JP2008072985A - Raw starch-degrading enzyme produced by eisenia fetida - Google Patents
Raw starch-degrading enzyme produced by eisenia fetida Download PDFInfo
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- DBTMGCOVALSLOR-DEVYUCJPSA-N (2s,3r,4s,5r,6r)-4-[(2s,3r,4s,5r,6r)-3,5-dihydroxy-6-(hydroxymethyl)-4-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]oxy-6-(hydroxymethyl)oxane-2,3,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](CO)O[C@H](O)[C@@H]2O)O)O[C@H](CO)[C@H]1O DBTMGCOVALSLOR-DEVYUCJPSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 1
- 240000006439 Aspergillus oryzae Species 0.000 description 1
- 241000230129 Eisenia <Phaeophyceae> Species 0.000 description 1
- KIWBPDUYBMNFTB-UHFFFAOYSA-N Ethyl hydrogen sulfate Chemical compound CCOS(O)(=O)=O KIWBPDUYBMNFTB-UHFFFAOYSA-N 0.000 description 1
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- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 102000013566 Plasminogen Human genes 0.000 description 1
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- 108010022999 Serine Proteases Proteins 0.000 description 1
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 125000000738 acetamido group Chemical group [H]C([H])([H])C(=O)N([H])[*] 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
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- PGLTVOMIXTUURA-UHFFFAOYSA-N iodoacetamide Chemical compound NC(=O)CI PGLTVOMIXTUURA-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Landscapes
- Processing Of Solid Wastes (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
本発明は一般的に生デンプン分解酵素に関する。より詳しくは、本発明はミミズの生デンプン分解酵素に関する。 The present invention relates generally to raw starch degrading enzymes. More particularly, the present invention relates to earthworm raw starch degrading enzymes.
ミミズは環形動物門貧毛目の生物で、地球上で3000〜7000種存在すると言われている。中国において、ミミズの乾燥粉末は、「地竜」と言う名で伝統的な漢方薬として知られており、解熱剤や利尿薬として長きに渡って用いられている。また、近年アカミミズ(Lumbrics rubellus)の凍結乾燥粉末から、血栓分解酵素が単離され、日本や韓国で研究が行われている(非特許文献1〜非特許文献3)。また、ミミズは重金属イオンを腸内に溜め込む習性があり、この習性を利用して汚染土壌のバイオマーカーとしても用いられている(非特許文献4、非特許文献5)。一方、日本の食品廃棄物は食品産業全体で1131万トン(2002年)と世界の食糧援助総量に匹敵するところまできている。また、環境省の2000年度の推計では、一般家庭からの食品廃棄物も1200万トンに上り、2001年に食品循環資源の再生利用等の促進に関する法律(食品リサイクル法)が施行され、食品廃棄物の発生抑制と再生利用などを促進することが義務づけられ、その効果的処理方法が模索されている。その方法の一つに、食品廃棄物を未利用資源としてとらえたバイオコンバージョンによるコンポスト化がある。 Earthworms are annelids of the annelid and are said to exist on the earth in 3000 to 7000 species. In China, the dry powder of earthworm is known as a traditional Chinese herb medicine under the name of “jiryu” and has long been used as an antipyretic and diuretic. In recent years, thrombolytic enzymes have been isolated from lyophilized powder of red earthworms (Lumbrics rubellus), and research is being conducted in Japan and Korea (Non-patent Documents 1 to 3). In addition, earthworms have the habit of accumulating heavy metal ions in the intestine, and are also used as biomarkers for contaminated soil using this habit (Non-patent Documents 4 and 5). On the other hand, Japan's food waste is 11.31 million tons (2002) in the food industry as a whole, comparable to the total amount of food aid in the world. In addition, the Ministry of the Environment estimates that the amount of food waste from households has reached 12 million tons. In 2001, the Law Concerning the Promotion of Recycling of Food Recycling Resources (Food Recycling Law) was enacted, and food waste It is obliged to promote the prevention and recycling of waste, and an effective treatment method is being sought. One of the methods is composting by bioconversion that treats food waste as an unused resource.
日本では微生物を用いたコンポスト化が一般的であるが、欧米諸国では実際に一般家庭でも有機ゴミを処理するのにミミズが用いられており、摂取された有機ゴミは腸内酵素と腸内微生物群によって分解され、非常に栄養価の高い糞を排泄する事が知られている(非特許文献6)。これは土壌改良剤として使用されている。コンポスト化に用いられるミミズは、ごく少数であり、中でもシマミミズ(Eisenia fetida)が最もよく用いられている(非特許文献7、非特許文献8)。しかしながら、シマミミズのどの腸内酵素、どの腸内微生物群のどの酵素がコンポスト化に寄与しているかについては充分に明らかにされているとは言えない。
そこで本研究では、コンポスト化において重要な働きをするであろうシマミミズ由来の加水分解酵素群のスクリーニングを行い、特に食品廃棄物処理に有用と考えられる、これまでミミズが生産することが知られていなかった生デンプン分解酵素をシマミミズから単離精製し、その性質を明らかにすることを目的とした。 Therefore, in this study, we screened the hydrolytic enzymes from the earthworm that will play an important role in composting, and it is known that earthworms have been produced so far, which is considered to be particularly useful for food waste treatment. The purpose of this study was to isolate and purify the raw starch-degrading enzyme from the earthworm and clarify its properties.
本発明は、従来知られていなかった、シマミミズが生産する生デンプン分解酵素を提供する。 The present invention provides a raw starch-degrading enzyme produced by an earthworm that has not been conventionally known.
本発明の生デンプン分解酵素Iは、一晩絶食させたシマミミズを凍結乾燥し、乳鉢で粉砕して得た粉末状シマミミズを50mMトリス塩酸(pH7.0)に懸濁し、遠心分離(15,000rpm、20分、4℃)した後上清を回収し、上清にプロテアーゼ阻害剤を加えて超遠心分離(100,000G、30分、4℃)した後上清を回収し、この上清に35%飽和になるよう硫酸アンモニウムを添加し、4℃で一晩保存し、続いて遠心分離(15,000rpm、20分、4℃)した後沈殿を回収し、得られた沈殿を少量の50mMトリス塩酸(pH7.0)に溶解して透析を行った。透析膜は透析用セルロースチューブ(三光純薬株式会社)、外液は20mMトリス塩酸(pH7.0)を用いた。その透析内液を、デンプン又は生デンプンに対する分解活性を指標とし、DEAE-TOYOPEARL 650Mを用いる陰イオン交換クロマトグラフィーにかけて先に溶出する活性画分を分取し、この画分を集めて次にSephacryl S-200を用いるゲルろ過クロマトグラフィーにかけて活性画分を分取し、この画分をさらにBUTYL-TOYOPEARL 650Sを用いる疎水クロマトグラフィーにかけて活性画分を分取することにより、生デンプン分解酵素活性画分として得られる。 The raw starch-degrading enzyme I of the present invention was prepared by lyophilizing an overnight fasted earthworm, pulverizing it in a mortar, suspending it in 50 mM Tris-HCl (pH 7.0), and centrifuging (15,000 rpm, The supernatant was collected after 20 minutes at 4 ° C., protease inhibitor was added to the supernatant, ultracentrifugation (100,000 G, 30 minutes, 4 ° C.) and the supernatant was collected, and 35% Ammonium sulfate was added to saturation and stored overnight at 4 ° C., followed by centrifugation (15,000 rpm, 20 minutes, 4 ° C.), and then the precipitate was recovered. The resulting precipitate was added to a small amount of 50 mM Tris-HCl (pH 7 0) and dialyzed. The dialysis membrane was a cellulose tube for dialysis (Sanko Junyaku Co., Ltd.), and the external solution was 20 mM Tris-HCl (pH 7.0). The dialysis internal solution is subjected to anion exchange chromatography using DEAE-TOYOPEARL 650M with the degradation activity on starch or raw starch as an index, and the active fraction eluting first is collected, and this fraction is collected and then separated by Sephacryl. The active fraction was separated by gel filtration chromatography using S-200, and this fraction was further subjected to hydrophobic chromatography using BUTYL-TOYOPEARL 650S to fractionate the active amylolytic enzyme active fraction. As obtained.
