Pediococcus cerevisiae var. dextrinicus）、ペディオコッカス・クラウセニ（Pediococcus claussenii）、ペディオコッカス・ダムノサス（Pediococcus damnosus）、ペディオコッカス・デキストリニカス（Pediococcus dextrinicus）、ペディオコッカス・ハロフィラス（Pediococcus halophilus）、ペディオコッカス・オマリ（Pediococcus homari）、ペディオコッカス・イノピタナス（Pediococcus inopinatus）、ペディオコッカス・パルバラス（Pediococcus parvulus）、ペディオコッカス・サブスピーシーズ・インテルメディウス（Pediococcus pentosaceus subsp. intermedius）、ペディオコッカス・ソヤ（Pediococcus soya）、ペディオコッカス・ソヤエ（Pediococcus soyae）、ペディオコッカス・エスピー（Pediococcus sp.）、ペディオコッカス・ウリナエッキー（Pediococcus urinaeequi）が挙げられる。後述する実施例では、これらのうち、ペディオコッカス・ペントサス(Pediococcus pentosaceus) TY-1573株（NITE P-91）を用いた例を示している。 The lactic acid bacteria used for the production of the hydrogen peroxide decomposing enzyme of the present invention, for example, among lactic acid bacteria belonging to the genus Pediococcus , exhibit degradation characteristics with respect to hydrogen peroxide in the presence of NAD (P) H. It is not particularly limited as long as it has a molecular weight of about 50 kDa as measured by SDS polyacrylamide gel electrophoresis (SDS-PAGE). As a species of lactic acid bacteria belonging to the genus Pediococcus (Pediococcus), for example, Pediococcus pentosus (Pediococcus pentosaceus), Pediococcus reeds di Lacty Shi (Pediococcus acidilactici), Pediococcus cerevisiae (Pediococcus cerevisiae), Pediococcus cereviche bar dextrinicus ( Pediococcus cerevisiae var. Dextrinicus ), Pediococcus claussenii , Pediococcus damnosus , Pediococcus dionocus tricus · halophilus (Pediococcus halophilus), Pediococcus Omari (Pediococcus homari), Pediococcus Inopitanasu (Pediococcus inopinatus), Pediococcus Parubarasu (Pediococcus parvulus), Pedio Kkasu subsp Intel intermedius (Pediococcus pentosaceus subsp. Intermedius), Pediococcus Soya (Pediococcus soya), Pediococcus sojae (Pediococcus soyae), Pediococcus sp (Pediococcus sp.), Pediococcus Urina Ecky ( Pediococcus urinaeequi ). In Examples described later, an example using Pediococcus pentosaceus TY-1573 strain (NITE P-91) is shown.
過酸化水素に対して分解特性を示す乳酸菌は次のようにしてスクリーニングすることができる。すなわち、まず、各種発酵食品を分離源とし、該分離源を固形培地で培養して乳酸菌を単離し、該単離した乳酸菌を液体培地で培養して得られる培養物から菌体を回収し、次いで該菌体を含有する菌体懸濁液を被検液として、該被検液が、過酸化水素に対する分解特性を有するか否か調べることによりスクリーニングすることができる。 Lactic acid bacteria exhibiting decomposition characteristics against hydrogen peroxide can be screened as follows. That is, first, various fermented foods as a separation source, the separation source is cultured in a solid medium to isolate lactic acid bacteria, and the cells are collected from a culture obtained by culturing the isolated lactic acid bacteria in a liquid medium, Subsequently, screening can be performed by examining whether or not the test liquid has a decomposition property with respect to hydrogen peroxide using the cell suspension containing the cells as a test liquid.
本発明において発酵食品とは、一般的に乳酸菌が含まれている動物性および植物性の発酵食品をいい、代表的な発酵食品を例示すれば、例えば、散麹、豆麹、餅麹、麹漬、味噌漬け、糠漬、野沢菜漬、すんき漬、ベッタラ漬、味噌、醤油、酒粕、納豆、米酢、バルサミコ酢、リンゴ酢、キムチ、腐乳、ナンプラー、とうふよう、ワイン粕、酒粕、ヨーグルト、ベジマイト、クワス、クミス、ギビヤック、ピクルス、クミス、サワークラウト等が挙げられる。そして、培養に際して、前記発酵食品を破砕し、例えば生理食塩水で希釈した食品懸濁液とすることが好ましい。 In the present invention, the fermented food generally refers to animal and plant fermented foods that contain lactic acid bacteria. Examples of typical fermented foods include, for example, miso, soybean cake, koji, koji. Pickles, miso pickles, pickles, nozawana pickles, sunki pickles, bettara pickles, miso, soy sauce, sake lees, natto, rice vinegar, balsamic vinegar, apple vinegar, kimchi, humor, nanpura, tofuyo, wine lees, sake lees, yogurt , Vegemite, Kvass, Kumis, Gibiyac, Pickles, Kumis, Sawarout. In the culture, the fermented food is preferably crushed and, for example, a food suspension diluted with physiological saline.
固形培地としては、乳酸菌の培養に通常用いられる培地であればよく、代表的なものを例示すれば、例えば、酵母エキスペプトン培地、ブドウ糖培地、フェネチルアルコール培地、アセテート培地、ＧＹＰ培地、ＭＲＳ培地、ＴＩＴＧ培地、ＳＬ培地、システイン・ミルク培地、ＬＢＳ培地、ＴＡＴＡＣ培地、ＭＧ培地、食塩18％-硝酸カリ培地、麹汁培地、ＢＣＰ培地、チオグリコレイト培地、稀釈ブドウ果汁培地、ＥＳＹ培地、吉栖氏培地、耐塩性乳酸菌用培地、耐塩性乳酸菌用醤油培地、上野培地、飯塚・山里培地、ＴＹＧ培地、ラクチック培地、Ｍ17培地、Lactic streptococciの分別培地、クエン酸を発酵するLactic streptococciの分別培地、PPYL培地、YPG培地、酸性トマト培地、Mayeux&ColmerのLeuconostoc検出培地、浜本らのLeuconostoc検出培地、Pearce&HaliganのLeuconostoc計測培地、APT培地、Briggsのトマトジュース培地、Rogosa培地、NAP培地等に、固形化剤として0.5〜2.0％の寒天を添加したものが挙げられる。また、培地上に乳酸菌を選択的に増殖させるため、好気性菌の発育を阻害する物質（例えば、アジ化ナトリウム）、グラム陰性菌の発育を阻害する物質（例えば、ポリミキシンＢ）、真菌の発育を阻害する物質（例えば、シクロヘキシミド）などを適宜組み合わせて前記固形培地に含有させてもよい。また、液体培地としては、乳酸菌の培養に通常用いられる培地であればよく、代表的なものを例示すれば、例えば、酵母エキスペプトン培地、ブドウ糖培地、フェネチルアルコール培地、アセテート培地、ＧＹＰ培地、ＭＲＳ培地、ＴＩＴＧ培地、ＳＬ培地、システイン・ミルク培地、ＬＢＳ培地、ＴＡＴＡＣ培地、ＭＧ培地、食塩18％-硝酸カリ培地、麹汁培地、ＢＣＰ培地、チオグリコレイト培地、稀釈ブドウ果汁培地、ＥＳＹ培地、吉栖氏培地、耐塩性乳酸菌用培地、耐塩性乳酸菌用醤油培地、上野培地、飯塚・山里培地、ＴＹＧ培地、ラクチック培地、Ｍ17培地、Lactic streptococciの分別培地、クエン酸を発酵するLactic streptococciの分別培地、PPYL培地、YPG培地、酸性トマト培地、Mayeux&ColmerのLeuconostoc検出培地、浜本らのLeuconostoc検出培地、Pearce&HaliganのLeuconostoc計測培地、APT培地、Briggsのトマトジュース培地、Rogosa培地、NAP培地等が挙げられる。 The solid medium may be any medium that is usually used for culturing lactic acid bacteria, and representative examples include, for example, yeast extract peptone medium, glucose medium, phenethyl alcohol medium, acetate medium, GYP medium, MRS medium, TITG medium, SL medium, cysteine milk medium, LBS medium, TATAC medium, MG medium, sodium chloride 18% -potassium nitrate medium, broth medium, BCP medium, thioglycolate medium, diluted grape juice medium, ESY medium, Yoshitake Mr. medium, salt-tolerant lactic acid bacteria medium, salt-tolerant lactic acid bacteria for soy sauce medium, Ueno medium, Iizuka-Yamazato medium, TYG medium, lactic medium, M17 medium, separation medium of lactic streptococci, separation medium of lactic streptococci to ferment citric acid, PPYL medium, YPG medium, acidic tomato medium, Mayeux & Colmer Leuconostoc detection medium, Hamamoto et al. Leuconostoc detection medium, Pearce & Hali Examples include gan's Leuconostoc measurement medium, APT medium, Briggs' tomato