JPH0678766A - New ascorbate oxidase - Google Patents
New ascorbate oxidaseInfo
- Publication number
- JPH0678766A JPH0678766A JP23472992A JP23472992A JPH0678766A JP H0678766 A JPH0678766 A JP H0678766A JP 23472992 A JP23472992 A JP 23472992A JP 23472992 A JP23472992 A JP 23472992A JP H0678766 A JPH0678766 A JP H0678766A
- Authority
- JP
- Japan
- Prior art keywords
- ascorbate oxidase
- ascorbic acid
- ascorbate
- composition
- present
- 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.)
- Granted
Links
- 108010024957 Ascorbate Oxidase Proteins 0.000 title claims abstract description 60
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims abstract description 41
- 235000010323 ascorbic acid Nutrition 0.000 claims abstract description 22
- 239000011668 ascorbic acid Substances 0.000 claims abstract description 22
- 229960005070 ascorbic acid Drugs 0.000 claims abstract description 21
- 230000000694 effects Effects 0.000 claims abstract description 11
- 102000004190 Enzymes Human genes 0.000 claims abstract description 7
- 108090000790 Enzymes Proteins 0.000 claims abstract description 7
- 239000000758 substrate Substances 0.000 claims abstract description 5
- SBJKKFFYIZUCET-JLAZNSOCSA-N Dehydro-L-ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(=O)C1=O SBJKKFFYIZUCET-JLAZNSOCSA-N 0.000 claims abstract description 3
- SBJKKFFYIZUCET-UHFFFAOYSA-N Dehydroascorbic acid Natural products OCC(O)C1OC(=O)C(=O)C1=O SBJKKFFYIZUCET-UHFFFAOYSA-N 0.000 claims abstract description 3
- 238000006243 chemical reaction Methods 0.000 claims abstract description 3
- 235000020960 dehydroascorbic acid Nutrition 0.000 claims abstract description 3
- 239000011615 dehydroascorbic acid Substances 0.000 claims abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 3
- 239000000203 mixture Substances 0.000 claims description 22
- 239000007788 liquid Substances 0.000 claims description 20
- 238000004458 analytical method Methods 0.000 claims description 16
- 239000000126 substance Substances 0.000 abstract description 3
- 238000004321 preservation Methods 0.000 abstract 1
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 12
- 239000003153 chemical reaction reagent Substances 0.000 description 12
- 241000219112 Cucumis Species 0.000 description 11
- 235000010071 Cucumis prophetarum Nutrition 0.000 description 11
- 240000001980 Cucurbita pepo Species 0.000 description 11
- 235000009852 Cucurbita pepo Nutrition 0.000 description 9
- 239000007853 buffer solution Substances 0.000 description 8
- 241000219122 Cucurbita Species 0.000 description 6
- 239000007990 PIPES buffer Substances 0.000 description 6
- 239000013504 Triton X-100 Substances 0.000 description 6
- 229920004890 Triton X-100 Polymers 0.000 description 6
- 230000002000 scavenging effect Effects 0.000 description 6
- 108090000854 Oxidoreductases Proteins 0.000 description 5
- 102000004316 Oxidoreductases Human genes 0.000 description 5
- 102000003992 Peroxidases Human genes 0.000 description 5
- 108040007629 peroxidase activity proteins Proteins 0.000 description 5
- 239000004094 surface-active agent Substances 0.000 description 5
- 235000009854 Cucurbita moschata Nutrition 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 238000005185 salting out Methods 0.000 description 4
- 230000008030 elimination Effects 0.000 description 3
- 238000003379 elimination reaction Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 102000009027 Albumins Human genes 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 2
- 235000000832 Ayote Nutrition 0.000 description 2
- 240000008067 Cucumis sativus Species 0.000 description 2
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 2
- 240000004244 Cucurbita moschata Species 0.000 description 2
- 235000009804 Cucurbita pepo subsp pepo Nutrition 0.000 description 2
- 239000006173 Good's buffer Substances 0.000 description 2
- 101710101148 Probable 6-oxopurine nucleoside phosphorylase Proteins 0.000 description 2
- 102000030764 Purine-nucleoside phosphorylase Human genes 0.000 description 2
- 108010093894 Xanthine oxidase Proteins 0.000 description 2
- 102100033220 Xanthine oxidase Human genes 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 230000003139 buffering effect Effects 0.000 description 2
- 239000002738 chelating agent Substances 0.000 description 2
