JPS63196300A - Determination of alpha-amylase activity - Google Patents
Determination of alpha-amylase activityInfo
- Publication number
- JPS63196300A JPS63196300A JP2894387A JP2894387A JPS63196300A JP S63196300 A JPS63196300 A JP S63196300A JP 2894387 A JP2894387 A JP 2894387A JP 2894387 A JP2894387 A JP 2894387A JP S63196300 A JPS63196300 A JP S63196300A
- Authority
- JP
- Japan
- Prior art keywords
- formula
- inositol
- amylase activity
- glucose
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000000694 effects Effects 0.000 title claims abstract description 26
- 102000004139 alpha-Amylases Human genes 0.000 title claims abstract description 21
- 108090000637 alpha-Amylases Proteins 0.000 title claims abstract description 21
- 229940024171 alpha-amylase Drugs 0.000 title claims abstract description 21
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims abstract description 22
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims abstract description 22
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims abstract description 21
- 229960000367 inositol Drugs 0.000 claims abstract description 21
- 239000000758 substrate Substances 0.000 claims abstract description 20
- FYGDTMLNYKFZSV-DZOUCCHMSA-N alpha-D-Glcp-(1->4)-alpha-D-Glcp-(1->4)-D-Glcp Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)O[C@H](O[C@@H]2[C@H](OC(O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-DZOUCCHMSA-N 0.000 claims abstract description 13
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims abstract description 7
- 102000004366 Glucosidases Human genes 0.000 claims abstract description 6
- 108010056771 Glucosidases Proteins 0.000 claims abstract description 6
- 101710088194 Dehydrogenase Proteins 0.000 claims abstract description 5
- 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 19
- 239000008103 glucose Substances 0.000 claims description 19
- 239000004382 Amylase Substances 0.000 claims description 6
- 102000013142 Amylases Human genes 0.000 claims description 6
- 108010065511 Amylases Proteins 0.000 claims description 6
- 235000019418 amylase Nutrition 0.000 claims description 6
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 4
- 229910052717 sulfur Inorganic materials 0.000 claims description 3
- 125000004434 sulfur atom Chemical group 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical group OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 2
- 229910052783 alkali metal Inorganic materials 0.000 claims description 2
- 150000001340 alkali metals Chemical class 0.000 claims description 2
- 125000000217 alkyl group Chemical group 0.000 claims description 2
- 125000004430 oxygen atom Chemical group O* 0.000 claims description 2
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 claims 2
- 238000000691 measurement method Methods 0.000 claims 2
- 229950006238 nadide Drugs 0.000 claims 2
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide 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](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 abstract description 5
- 238000002835 absorbance Methods 0.000 abstract description 4
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 abstract description 4
- 238000000034 method Methods 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 7
- 238000005259 measurement Methods 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 229920000858 Cyclodextrin Polymers 0.000 description 5
- 108010025880 Cyclomaltodextrin glucanotransferase Proteins 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 4
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 4
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 229920001353 Dextrin Polymers 0.000 description 3
- 239000004375 Dextrin Substances 0.000 description 3
- 235000019425 dextrin Nutrition 0.000 description 3
- 150000002303 glucose derivatives Chemical class 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 102000005548 Hexokinase Human genes 0.000 description 2
- 108700040460 Hexokinases Proteins 0.000 description 2
- 102100024295 Maltase-glucoamylase Human genes 0.000 description 2
- FTNIPWXXIGNQQF-UHFFFAOYSA-N UNPD130147 Natural products OC1C(O)C(O)C(CO)OC1OC1C(CO)OC(OC2C(OC(OC3C(OC(OC4C(OC(O)C(O)C4O)CO)C(O)C3O)CO)C(O)C2O)CO)C(O)C1O FTNIPWXXIGNQQF-UHFFFAOYSA-N 0.000 description 2
