JP2857788B2 - Enzyme amount measuring method and apparatus - Google Patents
Enzyme amount measuring method and apparatusInfo
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- JP2857788B2 JP2857788B2 JP6562190A JP6562190A JP2857788B2 JP 2857788 B2 JP2857788 B2 JP 2857788B2 JP 6562190 A JP6562190 A JP 6562190A JP 6562190 A JP6562190 A JP 6562190A JP 2857788 B2 JP2857788 B2 JP 2857788B2
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- culture
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Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は、培養により酵素を生産する系において、そ
の生産量を自動的に測定する方法及び測定装置に関する
ものである。Description: TECHNICAL FIELD The present invention relates to a method and an apparatus for automatically measuring the production amount in a system for producing an enzyme by culturing.
従来、特公昭56-54583号公報には、酵素反応の進行に
対し、３個以上の吸光度検出器を経時的に検出できるよ
うに配設し、第１検出器、第２検出器で得た値に基づい
て、算出に用いる検出値を選択することにより、酵素量
測定を行う方法及び装置が開示されている。Conventionally, Japanese Patent Publication No. 56-54583 discloses that three or more absorbance detectors are provided so as to be able to detect with time the progress of an enzyme reaction, and are obtained by a first detector and a second detector. A method and an apparatus for measuring an amount of an enzyme by selecting a detection value used for calculation based on the value are disclosed.
また、特公平1-21897号公報には、生化学成分の反応
速度測定法による成分分析において、反応過程にて複数
回測光を行い、最終回測光値を予め定めた基準値と比較
して取捨選択することにより、検液の成分分析を行う方
法が開示されている。In addition, Japanese Patent Publication No. 1-21897 discloses that in component analysis using a reaction rate measuring method for biochemical components, photometry is performed a plurality of times during the reaction process, and the final photometric value is compared with a predetermined reference value and discarded. A method for performing component analysis of a test solution by selection is disclosed.
上記２つの公報記載の技術は、いずれも吸光度を用い
た測定方法の改良に関するものであり、本発明の特徴で
ある「２点でpH値を測定することにより酵素量を推定す
る」ことについては、何ら記載されていない。The techniques described in the above two publications all relate to improvements in measurement methods using absorbance. Regarding the feature of the present invention, "estimating an enzyme amount by measuring a pH value at two points" is not described. Is not described at all.
従来の酵素量測定方法の主流は、培養液を採取し、こ
の試料を基質液と混合・反応させ、反応後の基質量減少
又は生成物の増加を、吸光度変化や滴定量変化として捉
えて推定するものであった。The mainstream of the conventional method for measuring the amount of enzyme is to collect a culture solution, mix and react this sample with a substrate solution, and estimate the decrease in substrate mass or increase in product after the reaction as a change in absorbance or a change in titer. Was to do.
しかし、この従来方法を用いる場合、以下のような問
題が生じる。However, when this conventional method is used, the following problems occur.
（１）希釈、測定等、人為に頼る部分が多く、作業が繁
雑であり時間がかかる。(1) There are many parts that depend on humans, such as dilution and measurement, and the work is complicated and time-consuming.
（２）サンプリングの際、雑菌の混入、培養液の減少等
の培養上の不都合が生じる。(2) At the time of sampling, inconveniences in culture such as contamination of various bacteria and reduction of culture solution occur.
（３）吸光度を利用した自動計測装置では、培養液中の
タンパク等による吸光部の汚れや、基質、生成物以外の
吸光度増大、光源の安定性、耐用時間等で維持管理、精
度に問題がある。(3) In an automatic measuring device using absorbance, there are problems in maintenance and accuracy due to contamination of the light absorbing portion due to proteins in the culture solution, increase in absorbance other than substrates and products, stability of the light source, life time, etc. is there.
（４）従来法の自動化及び連続測定を行うには、複雑な
機構が必要となる。(4) A complicated mechanism is required to perform the conventional method and perform continuous measurement.