また、本発明の生デンプン分解酵素IIは、一晩絶食させたシマミミズを凍結乾燥し、乳鉢で粉砕して得た粉末状シマミミズを50mMトリス塩酸(pH7.0)に懸濁し、遠心分離(15,000rpm、20分、4℃)した後、上清を回収し、上清にプロテアーゼ阻害剤を加え超遠心分離(100,000G、30分、4℃)した後上清を回収し、この上清に35%飽和になるよう硫酸アンモニウムを添加し、4℃で一晩保存し、続いて遠心分離(15,000rpm、20分、4℃)した後沈殿を回収し、得られた沈殿を少量の50mMトリス塩酸(pH7.0)に溶解して透析を行った。透析膜は透析用セルロースチューブ(三光純薬株式会社)、外液は20mMトリス塩酸(pH7.0)を用いた。その透析内液を、デンプン又は生デンプンに対する分解活性を指標とし、DEAE-TOYOPEARL 650Mを用いる陰イオン交換クロマトグラフィーにかけて後に溶出する活性画分を分取し、この画分を集めて次にSephacryl S-200を用いるゲルろ過クロマトグラフィーにかけて活性画分を分取し、この画分をさらにBUTYL-TOYOPEARL 650Sを用いる疎水クロマトグラフィーにかけて活性画分を分取することにより、生デンプン分解酵素活性画分として得られる。 In addition, the raw starch degrading enzyme II of the present invention was obtained by suspending a ground earthworm fasted overnight and freeze-drying and grinding it in a mortar in 50 mM Tris-HCl (pH 7.0) and centrifuging (15,000 (rpm, 20 minutes, 4 ° C), and the supernatant is collected. After adding a protease inhibitor to the supernatant and ultracentrifugating (100,000G, 30 minutes, 4 ° C), the supernatant is collected. Add ammonium sulfate to 35% saturation, store at 4 ° C overnight, and then centrifuge (15,000 rpm, 20 minutes, 4 ° C) to collect the precipitate, and collect the resulting precipitate in a small amount of 50 mM Tris-HCl. It was dissolved in (pH 7.0) and dialyzed. The dialysis membrane was a cellulose tube for dialysis (Sanko Junyaku Co., Ltd.), and the external solution was 20 mM Tris-HCl (pH 7.0). The dialysis internal solution is subjected to anion exchange chromatography using DEAE-TOYOPEARL 650M with the degradation activity on starch or raw starch as an index, and an active fraction that elutes later is collected. This fraction is collected and then separated by Sephacryl S The active fraction was separated by gel filtration chromatography using -200, and this fraction was further subjected to hydrophobic chromatography using BUTYL-TOYOPEARL 650S to obtain the active amylolytic enzyme active fraction. can get.
本発明の生デンプン分解酵素I及びIIは、その生デンプン分解様式からいずれもエンド型のα-アミラーゼである。 The raw starch degrading enzymes I and II of the present invention are both endo-type α-amylases because of their raw starch degrading mode.
本発明のデンプン分解酵素I及びIIの最適作用温度はいずれも約50℃であったが、30分の熱処理においては本発明のデンプン分解酵素Iは10〜60℃で安定であり、本発明のデンプン分解酵素IIは10〜50℃である。 The optimum working temperature of the amylolytic enzymes I and II of the present invention was both about 50 ° C., but the amylolytic enzyme I of the present invention is stable at 10 to 60 ° C. in the heat treatment for 30 minutes. Starch degrading enzyme II is 10-50 ° C.
本発明のデンプン分解酵素I及びIIの最適作用pHはいずれも5.5であり、4℃で24時間処理後の本発明のデンプン分解酵素IのpH安定性は7〜9であり、本発明のデンプン分解酵素IIのpH安定性は7〜8である。 The optimum action pH of the amylolytic enzymes I and II of the present invention is 5.5, and the pH stability of the amylolytic enzyme I of the present invention after treatment at 4 ° C. for 24 hours is 7 to 9, and the starch of the present invention The pH stability of the degrading enzyme II is 7-8.
本発明の生デンプン分解酵素Iは、4℃でのアミラーゼ活性が37℃のアミラーゼ活性の約40%を保持し、好ましくは37%の活性を保持する。 The raw starch degrading enzyme I of the present invention has an amylase activity at 4 ° C. of about 40% of the amylase activity at 37 ° C., preferably 37%.
本発明の生デンプン分解酵素IIは、4℃でのアミラーゼ活性が37℃のアミラーゼ活性の約25%を保持し、好ましくは21%の活性を保持する。 The raw starch degrading enzyme II of the present invention has an amylase activity at 4 ° C. that retains about 25% of the amylase activity at 37 ° C., and preferably 21%.
本発明はまた、前記の生デンプン分解酵素によりデンプンを糖化し、糖化物中で酵母を培養することを特徴とするアルコール発酵方法を提供する。前記デンプンは例えば生デンプン又は老化デンプンである。 The present invention also provides an alcoholic fermentation method characterized in that starch is saccharified with the raw starch degrading enzyme and yeast is cultured in the saccharified product. The starch is, for example, raw starch or aged starch.
以下、実施例により本発明をさらに詳しく説明するが、本発明はこれらの実施例等によりなんら限定されるものではない。本発明で使用した主材料及び酵素活性測定法は以下の通りである。 EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited at all by these Examples. The main materials and enzyme activity measurement methods used in the present invention are as follows.
1.実験材料
シマミミズ(Eisenia fetida)は、神奈川県伊勢原市三ノ宮116に住所を有する、相模浄化サービス(有)等から購入した。
1. The experimental material earthworm (Eisenia fetida) was purchased from Sagami Purification Service Co., Ltd., which has an address in 116 Ishinohara, Kanagawa Prefecture.
2.還元糖の測定法(ソモギーネルソン法(参考文献9))
試料、対照共に0.4%各基質を100μl、緩衝液を100μlずつ入れて37℃で5分プレインキュベートし、その後、試料にのみ酵素液を20μl加え、15分反応させた。反応後、試料、対照共にソモギー試薬を200μlずつ、対照にのみ酵素液を20μl加えた後、100℃で10分煮沸し反応を停止させた。試料、対照共に10分間水冷した後、ネルソン試薬を200μlずつ加え、20分間放置し発色させた。20分後、蒸留水2.0mlを加えてよく混和し、655nmの波長で吸光度を測定した。酵素活性はソモギーネルソンの検量線から得られた式を用いて求めた。なお、1単位は1分間に1μmolのグルコースに相当する還元糖を遊離する酵素量とした。基質には可溶化馬鈴薯デンプン、生馬鈴薯デンプン、生米デンプン、CMC(カルボキシルメチルセルロース)、キシラン、ラミナリンを用いた。
2. Measuring method of reducing sugar (Somogy Nelson method (Reference 9))
100 μl of 0.4% each substrate and 100 μl of buffer solution were added to each sample and control, and preincubated at 37 ° C. for 5 minutes. Then, 20 μl of enzyme solution was added only to the sample and reacted for 15 minutes. After the reaction, 200 μl of the somogenic reagent was added to each of the sample and the control, and 20 μl of the enzyme solution was added only to the control, followed by boiling at 100 ° C. for 10 minutes to stop the reaction. Both the sample and the control were water-cooled for 10 minutes, and then 200 μl of Nelson reagent was added and allowed to stand for 20 minutes for color development. After 20 minutes, 2.0 ml of distilled water was added and mixed well, and the absorbance was measured at a wavelength of 655 nm. Enzyme activity was determined using the formula obtained from the Somogy Nelson calibration curve. One unit is the amount of enzyme that releases reducing sugar corresponding to 1 μmol of glucose per minute. Solubilized potato starch, raw potato starch, raw rice starch, CMC (carboxyl methylcellulose), xylan, and laminarin were used as substrates.