- 108010028144 alpha-Glucosidases Proteins 0.000 description 2
- 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 description 2
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004040 coloring Methods 0.000 description 2
- 150000004001 inositols Chemical class 0.000 description 2
- FJCUPROCOFFUSR-UHFFFAOYSA-N malto-pentaose Natural products OC1C(O)C(OC(C(O)CO)C(O)C(O)C=O)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 FJCUPROCOFFUSR-UHFFFAOYSA-N 0.000 description 2
- FJCUPROCOFFUSR-GMMZZHHDSA-N maltopentaose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O[C@H]([C@H](O)CO)[C@H](O)[C@@H](O)C=O)O[C@H](CO)[C@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O[C@@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)O)[C@@H](CO)O2)O)[C@@H](CO)O1 FJCUPROCOFFUSR-GMMZZHHDSA-N 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 229920001542 oligosaccharide Polymers 0.000 description 2
- 150000002482 oligosaccharides Chemical class 0.000 description 2
- 150000002989 phenols Chemical class 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- WMXFNCKPYCAIQW-UHFFFAOYSA-N 1,2-dimethoxy-3-methylbenzene Chemical compound COC1=CC=CC(C)=C1OC WMXFNCKPYCAIQW-UHFFFAOYSA-N 0.000 description 1
- HEWZVZIVELJPQZ-UHFFFAOYSA-N 2,2-dimethoxypropane Chemical compound COC(C)(C)OC HEWZVZIVELJPQZ-UHFFFAOYSA-N 0.000 description 1
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 1
- 102100031126 6-phosphogluconolactonase Human genes 0.000 description 1
- 108010029731 6-phosphogluconolactonase Proteins 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000193752 Bacillus circulans Species 0.000 description 1
- 241000194107 Bacillus megaterium Species 0.000 description 1
- 239000001116 FEMA 4028 Substances 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 108010018962 Glucosephosphate Dehydrogenase Proteins 0.000 description 1
- LSDPWZHWYPCBBB-UHFFFAOYSA-N Methanethiol Chemical compound SC LSDPWZHWYPCBBB-UHFFFAOYSA-N 0.000 description 1
- 241000178960 Paenibacillus macerans Species 0.000 description 1
- 206010033645 Pancreatitis Diseases 0.000 description 1
- 206010033647 Pancreatitis acute Diseases 0.000 description 1
- 206010034038 Parotitis Diseases 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 238000011481 absorbance measurement Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000003377 acid catalyst Substances 0.000 description 1
- 201000003229 acute pancreatitis Diseases 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 150000001350 alkyl halides Chemical class 0.000 description 1
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 description 1
- 235000011175 beta-cyclodextrine Nutrition 0.000 description 1
- 229960004853 betadex Drugs 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000010531 catalytic reduction reaction Methods 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 230000001268 conjugating effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000005562 fading Methods 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 108010046301 glucose peroxidase Proteins 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- RJQKKZNUWRIHCS-VBNQBVOGSA-N maltodecaose Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@H]1[C@H](CO)O[C@@H](O[C@H]2[C@@H](O[C@@H](O[C@H]3[C@@H](O[C@@H](O[C@H]4[C@@H](O[C@@H](O[C@H]5[C@@H](O[C@@H](O[C@@H]6[C@H](O[C@H](O[C@@H]7[C@H](O[C@H](O[C@@H]8[C@H](O[C@H](O[C@@H]9[C@H](OC(O)[C@H](O)[C@H]9O)CO)[C@H](O)[C@H]8O)CO)[C@H](O)[C@H]7O)CO)[C@H](O)[C@H]6O)CO)[C@@H](O)[C@@H]5O)CO)[C@@H](O)[C@@H]4O)CO)[C@@H](O)[C@@H]3O)CO)[C@@H](O)[C@@H]2O)CO)[C@@H](O)[C@@H]1O RJQKKZNUWRIHCS-VBNQBVOGSA-N 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 150000003459 sulfonic acid esters Chemical class 0.000 description 1
- 150000003641 trioses Chemical class 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発面唸α−アミラーゼ活性測定方法に関するものであ
る。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for measuring α-amylase activity.
急性すい炎、耳下腺炎等の診断のために血清や尿中のα
−アミラーゼ活性を測定する手法がある。α in serum and urine for diagnosis of acute pancreatitis, parotitis, etc.
-There are methods to measure amylase activity.
アミラーゼ活性測定用基質として、従来はでんぷんが用
いられてきたが、精度の点で難点があった。Starch has conventionally been used as a substrate for measuring amylase activity, but there have been drawbacks in terms of accuracy.
