JP6697226B2 - New peptidase - Google Patents
New peptidase Download PDFInfo
- Publication number
- JP6697226B2 JP6697226B2 JP2015118315A JP2015118315A JP6697226B2 JP 6697226 B2 JP6697226 B2 JP 6697226B2 JP 2015118315 A JP2015118315 A JP 2015118315A JP 2015118315 A JP2015118315 A JP 2015118315A JP 6697226 B2 JP6697226 B2 JP 6697226B2
- Authority
- JP
- Japan
- Prior art keywords
- peptidase
- pseudozyma
- enzyme
- carboxypeptidase
- aminopeptidase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Description
本発明はペプチダーゼに関する。詳しくは、シュードザイマ・フベイエンシス（Pseudozyma hubeiensis）由来のペプチダーゼ及びその用途に関する。 The present invention relates to peptidases. Specifically, it relates to a peptidase derived from Pseudozyma hubeiensis and its use.
タンパク質分解酵素は作用様式によってエンドペプチダーゼ（プロテイナーゼ）とエキソペプチダーゼ（ペプチダーゼ）に大別される。エキソペプチダーゼには、ペプチドのN末端からのアミノ酸の遊離を触媒するアミノペプチダーゼと、同C末端からのアミノ酸の遊離を触媒するカルボキシペプチダーゼがある。これまでに、微生物由来の各種ペプチダーゼが知られており、様々な用途への適用・応用が試みられている。 Proteolytic enzymes are roughly classified into endopeptidases (proteinases) and exopeptidases (peptidases) depending on the mode of action. Exopeptidases include aminopeptidases that catalyze the release of amino acids from the N-terminus of peptides and carboxypeptidases that catalyze the release of amino acids from the same C-terminus. So far, various peptidases derived from microorganisms have been known, and their application to various uses has been attempted.
ペプチダーゼは酵母（非特許文献１、２）、糸状菌（非特許文献３、４）、バクテリア（非特許文献５、６）等からの単離の報告がある。特に、サッカロマイセス・セレビシエ由来のペプチダーゼの報告が多い（非特許文献７〜９）。サッカロマイセス・セレビシエ由来のアミノペプチダーゼはロイシルアミノペプチダーゼ（EC 3.4.11.1）、アミノペプチダーゼY（EC 3.4.11.15）、アミノペプチダーゼI（EC 3.4.11.22）等が報告され、カルボキシペプチダーゼではカルボキシペプチダーゼY（EC 3.4.16.5）等が知られている。
It has been reported that peptidases are isolated from yeast (Non-patent
ペプチダーゼは食品加工や発酵食品の製造に利用される。例えば、調味料やヨーグルトの風味の増強、チーズの熟成促進などに利用されている。 Peptidases are used in food processing and fermented food production. For example, it is used to enhance the flavor of seasonings and yogurt, and accelerate the ripening of cheese.
アスペルギルス属由来、リゾープス属由来、或いはバチルス属などのバクテリア由来のアミノペプチダーゼやカルボキシペプチダーゼが食品添加物として工業的規模で生産されている。しかしながら、これらのペプチダーゼ（組成物）にはプロテイナーゼが混在しているため、プロテイナーゼの作用によって意図しない影響が生ずる。例えば、食品の物性が変化したり、苦味ペプチドが生成する。ペプチダーゼに期待される効果の一つは、食品等に含まれる苦味ペプチドの分解による苦味の低減であるが（非特許文献１０）、プロテイナーゼの混在は当該効果を低減する。一方、サッカロマイセス・セレビシエ由来のペプチダーゼは苦味低減効果を発揮するものの、菌体内酵素であるため、工業的な利用（食品の製造など）を図る上で、生産性や経済性が課題となる。
そこで、食品の製造等の分野での利用に適した新規なペプチダーゼを提供することを課題とする。
BACKGROUND ART Aminopeptidases and carboxypeptidases derived from Aspergillus, Rhizopus, and bacteria such as Bacillus are produced on an industrial scale as food additives. However, since these peptidases (compositions) are mixed with proteinase, the action of the proteinase has an unintended effect. For example, the physical properties of foods change or bitter peptides are produced. One of the expected effects of peptidase is the reduction of bitterness due to the decomposition of bitter peptides contained in foods and the like (Non-Patent Document 10), but the inclusion of proteinase reduces the effect. On the other hand, although the peptidase derived from Saccharomyces cerevisiae exhibits a bitterness-reducing effect, it is an intracellular enzyme, and therefore, productivity and economical efficiency are problems in industrial use (such as food production).
Therefore, it is an object to provide a novel peptidase suitable for use in fields such as food production.
本発明者は上記課題に鑑み検討を重ねた。その結果、食虫植物ネペンテス（Nepenthes alata）の捕虫袋中の消化液から分離した酵母シュードザイマ・フベイエンシス31-B株が菌体外にアミノペプチダーゼとカルボキシペプチダーゼを産生することが明らかとなった。また、これらのペプチダーゼの精製に成功し、その性質を特定した。更には、これらのペプチダーゼが苦味ペプチドに対する分解活性を示し、優れた苦味低減効果を発揮することを明らかにした。一方、シュードザイマ・フベイエンシス31-B株は夾雑プロテイナーゼを殆ど産生しないことが判明した。この特徴はペプチダーゼを製剤化する上で大きなメリットとなる。
以下の発明は、主として上記の成果に基づく。
［１］下記の酵素化学的性質を有するペプチダーゼ：
(1)作用 ペプチド鎖のN末端アミノ酸を遊離する反応を触媒する；
(2)分子量 SDS-PAGEによる分子量が約75.3kDa；
(3)基質特異性 ペプチド鎖のN末端のロイシン、アルギニン、メチオニン、アラニンを遊離する。ペプチド鎖のN末端のグリシン、プロリンへの作用は弱い。プロリンに結合したN末端アミノ酸に作用しない；
(4)至適pH 約8；
(5)至適温度 約40℃。
［２］下記の酵素化学的性質を有するペプチダーゼ：
(1)作用 ペプチド鎖のC末端アミノ酸を遊離する反応を触媒する；
(2)分子量 SDS-PAGEによる分子量が約70.5kDa；
(3)基質特異性 ペプチド鎖のC末端のロイシン、フェニルアラニンによく作用する。ペプチド鎖のC末端のアラニン、プロリン、チロシンへの作用は弱い；
(4)至適pH 約5；
(5)至適温度 約50℃。
［３］シュードザイマ・フベイエンシス由来である、［１］又は［２］に記載のペプチダーゼ。
［４］シュードザイマ・フベイエンシスが、シュードザイマ・フベイエンシス31-B株である、［３］に記載のペプチダーゼ。
［５］［１］〜［４］のいずれか一項に記載のペプチダーゼを有効成分とする酵素剤。
［６］［１］に記載のペプチダーゼと、［２］に記載のペプチダーゼを含有する酵素剤。
［７］以下のステップ(1)及び(2)を含む、ペプチダーゼの製造法：
(1)シュードザイマ・フベイエンシスを培養するステップ；
(2)培養後の培養液より、ペプチダーゼを回収するステップ。
［８］シュードザイマ・フベイエンシスが、シュードザイマ・フベイエンシス31-B株である、［７］に記載の製造法。
［９］ステップ(2)のペプチダーゼとして、アミノペプチダーゼとカルボキシペプチダーゼが回収される、［７］又は［８］に記載の製造法。
［１０］ステップ(2)のペプチダーゼとして、アミノペプチダーゼ又はカルボキシペプチダーゼが回収される、［７］又は［８］に記載の製造法。
［１１］受託番号ＮＩＴＥ Ｐ−０２０４７で特定される、シュードザイマ・フベイエンシス31-B株。
［１２］食品の製造過程において、タンパク質若しくはペプチドを含有する原料又は中間加工品に対し、［１］〜［４］のいずれか一項に記載のペプチダーゼ、又は［５］若しくは［６］に記載の酵素剤を作用させることを特徴とする、食品の製造又は加工方法。
［１３］食品が乳製品である、［１２］に記載の方法。
［１４］［１］〜［４］のいずれか一項に記載のペプチダーゼ、又は［５］若しくは［６］に記載の酵素剤の作用によって苦味が低減した食品。
The present inventor has made repeated studies in view of the above problems. As a result, it was revealed that the yeast Pseudozyma fubeyensis 31-B strain isolated from the digestive juice in the insect bag of the carnivorous plant Nepenthes alata produced aminopeptidase and carboxypeptidase extracellularly. In addition, we succeeded in the purification of these peptidases and identified their properties. Furthermore, it was clarified that these peptidases exhibited a degrading activity for bitter peptides and exerted an excellent bitterness reducing effect. On the other hand, it was revealed that the Pseudozyma fuvayensis 31-B strain produced almost no contaminant proteinase. This feature is a great advantage in formulating peptidases.
The following inventions are mainly based on the above results.
[1] Peptidase having the following enzymatic chemistry:
(1) Action Catalyze the reaction that releases the N-terminal amino acid of the peptide chain;
(2) Molecular weight The molecular weight by SDS-PAGE is about 75.3 kDa;
(3) Substrate specificity Leucine, arginine, methionine and alanine at the N-terminal of the peptide chain are released. The action on the N-terminal glycine and proline of the peptide chain is weak. Does not act on the N-terminal amino acid bound to proline;
(4) Optimum pH of about 8;
(5) Optimum temperature about 40 ℃.
[2] Peptidase having the following enzymatic chemistry:
(1) Action Catalyze the reaction to release the C-terminal amino acid of the peptide chain;
(2) Molecular weight The molecular weight by SDS-PAGE is about 70.5 kDa;
(3) Substrate specificity It acts well on C-terminal leucine and phenylalanine of the peptide chain. Weak action on C-terminal alanine, proline and tyrosine of peptide chain;
(4) Optimum pH of about 5;
(5) Optimum temperature about 50 ℃.
[3] The peptidase according to [1] or [2], which is derived from Pseudozyma fuvayensis.
[4] The peptidase according to [3], wherein the Pseudozyma fuvayensis is Pseudozyma fuvayensis 31-B strain.