juice medium, Rogosa medium, NAP medium, etc., with 0.5 to 2.0% agar added as a solidifying agent. In addition, since lactic acid bacteria are selectively grown on the medium, substances that inhibit the growth of aerobic bacteria (for example, sodium azide), substances that inhibit the growth of gram-negative bacteria (for example, polymyxin B), and fungal growth A substance that inhibits (for example, cycloheximide) or the like may be appropriately combined and contained in the solid medium. The liquid medium may be any medium that is usually used for culturing lactic acid bacteria. Typical examples include, for example, yeast extract peptone medium, glucose medium, phenethyl alcohol medium, acetate medium, GYP medium, MRS. Medium, TITG medium, SL medium, cysteine milk medium, LBS medium, TATAC medium, MG medium, sodium chloride 18% -potassium nitrate medium, broth medium, BCP medium, thioglycolate medium, diluted grape juice medium, ESY medium, Gil栖氏medium, salt-tolerant lactic acid bacteria medium, salt-tolerant lactic acid bacteria for soy sauce medium, Ueno medium, Iizuka-Yamazato medium, TYG medium, lactic medium, M17 medium, lactic streptococci of the separation medium, separation of lactic streptococci to ferment citric acid Medium, PPYL medium, YPG medium, acidic tomato medium, Mayeux & Colmer Leuconostoc detection medium, Hamamoto et al. Leuconostoc detection medium, Pe arce &Haligan's Leuconostoc measuring medium, APT medium, Briggs' tomato juice medium, Rogosa medium, NAP medium and the like.
乳酸菌の培養は、常法にしたがって行えばよく、例えば、３０〜４０℃、１０〜４０時間の条件で好気的培養、静置培養または中和培養などを行えばよい。 The lactic acid bacteria may be cultured according to a conventional method, for example, aerobic culture, stationary culture or neutralization culture under conditions of 30 to 40 ° C. and 10 to 40 hours.
培養終了後、乳酸菌のコロニーを１コロニーずつ単離し、常法にしたがって純化することが好ましい。そして、前記単離した乳酸菌株を常法にしたがって培養し、得られた培養物を遠心分離して菌体を回収し、該菌体に緩衝液を加えてOD660nm＝1.5〜1.7に濃度調節した菌体懸濁液を調製し、該菌体懸濁液が、過酸化水素に対する分解特性を示すか否か調べ、過酸化水素に対する分解特性を示す乳酸菌株を本発明の目的に適した乳酸菌とする。 After completion of the culture, it is preferable to isolate colonies of lactic acid bacteria one by one and purify them according to a conventional method. Then, the isolated lactic acid strain is cultured according to a conventional method, and the obtained culture is centrifuged to recover the cells, and a buffer solution is added to the cells to adjust the concentration to OD 660 nm = 1.5 to 1.7. The microbial cell suspension is prepared, and it is examined whether or not the bacterial cell suspension exhibits a decomposition property with respect to hydrogen peroxide. And
過酸化水素に対する分解特性は、例えば、前記菌体懸濁液に過酸化水素（終濃度１．０〜３．０ｍＭ）、および必要に応じて糖類（たとえば、グルコース）（終濃度３０〜１００ｍＭ）をそれぞれ添加して、３７℃で３時間反応させ、反応終了後の反応液を遠心分離し、上清に含まれる過酸化水素残量を測定することにより評価される。なお、前記糖類は、乳酸菌株の代謝によるエネルギーを供与するために添加するものであるが、必ずしも添加しなくてもよい。過酸化水素残量の測定は、Sedewitzらの方法が適用される（Sedewitz et al.,Journal of Bacteriology,160,Oct.273-278. 1984）。 For example, hydrogen peroxide (final concentration: 1.0 to 3.0 mM) and, if necessary, sugars (for example, glucose) (final concentration: 30 to 100 mM) Each is added, reacted at 37 ° C. for 3 hours, the reaction solution after completion of the reaction is centrifuged, and the remaining amount of hydrogen peroxide contained in the supernatant is measured. In addition, although the said saccharide | sugar is added in order to provide the energy by metabolism of a lactic acid strain, it does not necessarily need to add. The method of Sedewitz et al. Is applied to the measurement of the remaining amount of hydrogen peroxide (Sedewitz et al., Journal of Bacteriology, 160, Oct. 273-278. 1984).
以上のようにして分離した乳酸菌株の過酸化水素分解特性を評価した結果、過酸化水素分解特性を示すものとして、例えば、後述する乳酸菌TY-1573株を選択することができた。そして、菌学的性質および遺伝学的特性の結果から、乳酸菌TY-1573株は、ペディオコッカス・ペントサス(Pediococcus pentosaceus)と同定された。上記菌株は、ペディオコッカス・ペントサス（Pediococcus pentosaceus）TY1573（NITE P-91）として独立行政法人製品評価技術基盤機構特許微生物寄託センターに寄託されている。 As a result of evaluating the hydrogen peroxide decomposition characteristics of the lactic acid strains isolated as described above, for example, the lactic acid bacteria TY-1573 strain described below could be selected as one that exhibits hydrogen peroxide decomposition characteristics. From the results of mycological properties and genetic characteristics, the lactic acid bacterium strain TY-1573 was identified as Pediococcus pentosaceus . The above strain is deposited as Pediococcus pentosaceus TY1573 (NITE P-91) at the National Institute of Technology and Evaluation Patent Microorganism Depositary.
本発明では、上述のようにして単離した乳酸菌株を常法にしたがって培養し、得られた培養物を遠心分離して菌体を回収し、該菌体に緩衝液を加えた菌体懸濁液や、該菌体懸濁液を機械的方法、酵素的方法等で破砕して得られる細胞抽出液など粗酵素液の状態で利用することができる。また、該粗酵素液を被覆保護材（例えば、レシチン、キチンなど）でミセル化してカプセルとして、あるいは賦形剤（例えば、デキストリン等）で吸着させて粉末として利用してもよい。好ましくは、本発明に係る過酸化水素分解酵素は精製した状態で利用される。また、該酵素は、必要に応じて常法にしたがい、凍結乾燥、噴霧乾燥などを行い、乾燥品としても利用可能である。 In the present invention, the lactic acid bacterial strain isolated as described above is cultured according to a conventional method, and the obtained culture is centrifuged to recover the bacterial cell, and the bacterial cell suspension obtained by adding a buffer solution to the bacterial cell is collected. It can be used in the state of a crude enzyme solution such as a suspension or a cell extract obtained by crushing the bacterial cell suspension by a mechanical method, an enzymatic method or the like. Further, the crude enzyme solution may be used as a capsule by being micellized with a coating protective material (eg, lecithin, chitin, etc.) or adsorbed with an excipient (eg, dextrin, etc.) and used as a powder. Preferably, the hydrogen peroxide decomposing enzyme according to the present invention is used in a purified state. In addition, the enzyme can be used as a dried product by lyophilization, spray drying and the like according to a conventional method if necessary.
