JPH01247085A - Soybean protein coagulation enzyme and production thereof - Google Patents
Soybean protein coagulation enzyme and production thereofInfo
- Publication number
- JPH01247085A JPH01247085A JP7198188A JP7198188A JPH01247085A JP H01247085 A JPH01247085 A JP H01247085A JP 7198188 A JP7198188 A JP 7198188A JP 7198188 A JP7198188 A JP 7198188A JP H01247085 A JPH01247085 A JP H01247085A
- Authority
- JP
- Japan
- Prior art keywords
- soybean protein
- enzyme
- coagulase
- activity
- producing
- 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.)
- Pending
Links
- 108010073771 Soybean Proteins Proteins 0.000 title claims abstract description 50
- 235000019710 soybean protein Nutrition 0.000 title claims abstract description 49
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 38
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 38
- 230000015271 coagulation Effects 0.000 title claims abstract description 20
- 238000005345 coagulation Methods 0.000 title claims abstract description 20
- 238000004519 manufacturing process Methods 0.000 title claims description 12
- 235000013322 soy milk Nutrition 0.000 claims abstract description 20
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims abstract description 8
- 241000193830 Bacillus <bacterium> Species 0.000 claims abstract description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 5
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 5
- 239000002253 acid Substances 0.000 claims abstract description 4
- 230000002378 acidificating effect Effects 0.000 claims abstract description 4
- 229940041514 candida albicans extract Drugs 0.000 claims abstract description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims abstract description 4
- 235000019341 magnesium sulphate Nutrition 0.000 claims abstract description 4
- 239000000203 mixture Substances 0.000 claims abstract description 4
- 239000001509 sodium citrate Substances 0.000 claims abstract description 4
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims abstract description 4
- 239000000126 substance Substances 0.000 claims abstract description 4
- 239000012138 yeast extract Substances 0.000 claims abstract description 4
- 235000019658 bitter taste Nutrition 0.000 claims abstract description 3
- 108010065152 Coagulase Proteins 0.000 claims description 32
- 230000000694 effects Effects 0.000 claims description 32
- 235000013305 food Nutrition 0.000 claims description 8
- 239000003112 inhibitor Substances 0.000 claims description 6
- 229910021645 metal ion Inorganic materials 0.000 claims description 6
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 claims description 6
- IZZYABADQVQHLC-UHFFFAOYSA-N 4-methylbenzenesulfonyl fluoride Chemical compound CC1=CC=C(S(F)(=O)=O)C=C1 IZZYABADQVQHLC-UHFFFAOYSA-N 0.000 claims description 5
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 5
- 241000894006 Bacteria Species 0.000 claims description 4
- 229910019142 PO4 Inorganic materials 0.000 claims description 4
- 230000004913 activation Effects 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 4
- 239000010452 phosphate Substances 0.000 claims description 4
- 102000005741 Metalloproteases Human genes 0.000 claims description 3
- 108010006035 Metalloproteases Proteins 0.000 claims description 3
- 102000012479 Serine Proteases Human genes 0.000 claims description 3
- 108010022999 Serine Proteases Proteins 0.000 claims description 3
- 150000007513 acids Chemical class 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 238000001962 electrophoresis Methods 0.000 claims 1
- 229940001941 soy protein Drugs 0.000 claims 1
- 239000002609 medium Substances 0.000 abstract description 11
- 238000012258 culturing Methods 0.000 abstract description 5
- 241000196324 Embryophyta Species 0.000 abstract description 3