本発明は上記の諸点に鑑みなされたもので、培養液を
基質緩衝液として適量混合し、その直後のpH値と、一定
時間反応後のpH値とを測定して、培養液中の酵素量を推
測するように構成することにより、簡便で、長期間、連
続測定可能である酵素量測定方法及びその装置を提供す
ることを目的とするものである。The present invention has been made in view of the above-mentioned points, and an appropriate amount of a culture solution is mixed as a substrate buffer, and the pH value immediately after the mixing and the pH value after a certain period of reaction are measured to determine the amount of the enzyme in the culture solution. It is an object of the present invention to provide a method for measuring the amount of enzyme which is simple and can be continuously measured for a long period of time, and an apparatus therefor.
上記の目的を達成するために、請求項１の酵素量測定
方法は、酵素生産培養系において、培地と同じ組成の基
質緩衝液を主成分とする反応液と、前記培地を主成分と
して培養した培養液とを混合するに際し、培養液から連
続的に培養液を抜き出して反応液と混合した後、混合液
を連続的に反応させ、混合時点でのpH値、及び一定反応
時間後のpH値を測定して、これらのpH値から酵素量を推
定することを特徴としている。In order to achieve the above object, the method for measuring the amount of enzyme according to claim 1 is characterized in that, in an enzyme-producing culture system, a reaction solution containing a substrate buffer having the same composition as a medium as a main component and a culture containing the medium as a main component. When mixing with the culture solution, the culture solution is continuously extracted from the culture solution and mixed with the reaction solution, and then the mixture solution is continuously reacted.The pH value at the time of mixing and the pH value after a certain reaction time Is measured, and the enzyme amount is estimated from these pH values.
請求項２の方法は、請求項１の方法において、反応液
と培養液とを混合し反応させた反応溶液を培養系に戻す
ことを特徴としている。According to a second aspect of the present invention, in the method of the first aspect, the reaction solution obtained by mixing and reacting the reaction solution and the culture solution is returned to the culture system.
請求項３の方法は、請求項１又は２の方法において、
酵素生産培養系が、タンナーゼ生産における麹菌培養系
であることを特徴としている。The method according to claim 3 is the method according to claim 1 or 2,
The enzyme production culture system is characterized in that it is a koji mold culture system for tannase production.
請求項４の酵素量測定装置は、第１図に示すように、
培養槽１と、反応液槽２と、この培養槽１及び反応液槽
２にそれぞれポンプ３、４を介して接続された混合槽５
と、この混合槽５にポンプ６を介して接続されたコイル
状細管７を備えた反応部８と、この反応部８に接続され
た最終槽10と、混合槽５に設けられた第1pH測定器11
と、最終槽10に設けられた第2pH測定器12と、第1pH測定
器11及び第2pH測定器12に接続された酵素量計測器13と
を包含することを特徴としている。As shown in FIG.
A culture tank 1, a reaction liquid tank 2, and a mixing tank 5 connected to the culture tank 1 and the reaction liquid tank 2 via pumps 3 and 4, respectively.
A reaction section 8 having a coiled thin tube 7 connected to the mixing tank 5 via a pump 6, a final tank 10 connected to the reaction section 8, and a first pH measurement provided in the mixing tank 5. Container 11
And a second pH measuring device 12 provided in the final tank 10, and an enzyme amount measuring device 13 connected to the first pH measuring device 11 and the second pH measuring device 12.
反応部８のコイル状細管７内を一定流速で液を流すこ
とにより、所定の反応時間を持たせる。培養液中の酵素
量変化に対して、流速や反応液との混合比を変化させる
ことにより、幅広い測定が可能となる。A predetermined reaction time is provided by flowing a liquid at a constant flow rate in the coiled thin tube 7 of the reaction section 8. By changing the flow rate and the mixing ratio with the reaction solution with respect to the change in the amount of the enzyme in the culture solution, a wide range of measurements can be performed.
請求項５の装置は、請求項４の装置において、反応部
８が培養槽１内に設置されていることを特徴としてい
る。この場合は、反応温度を一定に保つ機能を得るため
の温調装置を必要とせず、しかも、一定温度であること
から、効率よく、より正確な測定を行うことができる。An apparatus according to a fifth aspect is characterized in that, in the apparatus according to the fourth aspect, the reaction section 8 is provided in the culture tank 1. In this case, there is no need for a temperature control device for obtaining a function of keeping the reaction temperature constant, and since the temperature is constant, efficient and more accurate measurement can be performed.