過酸化水素分解酵素の精製にあたっては、例えば、前記菌体懸濁液を破砕して得られる細胞抽出液に遠心処理を行い、該細胞抽出液から未破砕菌体を除去したものを粗酵素液として用いる。そして、該粗酵素液に、例えば、硫酸アンモニウム、硫酸ナトリウム等の塩析処理、および疎水ブチルカラム、弱陰イオン交換カラム、強陰イオン交換カラム、ハイドロキシアパタイトカラム等のクロマトグラフィーを適宜組み合わせることで精製酵素を得ることができる。得られた精製酵素は、ＳＤＳ−ＰＡＧＥでほぼ単一のバンドを示す程度に純化されている。 In purifying the hydrogen peroxide-degrading enzyme, for example, the cell extract obtained by crushing the cell suspension is centrifuged, and the crude extract obtained by removing unbroken cells from the cell extract is used. Used as The crude enzyme solution can be purified by appropriately combining, for example, salting-out treatment of ammonium sulfate, sodium sulfate, etc., and chromatography such as hydrophobic butyl column, weak anion exchange column, strong anion exchange column, hydroxyapatite column, etc. Can be obtained. The obtained purified enzyme is purified to such an extent that it shows an almost single band by SDS-PAGE.
なお、上記の各種クロマト法を適用した際に得られる各画分については、各画分の過酸化水素分解活性、およびタンパク質のピークパターンを分析することで過酸化水素分解酵素を含有する画分を回収した。過酸化水素分解活性は、各画分に電子供与体であるＮＡＤＨまたはＮＡＤＰＨ（以下、「ＮＡＤ（Ｐ）Ｈ」という場合がある）および電子受容体である過酸化水素を添加し、３７℃で３分間反応させ、反応後におけるＮＡＤ（Ｐ）Ｈ濃度と過酸化水素濃度を測定することで評価した。ＮＡＤ（Ｐ）Ｈ濃度は３４０ｎｍの吸光度により測定され、過酸化水素濃度は公知のチオシアン酸−鉄錯体による発色法により測定される。 In addition, about each fraction obtained when applying said various chromatographic methods, the fraction containing hydrogen peroxide decomposing enzyme is analyzed by analyzing the hydrogen peroxide decomposing activity of each fraction and the peak pattern of protein. Was recovered. The hydrogen peroxide decomposition activity was determined by adding NADH or NADPH (hereinafter sometimes referred to as “NAD (P) H”) as an electron donor and hydrogen peroxide as an electron acceptor to each fraction at 37 ° C. The reaction was performed for 3 minutes, and the NAD (P) H concentration and the hydrogen peroxide concentration after the reaction were measured and evaluated. The NAD (P) H concentration is measured by absorbance at 340 nm, and the hydrogen peroxide concentration is measured by a color development method using a known thiocyanate-iron complex.
上記のようにして得られる過酸化水素分解酵素は、以下の理化学的性質を示す。
（１）基質特異性
ＮＡＤ（Ｐ）Ｈの存在下、過酸化水素に高い反応性を示し、かつ過酸化物には実質的に作用しない
（２）分子量
約５０ｋＤａ（ＳＤＳポリアクリルアミドゲル電気泳動による測定）
The hydrogen peroxide-degrading enzyme obtained as described above exhibits the following physicochemical properties.
(1) Substrate specificity High reactivity with hydrogen peroxide in the presence of NAD (P) H and substantially no action on peroxides (2) Molecular weight of about 50 kDa (by SDS polyacrylamide gel electrophoresis) Measurement)
なお、本発明において過酸化水素とは、水素原子に過酸基を有する化合物であり、過酸化物とは、通常の過酸化脂質（例えば、過酸化リノール酸など）の他、炭化水素基に過酸基を有する過酸化物（例えば、過酸化ブチル、過酸化クメンなど）を含む概念であり、過酸化水素は含まれない。また、上記の酵素反応において、過酸化水素は水に分解（還元）されていると推測される。 In the present invention, hydrogen peroxide is a compound having a peracid group on a hydrogen atom, and the peroxide is an ordinary lipid peroxide (for example, linoleic peroxide) or a hydrocarbon group. It is a concept that includes a peroxide having a peracid group (for example, butyl peroxide, cumene peroxide, etc.) and does not include hydrogen peroxide. In the above enzyme reaction, it is presumed that hydrogen peroxide is decomposed (reduced) into water.
さらに、前記過酸化水素分解酵素は、配列番号１に記載のアミノ酸配列をＮ末端に有する。得られた３０残基の配列に対し相同性の高いものとして、例えば、ペディオコッカス・ペントサス（Pediococcus pentosaceus）ATCC25745のUncharacterized NAD (FAD)-dependent dehydrogenase（８３％の相同性）やラクトバシラス・アシドフィルス（Lactobacillus acidophilus）のUncharacterized NADH peroxidase（７６％の相同性）を例示することができる。 Furthermore, the hydrogen peroxide degrading enzyme has the amino acid sequence shown in SEQ ID NO: 1 at the N-terminus. Examples of highly homologous to the obtained 30-residue sequence include, for example, Uncharacterized NAD (FAD) -dependent dehydrogenase (83% homology) of Pediococcus pentosaceus ATCC25745 and Lactobacillus acidophilus ( Lactobacillus acidophilus ) Uncharacterized NADH peroxidase (76% homology).
本発明に係る過酸化水素分解酵素は、例えば、飲食品、化粧品、医薬品に含有させた状態で用いることができる。前記酵素を飲食品等に含有させることにより、該酵素が有する過酸化水素除去能力を活用することが期待できる。 The hydrogen peroxide decomposing enzyme according to the present invention can be used, for example, in a state in which it is contained in foods, drinks, cosmetics, and pharmaceuticals. By including the enzyme in food or drink, it can be expected to utilize the hydrogen peroxide removing ability of the enzyme.
以下に実施例を示して本発明を詳細に説明するが、本発明は当該実施例に限定されるものではない。 Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to the examples.
１．乳酸菌のスクリーニング
1-1.食品懸濁液の調製
乳酸菌の分離源として、散麹、豆麹、餅麹、麹漬、味噌漬け、糠漬、野沢菜漬、すんき漬、ベッタラ漬、味噌、醤油、酒粕、納豆、米酢、バルサミコ酢、リンゴ酢、キムチ、腐乳、ナンプラー、とうふよう、ワイン粕、酒粕、ヨーグルト、ベジマイト、クワス、クミス、ギビヤック、ピクルス、クミス、サワークラウト、果実、野菜、海藻、魚類を入手し、前記食品をそれぞれ破砕した後、生理食塩水で希釈して食品懸濁液とした。
1. Screening for lactic acid bacteria
1-1. Preparation of food suspension As sources for separating lactic acid bacteria, Koji, bean paste, koji, koji pickled, miso pickled, koji pickled, Nozawa nazuke, sunki zuke, bettara pickled, miso, soy sauce, sake lees, Natto, rice vinegar, balsamic vinegar, apple vinegar, kimchi, roasted milk, nanpura, tofuyo, wine lees, sake lees, yogurt, vegemite, quas, cumis, gibiyac, pickles, kumis, sauerkraut, fruit, vegetable, seaweed, fish After obtaining and crushing each of the foods, it was diluted with physiological saline to obtain a food suspension.