3.アミラーゼ活性測定法(ヨウ素デンプン反応(参考文献10))
試料、対照共に0.4%デンプン溶液150μl、0.1Mトリス塩酸緩衝液(pH7.0)を220μl、0.2M CaCl2溶液を30μlずつ入れた。試料にのみ粗酵素液を200μl加え、37℃で15分反応させた。その後、試料、対照共に1.5N酢酸溶液を200μlずつ入れ、反応を停止させた。対照にのみ粗酵素液を200μl加えた後、試料、対照共にヨウ素ヨウ化カリウム溶液を200μl加えて発色させ、20分放置した。その後、蒸留水3.4mlを加えてよく混和し、690nmの波長で吸光度を測定した。このときの対照値-試料値をアミラーゼ活性とする。なお、1単位は、澱粉を基質として、1分間でA690を0.01低下させる酵素量と定義した。
3. Amylase activity measurement method (iodine starch reaction (reference document 10))
For both the sample and control, 150 μl of 0.4% starch solution, 220 μl of 0.1 M Tris-HCl buffer (pH 7.0), and 30 μl of 0.2 M CaCl 2 solution were added. Only 200 μl of the crude enzyme solution was added to the sample and reacted at 37 ° C. for 15 minutes. Thereafter, 200 μl of 1.5N acetic acid solution was added to each of the sample and control to stop the reaction. After adding 200 μl of the crude enzyme solution only to the control, 200 μl of potassium iodide iodide solution was added to both the sample and the control to develop a color, and the mixture was allowed to stand for 20 minutes. Thereafter, 3.4 ml of distilled water was added and mixed well, and the absorbance was measured at a wavelength of 690 nm. The control value-sample value at this time is defined as amylase activity. One unit was defined as the amount of enzyme that reduces A690 by 0.01 per minute using starch as a substrate.
4.タンパク質の定量法
精製過程におけるタンパク質は、ベックマンDU7400を用い、280nmの吸光度を測定した。また、ウシ血清アルブミンを標準タンパク質としてブラッドフォード法(参考文献12)により測定した。
4). Protein quantification method Proteins in the purification process were measured for absorbance at 280 nm using Beckman DU7400. Moreover, it measured by the Bradford method (reference document 12) using bovine serum albumin as a standard protein.
生デンプン分解活性の測定法(参考文献9)
試料、対照共に0.4%生デンプン懸濁液を100μl、緩衝液を100μlずつ入れて37℃で5分プレインキュベートし、その後試料にのみ酵素液を20μl加え、15分反応させた。反応後、試料、対照共にソモギー試薬を200μlずつ、対照にのみ酵素液を20μl加えた後、100℃で10分間煮沸し反応を停止させた。試料、対照共に10分間水冷した後ネルソン試薬を200μlずつ加え、20分間放置し発色させた。20分後、蒸留水2.0mlを加えてよく混和し、2500rpmで5分遠心分離を行った後、655nmの波長で吸光度を測定した。酵素活性はソモギーネルソンの検量線から得られた式を用いて求めた。なお、1単位は1分間に1μmolのグルコースに相当する還元糖を遊離する酵素量とした。
Method for measuring raw starch degrading activity (Reference 9)
100 μl of 0.4% raw starch suspension and 100 μl of buffer solution were added to each sample and control, and preincubated at 37 ° C. for 5 minutes. Then, 20 μl of enzyme solution was added to the sample and reacted for 15 minutes. After the reaction, 200 μl of the somogenic reagent was added to each of the sample and the control, and 20 μl of the enzyme solution was added only to the control, followed by boiling at 100 ° C. for 10 minutes to stop the reaction. Both the sample and the control were water-cooled for 10 minutes, and then 200 μl of Nelson reagent was added and allowed to stand for 20 minutes for color development. After 20 minutes, 2.0 ml of distilled water was added and mixed well. After centrifugation at 2500 rpm for 5 minutes, the absorbance was measured at a wavelength of 655 nm. Enzyme activity was determined using the formula obtained from the Somogy Nelson calibration curve. One unit is the amount of enzyme that releases reducing sugar corresponding to 1 μmol of glucose per minute.
シマミミズからの生デンプン分解酵素の単離
一晩絶食させたシマミミズ(Eisenia fetida)を凍結乾燥した後、10gを乳鉢で破砕した。粉末状にしたミミズを50mMトリス塩酸(pH7.0)に懸濁し、遠心分離(15,000rpm、20分、4℃)した後上清を回収した。上清にプロテアーゼ阻害剤カクテル(ナカライテクス)を2%(v/v)になるように加えた。そして、超遠心(100,000G、30分、4℃)後、上清を回収した。上清に35%飽和になるよう硫酸アンモニウムを添加し、4℃で一晩保存した。続いて遠心分離(15,000rpm、20分、4℃)し、沈殿を回収した。回収した沈殿を少量の50mMトリス塩酸(pH7.0)に溶解して得られた酵素液を透析した。透析膜は透析用セルロースチューブ(三光純薬株式会社)、外液は20mMトリス塩酸(pH7.0)を用いた。その透析内液を、陰イオン交換クロマトグラフィー(DEAE-TOYOPEARL 650M)、ゲルろ過クロマトグラフィー(Sephacryl S-200)、疎水クロマトグラフィー(BUTYL-TOYOPEARL 650S)の順で精製を行った(図1)。
Isolation of Raw Starch Degrading Enzyme from Earthworm Earthworm (Eisenia fetida) fasted overnight was freeze-dried, and 10 g was crushed in a mortar. The powdered earthworm was suspended in 50 mM Tris-HCl (pH 7.0), centrifuged (15,000 rpm, 20 minutes, 4 ° C.), and the supernatant was collected. To the supernatant, a protease inhibitor cocktail (Nacalai tex) was added to 2% (v / v). Then, after ultracentrifugation (100,000 G, 30 minutes, 4 ° C.), the supernatant was recovered. Ammonium sulfate was added to the supernatant to 35% saturation and stored overnight at 4 ° C. Subsequently, the precipitate was collected by centrifugation (15,000 rpm, 20 minutes, 4 ° C.). The enzyme solution obtained by dissolving the collected precipitate in a small amount of 50 mM Tris-HCl (pH 7.0) was dialyzed. The dialysis membrane was a cellulose tube for dialysis (Sanko Junyaku Co., Ltd.), and the external solution was 20 mM Tris-HCl (pH 7.0). The dialysis internal solution was purified in the order of anion exchange chromatography (DEAE-TOYOPEARL 650M), gel filtration chromatography (Sephacryl S-200), and hydrophobic chromatography (BUTYL-TOYOPEARL 650S) (FIG. 1).