- 238000003759 clinical diagnosis Methods 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 235000015136 pumpkin Nutrition 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- RXXOXPGVDNVIKC-UHFFFAOYSA-N 2-hydroxy-3-(3-methylanilino)propane-1-sulfonic acid Chemical compound CC1=CC=CC(NCC(O)CS(O)(=O)=O)=C1 RXXOXPGVDNVIKC-UHFFFAOYSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 241000219104 Cucurbitaceae Species 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 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 1
- 241000196324 Embryophyta Species 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 229930010555 Inosine Natural products 0.000 description 1
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 1
- 102000004195 Isomerases Human genes 0.000 description 1
- 108090000769 Isomerases Proteins 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 102000009097 Phosphorylases Human genes 0.000 description 1
- 108010073135 Phosphorylases Proteins 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 108010092464 Urate Oxidase Proteins 0.000 description 1
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 1
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 1
- LWZFANDGMFTDAV-BURFUSLBSA-N [(2r)-2-[(2r,3r,4s)-3,4-dihydroxyoxolan-2-yl]-2-hydroxyethyl] dodecanoate Chemical compound CCCCCCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O LWZFANDGMFTDAV-BURFUSLBSA-N 0.000 description 1
- 238000005377 adsorption chromatography Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 229940072107 ascorbate Drugs 0.000 description 1
- 229940079293 ascorbic acid 40 mg Drugs 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000004186 food analysis Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 229960003786 inosine Drugs 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- MWFOPMKUGZLPQA-UHFFFAOYSA-M sodium;3-(n-ethyl-3-methoxyanilino)propane-1-sulfonate Chemical compound [Na+].[O-]S(=O)(=O)CCCN(CC)C1=CC=CC(OC)=C1 MWFOPMKUGZLPQA-UHFFFAOYSA-M 0.000 description 1
- 235000011067 sorbitan monolaureate Nutrition 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 229940116269 uric acid Drugs 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は熱安定性、保存安定性の
良い、新規なアスコルビン酸オキシダーゼに関する。FIELD OF THE INVENTION The present invention relates to a novel ascorbate oxidase having good thermostability and storage stability.
【０００２】[0002]
【従来の技術】アスコルビン酸オキシダーゼは古くから
種々の高等植物に所在することが知られており、特に該
酵素含量の高いキュウリやカボチャなどのウリ科植物の
果実皮を給源にしての研究が行われ、これまで、アスコ
ルビン酸オキシダーゼの性質に関して詳細に検討され、
報告されている。これまで、その精製品は食品分析分野
において、アスコルビン酸の定量に、あるいは、臨床検
査分野において、検体中のアスコルビン酸が測定系に影
響を与える場合に、かかる系にアスコルビン酸オキシダ
ーゼを共存せしめ、アスコルビン酸を酸化分解して、そ
の妨害を排除する目的として用いられてきた。2. Description of the Related Art Ascorbate oxidase has long been known to be located in various higher plants, and in particular, studies have been conducted with the source of the skins of fruits of the Cucurbitaceae plants such as cucumber and pumpkin, which have a high enzyme content. So far, the properties of ascorbate oxidase have been studied in detail,
It has been reported. So far, the purified product, in the field of food analysis, for the determination of ascorbic acid, or in the field of clinical testing, when ascorbic acid in the sample affects the measurement system, coexist ascorbate oxidase in such system, It has been used for the purpose of oxidatively decomposing ascorbic acid to eliminate its interference.
【０００３】[0003]
【発明が解決しようとする課題】アスコルビン酸オキシ
ダーゼは既に知られているように、キュウリ（キュキュ
ミス エスピー、Cucumis sp. ：東洋紡社製）及び、カ
ボチャ（ククルビタ エスピー、Cucurbita sp. ：ベー
リンガーマンハイム社製）から生産され市販されてい
る。しかしながら、従来のアスコルビン酸オキシダーゼ
では、臨床診断用試薬、殊に液状分析用組成物として用
いる場合、保存安定性が不十分であった。また最近、臨
床診断の自動分析に於て、簡便化追求の一つの方向とし
て試薬溶解の手間を不要とする液状分析用組成物が注目
され、種々の試みがなされている。本発明の目的は、上
記現状に鑑み、液状分析用組成物に用いるのに好適な熱
安定性、保存安定性の良い、新規なアスコルビン酸オキ
シダーゼを提供することである。As is already known, ascorbate oxidase is known as cucumber (Cucumis sp .: manufactured by Toyobo Co., Ltd.) and pumpkin (Cucurbita sp., Cucurbita sp .: manufactured by Boehringer Mannheim Co., Ltd.). ) Produced and marketed. However, conventional ascorbate oxidase has insufficient storage stability when used as a reagent for clinical diagnosis, particularly as a liquid analysis composition. In recent years, in automatic analysis of clinical diagnosis, attention has been paid to a composition for liquid analysis which does not require labor for dissolving a reagent as one direction for pursuing simplification, and various attempts have been made. In view of the above situation, an object of the present invention is to provide a novel ascorbate oxidase having good heat stability and storage stability, which is suitable for use in a liquid analysis composition.