1-2.スクリーニング
ＧＹＰ培地（１％ グルコース，１％ 酵母エキス，0.5％ ペプトン，0.2％ 酢酸ナトリウム・3H20，20ppm MgSO4・7H20，1ppm MnSO4・4H20，1ppm FeSO4・7H20，1ppm NaCl，50ppm Tween80）にアジ化ナトリウム（終濃度30ppm）、ポリミキシンＢ（終濃度30ppm）およびシクロヘキシミド（終濃度30ppm）を含む選択寒天培地に前記「1-1.食品懸濁液の調製」で調製した食品懸濁液をそれぞれ添加し、３０℃で１６時間静置培養した。培養終了後、前記「1-1.食品懸濁液の調製」に記載の分離源から調製した食品懸濁液添加試料からコロニーが生育していることが確認され、１コロニーずつ単離した。そして、得られた乳酸菌株のコロニーをＧＹＰ寒天培地上に植菌して常法にしたがい純化した。そして、上記食品群から分離した乳酸菌株をTY-1573株と命名した。
1-2. Screening GYP medium (1% glucose, 1% yeast extract, 0.5% peptone, 0.2% sodium acetate · 3H 2 0,20ppm MgSO 4 · 7H 2 0,1ppm MnSO 4 · 4H 2 0,1ppm FeSO 4 · 7H 2 0,1ppm NaCl, 50ppm Tween80 sodium azide) (final concentration 30 ppm), polymyxin B (the on selective agar medium containing final concentration 30 ppm) and cycloheximide (final concentration 30 ppm) "1-1. food suspension Each of the food suspensions prepared in “Preparation” was added and statically cultured at 30 ° C. for 16 hours. After completion of the culture, it was confirmed that colonies had grown from the food suspension-added sample prepared from the separation source described in “1-1. Preparation of food suspension”, and each colony was isolated. The obtained colonies of lactic acid strains were inoculated on a GYP agar medium and purified according to a conventional method. And the lactic acid strain isolate | separated from the said food group was named TY-1573 strain | stump | stock.
２.乳酸菌の菌学的性質
前記「1-2.スクリーニング」で分離した乳酸菌株（TY-1573株）の菌学的性質を、乳酸菌実験マニュアル（小崎道雄監修、内村泰、岡田早苗著、朝倉書店）にしたがって検討した。結果を表１に示す。
2. Bacteriological properties of lactic acid bacteria The bacteriological properties of the lactic acid bacteria strain (TY-1573 strain) isolated in the above “1-2. Screening” are shown in the lactic acid bacteria experiment manual (supervised by Michio Kosaki, Yasushi Uchimura, Sanae Okada, Asakura). (Bookstore). The results are shown in Table 1.
３．乳酸菌の遺伝学的特性
前記「1-2.スクリーニング」で分離したTY-1573株について、常法にしたがい16S rDNAの塩基配列を決定し、BLASTプログラムおよびClustal Wプログラムを使用して、既存の乳酸菌のうちどの菌種の配列に最も近いか検索した。その結果、乳酸菌TY-1573株の16S rDNAの塩基配列がペディオコッカス・ペントサス(Pediococcus pentosaceus)の16S rDNAの塩基配列と100％一致した。以上の結果から、乳酸菌TY-1573株はペディオコッカス・ペントサス(Pediococcus pentosaceus)と同定された。
3. Genetic characteristics of lactic acid bacteria For TY-1573 strain isolated in “1-2. Screening”, the base sequence of 16S rDNA was determined according to the conventional method, and the existing lactic acid bacteria were analyzed using the BLAST program and Clustal W program. We searched which bacterial species was closest to the sequence. As a result, the base sequence of 16S rDNA of lactic acid bacteria strain TY-1573 was 100% identical with the base sequence of 16S rDNA of Pediococcus pentosaceus . From the above results, the lactic acid bacterium strain TY-1573 was identified as Pediococcus pentosaceus .
４．乳酸菌の過酸化水素分解特性
前記「３．乳酸菌の遺伝学的特性」で同定されたTY-1573株の菌体懸濁液に過酸化水素を添加し、所定時間経過後の反応液中に残存する過酸化水素の残量を測定して、過酸化水素に対する分解特性を調べた。
4). Hydrogen peroxide decomposition characteristics of lactic acid bacteria Hydrogen peroxide is added to the cell suspension of TY-1573 strain identified in “3. Genetic characteristics of lactic acid bacteria” and remains in the reaction solution after a predetermined time. The remaining amount of hydrogen peroxide to be measured was measured to investigate the decomposition characteristics against hydrogen peroxide.
4-1.菌体懸濁液の調製
TY1573株の菌株をそれぞれＧＹＰ培地に接種して３７℃で約２４時間静置培養した。培養終了後、培養物を8,000rpmで１０分間遠心分離し、上清を除去することにより菌体を集菌した。次いで、集菌した菌体をリン酸緩衝液に懸濁し、OD660nm＝1.6に濃度調節したものを菌体懸濁液とした。
4-1. Preparation of cell suspension
Each strain of TY1573 strain was inoculated into GYP medium and cultured at 37 ° C. for about 24 hours. After completion of the culture, the culture was centrifuged at 8,000 rpm for 10 minutes, and the cells were collected by removing the supernatant. Next, the collected bacterial cells were suspended in a phosphate buffer solution, and the concentration adjusted to OD 660 nm = 1.6 was used as the bacterial cell suspension.
4-2.過酸化水素に対する分解特性
前記「4-1.菌体懸濁液の調製」で調製した菌体懸濁液に過酸化水素（終濃度3.0mM）とグルコース（終濃度50mM）をそれぞれ加え、水平震盪機にて３７℃で３時間反応させた。反応終了後、卓上遠心機にて反応液を遠心処理し、上清に含まれる未反応の過酸化水素を測定した。
4-2. Degradation characteristics for hydrogen peroxide Hydrogen peroxide (final concentration: 3.0 mM) and glucose (final concentration: 50 mM) were added to the bacterial cell suspension prepared in “4-1. Preparation of bacterial cell suspension”. Each was added and reacted at 37 ° C. for 3 hours on a horizontal shaker. After completion of the reaction, the reaction solution was centrifuged using a tabletop centrifuge, and unreacted hydrogen peroxide contained in the supernatant was measured.
過酸化水素の濃度測定には、Sedewitzらの方法を用いた（Sedewitz et al.,Journal of Bacteriology,160,Oct.273-278. 1984）。具体的には、まず、前記反応液（上清部分）を50μl、４-アミノアンチピリン（濃度24.4mg/20ml）を400μl、3,5-ジクロロベンゼンスルホン酸（濃度111.4mg/20ml）を400μlそれぞれ混合し、該混合液に西洋ワサビ由来ペルオキシダーゼを4μl（500unit）添加し、３０℃で１５分間反応させた。反応終了後、得られた被検液の546nmにおける吸光度を測定し、該被検液中に含まれる過酸化水素の濃度を求めた。そして、得られたデータに基づいて過酸化水素の分解量を算出したところ、約2800μＭが得られた。 The method of Sedewitz et al. Was used to measure the concentration of hydrogen peroxide (Sedewitz et al., Journal of Bacteriology, 160, Oct. 273-278. 1984). Specifically, first, 50 μl of the reaction solution (supernatant part), 400 μl of 4-aminoantipyrine (concentration 24.4 mg / 20 ml) and 400 μl of 3,5-dichlorobenzenesulfonic acid (concentration 111.4 mg / 20 ml), respectively. After mixing, 4 μl (500 units) of horseradish peroxidase was added to the mixture and reacted at 30 ° C. for 15 minutes. After completion of the reaction, the absorbance at 546 nm of the obtained test solution was measured to determine the concentration of hydrogen peroxide contained in the test solution. When the amount of hydrogen peroxide decomposed was calculated based on the obtained data, about 2800 μM was obtained.
５．過酸化水素分解酵素の精製
過酸化水素分解酵素の精製を次のようにして行った。なお、各精製段階における、ＮＡＤＨ濃度、過酸化水素濃度、タンパク質濃度およびＳＤＳ−ＰＡＧＥは下記の方法にて測定した。
5). Purification of hydrogen peroxide-degrading enzyme Hydrogen peroxide-degrading enzyme was purified as follows. The NADH concentration, hydrogen peroxide concentration, protein concentration, and SDS-PAGE in each purification step were measured by the following methods.
