JPH0783720B2 - Method for inhibiting glycolysis and method for producing glycolytic inhibitor - Google Patents
Method for inhibiting glycolysis and method for producing glycolytic inhibitorInfo
- Publication number
- JPH0783720B2 JPH0783720B2 JP63210528A JP21052888A JPH0783720B2 JP H0783720 B2 JPH0783720 B2 JP H0783720B2 JP 63210528 A JP63210528 A JP 63210528A JP 21052888 A JP21052888 A JP 21052888A JP H0783720 B2 JPH0783720 B2 JP H0783720B2
- Authority
- JP
- Japan
- Prior art keywords
- mannose
- glucose
- blood
- glycolysis
- inhibitor
- 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.)
- Expired - Fee Related
Links
- 238000000034 method Methods 0.000 title claims description 26
- 230000034659 glycolysis Effects 0.000 title claims description 18
- 239000003112 inhibitor Substances 0.000 title claims description 17
- 230000002414 glycolytic effect Effects 0.000 title claims description 13
- 238000004519 manufacturing process Methods 0.000 title claims description 9
- 230000002401 inhibitory effect Effects 0.000 title claims description 8
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 claims description 83
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 70
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 60
- 239000008280 blood Substances 0.000 claims description 26
- 210000004369 blood Anatomy 0.000 claims description 26
- 108010001816 pyranose oxidase Proteins 0.000 claims description 22
- 102000030595 Glucokinase Human genes 0.000 claims description 16
- 108010021582 Glucokinase Proteins 0.000 claims description 16
- 108010050375 Glucose 1-Dehydrogenase Proteins 0.000 claims description 16
- 229920000057 Mannan Polymers 0.000 claims description 9
- 230000023555 blood coagulation Effects 0.000 claims description 8
- 239000012535 impurity Substances 0.000 claims description 7
- 239000000356 contaminant Substances 0.000 claims description 6
- 230000003301 hydrolyzing effect Effects 0.000 claims description 5
- 239000003130 blood coagulation factor inhibitor Substances 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 238000001953 recrystallisation Methods 0.000 claims description 4
- 238000007796 conventional method Methods 0.000 claims description 3
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 claims 2
- 239000008103 glucose Substances 0.000 description 28
- 239000000243 solution Substances 0.000 description 12
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- 229940088598 enzyme Drugs 0.000 description 8
- 238000005259 measurement Methods 0.000 description 8
- 239000000203 mixture Substances 0.000 description 7
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 239000013078 crystal Substances 0.000 description 6
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 230000007062 hydrolysis Effects 0.000 description 5
- 238000006460 hydrolysis reaction Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 241000346081 Kerriodoxa elegans Species 0.000 description 3
- 229920002752 Konjac Polymers 0.000 description 3
- 238000002425 crystallisation Methods 0.000 description 3
- 230000008025 crystallization Effects 0.000 description 3
- 235000013399 edible fruits Nutrition 0.000 description 3
- 235000010485 konjac Nutrition 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 239000006188 syrup Substances 0.000 description 3
- 235000020357 syrup Nutrition 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- 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 2
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- 108010015776 Glucose oxidase Proteins 0.000 description 2
- 229930195725 Mannitol Natural products 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 229960000583 acetic acid Drugs 0.000 description 2
- 102000020006 aldose 1-epimerase Human genes 0.000 description 2
- 108091022872 aldose 1-epimerase Proteins 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 235000013312 flour Nutrition 0.000 description 2
- 235000019420 glucose oxidase Nutrition 0.000 description 2
- 239000000594 mannitol Substances 0.000 description 2
- 235000010355 mannitol Nutrition 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- QXHFVXIQUAOLNU-UHFFFAOYSA-N 1-(3,5-dimethoxyanilino)propane-1-sulfonic acid Chemical compound CCC(S(O)(=O)=O)NC1=CC(OC)=CC(OC)=C1 QXHFVXIQUAOLNU-UHFFFAOYSA-N 0.000 description 1
- HNLXNOZHXNSSPN-UHFFFAOYSA-N 2-[2-[2-[2-[2-[2-[2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol Chemical compound CC(C)(C)CC(C)(C)C1=CC=C(OCCOCCOCCOCCOCCOCCOCCO)C=C1 HNLXNOZHXNSSPN-UHFFFAOYSA-N 0.000 description 1
- MSWZFWKMSRAUBD-CBPJZXOFSA-N 2-amino-2-deoxy-D-mannopyranose Chemical compound N[C@@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-CBPJZXOFSA-N 0.000 description 1
- 102100031126 6-phosphogluconolactonase Human genes 0.000 description 1
- 108010029731 6-phosphogluconolactonase Proteins 0.000 description 1
- 244000247812 Amorphophallus rivieri Species 0.000 description 1
- 235000001206 Amorphophallus rivieri Nutrition 0.000 description 1