結果:イオン交換クロマトグラフィー
シマミミズ乾燥重量10gから得られた粗酵素液を超遠心により、不溶性の物質を取り除いた後、35%硫安塩析により、目的とするアミラーゼのほとんどは沈殿した。この沈殿を少量の20mMトリス塩酸(pH7.0)に溶解したものを透析した。透析膜は透析用セルロースチューブ(三光純薬株式会社)、外液は20mMトリス塩酸(pH7.0)を用いた。その透析内液を、DEAE-TOYOPEARL 650Mを用いる陰イオン交換クロマトグラフィーにかけた。透析内液を20mMトリス塩酸(pH7.0)で平衡化したDEAE TOYOPEARL 650M(2.5×21cm、103ml)に負荷し、同緩衝液で洗浄後、0から0.5Mの濃度勾配をつけたNaClを含む同緩衝液で溶出を行った。その結果は図2に示すように、生デンプン分解活性のピークが2箇所見られた。このうち画分No.54〜61を生デンプン分解酵素I、画分No.66〜73を生デンプン分解酵素IIとして回収した。
Results: The crude enzyme solution obtained from 10 g of dry weight of ion-exchange chromatography castor earthworm was subjected to ultracentrifugation to remove insoluble substances, and most of the target amylase was precipitated by 35% ammonium sulfate salting out. The precipitate dissolved in a small amount of 20 mM Tris-HCl (pH 7.0) was dialyzed. The dialysis membrane was a cellulose tube for dialysis (Sanko Junyaku Co., Ltd.), and the external solution was 20 mM Tris-HCl (pH 7.0). The dialyzed internal solution was subjected to anion exchange chromatography using DEAE-TOYOPEARL 650M. Load the dialyzed internal solution into DEAE TOYOPEARL 650M (2.5 × 21cm, 103ml) equilibrated with 20mM Tris-HCl (pH7.0), and after washing with the same buffer, it contains NaCl with a concentration gradient of 0 to 0.5M. Elution was performed with the same buffer. As a result, as shown in FIG. 2, two peaks of raw starch degrading activity were observed. Of these, fractions Nos. 54 to 61 were recovered as raw starch degrading enzyme I, and fractions Nos. 66 to 73 were recovered as raw starch degrading enzyme II.
結果:ゲルろ過クロマトグラフィー
次いで、得られた生デンプン分解酵素Iの画分、及び生デンプン分解酵素IIの画分に、それぞれ80%飽和になるように硫酸アンモニウムを添加し、4℃で一晩放置した。続いて遠心分離(15,000rpm、20分、4℃)で得られた沈殿を8mlの20mMトリス塩酸(pH7.0)に溶解し、透析を行った。透析膜は透析用セルロースチューブ(三光純薬株式会社)、外液は20mMトリス塩酸(pH7.0)を用いた。その透析内液を、0.5M NaClを含む20mMトリス塩酸(pH7.0)で平衡化したSephacryl S-200(2.5×90cm、442ml)に負荷し、同緩衝液で溶出を行った。Sephacryl S-200を用いたゲルろ過クロマトグラフィーにより、図3及び図4に示すような結果を得た。このうち生デンプン分解酵素Iでは画分No.86〜100を、生デンプン分解酵素IIでは画分No.64〜80を活性画分として回収した。
Result: Gel filtration chromatography Then, ammonium sulfate was added to each of the obtained raw starch degrading enzyme I fraction and raw starch degrading enzyme II fraction so as to be 80% saturated, and left overnight at 4 ° C. did. Subsequently, the precipitate obtained by centrifugation (15,000 rpm, 20 minutes, 4 ° C.) was dissolved in 8 ml of 20 mM Tris-HCl (pH 7.0) and dialyzed. The dialysis membrane was a cellulose tube for dialysis (Sanko Junyaku Co., Ltd.), and the external solution was 20 mM Tris-HCl (pH 7.0). The dialyzed internal solution was loaded on Sephacryl S-200 (2.5 × 90 cm, 442 ml) equilibrated with 20 mM Tris-HCl (pH 7.0) containing 0.5 M NaCl, and eluted with the same buffer. The results as shown in FIGS. 3 and 4 were obtained by gel filtration chromatography using Sephacryl S-200. Among these, raw starch degrading enzyme I was recovered as fractions No. 86-100, and raw starch degrading enzyme II was recovered as fractions No. 64-80.
結果:疎水クロマトグラフィー
次いで、ゲルろ過クロマトグラフィー後の生デンプン分解酵素Iの活性画分、及び生デンプン分解酵素IIの活性画分にそれぞれ、0.5Mになるように硫酸アンモニウムを添加したものを、0.5M硫酸アンモニウムを含む20mMトリス塩酸(pH7.0)で平衡化したBUTYL-TOYOPEARL 650S(3.0×7.0cm、50ml)に負荷し、0.5から0Mの濃度勾配をつけた硫酸アンモニウムを含む20mMトリス塩酸(pH7.0)で溶出を行った。その結果を図4〜図6に示す。このうち生デンプン分解酵素Iでは画分No.30〜35を、生デンプン分解酵素IIでは画分No.40〜44を回収し、精製を終了した。以上の精製ステップを表1にまとめた。
Results: Hydrophobic chromatography Then, the active fraction of raw starch degrading enzyme I and the active fraction of raw starch degrading enzyme II after gel filtration chromatography were each added with ammonium sulfate to 0.5 M, Loaded onto a BUTYL-TOYOPEARL 650S (3.0 x 7.0 cm, 50 ml) equilibrated with 20 mM Tris-HCl (pH 7.0) containing M ammonium sulfate, 20 mM Tris-HCl (pH 7.5) containing ammonium sulfate with a concentration gradient of 0.5 to 0 M Elution was performed at 0). The results are shown in FIGS. Among these, raw starch degrading enzyme I recovered fractions No. 30 to 35, and raw starch degrading enzyme II recovered fractions No. 40 to 44, and purification was completed. The above purification steps are summarized in Table 1.
精製したアミラーゼの比活性は生デンプン分解酵素Iでは約85倍、生デンプン分解酵素IIでは約260倍に上昇し、回収率はおのおの3.6%、5.9%であった。上記の精製過程において、各画分のタンパク質量はベックマンDU7400を用い、280nmの吸光度を測定し、ウシ血清アルブミンを標準タンパク質としてブラッドフォード法(参考文献12)により定量した。 The specific activity of the purified amylase increased about 85 times for raw starch degrading enzyme I and about 260 times for raw starch degrading enzyme II, and the recoveries were 3.6% and 5.9%, respectively. In the above purification process, the protein amount of each fraction was determined by measuring the absorbance at 280 nm using Beckman DU7400, and quantifying it by the Bradford method (reference document 12) using bovine serum albumin as a standard protein.
結果:SDS-PAGEによる均一性分析
上記の精製により得られた本発明の生デンプン分解酵素をSDS-PAGEにより電気泳動を行なった。レムリーの方法(参考文献13)に従い、分離ゲルを10%、濃縮5%で行った。泳動はゲル1枚あたり20mAで行った。その結果は、図7に示すとおり電気泳動的に均一であり、生デンプン分解酵素I及びIIの分子量は共に約60kDaと推測された。
Result: Uniformity analysis by SDS-PAGE The raw starch degrading enzyme of the present invention obtained by the above purification was subjected to electrophoresis by SDS-PAGE. According to the method of Remley (Ref. 13), the separation gel was run at 10% and concentrated at 5%. Electrophoresis was performed at 20 mA per gel. The results were electrophoretically uniform as shown in FIG. 7, and the molecular weights of raw starch degrading enzymes I and II were both estimated to be about 60 kDa.
本発明の生デンプン分解酵素のデンプン分解様式
本発明の生デンプン分解酵素による生デンプン分解産物を、TLC(薄層クロマトグラフィー)により経時的に分析した。生米デンプンを精製したシマミミズ由来生デンプン分解酵素で、37℃の条件下で3時間、12時間、72時間反応させ、各時間の生成糖を薄層クロマトグラフィーにより分析した。薄層クロマトグラフィーはTLCプレートシリカゲル6(MERCK)に各時間の反応液をスポットし、常温で、クロロホルム:酢酸:水=5:7:1の展開溶媒で展開を行った後、ドラフト内で乾燥させ、20%硫酸エタノールを噴霧し、120℃で30分間放置し、糖検出を行った。
Starch Degradation Mode of the Raw Starch Degrading Enzyme of the Present Invention The raw starch degradation product by the raw starch degrading enzyme of the present invention was analyzed over time by TLC (thin layer chromatography). The raw starch was purified with the castor-degraded raw starch-degrading enzyme at 37 ° C for 3 hours, 12 hours and 72 hours, and the produced sugars at each time were analyzed by thin layer chromatography. For thin layer chromatography, spot the reaction solution for each hour on TLC plate silica gel 6 (MERCK), develop at room temperature with a developing solvent of chloroform: acetic acid: water = 5: 7: 1, and then dry in a fume hood. The mixture was sprayed with 20% ethanol sulfate and left at 120 ° C. for 30 minutes to detect sugar.