【０００４】[0004]
【課題を解決するための手段】本発明者らは、上記目的
を達成するために鋭意検討したところ、カボチャ クク
ルビタ モスカータ（Cucurbita moschata）由来のアス
コルビン酸オキシダーゼが、従来より知られているクク
ルビタ ペポ（Cucurbita pepo、米国特許第4,168,205
号明細書参照）及び、キュキュミス エスピー（Cucumi
s sp. 、J.Biochem .,Vol.64,No.2,P189−195,1968参
照）より保存安定性がよいことを見い出し、本発明を完
成した。Means for Solving the Problems The inventors of the present invention have made extensive studies to achieve the above-mentioned object. As a result, ascorbate oxidase derived from Cucurbita moschata has been found Cucurbita pepo, U.S. Pat.No. 4,168,205
No.) and Cucumi SP
s sp., J. Biochem., Vol. 64, No. 2, P189-195, 1968), the present invention was found to have better storage stability.
【０００５】すなわち、本発明の要旨は、下記の理化学
的性質を有するアスコルビン酸オキシダーゼに存する。 （１）作用：１モルのアスコルビン酸と１／２モルの酵
素より、１モルのデヒドロアスコルビン酸と１モルの水
を生ずる反応を触媒する。 （２）基質特異性：少なくともアスコルビン酸に特異的
に作用する。 （３）至適ｐＨ：５．５〜６．５ （４）ｐＨ安定性：６．０〜１０．０ （５）至適温度：約４５℃ （６）熱安定性：５０℃、１時間処理において、少なく
とも８０％の残存活性を有する。又、５５℃、１時間処
理において、少なくとも５０％の残存活性を有する。 なお、本発明のアスコルビン酸オキシダーゼの他の理化
学的性質は下記の通りである。 （７）分子量：約１４０，０００（ゲル濾過法） （８）Ｋｍ値：２．７×１０-4Ｍ付近 （９）等電点：５．５±０．２ （10) 界面活性剤の影響： 0.1%Triton X-100、Briji
35、Tween 20、Span 20等の存在下で１００％の残存活
性を有する。That is, the gist of the present invention resides in an ascorbate oxidase having the following physicochemical properties. (1) Action: Catalyze a reaction that produces 1 mol of dehydroascorbic acid and 1 mol of water from 1 mol of ascorbic acid and 1/2 mol of enzyme. (2) Substrate specificity: It acts specifically on at least ascorbic acid. (3) Optimum pH: 5.5 to 6.5 (4) pH stability: 6.0 to 10.0 (5) Optimum temperature: about 45 ° C (6) Thermal stability: 50 ° C, 1 hour It has a residual activity of at least 80% in the treatment. It also has a residual activity of at least 50% after treatment at 55 ° C. for 1 hour. The other physicochemical properties of the ascorbate oxidase of the present invention are as follows. (7) Molecular weight: about 140,000 (gel filtration method) (8) Km value: around 2.7 × 10 −4 M (9) Isoelectric point: 5.5 ± 0.2 (10) Surfactant Impact: 0.1% Triton X-100, Briji
It has 100% residual activity in the presence of 35, Tween 20, Span 20, etc.
【０００６】本発明のアスコルビン酸オキシダーゼは、
例えば「蛋白質・酵素の基礎実験法」（堀尾武一、山下
仁平編、南江堂、1982）に記載された方法に準じて、原
料であるククルビタ モスカータ（Cucurbita moschat
a）を破砕し、緩衝液等で抽出した後、硫安塩析、吸着
クロマトグラフィー等により精製することによって得ら
れる。The ascorbate oxidase of the present invention is
For example, in accordance with the method described in “Basic Experiments for Proteins and Enzymes” (Buichi Horio, Nihei Yamashita, Nankodo, 1982), the raw material Cucurbita moschat
It is obtained by crushing a), extracting with a buffer solution, etc., and then purifying by salting out with ammonium sulfate, adsorption chromatography and the like.
【０００７】又、本発明のもう一つの要旨は液状分析用
組成物において、ククルビタ モスカータ（Cucurubita
moschata ）由来のアスコルビン酸オキシダーゼを含有
することを特徴とする安定な液状分析用組成物に存す
る。Another aspect of the present invention is a composition for liquid analysis, which is Cucurubita.
moschata) -derived ascorbate oxidase, which is a stable composition for liquid analysis.