（ＮＡＤＨ濃度）
ＮＡＤＨの濃度測定には、該物質が有する吸光波長(λ＝340nm)の減少にて活性を測定した。具体的には、マイクロブラックセルに約10mM、pH無調製トリス緩衝液500μlに溶解した終濃度150μM ＮＡＤＨ、終濃度500μM 過酸化水素、1/100倍容 被検試料（細胞抽出液又は各精製段階で得られた各画分）を予め3分間37℃にて加温し、順次添加しその都度転換混和した。ダブルビーム吸光度計（ε340＝6.220 M−1cm−1）にて本酵素の活性を37℃で経時的にＮＡＤＨの減少量として測定した。過酸化水素の分解に伴うＮＡＤＨ減少活性は、過酸化水素を添加した際の初速から添加直後の前記被検試料の初速を引いて算出した。
(NADH concentration)
In measuring the concentration of NADH, the activity was measured by decreasing the absorption wavelength (λ = 340 nm) of the substance. Specifically, about 10 mM in a micro black cell, final concentration 150 μM NADH, final concentration 500 μM hydrogen peroxide dissolved in 500 μl of pH unadjusted Tris buffer, 1 / 100-fold test sample (cell extract or each purification step) Each fraction obtained in (1) was preheated at 37 ° C. for 3 minutes in advance, added sequentially, and mixed by inversion each time. The activity of the enzyme was measured as a decrease in NADH over time at 37 ° C. using a double beam absorptiometer (ε 340 = 6.220 M −1 cm −1 ). The NADH reduction activity associated with the decomposition of hydrogen peroxide was calculated by subtracting the initial speed of the test sample immediately after the addition from the initial speed when hydrogen peroxide was added.
（過酸化水素濃度）
過酸化水素の濃度測定は、チオシアン酸−鉄錯体による発色法を用いた。前記「（ＮＡＤＨ濃度）」と同じ実験系を用い、過酸化水素と反応させた過酸化水素分解酵素を適宜希釈し、500μl、2.0 mlエッペンドルフチューブに採取し、10%トリクロロ酢酸水溶液を500μl、0.01 N 硫酸にて溶解した硫酸アンモニウム鉄(II)10 mMを100μl、2.5 M チオシアン酸カリウムを500μl 順次添加し、赤色に反応したサンプルを、ダブルビーム分光光度計にてλ＝480nmにて測定した。過酸化水素の定量には、常法により測定値から検量線を作成し、被検試料（細胞抽出液又は各精製段階で得られた各画分）を測定し、過酸化水素濃度を算出した。
(Hydrogen peroxide concentration)
The concentration of hydrogen peroxide was measured by a color development method using a thiocyanate-iron complex. Using the same experimental system as the “(NADH concentration)”, the hydrogen peroxide-degrading enzyme reacted with hydrogen peroxide was appropriately diluted and collected in a 500 μl, 2.0 ml Eppendorf tube, and 10% trichloroacetic acid aqueous solution was added in 500 μl, 0.01 N 100 mM iron ammonium sulfate (II) dissolved in sulfuric acid and 100 μl 2.5 M potassium thiocyanate were sequentially added, and the sample that reacted in red was measured at λ = 480 nm with a double beam spectrophotometer. For the determination of hydrogen peroxide, a calibration curve was created from the measured values by a conventional method, and the test sample (cell extract or each fraction obtained at each purification stage) was measured to calculate the hydrogen peroxide concentration. .
（タンパク質濃度）
過酸化水素分解活性を有する各画分のタンパク質濃度は、ブラッドフォード法と呼ばれる常法を用いて算出した。測定原理はクマシーブリリアントブルー(CBB)という色素がタンパク質と結合した際の赤紫色から青への変色を利用したものである。本法では被検試料中に含まれるタンパク質濃度に比例し、濃い青色を示す。タンパク質の濃度の定量を行う際には、既知のタンパク質である鶏卵白アルブミンにて検量線を作製し、タンパク質濃度を算出した。
(Protein concentration)
The protein concentration of each fraction having hydrogen peroxide decomposing activity was calculated using a conventional method called the Bradford method. The measurement principle uses the color change from red purple to blue when a dye called Coomassie Brilliant Blue (CBB) binds to a protein. In this method, it is proportional to the protein concentration contained in the test sample and shows a deep blue color. When quantifying the protein concentration, a calibration curve was prepared with chicken egg white albumin, which is a known protein, and the protein concentration was calculated.
（ＳＤＳ−ＰＡＧＥ）
酵素精製過程における精製度の確認や、回収に際し有用な画分の判断には、ＳＤＳ−ＰＡＧＥを使用した。酵素の解析には、「初歩からのバイオ実験」（大山 徹、渡部 俊弘編著、三共出版）を参考に実施し、必要に応じて前著の変法を用いている。
(SDS-PAGE)
SDS-PAGE was used for confirmation of the degree of purification in the enzyme purification process and determination of fractions useful for collection. Enzyme analysis is performed with reference to “Bio-experiment from the beginning” (Toru Oyama, Toshihiro Watanabe, Sankyo Publishing), and uses the modified method of the previous book as necessary.
アクリルアミド29.2g、N,N’-メチレンビスアクリルアミド0.8g、100ml蒸留水にて調製したゲル作製ストック溶液Ａ液、pH8.8に調整した1.5Mトリス緩衝液に0.4％SDSを溶解せしめたゲル作製ストック溶液Ｂ液、pH6.8に調整した0.5Mトリス緩衝液に0.4％SDSを溶解せしめたゲル作製ストック溶液Ｃ液、および10％過硫酸アンモニウム溶液からなるゲル作製ストック溶液Ｄ液をそれぞれ調製した。上記ゲル作製ストック溶液と、N,N,N’N’-テトラメチルエチレンジアミン(TEMED)の組み合わせにてSDSポリアクリルアミドゲルの調製を行った。SDSポリアクリルアミドゲルの調製に際し、各種ストック溶液はアスピレーターにて溶存酸素を除去して実施した。各種ストック溶液の組み合わせについては後述する。 Gel preparation stock solution A solution prepared with 29.2 g of acrylamide, 0.8 g of N, N'-methylenebisacrylamide, 100 ml distilled water, and gel preparation with 0.4% SDS dissolved in 1.5 M Tris buffer adjusted to pH 8.8 Stock solution B, gel preparation stock solution C prepared by dissolving 0.4% SDS in 0.5 M Tris buffer adjusted to pH 6.8, and gel preparation stock solution D comprising 10% ammonium persulfate solution were prepared. An SDS polyacrylamide gel was prepared using a combination of the above gel preparation stock solution and N, N, N′N′-tetramethylethylenediamine (TEMED). In preparing the SDS polyacrylamide gel, various stock solutions were removed from the dissolved oxygen with an aspirator. The combination of various stock solutions will be described later.
分離用SDSポリアクリルアミドゲルの作製にあたっては、フラスコに、蒸留水6.0ml、ゲル作製ストック溶液Ｂ液4.5ml、ゲル作製ストック溶液Ａ液7.5ml、ゲル作製ストック溶液Ｄ液0.07ml、TEMED0.01mlの順番に添加し、撹拌後に作成用プレートに分注した。分離用SDSポリアクリルアミドゲルの硬化後、濃縮用SDSポリアクリルアミドゲルを重層した。 In preparation of the SDS polyacrylamide gel for separation, the flask was made up of 6.0 ml of distilled water, 4.5 ml of gel preparation stock solution B, 7.5 ml of gel preparation stock solution A, 0.07 ml of gel preparation stock solution D, 0.07 ml of TEMED 0.01 ml. They were added in order and dispensed into a preparation plate after stirring. After the separation SDS polyacrylamide gel was cured, the concentration SDS polyacrylamide gel was overlaid.
濃縮用SDSポリアクリルアミドゲルの作製にあたっては、フラスコに蒸留水3.6ml、ゲル作製ストック溶液Ｃ液1.5ml、ゲル作製ストック溶液Ａ液0.9ml、ゲル作製ストック溶液Ｄ液0.018ml、TEMED0.01mlを順番に添加し、撹拌後にSDSポリアクリルアミドゲル作成用プレートに重層した。重層後速やかにサンプルコームを挿入し、重合反応が終了するまで放置した。 In preparation of SDS polyacrylamide gel for concentration, 3.6ml of distilled water, 1.5ml of gel preparation stock solution C, 0.9ml of gel preparation stock solution A, 0.918ml of gel preparation stock solution D, 0.01ml of TEMED 0.01ml After stirring, it was layered on a plate for SDS polyacrylamide gel preparation. A sample comb was inserted immediately after the layering, and the sample was allowed to stand until the polymerization reaction was completed.