結果
図8から明らかなように、3時間後の分解産物はG2、G3、G4、G5そしてそれ以上のオリゴ糖が多く、G1はほとんど観測されなかった。12時間後では主にG2、G3が生成し、72時間後ではG1、G2が主要産物となっている。つまり、反応初期では重合度の大きなオリゴ糖が見られ、時間の経過に伴い重合度の小さなオリゴ糖が得られることより、これら2つの酵素はエンド型のα-アミラーゼであると決定した。
Results As is clear from FIG. 8, the degradation products after 3 hours were rich in G 2 , G 3 , G 4 , G 5 and higher oligosaccharides, and almost no G 1 was observed. After 12 hours, mainly G 2 and G 3 are produced, and after 72 hours, G 1 and G 2 are the main products. In other words, oligosaccharides with a high degree of polymerization were observed at the beginning of the reaction, and oligosaccharides with a low degree of polymerization were obtained with the passage of time, so that these two enzymes were determined to be endo-type α-amylases.
本発明のデンプン分解酵素活性に対する温度の影響
1.最適作用温度
0.1M CH3COOH/CH3COONa緩衝液(pH5.5)中で、30℃〜80℃の各温度で可溶化馬鈴薯デンプンを基質としてアミラーゼ活性を測定した。
Effect of temperature on amylolytic enzyme activity of the present invention
1. Optimal working temperature
Amylase activity was measured in 0.1M CH 3 COOH / CH 3 COONa buffer (pH 5.5) at 30 ° C. to 80 ° C. using solubilized potato starch as a substrate.
2.熱安定性
酵素液を10℃〜80℃の各温度で30分間インキュベートした後、直ちに氷冷し、0.1M CH3COOH/CH3COONa緩衝液(pH5.5)中で、37℃で可溶化馬鈴薯デンプンを基質として残存活性を測定した。
2. After the thermostable enzyme was incubated for 30 minutes at each temperature of 10 ° C. to 80 ° C., immediately cooled with ice, 0.1M CH 3 COOH / CH 3 COONa buffer in (pH 5.5), solubilized in 37 ° C. Residual activity was measured using potato starch as a substrate.
結果
可溶化した馬鈴薯デンプンに対する最適作用温度は、生デンプン分解酵素I及び生デンプン分解酵素II共に50℃であった(図9A)。熱安定性に関しては、生デンプン分解酵素Iは10〜60℃まで、生デンプン分解酵素IIは10〜50℃まで安定であった(図9B)。
Results The optimum action temperature for solubilized potato starch was 50 ° C. for both raw starch degrading enzyme I and raw starch degrading enzyme II (FIG. 9A). Regarding thermal stability, raw starch degrading enzyme I was stable up to 10-60 ° C., and raw starch degrading enzyme II was stable up to 10-50 ° C. (FIG. 9B).
本発明の生デンプン分解酵素活性に対するpHの影響
1.最適作用pH
37℃で、pH3.0〜9.0までの各緩衝液を用いて、可溶化馬鈴薯デンプンを基質としてアミラーゼ活性を測定した。用いた緩衝液は以下に示すとおりである。0.1M CH3COOH/HCl(pH3.0)、0.1M CH3COOH/CH3COONa(pH4.0〜6.0)、0.1M KH2PO4/NaOH(pH7.0〜8.0)、0.1M Na2CO3/NaHCO3(pH9.0)。
Effect of pH on the raw starch degrading enzyme activity of the present invention
1. Optimum pH
Amylase activity was measured using solubilized potato starch as a substrate at 37 ° C. using each buffer solution at pH 3.0 to 9.0. The buffer solution used is as shown below. 0.1M CH 3 COOH / HCl (pH 3.0), 0.1M CH 3 COOH / CH 3 COONa (pH 4.0 to 6.0), 0.1M KH 2 PO 4 / NaOH (pH 7.0 to 8.0), 0.1M Na 2 CO 3 / NaHCO 3 (pH 9.0).
2.pH安定性
pH4.0〜10.0までの各緩衝液に酵素液を加え、4℃で24時間放置した後、pHを5.5に戻し、37℃で可溶化馬鈴薯デンプンを基質として残存活性を測定した。用いた緩衝液は以下に示すとおりである。0.1M CH3COOH/CH3COONa(pH4.0〜6.0)、0.1Mトリス塩酸(pH7.0〜9.0)、0.1M Na2CO3/NaHCO3(pH10.0)。
2. pH stability
The enzyme solution was added to each buffer solution having a pH of 4.0 to 10.0 and allowed to stand at 4 ° C. for 24 hours. Then, the pH was returned to 5.5, and the residual activity was measured at 37 ° C. using solubilized potato starch as a substrate. The buffer solution used is as shown below. 0.1M CH 3 COOH / CH 3 COONa (pH4.0~6.0), 0.1M Tris-HCl (pH7.0~9.0), 0.1M Na 2 CO 3 / NaHCO 3 (pH10.0).
結果
可溶化馬鈴薯デンプンに対する最適作用pHは生デンプン分解酵素I及び生デンプン分解酵素II共に5.5であった(図10A)。pH安定性に関しては、生デンプン分解酵素IはpH7〜9まで、生デンプン分解酵素IIは7〜8まで安定であった(図10B)。
Results The optimum pH for solubilized potato starch was 5.5 for both raw starch degrading enzyme I and raw starch degrading enzyme II (FIG. 10A). Regarding pH stability, raw starch degrading enzyme I was stable up to pH 7-9, and raw starch degrading enzyme II was stable up to 7-8 (FIG. 10B).
本発明の生デンプン分解酵素活性に対する金属イオンの影響
0.1Mトリス塩酸(pH8.0)80μlに、10U/ml酵素液10μl、10mM金属イオン溶液10μlを加え、4℃で24時間放置した後、これを酵素液として、pH5.5、37℃で、可溶化馬鈴薯デンプンを基質としてアミラーゼ活性を測定し、金属イオンに対する影響を調べた。
Effect of metal ions on raw starch degrading enzyme activity of the present invention
To 80 μl of 0.1 M Tris-HCl (pH 8.0), 10 μl of 10 U / ml enzyme solution and 10 μl of 10 mM metal ion solution were added and allowed to stand at 4 ° C. for 24 hours, and this was used as the enzyme solution at pH 5.5 and 37 ° C. Amylase activity was measured using solubilized potato starch as a substrate, and the effect on metal ions was examined.
結果
1mMの金属イオンの存在下で、4℃で24時間インキュベートした後の残存活性を調べたところ、表2に示すような結果が得られた。Cu2+、Fe2+、Hg2+によって生デンプン分解酵素I、生デンプン分解酵素IIの活性は阻害を受けることが分かる。また、一般的にアミラーゼの安定化に関与すると言われているCa2+の存在下では、両者の活性はコントロールに比べてわずかであるが、上昇した。また生デンプン分解酵素IはAl3+、Mg2+の存在によって活性化もしくは安定化されたことが分かる。
result
When the residual activity after 24 hours of incubation at 4 ° C. in the presence of 1 mM metal ions was examined, the results shown in Table 2 were obtained. It can be seen that the activities of raw starch degrading enzyme I and raw starch degrading enzyme II are inhibited by Cu 2+ , Fe 2+ and Hg 2+ . In addition, in the presence of Ca 2+ , which is generally said to be involved in amylase stabilization, both activities were slightly higher than in the control, but increased. It can also be seen that raw starch degrading enzyme I was activated or stabilized by the presence of Al 3+ and Mg 2+ .