近年、でんぷんにかわって、マルトペンタオース（Ｇ５
）を代表とするマルトオリゴ塘がアミラーゼ活性測定用
基質として採用されつつある。In recent years, maltopentaose (G5
) is increasingly being adopted as a substrate for measuring amylase activity.
α−アミラーゼの共役酵素としてα−グルコシダーゼを
用いると、次の方法によりてα−アミラーゼの活性を測
定することができる。When α-glucosidase is used as the conjugate enzyme for α-amylase, the activity of α-amylase can be measured by the following method.
ｃ ５ ｄ−ＺコＥ？−−ｔｚ→マルトース（Ｇ２）
＋ツル上トリオース（Ｇ３）ｃｚ＋０３！！上ニア４２
己？望ｔ＝ｊ（−５ａ １ここに生成したグルコース（
Ｇｌ ）は、例えばグルコースオキシダーゼ／パーオキ
シダーゼ／色素系又はヘキソキナーゼ／水人ホグルコム
ターゼ／グルコース−６−ホスフェートデヒドロゲナー
ゼ／ＮＡＤＨ系等により定量される。c 5 d-ZkoE? --tz→maltose (G2)
+Triose on the vine (G3) cz+03! ! upper near 42
Myself? Desired t=j(-5a 1 Glucose produced here (
Gl ) is quantified, for example, by a glucose oxidase/peroxidase/dye system or a hexokinase/hydrophoglucomutase/glucose-6-phosphate dehydrogenase/NADH system.
更に最近、還元末端のグルコースにアグリゴントシてパ
ラニトロフェノール等のフェノール系化合物を導入し、
アブリボンを遊離させてそのスペクトル吸収を測定する
ことによりα−アミラーゼ活性を測定する方法も提案さ
れている（ｆＦ公昭５７−５３０７９）。Furthermore, recently, phenolic compounds such as para-nitrophenol have been introduced into the reducing terminal glucose,
A method has also been proposed for measuring α-amylase activity by releasing Alibibbon and measuring its spectral absorption (fF Publication No. 57-53079).
しかしながら、前述のマルトオリゴ糖をアミラーゼ活性
測定用基質として用いる場合には、試料である血清や尿
中に内因性のグルコースやマルトースが存在するため、
その影響を受け、測定誤差が生じることになる。従って
、マルトオリゴ糖を基質として用いる場合には試料中の
グルコース等を予めヘキソキナーゼ等を用いて処理する
必要がありた。However, when using the above-mentioned malto-oligosaccharide as a substrate for measuring amylase activity, endogenous glucose and maltose are present in the sample serum or urine.
Due to this influence, measurement errors will occur. Therefore, when maltooligosaccharide is used as a substrate, it is necessary to treat glucose and the like in the sample with hexokinase or the like in advance.
一方、アブリボンとしてフェノール系化合物を用いた場
合には、遊離した発色基は試料中の共存物質により作用
を受け、吸光度が変動しやすくなり、その結果、測定精
度が劣る場合があった。On the other hand, when a phenolic compound is used as the abribon, the free chromogenic group is affected by the coexisting substances in the sample, making the absorbance more likely to fluctuate, resulting in poor measurement accuracy.
本発明者は、これら従来技術の問題点を解決するために
研究を行ない、その結果、先にフルクトース等の単種類
を還元末端に転位させたマルトオリゴ糖を用いたα−ア
ミラーゼ活性測定方法を完成し、出願したｌ願昭６ｌ−
２７７７０２）。The present inventor conducted research to solve the problems of these conventional techniques, and as a result, completed a method for measuring α-amylase activity using a maltooligosaccharide in which a single type, such as fructose, was first rearranged to the reducing end. and filed an application in 1986.
277702).
しかし、この方法ではＮＡＤＨ−＋ＮＡＤの退色反応を
利用するものであり、発色反応と比べると感度や精度の
点で若干劣る場合がありた。However, this method utilizes a fading reaction of NADH-+NAD, and is sometimes slightly inferior in sensitivity and accuracy compared to a coloring reaction.