作製したゲルプレートを蒸留水1L当たりトリス緩衝液3.828g、グリシン14.411g、SDS 1gを含む泳動用緩衝液で満たした泳動槽にセットした。10%SDS 100μl、2-メルカプトエタノール 10μl、ゲル作製ストックC液 20μl、グリセリン 200μlからなるSDS処理緩衝液を5μlと粗酵素液や解析目的のサンプル10μlを添加混合後5分間煮沸処理し、処理サンプルを10μlとグリセリン12ml、蒸留水8ml、ブロモフェノールブルー10mgからなるマーカー色素2μlとを混合しサンプルコームを抜いたウェルに分注した。濃縮ゲル部分では7.5〜10ｍＡ、濃縮ゲル通過後は15〜20ｍＡに電流を印加し電気泳動を行った。電気泳動はゲルプレート下端手前約1cmにて泳動を停止し、染色工程に移行した。 The prepared gel plate was set in an electrophoresis tank filled with an electrophoresis buffer containing 3.828 g of Tris buffer, 14.411 g of glycine and 1 g of SDS per liter of distilled water. 10% SDS 100 μl, 2-mercaptoethanol 10 μl, gel preparation stock solution C 20 μl, glycerin 200 μl SDS treatment buffer 5 μl, crude enzyme solution and 10 μl sample for analysis are added and mixed, then boiled for 5 minutes. 10 μl, 12 ml of glycerin, 8 ml of distilled water and 2 μl of marker dye consisting of 10 mg of bromophenol blue were mixed and dispensed into wells from which the sample comb had been removed. Electrophoresis was performed by applying a current of 7.5 to 10 mA in the concentrated gel portion and 15 to 20 mA after passing through the concentrated gel. Electrophoresis stopped at about 1 cm before the lower end of the gel plate and moved to the staining step.
泳動終了後のポリアクリルアミドゲルをメタノール500ml、酢酸100ml、蒸留水400 ml、クマシーブリリアントブルー(CBB) R-250 2.5gからなる染色液にて30分染色し、明瞭な染色バンドが得られるまで、メタノール500ml、酢酸100ml、蒸留水400 mlからなる脱色液にて脱色作業を行った。得られた染色バンドについては、ゲルドライヤー、セロファン膜にて乾燥保管し、精製酵素の精製度や回収画分について考察を行った。 After the electrophoresis, the polyacrylamide gel is stained with a staining solution consisting of 500 ml of methanol, 100 ml of acetic acid, 400 ml of distilled water, and 2.5 g of Coomassie Brilliant Blue (CBB) R-250 until a clear staining band is obtained. Decolorization was performed with a decolorizing solution consisting of 500 ml of methanol, 100 ml of acetic acid and 400 ml of distilled water. About the obtained dyeing | staining band, it dried and stored with the gel dryer and the cellophane membrane, and the refinement | purification degree and collection | recovery fraction of purified enzyme were considered.
5-1.粗酵素液の調製
乳酸菌を培養する方法については、当業者が用いる常法に従って行った。乳酸菌の培養に通常用いられるGYP液体培地２０ lにペディオコッカス・ペントサス（Pediococcus pentosaceus）TY-1573株（NITE AP−91）を接種し、ジャーファーメンターにて３７℃、１３〜１４時間培養した。対数増殖期後期に培養菌体を遠心処理にて回収した。回収培養菌体についてはpH7.0、50ｍM リン酸緩衝液にて培養菌体を洗浄した。
5-1. Preparation of Crude Enzyme Solution The method for culturing lactic acid bacteria was performed according to a conventional method used by those skilled in the art. Pediococcus pentosaceus TY-1573 strain (NITE AP-91) was inoculated into 20 l of GYP liquid medium usually used for lactic acid bacteria culture, and cultured at 37 ° C. for 13 to 14 hours in a jar fermenter. . In the late logarithmic growth phase, the cultured cells were collected by centrifugation. The collected cultured cells were washed with a pH 7.0, 50 mM phosphate buffer.
上述の操作で得られた乳酸菌体を7.5g秤量し、リン酸緩衝液で５倍容に希釈した菌体を撹拌して菌体懸濁液とした。該菌体懸濁液に5mgリゾチームを添加し、３０分間撹拌した。その後、超音波処理機にて４分間、さらに超高圧細胞破砕機にて菌体破砕を４度実施した。こうして得られた懸濁液を0℃、7000rpm、20分間遠心処理して未破砕菌体を菌体破砕液から除去したものを粗酵素液とした。 7.5 g of the lactic acid bacteria obtained by the above operation was weighed, and the bacteria diluted 5 times with a phosphate buffer were stirred to obtain a cell suspension. 5 mg lysozyme was added to the cell suspension and stirred for 30 minutes. Then, microbial cell crushing was carried out four times with an ultrasonic treatment machine and further with an ultra-high pressure cell crusher four times. The suspension obtained in this manner was centrifuged at 0 ° C. and 7000 rpm for 20 minutes to remove unbroken cells from the cell disruption solution, which was used as a crude enzyme solution.
5-2.塩析処理およびクロマトグラフィー
上述の操作で得られた粗酵素液に終濃度23％硫酸アンモニウムを添加して不要なタンパクを沈殿せしめて除去し、さらに粗酵素溶液上清に対し10％ストレプトマイシン硫酸塩-トリス塩酸緩衝液溶液(pH 8.0、10mM)を1.0 ml添加し、不要な核酸を沈殿せしめて除去した。本工程で得られた上清を以下の順番にカラムに供して精製を実施した。
5-2. Salting-out treatment and chromatography Add the final concentration of 23% ammonium sulfate to the crude enzyme solution obtained by the above operation to precipitate and remove unnecessary proteins, and further 10% of the crude enzyme solution supernatant. 1.0 ml of streptomycin sulfate-Tris-HCl buffer solution (pH 8.0, 10 mM) was added to precipitate and remove unnecessary nucleic acids. The supernatant obtained in this step was applied to a column in the following order for purification.
上述の操作で得られた粗酵素液の上清を23% 硫酸アンモニウム含有、50ｍMリン酸緩衝液（pH7.0）で平衡化された疎水ブチルカラムに供し、画分を得た。精製工程における各画分の過酸化水素分解量を測定し、精製工程で得られたタンパク質のピークパターンと過酸化水素分解活性から必要な画分を回収後、回収画分に対して透析処理を24〜48時間行った。 The supernatant of the crude enzyme solution obtained by the above operation was applied to a hydrophobic butyl column containing 23% ammonium sulfate and equilibrated with 50 mM phosphate buffer (pH 7.0) to obtain a fraction. Measure the amount of hydrogen peroxide decomposed in each fraction in the purification process, collect the required fraction from the protein peak pattern and hydrogen peroxide decomposition activity obtained in the purification process, and then dialyze the recovered fraction. 24 to 48 hours.
疎水ブチルカラムにて精製した前記画分を、pH 7.0、50mM リン酸緩衝液で平衡化済みの弱陰イオン交換カラムに供した。精製工程では250mM NaCl含有 50mM リン酸緩衝液で濃度勾配溶出を3カラム容量分実施し、同じく250mM NaCl含有リン酸緩衝液にて3カラム容量分溶出した後、1M NaCl含有リン酸緩衝液にて濃度勾配溶出し精製画分を得た。精製工程における各画分の過酸化水素分解量を測定し、精製工程で得られたタンパク質のピークパターンと過酸化水素分解活性から必要な画分を回収後、回収画分に対して透析処理を24〜48時間行った。 The fraction purified on a hydrophobic butyl column was applied to a weak anion exchange column that had been equilibrated with a pH 7.0, 50 mM phosphate buffer. In the purification step, concentration gradient elution was carried out for 3 column volumes with 50 mM phosphate buffer containing 250 mM NaCl, and 3 column volumes were eluted with the same phosphate buffer containing 250 mM NaCl, and then with phosphate buffer containing 1 M NaCl. A purified fraction was obtained by concentration gradient elution. Measure the amount of hydrogen peroxide decomposed in each fraction in the purification process, collect the required fraction from the protein peak pattern and hydrogen peroxide decomposition activity obtained in the purification process, and then dialyze the recovered fraction. 24 to 48 hours.