- 238000000034 method Methods 0.000 abstract description 3
- 239000001963 growth medium Substances 0.000 abstract description 2
- 239000002689 soil Substances 0.000 abstract description 2
- 230000000813 microbial effect Effects 0.000 abstract 2
- 230000002255 enzymatic effect Effects 0.000 abstract 1
- 210000003254 palate Anatomy 0.000 abstract 1
- 150000003016 phosphoric acids Chemical class 0.000 abstract 1
- 238000012216 screening Methods 0.000 abstract 1
- 210000003323 beak Anatomy 0.000 description 15
- 238000010586 diagram Methods 0.000 description 9
- 230000001112 coagulating effect Effects 0.000 description 8
- 235000010469 Glycine max Nutrition 0.000 description 6
- 244000068988 Glycine max Species 0.000 description 6
- 238000010828 elution Methods 0.000 description 5
- 244000005700 microbiome Species 0.000 description 4
- 235000013336 milk Nutrition 0.000 description 4
- 239000008267 milk Substances 0.000 description 4
- 210000004080 milk Anatomy 0.000 description 4
- 108091005804 Peptidases Proteins 0.000 description 3
- 239000004365 Protease Substances 0.000 description 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108010082495 Dietary Plant Proteins Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- KRHYYFGTRYWZRS-UHFFFAOYSA-M Fluoride anion Chemical compound [F-] KRHYYFGTRYWZRS-UHFFFAOYSA-M 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 239000005909 Kieselgur Substances 0.000 description 1
- -1 Phenyl methylsulfonyl Chemical group 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- VJHCJDRQFCCTHL-UHFFFAOYSA-N acetic acid 2,3,4,5,6-pentahydroxyhexanal Chemical compound CC(O)=O.OCC(O)C(O)C(O)C(O)C=O VJHCJDRQFCCTHL-UHFFFAOYSA-N 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 208000011775 arteriosclerosis disease Diseases 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000021135 plant-based food Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000002250 progressing effect Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は大豆乳を？凝固せしめる大豆蛋白凝固酵素及び
その１！遣方法に関し、詳しくは、大豆乳を凝固させ、
苦味のな１％滑らかな口あたｔ）の食品を得ることので
きる大豆蛋白凝固酵素及びその製造方法に関するもので
ある。[Detailed Description of the Invention] (Field of Industrial Application) The present invention uses soy milk? Soybean protein coagulase that causes coagulation and part 1! For details on the method, see coagulating soy milk,
The present invention relates to a soybean protein coagulating enzyme and a method for producing the same, which can produce a food with a 1% smooth mouthfeel and no bitterness.
（従来の技術）
１口、健康上の理由、すなわち心ｉ病および動脈硬化等
の防止から植物性食品が注目されている。特に、大豆蛋
白を利用した食品の開発が進められている。(Prior Art) Plant-based foods are attracting attention for health reasons, ie, prevention of heart disease and arteriosclerosis. In particular, the development of foods using soybean protein is progressing.
（発明が解決しようとする課題）
上記のように、植物性食品を得るため、大豆蛋白を利用
した食品開発が行なわれているが、これまでに牛乳から
チーズを作成する場合に必要となる凝固酵素は存在する
が、大豆蛋白を；凝固せしめる凝固酵素は見つかってい
ないため、その成果が上がっていない。(Problems to be Solved by the Invention) As mentioned above, food development using soybean protein has been carried out in order to obtain plant foods, but until now, the coagulation required when making cheese from milk has been Although enzymes exist, the coagulation enzyme that coagulates soybean protein has not been found, so no results have been achieved.
本発明者は食品素材として有用な大豆蛋白食品を！Ｊｌ
造するため、豆乳の凝固酵素を求めて研究し、その結果
、自然界より分離した微生物よ 、り大豆蛋白
凝固酵素が分泌されることを見いだし、この大豆蛋白凝
固酵素生産菌を単離して、二の菌の生産する大豆蛋白凝
固酵素を精製した。The inventor has created a soybean protein food that is useful as a food material! Jl
In order to produce soybean milk, they conducted research to find the coagulating enzyme in soymilk. As a result, they discovered that soybean protein coagulating enzyme was secreted by microorganisms isolated from nature. Soybean protein coagulase produced by this fungus was purified.