低温条件下での生デンプン加水分解率の測定
基質である米デンプンは、3度水で洗浄し、遠心分離後、凍結乾燥したものを使用した。0.4%生米デンプン1.0ml、0.1M酢酸緩衝液(pH6.0)980μl、酵素液200μl、2%アジ化ナトリウム20μlを含む反応液を37℃でインキュベートし、24時間、48時間、72時間、96時間の各時間で測定を行った。酵素は、シマミミズ由来生デンプン分解酵素(生デンプン分解酵素I、生デンプン分解酵素II)、A. oryzae由来α-アミラーゼ(SIGMA)、ブタ膵臓由来α-アミラーゼ(SIGMA)をそれぞれ2U/ml(基質・可溶化馬鈴薯デンプン)に調製して用いた。
Rice starch, which is a substrate for measuring the raw starch hydrolysis rate under low temperature conditions, was washed three times with water, centrifuged, and freeze-dried. Incubate a reaction solution containing 1.0 ml of 0.4% raw rice starch, 980 μl of 0.1M acetate buffer (pH 6.0), 200 μl of enzyme solution, 20 μl of 2% sodium azide at 37 ° C., 24 hours, 48 hours, 72 hours, Measurements were taken at each time of 96 hours. Enzymes are raw earth-degrading enzyme derived from earthworms (raw starch-degrading enzyme I, raw starch-degrading enzyme II), α-amylase derived from A. oryzae (SIGMA), and α-amylase derived from porcine pancreas (SIGMA) each at 2U / ml (substrate) -Solubilized potato starch) was used.
加水分解率(Rd)は以下の式により定義した。
Rd(%)=(A1/A0)×100
ここで、A1は反応後の遊離糖のモル濃度であり、A0は反応前の生デンプンのモル濃度である。
The hydrolysis rate (R d ) was defined by the following formula.
R d (%) = (A 1 / A 0 ) × 100
Here, A 1 is the molar concentration of free sugar after the reaction, and A 0 is the molar concentration of raw starch before the reaction.
結果
本発明の生デンプン分解酵素I及び生デンプン分解酵素IIの生米デンプンに対する加水分解率を生デンプン分解能を有するブタ膵臓由来α-アミラーゼ(PPA)(参考文献14、19)と、タカアミラーゼ(TAA)を用いて比較した(図11)。本発明の生デンプン分解酵素Iは4日で37%、生デンプン分解酵素IIは43.5%であった。これは一般的に生デンプンを分解するPPAよりは若干劣っているが、同程度の加水分解能を有していると考えられる。本酵素は4℃でも、37℃の37%(生デンプン分解酵素I)、20%(生デンプン分解酵素II)の活性が維持されることから、低温条件下での利用が可能であると考えられる。
Results The rate of hydrolysis of raw starch degrading enzyme I and raw starch degrading enzyme II of the present invention with respect to raw rice starch was derived from porcine pancreas-derived α-amylase (PPA) (PPs 14 and 19) and taka amylase ( TAA) was used for comparison (FIG. 11). The raw starch degrading enzyme I of the present invention was 37% in 4 days, and the raw starch degrading enzyme II was 43.5%. This is generally slightly inferior to PPA that degrades raw starch, but is believed to have comparable hydrolytic ability. Since this enzyme maintains 37% (raw starch degrading enzyme I) and 20% (raw starch degrading enzyme II) at 37 ° C even at 4 ° C, it can be used under low temperature conditions. It is done.
低温条件下でのアミラーゼ活性
0.1M CH3COOH/CH3COONa緩衝液(pH6.0)中で、4℃と37℃での可溶化馬鈴薯デンプンを基質としてアミラーゼ活性を比較した。
Amylase activity under low temperature conditions
Amylase activity was compared in 0.1M CH 3 COOH / CH 3 COONa buffer (pH 6.0) using solubilized potato starch at 4 ° C and 37 ° C as a substrate.
結果
4℃と37℃での可溶化馬鈴薯デンプンに対する本発明の酵素のアミラーゼ活性の比較を行ったところ、生デンプン分解酵素Iでは4℃で37℃の36.8%、生デンプン分解酵素IIでは20.7%の活性が維持されていた(図12A、12B)。
result
When the amylase activity of the enzyme of the present invention was compared to solubilized potato starch at 4 ° C and 37 ° C, 36.8% at 37 ° C at 4 ° C for raw starch degrading enzyme I and 20.7% for raw starch degrading enzyme II The activity was maintained (FIGS. 12A and 12B).
N-末端をブロックされているミミズ由来のα-アミラーゼのN-末端アミノ酸配列解析
(1)N末端ブロックタンパク質デブロッキングのストラテジー
ミミズ由来のα-アミラーゼはN-末端をブロックされているので、以下に示すストラテジーに基づいて解析を行った。
N-terminal amino acid sequence analysis of earthworm-derived α-amylase blocked at the N-terminus (1) N-terminal blocking protein deblocking strategy Since earthworm-derived α-amylase is blocked at the N-terminus, Analysis was performed based on the strategy shown in.
(2)方法I N-formyl基のデブロッキング
タンパク質(100 pmol-1 nmol)を0.6 M HCIに溶解する(PVDF膜にエレクトロブロットされた試料の場合は、膜をこの溶液100μlに浸す)。25℃にて24時間インキュベートする.処理したPVDF膜をプロテインシーケンサーにかけたが、AmyI、 AmyIIとも解析できなかった。
(2) Method I Deblocking protein of N-formyl group (100 pmol-1 nmol) is dissolved in 0.6 M HCI (in the case of a sample electroblotted on a PVDF membrane, the membrane is immersed in 100 μl of this solution). Incubate at 25 ° C for 24 hours. The treated PVDF membrane was applied to a protein sequencer, but neither AmyI nor AmyII could be analyzed.
(3)方法II N-pyroglutamyl基のデブロッキング
方法Iでシーケンスを決定できなかったので、Pfu Pyroglutamate Aminopeptidaseを用いてN末端Pyroglutamyl基のデブロッキングを試みた。PVDF膜に固定させたタンパク質(100 pmol-1 nmol)を60%メタノール 1 ml×2回、90%メ タノール 1 ml×1回、手で振とうして洗浄する。0.5%(w/v)のpolyvinylpyrrolidone(PVP)-55 含有100 mM酢酸溶液に500μlに37℃で30分間浸し、膜を蒸留水で少なくとも10回以上撹拌洗浄した。膜をメタノールで湿らせて、酵素消化を行った。Pfu Pyroglutamate Aminopeptidase(2 mU)5 μl、100 mMリン酸ナトリウム緩衝液(pH7.0)100 μl、100 mM DTT 20 μl、 蒸留水 75 μl, 合計200 μlとし,50℃で5時間インキュベートした。膜を蒸留水で3回撹拌洗浄後、シーケンサーでアミノ酸配列分析を行った。その結果、AmyI、AmyIIとも解析できなかった。
(3) Method II Deblocking of N-pyroglutamyl group Since the sequence could not be determined by Method I, deblocking of the N-terminal Pyroglutamyl group was attempted using Pfu Pyroglutamate Aminopeptidase. Wash the protein (100 pmol-1 nmol) immobilized on the PVDF membrane by shaking with 60% methanol 1 ml x 2 and 90% methanol 1 ml x 1 by hand. The membrane was immersed in 100 μl acetic acid solution containing 0.5% (w / v) polyvinylpyrrolidone (PVP) -55 in 500 μl at 37 ° C. for 30 minutes, and the membrane was washed by stirring at least 10 times with distilled water. Enzymatic digestion was performed by moistening the membrane with methanol. Pfu Pyroglutamate Aminopeptidase (2 mU) 5 μl, 100 mM sodium phosphate buffer (pH 7.0) 100 μl, 100 mM DTT 20 μl, distilled water 75 μl, total 200 μl, and incubated at 50 ° C. for 5 hours. The membrane was stirred and washed with distilled water three times, and amino acid sequence analysis was performed with a sequencer. As a result, neither AmyI nor AmyII could be analyzed.