【０００８】本発明の安定な液状分析用組成物は、クク
ルビタ モスカータ（Cucurubita moschata ）由来のア
スコルビン酸オキシダーゼと生化学分析に用いられる通
常の緩衝液を含有し、使用目的に応じて、緩衝液中にキ
レート剤、無機塩類、アルブミン、アミノ酸、界面活性
剤、抗生物質、４-アミノアンチピリン、トリンダー試
薬（酸化発色色素）、ロイコ色素、ホルマザン色素、酸
化酵素、脱水素酵素、ペルオキシダーゼ、異性化酵素、
りん酸化酵素、ヒドロラーゼなどを含有することもでき
る。The stable composition for liquid analysis of the present invention contains an ascorbate oxidase derived from Cucurubita moschata and an ordinary buffer solution used for biochemical analysis. Chelating agents, inorganic salts, albumin, amino acids, surfactants, antibiotics, 4-aminoantipyrine, Trinder's reagent (oxidative coloring dye), leuco dye, formazan dye, oxidase, dehydrogenase, peroxidase, isomerase,
It can also contain phosphorylase, hydrolase, and the like.
【０００９】本発明に用いる緩衝液種及びその濃度は特
に限定されるものではないが、pH5.5 〜8.5 の間で緩衝
能を有し、且つ必要十分な緩衝能を保つ濃度に設定され
ていることが望ましい。この様な緩衝液種として汎用的
なリン酸バッファーやトリスバッファーを使用すること
もできるし、ＢＥＳ，ＨＥＰＥＳ，ＴＥＳ等のグッドバ
ッファーを使用することもできる。緩衝液濃度は好まし
くは10ｍＭ〜0.5 Ｍ、更に好ましくは50ｍＭ〜0.1 Ｍで
ある。キレート剤としては、EDTA等を、無機塩類として
はNaCl、MgCl2等を、アルブミンとしては、牛血清、人
血清、馬血清などの由来のものを、アミノ酸としては、
アルギニン、リジン、ヒスチジン等を、含有することが
できる。The type of the buffer solution used in the present invention and the concentration thereof are not particularly limited, but they are set to a concentration having a buffering capacity between pH 5.5 and 8.5 and maintaining a necessary and sufficient buffering capacity. Is desirable. A general-purpose phosphate buffer or Tris buffer can be used as such a buffer solution type, and a good buffer such as BES, HEPES, TES can also be used. The buffer concentration is preferably 10 mM to 0.5 M, more preferably 50 mM to 0.1 M. As the chelating agent, EDTA or the like, as the inorganic salt, NaCl, MgCl 2 or the like, as the albumin, those derived from bovine serum, human serum, horse serum, etc., as the amino acid,
Arginine, lysine, histidine and the like can be included.
【００１０】又、本発明は、場合によって、Ｉ液組成物
でも、ＩＩ液組成物、ＩＩＩ液組成物でもよく、その選
択は、測定する物質や、測定を実施する自動分析機の性
能に従って、好的に組み合わせることが出来る。又、該
液状分析用組成物は液状分析用組成物に限らず、通常の
分析用試薬に用いられている凍結乾燥品等の、固形物と
しても好適に利用できる。Further, the present invention may be a liquid I composition, a liquid II composition, or a liquid III composition depending on the case, and the selection thereof depends on the substance to be measured and the performance of the automatic analyzer for carrying out the measurement. It can be combined favorably. Further, the liquid analysis composition is not limited to the liquid analysis composition, and can be suitably used as a solid substance such as a freeze-dried product which is used as a usual analysis reagent.
【００１１】[0011]
【実施例】以下、本発明を実施例により詳細に説明す
る。 実施例１ 原料であるククルビタ モスカータ（Cucurubita mosch
ata ）を破砕し、ホウ酸緩衝液で抽出した後、濾過を行
い固形物を除き、上澄みを得た。次に硫酸アンモニウム
で塩析処理し、塩析沈殿物を得た。これを緩衝液にて再
懸濁し、更に塩析処理を行い、塩析沈殿物を得た。これ
を緩衝液にて再懸濁し、同緩衝液で平衡化したイオン交
換クロマトグラフィー、疎水クロマトグラフィーに供
し、NaClグラジエント、飽和度を下げるなどしてアスコ
ルビン酸オキシダーゼ画分を得た。このアスコルビン酸
オキシダーゼ画分をセファデックスＧ−２５などで脱塩
し、酵素を得た。EXAMPLES The present invention will be described in detail below with reference to examples. Example 1 Cucurubita mosch (raw material)
ata) was crushed and extracted with a borate buffer solution, followed by filtration to remove solids and obtain a supernatant. Next, salting-out treatment was performed with ammonium sulfate to obtain a salting-out precipitate. This was resuspended in a buffer solution and subjected to salting-out treatment to obtain a salted-out precipitate. This was resuspended in a buffer solution, subjected to ion exchange chromatography equilibrated with the same buffer solution, and subjected to hydrophobic chromatography to obtain an ascorbate oxidase fraction by decreasing the NaCl gradient and the degree of saturation. This ascorbate oxidase fraction was desalted with Sephadex G-25 or the like to obtain an enzyme.