この発明は純植物性蛋白質たる大豆蛋白をイ反固させる
大豆蛋白凝固酵素を得ることを目的としており、さらに
、この大豆蛋白凝固酵素を効率的に生産しえる大豆蛋白
凝固酵素の製造方法を提供することを目的としている。The purpose of this invention is to obtain a soybean protein coagulase that can solidify soybean protein, which is a pure vegetable protein, and also to provide a method for producing soybean protein coagulase that can efficiently produce the soybean protein coagulase. It is intended to.
上記大豆蛋白凝固Ｂ￥素生産菌は２６Ｄ７と表示され徽
エモ舅にＦＥＲＭ ＢＰ−１７７８としで寄託されてい
る。The above-mentioned soybean protein coagulation B yen-producing bacterium is designated as 26D7 and has been deposited with Hui Emo-fu as FERM BP-1778.
（課題を解決するための手段及び作用）本発明にかかる
大豆蛋白凝固酵素は、ＳＤＳ電気電気的動的一なバンド
となり、分子量は３００００前後であった。次にこの酵
素の至適温度および温度安定性を調べた。ビーク■の活
性至適温度は約８０℃であった。また、ビーク■（粗６
ｙ素）の活性至適温度は約６５℃であった。(Means and effects for solving the problems) The soybean protein coagulase according to the present invention exhibited an SDS electroelectrically dynamic band and had a molecular weight of around 30,000. Next, we investigated the optimal temperature and temperature stability of this enzyme. The optimum temperature for activation of Beak II was approximately 80°C. Also, beak ■ (rough 6
The optimum temperature for activation of y element) was about 65°C.
精製された酵素の熱にたいする安定性は３５ないし４０
゛ｃで３０分１１１安定であった。同時に各Ｐ Ｈにた
いする酵素の安定性を調べた結果（第６図）、Ｐ Ｈ４
からＰＨ９（１７時間 ４゜Ｃ）までほぼ７０％の活性
が残存していた。また、至適ＰＩ■は酸性側に存在して
おりＰ Ｉｆ ７を越えるアルカリ性側では酵素活性が
ほとんど消失［る。The heat stability of the purified enzyme is 35 to 40.
It was stable at 111 °C for 30 minutes. At the same time, as a result of examining the stability of the enzyme against each PH (Fig. 6), PH4
Approximately 70% activity remained from pH 9 to pH 9 (17 hours at 4°C). Further, the optimum PI is present on the acidic side, and the enzyme activity almost disappears on the alkaline side exceeding P If 7.
阻害剤および金属イオンにだいする影響をこの酵素で調
べた。第９図に示すように金属イオンにたいしてビーク
Ｉおよびビーク■の酵素活性はほとんど影響を受けなか
った。阻害剤実験ではフェニル メチルスルフォニル
フルオリド（ＰＭＳＦ）およびトシルフルオライド（Ｔ
ＳＦ）の添加によってビークＩの凝固活性は完全に抑制
された。また、ビーク■の凝固活性はエチレンジアミン
四酢酸（Ｅ Ｄ Ｔ Ａ ）の添加により、完全に抑制
された。すなわち、ビークＩはセリンプロテアーゼの一
種と考えられ、ビーク■は金属プロテアーゼの一種と認
められる。The effects of inhibitors and metal ions on this enzyme were investigated. As shown in FIG. 9, the enzyme activities of Beak I and Beak II were hardly affected by metal ions. Phenyl methylsulfonyl in inhibitor experiments
fluoride (PMSF) and tosyl fluoride (T
The coagulation activity of Beak I was completely inhibited by the addition of SF). Furthermore, the coagulation activity of Beak (■) was completely suppressed by the addition of ethylenediaminetetraacetic acid (EDTA). That is, Beak I is considered to be a type of serine protease, and Beak ■ is recognized to be a type of metalloprotease.
また、この大豆蛋白凝固酵素の製造方法は、バチルス属
の大豆蛋白凝固酵素生産菌を培養し、大豆蛋白凝固酵素
を取得するものである。Furthermore, this method for producing soybean protein coagulase involves culturing soybean protein coagulase-producing bacteria belonging to the genus Bacillus to obtain soybean protein coagulase.