本発明は、先願発明を更に改良すべく、研究を継続した
結果、完成されたもので、下記の一般式（Ｈで表わされ
るマルトオリゴ糖を含む基質と試料とをグルコシダーゼ
の共存下に接触させ、遊離するイノシトール又はその誘
導体をＮＡＤの存在下、イノシトール脱水素酵素と接触
させることにより、試料中のα−アミラーゼ活性を測定
することを特徴とするα−アミラーゼ活性測定方法であ
る。The present invention was completed as a result of continued research in order to further improve the prior invention. is a method for measuring α-amylase activity, which comprises measuring α-amylase activity in a sample by contacting liberated inositol or its derivative with inositol dehydrogenase in the presence of NAD.
ｋ−Ｇｎ−１ｔＩ）
〔ここでＡは
Ｃ）Ｉｓ−Ｘｓ−鳥
を、■はイノシトール又はその誘導体を、Ｇはグルコー
スを、ｎは３〜１５の整数をそれぞれ表わす。k-Gn-1tI) [Here, A represents C) Is-Xs-bird, ■ represents inositol or its derivative, G represents glucose, and n represents an integer from 3 to 15, respectively.
（「１式又は［相］式において、Ｒ１〜Ｒ４は水素原子
、低級アルキル基、又は（ＣＨｚ ） ｙ ＣＯＯＭ
（７はＯ〜ｚ ｔ Ｍは水素原子又はアルカリ金属を表
わす。〕を、Ｘ、〜Ｘ４は酸素原子又はイオウ原子をそ
れぞれ表わす。〕
本発明会提供されるα−アミラーゼ活性測定用基質であ
る一般式中で表わされるマルトオリゴ糖において、非還
元末端であるＡは未置換グルコースでもよいし、グルコ
ースの４位及び／又は６位を置換したものでよい（前記
「式）。さらにグルコースの４位と６位が−緒になりて
アルキレン橋を形成していてもよい（前記■式）。(In the formula 1 or [phase] formula, R1 to R4 are hydrogen atoms, lower alkyl groups, or (CHz) y COOM
(7 represents O~z t M represents a hydrogen atom or an alkali metal), and X and ~X4 represent an oxygen atom or a sulfur atom, respectively.] Substrate for measuring α-amylase activity provided by the present invention group In the malto-oligosaccharide represented by the general formula, A, which is the non-reducing end, may be unsubstituted glucose or may be substituted at the 4-position and/or 6-position of glucose (formula above). and the 6th position may be joined together to form an alkylene bridge (formula ① above).
一般式（１１において、還元末端となる■はイノシトー
ル又はその誘導体である。■がイノシトールであること
によって前述の通り内因性のグルコースと区別すること
ができる。In the general formula (11), the reducing terminal (■) is inositol or a derivative thereof. By the fact that (■) is inositol, it can be distinguished from endogenous glucose as described above.
一般式（Ｉ）において、Ａ−Ｇｎは具体的にはマルトペ
ンタオース（Ｇ５）％マルトオクタツース（Ｇ８）、マ
ルトデカオース（ＧＩＯ）、マルトヘキサデカオース（
Ｇｌ ６ ）等が例示される。このうち、０５〜Ｇ８は
水溶性に優れｌ
るうえに、２種のアイ妙エンザイムの作用を均等に受け
る可能性が高いため、基質として好ましい。In general formula (I), A-Gn is specifically maltopentaose (G5)% maltooctatose (G8), maltodecaose (GIO), maltohexadecose (
Gl 6 ), etc. are exemplified. Among these, 05 to G8 are preferable as substrates because they have excellent water solubility and are likely to be equally affected by the two types of enzymes.
一般式＋１３において、Ａは前述のとおり、未修飾され
たグルコースを含むものである。無修飾の場合でも本−
発明の基質は従来の基質に比べて、はるかに精度よくα
−アミラーゼ活性を測定し得るものであるが、下記の理
由により修飾グルコースが好ましい。In general formula +13, A includes unmodified glucose as described above. Book even if unqualified
The substrate of the invention has much higher accuracy than conventional substrates.
- Although amylase activity can be measured, modified glucose is preferred for the following reasons.