- 101001053401 Arabidopsis thaliana Acid beta-fructofuranosidase 3, vacuolar Proteins 0.000 description 1
- 150000000780 D-glucose derivatives Chemical class 0.000 description 1
- 150000000828 D-mannose derivatives Chemical class 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 206010018429 Glucose tolerance impaired Diseases 0.000 description 1
- 108010018962 Glucosephosphate Dehydrogenase Proteins 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 229920000161 Locust bean gum Polymers 0.000 description 1
- ACFIXJIJDZMPPO-NNYOXOHSSA-N NADPH Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 ACFIXJIJDZMPPO-NNYOXOHSSA-N 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 208000001280 Prediabetic State Diseases 0.000 description 1
- 239000012445 acidic reagent Substances 0.000 description 1
- WQZGKKKJIJFFOK-PQMKYFCFSA-N alpha-D-mannose Chemical compound OC[C@H]1O[C@H](O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-PQMKYFCFSA-N 0.000 description 1
- AYJRCSIUFZENHW-DEQYMQKBSA-L barium(2+);oxomethanediolate Chemical compound [Ba+2].[O-][14C]([O-])=O AYJRCSIUFZENHW-DEQYMQKBSA-L 0.000 description 1
- 238000011325 biochemical measurement Methods 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 235000008504 concentrate Nutrition 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000010908 decantation Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- ZYBWTEQKHIADDQ-UHFFFAOYSA-N ethanol;methanol Chemical compound OC.CCO ZYBWTEQKHIADDQ-UHFFFAOYSA-N 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000000252 konjac Substances 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 239000000711 locust bean gum Substances 0.000 description 1
- 235000010420 locust bean gum Nutrition 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 201000009104 prediabetes syndrome Diseases 0.000 description 1
- 150000003214 pyranose derivatives Chemical class 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
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
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Enzymes And Modification Thereof (AREA)
Description
【発明の詳細な説明】 〈産業上の利用分野〉 この発明は、従来容易に得られなかった高純度のＤ−マ
ンノースを使用する、血液の解糖（Ｄ−グルコースの解
糖）を阻止する方法、及び解糖阻止剤の製造方法に関す
るものである。DETAILED DESCRIPTION OF THE INVENTION <Industrial field of application> The present invention inhibits blood glycolysis (glycolysis of D-glucose) using high-purity D-mannose, which has hitherto not been easily obtained. The present invention relates to a method and a method for producing a glycolytic inhibitor.
〈従来の技術と発明が解決しようとする問題点〉 臨床化学検査の分野で最も重要なものの一つである血液
中のグルコースの測定に際しては、採血から測定までの
時間中にグルコースが分解しないように、通常採血時に
解糖阻止剤といわれる製剤を血液に溶解しグルコースの
解糖を抑制している。<Problems to be solved by conventional techniques and inventions> When measuring glucose in blood, which is one of the most important things in the field of clinical chemistry testing, make sure that glucose is not decomposed during the time from blood collection to measurement. In addition, a preparation called a glycolysis inhibitor is usually dissolved in blood at the time of blood collection to suppress glucose glycolysis.
近年、本発明者等はＤ−マンノース及びその誘導体が従
来までの解糖阻止剤の欠点を解消する優れた効果を持つ
ことを明らかにし、先に特許出願した（特願昭61−1480
36号、特願昭62−112289号、及び特願昭62−287014
号）。In recent years, the present inventors have revealed that D-mannose and its derivatives have an excellent effect of eliminating the drawbacks of conventional glycolytic inhibitors, and previously filed a patent application (Japanese Patent Application No. 61-1480).
36, Japanese Patent Application No. 62-112289, and Japanese Patent Application No. 62-287014
issue).
従来、Ｄ−マンノースは次の（Ａ）〜（Ｅ）に記載する
方法により製造されている。Conventionally, D-mannose has been produced by the methods described in the following (A) to (E).
（Ａ）マンナンを多量に含むゾウゲヤシの実、コンニャ
ク粉粒、イナゴマメのゴム等の加水分解 （Ｂ）ホルムアルデヒドの重合 （Ｃ）マンニット（マンニトール）の酸化 （Ｄ）エミールフィシャー（E.Fischer）法（化学大辞
典 第１巻 第44ページ：共立出版 昭和35年発行参
照） （Ｅ）Lobry de Bruyn−van Ekenstein転移 しかしこれらの方法の内、（Ａ）のマンナンを加水分解
する方法は、マンナンには量的な差はあってもグルコー
スがその構成成分として含まれているので、通常の物理
・化学的精製処理では微量のＤ−グルコースの混入（残
存）を避けるのは困難であり、また（Ｂ）〜（Ｅ）の方
法においても、Ｄ−グルコースの共存溶液に、物理・化
学的精製処理を施してＤ−マンノースを得るものである
ため微量のＤ−グルコースの混入（残存）を避けるのは
不可能である。(A) Hydrolysis of elephant palm fruit, konjak powder, locust bean gum, etc. containing a large amount of mannan (B) Polymerization of formaldehyde (C) Oxidation of mannitol (mannitol) (D) Emile Fischer (E. Fischer) method (See The Chemical Dictionary, Volume 1, Page 44: Kyoritsu Shuppan, published in 1960.) (E) Lobry de Bruyn-van Ekenstein transition However, among these methods, the method of hydrolyzing mannan of (A) is mannan. Since glucose is contained as its constituent component even though there is a difference in quantity, it is difficult to avoid mixing (remaining) of a very small amount of D-glucose by ordinary physical and chemical purification treatments. Also in the methods B) to (E), since a coexisting solution of D-glucose is subjected to a physical / chemical purification treatment to obtain D-mannose, a trace amount of D-glucose is mixed (residual). It is impossible to avoid.