請求項６の装置は、請求項４の装置において、反応部
８、混合槽５及び最終槽10が培養槽１内に設置されてい
ることを特徴としている。この場合は、反応前の溶液温
度から反応温度、反応後の温度まで一定に保つことがで
き、酵素反応を安定化できるため、さらに、より正確な
測定を行うことができる。The apparatus of claim 6 is characterized in that, in the apparatus of claim 4, the reaction unit 8, the mixing tank 5, and the final tank 10 are installed in the culture tank 1. In this case, since the temperature from the solution temperature before the reaction to the reaction temperature and the temperature after the reaction can be kept constant and the enzyme reaction can be stabilized, more accurate measurement can be performed.
請求項７の装置は、請求項４、５又は６の装置におい
て、最終槽10と培養槽１とを、反応溶液戻しライン14を
介して接続したことを特徴としている。この場合は、培
養液の損失を防ぐとともに、栄養分の補充を行うことが
できる。The apparatus of claim 7 is characterized in that, in the apparatus of claim 4, 5 or 6, the final tank 10 and the culture tank 1 are connected via a reaction solution return line 14. In this case, nutrients can be replenished while preventing the loss of the culture solution.
以下、本発明の酵素量測定方法及びその装置の一実施
例を第１図に基づいて説明する。１は培養槽で、内部に
菌体を加えた培養液15を満たしている。２は反応液槽
で、内部に培養液の培地と同じ組成の基質緩衝液を主成
分とする反応液16を満たしている。培養槽１は、図示し
ていないが、通常培養では恒温装置等により、一定の温
度を維持できるように構成されている。また、一般に、
培養槽の環境は、液温に近く維持されているので、反応
液自体も反応温度に近い温度となっている。Hereinafter, an embodiment of the method and apparatus for measuring the amount of enzyme of the present invention will be described with reference to FIG. Reference numeral 1 denotes a culture tank, which is filled with a culture solution 15 containing cells therein. Reference numeral 2 denotes a reaction solution tank, which is filled with a reaction solution 16 containing a substrate buffer having the same composition as the culture medium as a main component. Although not shown, the culture tank 1 is configured to be able to maintain a constant temperature by a constant temperature device or the like in normal culture. Also, in general,
Since the environment of the culture tank is maintained close to the liquid temperature, the reaction liquid itself has a temperature close to the reaction temperature.
培養液15をポンプ３により混合槽５へ導くとともに、
反応液16をポンプ４により混合槽５へ導き、攪拌機17に
より攪拌する。そして、混合槽５内の液のpH値を第1pH
測定器11で測定する。While the culture solution 15 is guided to the mixing tank 5 by the pump 3,
The reaction solution 16 is guided to the mixing tank 5 by the pump 4 and stirred by the stirrer 17. Then, the pH value of the liquid in the mixing tank 5 is set to the first pH.
The measurement is performed by the measuring device 11.
ついで、混合液をポンプ６により反応部８へ送り、コ
イル状細管７内を一定流速で流し、最終槽10に導く。そ
して、この液のpH値を第2pH測定器12で測定する。このp
H値及び第1pH測定器11のpH値を酵素量計測器13に導き、
酵素量を推定する。Next, the mixed liquid is sent to the reaction section 8 by the pump 6, flows through the coiled thin tube 7 at a constant flow rate, and is guided to the final tank 10. Then, the pH value of the liquid is measured by the second pH measuring device 12. This p
The H value and the pH value of the first pH measuring device 11 are led to the enzyme amount measuring device 13,
Estimate the amount of enzyme.
つぎに、酵素量の測定方法について、実験例に基づい
て説明する。Next, a method for measuring the amount of enzyme will be described based on experimental examples.
下記の組成、性状の基本培地液、酵素液、反応液を調
製した。A basic medium solution, an enzyme solution, and a reaction solution having the following composition and properties were prepared.
