JPH04158798A - Measurement of enzyme activity - Google Patents
Measurement of enzyme activityInfo
- Publication number
- JPH04158798A JPH04158798A JP28134790A JP28134790A JPH04158798A JP H04158798 A JPH04158798 A JP H04158798A JP 28134790 A JP28134790 A JP 28134790A JP 28134790 A JP28134790 A JP 28134790A JP H04158798 A JPH04158798 A JP H04158798A
- Authority
- JP
- Japan
- Prior art keywords
- reaction
- enzyme
- measuring
- substrate
- enzyme activity
- 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
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 35
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 35
- 230000000694 effects Effects 0.000 title claims abstract description 14
- 238000005259 measurement Methods 0.000 title abstract description 11
- 238000006243 chemical reaction Methods 0.000 claims abstract description 38
- 239000000758 substrate Substances 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims description 14
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 abstract description 2
- 102100022624 Glucoamylase Human genes 0.000 abstract description 2
- 108700040099 Xylose isomerases Proteins 0.000 abstract description 2
- 108090000604 Hydrolases Proteins 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 27
- 239000003708 ampul Substances 0.000 description 7
- 239000011521 glass Substances 0.000 description 5
- 229920002472 Starch Polymers 0.000 description 4
- 238000006911 enzymatic reaction Methods 0.000 description 4
- 239000008107 starch Substances 0.000 description 4
- 235000019698 starch Nutrition 0.000 description 4
- 102000013142 Amylases Human genes 0.000 description 3
- 108010065511 Amylases Proteins 0.000 description 3
- 229940111205 diastase Drugs 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 108010093096 Immobilized Enzymes Proteins 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 238000005538 encapsulation Methods 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 239000006087 Silane Coupling Agent Substances 0.000 description 1
- 239000004809 Teflon Substances 0.000 description 1
- 229920006362 Teflon® Polymers 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003094 microcapsule Substances 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 229920002635 polyurethane Polymers 0.000 description 1
- 239000004814 polyurethane Substances 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 229920005612 synthetic inorganic polymer Polymers 0.000 description 1
- 229920005613 synthetic organic polymer Polymers 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 230000004580 weight loss Effects 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〕
本発明は酵素活性の測定方法に関するものであり、更に
詳しくは酵素反応における反応熱量を測定することによ
り酵素活性の測定を行う方法に関する。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for measuring enzyme activity, and more particularly to a method for measuring enzyme activity by measuring the amount of reaction heat in an enzyme reaction.
生化学的反応における酵素活性を測定及び評価する方法
としては、従来、例えば、検圧法、酸素電極法、分光光
度法１、蛍光法、滴定法等が知られている。Conventionally, methods for measuring and evaluating enzyme activity in biochemical reactions include, for example, a pressure detection method, an oxygen electrode method, a spectrophotometry method, a fluorescence method, a titration method, and the like.
〔発明が解決しようとする課題］
しかし、上記の方法には、測定結果が温度、溶媒、ｐＨ
等の測定条件などの影響を受けるため不安定である、経
時的な連続測定が不可能である、測定の対象となる酵素
の種類が限定される等の欠点がある。[Problems to be Solved by the Invention] However, the above method has problems in that the measurement results are dependent on temperature, solvent, pH
This method has disadvantages such as being unstable due to the influence of measurement conditions such as, etc., making continuous measurement over time impossible, and limiting the types of enzymes that can be measured.
そこで、本発明は、反応条件、測定条件等の影響を受け
ず、経時的に連続測定が可能で、しかも再現性の高い測
定結果が得られる酵素活性の測定方法を提供することを
課題とするものである。Therefore, an object of the present invention is to provide a method for measuring enzyme activity that is not affected by reaction conditions, measurement conditions, etc., allows continuous measurement over time, and provides highly reproducible measurement results. It is something.
