阅读说明：本技术 基于恒温催化反应过程中实时测定吸光度的酶活测定方法 (Enzyme activity determination method based on real-time absorbance determination in constant-temperature catalytic reaction process ) 是由 姜文侠 田晓丽 张笑然 孙瑞雪 于 2019-11-28 设计创作，主要内容包括：本发明公开了一种基于恒温催化反应过程中实时测定吸光度的酶活测定方法。该方法包括：在待测酶恒温催化反应的同时实时测定反应体系吸光度；如吸光度随时间变化曲线为直线,则任意选择两个时间点测定的吸光度计算待测酶酶活；如包含直线部分,则选择此直线部分中任意两个时间点测定的吸光度计算待测酶酶活；如不为直线且不包含直线部分,则改变或优化测定条件,重新测定,直至变化曲线为直线或包含直线部分止；以上所有直线部分均至少有三个吸光度值且斜率不为零。本发明实现酶催化温度及时间的精确计量,简化操作。且以初始催化反应速率计算酶活,缩短检测时间,降低底物和待测酶液浓度,节省成本。该方法无终止催化反应操作,缩短检测总时间。(The invention discloses an enzyme activity determination method based on real-time absorbance determination in a constant-temperature catalytic reaction process. The method comprises the following steps: measuring the absorbance of a reaction system in real time while carrying out constant-temperature catalytic reaction on the enzyme to be measured; if the time-varying curve of the absorbance is a straight line, the absorbance measured at two time points is arbitrarily selected to calculate the enzyme activity of the enzyme to be measured; if the linear part is included, selecting the absorbance measured at any two time points in the linear part to calculate the enzyme activity of the enzyme to be measured; if the curve is not a straight line and does not contain a straight line part, changing or optimizing the measurement condition, and re-measuring until the curve is a straight line or contains a straight line part; all the above straight-line portions have at least three absorbance values and the slope is not zero. The invention realizes the accurate measurement of enzyme catalysis temperature and time and simplifies the operation. And the enzyme activity is calculated according to the initial catalytic reaction rate, the detection time is shortened, the concentrations of a substrate and the enzyme solution to be detected are reduced, and the cost is saved. The method has no termination of catalytic reaction operation, and shortens the total detection time.)

1. A method for measuring enzyme activity based on a spectrophotometry method comprises the following steps:

(1) carrying out real-time measurement on the absorbance value of a reaction system while carrying out constant-temperature catalytic reaction on the enzyme to be measured;

(2) drawing a variation curve of the absorbance value along with time; if the change curve of the absorbance value along with the time is a straight line with invariable slope, the absorbance values measured at two time points are selected randomly to calculate the enzyme activity of the enzyme to be measured; if the variation curve of the absorbance value along with the time comprises a straight line part, selecting the absorbance values measured at any two time points in the straight line part to calculate the enzyme activity of the enzyme to be measured; if the change curve of the absorbance value along with the time is not a straight line with a constant slope and does not contain a straight line part, changing or optimizing the measuring condition, and repeating the step (1) until the change curve of the absorbance value along with the time is a straight line with a constant slope or contains a straight line part; all of the above straight-line portions have at least three absorbance values and a slope different from zero.

2. The method of claim 1, wherein: in the step (1), the real-time measurement of the absorbance value is to measure the absorbance value at least at 3 time points.

3. The method according to claim 1 or 2, characterized in that: in the step (2), the straight line or the straight line portion is obtained under the condition that the enzyme to be tested is maintained at the initial catalytic reaction rate.

4. The method of claim 3, wherein: in the step (2), the absorbance value corresponding to the straight line or the straight line part is determined under the condition that the substrate concentration in the reaction system is not lower than the lowest concentration which enables the enzyme to be detected to keep the initial catalytic reaction rate; and/or

In the step (2), the absorbance value corresponding to the straight line or the straight line portion is measured within a maximum time length range in which the enzyme to be measured is kept at the initial catalytic reaction rate in the reaction process.