しかしながら、解糖阻止剤として使用するＤ−マンノー
スは、この中に夾雑物質としてＤ−グルコースが混入し
ていると、このＤ−グルコースは血糖の測定値に直接影
響（正誤差）を与えるから、Ｄ−マンノースを可能な限
り精製して、Ｄ−グルコースを除去しなければ、解糖阻
止剤として致命的な欠点を有することになる。However, D-mannose used as a glycolytic inhibitor, when D-glucose is mixed as a contaminant in the D-mannose, since this D-glucose directly affects the blood glucose measurement value (positive error), If D-mannose is purified as much as possible and D-glucose is not removed, it will have a fatal defect as a glycolysis inhibitor.
これは、例えばＤ−グルコース１％を含有するＤ−マン
ノースを解糖阻止剤として、通常の濃度の300mg/dl血液
で使用すれば、血液中のグルコースの値を３％以上高く
する結果を招くことになり、糖尿病前状態の受信に対し
て誤診を招く結果にもなりかねない。For example, if D-mannose containing 1% D-glucose is used as a glycolytic inhibitor at a normal concentration of 300 mg / dl blood, the glucose level in blood is increased by 3% or more. This may result in misdiagnosis for receiving the pre-diabetes condition.
〈問題を解決するための手段〉 そこで、本願は前記（Ａ）〜（Ｅ）の方法で得られるＤ
−マンノースを含む溶液を従来の精製法で精製し、Ｄ−
グルコース含有量を一定の値以下とした後、従来の精製
法では除去することが極めて困難であった残存する微量
のＤ−グルコースを、ピラノース酸化酵素、グルコース
脱水素酵素、グルコキナーゼのいずれか一種、またはこ
れらを組み合せて作用させて効率よく分解し、Ｄ−グル
コース含有量を血糖測定値に影響を及ぼさないレベルに
まで低下させて得られる高純度のＤ−マンノースを使用
した、血液の解糖を阻止する方法、及び解糖阻止剤の製
造方法を提供しようとするものである。<Means for Solving Problems> Therefore, the present application provides D obtained by the methods (A) to (E) described above.
-The solution containing mannose was purified by a conventional purification method, and D-
After reducing the glucose content to a certain value or less, the remaining trace amount of D-glucose, which was extremely difficult to remove by the conventional purification method, was used as a pyranose oxidase, glucose dehydrogenase, or glucokinase. , Or a high-purity D-mannose obtained by reducing the D-glucose content to a level that does not affect the blood glucose measurement value by efficiently acting by combining these, and glycolysis of blood. The present invention is intended to provide a method for inhibiting the above, and a method for producing a glycolytic inhibitor.
本願は次の（１）〜（４）に記載する４個の請求項から
構成されている。This application consists of four claims described in the following (1) to (4).
（１）不純物（夾雑物）として微量のＤ−グルコースを
含有するＤ−マンノースに対して、ピラノース酸化酵
素、グルコース脱水素酵素、グルコキナーゼのいずれか
一種、またはこれらを組み合せて作用させて、Ｄ−グル
コース含有量をＤ−マンノースに対して0.5重量％以下
としたＤ−マンノースを血液に添加することを特徴とす
る解糖を阻止する方法。(1) D-mannose containing a small amount of D-glucose as an impurity (contaminant) is allowed to act on any one of pyranose oxidase, glucose dehydrogenase, and glucokinase, or a combination thereof to act D -A method for inhibiting glycolysis, which comprises adding D-mannose to blood having a glucose content of 0.5% by weight or less based on D-mannose.
（２）不純物（夾雑物）として微量のＤ−グルコースを
含有するＤ−マンノースに対して、ピラノース酸化酵
素、グルコース脱水素酵素、グルコキナーゼのいずれか
一種、またはこれらを組み合せて作用させて、Ｄ−グル
コース含有量をＤ−マンノースに対して0.5重量％以下
としたＤ−マンノースに、必要に応じて血液凝固阻止剤
または血液凝固促進剤を添加することを特徴とする解糖
阻止剤の製造方法。(2) D-mannose containing a small amount of D-glucose as an impurity (contaminant) is allowed to act on one or a combination of pyranose oxidase, glucose dehydrogenase, and glucokinase, and D -A method for producing a glycolysis inhibitor, which comprises adding a blood coagulation inhibitor or a blood coagulation promoter to D-mannose having a glucose content of 0.5% by weight or less based on D-mannose, if necessary. .
（３）マンナンを加水分解した後、この加水分解を精製
してＤ−マンノースを製造し、このＤ−マンノースを血
液に添加して解糖を阻止する方法において、Ｄ−マンノ
ースの不純物（夾雑物）として含まれるＤ−グルコース
含有量をＤ−マンノースに対して２重量％以下まで精製
した粗Ｄ−マンノースに対し、ピラノース酸化酵素、グ
ルコース脱水素酵素、グルコキナーゼのいずれか一種、
またはこれらを組み合せて作用させて、Ｄ−グルコース
含有量をＤ−マンノースに対して0.5重量％以下とした
Ｄ−マンノースを血液に添加することを特徴とする解糖
を阻止する方法。(3) After hydrolyzing mannan, the hydrolysis is purified to produce D-mannose, and the D-mannose is added to blood to inhibit glycolysis. ), The crude D-mannose purified to a D-glucose content of 2% by weight or less with respect to D-mannose, pyranose oxidase, glucose dehydrogenase, or any one of glucokinase,
Alternatively, a method for inhibiting glycolysis, which is characterized in that D-mannose having a D-glucose content of 0.5% by weight or less with respect to D-mannose is added to blood by acting these in combination.