本発明は上記の課題を解決するため、
酵素と基質の反応において発生する反応熱量を測定する
ことからなる酵素活性の測定方法を提供するものである
。In order to solve the above problems, the present invention provides a method for measuring enzyme activity, which comprises measuring the amount of reaction heat generated in the reaction between an enzyme and a substrate.
酵素反応に伴う反応熱の存在は従来から知られているが
、本発明は、この反応熱量を酵素活性の測定に利用する
ことにより、時間とともに変化する酵素活性を経時的か
つ連続的に評価することが可能である。The existence of reaction heat associated with enzymatic reactions has been known for a long time, but the present invention utilizes this reaction heat to measure enzyme activity, thereby continuously and chronologically evaluating enzyme activity that changes over time. Is possible.
■素
本発明の方法は、酵素反応に発熱あるいは吸熱の反応熱
を伴う酵素に適用可能である。また反応基質は、測定す
る酵素に対して反応する基質が使用可能である。(1) The method of the present invention is applicable to enzymes whose enzymatic reactions involve exothermic or endothermic reaction heat. Further, as the reaction substrate, a substrate that reacts with the enzyme to be measured can be used.
本発明の方法によって酵素活性を測定できる酵素として
は、例えば、グルコアミラーゼ等の加水分解酵素、グル
コースイソメラーゼ等の添加酵素、グルコースオキシダ
ーゼ等の酸化酵素などが挙げられる。Examples of enzymes whose enzyme activity can be measured by the method of the present invention include hydrolytic enzymes such as glucoamylase, additive enzymes such as glucose isomerase, and oxidizing enzymes such as glucose oxidase.
測定される酵素は、固定化されていても、また固定化さ
れていなくてもよい。固定化する場合、固定化方法は公
知の何れの方法でもよく、例えば担体結合型、格子内包
捨型、架橋型、ミクロカプセル内包括型等がある。その
際に用いられる担体には、例えばポリスチレン、ポリウ
レタン、シリコン等の合成有機高分子、または例えばガ
ラス、シリカ、セラミックス等の合成無機高分子等が挙
げられるが、その他酵素や反応基質影響を与えない限り
いずれの担体も使用可能である。The enzyme to be measured may be immobilized or non-immobilized. In the case of immobilization, any known immobilization method may be used, such as carrier binding type, lattice encapsulation type, crosslinking type, microcapsule encapsulation type, etc. Examples of carriers used in this case include synthetic organic polymers such as polystyrene, polyurethane, and silicon, and synthetic inorganic polymers such as glass, silica, and ceramics, but they do not affect other enzymes or reaction substrates. Any carrier can be used as long as it is suitable.
勇足１！
反応熱の測定手段としては、例えば熱電対、サーミスタ
、白金測温抵抗体等の測温体を使用することができる０
代表的な測定装置は、恒温槽中で酵素を反応させ、その
反応熱量を測定する装置である。Brave leg 1! As a means for measuring the heat of reaction, a thermometer such as a thermocouple, a thermistor, or a platinum resistance thermometer can be used.
A typical measuring device is a device that reacts an enzyme in a constant temperature bath and measures the amount of heat of the reaction.
測温体は、１０−４℃以上の精度で温度変化を検出可能
であることを必要とし、好ましくはサーミスタである。The temperature measuring body needs to be able to detect temperature changes with an accuracy of 10-4°C or higher, and is preferably a thermistor.
恒温槽は槽内温度を精密に制御可能であることが必要で
あり、２５°Ｃ±０．１ ”Ｃの範囲で制御可能である
ことが好ましい。It is necessary that the temperature inside the constant temperature bath can be precisely controlled, and it is preferable that the temperature within the bath can be controlled within a range of 25°C±0.1''C.