(4)方法III Acyl-amino releasing enzymeを用いたN-末端アミノ酸列の解析
方法Iおよび方法IIで配列を解析できなかったので、N末端アセチル化ペプチドからアセチルアミノ酸を遊離するAcylamino-acid releasing enzymeを用いてデブロッキングを行った。AmyIおよびAmyIIをSDS-PAGE後、PVDF膜にエレクトロブロッティングし、ブロットされている膜画分を切り出し、0.5%(w/v)のpolyvinylpyrrolidone(PVP)-40を含む100 mM酢酸に浸し、37℃で30分間反応させた(この操作により膜のunbound protein-binding siteがブロックされる)。
(4) Method III Analysis of N-terminal amino acid sequence using Acyl-amino releasing enzyme Since the sequence could not be analyzed by Method I and Method II, acylamino-acid releasing enzyme that releases acetylamino acid from N-terminal acetylated peptide Was used for deblocking. After SDS-PAGE of AmyI and AmyII, electroblotting on PVDF membrane, excising the blotted membrane fraction, soaking in 100 mM acetic acid containing 0.5% (w / v) polyvinylpyrrolidone (PVP) -40, 37 ° C For 30 minutes (this operation blocks the unbound protein-binding site of the membrane).
1 mlの蒸留水で10回膜を洗浄した.膜を5〜10 μgトリプシンを含む0.1 M重炭酸アンモニウム緩衝液(pH8.0、10%アセトニトリル含有)に浸し、37℃、24時間、振とうしながら反応させた。可溶化したプロテアーゼ分解物をマイクロチューブに回収した。膜は100 μlの蒸留水で洗浄し洗液をプロテアーゼ分解液と一緒にした。減圧乾固後、Acylamino-acid releasing enzymeを用いて、N末端アセチル基のデブロックを行った。デブロック後プロテインシーケンサーにより分析を行ったところ、AmyI、AmyIIとも表4に示すような結果を得た。
AmyIはAmyIIと相同性が見られた。この配列は、ブタの唾液由来のリポカリンと相同性が見られた。
The membrane was washed 10 times with 1 ml of distilled water. The membrane was immersed in 0.1 M ammonium bicarbonate buffer (pH 8.0, containing 10% acetonitrile) containing 5 to 10 μg trypsin, and reacted at 37 ° C. for 24 hours with shaking. The solubilized protease degradation product was collected in a microtube. The membrane was washed with 100 μl of distilled water and the washing was combined with the protease digestion solution. After drying under reduced pressure, N-terminal acetyl group was deblocked using acylamino-acid releasing enzyme. After deblocking and analysis with a protein sequencer, the results shown in Table 4 were obtained for both AmyI and AmyII.
AmyI showed homology with AmyII. This sequence was found to be homologous with lipocalin from porcine saliva.
内部アミノ酸配列解析
In gel digestion法を用いた内部アミノ酸配列の決定
(1)In gel digestion
1) ゲル断片を1mm角に細断した。
2) ゲル断片をmilliQ水で洗浄し、アセトニトリルを加えて脱水後、遠心乾固
を行った。
3) 10mM DTT/ 100mM NH4HCO3を加えて56℃で還元を行った。
Internal amino acid sequence analysis
Determination of internal amino acid sequence using In gel digestion method (1) In gel digestion
1) The gel piece was cut into 1 mm square.
2) The gel fragment was washed with milliQ water, dehydrated by adding acetonitrile, and centrifuged to dryness.
3) Reduction was performed at 56 ° C by adding 10 mM DTT / 100 mM NH4HCO3.
4) 55mM ヨードアセトアミド/ 100mM NH4HCO3を加えて室温、遮光下で攪拌を行った。
5) 100mM NH4HCO3で洗浄後、アセトニトリルで脱水し、遠心乾固を行った。
6) ゲル片の入ったチューブを氷上で冷却し、トリプシン溶液を加え、膨潤を
行った。
7) 50mM NH4HCO3, 5mM CaCl2 を加えて、37℃, O/Nでインキュベーションを行った。
8) チューブ内の溶液を回収し、20mM NH4HCO3、50%ギ酸を加えて攪拌後、溶液を回収した。
9) 回収したサンプルを遠心乾固し、HPLC分離に使用するサンプルとした。
4) 55 mM iodoacetamide / 100 mM NH 4 HCO 3 was added and stirred at room temperature under light shielding.
5) Washed with 100 mM NH 4 HCO 3 , dehydrated with acetonitrile, and centrifuged to dryness.
6) The tube containing the gel pieces was cooled on ice, added with a trypsin solution, and swollen.
7) 50 mM NH 4 HCO 3 and 5 mM CaCl 2 were added, and incubation was performed at 37 ° C. and O / N.
8) The solution in the tube was collected, 20 mM NH 4 HCO 3 and 50% formic acid were added, and the solution was collected after stirring.
9) The collected sample was centrifuged to dryness and used as a sample for HPLC separation.
(2)逆相カラムによる分離
(条件)
Column : Inertsila ODS-SP 5mm 2.1×150 mm (GL Sciences)
Solvent A : 0.1% TFA / H2O
B : 0.07% TFA / CH3CN
Flow rate : 0.2 ml / min
Detect : 216 nm
(2) Separation by reversed phase column (Conditions)
Column: Inertsila ODS-SP 5mm 2.1 × 150 mm (GL Sciences)
Solvent A: 0.1% TFA / H 2 O
B: 0.07% TFA / CH 3 CN
Flow rate: 0.2 ml / min
Detect: 216 nm
上記の条件で分析したところ、41.4 分、42.3 分、43.3 分、44.3 分、51.2 分、53.7 分、55.5 分、56.4 分、57.3 分にピークが見られ (図13)、この部分の溶出液を回収し、島津プロテインシーケンサーPPSQ-23を用いて内部アミノ酸配列を明らかにした。その結果42.3 分、44.3 分、51.2 分、53.7 分、57.3 分の部分のアミノ酸配列を明らかにした(表5)。 When analyzed under the above conditions, peaks were observed at 41.4 minutes, 42.3 minutes, 43.3 minutes, 44.3 minutes, 51.2 minutes, 53.7 minutes, 55.5 minutes, 56.4 minutes, and 57.3 minutes (Figure 13). It was collected and the internal amino acid sequence was clarified using Shimadzu protein sequencer PPSQ-23. As a result, amino acid sequences of 42.3 minutes, 44.3 minutes, 51.2 minutes, 53.7 minutes, and 57.3 minutes were revealed (Table 5).
生デンプン(米)ならびに老化デンプン(米)の常温下での糖化とアルコール発酵
(1)シマミミズ由来の生デンプン分解酵素と他起源の生デンプン分解酵素との比較
シマミミズ由来の生デンプン分解酵素 (Amy I, Amy II)と生デンプン分解能を有するカビ由来のグルコアミラーゼ(グルクザイム AF6,天野エンザイム)を用いて30℃と20℃での生デンプン分解酵素活性の比較を行った。その結果、ミミズの酵素のAmyIを用いると20℃での活性は0.082 U/mlで、37℃での活性の78.2%の活性を示した。またAmyIIでは20℃においても37℃とほぼ同程度の活性を示した(表6)。また、カビ由来のアミラーゼは20℃での活性は37℃での活性の14%しか示さず、低温条件下での糖化には適していない事を明らかにした。
今回の実験より,37℃でも20℃でも効率よく生デンプン(米)を分解できるのはシマミミズ由来の酵素だけであることを明らかにした。
Saccharification and alcoholic fermentation of raw starch (rice) and aged starch (rice) at room temperature (1) Comparison of raw starch-degrading enzyme derived from earthworm and other raw starch-degrading enzymes Raw starch-degrading enzyme derived from earthworm (Amy I, Amy II) and fungal-derived glucoamylase (Gluczyme AF6, Amano Enzyme) having a raw starch-degrading ability were compared at 30 ° C and 20 ° C. As a result, when using the earthworm enzyme AmyI, the activity at 20 ° C. was 0.082 U / ml, which was 78.2% of the activity at 37 ° C. AmyII showed almost the same activity at 20 ° C. as at 37 ° C. (Table 6). In addition, fungi-derived amylase showed only 14% of the activity at 20 ° C. at 37 ° C., indicating that it is not suitable for saccharification under low temperature conditions.
