JP2011010608A - Phospholipase c and method for decomposing phospholipid using the same - Google Patents
Phospholipase c and method for decomposing phospholipid using the same Download PDFInfo
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- 108010079194 Type C Phospholipases Proteins 0.000 title claims abstract description 59
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- 238000000034 method Methods 0.000 title claims abstract description 13
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- 230000007935 neutral effect Effects 0.000 abstract description 5
- 239000006228 supernatant Substances 0.000 abstract description 4
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- 239000003960 organic solvent Substances 0.000 abstract description 3
- 239000002244 precipitate Substances 0.000 abstract description 3
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- 239000012530 fluid Substances 0.000 abstract 2
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- 238000006460 hydrolysis reaction Methods 0.000 description 14
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- 239000002994 raw material Substances 0.000 description 10
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 9
- 241000196324 Embryophyta Species 0.000 description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 6
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- 240000005809 Prunus persica Species 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 235000002566 Capsicum Nutrition 0.000 description 5
- 239000006002 Pepper Substances 0.000 description 5
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- 238000006243 chemical reaction Methods 0.000 description 4
- 235000013399 edible fruits Nutrition 0.000 description 4
- 230000003301 hydrolyzing effect Effects 0.000 description 4
- PORPENFLTBBHSG-MGBGTMOVSA-N 1,2-dihexadecanoyl-sn-glycerol-3-phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(O)=O)OC(=O)CCCCCCCCCCCCCCC PORPENFLTBBHSG-MGBGTMOVSA-N 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 3
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 3
- 240000006108 Allium ampeloprasum Species 0.000 description 3
- 235000005254 Allium ampeloprasum Nutrition 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 3
- 244000300264 Spinacia oleracea Species 0.000 description 3
- 235000009337 Spinacia oleracea Nutrition 0.000 description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 235000013311 vegetables Nutrition 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
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- 230000036039 immunity Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 2
- YHHSONZFOIEMCP-UHFFFAOYSA-O phosphocholine Chemical compound C[N+](C)(C)CCOP(O)(O)=O YHHSONZFOIEMCP-UHFFFAOYSA-O 0.000 description 2
- 201000010153 skin papilloma Diseases 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
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- 241000228245 Aspergillus niger Species 0.000 description 1
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- 241000193755 Bacillus cereus Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 235000011331 Brassica Nutrition 0.000 description 1
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- 241000219193 Brassicaceae Species 0.000 description 1
- 241000222122 Candida albicans Species 0.000 description 1
- 241001249699 Capitata Species 0.000 description 1
- 235000002568 Capsicum frutescens Nutrition 0.000 description 1
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- 238000005377 adsorption chromatography Methods 0.000 description 1
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- 235000013614 black pepper Nutrition 0.000 description 1
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- 230000015556 catabolic process Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000010411 cooking Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000030609 dephosphorylation Effects 0.000 description 1
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- 238000000502 dialysis Methods 0.000 description 1
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- 238000010438 heat treatment Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 238000001155 isoelectric focusing Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
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- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 150000003905 phosphatidylinositols Chemical class 0.000 description 1
- LFGREXWGYUGZLY-UHFFFAOYSA-N phosphoryl Chemical group [P]=O LFGREXWGYUGZLY-UHFFFAOYSA-N 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 235000021110 pickles Nutrition 0.000 description 1
- 239000001931 piper nigrum l. white Substances 0.000 description 1
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- CIJQGPVMMRXSQW-UHFFFAOYSA-M sodium;2-aminoacetic acid;hydroxide Chemical compound O.[Na+].NCC([O-])=O CIJQGPVMMRXSQW-UHFFFAOYSA-M 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000758 substrate Substances 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
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
本発明は、新規なホスホリパーゼＣおよびそれを用いたりん脂質の分解方法に関する。 The present invention relates to a novel phospholipase C and a phospholipid degradation method using the same.