かかる測定装置の具体例を第１図に示す。この装置は、
恒温槽１内に反応容器２が収納され、該反応容器２の底
に測温体３が接続されている。反応容器２内には反応基
質を溶解した溶液４が入れられ、その中に酵素封入アン
プル５を配置しである。該装置には、攪拌機６が備わり
溶液４を攪拌できるようになっている。酵素封入アンプ
ル中５には、例えば固定化されたジアスターゼのような
酵素が封入されており、このアンプルを破壊することに
より酵素と基質を接触させ、反応を開始させるものであ
る。以下に実施例を示す。A specific example of such a measuring device is shown in FIG. This device is
A reaction vessel 2 is housed in a constant temperature bath 1, and a temperature measuring element 3 is connected to the bottom of the reaction vessel 2. A solution 4 in which a reaction substrate is dissolved is placed in the reaction container 2, and an enzyme-filled ampoule 5 is placed therein. The device is equipped with a stirrer 6 so that the solution 4 can be stirred. An enzyme such as immobilized diastase is enclosed in the enzyme-enclosed ampoule 5, and by breaking the ampoule, the enzyme and the substrate are brought into contact to initiate a reaction. Examples are shown below.
〔実施例１〕
固定化する担体としてシャモットを用い、その表面にシ
ランカップリング剤としてＴ−アミノプロピルトリエト
キシシランを付与し、カップリング反応が終了した後、
該表面上にジアスターゼを固定化した。この固定化酵素
を５ ｃｃのガラスアンプル中に封入した。一方、反応
基質としてデンプンを濃度３重量％で含む水溶液２０ｃ
ｃ （デンプンとして６００■）をステンレス製の容量
１００ｃｃの反応容器に入れ、この中に前記の該ガラス
アンプルを置いた０次に、この反応容器を恒温槽内に装
填した。[Example 1] Using chamotte as a carrier to be immobilized, T-aminopropyltriethoxysilane was applied as a silane coupling agent to the surface of the carrier, and after the coupling reaction was completed,
Diastase was immobilized on the surface. This immobilized enzyme was sealed in a 5 cc glass ampoule. On the other hand, an aqueous solution 20c containing starch as a reaction substrate at a concentration of 3% by weight.
(600 μm as starch) was placed in a stainless steel reaction vessel with a capacity of 100 cc, and the glass ampoule was placed therein. Next, this reaction vessel was loaded into a constant temperature bath.
恒温槽により反応容器中の温度が２５°Ｃ±０．１℃の
範囲に保たれたことを確認した後、テフロン棒によりガ
ラスアンプルを破壊した。攪拌機により反応溶液を攪拌
しながら、反応熱によるサーミスタの抵抗の経時の変化
を電圧に変換して測定した。After confirming that the temperature in the reaction vessel was maintained within the range of 25°C±0.1°C using a constant temperature bath, the glass ampoule was broken with a Teflon rod. While stirring the reaction solution with a stirrer, the change over time in the resistance of the thermistor due to reaction heat was converted into voltage and measured.
結果を第２図に示す、サーミスタの電圧変化曲線と横軸
（時間軸）に囲まれた部分の面積を計算することにより
、発生した全反応熱量は１ 、０４ｃａ ｌであること
がわかった。The results are shown in FIG. 2, and by calculating the area surrounded by the voltage change curve of the thermistor and the horizontal axis (time axis), it was found that the total amount of reaction heat generated was 1.04 cal.
［実施例２］
実施例１と同様にしてジアスターゼをシャモットに固定
化した後、これを３重量％のデンプン水溶液５０ｇ（デ
ンプンとして１５００■）と十分に反応させた。その後
、該固定化酵素をよく洗浄した後にガラスアンプルに封
入して使用した以外は、実施例１と同様にして反応熱に
よるサーミスタの電圧の経時的変化を測定した。[Example 2] After diastase was immobilized on chamotte in the same manner as in Example 1, it was sufficiently reacted with 50 g of a 3% by weight starch aqueous solution (1500 μm as starch). Thereafter, the change in voltage of the thermistor over time due to reaction heat was measured in the same manner as in Example 1, except that the immobilized enzyme was thoroughly washed and then sealed in a glass ampoule.