上記大豆蛋白凝固酵素生産菌の培養地としては、その組
成を下記の比率とすると大豆蛋白凝固酵素の生産に最も
適する。The culture medium for the above-mentioned soybean protein coagulase-producing bacteria is most suitable for the production of soybean protein coagulase if its composition is set to the following ratio.
酵母エキス ０．１％
カザミノ酸 ０．０２％
硫酸アンモニウム ０．１％
リン酸塩 １．０％
クエン酸ナトリウム ０．０５％
硫酸マグネシウム ０．０１％
大豆乳 ５．０９６
Ｋ ＯＨでＰＩ−１６，０に調整
（実施例）
最初に、豆乳を凝固せしめる大豆蛋白凝固酵素を分泌す
る微生物のスクリーニングを行なった。この人クリーニ
ングは主に植物および土壌より常法どおり行ない、その
結果、大豆蛋白凝固酵素を分泌する微生物を単離した。Yeast extract 0.1% Casamino acids 0.02% Ammonium sulfate 0.1% Phosphate 1.0% Sodium citrate 0.05% Magnesium sulfate 0.01% Soy milk 5.096 K PI-16,0 in OH (Example) First, microorganisms that secrete soybean protein coagulase, which coagulates soymilk, were screened. This human cleaning was carried out in a conventional manner mainly from plants and soil, and as a result, a microorganism that secreted soybean protein coagulase was isolated.
そして、この微生物を常法によりニトロングアニノン（
ＮＴに）処理を行ない、得られた大豆蛋白凝固酵素生産
菌は２６Ｄ７と命名する。Then, this microorganism was treated with nitronganinon (
NT), and the obtained soybean protein coagulase-producing strain is named 26D7.
この２６Ｄ７の菌学的性質を示す。The mycological properties of this 26D7 are shown.
形態 かん菌
芽胞形成 士
ダラム染色 十
４５° Ｃの発育 士
６５℃の発育 −
７％Ｎａ１ｌの発育 士
ｖｐ反応 十
カタラーゼ ＋
嫌気下での発Ｈ＋
運動性 十十＋
糖資化粧
グルコース ±
以上の結果より２６Ｄ７はＢａｃｉｌｌｕｓ属と認めら
れる。Morphology Bacillus spore formation Duram staining Growth at 145°C Growth at 65°C - Growth at 7% Na1L Vp reaction Ten catalase + H production under anaerobic conditions Motility Ten + Sugar supply glucose ± Results above Therefore, 26D7 is recognized as belonging to the genus Bacillus.
この結果は次に示す培地で菌株を培養したらのである。This result was obtained by culturing the bacterial strain in the following medium.
Ｂ−２培地 牛肉エキス １０ｇ ポリペプトン １０ｇ 塩化ナトリウム ５ｇ 蒸留水 １０１００Ｏ プレー１にする場合は１．５％の寒天末を加える。B-2 medium Beef extract 10g Polypeptone 10g Sodium chloride 5g Distilled water 10100O For play 1, add 1.5% agar powder.
この菌株２６Ｄ７は大豆蛋白凝固酵素生産のために培地
で培１！される。この培地としては、資化しうる炭素源
、窒素源および栄ｔＴＡ４！Ｐを含有するもので、豆乳
凝固酵素を分泌するものであれぽいかなる培地でもかま
わない。ただし、培地には豆乳（大豆固形分として１０
％）ある（・はリン酸塩を添加する必要がある。また、
その添加豆乳量も豆乳凝固酵素の分泌酵素活性１こ大き
く影響する。以下に豆乳凝固酵素の生産に最も適した培
地組成を記す。This strain 26D7 was cultured in a medium for the production of soybean protein coagulase. be done. This medium includes an assimilable carbon source, a nitrogen source, and Sakae tTA4! Any medium may be used as long as it contains P and secretes soymilk coagulation enzyme. However, the medium contains soymilk (10% as soybean solids).