（４）次の（Ａ）、（Ｂ）、（Ｃ）工程を順次経て製造
される解糖阻止剤の製造方法において、（Ｂ）工程に対
し、この工程における精製中のＤ−マンノースの純度
が、不純物としてＤ−グルコースをＤ−マンノースに対
して２重量％〜0.5重量％含有する範囲に達したとき、
この粗Ｄ−マンノースに対し、ピラノース酸化酵素、グ
ルコース脱水素酵素、グルコキナーゼのいずれか一種、
またはこれらを組み合せて作用させ、Ｄ−グルコース含
有量をＤ−マンノースの0.5重量％以下とする工程を付
加することを特徴とする解糖阻止剤の製造方法。(4) In the method for producing a glycolytic inhibitor, which is sequentially produced through the following steps (A), (B), and (C), the purity of D-mannose during purification in this step is different from that in the step (B). Reaches a range containing 2 wt% to 0.5 wt% of D-glucose as an impurity with respect to D-mannose,
For this crude D-mannose, any one of pyranose oxidase, glucose dehydrogenase, and glucokinase,
Alternatively, a method for producing a glycolytic inhibitor, which comprises adding a step of causing these to act in combination to make the D-glucose content 0.5% by weight or less of D-mannose.
（Ａ）工程：マンナンを加水分解する工程 （Ｂ）工程：（Ａ）工程により得られる加水分解液を常
法（再結晶法）により精製してＤ−マンノースを製造す
る工程 （Ｃ）工程：（Ｂ）工程により得られるＤ−マンノース
に必要に応じて血液凝固阻止剤または血液凝固促進剤を
添加する工程 本願発明は、Ｄ−マンノース中のＤ−グルコースを分解
除去できるばかりではなく、Ｄ−マンノースと同様に血
液の解糖阻止剤として使用されるＤ−マンノース誘導体
（例えばＤ−マンノサミン）中のＤ−グルコースを同様
に分解除去することも可能である。また、血糖測定ばか
りではなく、広くグルコース測定系を利用した他の項目
測定用の、干渉を与えない血液試料を提供できるという
点で重量である。Step (A): Step of hydrolyzing mannan Step (B): Step of purifying the hydrolyzed liquid obtained in the step (A) by a conventional method (recrystallization method) to produce D-mannose (C) Step: (B) A step of adding a blood coagulation inhibitor or a blood coagulation promoter to the D-mannose obtained in the step as needed. The present invention can not only decompose and remove D-glucose in D-mannose, but also D-mannose. It is also possible to decompose and remove D-glucose in a D-mannose derivative (for example, D-mannosamine) used as an inhibitor of blood glycolysis in the same manner as mannose. In addition, it is a weight because it can provide a blood sample not only for blood glucose measurement but also for measuring other items using a glucose measurement system widely, and not giving interference.
本願発明に使用可能な酵素は、ピラノース酸化酵素（Yo
go Machida and Toru Nakanishi,Agric.Biol Chem.,48
（10）,2463,1984）、グルコース脱水素酵素（H.U.Berg
meyer et al.,Method of Enzymatic Analysis,vol.VI 3
rd ed.,pp 172,1984）、グルコキナーゼ（富田耕右,Bio
Industry,3（９）,5,1986）であり、好ましくはピラノ
ース酸化酵素である。その理由は次の通りである。The enzyme usable in the present invention is pyranose oxidase (Yo
go Machida and Toru Nakanishi, Agric.Biol Chem., 48
(10), 2463,1984), glucose dehydrogenase (HUBerg
meyer et al., Method of Enzymatic Analysis, vol.VI 3
rd ed., pp 172,1984), Glucokinase (Koji Tomita, Bio
Industry, 3 (9), 5, 1986), preferably pyranose oxidase. The reason is as follows.
（イ）すべてのグルコース酸化酵素はＤ−マンノースと
反応するが、ピラノース酸化酵素、グルコース脱水素酵
素、グルコキナーゼはＤ−マンノースと反応しない（第
５表参照）。(A) All glucose oxidases react with D-mannose, but pyranose oxidase, glucose dehydrogenase, and glucokinase do not react with D-mannose (see Table 5).
（ロ）しかし、グルコース脱水素酵素には、遊離のNAD
（補酵素）、グルコキナーゼには、同様にATPが必要と
なる。従って、酵素処理後に未反応物、及び反応生成物
をＤ−マンノースから分離除去する工程が必要となり、
剤型化までの操作過程が幾分複雑なものとなりコスト高
となり易い。(B) However, glucose dehydrogenase contains free NAD
ATP is also required for (coenzyme) and glucokinase. Therefore, a step of separating and removing unreacted substances and reaction products from D-mannose is required after the enzyme treatment,
The operation process until formulation is somewhat complicated and the cost tends to increase.