From this experiment, it was clarified that only the enzyme derived from earthworms can decompose raw starch (rice) efficiently at 37 ℃ or 20 ℃.
(2)ミミズ由来の生デンプン分解酵素を用いた生デンプンの糖化
DEAE-toyopearl 650Mクロマトグラフィー後の酵素液 (濃縮し0.514 U/mlとした。基質:生米デンプン)をもちいて、25℃にてpH 5.0の条件で生デンプンの糖化を行った。0.4% 生米デンプン溶液 3.0 ml、0.1 M 酢酸緩衝液 (pH 5.0)、酵素液 0.6 mlを用いて反応させた。その結果、反応3日目には分解率が90%に達した(図14)。
(2) Raw starch saccharification using earthworm-derived raw starch degrading enzyme
DEAE-toyopearl 650M chromatographic enzyme solution (concentrated to 0.514 U / ml; substrate: raw rice starch) was used to saccharify raw starch at 25 ° C and pH 5.0. The reaction was performed using 3.0 ml of 0.4% raw rice starch solution, 0.1 M acetate buffer (pH 5.0), and 0.6 ml of enzyme solution. As a result, the degradation rate reached 90% on the third day of the reaction (FIG. 14).
(3)生デンプン分解物からのアルコール発酵
ミミズ由来の生デンプン分解酵素を用いて糖化させ分解産物を用いてアルコール発酵を行った。YPD培地(グルコース 20g, ポリペプトン20g、酵母エキス 10gを1リットル中に含む.pH 6.0)を用いて培養した酵母(Saccharomyces cerevisiae)溶液60μlを生デンプン分解産物(上記の72時間反応させた分解産物 6.6ml)に添加した。その結果,反応24時間後にはアルコール濃度 0.18 g/literにまで達した(図15)。このとき,反応液中のグルコースの濃度はアルコール濃度の上昇に伴い,急激に低下した。
(3) Alcohol fermentation from raw starch degradation product Alcohol fermentation was performed using a degradation product by saccharification using a raw starch-degrading enzyme derived from earthworms. 60 μl of yeast (Saccharomyces cerevisiae) solution cultured in YPD medium (glucose 20 g, polypeptone 20 g, yeast extract 10 g in 1 liter, pH 6.0) is a raw starch degradation product (the degradation product obtained by reacting the above for 72 hours 6.6 ml). As a result, the alcohol concentration reached 0.18 g / liter after 24 hours of reaction (FIG. 15). At this time, the glucose concentration in the reaction solution decreased rapidly as the alcohol concentration increased.
(4)ミミズ由来の生デンプン分解酵素を用いた老化デンプンの糖化
DEAE-toyopearl 650Mクロマトグラフィー後の酵素液 (濃縮し0.514 U/mlとした。基質:生米デンプン)をもちいて、25℃,pH 5.0の条件で老化デンプンの糖化を行った。老化米飯(55.3mg)、蒸留水 3.0 ml、0.1 M 酢酸緩衝液(pH 5.0)3.0 ml、酵素液 0.6 mlを25℃で反応させた。その結果,反応が進むに従って分解率が上昇し,72時間後には分解率が約80%まで達した(図16)。
(4) Saccharification of aging starch using raw starch-degrading enzyme derived from earthworm
Aged starch was saccharified using DEAE-toyopearl 650M chromatographic enzyme solution (concentrated to 0.514 U / ml, substrate: raw rice starch) at 25 ° C and pH 5.0. Aged cooked rice (55.3 mg), distilled water 3.0 ml, 0.1 M acetic acid buffer (pH 5.0) 3.0 ml, and enzyme solution 0.6 ml were reacted at 25 ° C. As a result, the decomposition rate increased as the reaction progressed, and the decomposition rate reached about 80% after 72 hours (Fig. 16).
(5)老化デンプン分解物からのアルコール発酵
ミミズ由来の生デンプン分解酵素を用いて老化デンプンを糖化させ、その分解産物を用いてアルコール発酵を行った。YPD培地を用いて培養した酵母(S. cerevisiae)溶液60μlを老化デンプン分解産物(上記の72時間反応させた分解産物 6.6ml)に添加した.その結果,反応24時間後にはアルコール濃度 0.19 g/literにまで達し,その後濃度は低下した(図17)。このとき、反応液中のグルコースの濃度はアルコール濃度の上昇に伴い、急激な低下が見られた。
(6)シマミミズ由来の生デンプン分解酵素並びにカビ由来のグルコアミラーゼを用いた効率的な糖化方法の開発
DEAE-toyopearl 650Mクロマトグラフィー後の酵素液 (濃縮し0.514U/mlとした。基質:生米デンプン)をもちいて,25℃,pH 5.0で12時間処理し、その後、カビ由来のグルコアミラーゼ(グルクザイムAF6,0.5U/ml)を添加し、37℃に加温して処理を続けて行った。0.4% 生米デンプン溶液 3.0 ml、0.1 M 酢酸緩衝液 (pH 5.0)、シマミミズ由来の酵素液 0.3 mlを用いて反応させ、12時間後、グルコアミラーゼを 0.3 ml添加した。その結果、グルコアミラーゼ添加と加温することによって糖化が促進され、72時間後にはほぼ糖化が終了した(図18)。グルコアミラーゼを用いることと、加温することにより効率的な加水分解が可能であることを明らかにした。
(5) Alcohol fermentation from aged starch degradation product Aged starch was saccharified using a raw starch-degrading enzyme derived from earthworms, and alcohol fermentation was performed using the degradation product. 60 μl of yeast (S. cerevisiae) solution cultured in YPD medium was added to the aged starch degradation product (degradation product reacted for 72 hours described above, 6.6 ml). As a result, the alcohol concentration reached 0.19 g / liter after 24 hours of reaction, and then the concentration decreased (Figure 17). At this time, the concentration of glucose in the reaction solution was drastically decreased as the alcohol concentration increased.
(6) Development of an efficient saccharification method using raw starch-degrading enzyme derived from earthworm and mold-derived glucoamylase
DEAE-toyopearl 650M Chromatographic enzyme solution (concentrated to 0.514 U / ml. Substrate: raw rice starch) treated at 25 ° C and pH 5.0 for 12 hours, then mold-derived glucoamylase (gluczyme) AF6, 0.5 U / ml) was added, and the treatment was continued by heating to 37 ° C. The reaction was performed using 0.4 ml of fresh rice starch solution (3.0 ml), 0.1 M acetic acid buffer (pH 5.0), and 0.3 ml of the enzyme solution derived from earthworm, and after 12 hours, 0.3 ml of glucoamylase was added. As a result, saccharification was accelerated by adding glucoamylase and heating, and saccharification was almost completed after 72 hours (FIG. 18). It was clarified that efficient hydrolysis is possible by using glucoamylase and heating.
参考文献
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シマミミズが生産する本発明の生デンプン分解酵素は生デンプンを含む廃棄物の処理に、特に低温での処理に有用である。 The raw amylolytic enzyme of the present invention produced by the earthworm is useful for the treatment of waste containing raw starch, particularly at low temperatures.
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JP2019041722A (en) * | 2017-09-06 | 2019-03-22 | 公立大学法人大阪府立大学 | Starch degrading enzymes, nucleic acids encoding them and uses thereof |
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JP2019041722A (en) * | 2017-09-06 | 2019-03-22 | 公立大学法人大阪府立大学 | Starch degrading enzymes, nucleic acids encoding them and uses thereof |
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