【００１２】このように精製した本発明酵素と同様の精
製法で調整したククルビタ ペポ（Cucurbita pepo）及
びキュキュミス エスピー（Cucumis sp. ）由来のアス
コルビン酸オキシダーゼを熱安定性について比較した。
各アスコルビン酸オキシダーゼを５U/mlに0.1% Triton
X-100を含む 50mM PIPESbuffer(pH7.0)で溶解し、各温
度で１時間処理した後の残存活性を測定した。その結果
を、図１に示す。図１中の３種のアスコルビン酸オキシ
ダーゼを比較すると、50℃、55℃において、本発明のア
スコルビン酸オキシダーゼが他のアスコルビン酸オキシ
ダーゼより熱安定性に優れていることがわかる。The ascorbate oxidase derived from Cucurbita pepo and Cucumis sp. Prepared by the same purification method as the enzyme of the present invention thus purified was compared for thermal stability.
Add each ascorbate oxidase to 5 U / ml with 0.1% Triton
It was dissolved in 50 mM PIPES buffer (pH 7.0) containing X-100, and the residual activity after treatment at each temperature for 1 hour was measured. The result is shown in FIG. Comparing the three types of ascorbate oxidase in FIG. 1, it can be seen that the ascorbate oxidase of the present invention is superior in thermal stability to other ascorbate oxidases at 50 ° C. and 55 ° C.
【００１３】実施例２ 本発明のアスコルビン酸オキシダーゼとククルビタ ペ
ポ（Cucurbita pepo）及びキュキュミス エスピー（Cu
cumis sp. ）由来のアスコルビン酸オキシダーゼの保存
安定性を比較した。各アスコルビン酸オキシダーゼを５
U/mlに0.1% Triton X-100 を含む 50mM PIPESbuffer(pH
7.0)で溶解し、各温度で一週間保存し、アスコルビン酸
オキシダーゼの残存活性の経過を測定した。その結果を
図２に示す。図２中の３種のアスコルビン酸オキシダー
ゼを比較すると、4 ℃、37℃、40℃のいずれの温度にお
いても本発明のアスコルビン酸オキシダーゼの保存安定
性が優れていることがわかる。Example 2 Ascorbate oxidase of the present invention and Cucurbita pepo and Cucumis sp (Cu)
The storage stability of ascorbate oxidase derived from cumis sp.) was compared. 5 each ascorbate oxidase
U / ml containing 0.1% Triton X-100 50 mM PIPES buffer (pH
It was dissolved in 7.0) and stored at each temperature for 1 week, and the course of residual activity of ascorbate oxidase was measured. The result is shown in FIG. Comparing the three types of ascorbate oxidase in FIG. 2, it can be seen that the storage stability of the ascorbate oxidase of the present invention is excellent at any temperature of 4 ° C., 37 ° C., and 40 ° C.
【００１４】実施例３ 液状分析用組成物の一例として、グッドバッファー、ペ
ルオキシダーゼ、４−アミノアンチピリン、トリンダー
試薬、基質、酸化酵素、界面活性剤などから構成される
アスコルビン酸オキシダーゼを含有する下記組成の無機
リン測定液状試薬を調製し、９、３０、４０℃で一週間
保存し、アスコルビン酸オキシダーゼの残存活性を測定
した。また、本発明のアスコルビン酸オキシダーゼの他
に、ククルビタ ペポ（Cucurbita pepo）、キュキュミ
ス エスピー（Cucumis sp. ）及びククルビタ エスピ
ー（Cucurbita sp. ）由来のアスコルビン酸オキシダー
ゼを用いて、同様に液状分析用組成物を調製し、同様に
保存し、残存活性を測定した。また、アスコルビン酸消
去能を測定し、比較した。Example 3 As an example of the liquid analysis composition, the following composition containing ascorbate oxidase composed of Good buffer, peroxidase, 4-aminoantipyrine, Trinder reagent, substrate, oxidase, surfactant and the like was prepared. Inorganic phosphorus measurement A liquid reagent was prepared and stored at 9, 30, and 40 ° C for one week, and the residual activity of ascorbate oxidase was measured. Further, in addition to the ascorbate oxidase of the present invention, ascorbate oxidase derived from Cucurbita pepo, Cucumis sp. And Cucurbita sp. The product was prepared and stored in the same manner, and the residual activity was measured. Moreover, the ascorbic acid elimination ability was measured and compared.