（ハ）一方、ピラノース酸化酵素は単独で触媒活性を発
現するから酵素とＤ−マンノース溶液との接触のみで目
的を達成できる。(C) On the other hand, pyranose oxidase expresses catalytic activity by itself, so that the purpose can be achieved only by contacting the enzyme with a D-mannose solution.
本願発明において、ピラノース酸化酵素、グルコース脱
水素酵素、グルコキナーゼ等を作用させる、微量のＤ−
グルコースを含有するＤ−マンノース（粗Ｄ−マンノー
ス）は、マンナンの加水分解に由来するものに限らず、
他の如何なる方法に由来するものであってもよい。In the present invention, a trace amount of D- which causes pyranose oxidase, glucose dehydrogenase, glucokinase, etc. to act.
D-mannose containing glucose (crude D-mannose) is not limited to that derived from the hydrolysis of mannan,
It may be derived from any other method.
〈実施例〉 マンナンの原料として、ゾウゲヤシの実とコンニャク粉
を原料として使用し、下記に記載する方法で加水分解、
結晶化、再結晶化を繰り返しＤ−マンノース（試料Ａ、
試料Ｂ）を製造した。<Example> As a raw material of mannan, using elephant palm fruit and konjak flour as raw materials, hydrolysis by the method described below,
Crystallization and recrystallization were repeated and D-mannose (Sample A,
Sample B) was produced.
（ａ）加水分解：ゾウゲヤシの実、またはコンニャク粉
1000gに75％硫酸を1000g加えてミキサーにかけスラリー
とした後35〜40℃で12時間放置した。これに水10lを加
えて攪拌し、布でロカしてロ液を得た。このロ液の容量
が減らないように水を加えつつ６時間煮沸した。(A) Hydrolysis: Elephant palm fruit or konjac flour
To 1000 g, 1000 g of 75% sulfuric acid was added, and the mixture was mixed with a mixer to form a slurry, which was then left at 35 to 40 ° C for 12 hours. To this, 10 l of water was added, and the mixture was stirred and located with a cloth to obtain a solution. The solution was boiled for 6 hours while adding water so that the volume of the solution did not decrease.
（ｂ）結晶化：この煮沸液を炭酸バリウムで中和し、活
性炭を加えてロカし脱色した。ロ液を約85％に濃縮し、
温メタノール100mlを加えた後、更にイソプロピルアル
コール2000mlで稀釈した。デカンテイションにより上澄
みを採取し、ゴム状残分はメタノールと、イソプロピル
アルコールで数回抽出し、上澄みを合せ、活性炭を加え
てロカ脱色し、約60％のシロップに濃縮した。このシロ
ップを氷酢酸250mlに溶かし、Ｄマンノースの種を加え
て、室温で数日放置し、生成した結晶をエタノール−メ
タノール混液で洗浄した後結晶を採取した。母液は再濃
縮し、前記と同様な操作でＤ−マンノースの結晶を採取
した。両者を合せて約350gのＤ−マンノースの結晶（粗
結晶）が得られた。(B) Crystallization: The boiling liquid was neutralized with barium carbonate, activated carbon was added, and the mixture was bleached to remove the color. Concentrate the liquid to about 85%,
After adding 100 ml of warm methanol, the mixture was further diluted with 2000 ml of isopropyl alcohol. The supernatant was collected by decantation, and the rubbery residue was extracted several times with methanol and isopropyl alcohol. The supernatants were combined, activated carbon was added to decolorize the roca, and the mixture was concentrated to about 60% syrup. This syrup was dissolved in 250 ml of glacial acetic acid, seeds of D-mannose were added, the mixture was allowed to stand at room temperature for several days, and the formed crystals were washed with an ethanol-methanol mixed solution and then collected. The mother liquor was re-concentrated and D-mannose crystals were collected by the same procedure as above. Both were combined to obtain about 350 g of D-mannose crystals (crude crystals).
（ｃ）再結晶:D−マンノースの粗結晶に水を同重量加え
て溶解し、酢酸１滴と脱色炭0.5gを加えてロカし、ロ液
を約85％に減圧濃縮した。このシロップを前記結晶化の
項に記載したと同様な方法でＤ−マンノースを結晶化さ
せた。(C) Recrystallization: The same weight of water was added to the crude crystal of D-mannose to dissolve it, and 1 drop of acetic acid and 0.5 g of decolorizing carbon were added to the solution for localization, and the solution was concentrated under reduced pressure to about 85%. D-mannose was crystallized from this syrup by the same method as described in the above crystallization section.
得られた試料Ａ、試料ＢのＤ−マンノースの結晶のＤ−
グルコースの含量と、参考として市販のＤ−（＋）−マ
ンノース（試薬）中のＤ−グルコースの含量を第１表
（巻末）に示す。The obtained sample A and sample B D-mannose crystal D-
The content of glucose and the content of D-glucose in commercially available D-(+)-mannose (reagent) are shown in Table 1 (at the end) as a reference.
第１表によれば、Ｄ−マンノース中のＤ−グルコースの
含量は非常に高いものもあり、これをこのまま解糖阻止
剤として使用すれば、血糖の測定値に大きな影響を与
え、無視することができないことがわかる。According to Table 1, the content of D-glucose in D-mannose is very high, and if it is used as it is as a glycolytic inhibitor, it will have a great influence on the blood glucose measurement value and should be ignored. I understand that I can not do it.