ホスホリパーゼＣは、リン脂質のリン酸ジエステル結合を加水分解する酵素である。これまで、各種ホスホリパーゼＣが知られており、例えば、細菌由来のホスホリパーゼＣでは、シュードモーナス・シュルキリエンシス（Ｐｓｕｄｏｍｏｎｕｓ ｓｃｈｕｙｌｋｉｌｌｉｅｎｓｉｓ）、ブルコールデリア・シュードマレイ（Ｂｕｌｋｈｏｌｄｅｒｉａ ｐｓｅｕｄｏｍａｌｌｅｉ）、バチラス・セレウス（Ｂａｃｉｌｌｕｓ ｃｅｒｅｕｓ）、スタフィロコッカス・アウレウス（Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ａｕｒｅｕｓ）、クロストリディウム・ぺルフリンゲンス（Ｃｌｏｓｔｒｉｄｉｕｍ ｐｅｒｆｒｉｎｇｅｎｓ）が報告されている（例えば、非特許文献１〜４、特許文献１参照）。また、放線菌由来のホスホリパーゼＣでは、ストレプトマイセス・ハチジョウエンシス（Ｓｔｒｅｐｔｏｍｙｃｅｓ ｈａｃｈｉｊｙｏｅｎｓｉｓ）が報告されている（例えば、特許文献２参照）。更に、酵母由来のホスホリパーゼＣでは、キャンディダ・アルビカンス（Ｃａｎｄｉｄａ ａｌｂｉｃａｎｓ）、サッカロマイセス・セレビシアエ（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ）が報告されている（例えば、非特許文献５，６参照）。加えて、カビ由来のホスホリパーゼＣでは、アスペルギルス・ニガー（Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ）、アスペルギルス・サイトイ（Ａｓｐｅｒｇｉｌｌｕｓ ｓａｉｔｏｉ）が報告されている（例えば、非特許文献７、特許文献３参照）。 Phospholipase C is an enzyme that hydrolyzes the phosphodiester bond of phospholipids. Various phospholipases C have been known so far. For example, phospholipases C derived from bacteria include Pseudomonus schuylkilliensis, Bulkolderia pseudomallei, and Bacillus cerus ), Staphylococcus aureus and Clostridium perfringens have been reported (for example, see Non-Patent Documents 1 to 4 and Patent Document 1). In addition, Streptomyces hajiyoensis has been reported for phospholipase C derived from actinomycetes (see, for example, Patent Document 2). Furthermore, in phospholipase C derived from yeast, Candida albicans and Saccharomyces cerevisiae have been reported (for example, refer to Non-Patent Documents 5 and 6). In addition, aspergillus niger and Aspergillus saitoi have been reported for phospholipase C derived from fungi (see, for example, Non-Patent Document 7 and Patent Document 3).
しかしながら、これらのホスホリパーゼＣを生産するに際して、各種の問題が内在しており、特に微生物由来のホスホリパーゼＣは、一部のものについて病原性が指摘されており、すべてのものについて安全であるということはできず、特に、食品分野に利用するには問題があった。 However, various problems are inherent in the production of these phospholipases C. In particular, microbial phospholipase C has been pointed out to be pathogenic and is safe for all. In particular, there was a problem in using it in the food field.
また、これらのホスホリパーゼＣは、ホスファチジルコリン、ホスファチジルイノシトールのみに有効であり、また、酸性領域でのみ活性を示すという問題があった。更に、動物由来のホスホリパーゼＣは、宗教的に受け入れられない国や地域があり、汎用性の観点からも問題があった。 Further, these phospholipases C are effective only for phosphatidylcholine and phosphatidylinositol, and have the problem that they exhibit activity only in the acidic region. Furthermore, animal-derived phospholipase C has some countries and regions that are not religiously acceptable and has a problem from the viewpoint of versatility.