[5] An enzyme preparation containing the peptidase according to any one of [1] to [4] as an active ingredient.
[6] An enzyme preparation containing the peptidase according to [1] and the peptidase according to [2].
[7] A method for producing peptidase, which comprises the following steps (1) and (2):
(1) Step of culturing Pseudozyma fuvayensis;
(2) A step of recovering peptidase from the culture medium after culturing.
[8] The production method according to [7], wherein Pseudozyma huveyensis is Pseudozyma huveyensis 31-B strain.
[9] The production method according to [7] or [8], wherein aminopeptidase and carboxypeptidase are recovered as the peptidase of step (2).
[10] The production method according to [7] or [8], wherein aminopeptidase or carboxypeptidase is recovered as the peptidase of step (2).
[11] Pseudozyma fubeiensis 31-B strain identified by accession number NITE P-02047.
[12] The peptidase according to any one of [1] to [4], or [5] or [6] for a raw material or an intermediate processed product containing a protein or a peptide in the process of producing a food. A method for producing or processing a food, characterized in that the enzyme agent of 1.
[13] The method according to [12], wherein the food is a dairy product.
[14] A food in which bitterness is reduced by the action of the peptidase according to any one of [1] to [4] or the enzyme preparation according to [5] or [6].
１．ペプチダーゼ及びその生産菌
本発明の第1の局面はペプチダーゼ及びその生産菌を提供する。後述の実施例に示す通り、本発明者らは鋭意検討の結果、シュードザイマ・フベイエンシス（Pseudozyma hubeiensis）31-B株が２種類の特徴的なペプチダーゼ（アミノペプチダーゼ及びカルボキシペプチダーゼ）を産生することを見出した。また、これらのペプチダーゼの精製に成功し、以下に示す通り、その酵素化学的性質を決定した。以下、各ペプチダーゼの酵素化学的性質を説明する。
1. Peptidase and its producing bacterium The first aspect of the present invention provides a peptidase and its producing bacterium. As shown in Examples below, as a result of intensive studies, the present inventors have found that Pseudozyma hubeiensis 31-B strain produces two types of characteristic peptidases (aminopeptidase and carboxypeptidase). It was We also succeeded in purifying these peptidases and determined their enzymatic chemistry as shown below. The enzymatic chemistry of each peptidase will be described below.
＜アミノペプチダーゼ＞
（１）作用
本酵素はアミノペプチダーゼであり、ペプチド鎖のN末端アミノ酸を遊離する反応を触媒する。
（２）分子量
本酵素の分子量は約75.3kDa（SDS-PAGEによる）である。
（３）基質特異性
本酵素はペプチド鎖のN末端のロイシン、アルギニン、メチオニン、アラニン等を遊離する。ペプチド鎖のN末端のグリシン、プロリンへの作用は弱い。プロリンに結合したN末端アミノ酸に作用しない。
（４）至適pH
本酵素の至適pHは約8である。本酵素はpH約6.5〜約9.5において高い活性を示す。至適pHの測定には後述のLAP法（反応時の温度は37℃）を利用した。マッキルバイン（McIlvaine）緩衝液（pH 4〜6）、リン酸緩衝液（pH 6〜7）、Tris-HCl緩衝液（pH 7〜9）、Atkins-Pantin緩衝液（pH 9〜10）を使用した。
（５）至適温度
本酵素の至適温度は約40℃である。本酵素は約30℃〜約55℃において高い活性を示す。至適温度の測定には、後述のLAP法（反応時のpHは8.0）を利用した。
（６）その他の特性
亜鉛イオン（Zn2+）によって活性が完全に阻害され、二価鉄イオン（Fe2+）、銅イオン（Cu2+）及び三価鉄イオン（Fe3+）によっても活性が阻害される。また、SDS、EDTA及びPCMBによっても活性が阻害される。E-64、Pepstatin A及びPetablocによっては活性が阻害されない。一方、カリウムイオン（K+）は活性化を促す。
<Aminopeptidase>
(1) Action This enzyme is an aminopeptidase and catalyzes the reaction of releasing the N-terminal amino acid of the peptide chain.
(2) Molecular weight The molecular weight of this enzyme is about 75.3 kDa (by SDS-PAGE).
(3) Substrate specificity This enzyme releases leucine, arginine, methionine, alanine, etc. at the N-terminus of the peptide chain. The action on the N-terminal glycine and proline of the peptide chain is weak. It does not act on the N-terminal amino acid bound to proline.
(4) Optimal pH
The optimum pH of this enzyme is about 8. The enzyme exhibits high activity at pH of about 6.5 to about 9.5. The LAP method described later (the temperature during the reaction was 37 ° C.) was used to measure the optimum pH. McIlvaine buffer (pH 4-6), phosphate buffer (pH 6-7), Tris-HCl buffer (pH 7-9), Atkins-Pantin buffer (pH 9-10) were used. .
(5) Optimal temperature The optimal temperature for this enzyme is about 40 ° C. The enzyme exhibits high activity at about 30 ° C to about 55 ° C. The LAP method described later (pH during the reaction was 8.0) was used to measure the optimum temperature.
(6) Other properties Zinc ion (Zn 2+ ) completely inhibits the activity, and divalent iron ion (Fe 2+ ), copper ion (Cu 2+ ) and trivalent iron ion (Fe 3+ ) also The activity is inhibited. In addition, the activity is also inhibited by SDS, EDTA and PCMB. The activity is not inhibited by E-64, Pepstatin A and Petabloc. On the other hand, potassium ion (K + ) promotes activation.
＜カルボキシペプチダーゼ＞
（１）作用
本酵素はカルボキシペプチダーゼであり、ペプチド鎖のC末端アミノ酸を遊離する反応を触媒する。
（２）分子量
本酵素の分子量は約70.5kDa（SDS-PAGEによる）である。
（３）基質特異性
本酵素はペプチド鎖のC末端のロイシン、フェニルアラニンによく作用する。ペプチド鎖のC末端のアラニン、プロリン、チロシンへの作用は弱い。
（４）至適pH
本酵素の至適pHは約5である。本酵素はpH約3.0〜約5.5において高い活性を示す。至適pHの測定には後述のCP法（反応時の温度は40℃）を利用した。マッキルバイン（McIlvaine）緩衝液（pH 3〜６）、リン酸緩衝液（pH 6〜7）、Tris-HCl緩衝液（pH 7〜9）を使用した。
（５）至適温度
本酵素の至適温度は約50℃である。本酵素は約30℃〜約55℃において高い活性を示す。至適温度の測定には、後述のACP法（反応時のpHは5.0）を利用した。
（６）その他の特性
カリウムイオン（K+）、マンガンイオン（Mn2+）及び二価鉄イオン（Fe2+）によって活性が阻害される。また、SDS及びPCMBによっても活性が阻害される。
<Carboxypeptidase>
(1) Action This enzyme is a carboxypeptidase and catalyzes the reaction of releasing the C-terminal amino acid of the peptide chain.
(2) Molecular weight The molecular weight of this enzyme is about 70.5 kDa (by SDS-PAGE).
(3) Substrate specificity This enzyme acts well on leucine and phenylalanine at the C-terminal of the peptide chain. The action of C-terminal alanine, proline and tyrosine of the peptide chain is weak.
(4) Optimal pH
The optimum pH of this enzyme is about 5. This enzyme exhibits high activity at pH of about 3.0 to about 5.5. The CP method described below (the temperature during the reaction was 40 ° C.) was used to measure the optimum pH. A McIlvaine buffer solution (
(5) Optimum temperature The optimum temperature of this enzyme is about 50 ° C. The enzyme exhibits high activity at about 30 ° C to about 55 ° C. The below-mentioned ACP method (pH at the time of reaction was 5.0) was used to measure the optimum temperature.
(6) Other properties The activity is inhibited by potassium ion (K + ), manganese ion (Mn 2+ ) and divalent iron ion (Fe 2+ ). Moreover, the activity is also inhibited by SDS and PCMB.
本発明のペプチダーゼは、好ましくはシュードザイマ・フベイエンシス、更に好ましくはシュードザイマ・フベイエンシス31-B株に由来する酵素である。ここでの「シュードザイマ・フベイエンシス31-B株に由来するペプチダーゼ」とは、シュードザイマ・フベイエンシス31-B株が産生するペプチダーゼ、或いはシュードザイマ・フベイエンシス31-B株（野生株であっても変異株であってもよい）のペプチダーゼ遺伝子を利用して遺伝子工学的手法によって得られたペプチダーゼであることを意味する。従って、シュードザイマ・フベイエンシス31-B株より取得したペプチダーゼ遺伝子（又は当該遺伝子を改変した遺伝子）を導入した宿主微生物によって生産された組み換え体も、「シュードザイマ・フベイエンシス31-B株に由来するペプチダーゼ」に該当する。 The peptidase of the present invention is preferably an enzyme derived from Pseudozyma fuvayensis, more preferably Pseudozyma fuvayensis 31-B strain. As used herein, the term "peptidase derived from Pseudozyma huveyensis 31-B strain" refers to a peptidase produced by Pseudozyma huveyensis 31-B strain, or Pseudozyma huveyensis 31-B strain (even a wild strain is a mutant strain. It may be a peptidase gene obtained by a genetic engineering technique using the peptidase gene (may be). Therefore, the recombinant produced by the host microorganism into which the peptidase gene (or the gene obtained by modifying the gene) obtained from the Pseudozyma huveyensis 31-B strain was also converted into the "peptidase derived from the Pseudozyma huveyensis 31-B strain". Applicable
本発明のペプチダーゼの由来であるシュードザイマ・フベイエンシス31-B株のことを、説明の便宜上、本発明のペプチダーゼの生産菌という。シュードザイマ・フベイエンシス31-B株は以下の通り所定の寄託機関に寄託されており、容易に入手可能である。
寄託機関：独立行政法人製品評価技術基盤機構 バイオテクノロジーセンター 特許微生物寄託センター(NPMD)（〒292-0818 日本国千葉県木更津市かずさ鎌足2-5-8 122号室）
寄託日：２０１５年５月１１日
受託番号：ＮＩＴＥ Ｐ−０２０４７
For the sake of convenience of explanation, the Pseudozyma huveyensis 31-B strain from which the peptidase of the present invention is derived is referred to as the peptidase-producing bacterium of the present invention. Pseudozyma huveyensis 31-B strain has been deposited with the following depositary institutions and is easily available.