（１）基本培地 NH4H2PO41.4％（W/V） KH2PO40.2％ 〃 MgSO4・7H2O0.05％（W/V） グルコース1.0 〃 pH5.8（NaOHで調整） （２）酵素液 タンナーゼ（三共製薬（株）製の酵素） 20.0％（W/V） 基本培地 残部 pH5.8（NaOHで調整） （３）反応液 タンニン酸2.5％（W/V） 基本培地残部 酵素液の酵素活性を、通常の紫外（UV310）吸光測定
により測定した後、目的の５倍相当の希釈酵素液を、酵
素液と基本培地液とから60ml調製した。この希釈酵素液
60mlと基質液（反応液）240mlとを、ウォーターバス内
で30℃に加温した後、溶液を混合し、液温を保ちなが
ら、各時間のpH値を測定した。タンナーゼ活性量5.5u/m
l、8.2u/ml、18.9u/ml、22.6u/mlの場合における時間と
pH値との関係は、第２図に示す如くであった。なお、u/
mlはunit/ml（力価）のことである。(1) basal medium NH 4 H 2 PO 4 1.4% (W / V) KH 2 PO 4 0.2% 〃 MgSO 4 · 7H 2 O0.05% ( W / V) ( adjusted with NaOH) glucose 1.0 〃 pH5.8 (2) Enzyme solution tannase (enzyme manufactured by Sankyo Pharmaceutical Co., Ltd.) 20.0% (W / V) Basic medium Remainder pH 5.8 (adjusted with NaOH) (3) Reaction solution 2.5% tannic acid (W / V) Basic medium the enzymatic activity of the remainder enzyme solution was measured by a conventional ultraviolet (UV 310) absorption spectroscopy, a 5-fold equivalent diluted enzyme solution of interest was 60ml prepared from an enzyme solution and the basal medium solution. This diluted enzyme solution
After heating 60 ml and 240 ml of the substrate solution (reaction solution) to 30 ° C. in a water bath, the solutions were mixed, and the pH value was measured at each time while maintaining the solution temperature. Tannase activity 5.5u / m
l, time at 8.2 u / ml, 18.9 u / ml and 22.6 u / ml
The relationship with the pH value was as shown in FIG. Note that u /
ml is unit / ml (titer).
タンナーゼを生産する場合は、培養液に予め菌株とし
てアスペルギルス オリゼ（Aspergillus oryzae）を加
える必要があるが、上記の実験では、菌株を加えずに、
生産物であるタンナーゼを加えて行った。When producing tannase, it is necessary to add Aspergillus oryzae as a strain to the culture solution in advance, but in the above experiment, without adding the strain,
Performed with the addition of tannase, a product.
第２図における所定時間（５分、10分、20分）での酵
素量（タンナーゼ活性量）とpH値との関係をプロットす
ると、第３図のようになる。この第３図から、反応時間
とpH値とがわかれば、タンナーゼ活性量を求めることが
できる。FIG. 3 is a plot of the relationship between the amount of enzyme (tannase activity) and the pH value at predetermined times (5 minutes, 10 minutes, 20 minutes) in FIG. From FIG. 3, if the reaction time and the pH value are known, the amount of tannase activity can be determined.
第４図は、反応液（タンニン酸2.5％（W/V）、基本培
地）、タンナーゼ５、10、15、20％（W/V）の４種の酵
素液（タンナーゼ＋基本培地）における時間とpH値との
関係を示している。上側の曲線におけるイン側、、
、はそれぞれ５、10、15、20％（W/V）タンナーゼ
液が、第１図における混合槽５に対し、酵素液の1/4倍
量ポンプにより流入・混合され始めた点を示している。
下側の曲線におけるアウト側、、、は、イン側
の、、、で流され始めた溶液が、反応後最終槽
10に入り、定常化している点を示している。なお、反応
部８の通過時間は約10分であった。FIG. 4 shows the time in the reaction solution (tannic acid 2.5% (W / V), basal medium) and four enzyme solutions of tannase 5, 10, 15, and 20% (w / v) (tannase + basal medium). And shows the relationship between pH and pH value. The in side of the upper curve,
Indicates the point at which the 5, 10, 15, and 20% (W / V) tannase solution respectively began to flow and mix into the mixing tank 5 in FIG. 1 by a 1/4 volume pump of the enzyme solution. I have.