本発明は、このような問題に基づきなされたものであり、安全性が高く、食品分野にも安心して利用することができ、中性領域でも活性を示すホスホリパーゼＣおよびこれを用いてリン脂質を分解する方法を提供することを目的とする。 The present invention has been made on the basis of such problems, and has high safety, can be safely used in the food field, and exhibits activity in the neutral region, and phospholipids can be obtained using the same. The object is to provide a method of disassembling.
本発明のホスホリパーゼＣは、モモ、レタス、トマト、キャベツ、ナスまたはキュウリから抽出されるものである。 The phospholipase C of the present invention is extracted from peach, lettuce, tomato, cabbage, eggplant or cucumber.
本発明のリン脂質の分解方法は、リン脂質に、モモ、レタス、トマト、キャベツ、ナスまたはキュウリから抽出されるホスホリパーゼＣを加えて、リン酸ジエステル結合を加水分解するものである。 In the method for decomposing phospholipids of the present invention, phospholipase C extracted from peach, lettuce, tomato, cabbage, eggplant or cucumber is added to phospholipid to hydrolyze the phosphodiester bond.
本発明のホスホリパーゼＣによれば、モモ、レタス、トマト、キャベツ、ナスまたはキュウリから抽出されたものであるので、安全性が高く、食品分野にも安心して利用することができる。また、中性領域においても活性を有するので、本発明のリン脂質の分解方法によれば、種々のリン脂質を効率よく分解することができる。 Since the phospholipase C of the present invention is extracted from peach, lettuce, tomato, cabbage, eggplant or cucumber, it is highly safe and can be used safely in the food field. Moreover, since it has activity also in a neutral region, according to the method for decomposing phospholipid of the present invention, various phospholipids can be efficiently decomposed.
以下、本発明の実施の形態について詳細に説明する。 Hereinafter, embodiments of the present invention will be described in detail.
本発明の一実施の形態に係るホスホリパーゼＣは、モモ、レタス、トマト、キャベツ、ナスまたはキュウリから抽出されるものである。モモ、レタス、トマト、キャベツ、ナスまたはキュウリから抽出されるものであるので、安全性が高く、食品分野にも安心して利用できるようになっている。 The phospholipase C according to one embodiment of the present invention is extracted from peach, lettuce, tomato, cabbage, eggplant or cucumber. Since it is extracted from peach, lettuce, tomato, cabbage, eggplant or cucumber, it is highly safe and can be used safely in the food field.
なお、モモは、桃（学名 Ａｍｙｇｄａｌｕｓ ｐｅｒｓｉｃａ）であり、バラ科モモ属の落葉小高木である。春には五弁または多重弁の花を咲かせ、夏には水分が多く甘い球形の果実を実らせる中国原産の植物である。モモは食用あるいは観賞用として世界各地で栽培されている。本実施の形態に係るホスホリパーゼＣは、モモの果実を原料とする。モモの品種としては、例えば、日本の市場に出回っているものでは、白桃系の桃、白鳳系の桃、果肉の黄色い黄桃、あかつき、ゆうぞら、川中島、白桃、まどか、名月などがあり、これらのうち１種または２種以上を原料とすることができる。 The peach is a peach (scientific name: Amygdalus persica), a deciduous small tree of the genus Rosaceae. It is a plant native to China that blooms five-petal or multi-petal flowers in the spring and produces sweet and spherical fruits in the summer. Peach is cultivated around the world for food and ornamental use. Phospholipase C according to the present embodiment uses peach fruit as a raw material. As peach varieties, for example, those in the Japanese market are white peaches, white peaches, yellow yellow peaches, Akatsuki, Yuzora, Kawanakajima, white peaches, Madoka, Meigetsu, etc. Of these, one or more of these can be used as raw materials.