%) Yes (・ means it is necessary to add phosphate. Also,
The amount of soymilk added also has a large effect on the secreted enzyme activity of soymilk coagulating enzyme. The most suitable medium composition for the production of soymilk coagulating enzyme is described below.
酵母エキス ０．１％ カザミノ酸 ０．０２％ 硫酸アンモニウム ０，１％ リン酸塩 １．ｏ９６ クエン酸ナトリウム ０．０５％ 硫酸マグネシウム ０．０１％ 大豆孔 ５・０％ Ｋ ＯＨでＰ）１６．０に、１１！整 培地に添加する豆乳量は約５％が適量である。Yeast extract 0.1% Casamino acid 0.02% Ammonium sulfate 0.1% Phosphate 1. o96 Sodium citrate 0.05% Magnesium sulfate 0.01% Soybean hole 5.0% K OH P) 16.0, 11! Adjustment The appropriate amount of soymilk to be added to the medium is approximately 5%.
それより多（でも、あるいは少なくても培地への豆乳凝
固酵素の生産量は減少する。培地中への豆乳添加量と豆
乳凝固酵素の生産量の関係は第１図に示した。Even if the amount is more (or less) than that, the amount of soymilk coagulating enzyme produced in the medium will decrease. The relationship between the amount of soymilk added to the medium and the amount of soymilk coagulating enzyme produced is shown in Figure 1.
培養は、４５°ｃ１４８時間から７２時間、振とう、通
気度はん等で好気的に）３１ ｒることができる。しか
し、大豆蛋白凝固酵素の生産量から考えるとあまり空気
供給量が多すぎても、少なすぎても良（ない０通常攪は
ん回転数２００から４０ Ｏｒｐｍ、空気供給ｌＯ，５
ＶＶＩ６から０．８ＶＶｍで行なうのが望ましい。なお
、上記の培養条件で培養した結果を第２図に示した。The culture can be carried out aerobically (with shaking, ventilation, etc.) for 148 to 72 hours at 45°C. However, considering the production amount of soybean protein coagulase, it is okay to supply too much air or too little (no, no, no, no, no, no. 0. Normal stirring speed: 200 to 40 Orpm, air supply: 1O, 5
It is desirable to perform this at 0.8VVm from VVI6. The results of culturing under the above culture conditions are shown in FIG.
培養液は、５ＳＣ（珪藻土）ろ過した後硫安分画（５５
％飽和）を行ない、親塩を行なって４■酵素画分（大豆
蛋白凝固酵素）を得ることができた。The culture solution was filtered through 5SC (diatomaceous earth) and then subjected to ammonium sulfate fraction (55
% saturation) and parent salt analysis, it was possible to obtain a 4-inch enzyme fraction (soybean protein coagulase).
この粗酵素画分をＣＭ−セルロースカラムに０（し、Ｎ
ａＣｌ溶液の直ｍｅ度勾配により２つの大豆蛋白凝固＾
Ｙ素活性を持つ７ラクシヨンを得た。This crude enzyme fraction was applied to a CM-cellulose column with 0
Coagulation of two soybean proteins by a linear gradient of aCl solution
A 7-lactone with Y-element activity was obtained.
第３図にその溶出パターンを示した。ビークＩとビーク
■が大豆蛋白凝固活性の存在するビークである。さらに
このビークをゲルろ過に供して、大豆蛋白凝固酵素を単
離、精製した。第４図にその溶出パターンを示した。Figure 3 shows the elution pattern. Beak I and beak ■ are beaks in which soybean protein coagulation activity exists. Furthermore, this beak was subjected to gel filtration to isolate and purify soybean protein coagulase. Figure 4 shows the elution pattern.
次に、本発明の大豆蛋白凝固酵素の理化学的性′Ｌ１を
示す。Next, the physicochemical properties 'L1 of the soybean protein coagulase of the present invention are shown.