即ち、マルトオリゴ糖から成る基質において、非還元末
端がグルコースそのものであると、α−アミラーゼ活性
測定時に使用する共役酵素であるグルコシダーゼが一部
の非還元末端のグルコースを切断し、α−アミラーゼ活
性測定に誤差を与えてしまうのである。That is, in a substrate consisting of maltooligosaccharide, if the non-reducing end is glucose itself, glucosidase, which is the conjugate enzyme used for α-amylase activity measurement, will cleave some of the glucose at the non-reducing end, and the α-amylase activity measurement will be interrupted. This gives an error.
修飾された非還元末端としては第１表に示したような置
換基を導入したものが例示される。Examples of modified non-reducing terminals include those introduced with substituents as shown in Table 1.
第１表
一般式（Ｉｌで表わされる化合物のうち、α−アミラー
ゼ活性測定用基質としてもっとも好ましいのは次の■］
式で表わされる化合物である。Table 1 General formula (Among the compounds represented by Il, the following ■ is the most preferable as a substrate for measuring α-amylase activity]
It is a compound represented by the formula.
（Ｚｌ−Ｚ、は水素原子又はリン酸基を表わす。）Ｎ式
の化合物を以下ＭｅＧｓｉと略すことがある。(Zl-Z represents a hydrogen atom or a phosphoric acid group.) The compound of formula N may be hereinafter abbreviated as MeGsi.
一般式（Ｉ）のマルトオリゴ糖の製造方法の一例を述べ
る。An example of a method for producing maltooligosaccharide of general formula (I) will be described.
先ず、サイクロデキストリン（α、β、γのいずれでも
よい。）とイノジ−トールとをサイクロデキストリング
ルカノトランスフェラーゼの共存下に反応させる。その
結果、サイクロデキストリンが開環し、その還元末端に
イノシトールが転移し、新たな還元末端がイノシトール
となる。もつとも、この際デキストリンの分断も行なわ
れるので得られるデキストリンは原料のサイクロデキス
トリンのグルコース数のものに限られず、それ以下のグ
ルコース数のものも生成する。マルトオリゴ糖のグルコ
ース数が、この操作で所望のものよりも少なければ、マ
ルトース以上のオリゴ糖と、得られた転位デキストリン
とを再度サイクロデキストリングルカノトランスフェラ
ーゼの共存下に反応させればよい。First, cyclodextrin (any of α, β, and γ) and inoditol are reacted in the presence of cyclodextrin glucanotransferase. As a result, the cyclodextrin opens, inositol is transferred to its reducing end, and the new reducing end becomes inositol. However, since the dextrin is also split at this time, the obtained dextrin is not limited to the number of glucose in the cyclodextrin used as the raw material, but also those having a lower number of glucose are also produced. If the number of glucose in the malto-oligosaccharide is lower than desired in this operation, the oligosaccharide containing more than maltose and the obtained rearranged dextrin may be reacted again in the presence of cyclodextrin glucanotransferase.
なお、非還元末端のグルコースは、前述の通り、その４
位及び／又は６位が修飾されている方が測定誤差が少な
くなるので好ましいものであるが、非還元末端の４位及
び／又は６位を修飾する場合には、上述の方法で得られ
たイノシトール転移マルトオリゴ塘を酸触媒の存在下に
ジメトキシトルエンやジメトキシプロパンと反応させる
。その結果、■式で示される非還元末端が得られる。As mentioned above, the non-reducing terminal glucose is
It is preferable that the 4th and/or 6th positions of the non-reducing end are modified, as this reduces measurement errors. The inositol-transferred maltooligotox is reacted with dimethoxytoluene or dimethoxypropane in the presence of an acid catalyst. As a result, a non-reducing end represented by the formula (2) is obtained.
一方、■式で示される非還元末端を導入する場合には、
■式の化合物の６位及び／又は４位を触媒存在下にＯＨ
とし、次いで、ハロゲン化アルキルを反応させ、接触還
元すればよい。On the other hand, when introducing a non-reducing end represented by the formula ■,
OH in the presence of a catalyst at the 6th and/or 4th positions of the compound of formula
and then react with an alkyl halide to carry out catalytic reduction.