Depositary Organisation: Institute for Product Evaluation Technology, Biotechnology Center, Patent Microorganism Depositary Center (NPMD) (Room 2-5-8, Kazusa, Kazusa, Kisarazu, Chiba Prefecture, 292-0818 Japan)
Deposit Date: May 11, 2015 Deposit Number: NITE P-02047
２．酵素剤
本発明のペプチダーゼは例えば酵素剤の形態で提供される。酵素剤は、有効成分（ペプチダーゼ）の他、賦形剤、緩衝剤、懸濁剤、安定剤、保存剤、防腐剤、生理食塩水などを含有していてもよい。賦形剤としては乳糖、ソルビトール、D-マンニトール、白糖等を用いることができる。緩衝剤としてはリン酸塩、クエン酸塩、酢酸塩等を用いることができる。安定剤としてはプロピレングリコール、アスコルビン酸等を用いることができる。保存剤としてはフェノール、塩化ベンザルコニウム、ベンジルアルコール、クロロブタノール、メチルパラベン等を用いることができる。防腐剤としては塩化ベンザルコニウム、パラオキシ安息香酸、クロロブタノール等を用いることができる。
2. Enzyme Agent The peptidase of the present invention is provided in the form of an enzyme agent, for example. The enzyme preparation may contain, in addition to the active ingredient (peptidase), an excipient, a buffer, a suspension, a stabilizer, a preservative, a preservative, physiological saline and the like. As the excipient, lactose, sorbitol, D-mannitol, sucrose and the like can be used. As the buffer, phosphate, citrate, acetate or the like can be used. As the stabilizer, propylene glycol, ascorbic acid or the like can be used. As the preservative, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben and the like can be used. As the preservative, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.
本発明の酵素剤には上記のアミノペプチダーゼ又はカルボキシペプチダーゼ、或いはこれらの両者が有効成分として含まれる。後者の態様、即ち、アミノペプチダーゼとカルボキシペプチダーゼの両者を有効成分とする酵素剤は本願発明に特徴的である。後述の実施例に示す通り、本発明者らの検討の結果、シュードザイマ・フベイエンシス31-B株が上記二つの酵素を菌体外に産生することが明らかになった。菌体外に産生することから、高い生産性を実現できる。一方、シュードザイマ・フベイエンシス31-B株が菌体外にプロテイナーゼを実質的に含まないことも明らかになった。この事実は、複雑な精製工程を経なくとも、夾雑プロテイナーゼを実質的に含まない、アミノペプチダーゼとカルボキシペプチダーゼを有効成分とするペプチダーゼ剤を調製できることを意味する。即ち、製剤化が容易である。また、精製するとしても、そのための操作、処理が簡便なものとなる。 The enzyme preparation of the present invention contains the above aminopeptidase or carboxypeptidase, or both of them as an active ingredient. The latter aspect, that is, the enzyme agent containing both aminopeptidase and carboxypeptidase as active ingredients is characteristic of the present invention. As shown in the Examples below, as a result of the study by the present inventors, it was revealed that Pseudozyma huveyensis 31-B strain produced the above two enzymes extracellularly. Since it is produced outside the cells, high productivity can be realized. On the other hand, it was also clarified that the Pseudozyma fuvayensis 31-B strain contained substantially no proteinase outside the cells. This fact means that it is possible to prepare a peptidase agent containing aminopeptidase and carboxypeptidase as an active ingredient, which is substantially free of contaminant proteinases, even without complicated purification steps. That is, formulation is easy. Further, even if it is purified, the operation and treatment therefor are simple.
アミノペプチダーゼとカルボキシペプチダーゼの両者を有効成分とする酵素剤は、例えば、シュードザイマ・フベイエンシス31-B株の培養上清をろ過、遠心処理等に供して不溶物を除去した後に濃縮することによって得ることができる。或いは、不純物の除去、濃縮、希釈、塩析、透析、溶解及び乾燥からなる群より選択される一以上の精製工程によって培養上清を精製することによっても得ることができる。シュードザイマ・フベイエンシス31-B株の培養上清にはプロテイナーゼを実質的に含まないことから、プロテイナーゼを除去するための精製工程（例えばカラムクロマトグラフィー）を経ずとも、夾雑プロテイナーゼを実質的に含まない、アミノペプチダーゼとカルボキシペプチダーゼを有効成分とするペプチダーゼ剤を調製可能となる。 Enzymatic agents containing both aminopeptidase and carboxypeptidase as active ingredients are obtained by, for example, filtering the culture supernatant of Pseudozyma fubeiensis 31-B strain, subjecting it to centrifugation, etc. to remove insolubles, and then concentrating it. You can Alternatively, it can also be obtained by purifying the culture supernatant by one or more purification steps selected from the group consisting of removal of impurities, concentration, dilution, salting out, dialysis, dissolution and drying. Since the culture supernatant of Pseudozyma fubeiensis 31-B strain does not substantially contain proteinase, it does not substantially contain contaminating proteinase even without a purification step (eg, column chromatography) for removing proteinase. It becomes possible to prepare a peptidase agent containing aminopeptidase and carboxypeptidase as active ingredients.
３．ペプチダーゼの製造法
本発明の更なる局面はペプチダーゼの製造法を提供する。本発明では、(1)シュードザイマ・フベイエンシスを培養するステップと(2)培養後の培養液より、ペプチダーゼを回収するステップを行う。回収の一態様として単離・精製を行っても良い。
3. Method of Producing Peptidase A further aspect of the present invention provides a method of producing peptidase. In the present invention, (1) a step of culturing Pseudozyma fuvayensis and (2) a step of recovering peptidase from the culture medium after the culturing are performed. Isolation / purification may be performed as one mode of recovery.
ステップ（1）では、シュードザイマ・フベイエンシスを培養する。シュードザイマ・フベイエンシスは、目的のペプチダーゼの生産能を有する限り特に限定されない。例えば、上記のシュードザイマ・フベイエンシス31-B株を用いることができる。培養法及び培養条件は、目的の酵素（ペプチダーゼ）が生産されるものである限り特に限定されない。即ち、本発明のペプチダーゼが生産されることを条件として、培養に適合した方法や培養条件を適宜設定できる。培養法としては液体培養、固体培養のいずれでも良いが、好ましくは液体培養が利用される。液体培養を例にとり、その培養条件を説明する。 In step (1), Pseudozyma fuvayensis is cultured. Pseudozyma fuvayensis is not particularly limited as long as it has the ability to produce the desired peptidase. For example, the above-mentioned Pseudozyma huveyensis 31-B strain can be used. The culturing method and culturing conditions are not particularly limited as long as the target enzyme (peptidase) is produced. That is, on condition that the peptidase of the present invention is produced, a method and culture conditions suitable for culture can be appropriately set. The culture method may be either liquid culture or solid culture, but liquid culture is preferably used. The culture conditions will be described by taking liquid culture as an example.
培地としては、シュードザイマ・フベイエンシスが生育可能な培地であれば、特に限定されない。例えば、グルコース、シュクロース、ゲンチオビオース、可溶性デンプン、グリセリン、デキストリン、糖蜜、有機酸等の炭素源、硫酸アンモニウム、炭酸アンモニウム、リン酸アンモニウム、酢酸アンモニウム、あるいは、ペプトン、酵母エキス、コーンスティープリカー、カゼイン加水分解物、ふすま、肉エキス等の窒素源、カリウム塩、マグネシウム塩、ナトリウム塩、リン酸塩、マンガン塩、鉄塩、亜鉛塩等の無機塩を添加した培地を用いることができる。シュードザイマ・フベイエンシスの生育を促進するためにビタミン、アミノ酸などを培地に添加してもよい。培地のpHは例えば約４〜７、好ましくは約６〜７程度に調整し、培養温度は通常約２０℃〜３５℃、好ましくは約２５℃〜３０℃程度で、１日間〜５日間、好ましくは２日間〜３日間程度、好気的条件下で培養する。培養法としては例えば振盪培養法、ジャー・ファーメンターによる好気的深部培養法が利用できる。 The medium is not particularly limited as long as it can grow Pseudozyma fubeiensis. For example, glucose, sucrose, gentiobiose, soluble starch, glycerin, dextrin, molasses, carbon sources such as organic acids, ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium acetate, or peptone, yeast extract, corn steep liquor, casein hydrolyzate. It is possible to use a medium to which a nitrogen source such as a decomposed product, bran or meat extract, and an inorganic salt such as potassium salt, magnesium salt, sodium salt, phosphate, manganese salt, iron salt or zinc salt is added. Vitamins, amino acids and the like may be added to the medium in order to promote the growth of Pseudozyma fuvayensis. The pH of the medium is adjusted to, for example, about 4 to 7, preferably about 6 to 7, and the culture temperature is usually about 20 ° C to 35 ° C, preferably about 25 ° C to 30 ° C, for 1 day to 5 days, preferably Are cultured under aerobic conditions for about 2 to 3 days. As the culturing method, for example, a shaking culturing method and an aerobic deep culturing method using a jar fermenter can be used.