得られた大豆蛋白凝固酵素（ビークｌ）はＳＤ３７１１
気泳動的にＩｉ−なハンドとなり、分子−ハは３ｏｏｏ
ｏ曲後であった。次にこの酵素の至適温度および温度安
定性を調べた。第５図に示したように、ビークＩの活性
至適温度は約８０゜Ｃであった。また、ビーク■（粗酵
素）の活性至ｊ轟γ是度は約６５℃であった。精製され
た酵素の熱にたいする安定性は３５ないし４０℃で３０
分間安定であった。同時に各ｐＨにたいする酵素の安定
性を調べた結果（第６図）、４℃でＰＨ４からＰＨ９（
１７時間）までほぼ７０９６の活性が残存していた。ま
た、至適ＰＨは酸推測に存在しておりＰ Ｉ−１７を越
えるアルカリ性側では酵素活性がほとんど消失する。The obtained soybean protein coagulase (beak l) was SD3711
Aerophoretically it becomes an Ii- hand, and the molecule-Ha is 3ooo
It was after the o song. Next, we investigated the optimal temperature and temperature stability of this enzyme. As shown in FIG. 5, the optimum temperature for activation of Beak I was about 80°C. In addition, the activity level of Beak (crude enzyme) was approximately 65°C. The thermal stability of the purified enzyme is 30°C at 35-40°C.
It was stable for minutes. At the same time, we investigated the stability of the enzyme at various pH values (Figure 6), and found that at 4°C, pH 4 to 9 (
Approximately 7096 activity remained until 17 hours). Furthermore, the optimum pH is assumed to be acidic, and enzyme activity almost disappears at alkaline levels exceeding PI-17.
阻害剤および金属イオンにたいｒる影響をこの酵素で調
べた。第９図に示すように金属イオンにたいしてビーク
Ｉおよびビーク■の酵素活性はほとんど影響を受けなか
った。阻害剤実験ではフェニル メチルスル７オニルフ
ルオリドおよびトシルフルオライド（ＴＳＦ）の添加に
よってビーク■の凝固活性は完全に抑制された。The effects of inhibitors and metal ions on this enzyme were investigated. As shown in FIG. 9, the enzyme activities of Beak I and Beak II were hardly affected by metal ions. In inhibitor experiments, the coagulation activity of Beak ■ was completely inhibited by the addition of phenylmethylsulfonyl fluoride and tosyl fluoride (TSF).
また、ビーク■の凝固活性はエチレンジアミン四酢酸（
ＥＤＴΔ）の添加により完全に抑制された。すなわち、
ビーク■はセリンプロテアーゼの一種と考えられ、ビー
ク■は金属プロテアーゼの一種と認められる。In addition, the coagulation activity of Beak ■ is ethylenediaminetetraacetic acid (
It was completely inhibited by the addition of EDTΔ). That is,
Beak ■ is considered to be a type of serine protease, and Beak ■ is recognized as a type of metalloprotease.
（発明の効果）
本発明によれば純植物性蛋白質たる大豆乳を凝固させる
大豆蛋白２疑固酵素を得ることができるとともに、この
酵素を効率的に生産することができる。(Effects of the Invention) According to the present invention, it is possible to obtain a soybean protein 2 pseudocoagulase that coagulates soybean milk, which is a pure vegetable protein, and to efficiently produce this enzyme.