（ｄ）ピラノース酸化酵素によるＤ−マンノース中のＤ
−グルコースの酸化： 1mMリン酸緩衝液（pH7.0）に溶解した10mMのＤ−マンノ
ース溶液（試料Ａから調製）100mlを、予じめ37℃に加
温しておき、ピラノース酸化酵素（100U/ml）1mlを加
え、攪拌下、グルコース量とマンノース量の経時変化を
調べた。酵素添加後、20分、40分、60分、120分、の各
時点で一部を採取し下記の試薬（Ｉ）、（II）を使用す
る酵素法により比色定量（GOD法）した。(D) D in D-mannose by pyranose oxidase
-Glucose oxidation: 100 ml of a 10 mM D-mannose solution (prepared from sample A) dissolved in 1 mM phosphate buffer (pH 7.0) was preliminarily heated to 37 ° C and pyranose oxidase (100 U was added). / ml) 1 ml was added, and changes with time of glucose amount and mannose amount were examined under stirring. After addition of the enzyme, a part was sampled at each of 20 minutes, 40 minutes, 60 minutes, and 120 minutes, and colorimetric determination (GOD method) was carried out by an enzyme method using the following reagents (I) and (II).
すなわち、下記の試薬（Ｉ）、（II）各1mlを37℃に予
め加温しておき、前記夫々の酵素反応後の試料200μｌ
を添加して反応を始めた。580nmで１分後の１分間当り
の吸光度変化量と、３分後と４分後の１分間当りの吸光
度変化量を測定した。同様に試料を検量用標準液に変え
て測定を行ない、各々Ｄ−グルコース量とＤ−マンノー
ス量を算出した。その結果を第２表（巻末）に示す。That is, 1 ml each of the following reagents (I) and (II) was preheated to 37 ° C., and 200 μl of the sample after each enzyme reaction was performed.
Was added to start the reaction. At 580 nm, the change in absorbance per minute after 1 minute and the change in absorbance per minute after 3 minutes and 4 minutes were measured. Similarly, the sample was changed to the standard solution for calibration and the measurement was performed to calculate the D-glucose amount and the D-mannose amount, respectively. The results are shown in Table 2 (at the end of this document).
第２表の結果によれば、ピラノース酸化酵素を添加後、
60分で約90％のＤ−グルコースを分解させることができ
る。According to the results in Table 2, after adding pyranose oxidase,
About 60% of D-glucose can be decomposed in 60 minutes.
試薬（Ｉ）:1.7mM 3,5−ジメトキシアニリノプロパンス
ルホン酸を含む0.15Mリン酸緩衝液（pH6.0） 試薬（II）：ムタロターゼ1.6U/ml、グルコース酸化酵
素1.7×104U/ml、ペルオキシターゼ5600U/ml、1.0mM 4
−アミノアンチピリン、0.2％Triton×100を含む50mM
リン酸緩衝液（pH7.2） 検量用標準液は、Ｄ−マンノースの6mg/mlと、Ｄ−マン
ノースの0.2mg/mlを使用した。Reagent (I): 0.15 M phosphate buffer (pH 6.0) containing 1.7 mM 3,5-dimethoxyanilinopropanesulfonic acid Reagent (II): Mutarotase 1.6 U / ml, glucose oxidase 1.7 × 10 4 U / ml, peroxidase 5600 U / ml, 1.0 mM 4
-Aminoantipyrine, 50 mM containing 0.2% Triton x 100
Phosphate buffer (pH 7.2) As the standard solution for calibration, 6 mg / ml of D-mannose and 0.2 mg / ml of D-mannose were used.
（ｅ）グルコキナーゼによるＤ−マンノース中のＤ−グ
ルコースの分解： 8mM MgCl2,2mM NADPH,2mM ATPを含む20mM トリス塩酸
緩衝液（pH8.5）に溶解した10mMのＤ−マンノース溶液
（試料１から調製）100mlを37℃に予じめ加温し、グル
コキナーゼ（188U/ml）1mlとグルコース−６−リン酸脱
水素酵素（126U/ml）1mlを加え攪拌下に、グルコース量
とマンノース量の経時変化を調べた。その結果を第３表
に示す。酵素添加後、120分で約90％のＤ−グルコース
を分解させることができる。(E) Degradation of D-glucose in D-mannose by glucokinase: 10 mM D-mannose solution (Sample 1) dissolved in 20 mM Tris-HCl buffer (pH 8.5) containing 8 mM MgCl 2 , 2 mM NADPH, 2 mM ATP. 100 ml) was preheated to 37 ° C and warmed, 1 ml of glucokinase (188U / ml) and 1 ml of glucose-6-phosphate dehydrogenase (126U / ml) were added, and the amount of glucose and mannose were stirred. Was examined over time. The results are shown in Table 3. About 90% of D-glucose can be decomposed 120 minutes after the addition of the enzyme.