以上の条件で培養した後、培養液よりペプチダーゼを回収する（ステップ（2））。例えば、培養液をろ過、遠心処理等することによって不溶物を除去し、目的のペプチダーゼ液を得る。更に、濃縮、希釈、塩析、透析、溶解、乾燥等の精製工程を行い、純度の高いペプチダーゼを得ることにしてもよい。以上のごとき方法によると、アミノペプチダーゼとカルボキシペプチダーゼの両者を含む組成物が得られることになる。一方、各種クロマトグラフィー（イオン交換クロマトグラフィー、吸着クロマトグラフィー、疎水性クロマトグラフィーなど）や、溶媒分画、ｐH処理、熱処理等を適用することによって、片方のペプチダーゼを除去し、アミノペプチダーゼ又はカルボキシペプチダーゼを選択的に回収することにしてもよい。 After culturing under the above conditions, peptidase is recovered from the culture medium (step (2)). For example, the culture solution is filtered, centrifuged, etc. to remove the insoluble matter to obtain the target peptidase solution. Further, purification steps such as concentration, dilution, salting out, dialysis, dissolution, and drying may be performed to obtain a highly pure peptidase. According to the method as described above, a composition containing both aminopeptidase and carboxypeptidase can be obtained. On the other hand, by applying various chromatographies (ion exchange chromatography, adsorption chromatography, hydrophobic chromatography, etc.), solvent fractionation, pH treatment, heat treatment, etc., one peptidase is removed and aminopeptidase or carboxypeptidase. May be selectively collected.
回収したペプチダーゼの精製度は特に限定されないが、例えば比活性が10〜500（U/mg）の状態に精製することができる。また、最終的な形態は液体状であっても固体状（粉体状を含む）であってもよい。回収したペプチダーゼを、例えば凍結乾燥や真空乾燥或いはスプレードライなどにより粉末化して提供することも可能である。その際、精製酵素を予めリン酸緩衝液、トリエタノールアミン緩衝液、トリス塩酸緩衝液やGOODの緩衝液に溶解させておいてもよい。好ましくは、リン酸緩衝液、トリス塩酸緩衝液を使用することができる。尚、ここでGOODの緩衝液としてはPIPES、MES又はMOPSが挙げられる。 The degree of purification of the recovered peptidase is not particularly limited, but it can be purified to a specific activity of 10 to 500 (U / mg), for example. The final form may be liquid or solid (including powder). It is also possible to provide the recovered peptidase in the form of powder by freeze drying, vacuum drying, spray drying, or the like. At that time, the purified enzyme may be previously dissolved in a phosphate buffer solution, a triethanolamine buffer solution, a tris-hydrochloric acid buffer solution, or a GOOD buffer solution. Preferably, a phosphate buffer solution and a Tris-HCl buffer solution can be used. The GOOD buffer may be PIPES, MES or MOPS.
４．ペプチダーゼの用途
本発明の更なる局面は上記ペプチダーゼの用途として食品の製造・加工方法を提供する。本発明の方法では、食品の製造過程又は加工過程において、その原料又は中間加工品に対して本発明のペプチダーゼを作用させる。ペプチダーゼに代えてそれを含む酵素剤を用いてもよい。
4. Use of Peptidase A further aspect of the present invention provides a method for producing and processing a food as a use of the above peptidase. In the method of the present invention, the peptidase of the present invention is allowed to act on a raw material or an intermediate processed product in the manufacturing process or processing process of food. Instead of peptidase, an enzyme preparation containing it may be used.
典型的には、処理対象物となる原料又は中間加工品に対して、本発明のペプチダーゼ又は酵素剤を添加し、ペプチダーゼが作用する条件で処理する。通常の製造／加工過程においてペプチダーゼが作用する条件が形成されるのであれば、ペプチダーゼを作用させるための専用の工程を設けなくてもよい。即ち、一態様では、ペプチダーゼを添加すること以外は通常の製造／加工過程によって食品を製造する。尚、ペプチダーゼの添加によるのではなく、例えば、ペプチダーゼを含有する溶液に食品の原料又は中間加工品を浸漬するなどして、ペプチダーゼを作用させることにしてもよい。 Typically, the peptidase or enzyme agent of the present invention is added to a raw material or an intermediate processed product to be treated, and treatment is carried out under the condition that the peptidase acts. As long as the conditions under which the peptidase acts are formed in the usual production / processing process, a dedicated step for acting the peptidase need not be provided. That is, in one aspect, the food product is manufactured by a conventional manufacturing / processing process except that peptidase is added. Instead of adding peptidase, the peptidase may be allowed to act by, for example, immersing a raw material of food or an intermediate processed product in a solution containing peptidase.
本発明の適用可能な食品として、乳製品（乳、チーズ、ヨーグルトなど）、食肉、食肉加工品、肉エキス、魚肉製、魚肉加工品、魚肉エキス、酵母エキス、酵母エキス加工品（ベジマイト、マーマイト等）、ブイヨン、コンソメ、野菜、野菜加工品、野菜エキス、果実、果実加工品、果汁、果汁加工品、穀類、穀類粉末、穀類加工品、レトルト食品、調理加工済み食品、栄養補助食品（サプリメント、栄養ドリンク等）、食品添加物、ペットフード、醤油、味噌等の発酵食品を例示することができる。 As foods to which the present invention is applicable, dairy products (milk, cheese, yogurt, etc.), meat, processed meat products, meat extract, fish meat, processed fish products, fish meat extract, yeast extract, yeast extract processed products (vemite, marmite). Etc.), bouillon, consomme, vegetables, processed vegetable products, vegetable extracts, fruits, processed fruit products, fruit juice, processed fruit juice products, cereals, cereal powder, processed cereal products, retort foods, cooked foods, dietary supplements (supplements) , Nutritional drinks, etc.), food additives, pet foods, soy sauce, and fermented foods such as miso.
本発明の方法によれば、ペプチダーゼの作用によって苦味ペプチドが分解され、苦味の低減した食品を得ることができる。従って、本発明は、苦味がその品質に負の影響を与える食品（苦味の少ないことが好まれる食品）の製造又は加工に特に有用である。また、その使用によって苦味が付与されることが好ましくない食品・食品添加物（例えば肉エキス、魚肉エキス、酵母エキスなど）の製造又は加工においても有用性が高い。 According to the method of the present invention, a bitterness peptide is decomposed by the action of peptidase, and a food with reduced bitterness can be obtained. Therefore, the present invention is particularly useful for the production or processing of foods in which bitterness negatively affects their quality (foods in which less bitterness is preferred). Further, it is also highly useful in the production or processing of foods and food additives (for example, meat extract, fish meat extract, yeast extract, etc.) whose use does not impart bitterness.
酵母のタンパク質分解酵素としてエンドペプチダーゼ（プロテイナーゼ）、アミノペプチダーゼ類、カルボキシペプチダーゼ類が知られている。これらのプロテイナーゼやペプチダーゼ類は酵母菌体の自己消化液中に認められる。従って、菌体内からの抽出する必要があり、産業用酵素として見た場合、生産性には疑問がある。これまでに、菌体外へ産生されるペプチダーゼの報告は少ない。そこで、菌体外に産生される、酵母由来の新規ペプチダーゼの取得を目指し、以下の検討を行った。 Endopeptidases (proteinases), aminopeptidases, and carboxypeptidases are known as yeast proteolytic enzymes. These proteinases and peptidases are found in the autolysate of yeast cells. Therefore, it is necessary to extract from the microbial cells, and there is a question of productivity when viewed as an industrial enzyme. To date, there have been few reports of peptidases produced outside the cells. Therefore, the following studies were carried out with the aim of obtaining a novel yeast-derived peptidase produced outside the cells.
＜新規ペプチダーゼの取得＞
Ａ．材料と方法
１．菌株
東山植物園（日本、名古屋市）にて、食虫植物ネペンテス（Nepenthes alata）の捕虫袋中の消化液から分離した酵母の中から、菌体外にプロテイナーゼを産生せずエキソペプチダーゼ産生能の高い一菌株（31-B株）を見出し、研究に供した。
<Acquisition of new peptidase>
A. Materials and
２．酵母の培養
13％のグリセリン溶液に懸濁して-85℃にて保存した酵母31-B株懸濁液を、表１のAに示す斜面培地に塗布し、25℃で2日間培養した。次に、表１のBに示す500 mL容振とうフラスコに入れた種培地140 mLに、斜面培養から１白金耳接種し、25℃で24時間振とう培養(180 rpm)した。続いて、種培養液140 mLを、表１のCに示す培養液7 Lを入れた10 L容ジャー・ファーメンターに接種し、25℃で通気撹拌培養（0.5 vvm, 500 rpm, 72 hr)した。
The yeast 31-B strain suspension suspended in a 13% glycerin solution and stored at -85 ° C was applied to the slant medium shown in A of Table 1 and cultured at 25 ° C for 2 days. Next, 1 platinum loop was inoculated from the slope culture into 140 mL of the seed medium placed in the 500 mL shake flask shown in B of Table 1, and shake-cultured (180 rpm) at 25 ° C. for 24 hours. Subsequently, 140 mL of the seed culture was inoculated into a 10 L jar fermenter containing 7 L of the culture shown in C of Table 1, and aerated and agitated at 25 ° C (0.5 vvm, 500 rpm, 72 hr). did.
３．活性測定法
３−１．カルボキシペプチダーゼ活性測定法
３−１−１．ACP(酸性カルボキシペプチダーゼ）法
酸性カルボキシペプチダーゼ測定キット（キッコーマンバイオケミファ株式会社）を用い、キット仕様に従い37℃の反応条件で測定した。活性表示はキット仕様に従い、1分間に基質Cbz-Tyr-Alaから1μmolのL-アラニンを遊離する酵素量を1単位とした。
3. Activity measurement method 3-1. Method for measuring carboxypeptidase activity 3-1-1. ACP (acidic carboxypeptidase) method Using an acidic carboxypeptidase measurement kit (Kikkoman Biochemifa Co., Ltd.), measurement was carried out at 37 ° C. according to the kit specifications. The activity was displayed according to the kit specifications, and the amount of enzyme that liberates 1 μmol of L-alanine from the substrate Cbz-Tyr-Ala in 1 minute was defined as 1 unit.