レタスは、キク科アキノノゲシ属の一年草または二年草であり、野菜として利用され、和名をチシャと呼ぶ植物である。レタスの品種としては、例えば、ヘッドレタス（Ｌ. ｓ. ｖａｒ. ｃａｐｉａｔａ)−タマチシャ、リーフレタス（Ｌ. ｓ. ｖａｒ. ｃｒｉｓｐａ)−葉チシャ、チリメンヂシャ、立ちレタス（Ｌ. ｓ. ｖａｒ. ｌｏｎｇｉｆｏｌｉａ)−立ちヂシャ、カッティングレタス（Ｌ. ｓ. ｖａｒ. ｃｒｉｓｐａ)−カキヂシャ、ステムレタス（Ｌ. ｓ. ｖａｒ. ａｎｇｕｓｔａｎａ)−茎チシャがあり、これらのうち１種または２種以上を原料とすることができる。 Lettuce is an annual or biennial plant belonging to the genus Asteraceae, and is a plant that is used as a vegetable and its Japanese name is called chisha. Examples of the varieties of lettuce include head lettuce (L. s. Var. Capitata)-Tamachisha, leaf lettuce (L. s. Var. Crispa)-leaf chisha, chili mushroom, standing lettuce (L. s. Var. Longifolia) -Standing shears, cutting lettuce (L. s. Var. Crispa)-Oysters, stem lettuce (L. s. Var. Angstana)-Stem tissue, one or more of which may be used as a raw material it can.
トマトは、ナス科ナス属の多年生であり、原産地はアンデス地方とされる緑黄色野菜の一種である。トマトの品種としては、例えば、桃色系トマト、ファースト系トマト、ミニトマト、高糖度系トマト、ミディトマト、調理用トマトがあり、これらのうち１種または２種以上を原料とすることができる。 Tomatoes are perennials of the solanaceous genus Solanum and are a kind of green-yellow vegetables whose origin is the Andes region. Examples of tomato varieties include pink tomatoes, fast tomatoes, mini tomatoes, high sugar tomatoes, midi tomatoes, and cooking tomatoes, and one or more of these can be used as raw materials.
キャベツは、アブラナ科アブラナ属の多年草であり、野菜として広く利用されている。キャベツの品種としては、例えば、日本国内で一般に消費されている玉に加え、ムラサキキャベツ、サボイキャベツ、ハボタンがあり、これらのうち１種または２種以上を原料とすることができる。 Cabbage is a perennial plant of the Brassicaceae genus Brassica and is widely used as a vegetable. Examples of the cabbage varieties include, in addition to balls generally consumed in Japan, murasaki cabbage, savoy cabbage, and ha button, and one or more of these can be used as a raw material.
ナスは、ナス科ナス属の植物の果実である。ナスの品種としては山科なす、賀茂なす、田屋なす、十市なす、水なす、下田なす、民田なす、ていざなす、やきなすなどがあり、これらのうち１種または２種以上を原料とすることができる。 Eggplant is a fruit of a plant belonging to the genus Solanum. Eggplant varieties include Yamashina eggplant, Kamo eggplant, Taya eggplant, Juichi eggplant, Mizusu eggplant, Shimoda eggplant, Tamita eggplant, handmade eggplant, yakinas, etc. Use one or more of these as raw materials Can do.
キュウリは、ウリ科キュウリ属のつる性一年草の果実である。キュウリの品種としては、例えば、白イボ系、黒イボ系、四葉（スーヨー）胡瓜、四川胡瓜、馬込半白胡瓜、高井戸節成胡瓜、加賀太胡瓜、聖護院胡瓜、毛馬胡瓜、大和三尺、ピクルス用キュウリがあり、これらのうち１種または２種以上を原料とすることができる。 Cucumber is the fruit of the vine genus annual plant of the genus Cucumber. The cucumber varieties include, for example, white wart, black wart, Suyo pepper, Sichuan pepper, Umagome half white pepper, Takaido Bunsei pepper, Kaga Taiko pepper, Shogoin pepper, Mao pepper, Yamato three scales, pickles There are cucumbers for use, and one or more of these can be used as raw materials.