結果を第２図に示す、実施例１と同様にして、発生した
全反応熱量は１．０７ｃａｌであることがわがった。The results are shown in FIG. 2. In the same manner as in Example 1, it was found that the total reaction heat generated was 1.07 cal.
また、反応に関与した酵素の量を測定するため、反応終
了後のシャモットを室温から１０００℃まで加熱するこ
とにより得られた重量減少量は、実施例１ではシャモッ
ト１ｇ当り５９．８■、実施例２ではシャモット１ｇ当
り３４．０■であった。In addition, in order to measure the amount of enzyme involved in the reaction, the amount of weight loss obtained by heating the chamotte from room temperature to 1000°C after the completion of the reaction was 59.8 cm per gram of chamotte in Example 1; In Example 2, the amount was 34.0 μ/g of chamotte.
以上のことから、第２図において測温体の電圧の変化の
曲線をｆ（ｔ）とすると、ｆ（ｔ）は時間ｔにおいて基
質と反応している全酵素量（活性部位数が２以上のとき
は全活性部位量）に比例し、反応初期のｄｆ（ｔ）／ｄ
ｔは酵素の反応速度に比例し、全反応熱量５ｆ（ｔ）ｄ
ｔ（ｔ・０〜ｏｏ）は、基質量または反応生成物量に比
例することわかった。From the above, if the curve of voltage change of the thermometer in Figure 2 is f(t), then f(t) is the total amount of enzyme reacting with the substrate at time t (the number of active sites is 2 or more). df(t)/d at the initial stage of the reaction.
t is proportional to the reaction rate of the enzyme, and the total reaction heat 5f(t)d
It was found that t (t·0~oo) is proportional to the amount of substrate or reaction product.
本発明の酵素活性の測定方法は、反応条件、測定条件等
の影響を受けず、経時的に連続測定が可能で、しかも再
現性の高い測定結果が得られる。The method for measuring enzyme activity of the present invention is not affected by reaction conditions, measurement conditions, etc., allows continuous measurement over time, and provides highly reproducible measurement results.
例えば研究または生産の手段等に用いられる酵素反応に
おいて、それらに使用される酵素の活性を評価し判断す
るのに有用である。For example, it is useful for evaluating and determining the activity of enzymes used in enzyme reactions used in research or production methods.
第１図は、本発明の方法の実施に用いる装置例の概念図
であり、第２図は、実施例１及び２により得られた、時
間経過に対する測温体の電圧変化を示したグラフである
。FIG. 1 is a conceptual diagram of an example of an apparatus used to carry out the method of the present invention, and FIG. 2 is a graph showing voltage changes of the temperature measuring element over time obtained in Examples 1 and 2. be.
Claims (1)
ことからなる酵素活性の測定方法。A method for measuring enzyme activity that consists of measuring the amount of reaction heat generated in the reaction between an enzyme and a substrate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP28134790A JPH04158798A (en) | 1990-10-19 | 1990-10-19 | Measurement of enzyme activity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP28134790A JPH04158798A (en) | 1990-10-19 | 1990-10-19 | Measurement of enzyme activity |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH04158798A true JPH04158798A (en) | 1992-06-01 |
Family
ID=17637845
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP28134790A Pending JPH04158798A (en) | 1990-10-19 | 1990-10-19 | Measurement of enzyme activity |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH04158798A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6720675B2 (en) | 2001-03-30 | 2004-04-13 | Mitsubishi Denki Kabushiki Kaisha | Power converter |
| US7087413B2 (en) | 1994-07-01 | 2006-08-08 | The Board Of Regents Of The University Of Oklahoma | Hyaluronan synthase gene and uses thereof |
-
1990
- 1990-10-19 JP JP28134790A patent/JPH04158798A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7087413B2 (en) | 1994-07-01 | 2006-08-08 | The Board Of Regents Of The University Of Oklahoma | Hyaluronan synthase gene and uses thereof |
| US6720675B2 (en) | 2001-03-30 | 2004-04-13 | Mitsubishi Denki Kabushiki Kaisha | Power converter |
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