３−１−２．CP（カルボキシペプチダーゼ）法
Z-Phe-Leu( 20.6 mg/ 0.05M 酢酸緩衝液,pH 5.0 )溶液0.9 mLに酵素液0.1 mLを加え、37℃で30分間反応させた。ニンヒドリン溶液1 mLを添加後、沸騰水中に15分間放置し、遊離したアミノ酸を呈色した。流水中で冷却後、5 mLの50% エタノール溶液を加え、十分撹拌後、570 nmの吸光度を測定した。上記条件で1分間に1μmolのロイシンを遊離する酵素量を1単位とした。
3-1-2. CP (carboxypeptidase) method
0.1 mL of the enzyme solution was added to 0.9 mL of the Z-Phe-Leu (20.6 mg / 0.05 M acetate buffer, pH 5.0) solution, and the mixture was reacted at 37 ° C. for 30 minutes. After adding 1 mL of the ninhydrin solution, it was left in boiling water for 15 minutes, and the released amino acid was colored. After cooling in running water, 5 mL of 50% ethanol solution was added, and after sufficiently stirring, the absorbance at 570 nm was measured. Under the above conditions, the amount of enzyme that releases 1 μmol of leucine per minute was defined as 1 unit.
３−２．アミノペプチダーゼ（LAP）活性測定法
L-ロイシル-p-ニトロアニリド塩酸塩溶液(71.5 mg/ 100 mL 精製水) 0.8 mLに200mMトリス（Tris-HCl）緩衝液（pH8.0)を1 mL加え、37℃に5分間予熱した。酵素液0.2 mLを添加、37℃に30分間反応後、15% 酢酸溶液を加え反応を停止し、410nmの吸光度を測定した。上記方法で、1分間に1μmolのp-ニトロアニリンを遊離する酵素量を1単位とした。
3-2. Method for measuring aminopeptidase (LAP) activity
1 mL of 200 mM tris (Tris-HCl) buffer solution (pH 8.0) was added to 0.8 mL of L-leucyl-p-nitroanilide hydrochloride solution (71.5 mg / 100 mL purified water), and preheated to 37 ° C. for 5 minutes. After adding 0.2 mL of the enzyme solution and reacting at 37 ° C for 30 minutes, a 15% acetic acid solution was added to stop the reaction, and the absorbance at 410 nm was measured. In the above method, the amount of enzyme that liberates 1 μmol of p-nitroaniline in 1 minute was defined as 1 unit.
３−３．プロテイナーゼ活性測定法
0.6% アゾカゼイン（シグマ）水溶液0.9 mLに0.1Mリン酸緩衝液（pH 7.0) 0.3 mLを加え、0.4 mLの酵素液を添加した。37℃で30分間反応後、2.4 mLの10% トリクロロ酢酸を加えて反応を停止した。東洋ろ紙No.131で濾過後、400 nmの吸光度を測定した。上記条件で吸光度を1分間当たり1.0上昇させる酵素量を1単位とした。
3-3. Proteinase activity assay
0.3 mL of 0.1 M phosphate buffer (pH 7.0) was added to 0.9 mL of 0.6% azocasein (Sigma) aqueous solution, and 0.4 mL of enzyme solution was added. After reacting at 37 ° C for 30 minutes, 2.4 mL of 10% trichloroacetic acid was added to stop the reaction. After filtering through Toyo Filter Paper No. 131, the absorbance at 400 nm was measured. One unit was the amount of enzyme that increased the absorbance by 1.0 per minute under the above conditions.
４．タンパク質量の測定
牛血清アルブミンを標準として、Bio-Rad社のプロテインアッセイ試薬を用いて測定した。クロマトグラフィーの際には、280nmの吸光度を測定し、タンパク質をモニターした。
4. Measurement of protein amount Using bovine serum albumin as a standard, a protein assay reagent from Bio-Rad was used for measurement. During chromatography, the absorbance at 280 nm was measured to monitor the protein.
５．ペプチダーゼの精製
72時間培養したジャー・ファーメンター培養により得られた培養液上清を限外濾過膜（AIV-1010；旭化成ケミカルズ株式会社）により濃縮し、以下のペプチダーゼの精製に用いた。
5. Purification of peptidase
The culture solution supernatant obtained by the jar fermenter culture that was cultured for 72 hours was concentrated with an ultrafiltration membrane (AIV-1010; Asahi Kasei Chemicals Corporation) and used for the purification of the following peptidase.
５−１．塩析
濃縮液に固体硫酸アンモニウムを80飽和濃度となるよう添加、溶解した。5℃で20時間放置後、濾過で粗酵素を集め、精製水に溶解後、水道水に8時間、0.01M リン酸緩衝液（pH7.0) に16時間透析した。
5-1. Salting out To the concentrated solution, solid ammonium sulfate was added and dissolved so as to have a saturated concentration of 80. After standing at 5 ° C for 20 hours, the crude enzyme was collected by filtration, dissolved in purified water, dialyzed against tap water for 8 hours and against 0.01M phosphate buffer (pH 7.0) for 16 hours.
５−２．DEAE-Toyopearl 650 Mカラムクロマトグラフィー
0.01 Mリン酸緩衝液（pH 7.0）に透析した濃縮液を、同緩衝液で平衡化したDEAE-Toyopearl 650 M カラム（4.0 × 50 cm)に乗せ、吸着した画分は食塩濃度を直線的に上げ(0〜0.5M)、溶出(60mL/h)した。溶出した活性画分を集め、硫酸アンモニウムを20%溶液濃度になるよう溶解し、次のクロマトグラフィーに供した。
5-2. DEAE-Toyopearl 650 M column chromatography
The concentrated solution dialyzed against 0.01 M phosphate buffer (pH 7.0) was placed on a DEAE-Toyopearl 650 M column (4.0 × 50 cm) equilibrated with the same buffer, and the adsorbed fraction showed a linear salt concentration. Raised (0-0.5M) and eluted (60 mL / h). The active fractions thus eluted were collected, dissolved with ammonium sulfate to a 20% solution concentration, and subjected to the next chromatography.
５−３．Butyl-Toyopearl 650Mカラムクロマトグラフィー
20%濃度の硫酸アンモニウムを含む0.01Mリン酸緩衝液（pH7.0）で平衡化したButyl-Toyopearl 650Mカラム(4.0 × 30 cm）に、活性画分を乗せ、吸着した活性画分を、硫安濃度を直線的に下げ（20〜0％）、溶出(50mL/h)した。溶出した活性画分を集め、0.01Mリン酸緩衝液(pH7.0)に対し透析した後、次のクロマトグラフィーに供した。
5-3. Butyl-Toyopearl 650M column chromatography
The active fraction was loaded onto a Butyl-Toyopearl 650M column (4.0 × 30 cm) equilibrated with 0.01 M phosphate buffer (pH 7.0) containing 20% ammonium sulfate, and the adsorbed active fraction was concentrated with ammonium sulfate. Was reduced linearly (20-0%) and eluted (50 mL / h). The eluted active fractions were collected, dialyzed against 0.01M phosphate buffer (pH 7.0), and then subjected to the next chromatography.
５−４．Hydroxylapatite カラムクロマトグラフィー
0.01Mリン酸緩衝液 (pH7.0 )で平衡化したカラム（4.5 × 30 cm）に、得られた活性画分を乗せ、リン酸濃度を直線的に高め（10〜250mM）、吸着した活性画分を溶出(20mL/h)した。溶出した活性画分を集め、0.5M NaCl含有0.02Mリン酸緩衝液（pH7.0）に対して透析した後、次のクロマトグラフィーに供した。
5-4. Hydroxylapatite column chromatography
The active fraction thus obtained was placed on a column (4.5 × 30 cm) equilibrated with 0.01 M phosphate buffer (pH 7.0) to increase the phosphate concentration linearly (10 to 250 mM) and the adsorbed activity. Fractions were eluted (20 mL / h). The eluted active fractions were collected, dialyzed against 0.02 M phosphate buffer (pH 7.0) containing 0.5 M NaCl, and then subjected to the next chromatography.
５−５．Toyopearl HW-55F カラムクロマトグラフィー
0.5 MのNaClを含む0.02 Mリン酸緩衝液 ( pH7.0 )で平衡化したカラム（2.2 × 90 cm)に、活性画分を乗せ、流速15 mL/hでゲル濾過を行った。溶出した活性画分を集め、0.01Mリン酸緩衝液（pH7.0）に対して透析した後、精製酵素として以下の実験に用いた。以下、精製したカルボキシペプチダーゼはCP31-B、アミノペプチダーゼはAP31-Bと表す。
5-5. Toyopearl HW-55F column chromatography
The active fraction was loaded on a column (2.2 × 90 cm) equilibrated with 0.02 M phosphate buffer (pH 7.0) containing 0.5 M NaCl, and gel filtration was performed at a flow rate of 15 mL / h. The eluted active fractions were collected, dialyzed against 0.01M phosphate buffer (pH 7.0), and then used as a purified enzyme in the following experiments. Hereinafter, the purified carboxypeptidase will be referred to as CP31-B and the aminopeptidase will be referred to as AP31-B.
６．精製酵素の特性の検討
６−１．SDS-PAGE
精製したペプチダーゼは、それぞれSDS-PAGEにより純度及び分子量の確認を行った。分子量マーカーとしてホスホリラーゼ b（97.2 kDa)、牛血清アルブミン(66.4 kDa)、オボアルブミン(45.0 kDa)、炭酸脱水素酵素II(29.0 kDa)、大豆トリプシンインヒビター（21.0 kDa）及びリゾチーム(14.3 kDa)を用い、C-PAGELを用いCompact PAGE AE-7305（アトー株式会社）により電気泳動後、クマシーブリリアントブルーで染色した。
6. Examination of characteristics of purified enzyme 6-1. SDS-PAGE
Purity and molecular weight of the purified peptidases were confirmed by SDS-PAGE. Phosphorylase b (97.2 kDa), bovine serum albumin (66.4 kDa), ovalbumin (45.0 kDa), carbonic anhydrase II (29.0 kDa), soybean trypsin inhibitor (21.0 kDa) and lysozyme (14.3 kDa) were used as molecular weight markers. , C-PAGEL was used for electrophoresis by Compact PAGE AE-7305 (Ato Co., Ltd.), and then stained with Coomassie Brilliant Blue.