このホスホリパーゼＣは、例えば、モモ、レタス、トマト、キャベツ、ナスあるいはキュウリを粉砕して濾過した濾液、あるいは遠心分離した際の上澄み液を、アセトンなどの有機溶媒と混合したのち、沈殿物を必要に応じて、凍結乾燥等することにより、粗酵素液あるいは粗酵素粉末として抽出することができる。更に、例えば、この粗酵素を塩析、有機溶媒沈殿、透析、限外濾過、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、ゲル濾過、凍結乾燥、等電点電気泳動等の方法を、ホスホリパーゼＣの理化学的性質を考慮した条件下で行うことにより、濃縮して採取することができる。なお、ホスホリパーゼＣの抽出は、このような方法に限定されるものではなく、適宜専門業者が行う他の方法により行うようにしてもよい。 This phospholipase C requires, for example, a precipitate obtained by mixing a filtrate obtained by pulverizing and filtering peach, lettuce, tomato, cabbage, eggplant or cucumber, or a supernatant after centrifugation with an organic solvent such as acetone. Depending on the condition, it can be extracted as a crude enzyme solution or a crude enzyme powder by freeze-drying or the like. Further, for example, the crude enzyme is subjected to methods such as salting out, organic solvent precipitation, dialysis, ultrafiltration, ion exchange chromatography, adsorption chromatography, gel filtration, lyophilization, isoelectric focusing, and physicochemical analysis of phospholipase C. It can be concentrated and collected by carrying out under conditions that take into account the physical properties. The extraction of phospholipase C is not limited to such a method, and may be performed by other methods appropriately performed by a specialist.
モモ、レタス、トマト、キャベツ、ナスまたはキュウリから抽出されるホスホリパーゼＣは、中性領域においても加水分解活性を有しており、リン脂質を効率的に加水分解することができる。例えば、ホスファチジルコリンを原料として、１，２−ジグリセリドとホスホコリンとを効率的に製造することができる。なお、このホスホリパーゼＣを用いてリン脂質の加水分解反応を行う際に、エチレンジアミン四酢酸（ＥＤＴＡ）を添加するようにすれば、反応性を高めることができるので好ましい。また、例えば、ホスファチジルコリンを原料として、１，２−ジグリセリドとホスホコリンとを製造したのち、水洗あるいはカラム精製などにより、ホスファチジルコリンを除去することにより、リン脂質を除去することができる。 Phospholipase C extracted from peach, lettuce, tomato, cabbage, eggplant or cucumber has hydrolytic activity even in the neutral region, and can efficiently hydrolyze phospholipids. For example, 1,2-diglyceride and phosphocholine can be efficiently produced using phosphatidylcholine as a raw material. In addition, when performing a phospholipid hydrolysis reaction using this phospholipase C, it is preferable to add ethylenediaminetetraacetic acid (EDTA) because the reactivity can be increased. Further, for example, phosphatidylcholine can be removed by producing phosphatidylcholine using phosphatidylcholine as a raw material and then removing phosphatidylcholine by washing with water or column purification.
このように本実施の形態のホスホリパーゼＣによれば、モモ、レタス、トマト、キャベツ、ナスまたはキュウリから抽出されたものであるので、安全性が高く、食品分野にも安心して利用することができる。また、中性領域においても活性を有するので、種々のリン脂質を効率よく分解することができる。よって、例えば、ホスファチジルコリンを原料として、１，２−ジグリセリドとホスホコリンとを効率的に製造することができる。 Thus, according to the phospholipase C of the present embodiment, since it is extracted from peach, lettuce, tomato, cabbage, eggplant or cucumber, it is highly safe and can be used safely in the food field. . Moreover, since it also has activity in the neutral region, various phospholipids can be efficiently decomposed. Therefore, for example, 1,2-diglyceride and phosphocholine can be efficiently produced using phosphatidylcholine as a raw material.