【００１５】 試薬組成 Ｒ１ ＰＩＰＥＳバッファー（ｐＨ６．８） ５０ｍＭ トリトンＸ−１００（界面活性剤） ０．１ ％ アスコルビン酸オキシダーゼ（ククルビタ モスカータ、Cucurbita moschata由来） ７．０ Ｕ／ｍｌ ＸＴＯ（キサンチンオキシダーゼ） ２．０Ｕ／ｍｌ ＰＥＯ（ペルオキシダーゼ） ５．０Ｕ／ｍｌ ＡＤＰＳ（トリンダ−試薬） ０．２ｍｇ／ｍｌ Ｒ２ ＰＩＰＥＳバッファー（ｐＨ６．８） ５０ｍＭ ＰＮＰ（プリンヌクレオシドりん酸化酵素）１．０Ｕ／ｍｌ ４−ＡＡ（４−アミノアンチピリン） ０．２ｍｇ／ｍｌ イノシン（基質） ８．０ｍｇ／ｍｌ トリトンＸ−１００（界面活性剤） ０．１％Reagent composition R1 PIPES buffer (pH 6.8) 50 mM Triton X-100 (surfactant) 0.1% Ascorbate oxidase (from Cucurbita moschata) 7.0 U / ml XTO (xanthine oxidase) 2 0.0 U / ml PEO (peroxidase) 5.0 U / ml ADPS (Trinda reagent) 0.2 mg / ml R2 PIPES buffer (pH 6.8) 50 mM PNP (purine nucleoside phosphorylase) 1.0 U / ml 4-AA ( 4-aminoantipyrine) 0.2 mg / ml inosine (substrate) 8.0 mg / ml Triton X-100 (surfactant) 0.1%
【００１６】上記３種のアスコルビン酸オキシダーゼ
（ＡＳＯ）の保存後の液状試薬中における残存活性を表
１に示す。また、アスコルビン酸消去能を表２に示す。
表１中の３種のアスコルビン酸オキシダーゼを比較する
と、３０℃３日で差は顕著であり、４０℃、１週間で
は、ククルビタ ペポ（Cucurbita pepo）由来のアスコ
ルビン酸オキシダーゼは２％、キュキュミス エスピー
（Cucumis sp. ）由来のアスコルビン酸オキシダーゼは
５％しか残存しないが、本発明のアスコルビン酸オキシ
ダーゼは２０％残存していた。また、表２より、本発明
のアスコルビン酸オキシダーゼの場合、４０℃、２週間
後においてもアスコルビン酸の消去に十分なアスコルビ
ン酸オキシダーゼが残存しており、本発明のアスコルビ
ン酸オキシダーゼが保存安定性において優れていること
が認められる。Table 1 shows the residual activities of the above three types of ascorbate oxidase (ASO) in the liquid reagent after storage. Table 2 shows the ascorbic acid scavenging ability.
Comparing the three ascorbate oxidases in Table 1, the difference was remarkable at 30 ° C. for 3 days, and at 40 ° C. for 1 week, 2% of the ascorbate oxidase derived from Cucurbita pepo was found, and Cucumis sp. Only 5% of ascorbate oxidase derived from (Cucumis sp.) Remained, but 20% of the ascorbate oxidase of the present invention remained. In addition, from Table 2, in the case of the ascorbate oxidase of the present invention, sufficient ascorbate oxidase for scavenging ascorbic acid remains even after 40 ° C. and 2 weeks, and the ascorbate oxidase of the present invention shows storage stability. It is recognized that it is excellent.
【００１７】[0017]
【表１】 各温度で保存後のアスコルビン酸オキシダーゼの残存活
性（％）[Table 1] Residual activity (%) of ascorbate oxidase after storage at each temperature
【００１８】[0018]
【表２】 ４０℃、２週間保存後のアスコルビン酸消去能（％） （アスコルビン酸 40mg/dl 添加時）[Table 2] Ascorbic acid scavenging ability (%) after storage at 40 ° C for 2 weeks (when 40 mg / dl of ascorbic acid is added)
【００１９】実施例４ 実施例３と同様にアスコルビン酸オキシダーゼを含有す
る下記組成の尿酸測定液状試薬を調製し、４０℃で６日
間保存し、３日目、６日目にアスコルビン酸消去能を測
定した。また、本発明の他に、比較例として、本発明の
アスコルビン酸オキシダーゼと同様の精製法で調製した
ククルビタ ペポ（Cucurbita pepo）、キュキュミス
エスピー（Cucumis sp. ）及びククルビタ エスピー
（Cucurbita sp. ）由来のアスコルビン酸オキシダーゼ
を用いて、同様に液状分析用組成物を調製し、同様に保
存し、アスコルビン酸消去能を測定し、比較した。Example 4 A liquid reagent for measuring uric acid having the following composition containing ascorbic acid oxidase was prepared in the same manner as in Example 3 and stored at 40 ° C. for 6 days, and the ascorbic acid elimination ability was determined on the 3rd and 6th days. It was measured. In addition to the present invention, as a comparative example, Cucurbita pepo and Cucumis prepared by the same purification method as that of the ascorbate oxidase of the present invention.