６−２．至適pH、至適温度の測定
AP31-Bは活性測定法(LAP法）に準じて、CP31-Bの至適pHはCP法で、至適温度はACP法で測定を行った。
6-2. Measurement of optimum pH and temperature
AP31-B was measured according to the activity measurement method (LAP method) by measuring the optimum pH of CP31-B by the CP method and the optimum temperature by the ACP method.
６−３．基質特異性
AP31-Bはアミノ酸-pNA（L-amio p-nitroanilide）を基質として、活性測定法(LAP法）に準じて測定した。
6-3. Substrate specificity
AP31-B was measured according to the activity measurement method (LAP method) using amino acid-pNA (L-amio p-nitroanilide) as a substrate.
CP31-Bは各種ベンジルオキシカルボニルジペプチド（Ｚ−ジペプチド）を基質に用い、CP法で測定した。 CP31-B was measured by the CP method using various benzyloxycarbonyl dipeptides (Z-dipeptides) as substrates.
７．ペプチダーゼの分解様式の検討
７−１．基質溶液の調製
0.8 m molのテトラペプチド（タフトシン；（株）ペプチド研究所）、トリペプチド又はジペプチドを0.1 M マッキルバイン緩衝液（ pH 5.0 ）1 mLに精製水2 mLを加えた溶液に溶解した（以上CP31-Bの場合)。AP31-Bの場合は、緩衝液として0.1 Mトリス緩衝液（pH 7.0）を用いた。
7. Examination of degradation mode of peptidase 7-1. Preparation of substrate solution
0.8 mmol of tetrapeptide (Taftosin; Peptide Research Co., Ltd.), tripeptide or dipeptide was dissolved in a solution prepared by adding 2 mL of purified water to 1 mL of 0.1 M McILVINE buffer (pH 5.0) (above CP31-B). in the case of). In the case of AP31-B, 0.1 M Tris buffer (pH 7.0) was used as a buffer.
７−２．反応
AP31-B溶液（2.28単位）1 mLを上記の各基質液に加え、40℃で分解反応を行った。CP31-Bの場合、1 mL（ACP活性で3.7単位）を加え、50℃で分解反応を行った。
7-2. reaction
1 mL of AP31-B solution (2.28 units) was added to each of the above substrate solutions, and a decomposition reaction was performed at 40 ° C. In the case of CP31-B, 1 mL (3.7 units of ACP activity) was added and the decomposition reaction was performed at 50 ° C.
７−３．遊離アミノ酸の定性
各反応時間で少量をサンプリングし、限外濾過膜付き遠心チューブ（Spin-X UF, CORNING製）で酵素を除去後、TLCで遊離アミノ酸を確認した。TLCプレートはシリカゲル60F 254（メルク社）を、展開溶媒はブタノール：酢酸：水（4：1：2.5 (V/V)）を用い、0.2％ニンヒドリンの95％ n-ブタノール溶液を噴霧し、90℃で発色した。
7-3. Qualitative analysis of free amino acid A small amount was sampled at each reaction time, the enzyme was removed by a centrifugal tube with an ultrafiltration membrane (Spin-X UF, manufactured by CORNING), and the free amino acid was confirmed by TLC. Silica gel 60 F 254 (Merck) is used for the TLC plate, butanol: acetic acid: water (4: 1: 2.5 (V / V)) is used as the developing solvent, and a 95% n-butanol solution of 0.2% ninhydrin is sprayed onto the plate. Color developed at ° C.
８．金属イオン及び酵素阻害剤の影響
各種金属イオン及び酵素阻害剤の存在下（1mM）でカルボキシペプチダーゼ活性（CP法）及びアミノペプチダーゼ活性（LAP法）を測定した。
8. Effect of Metal Ions and Enzyme Inhibitors Carboxypeptidase activity (CP method) and aminopeptidase activity (LAP method) were measured in the presence of various metal ions and enzyme inhibitors (1 mM).
９．苦味低減試験１（苦味ペプチドに対する効果）
９−１．苦味ペプチドの調製
ミルクカゼイン（和光純薬工業（株）製）2 gに0.1 M NaOH溶液10 mLを加え、加温溶解した。冷却後、0.1 M トリス緩衝液（pH 7.0)10 mL及び精製水10 mLを加え、pHを7.0に調製後、精製水で50 mLとした。更に、プロチンSD-AY10（天野エンザイム（株）製）100 mgを加え、50℃で3.5時間反応後、121℃、１分間の殺菌処理をした。CP31-B試験では、プロチンSD-AY10の代わりに、プロレザーFG-F（天野エンザイム（株）製）を用い、同様に苦味ペプチドを調製した。
9. Bitterness reduction test 1 (effect on bitterness peptides)
9-1. Preparation of Bitter Peptide To 2 g of milk casein (manufactured by Wako Pure Chemical Industries, Ltd.), 10 mL of 0.1 M NaOH solution was added and dissolved by heating. After cooling, 10 mL of 0.1 M Tris buffer (pH 7.0) and 10 mL of purified water were added to adjust the pH to 7.0, and then 50 mL was added with purified water. Further, 100 mg of Protin SD-AY10 (manufactured by Amano Enzyme Co., Ltd.) was added and reacted at 50 ° C. for 3.5 hours and then sterilized at 121 ° C. for 1 minute. In the CP31-B test, Proleather FG-F (manufactured by Amano Enzyme Co., Ltd.) was used in place of Protin SD-AY10, and a bitter peptide was similarly prepared.
９−２．苦味標準液の調製
カフェイン（和光純薬工業（株））0から20 g / Lの各種濃度の 精製水溶液を調製した。閾値はおよそ200 mg/Lであった。
9-2. Preparation of bitterness standard solution Caffeine (Wako Pure Chemical Industries, Ltd.) Prepared purified aqueous solutions with various concentrations of 0 to 20 g / L. The threshold was approximately 200 mg / L.
９−３．ペプチド分解率の算出
ケルダール法による全窒素に対するホルモール滴定法によるホルモール態窒素の割合で、分解率を表示した。
9-3. Calculation of Peptide Degradation Rate Degradation rate was expressed by the ratio of formol nitrogen by the formol titration method to total nitrogen by the Kjeldahl method.
９−４．苦味低減反応
苦味ペプチド溶液27 mLに酵素液3 mLを加え、1、3又は5時間反応させ、官能試験により苦味を測定した。CP31-Bの場合、ACP活性で3.22単位を加え、50℃（pH6.0）で反応させた。AP31-Bの場合、6.81単位を加え、40℃（pH7.0）で反応させた。
9-4. Bitterness reduction reaction To 27 mL of the bitterness peptide solution, 3 mL of the enzyme solution was added, reacted for 1, 3 or 5 hours, and the bitterness was measured by a sensory test. In the case of CP31-B, 3.22 units of ACP activity was added and reacted at 50 ° C (pH 6.0). In the case of AP31-B, 6.81 units were added and reacted at 40 ° C (pH 7.0).
１０．苦味低減試験２（ヨーグルトに対する効果）
１０−１．ヨーグルトの調製
AP31-B添加試験では、250mLの牛乳（明治おいしい牛乳、（株）明治）に種菌0.5 g（ヨーグルトの願い：（株）リジエールドーレＲＤ）とAP31-B(1.07、2.15、又は4.30単位）をそれぞれ加え、28℃、24h放置後、5℃に1h放置し、官能評価とグルタミン酸生成量の測定を行った。CP31-B添加試験では、牛乳（明治おいしい牛乳、（株）明治）250 mLに種菌（ROYAL KEFIR PRO；（株）ロイヤルユキ）0.5 g及びペプチダーゼ（1.03、3.09又は5.15単位）をそれぞれ加え、25℃で16時間発酵、その後5℃で１時間冷却後、官能評価とグルタミン酸生成量の測定を行った。グルタミン酸量の測定にはヤマサＬ−グルタミン酸測定キットII（ヤマサ醤油（株））を用いた。
10. Bitterness reduction test 2 (effect on yogurt)
10-1. Preparation of yogurt
In the AP31-B addition test, 250 mL of milk (Meiji Tasty Milk, Meiji Co., Ltd.) was inoculated with 0.5 g of inoculum (hope of yogurt: Lisier Dore RD Co., Ltd.) and AP31-B (1.07, 2.15, or 4.30 units), respectively. In addition, after left at 28 ° C. for 24 hours, left at 5 ° C. for 1 hour to perform sensory evaluation and measurement of glutamic acid production. In the CP31-B addition test, 250 g of milk (Meiji Tasty Milk, Meiji Co., Ltd.) was added with 0.5 g of inoculum (ROYAL KEFIR PRO; Royal Yuki Co., Ltd.) and peptidase (1.03, 3.09 or 5.15 units), respectively, and 25 After 16 hours of fermentation at 0 ° C. and then cooling at 5 ° C. for 1 hour, sensory evaluation and measurement of glutamic acid production amount were performed. Yamasa L-glutamic acid measurement kit II (Yamasa Shoyu Co., Ltd.) was used to measure the amount of glutamic acid.
１０−２．ヨーグルトの官能評価
椙山女学園大学 大学生（21±1歳）10名により試食し、順位法で評価項目ごとに順位を付け、合計値からクレーマー検定（Kramer, A. (1960). Rapid method for determination significance of difference from ranking. Food Technol., 14,576-582.）により有意差を評価した。
10-2. Sensory evaluation of
Ｂ．結果と考察
１．31-B株の同定
ネペンテス消化液から分離した酵母31-B株を(株）テクノスルガ（静岡市、日本）にて26S rDNA-D1/D2塩基配列解析に供した。26S rDNA-D1/D2の塩基配列は、シュードザイマ・フベイエンシス CBS 10077T(DQ008953)の配列に100%の相同性を示した。31-B株はyeast extract-malt extract agar培地（ベクトン・ディッキンソン）上で好気的に生育した（25℃、7日間）。卵形又は楕円形の出芽を示し、３週間の観察では生殖器官は認められなかった。コロニーは白色から黄白色がかったクリーム色であり、いくぶん、表面が滑らかな粘液様且つ凸状を呈した。これらの特性はシュードザイマ・フベイエンシスの特性に該当する（Wang, Q-M., Jia, J-H. and Bai, F-Y. (2006) Int. J. Syst. Evol. Microbiol., 56, 289-293.）。以上の結果から、31-B株はシュードザイマ・フベイエンシス（Pseudozuma hubeiensis）と同定された。
B. Results and
２．31-B株の培養
培養72時間まで両ペプチダーゼとも生産が伸び、安定に回収できた（データ示さず）。
2. Culturing of 31-B strain Production of both peptidases increased up to 72 hours of cultivation, and stable recovery was possible (data not shown).