（実施例１）
レタス（茨城県産）４００ｇに純水６００ｍｌを加え、氷冷下にてワーリングブレンダー（ハイパワーホモジナイザー，広沢鉄工所社製）でホモジナイズ（１００００ｒｐｍ、１ｍｉｎ、５回）した。これをガーゼ濾過したのち、濾液を遠心分離機（ＣＸ−２５０、Ｔｏｍｙ社製）で遠心分離（１００００ｒｐｍ、４℃、３０ｍｉｎ）して、上澄み液を回収した。続いて、回収した上澄み液に対して２倍量の冷アセトンを撹拌しながら加え、氷冷下で１時間静置したのち、生じた沈殿を遠心分離（１００００ｒｐｍ，４℃，３０ｍｉｎ）して回収し、真空凍結乾燥機（ＶＤ−８００Ｆ、Ｔａｉｔｅｃ社製）で凍結乾燥した。凍結乾燥物を氷冷させた乳鉢で微粉末にし、得られた粉末をレタスホスホリパーゼＣとして、−２０℃で保存した。得られたレタスホスホリパーゼＣについて加水分解活性を測定したところ、８１．７Ｕ／ｇであった。
Example 1
600 ml of pure water was added to 400 g of lettuce (produced in Ibaraki Prefecture), and homogenized (10000 rpm, 1 min, 5 times) with a Waring blender (High Power Homogenizer, manufactured by Hirosawa Iron Works Co., Ltd.) under ice cooling. After gauze filtration, the filtrate was centrifuged (10000 rpm, 4 ° C., 30 min) with a centrifuge (CX-250, manufactured by Tomy), and the supernatant was collected. Subsequently, twice the amount of cold acetone is added to the collected supernatant while stirring, and the mixture is allowed to stand for 1 hour under ice-cooling, and the resulting precipitate is collected by centrifugation (10000 rpm, 4 ° C., 30 min). And lyophilized with a vacuum freeze dryer (VD-800F, manufactured by Taitec). The freeze-dried product was made into fine powder in an ice-cooled mortar, and the obtained powder was stored as lettuce phospholipase C at -20 ° C. The hydrolysis activity of the obtained lettuce phospholipase C was measured and found to be 81.7 U / g.
また、レタスに代えてトマト（桃太郎：熊本県産）、モモ（福島県産）、キャベツ（愛知県産）、ナス（会津丸：福島県産）、又はキュウリを用いたことを除き、他はレタスと同様にしてトマトホスホリパーゼＣ、モモホスホリパーゼＣ、キャベツホスホリパーゼＣ、ナスホスホリパーゼＣ、又はキュウリホスホリパーゼＣをそれぞれ得た。得られた各ホスホリパーゼＣについて加水分解活性を測定したところ、トマトホスホリパーゼＣが４９．４Ｕ／ｇ、モモホスホリパーゼＣが２１３Ｕ／ｇ、キャベツホスホリパーゼＣが２８．１Ｕ／ｇ、ナスホスホリパーゼＣが３２．６Ｕ／ｇ、キュウリホスホリパーゼＣが２０３Ｕ／ｇであった。 Other than using tomato (Momotaro: from Kumamoto Prefecture), peach (from Fukushima Prefecture), cabbage (from Aichi Prefecture), eggplant (Aizu Maru: from Fukushima Prefecture), or cucumber, instead of lettuce, Tomato phospholipase C, peach phospholipase C, cabbage phospholipase C, eggplant phospholipase C, or cucumber phospholipase C were obtained in the same manner as lettuce. When hydrolytic activity was measured for each of the obtained phospholipases C, tomato phospholipase C was 49.4 U / g, peach phospholipase C was 213 U / g, cabbage phospholipase C was 28.1 U / g, and eggplant phospholipase C was 32.6 U. / G, cucumber phospholipase C was 203 U / g.