In the lower curve, the solution started to flow on the out side, on the in side, is the final tank after the reaction.
Entering 10 shows the steady state. The passage time of the reaction section 8 was about 10 minutes.
白丸をプロットした線、すなわち混合槽５におけるpH
値は、第1pH測定器11で測定した値であり、黒丸をプロ
ットした線、すなわち最終槽10におけるpH値は、第2pH
測定器12で測定した値である。第４図におけるアウト側
、、、の上に示した範囲が各条件に対する測定
域である。The line in which the white circles are plotted, ie, the pH in the mixing tank 5
The value is a value measured by the first pH meter 11, and the line plotted with a black circle, that is, the pH value in the final tank 10 is the second pH value.
This is a value measured by the measuring device 12. The range shown on the out side in FIG. 4 is the measurement area for each condition.
本発明は上記のように構成されているので、つぎのよ
うな効果を奏する。Since the present invention is configured as described above, the following effects can be obtained.
（１）複雑な前処理工程を要せず、連続的に酵素量を測
定することができる。(1) The amount of enzyme can be measured continuously without a complicated pretreatment step.
（２）得られる出力が連続であるため、コンピュータ制
御に使用することができる。(2) Since the obtained output is continuous, it can be used for computer control.
（３）測定を培養系内で完全に行えるため、無菌的操作
が可能となる。(3) Since the measurement can be performed completely in the culture system, aseptic operation becomes possible.
（４）測定にはpH電極が用いられ、簡便で長期間、連続
測定が可能である。(4) A pH electrode is used for the measurement, and simple and continuous measurement is possible for a long time.
（５）反応液を培地組成に準ずることにより、反応終了
液を培養系に戻すことができる。(5) The reaction-terminated liquid can be returned to the culture system by making the reaction liquid conform to the medium composition.
（６）酵素量の変化に対し、流速、混合比を変化させる
ことで対応できる。(6) A change in the amount of enzyme can be handled by changing the flow rate and the mixing ratio.
（７）細管部を通して反応させることにより、連続して
正確な測定ができる。(7) Accurate measurement can be continuously performed by reacting through a thin tube portion.
（８）請求項５、６では、細管部をコイル状にし、培養
槽内で一定温度に保つことにより、装置をコンパクトに
することができる。(8) In the fifth and sixth aspects, the device can be made compact by forming the thin tube portion into a coil shape and maintaining the temperature at a constant temperature in the culture tank.
第１図は本発明の酵素量測定装置の一実施例を示す概念
図、第２図は酵素反応に伴うpHの経時変化を示すグラ
フ、第３図は所定時間での酵素量（タンナーゼ活性量）
とpH値との関係を示すグラフ、第４図は混合槽及び最終
槽におけるpHの経時変化を示すグラフである。 １……培養槽、２……反応液槽、３、４、６……ポン
プ、５……混合槽、７……コイル状細管、８……反応
部、10……最終槽、11……第1pH測定器、12……第2pH測
定器、13……酵素量計測器、14……反応溶液戻しライ
ン、15……培養液、16……反応液、17……攪拌機FIG. 1 is a conceptual diagram showing one embodiment of the enzyme amount measuring apparatus of the present invention, FIG. 2 is a graph showing a change with time of pH accompanying the enzyme reaction, and FIG. 3 is an enzyme amount (tannase activity amount) at a predetermined time. )
FIG. 4 is a graph showing a change in pH over time in the mixing tank and the final tank. DESCRIPTION OF SYMBOLS 1 ... Culture tank, 2 ... Reaction tank, 3, 4, 6 ... Pump, 5 ... Mixing tank, 7 ... Coiled tubule, 8 ... Reaction part, 10 ... Final tank, 11 ... 1st pH measuring device, 12… 2nd pH measuring device, 13… Enzyme amount measuring device, 14… Reaction solution return line, 15… Culture solution, 16… Reaction solution, 17… Stirrer
───────────────────────────────────────────────────── フロントページの続き (56)参考文献 特開 昭53−121698（ＪＰ，Ａ) (58)調査した分野(Int.Cl.6，ＤＢ名) C12N 9/00 C12Q 1/25────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-53-121698 (JP, A) (58) Fields investigated (Int. Cl. 6 , DB name) C12N 9/00 C12Q 1/25
Claims (7)
の基質緩衝液を主成分とする反応液と、前記培地を主成
分として培養した培養液とを混合するに際し、培養液か
ら連続的に培養液を抜き出して反応液と混合した後、混