３．ペプチダーゼの精製
培地組成Cにより72時間培養した培養液から、遠心分離( 6000rpm, 4℃, 10min )により31-B株菌体を含む不溶物を除去した。上清は限外濾過膜（AIV 1010；旭化成ケミカルズ株式会社）により約５倍に濃縮した。濃縮液に硫酸アンモニウムを固体のまま、80飽和濃度になるよう添加溶解し、5℃で20時間放置し、沈殿を集め精製水に溶解、透析による脱塩の後、0.01Mリン酸緩衝液に透析、同緩衝液で平衡化したDEAE-Toyopearl 650Mクロマトグラフィーに供した。 DEAE-Toyopearl 650Mクロマトグラフィーの結果(図１）、アミノペプチダーゼとカルボキシペプチダーゼはそれぞれの活性画分に分取され、以降の精製はそれぞれのペプチダーゼについて実施した。
3. Purification of peptidase The insoluble matter containing the 31-B strain bacterial cells was removed from the culture broth cultured for 72 hours with medium composition C by centrifugation (6000 rpm, 4 ° C, 10 min). The supernatant was concentrated about 5 times with an ultrafiltration membrane (AIV 1010; Asahi Kasei Chemicals Corporation). Ammonium sulfate was added to the concentrate as a solid to dissolve it to a saturated concentration of 80, left to stand at 5 ° C for 20 hours, collected and dissolved in purified water, desalted by dialysis, and dialyzed against 0.01M phosphate buffer. It was subjected to DEAE-Toyopearl 650M chromatography equilibrated with the same buffer. As a result of DEAE-Toyopearl 650M chromatography (FIG. 1), aminopeptidase and carboxypeptidase were fractionated into respective active fractions, and subsequent purification was carried out for each peptidase.
３−１．アミノペプチダーゼの精製
アミノペプチダーゼの精製結果を表２に示す。
31-B株の培養上清にはプロテイナーゼは検出されなかった。また、精製過程を通して、アミノペプチダーゼのアイソザイムも認められなかった。最終的に、アミノペプチダーゼ（AP31-B）は8.0%の収率で、比活性約107倍に精製できた。 No proteinase was detected in the culture supernatant of 31-B strain. Also, no aminopeptidase isozyme was observed throughout the purification process. Finally, aminopeptidase (AP31-B) could be purified with a yield of 8.0% and a specific activity of about 107 times.
３−２．カルボキシペプチダーゼの精製
カルボキシペプチダーゼの精製結果を表３に示す。
４．精製酵素のSDS-PAGE
図２（AP31-B）及び図３（CP31-B）にSDS-PAGEの結果を示した。いずれの酵素もSDS-PAGEで単一バンドとして精製された。分子量は、AP31-Bが分子量75.3 kDa、CP31-Bが分子量70.5 kDaであった。
4. SDS-PAGE of purified enzyme
The results of SDS-PAGE are shown in Fig. 2 (AP31-B) and Fig. 3 (CP31-B). Both enzymes were purified by SDS-PAGE as a single band. Regarding the molecular weight, AP31-B had a molecular weight of 75.3 kDa and CP31-B had a molecular weight of 70.5 kDa.
５．精製酵素の酵素学的検討
５−１．温度と活性
カルボキシペプチダーゼ（CP31-B）の測定結果を図４に示す。カルボキシペプチダーゼ活性はACP活性測定法に従いpH 5.0の条件で測定した。至適温度は50℃であった。一方、アミノペプチダーゼ（AP31-B）の結果を図５に示す。至適温度は40℃（pH8.0)であった。
5. Enzymatic study of purified enzyme 5-1. Temperature and activity The measurement results of carboxypeptidase (CP31-B) are shown in Fig. 4. Carboxypeptidase activity was measured according to the ACP activity assay method at pH 5.0. The optimum temperature was 50 ° C. On the other hand, the result of aminopeptidase (AP31-B) is shown in FIG. The optimum temperature was 40 ° C (pH 8.0).
５−２．pHと活性
カルボキシペプチダーゼについては、CP法で40℃で測定した(図６）。至適pHは5であった。アミノペプチダーゼの至適pHは8（37℃）であった（図７）。
5-2. pH and activity Carboxypeptidase was measured by the CP method at 40 ° C (Fig. 6). The optimum pH was 5. The optimum pH of aminopeptidase was 8 (37 ° C) (Fig. 7).
５−３．酵素活性に対する金属イオン及び酵素阻害剤の影響
アミノペプチダーゼ活性の測定では、pH 8.0、40℃、30分反応の条件とし、反応系に1 mMの濃度の金属塩又は酵素阻害剤を添加した。一方、カルボキシペプチダーゼ活性はCP活性測定法（pH 5.0、30分、50℃）で測定し、反応系に1 mMの濃度の金属塩又は酵素阻害剤を添加した。アミノペプチダーゼについての結果を表５に示す。
アミノペプチダーゼは、亜鉛イオン（Zn2+）によって活性が完全に阻害され、二価鉄イオン（Fe2+）、銅イオン（Cu2+）及び三価鉄イオン（Fe3+）によっても活性が阻害された。また、SDS、EDTA及びPCMBによっても活性が阻害された。E-64、Pepstatin A及びPetablocによっては活性が阻害されない。一方、カリウムイオン（K+）は活性化を促した。 Aminopeptidase is completely inhibited by zinc ion (Zn 2+ ), and is also activated by divalent iron ion (Fe 2+ ), copper ion (Cu 2+ ), and trivalent iron ion (Fe 3+ ). Was blocked. The activity was also inhibited by SDS, EDTA, and PCMB. The activity is not inhibited by E-64, Pepstatin A and Petabloc. On the other hand, potassium ion (K + ) promoted activation.
カルボキシペプチダーゼについての結果を表６に示す。
カルボキシペプチダーゼはカリウムイオン（K+）、マンガンイオン（Mn2+）及び二価鉄イオン（Fe2+）によって活性が阻害された。また、SDS及びPCMBによっても活性が阻害された。 The activity of carboxypeptidase was inhibited by potassium ion (K + ), manganese ion (Mn 2+ ) and divalent iron ion (Fe 2+ ). The activity was also inhibited by SDS and PCMB.
５−４．基質特異性
アミノペプチダーゼ活性はLAP法に準じて測定した。カルボキシペプチダーゼ活性はCP法で測定した。アミノペプチダーゼの測定結果（基質特異性）を表７に示す。
アミノペプチダーゼ（AP31-B）はN末端のロイシン、アルギニン、メチオニン、アラニンに作用した。一方、ペプチド鎖のN末端のグリシン、プロリンには僅かに作用した。また、オリゴペプチドに対しては、アミノペプチダーゼ（AP31-B）はtuftsin(Thr-Lys-Pro-Arg)からスレオニンのみを遊離し、トリペプチド(Leu-Ala-Pro)からはロイシンのみを遊離した。トリペプチド(Leu-Leu-Ala)からはロイシン及びアラニンを遊離した。これらの結果から、アミノペプチダーゼ（AP31-B）は、プロリンに結合したN末端アミノ酸には作用しないことが推測された。このように、アミノペプチダーゼ（AP31-B）は既報のアミノペプチダーゼに類似した基質特異性を示した。 Aminopeptidase (AP31-B) acted on N-terminal leucine, arginine, methionine and alanine. On the other hand, it slightly acted on N-terminal glycine and proline of the peptide chain. For oligopeptides, aminopeptidase (AP31-B) released only threonine from tuftsin (Thr-Lys-Pro-Arg) and only leucine from tripeptide (Leu-Ala-Pro). .. Leucine and alanine were released from the tripeptide (Leu-Leu-Ala). From these results, it was inferred that aminopeptidase (AP31-B) did not act on the N-terminal amino acid bound to proline. Thus, aminopeptidase (AP31-B) showed a substrate specificity similar to that of previously reported aminopeptidases.
カルボキシペプチダーゼの測定結果を表８に示す。
カルボキシペプチダーゼ（CP31-B）はC末端のロイシン、フェニルアラニンに作用した。一方、ペプチド鎖のC末端のアラニン、プロリン、チロシンに対する作用は僅かであった。このように、カルボキシペプチダーゼ（CP31-B）の基質特異性は概ねCPYに似た傾向にあった。 Carboxypeptidase (CP31-B) acted on C-terminal leucine and phenylalanine. On the other hand, the action on the C-terminal alanine, proline and tyrosine of the peptide chain was slight. Thus, the substrate specificity of carboxypeptidase (CP31-B) tended to be similar to CPY.
６．ペプチダーゼによる苦みペプチドの苦み低減
調製した苦みペプチドは分解率8.5％（AP31-B試験に用いた）或いは11%（CP31-Bに用いた試験）で、いずれもカフェイン20g/L相当の苦みを呈した。分解経過とそれに伴う苦味の低下経過を図８及び図９に示した。ペプチドの苦みは、いずれの場合も10分の１以下に低減した。
6. Reduction of bitterness of bitter peptides by peptidases The prepared bitter peptides have a decomposition rate of 8.5% (used for AP31-B test) or 11% (test used for CP31-B), and all have bitterness equivalent to 20 g / L of caffeine. Presented. The decomposition process and the accompanying decrease in bitterness are shown in FIGS. 8 and 9. The bitterness of the peptide was reduced to less than 1/10 in each case.