そして、透析処理した画分を限外濾過膜で処理して9kDa以下の莢雑タンパク質、ペプチドを除去し、透析画分容量から6倍の濃度になるように濃縮処理を実施し濃縮画分とした。次に濃縮画分に対して透析処理を24〜48時間行った。 The dialyzed fraction is treated with an ultrafiltration membrane to remove contaminating proteins and peptides of 9 kDa or less, and concentrated so that the concentration is 6 times the dialysis fraction volume. did. The concentrated fraction was then dialyzed for 24-48 hours.
次に、濃縮画分に対して透析処理を行った前記画分をpH7.0、50mM リン酸緩衝液で平衡化済の強陰イオン交換カラムに供した。250mM NaCl含有50mM リン酸緩衝液で濃度勾配溶出を実施し1M 塩化ナトリウム含有リン酸緩衝液で切り替え溶出を実施した。精製工程で得られたタンパク質のピークパターンと過酸化水素分解活性から必要な画分を回収後、回収画分に対して透析処理を24〜48時間行った。 Next, the concentrated fraction was dialyzed and applied to a strong anion exchange column equilibrated with 50 mM phosphate buffer at pH 7.0. Concentration elution was carried out with 50 mM phosphate buffer containing 250 mM NaCl, and switching elution was carried out with phosphate buffer containing 1 M sodium chloride. The necessary fraction was collected from the protein peak pattern and hydrogen peroxide decomposing activity obtained in the purification step, and the collected fraction was dialyzed for 24-48 hours.
続いて、得られた画分を50mM 塩化ナトリウム含有、pH7.0、50 mM リン酸緩衝液で平衡化済のハイドロキシアパタイトカラムに供した。100 mM 塩化ナトリウム含有pH7.0、50 mM リン酸緩衝液、200 mM 塩化ナトリウム含有 pH7.0、50mM リン酸緩衝液、400 mM 塩化ナトリウム含有 pH 7.0、50 mM リン酸緩衝液で順次溶出を実施した後、1 M NaCl含有pH 7.0、50mMリン酸緩衝液に切り替え溶出し精製画分を得た。精製工程で得られたタンパク質のピークと過酸化水素分解活性から必要な画分を回収後、回収画分に対して透析処理を24〜48時間行った。 Subsequently, the obtained fraction was applied to a hydroxyapatite column containing 50 mM sodium chloride, pH 7.0, and equilibrated with 50 mM phosphate buffer. Elution was performed sequentially with 100 mM sodium chloride-containing pH 7.0, 50 mM phosphate buffer, 200 mM sodium chloride-containing pH 7.0, 50 mM phosphate buffer, 400 mM sodium chloride-containing pH 7.0, and 50 mM phosphate buffer. Then, the purified fraction was obtained by switching to 1 mM NaCl-containing pH 7.0, 50 mM phosphate buffer elution. The necessary fraction was collected from the protein peak and hydrogen peroxide decomposing activity obtained in the purification step, and the collected fraction was dialyzed for 24-48 hours.
回収画分中に特に過酸化水素の分解能力が高く、精製されているバンドをＳＤＳ−ＰＡＧＥにかけたところ、約５０ｋＤａの位置に単一バンドの存在を確認し、本画分を精製酵素とした。また、ＮＡＤＨ減少活性値、過酸化水素分解活性値およびタンパク質量から算出したところ、前記精製酵素の過酸化水素分解活性は４０Ｕ／ｍｇタンパク質量であった。 In the recovered fraction, when the hydrogen peroxide decomposition ability was particularly high and the purified band was subjected to SDS-PAGE, the presence of a single band was confirmed at about 50 kDa, and this fraction was used as a purified enzyme. . Further, when calculated from the NADH decreasing activity value, the hydrogen peroxide decomposing activity value, and the protein amount, the hydrogen peroxide decomposing activity of the purified enzyme was 40 U / mg protein amount.
６．Ｎ末端アミノ酸配列の分析
精製酵素が確認されたSDSポリアクリルアミドゲルプレートは、セミドライブロティング装置を用いて、PVDF膜にブロッティングし、Ｎ末端アミノ酸配列分析に供した。
6). Analysis of N-terminal amino acid sequence The SDS polyacrylamide gel plate in which the purified enzyme was confirmed was blotted on a PVDF membrane using a semi-driving apparatus and subjected to N-terminal amino acid sequence analysis.
メタノール50ml、蒸留水450ml、トリスアミノメタン18.15g、10％SDS 2.5mlを混合し、ブロッティングＡ液とした。メタノール50ml、蒸留水450ml、トリスアミノメタン1.5g、10％SDS 2.5mlを混合し、ブロッティングＢ液とした。メタノール100ml、蒸留水900ml、トリスアミノメタン3.0g、6-アミノ-n-カプロン酸5.2g、10％SDS 5.0mlを混合し、ブロッティングＣ液とした。また、アミノ酸配列分析の為に1Mチオグリコール酸ナトリウム水溶液と、1Mチオグリコール酸ナトリウム水溶液10μl、前述の色素マーカー90μlからなるブロッティング用色素マーカーを用意した。次にCBB R-250 0.12g、メタノール20ml、酢酸 50ml、蒸留水50mlからなるPVDF膜用染色液と、メタノール20ml、酢酸 50ml、蒸留水50mlからなるPVDF膜用脱色液を準備し、以下の実験に供した。 50 ml of methanol, 450 ml of distilled water, 18.15 g of trisaminomethane, and 2.5 ml of 10% SDS were mixed to prepare a blotting A solution. 50 ml of methanol, 450 ml of distilled water, 1.5 g of trisaminomethane, and 2.5 ml of 10% SDS were mixed to prepare Blotting B solution. Blotting solution C was prepared by mixing 100 ml of methanol, 900 ml of distilled water, 3.0 g of trisaminomethane, 5.2 g of 6-amino-n-caproic acid, and 5.0 ml of 10% SDS. For amino acid sequence analysis, a 1M sodium thioglycolate aqueous solution, 10 μl of 1M sodium thioglycolate aqueous solution, and 90 μl of the aforementioned dye marker were prepared for blotting. Next, prepare a staining solution for PVDF membrane consisting of 0.12 g of CBB R-250, 20 ml of methanol, 50 ml of acetic acid and 50 ml of distilled water, and a decolorizing solution for PVDF membrane consisting of 20 ml of methanol, 50 ml of acetic acid and 50 ml of distilled water. It was used for.
精製酵素を含むSDSポリアクリルアミドゲルをブロッティングＣ液に浸し、5分間浸透した。次に、PVDF膜をメタノールに浸した後、Ｃ液に浸したゲル容器にPVDF膜を浸し、5分間浸透した。下から、ブロッティングＡ液に浸した濾紙、ブロッティングＡ液に浸した濾紙、ブロッティングＢ液に浸した濾紙、ブロッティングＢ液に浸した濾紙、PVDF膜、SDSポリアクリルアミドゲル、ブロッティングＣ液に浸した濾紙、ブロッティングＣ液に浸した濾紙の順番に重層し、ゲルの面積×1mA分、ブロッティング泳動を実施した。PVDF膜、ゲルについてはそれぞれCBB染色後、精製酵素に該当するバンドを切り出して、アミノ酸配列分析を実施した。アミノ酸配列分析には、常法であるPITCもしくはエドマン法にて自動解析を行った。アミノ酸配列分析の結果、配列番号１のアミノ酸配列を得ることができた。 SDS polyacrylamide gel containing the purified enzyme was immersed in Blotting C solution and permeated for 5 minutes. Next, after immersing the PVDF membrane in methanol, the PVDF membrane was immersed in a gel container immersed in the liquid C and allowed to permeate for 5 minutes. From below, filter paper soaked in Blotting A liquid, filter paper soaked in Blotting A liquid, filter paper soaked in Blotting B liquid, filter paper soaked in Blotting B liquid, PVDF membrane, SDS polyacrylamide gel, filter paper soaked in Blotting C liquid The filter paper soaked in Blotting C solution was layered in order, and blotting electrophoresis was performed for the gel area × 1 mA. For PVDF membranes and gels, after CBB staining, bands corresponding to purified enzymes were cut out and amino acid sequence analysis was performed. For amino acid sequence analysis, automatic analysis was performed by the conventional method PITC or Edman method. As a result of amino acid sequence analysis, the amino acid sequence of SEQ ID NO: 1 could be obtained.