A liquid analysis composition was similarly prepared using ascorbic acid oxidase derived from SP (Cucumis sp.) And Cucurbita sp. (Cucurbita sp.), And similarly stored, and the ascorbic acid elimination ability was measured and compared.
【００２０】 試薬組成 Ｒ１ ＰＩＰＥＳバッファー（ｐＨ６．８） ５０ｍＭ トリトンＸ−１００ ０．１％ アスコルビン酸オキシダーゼ ７．０Ｕ／ｍｌ ＰＥＯ ５．０Ｕ／ｍｌ ４−ＡＡ（４−アミノアンチピリン） ０．２ｍｇ／ｍｌ Ｒ２ ＰＩＰＥＳバッファー（ｐＨ６．８） ５０ｍＭ ＵＡＯ（ウリカーゼ） ０．２Ｕ／ｍｌ ＥＨＳＰＴ ０．２Ｕ／ｍｌ （N-エチル-N-(2-ヒドロキシ-3-スルホプロピル-m-トルイジン):トリン ダー試薬） トリトンＸ−１００ ０．１％Reagent composition R1 PIPES buffer (pH 6.8) 50 mM Triton X-100 0.1% Ascorbate oxidase 7.0 U / ml PEO 5.0 U / ml 4-AA (4-aminoantipyrine) 0.2 mg / ml R2 PIPES buffer (pH 6.8) 50 mM UAO (uricase) 0.2 U / ml EHSPT 0.2 U / ml (N-ethyl-N- (2-hydroxy-3-sulfopropyl-m-toluidine): Trinder reagent) Triton X-100 0.1%
【００２１】４種のアスコルビン酸オキシダーゼの保存
後のおけるアスコルビン酸消去能を表３に示す。表３に
示すように、本発明のアスコルビン酸オキシダーゼは４
０℃で６日保存後も９５．１７％と充分なアスコルビン
酸消去能が存在するのに対し、他のアスコルビン酸オキ
シダーゼ、例えばククルビタ ペポ（Cucurbita pepo）
由来のアスコルビン酸オキシダーゼの場合、８５．９６
％の消去能しかなく、充分とは言えず、本発明のアスコ
ルビン酸オキシダーゼが保存安定性において優れている
ことが認められる。Table 3 shows the ascorbic acid scavenging ability of the four kinds of ascorbic acid oxidase after storage. As shown in Table 3, the ascorbate oxidase of the present invention has 4
It has a sufficient ascorbate scavenging capacity of 95.17% even after storage at 0 ° C for 6 days, while other ascorbate oxidases such as Cucurbita pepo
85.96 for ascorbate oxidase from
It has only an erasing capacity of%, which is not sufficient, and it is recognized that the ascorbate oxidase of the present invention is excellent in storage stability.
【００２２】[0022]
【表３】 40 ℃, ３、６日保存後のアスコルビン酸消去能（％） （アスコルビン酸 40mg/dl 添加時）[Table 3] Ascorbic acid scavenging capacity after storage at 40 ° C for 3 or 6 days (%) (when ascorbic acid 40 mg / dl is added)
【００２３】[0023]
【発明の効果】本発明により熱安定性、保存安定性の優
れたアスコルビン酸オキシダーゼが提供され、又本発明
のアスコルビン酸オキシダーゼを液状分析用組成物に用
いることにより、該組成物の保存安定性を飛躍的に向上
させることが出来る。INDUSTRIAL APPLICABILITY The present invention provides an ascorbate oxidase excellent in heat stability and storage stability, and by using the ascorbate oxidase of the present invention in a liquid analysis composition, the storage stability of the composition is improved. Can be dramatically improved.
【図面の簡単な説明】[Brief description of drawings]
【図１】実施例１における本発明のアスコルビン酸オキ
シダーゼとククルビタ ペポ（Cucurbita pepo）及びキ
ュキュミス エスピー（Cucumis sp. ）由来のアスコル
ビン酸オキシダーゼとの熱安定性の比較を示す。1 shows a comparison of the thermostability of the ascorbate oxidase of the present invention in Example 1 with the ascorbate oxidase derived from Cucurbita pepo and Cucumis sp.