７．ヨーグルト調製時のペプチダーゼ添加の影響
アミノペプチダーゼ（AP31-B）についての評価結果を表９及び表１０に示す。
アミノペプチダーゼ（AP31-B）を添加したヨーグルトは、いずれの添加量においても、苦味、うま味において対照（コントロール）よりも評価が高い傾向にあり、対照（コントロール）の苦味が低減している傾向にあった。アミノペプチダーゼ（AP31-B）を添加したヨーグルトは、総合評価でも好まれる傾向にあった。 Aminopeptidase (AP31-B) -added yogurt tended to have a higher evaluation of bitterness and umami than the control (control) at any addition amount, and the bitterness of the control (control) tended to decrease. there were. The yogurt to which aminopeptidase (AP31-B) was added tended to be preferred in the comprehensive evaluation.
カルボキシペプチダーゼ（CP31-B）についての評価結果を表１１及び表１２に示す。
カルボキシペプチダーゼ（CP31-B）を添加したヨーグルト（1.03及び3.09 U/250mL 牛乳）は色、甘味に関して評価が高い傾向にあった。また、ヨーグルト（3.09U/250mL 牛乳）では、苦味、塩味が優位に好まれた。いずれの添加量においても、対照（コントロール）の苦味が低減される傾向にあり、総合点においても対照（コントロール）よりも評価が高い傾向にあった。 Yogurt (1.03 and 3.09 U / 250 mL milk) supplemented with carboxypeptidase (CP31-B) tended to be highly evaluated for color and sweetness. For yogurt (3.09U / 250mL milk), bitterness and saltiness were preferred. At any addition amount, the bitterness of the control tended to be reduced, and the overall score tended to be higher than that of the control.
＜食品加工への適用＞
ペプチダーゼとしての食品添加物酵素には既にいくつかの酵素剤が登録されているが、その多くはプロテイナーゼを主体とした酵素剤である。蛋白系食品加工において、主に用いられるのはプロテイナーゼではあるが、アミノ酸のみを遊離する目的、例えばペプチドの苦み消去などの目的に使用すれば、素材タンパク質の物性などに影響を及ぼすため、プロテイナーゼを含まないペプチダーゼ剤は有用である。上記の実験によって、シュードザイマ・フベイエンシス31-B株が産生するペプチダーゼの精製に成功し、その諸性質を明らかにした。以下では、31-B株が産生するペプチダーゼの食品加工分野での有用性を検討することにした。
<Application to food processing>
Some enzyme agents have already been registered as food additive enzymes as peptidases, but most of them are proteinase-based enzyme agents. Proteinases are mainly used in protein-based food processing, but if they are used for the purpose of releasing only amino acids, for example, for the purpose of eliminating bitterness of peptides, etc., they will affect the physical properties of the raw material proteins. Peptidase agents that do not contain are useful. Through the above experiments, the peptidase produced by Pseudozyma fubeiensis 31-B was successfully purified, and its properties were clarified. Below, we decided to examine the usefulness of the peptidase produced by the 31-B strain in the food processing field.
Ａ．材料と方法
１．酵素
シュードザイマ・フベイエンシス31-B株を培養、遠心上清、塩析、溶解した粗酵素液（上記実験でのペプチダーゼ精製工程の前のもの）をサンプル（以下、「PP31」と呼ぶ）とし、以下の検討に用いた。比較対象として、市販食品添加物酵素剤である、Rhizopus起源のペプチダーゼＲ（天野エンザイム株式会社製）とAspergillus起源のプロテアックス（天野エンザイム株式会社製）を用いた。
A. Materials and
２．苦味ペプチドの苦味低減試験
上記実験に準じて実施したが、ペプチド調製には枯草菌プロテアーゼであるプロレザーFG-F（天野エンザイム株式会社製）を100 mg用いた。ミルクカゼインの分解率は15〜17％であった。苦味低減反応は、苦みペプチド溶液27 mLに酵素液3 mL（ACP活性で5 単位）加え、37℃で反応させた。苦味の評価は椙山女学園大学 大学生（21±1歳）パネル３名で行い、各種濃度のカフェイン溶液と比較した。
2. Bitterness Peptide Bitterness Reduction Test This was carried out according to the above experiment, but 100 mg of Bacillus subtilis protease Proleather FG-F (manufactured by Amano Enzyme Inc.) was used for peptide preparation. The decomposition rate of milk casein was 15-17%. For the bitterness reduction reaction, 3 mL of the enzyme solution (5 units of ACP activity) was added to 27 mL of the bitterness peptide solution, and the reaction was carried out at 37 ° C. The bitterness was evaluated by 3 panelists (21 ± 1 years old) from Sugiyama Jogakuen University and compared with caffeine solutions of various concentrations.
Ｂ．結果と考察
１．酵素活性測定結果
結果を表１３に示す。
PP31（塩析溶解液）のプロテイナーゼ活性は実質的にゼロであった。即ち、夾雑するプロテイナーゼは実質的にないことが明らかとなった。 The proteinase activity of PP31 (salting out solution) was substantially zero. That is, it was revealed that there is substantially no contaminating proteinase.
２．酵素性質の検討
２−１．アミノペプチダーゼ活性に及ぼすｐＨの影響
実験結果を図１０に示す。
2. Examination of enzymatic properties 2-1. Effect of pH on aminopeptidase activity Experimental results are shown in FIG.
２−２．ペプチダーゼ活性に及ぼす温度の影響
２−２−１．アミノペプチダーゼ
実験結果を図１１に示す。
2-2. Effect of temperature on peptidase activity 2-2-1. The aminopeptidase experimental results are shown in FIG.
２−２−２．カルボキシペプチダーゼ
実験結果を図１２に示す。
2-2-2. The results of the carboxypeptidase experiment are shown in FIG.
３．酵素剤による苦味ペプチドの苦味低減試験
３−１．PP31による苦味低減効果
pH7.0で反応させた場合の実験結果を図１３に示す。分解率は7.40％上昇し、苦味は５分の１以下に低減した。pH5.0で反応させた場合の実験結果を図１４に示す。分解率は12.06％上昇し、苦味は半分以下となった。尚、ペプチダーゼRでは分解率は17.4％上昇したが、苦味は３時間以降むしろ増加した（データを示さず）。プロテアックスについても、分解率は13.4％上昇したが、苦味の減少は僅かであった（データ示さず）。
3. Bitterness peptide bitterness reduction test 3-1. Bitterness reduction effect of PP31
FIG. 13 shows the experimental result when the reaction was performed at pH 7.0. The decomposition rate increased by 7.40%, and the bitterness was reduced to less than 1/5. FIG. 14 shows the experimental result when the reaction was performed at pH 5.0. The decomposition rate increased by 12.06% and bitterness was less than half. The degradation rate of peptidase R increased by 17.4%, but the bitterness increased rather after 3 hours (data not shown). Regarding Proteax, the decomposition rate increased by 13.4%, but the bitterness was slightly reduced (data not shown).
シュードザイマ・フベイエンシス由来の新規ペプチダーゼが提供される。本発明のペプチダーゼは例えば、各種食品の製造、加工に利用され得る。 Provided is a novel peptidase from Pseudozyma fuvayensis. The peptidase of the present invention can be used, for example, in the production and processing of various food products.
この発明は、上記発明の実施の形態及び実施例の説明に何ら限定されるものではない。特許請求の範囲の記載を逸脱せず、当業者が容易に想到できる範囲で種々の変形態様もこの発明に含まれる。本明細書の中で明示した論文、公開特許公報、及び特許公報などの内容は、その全ての内容を援用によって引用することとする。 The present invention is not limited to the description of the embodiments and examples of the invention. The present invention also includes various modifications within the scope that can be easily conceived by those skilled in the art without departing from the scope of the claims. The contents of the papers, published patent publications, patent publications, and the like, which are specified in this specification, are all incorporated by reference.
Claims (11)
(1)作用 ペプチド鎖のN末端アミノ酸を遊離する反応を触媒する；
(2)分子量 SDS-PAGEによる分子量が75.3kDa；
(3)基質特異性 ペプチド鎖のN末端のロイシン、アルギニン、メチオニン、アラニンを遊離する。ペプチド鎖のN末端のグリシン、プロリンへの作用は弱い。プロリンに結合したN末端アミノ酸に作用しない；
(4)至適pH 8；
(5)至適温度 40℃。 Has enzymatic properties below, as an active ingredient peptidase is derived from Pseudozyma Fubeienshisu, enzymatic agent flop Roteinaze is not contaminated:
(1) Action Catalyze the reaction that releases the N-terminal amino acid of the peptide chain;
(2) Molecular weight The molecular weight by SDS-PAGE is 75.3 kDa;
(3) Substrate specificity Leucine, arginine, methionine and alanine at the N-terminal of the peptide chain are released. The action on the N-terminal glycine and proline of the peptide chain is weak. Does not act on the N-terminal amino acid bound to proline;
(4) Optimum pH 8;
(5) Optimum temperature 40 ℃.
(1)作用 ペプチド鎖のC末端アミノ酸を遊離する反応を触媒する；
(2)分子量 SDS-PAGEによる分子量が70.5kDa；
(3)基質特異性 ペプチド鎖のC末端のロイシン、フェニルアラニンによく作用する。ペプチド鎖のC末端のアラニン、プロリン、チロシンへの作用は弱い；
(4)至適pH 5；
(5)至適温度 50℃。 Has enzymatic properties below, as an active ingredient peptidase is derived from Pseudozyma Fubeienshisu, enzymatic agent flop Roteinaze is not contaminated:
(1) Action Catalyze the reaction to release the C-terminal amino acid of the peptide chain;
(2) Molecular weight The molecular weight by SDS-PAGE is 70.5 kDa;
(3) Substrate specificity It acts well on C-terminal leucine and phenylalanine of the peptide chain. Weak action on C-terminal alanine, proline and tyrosine of peptide chain;
(4) Optimum pH 5;
(5) Optimum temperature 50 ℃.
(1)シュードザイマ・フベイエンシス31-B株を培養するステップ；
(2)培養後の培養液より、ペプチダーゼを回収するステップ。 The following steps (1) and (2) including, peptidases flop Roteinaze is not contaminated preparation:
(1) a step of culturing Pseudozyma fubeiensis 31-B strain;
(2) A step of recovering peptidase from the culture medium after culturing.
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