なお、加水分解活性は、リン脂質（代表的には、ホスファチジルコリン）を基質として、酵素反応により遊離するリン酸を定量することによって測定した。具体的には、まず、リン脂質として１％（ｗ／ｖ）のホスファチジルコリン５μＬと、０．２ＭのＥＤＴＡ３μＬと、５０ｍＭトリス−塩酸緩衝液（ｐＨ７．２）２８２μＬとを混合し、３７℃で５分間加熱した後、酵素液を１０μＬ加えて撹拌し、３７℃で１０分間反応させた。その後、反応液を１００℃で１０分間加熱して、反応を停止させた。反応終了後、この反応液１０μＬをアルカリホスファターゼ溶液（０．２ｕｎｉｔｓのアルカリホスファターゼと、１ｍＭのＭｇＣｌ２と、０．１ｍＭのＺｎＣｌ２と、５０ｍＭのグリシン−水酸化ナトリウム緩衝液（ｐＨ９．６）との混合溶液）１９０μＬに添加し、３７℃にて２０分間脱リン酸化反応を行った。その後、遊離したリン酸を定量することにより、ホスホリパーゼＣの加水分解活性を求めた。これにより求めたホスホリパーゼＣの１Ｕは、１分間に１μｍｏｌのホスホリル塩基を遊離する酵素量である。 The hydrolysis activity was measured by quantifying phosphoric acid released by an enzymatic reaction using phospholipid (typically phosphatidylcholine) as a substrate. Specifically, first, 5 μL of 1% (w / v) phosphatidylcholine as a phospholipid, 3 μL of 0.2 M EDTA, and 282 μL of 50 mM Tris-HCl buffer (pH 7.2) were mixed, and 5 ° C. at 37 ° C. After heating for 10 minutes, 10 μL of enzyme solution was added and stirred, and reacted at 37 ° C. for 10 minutes. Thereafter, the reaction solution was heated at 100 ° C. for 10 minutes to stop the reaction. After completion of the reaction, 10 μL of this reaction solution was added to an alkaline phosphatase solution (0.2 units alkaline phosphatase, 1 mM MgCl 2 , 0.1 mM ZnCl 2 , 50 mM glycine-sodium hydroxide buffer (pH 9.6)). The mixed solution was added to 190 μL and subjected to dephosphorylation at 37 ° C. for 20 minutes. Thereafter, phospholipase C hydrolysis activity was determined by quantifying the released phosphoric acid. 1 U of phospholipase C thus determined is the amount of enzyme that liberates 1 μmol of phosphoryl base per minute.
（比較例１）
レタスに代えてホウレンソウ（北海道産）又はネギ（埼玉県産）を用い、実施例１と同様にして、ホウレンソウホスホリパーゼＣ又はネギホスホリパーゼＣを調製した。得られたホウレンソウホスホリパーゼＣおよびネギホスホリパーゼＣについても、実施例１と同様にして加水分解活性を測定したところ、どちらも加水分解活性は微弱であった。
(Comparative Example 1)
Spinach phospholipase C or leek phospholipase C was prepared in the same manner as in Example 1 using spinach (produced in Hokkaido) or leek (produced in Saitama Prefecture) instead of lettuce. Regarding the obtained spinach phospholipase C and leek phospholipase C, the hydrolysis activity was measured in the same manner as in Example 1. As a result, the hydrolysis activity was weak.
（実施例１と比較例１との比較）
実施例１によれば高い加水分解活性が得られたのに対して、比較例１では微弱であった。すなわち、レタス、トマト、モモ、キャベツ、ナス、又はキュウリから抽出されたホスホリパーゼＣによれば、高い加水分解活性を得ることができ、リン脂質を高い効率で分解することができることがわかった。
(Comparison between Example 1 and Comparative Example 1)
According to Example 1, high hydrolysis activity was obtained, whereas in Comparative Example 1, it was weak. That is, according to phospholipase C extracted from lettuce, tomato, peach, cabbage, eggplant, or cucumber, it was found that high hydrolytic activity can be obtained and phospholipids can be decomposed with high efficiency.