（ｆ）グルコース脱水素酵素によるＤ−マンノース中の
Ｄ−グルコースの酸化 0.5mMNADを含む20mMトリス塩酸緩衝液8pH8.2）に溶解し
た10mMのＤ−マンノース溶液（試料１から調製）100ml
を37℃に予じめ加温し、グルコース脱水素酵素（500U/m
l）1mlとムタロターゼ（80U/ml）1mlを加え攪拌下に、
グルコース量とマンノース量の経時変化を（ｄ）と同様
に調べた。その結果を第４表に示す。この結果によれ
ば、酵素添加後、120分で約70％のＤ−グルコースを分
解させることができる。(F) Oxidation of D-glucose in D-mannose by glucose dehydrogenase 100 ml of a 10 mM D-mannose solution (prepared from sample 1) dissolved in 20 mM Tris-HCl buffer 8 pH 8.2 containing 0.5 mM NAD
Preheat to 37 ℃ and heat it to glucose dehydrogenase (500 U / m
l) 1 ml and mutarotase (80 U / ml) 1 ml are added and stirred,
The changes with time of the glucose amount and the mannose amount were examined as in (d). The results are shown in Table 4. According to this result, about 70% of D-glucose can be decomposed 120 minutes after the addition of the enzyme.
（ｇ）ピラノース酸化酵素処理済みＤ−マンノースを使
用した解糖阻止効果： 前記（ｄ）で120分間ピラノース酸化酵素処理したＤ−
マンノース溶液を70℃で30分間加温し酵素を失活させた
後、冷却し、その後凍結乾燥した（−60℃,12時間）。(G) Glycolytic inhibitory effect using D-mannose treated with pyranose oxidase: D-treated with pyranose oxidase for 120 minutes in the above (d)
The mannose solution was heated at 70 ° C for 30 minutes to inactivate the enzyme, cooled, and then freeze-dried (-60 ° C, 12 hours).
この凍結乾燥品100mg（88mgのＤ−マンノースを含有）
を蒸留水880μｌで溶解し、その150μｌを、予め採血し
た血液（ヘパリン30IU/ml含有）5mlに加え、室温に放置
した。100 mg of this lyophilized product (containing 88 mg of D-mannose)
Was dissolved in 880 μl of distilled water, and 150 μl thereof was added to 5 ml of blood (containing 30 IU / ml of heparin) collected in advance and left at room temperature.
同様にピラノース酸化酵素未処理標品を添加したもの
（試料１）及びＤ−マンノース標品無添加（試料２）の
各血漿を作製した。Similarly, each plasma of the sample to which the pyranose oxidase untreated sample was added (Sample 1) and the D-mannose sample not added (Sample 2) was prepared.
１時間、２時間、３時間、８時間後、各々その1mlを採
取し、12000r.p.m.で６分間の遠心分離を行ない、その
上清を使って血漿中のグルコース含量を測定した。同様
にピラノース酸化酵素未処理標品（試料１）、及びＤ−
マンノース無添加の試料（試料２）を用いて血漿中のグ
ルコース含量を測定し、三者を比較した。その結果図面
（巻末）に示す。After 1 hour, 2 hours, 3 hours, and 8 hours, 1 ml of each was collected, centrifuged at 12000 rpm for 6 minutes, and the supernatant was used to measure the glucose content in plasma. Similarly, a pyranose oxidase-untreated preparation (Sample 1), and D-
The glucose content in plasma was measured using a sample without mannose addition (Sample 2), and the three were compared. The results are shown in the drawing (at the end of this document).
図面によれば、ピラノース酸化酵素処理を行なったＤ−
マンノースは、未処理標品（試料１）と比較して、全て
の放置時間経過後の測定区分において、一定の低いグル
コースの値（血糖値）を示した。これは、処理品はグル
コースを含まないため真の値に近く、一方未処理品はグ
ルコースを含有しているため真の値より大きい値を示し
たものと考えられる。According to the drawing, D- treated with pyranose oxidase
Mannose showed a constant low glucose level (blood glucose level) in all the measurement categories after the passage of time, as compared with the untreated standard (Sample 1). It is considered that this is because the treated product was close to the true value because it did not contain glucose, whereas the untreated product contained glucose and was higher than the true value.
また、ピラノース酸化酵素処理品もＤ−マンノース無添
加（試料２）に比較して充分な解糖阻止効果を維持して
いた。In addition, the pyranose oxidase-treated product also maintained a sufficient glycolytic inhibition effect as compared with the D-mannose-free product (Sample 2).
なお、グルコキナーゼ、或いはグルコース脱水素酵素を
用いたときも同様な効果が認められた。Similar effects were observed when glucokinase or glucose dehydrogenase was used.
〈発明の効果〉 本願発明は、以上のように構成したから解糖阻止作用を
有するＤ−マンノースを使用しても、Ｄ−グルコースが
混入することがなく、血糖及びグルコース反応系を利用
した他の生化学的測定に干渉を与えない血液試料を提供
できるという効果を有する。<Effects of the Invention> Since the present invention is configured as described above, even when D-mannose having a glycolysis-inhibiting action is used, D-glucose is not contaminated and a blood glucose and glucose reaction system is used. It has the effect of providing a blood sample that does not interfere with the biochemical measurement of.
図面は、解糖阻止剤を使用した血液中の血糖値を示すグ
ラフである。The drawing is a graph showing the blood glucose level in blood using a glycolytic inhibitor.