【図２】実施例２における本発明のアスコルビン酸オキ
シダーゼとククルビタ ペポ（Cucurbita pepo）及びキ
ュキュミス エスピー（Cucumis sp. ）由来のアスコル
ビン酸オキシダーゼとの保存安定性の比較を示す。FIG. 2 shows a comparison of storage stability between the ascorbate oxidase of the present invention in Example 2 and ascorbate oxidase derived from Cucurbita pepo and Cucumis sp.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 愛水 重典 福井県敦賀市東洋町10番24号 東洋紡績株 式会社敦賀バイオ研究所内 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shigenori Aisui 10-24 Toyomachi, Tsuruga City, Fukui Prefecture Toyobo Co., Ltd. Tsuruga Bio Research Institute
Claims (3)
ン酸オキシダーゼ （１）作用：１モルのアスコルビン酸と１／２モルの酵
素より、１モルのデヒドロアスコルビン酸と１モルの水
を生ずる反応を触媒する。 （２）基質特異性：少なくともアスコルビン酸に特異的
に作用する。 （３）至適ｐＨ：５．５〜６．５ （４）ｐＨ安定性：６．０〜１０．０ （５）至適温度：約４５℃ （６）熱安定性：５０℃、１時間処理において、少なく
とも８０％の残存活性を有する。1. An ascorbate oxidase having the following physicochemical properties (1) Action: Catalyzing a reaction that produces 1 mol of dehydroascorbic acid and 1 mol of water from 1 mol of ascorbic acid and 1/2 mol of enzyme. To do. (2) Substrate specificity: It acts specifically on at least ascorbic acid. (3) Optimum pH: 5.5 to 6.5 (4) pH stability: 6.0 to 10.0 (5) Optimum temperature: about 45 ° C (6) Thermal stability: 50 ° C, 1 hour It has a residual activity of at least 80% in the treatment.
モスカータ（Cucurubita moschata ）由来のアスコル
ビン酸オキシダーゼを含有することを特徴とする安定な
液状分析用組成物。2. A stable composition for liquid analysis, which comprises an ascorbate oxidase derived from Cucurubita moschata in the composition for liquid analysis.
oschata ）由来のアスコルビン酸オキシダーゼが請求項
１記載のアスコルビン酸オキシダーゼである請求項２記
載の安定な液状分析用組成物。3. Cucurubita m
The stable liquid composition for analysis according to claim 2, wherein the ascorbate oxidase derived from Oschata is the ascorbate oxidase according to claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP23472992A JP3216735B2 (en) | 1992-09-02 | 1992-09-02 | Novel ascorbate oxidase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP23472992A JP3216735B2 (en) | 1992-09-02 | 1992-09-02 | Novel ascorbate oxidase |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2001068808A Division JP2001299387A (en) | 2001-03-12 | 2001-03-12 | Stable liquid composition for analysis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0678766A true JPH0678766A (en) | 1994-03-22 |
| JP3216735B2 JP3216735B2 (en) | 2001-10-09 |
Family
ID=16975454
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP23472992A Expired - Lifetime JP3216735B2 (en) | 1992-09-02 | 1992-09-02 | Novel ascorbate oxidase |
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| Country | Link |
|---|---|
| JP (1) | JP3216735B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0682116A1 (en) * | 1994-05-11 | 1995-11-15 | Amano Pharmaceutical Co., Ltd. | Ascorbate oxidase, gene encoding the same, process for producing the same, and reagent composition using the same |
| JP2007509098A (en) * | 2003-10-24 | 2007-04-12 | ウエラ アクチェンゲゼルシャフト | Compositions for oxidative treatment of hair or skin, and fixing compositions and methods for permanent deformation of hair |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110051717A (en) * | 2019-05-25 | 2019-07-26 | 广东大鹏医药科技有限公司 | A kind of honeysuckle VC effervescent tablet and preparation method thereof |
-
1992
- 1992-09-02 JP JP23472992A patent/JP3216735B2/en not_active Expired - Lifetime
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0682116A1 (en) * | 1994-05-11 | 1995-11-15 | Amano Pharmaceutical Co., Ltd. | Ascorbate oxidase, gene encoding the same, process for producing the same, and reagent composition using the same |
| JP2007509098A (en) * | 2003-10-24 | 2007-04-12 | ウエラ アクチェンゲゼルシャフト | Compositions for oxidative treatment of hair or skin, and fixing compositions and methods for permanent deformation of hair |
Also Published As
| Publication number | Publication date |
|---|---|
| JP3216735B2 (en) | 2001-10-09 |
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