合液を連続的に反応させ、混合時点でのpH値、及び一定
反応時間後のpH値を測定して、これらのpH値から酵素量
を推定することを特徴とする酵素量測定方法。In an enzyme-producing culture system, a reaction solution containing a substrate buffer having the same composition as a medium as a main component and a culture solution cultured using the medium as a main component are continuously mixed from the culture solution. After extracting the culture solution and mixing it with the reaction solution, the mixture is allowed to react continuously, and the pH value at the time of mixing and the pH value after a certain reaction time are measured, and the enzyme amount is estimated from these pH values. A method for measuring the amount of an enzyme, comprising:
溶液を培養系に戻すことを特徴とする請求項１記載の酵
素量測定方法。2. The method according to claim 1, wherein the reaction solution obtained by mixing and reacting the reaction solution and the culture solution is returned to the culture system.
る麹菌培養系であることを特徴とする請求項１又は２記
載の酵素量測定方法。3. The method according to claim 1, wherein the enzyme production culture system is a koji mold culture system for tannase production.
培養槽（１）及び反応液槽（２）にそれぞれポンプ
（３、４）を介して接続された混合槽（５）と、この混
合槽（５）にポンプ（６）を介して接続されたコイル状
細管（７）を備えた反応部（８）と、この反応部（８）
に接続された最終槽（10）と、混合槽（５）に設けられ
た第1pH測定器（11）と、最終槽（10）に設けられた第2
pH測定器（12）と、第1pH測定器（11）及び第2pH測定器
（12）に接続された酵素量計測器（13）とを包含するこ
とを特徴とする酵素量測定装置。4. A culture tank (1), a reaction liquid tank (2), and a mixing tank (3) connected to the culture tank (1) and the reaction liquid tank (2) via pumps (3, 4), respectively. 5), a reaction section (8) having a coiled thin tube (7) connected to the mixing tank (5) via a pump (6), and the reaction section (8)
, A first pH meter (11) provided in the mixing tank (5), and a second pH meter (11) provided in the final tank (10).
An enzyme amount measuring device comprising: a pH measuring device (12); and an enzyme amount measuring device (13) connected to the first pH measuring device (11) and the second pH measuring device (12).
ていることを特徴とする請求項４記載の酵素量測定装
置。5. The apparatus according to claim 4, wherein the reaction section (8) is provided in the culture tank (1).
（10）が培養槽（１）内に設置されていることを特徴と
する請求項４記載の酵素量測定装置。6. The apparatus according to claim 4, wherein the reaction section (8), the mixing tank (5) and the final tank (10) are provided in the culture tank (1).
液戻しライン（14）を介して接続したことを特徴とする
請求項４、５又は６記載の酵素量測定装置。7. The apparatus according to claim 4, wherein the final tank (10) and the culture tank (1) are connected via a reaction solution return line (14).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6562190A JP2857788B2 (en) | 1990-03-16 | 1990-03-16 | Enzyme amount measuring method and apparatus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6562190A JP2857788B2 (en) | 1990-03-16 | 1990-03-16 | Enzyme amount measuring method and apparatus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03266997A JPH03266997A (en) | 1991-11-27 |
| JP2857788B2 true JP2857788B2 (en) | 1999-02-17 |
Family
ID=13292282
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP6562190A Expired - Fee Related JP2857788B2 (en) | 1990-03-16 | 1990-03-16 | Enzyme amount measuring method and apparatus |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2857788B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP7515447B2 (en) * | 2021-09-16 | 2024-07-12 | 株式会社東芝 | Apparatus and method for evaluating binding ability |
-
1990
- 1990-03-16 JP JP6562190A patent/JP2857788B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH03266997A (en) | 1991-11-27 |
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