！＠１（７Ｉは本発明にかかる大豆蛋白凝固酵素を生産
するための培地における豆乳添加量と大豆蛋白凝固酵素
活性との関係図、第２図は２６Ｄ７を培養した場合の凝
固活性、プロテアーゼ活性、生産置数を示す図、第３図
は大Ｆ２蛋白凝固酵素を単離精製した際の溶出パターン
を示すＩＩＩ係図、第４図は大豆蛋白凝固酵素をデルろ
過に供して単離精製した際の溶出パターンを示す関係図
、第５図（Ａ）は大豆蛋白凝固酵素の至適温度、（［３
）は熱安定性を示す関係図、第６図（Ａ）は大豆蛋白凝
固酵素のＰト■に対する安定性を示し、（Ｂ）は至適Ｐ
Ｈを示す関係図、第７図は大Ｆ２蛋白凝固酵素のビーク
Ｉのプロテアーゼ活性とＰ Ｈとの関係図、第８図は第
７図と同時にビーク【のプロテアーゼ活性と温度との関
係図、！ｆＳ！３図は大豆蛋白５迂固醇素のビーク■、
ビーク■と金属塩及び阻害剤の影響を水ｒ関係図である
。
１ｑ；１出ゑ１へ 体−’Ｓ ３．： ｔＬ −
＋し海道［］清第１図
一閂（Ｊｔ）
第３図
フラ′：）Σ−ＩＬ！Ｏ。
第４図
フラグ昂；仔０９
（Ｐ）Ｉ］ ［
Ｐ用４ ５ ６ ７ ８ ９ ｉｏ １！
：５４５ ５５ ６５ ７５第９図
Ｃｈｅｍｉｃａｌｓ Ｃｏｎｃ、（Ｍ）
Ｒｅ１ａｔｉｖｅ ａｃｔｉｖｉｔｙ （：）（Ｉ）
（ＩＩ）
ＺｎＳＯ４７）１２０ １ ｘ １０−３１００
ＬＯＯＣｕＳＯ４５Ｂ２０ １００
９３．３ＭｇＣ１２６）１２０
９４．３ ９６．４Ｍｎ５Ｏ４４）＋２０
１００ １００ＣａＣ１２２８２０９
１，６１００
ＢａＣ１２２Ｈ２０コ００ ）ＯＯＦｅ
ＳＯ４７）！２０ ：つ０
９６．２ＬｉＯＨ）１２０ ＥＯ
＋００ＨｇＣ１２１００９０，２! @1 (7I is a diagram of the relationship between the amount of soybean milk added and soybean protein coagulase activity in the medium for producing the soybean protein coagulase according to the present invention, and Figure 2 is a diagram showing the coagulation activity, protease activity, and protease activity when culturing 26D7. Figure 3 is a diagram showing the production number, Figure 3 is a III diagram showing the elution pattern when large F2 protein coagulase was isolated and purified, and Figure 4 is a diagram showing the elution pattern when soybean protein coagulase was isolated and purified by Delfiltration. Figure 5 (A) is a relationship diagram showing the elution pattern of soybean protein coagulase, ([3
) is a relationship diagram showing thermal stability, Figure 6 (A) shows the stability of soybean protein coagulase against P and (B) shows the optimum P
Figure 7 is a diagram showing the relationship between the protease activity of beak I of the large F2 protein coagulase and PH. ! fS! Figure 3 shows the beak of soybean protein 5-diluted soybean.
It is a water r relationship diagram showing the influence of beak (■), metal salts, and inhibitors. 1q; 1 out 1 to body-'S 3. :tL-
+Shi Kaido [] Qing 1st figure one bolt (Jt) 3rd figure Fra':) Σ-IL! O. Fig. 4 flag 09 (P)I] [
For P 4 5 6 7 8 9 io 1!