また、Ｘ１〜Ｘ４のいずれかをイオウ原子にしたい場合
には■弐又は１■式の化合物をスルホン酸エステル化し
、メルカプタン類を反応させればよい。なお、サイクロ
デキストリンのかわりに市販のマルトオリゴ糖を用いて
もよい。Furthermore, if any of X1 to X4 is desired to be a sulfur atom, the compound of formula (2) or (1) may be converted into a sulfonic acid ester and reacted with a mercaptan. Note that a commercially available maltooligosaccharide may be used instead of cyclodextrin.
前述の反応で用い北サイクロデキストリングルカノトラ
ンスフェラーゼはバチルス・マセランス、バチルス・メ
ガテリウム、バチルス・サーキュランス及ヒバチルス・
オーペンシス等を起源とするものを使用できる。The northern cyclodextrin glucanotransferases used in the aforementioned reactions were Bacillus macerans, Bacillus megaterium, Bacillus circulans, and Hibacillus.
Those originating from Orpensis etc. can be used.
反応液から所望のマルトオリゴ糖を分離精製する手段と
してはＧＰＣ，イオン交換クロマトグラフィー、合成吸
着剤による方法等が挙げられる。Examples of means for separating and purifying the desired maltooligosaccharide from the reaction solution include GPC, ion exchange chromatography, and a method using a synthetic adsorbent.
次に、（ＩＪ式で示される基質を用いて試料中のα−ア
ミラーゼ活性を測定する方法を述べる。Next, a method for measuring α-amylase activity in a sample using a substrate represented by the formula (IJ) will be described.
体液等の試料に基質及び共役酵素として、グルコシダー
ゼを加えると下記のように反応が進む。When glucosidase is added as a substrate and a conjugate enzyme to a sample such as a body fluid, the reaction proceeds as shown below.
ＭｅＧ、 ｌ ” ”−” ＭｅＧ１＋Ｇ、 ■ｃ、
Ｉ ａゴ竺で１−２Ｇ、＋１
ここで遊離したイノシトール又はその誘導体はイノシト
ール脱水素酵素（ＩＤＨ）と、ＮＡＬ）共存下に反応さ
せることによりＮＡＤＨを生成させ、その吸光度の変化
によりイノシトール量が測定できる。MeG, l ” ”-” MeG1+G, ■c,
1-2G, +1 The liberated inositol or its derivative is reacted with inositol dehydrogenase (IDH) in the presence of NAL to generate NADH, and the amount of inositol is determined by the change in absorbance. Can be measured.
一般式ＩＩ）で表わされる本発明で用いる基質は試料中
のα−アミラーゼ及び共役酵素のグルコシダーゼにより
切断されてイノシトール又はその誘導体を生じる。この
イノシトール類を例えばＩＤＨ等で脱水素することによ
り、ＮＡＤ−＋ＮＡＤＩ（の発色反応を起こさせ、生成
し九ＮＡＤＨ量を測定することによりα−アミラーゼ活
性を測定することができる。The substrate used in the present invention represented by general formula II) is cleaved by α-amylase in the sample and glucosidase, which is a conjugating enzyme, to produce inositol or a derivative thereof. By dehydrogenating these inositols with, for example, IDH, a coloring reaction of NAD-+NADI (NAD-+NADI) is caused, and α-amylase activity can be measured by measuring the amount of NAD-NADH produced.
以下に実施例ｔ−揚げて本発明を更に説明する。The present invention will be further explained with reference to Example t below.
実施例１
ａ、 Ｉの製造
β−サイクロデキストリン２０９−とイノシトール２０
５Ｆ−を溶解した液中に、バチルス・オーペンシスを起
源とするサイクロデキストリングルカノトランスフェラ
ーゼｆｔ２００１Ｌ／／ を基質添加後、６０ＣｐＨ６
，ｏの条件下で２５時間反応させた。Example 1 a, Preparation of I β-cyclodextrin 209- and inositol 20
After adding a substrate of cyclodextrin glucanotransferase ft2001L, which originated from Bacillus opensis, to the solution containing 5F-, 60CpH6
, o for 25 hours.
次いで、この反応液をシリカ充填カラムによりクロマト
分離し、Ｇ、Ｉを８．８ｙ−得た。Next, this reaction solution was chromatographically separated using a silica-packed column to obtain 8.8y of G and I.