（実施例２）
実施例１において調製したレタスホスホリパーゼＣ、キャベツホスホリパーゼＣおよびキュウリホスホリパーゼＣについて、リン脂質として１０％（ｗ／ｖ）のホスファチジン酸、又は１０％（ｗ／ｖ）のホスファチジルエタノールアミンを用い、実施例１と同様にして加水分解反応を行い、加水分解活性を測定した。なお、リン脂質としてホスファチジン酸を用いた場合には、酵素反応させた後、アルカリホスファターゼ溶液で処理せずに、純水１９０μＬに加えて遊離したリン酸を定量した。また、実施例１において調製したキュウリホスホリパーゼＣについて、リン脂質としてスフィンゴミエリンを用い、実施例１と同様にして加水分解反応を行い、加水分解活性を測定した。
(Example 2)
For lettuce phospholipase C, cabbage phospholipase C and cucumber phospholipase C prepared in Example 1, 10% (w / v) phosphatidic acid or 10% (w / v) phosphatidylethanolamine was used as the phospholipid. The hydrolysis reaction was carried out in the same manner as in 1, and the hydrolysis activity was measured. When phosphatidic acid was used as the phospholipid, the phosphoric acid liberated in addition to 190 μL of pure water was quantified without being treated with an alkaline phosphatase solution after the enzyme reaction. Moreover, about the cucumber phospholipase C prepared in Example 1, sphingomyelin was used as a phospholipid, it hydrolyzed like Example 1, and the hydrolysis activity was measured.
その結果、リン脂質としてホスファチジン酸を用いた場合の加水分解活性は、レタスホスホリパーゼＣが９５．９Ｕ／ｇ、キャベツホスホリパーゼＣが５６．５Ｕ／ｇ、キュウリホスホリパーゼＣが１４４Ｕ／ｇであった。また、リン脂質としてホスファチジルエタノールアミンを用いた場合の加水分解活性は、レタスホスホリパーゼＣが１１０Ｕ／ｇ、キャベツホスホリパーゼＣが７０．６Ｕ／ｇ、キュウリホスホリパーゼＣが１３１Ｕ／ｇであった。更に、リン脂質としてスフィンゴミエリンを用いた場合におけるキュウリホスホリパーゼＣの加水分解活性は、２７５Ｕ／ｇであった。すなわち、様々なリン脂質について、高い加水分解活性をえら得ることが分かった。 As a result, when phosphatidic acid was used as the phospholipid, the lettuce phospholipase C was 95.9 U / g, the cabbage phospholipase C was 56.5 U / g, and the cucumber phospholipase C was 144 U / g. The hydrolysis activity when phosphatidylethanolamine was used as the phospholipid was 110 U / g for lettuce phospholipase C, 70.6 U / g for cabbage phospholipase C, and 131 U / g for cucumber phospholipase C. Furthermore, the hydrolysis activity of cucumber phospholipase C when sphingomyelin was used as the phospholipid was 275 U / g. That is, it was found that high hydrolytic activity can be obtained for various phospholipids.
以上、実施の形態および実施例を挙げて本発明を説明したが、本発明は、上記実施の形態および実施例に限定されるものではなく、種々変形可能である。例えば、上記実施の形態および上記実施例では、ホスホリパーゼＣの抽出方法、精製方法およびホスホリパーゼＣを用いたリン脂質の加水分解方法について具体的に説明したが、他の方法により行うようにしてもよい。 While the present invention has been described with reference to the embodiments and examples, the present invention is not limited to the above embodiments and examples, and various modifications can be made. For example, in the above embodiment and the above examples, the phospholipase C extraction method, purification method, and phospholipid hydrolysis method using phospholipase C have been specifically described. However, other methods may be used. .
リン脂質の加水分解に用いることができる。 It can be used for the hydrolysis of phospholipids.
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