:545 55 65 75Figure 9 Chemicals Conc, (M)
Reactive activity (:) (I)
(II) ZnSO47) 120 1 x 10-3100
LOOCuSO45B20 100
93.3MgC126)120
94.3 96.4Mn5O44)+20
100 100CaC1228209
1,6100 BaC122H20ko00)OOFe
SO47)! 20: 0
96.2LiOH)120EO
+00HgC1210090,2
Claims (3)
品素材に適している。チーズ様、クリーム様若しくはヨ
ーグルト様の食品ができる。 ［３］ＳＤＳ電気泳動的に単一なバンドとなり、分子量
は３００００前後となる。 ［４］活性至適温度は約８０℃である。 ［５］熱安定性は３５℃ないし４０℃で３０分間安定で
ある。 ［６］４℃、１７時間処理でＰＨ４からＰＨ９までは７
０％の活性が残存する。 ［７］至適ＰＨは酸性側に存在しており、ＰＨ７を越え
るアルカリ性側では活性はほとんど消失する。 ［８］本酵素にはフェニルメチルスルフォニルフルオリ
ド（ＰＭＳＦ）およびトシルフルオライド（ＴＳＦ）の
添加により、凝固活性は完全に抑制される。本酵素はセ
リンプロテアーゼの一種を含む。 ［９］金属イオンに対して酵素活性はほとんど影響を受
けない。 ［１０］本酵素はその活性至適温度が約６５℃、金属イ
オンに対して酵素活性はほとんど影響を受けなく、阻害
剤に対して凝固活性がエチレンジアミン四酢酸（ＥＤＴ
Ａ）の添加により完全に抑制される金属プロテアーゼの
一種をわずかに含む。(1) A soybean protein coagulase having the following physical and chemical properties. [1] When it acts on soy milk, it coagulates soy protein. [2] Soy milk curd has no bitter taste and has a smooth mouthfeel, making it suitable as a food material. Cheese-like, cream-like or yogurt-like foods can be produced. [3] A single band appears in SDS electrophoresis, and the molecular weight is around 30,000. [4] The optimum temperature for activation is about 80°C. [5] Thermal stability is stable at 35°C to 40°C for 30 minutes. [6] PH4 to PH9 is 7 after treatment at 4℃ for 17 hours
0% activity remains. [7] The optimum pH exists on the acidic side, and activity almost disappears on the alkaline side exceeding PH7. [8] Coagulation activity is completely suppressed by adding phenylmethylsulfonyl fluoride (PMSF) and tosyl fluoride (TSF) to this enzyme. This enzyme includes a type of serine protease. [9] Enzyme activity is hardly affected by metal ions. [10] The optimum temperature for this enzyme's activity is approximately 65°C, the enzyme activity is almost unaffected by metal ions, and the coagulation activity is less affected by inhibitors than ethylenediaminetetraacetic acid (EDT).
Contains a small amount of metalloprotease, which is completely inhibited by the addition of A).
大豆蛋白凝固酵素を取得することを特徴とする大豆蛋白
凝固酵素の製造方法。(2) Cultivating soybean protein coagulase-producing bacteria of the genus Bacillus,
A method for producing soybean protein coagulase, which comprises obtaining soybean protein coagulase.
許請求の範囲第２項記載の大豆蛋白凝固酵素の製造方法
。 酵母エキス０．１％ カザミノ酸０．０２％ 硫酸アンモニウム０．１％ リン酸塩１．０％ クエン酸ナトリウム０．０５％ 硫酸マグネシウム０．０１％ 大豆乳５．０％ （ＫＯＨでＰＨ６．０に調整）(3) The method for producing a soybean protein coagulase according to claim 2, characterized in that the composition of the culture is as follows. Yeast extract 0.1% Casamino acids 0.02% Ammonium sulfate 0.1% Phosphate 1.0% Sodium citrate 0.05% Magnesium sulfate 0.01% Soy milk 5.0% (pH 6.0 with KOH) adjustment)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7198188A JPH01247085A (en) | 1988-03-28 | 1988-03-28 | Soybean protein coagulation enzyme and production thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7198188A JPH01247085A (en) | 1988-03-28 | 1988-03-28 | Soybean protein coagulation enzyme and production thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH01247085A true JPH01247085A (en) | 1989-10-02 |
Family
ID=13476149
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP7198188A Pending JPH01247085A (en) | 1988-03-28 | 1988-03-28 | Soybean protein coagulation enzyme and production thereof |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01247085A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61282074A (en) * | 1985-06-07 | 1986-12-12 | Kibun Kk | Novel protein coagulating enzyme and production thereof |
-
1988
- 1988-03-28 JP JP7198188A patent/JPH01247085A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61282074A (en) * | 1985-06-07 | 1986-12-12 | Kibun Kk | Novel protein coagulating enzyme and production thereof |
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