次に、このＧ、１８５４と市販のＧ４オリゴ糖１２５Ｌ
とを４ｏｏｍｌのバッファ液（ｐＨ６，０）に溶解させ
、サイクロデキストリングルカノトランスフェラーゼ２
ｏｏｍ／Ｐ基質を添加し、３７Ｃで２０分間反応させた
。次いで再度クロマト分離したところＧ、Ｉが１．８）
得られ友。Next, this G, 1854 and commercially available G4 oligosaccharide 125L
and cyclodextrin glucanotransferase 2 were dissolved in 4 ooml of buffer solution (pH 6,0).
oom/P substrate was added and reacted at 37C for 20 minutes. Then, when chromatographic separation was performed again, G and I were 1.8)
A friend gained.
α−アミラーゼ活性測定例
試薬Ｉ
上記で得られた基質 １ ｍ ｏ ｌ／Ｉ３（最終
濃度）α−グルコシダーゼ ２５ＵＡｎｌ（〃）
イノシト−欠デ？ト５クシーービ ４０ＵＡｌ
（ｔｔ ）ＮＡＤ
４，０’ｍｍａＶ１３ｃ ” ）ＰＩ
ＰＥＣ−（ｙ）７−（ｐＩ（７，０） １ｏｏｍｍｏｌ
／＃（〃 ）上記試薬８００μｌに、α−アミラー
ゼ活性が５５ ｍＴＪ／ｍｌ及び２２０ ｍＵ／ｎｔｌ
である検体血清１００μｍを加えて３７Ｃで１０分間イ
ンキュベーションしなから３４ｏ」で吸光度変化を測定
した。α-amylase activity measurement example Reagent I Substrate obtained above 1 mol/I3 (final concentration) α-glucosidase 25 UAnl (〃)
Inosito - Missing De? To5Kushibi 40UAl
(tt)NAD
4,0'mmaV13c'')PI
PEC-(y)7-(pI(7,0) 1oommol
/#(〃) 800 μl of the above reagent contains α-amylase activity of 55 mTJ/ml and 220 mU/ntl.
100 μm of sample serum was added and incubated at 37C for 10 minutes, and then the change in absorbance was measured at 34o.
結果を図に示す。The results are shown in the figure.
図から本発明においては４分以降にお
いて、いずれの濃度でも良好な直線関係が得られている
ことがわかる。From the figure, it can be seen that in the present invention, a good linear relationship is obtained at any concentration after 4 minutes.
実施例２
実施例２において、各検体血清にさら
にグルコースを１００ ｍ９７ｍ１添加した他は同様の
手順で測定を行なった。Example 2 Measurements were carried out in the same manner as in Example 2, except that 100 m97 ml of glucose was further added to each sample serum.
その結果、図とほとんど同一の結果が 得られた。As a result, the result is almost the same as the one shown in the figure. Obtained.
従って、本発明は内因性のグルコース 等によりては全く影響を受けないことがわかる。Therefore, the present invention provides endogenous glucose It can be seen that there is no influence at all.
本発明のマルトオリゴ糖を含む基質は還元末端にイノシ
トール類を転位させたので、試料中に含まれる内因性グ
ルコースやマルトース等の影響を受けることなく正確に
効率よ〈α−アミラーゼ活性を測定することができる。Since the malto-oligosaccharide-containing substrate of the present invention has inositols transferred to the reducing end, α-amylase activity can be measured accurately and efficiently without being affected by endogenous glucose, maltose, etc. contained in the sample. Can be done.
さらに、非遺元末端に６飾したグルコースを導入すると
、さらに精度が上がる。また、イノシトールをＮＡＤ共
存下酵素処理することによりＮＡＤＨが得られるが、こ
の際の吸光度測定は、試料中の他の夾雑物により影響を
受けず、安定して測定することができる。Furthermore, introducing a hexa-decorated glucose at the non-primary end further improves accuracy. Furthermore, NADH can be obtained by treating inositol with an enzyme in the presence of NAD, and the absorbance measurement at this time is not affected by other impurities in the sample and can be stably measured.