Claims (4)
ースを含有するＤ−マンノースに対して、ピラノース酸
化酵素、グルコース脱水素酵素、グルコキナーゼのいず
れか一種、またはこれらを組み合せて作用させて、Ｄ−
グルコース含有量をＤ−マンノースに対して0.5重量％
以下としたＤ−マンノースを血液に添加することを特徴
とする解糖を阻止する方法。1. A D-mannose containing a trace amount of D-glucose as an impurity (contaminant) is allowed to act on any one of pyranose oxidase, glucose dehydrogenase, glucokinase, or a combination thereof. , D-
Glucose content is 0.5% by weight based on D-mannose
The following method for inhibiting glycolysis, which comprises adding D-mannose to blood.
ースを含有するＤ−マンノースに対して、ピラノース酸
化酵素、グルコース脱水素酵素、グルコキナーゼのいず
れか一種、またはこれらを組み合せて作用させて、Ｄ−
グルコース含有量をＤ−マンノースに対して0.5重量％
以下としたＤ−マンノースに、必要に応じて血液凝固阻
止剤または血液凝固促進剤を添加することを特徴とする
解糖阻止剤の製造方法。2. A D-mannose containing a trace amount of D-glucose as an impurity (contaminant) is allowed to act on any one of pyranose oxidase, glucose dehydrogenase, glucokinase, or a combination thereof. , D-
Glucose content is 0.5% by weight based on D-mannose
A method for producing a glycolysis inhibitor, which comprises adding a blood coagulation inhibitor or a blood coagulation promoter to the following D-mannose as needed.
液を精製してＤ−マンノースを製造し、このＤ−マンノ
ースを血液に添加して解糖を阻止する方法において、Ｄ
−マンノースの不純物（夾雑物）として含まれるＤ−グ
ルコース含有量をＤ−マンノースに対して２重量％以下
まで精製した粗Ｄ−マンノースに対し、ピラノース酸化
酵素、グルコース脱水素酵素、グルコキナーゼのいずれ
か一種、またはこれらを組み合せて作用させて、Ｄ−グ
ルコース含有量をＤ−マンノースに対して0.5重量％以
下としたＤ−マンノースを血液に添加することを特徴と
する解糖を阻止する方法。3. A method of hydrolyzing mannan, purifying the hydrolyzed solution to produce D-mannose, and adding the D-mannose to blood to inhibit glycolysis.
Any of pyranose oxidase, glucose dehydrogenase, and glucokinase for crude D-mannose purified to a content of D-glucose contained as impurities (contaminants) of mannose to 2% by weight or less with respect to D-mannose A method for inhibiting glycolysis, which comprises adding D-mannose having a D-glucose content of 0.5% by weight or less to D-mannose to blood by acting one kind or a combination thereof.
て製造される解糖阻止剤の製造方法において、（Ｂ）工
程に対し、この工程における精製中のＤ−マンノースの
純度が、不純物としてＤ−グルコースをＤ−マンノース
に対して２重量％〜0.5重量％含有する範囲に達したと
き、この粗Ｄ−マンノースに対し、ピラノース酸化酵
素、グルコース脱水素酵素、グルコキナーゼのいずれか
一種、またはこれらを組み合せて作用させ、Ｄ−グルコ
ース含有量をＤ−マンノースの0.5重量％以下とする工
程を付加することを特徴とする解糖阻止剤の製造方法。 （Ａ）工程：マンナンを加水分解する工程 （Ｂ）工程：（Ａ）工程により得られる加水分解液を常
法（再結晶法）により精製してＤ−マンノースを製造す
る工程 （Ｃ）工程：（Ｂ）工程により得られるＤ−マンノース
に必要に応じて血液凝固阻止剤または血液凝固促進剤を
添加する工程4. A method for producing a glycolytic inhibitor, which is produced by sequentially performing the following steps (A), (B) and (C), wherein D-mannose being purified in this step is used in contrast to step (B). The purity of D-glucose as an impurity reaches a range containing 2% by weight to 0.5% by weight of D-mannose, pyranose oxidase, glucose dehydrogenase and glucokinase are added to the crude D-mannose. A method for producing a glycolysis inhibitor, which comprises adding any one of the above or a combination thereof to make the D-glucose content 0.5% by weight or less of D-mannose. Step (A): Step of hydrolyzing mannan Step (B): Step of purifying the hydrolyzed liquid obtained in the step (A) by a conventional method (recrystallization method) to produce D-mannose (C) Step: (B) A step of adding a blood coagulation inhibitor or a blood coagulation promoter to the D-mannose obtained in the step, if necessary.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63210528A JPH0783720B2 (en) | 1988-08-26 | 1988-08-26 | Method for inhibiting glycolysis and method for producing glycolytic inhibitor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63210528A JPH0783720B2 (en) | 1988-08-26 | 1988-08-26 | Method for inhibiting glycolysis and method for producing glycolytic inhibitor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0259668A JPH0259668A (en) | 1990-02-28 |
| JPH0783720B2 true JPH0783720B2 (en) | 1995-09-13 |
Family
ID=16590857
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63210528A Expired - Fee Related JPH0783720B2 (en) | 1988-08-26 | 1988-08-26 | Method for inhibiting glycolysis and method for producing glycolytic inhibitor |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0783720B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FI114553B (en) * | 2001-12-31 | 2004-11-15 | Danisco Sweeteners Oy | Procedure for collecting sugar |
-
1988
- 1988-08-26 JP JP63210528A patent/JPH0783720B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0259668A (en) | 1990-02-28 |
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