得られたＮ末端アミノ酸配列３０残基の配列は、ペディオコッカス・ペントサス（Pediococcus pentosaceus） ATCC25745のUncharacterized NAD (FAD)-dependent dehydrogenaseのＮ末端アミノ酸配列３０残基と８３％の相同性を、ラクトバシラス・アシドフィルス（Lactobacillus acidophilus）のUncharacterized NADH peroxidaseのＮ末端アミノ酸配列３０残基と７６％の相同性を示した。 The obtained 30-residue N-terminal amino acid sequence was found to have 83% homology with the 30-character N-terminal amino acid sequence of Uncharacterized NAD (FAD) -dependent dehydrogenase of Pediococcus pentosaceus ATCC25745. -It showed 76% homology with 30 residues of the N-terminal amino acid sequence of Lactobacillus acidophilus Uncharacterized NADH peroxidase.
７．基質特異性
「５．過酸化水素分解酵素の精製 （ＮＡＤＨ濃度）」の実験系のうち、被検試料として精製した過酸化水素分解酵素を使用し、過酸化水素に代えて過酸化クメン（過酸化物）を使用した以外は前記と同様に酵素反応を行わせることにより、過酸化クメン濃度を測定し、前記過酸化水素分解酵素が過酸化クメンに対する分解活性を有するか否かを調べた。過酸化クメンの濃度測定は、前記「５．過酸化水素分解酵素の精製 （過酸化水素濃度）」に準じて行った。結果、反応前後で過酸化クメン濃度は変化せず、このことから、本発明に係る過酸化水素分解酵素は過酸化クメンには実質的には作用しないことが分かった。
7). Substrate specificity In the experimental system of “5. Purification of hydrogen peroxide-degrading enzyme (NADH concentration)”, purified hydrogen peroxide-degrading enzyme was used as a test sample, and hydrogen peroxide was replaced with cumene peroxide (peroxide). By using the enzyme reaction in the same manner as described above except that (oxide) was used, the concentration of cumene peroxide was measured to determine whether or not the hydrogen peroxide-degrading enzyme had a decomposition activity for peroxide cumene. The concentration of cumene peroxide was measured according to the above-mentioned “5. Purification of hydrogen peroxide-degrading enzyme (hydrogen peroxide concentration)”. As a result, the concentration of cumene peroxide did not change before and after the reaction. From this, it was found that the hydrogen peroxide-degrading enzyme according to the present invention does not substantially act on the peroxide cumene.
８．過酸化水素分解酵素の用途
8-1.過酸化水素分解粗酵素液の製造例
水97.5重量部、ブドウ糖1重量部、大豆ペプチド0.5重量部および酵母エキス1重量部から構成される培地にTY-1573株を接種して、３０℃で１６時間静置培養し、培養後の培養物を遠心処理によって菌体を採取した。この菌体を湿重量に対し５倍容のリン酸緩衝液に懸濁し、２７０ＭＰａ以上の高圧処理にて菌体を破砕し、これを過酸化水素分解粗酵素液とした。
8). Applications of hydrogen peroxide degrading enzymes
8-1. Production Example of Hydrogen Peroxide Degradation Crude Enzyme Solution Inoculate TY-1573 strain on a medium composed of 97.5 parts by weight of water, 1 part by weight of glucose, 0.5 part by weight of soybean peptide and 1 part by weight of yeast extract, The cells were statically cultured at 30 ° C. for 16 hours, and the cultured cells were collected by centrifugation. This microbial cell was suspended in a phosphate buffer solution having a volume 5 times the wet weight, and the microbial cell was crushed by a high-pressure treatment of 270 MPa or more to obtain a hydrogen peroxide-decomposing crude enzyme solution.
8-2.ヨーグルトの製造例
水44.36重量部、ゼラチン0.1重量部、砂糖8.2重量部、乳酸菌スターター0.05重量部、脱脂粉乳5.81重量部、牛乳41.4重量部、過酸化水素分解粗酵素液0.03重量部、香料0.05重量部を混合してヨーグルトを得た。なお、過酸化水素分解粗酵素液は前記で製造したものを使用した。
8-2.Production example of yogurt 44.36 parts by weight of water, 0.1 part by weight of gelatin, 8.2 parts by weight of sugar, 0.05 part by weight of lactic acid bacteria starter, 5.81 parts by weight of skim milk powder, 41.4 parts by weight of milk, 0.03 part by weight of hydrogen peroxide-degrading crude enzyme solution Then, 0.05 part by weight of a fragrance was mixed to obtain yogurt. The hydrogen peroxide-decomposing crude enzyme solution used was the one produced above.
8-3.アイスクリームの製造例
水55.85重量部、砂糖8.0重量部、水飴8.0重量部、液糖4.0重量部、植物油脂13.0重量部、脱脂粉乳8.5重量部、加糖卵黄（２０％卵黄分）2.0重量部、過酸化水素分解粗酵素液0.05重量部、安定剤0.5重量部および香料0.1重量部を混合してアイスクリームを得た。なお、過酸化水素分解粗酵素液は前記で製造したものを使用した。
8-3. Production Example of Ice Cream 55.85 parts by weight of water, 8.0 parts by weight of sugar, 8.0 parts by weight of starch syrup, 4.0 parts by weight of liquid sugar, 13.0 parts by weight of vegetable oil and fat, 8.5 parts by weight of skim milk powder, sweetened egg yolk (20% egg yolk content) An ice cream was obtained by mixing 2.0 parts by weight, 0.05 parts by weight of a hydrogen peroxide-degrading crude enzyme solution, 0.5 parts by weight of a stabilizer and 0.1 parts by weight of a fragrance. The hydrogen peroxide-decomposing crude enzyme solution used was the one produced above.
8-4.化粧水の製造例
水73.89重量部、グリセリン5重量部、可溶化剤5重量部、界面活性剤1重量部、エタノール15.0重量部、過酸化水素分解粗酵素液0.1重量部、防腐剤0.005重量部および香料0.005重量部を混合して化粧水を製造した。なお、過酸化水素分解粗酵素液は前記で製造したものを使用した。
8-4. Example of lotion preparation 73.89 parts by weight of water, 5 parts by weight of glycerin, 5 parts by weight of solubilizer, 1 part by weight of surfactant, 15.0 parts by weight of ethanol, 0.1 part by weight of hydrogen peroxide-degrading crude enzyme solution, antiseptic A lotion was prepared by mixing 0.005 parts by weight of the agent and 0.005 parts by weight of the fragrance. The hydrogen peroxide-decomposing crude enzyme solution used was the one produced above.
Claims (4)
（１）基質特異性
ＮＡＤ（Ｐ）Ｈの存在下、過酸化水素に高い反応性を示し、かつ過酸化物には実質的に作用しない。
（２）分子量
５０ｋＤａ（ＳＤＳポリアクリルアミドゲル電気泳動による測定）。 A hydrogen peroxide-degrading enzyme derived from Pediococcus pentosaceus TY-1573 strain (NITE P-91) and having the following physicochemical properties:
(1) Substrate specificity In the presence of NAD (P) H, it exhibits high reactivity with hydrogen peroxide and does not substantially act on peroxide.
(2) Molecular weight
50 kDa (measured by SDS polyacrylamide gel electrophoresis).
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