図面は本発明の実施例の結果を示す図面である。 The drawings are diagrams showing the results of the embodiments of the present invention.
Claims (1)
を含む基質と試料とをグルコシダーゼの共存下に接触さ
せ、遊離するイノシトール又はその誘導体をＮＡＤ（ニ
コチン酸アミドアデニンジヌクレオチド）の存在下、イ
ノシトール脱水素酵素と接触させることにより、試料中
のα−アミラーゼ活性を測定することを特徴とするα−
アミラーゼ活性測定方法。 Ａ−Ｇｎ−Ｉ（ I ） 〔ここでＡは ▲数式、化学式、表等があります▼（II）又は▲数式、
化学式、表等があります▼（III）を、Ｉはイノシトー
ル又はその誘導体を、Ｇはグルコースを、ｎは３〜１５
の整数をそれぞれ表わす。 （II）式又は（III）式において、Ｒ＿１〜Ｒ＿４は水
素原子、低級アルキル基、又は（ＣＨ＿２）ｙＣＯＯＭ
〔ｙは０〜２、Ｍは水素原子又はアルカリ金属を表わす
〕を、Ｘ＿１〜Ｘ＿４は酸素原子又はイオウ原子をそれ
ぞれ表わす。〕 ２）（ I ）式が下式で表わされる特許請求の範囲第１
項記載の測定方法。 ▲数式、化学式、表等があります▼ （Ｚ＿１〜Ｚ＿５はそれぞれ水素原子又はリン酸基を表
わす。）[Claims] 1) A substrate containing a maltooligosaccharide represented by the following general formula (I) is brought into contact with a sample in the presence of glucosidase, and the liberated inositol or its derivative is converted into NAD (nicotinamide adenine dinucleotide). ), the α-amylase activity in the sample is measured by contacting it with inositol dehydrogenase.
Amylase activity measurement method. A-Gn-I (I) [Here, A is ▲Mathematical formula, chemical formula, table, etc.▼(II) or ▲Mathematical formula,
There are chemical formulas, tables, etc. ▼ (III), I is inositol or its derivative, G is glucose, n is 3 to 15
each represents an integer. In formula (II) or formula (III), R_1 to R_4 are hydrogen atoms, lower alkyl groups, or (CH_2)yCOOM
[y represents 0 to 2, M represents a hydrogen atom or an alkali metal], and X_1 to X_4 represent an oxygen atom or a sulfur atom, respectively. ] 2) Claim 1 in which formula (I) is represented by the following formula:
Measurement method described in section. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (Z_1 to Z_5 each represent a hydrogen atom or a phosphoric acid group.)
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2894387A JPS63196300A (en) | 1987-02-10 | 1987-02-10 | Determination of alpha-amylase activity |
| US07/116,617 US5043436A (en) | 1986-11-20 | 1987-11-03 | Substrate for measurement of alpha-amylase activity |
| DE19873738477 DE3738477A1 (en) | 1986-11-20 | 1987-11-12 | SUBSTRATE FOR MEASURING (ALPHA) AMYLASE ACTIVITY |
| US07/637,135 US5068183A (en) | 1986-11-20 | 1991-01-03 | Method for measurement of α-amylase activity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2894387A JPS63196300A (en) | 1987-02-10 | 1987-02-10 | Determination of alpha-amylase activity |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS63196300A true JPS63196300A (en) | 1988-08-15 |
Family
ID=12262488
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2894387A Pending JPS63196300A (en) | 1986-11-20 | 1987-02-10 | Determination of alpha-amylase activity |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS63196300A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9340134B2 (en) | 2010-08-30 | 2016-05-17 | Johnson Controls Technology Company | Vehicle seat cover |
| US11013342B2 (en) | 2018-08-09 | 2021-05-25 | Toyota Boshoku Kabushiki Kaisha | Conveyance seat and seat cover for the same |
-
1987
- 1987-02-10 JP JP2894387A patent/JPS63196300A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9340134B2 (en) | 2010-08-30 | 2016-05-17 | Johnson Controls Technology Company | Vehicle seat cover |
| US11013342B2 (en) | 2018-08-09 | 2021-05-25 | Toyota Boshoku Kabushiki Kaisha | Conveyance seat and seat cover for the same |
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