5. The method according to any one of claims 1-4, wherein: in the step (2), the change or optimization of the determination conditions is as follows: diluting the enzyme solution of the enzyme to be detected and/or increasing the concentration of the substrate.

6. The method according to any one of claims 1-5, wherein: in the step (1), the catalytic reaction of the enzyme to be detected is carried out in a container which can transmit ultraviolet light and/or visible light in a device capable of measuring absorbance, and the temperature of an enzyme catalytic reaction system in the container is accurately controlled by a medium; and (3) carrying out constant-temperature catalytic reaction on the enzyme to be detected and simultaneously measuring the absorbance of the catalytic system in real time.

7. The method according to any one of claims 1-6, wherein: in the step (1), the temperature of the enzyme solution of the enzyme to be detected and the temperature of the test solution for enzyme activity determination are made to be the same as the catalytic reaction temperature in advance.

8. The method according to any one of claims 1-7, wherein: the enzyme to be detected is an enzyme capable of detecting the change of the concentration of a substrate or a product thereof by a spectrophotometric method.

9. The method of claim 8, wherein: the enzyme to be detected is an enzyme with a catalytic substrate containing dissolved oxygen and an enzyme with a catalytic substrate not containing dissolved oxygen;

further, the enzyme that catalyzes the substrate, including dissolved oxygen, is an oxidase;

still further, the oxidase is a laccase.

10. Use of the method according to any one of claims 1 to 9, characterized in that: the application is to compare the activity of different enzymes or different enzyme preparations.

Technical Field

The invention relates to the field of enzyme activity determination, in particular to an enzyme activity determination method based on real-time absorbance determination in a constant-temperature catalytic reaction process.

Background

Enzyme activity is an essential parameter for the discovery, creation, research, production, isolation, purification and use of enzymes and enzyme preparations, expressed as the rate of reaction catalyzed by the enzyme under test conditions such as a specific temperature.

Spectrophotometry is the most commonly used enzyme activity determination technique, and the process of measuring enzyme activity by spectrophotometry generally comprises maintaining an enzyme catalysis reaction system at a certain set temperature for a certain time, then inactivating the enzyme by heating to boil or adding an enzyme inhibitor to terminate the reaction, determining the generation amount of an enzyme catalysis product or the consumption amount of an enzyme catalysis substrate by the change of absorbance (OD) of the reaction system before and after the enzyme catalysis reaction, and further calculating to obtain the enzyme activity [ Miyabe R, Takahashi Y, Matsufuji H, Ogihara J, Itou K, Kawai Y, et al. 21(3) 445-51.] [ Mejias L, Cerda A, BarrenaR, Gea T, Sanchez A. microbiological protocols for cellular and xylanase production through soluble-state transfer of differential from biological. Sustainability.2018; https:// doi.org/10.3390/su10072433.] [ Alhelli AM, Abdul Man MY, Mohammed AS, Mirhosseini H, Suliman E, Shad Z, et al.Response surface methodology of an aqueous two-phase system for publication of protein from peptide library candidate (PCA1/TT031) under solid state conversion and is biological chemical conversion. International Journal of molecular science.2016; https:// doi. org/10.3390/ijms17111872 ]. The method is called as a two-point method because only the absorbance values of two time points before and after the catalytic reaction are measured. In such a measurement process, since the catalytic reaction process of the enzyme and the detection of absorbance are separated from each other in time and space, that is, the reaction and the detection are not simultaneously performed in the same vessel, the operation is complicated, and errors are liable to occur.

The measurement of the reaction temperature and reaction time of enzyme catalysis is an important influence factor for the accuracy of enzyme activity detection. In the existing 'two-point method', the reagent mixing and temperature control processes of an enzyme catalysis reaction system at the initial stage of the reaction have larger system errors and accidental errors at the stage of terminating the enzyme catalysis reaction. The concrete expression is as follows: in the initial stage of the reaction, the temperature of a reagent solution of the enzyme catalysis reaction and the temperature of an enzyme solution to be detected are generally room temperature, and the set temperature of the enzyme catalysis reaction is mostly not room temperature; mixing the above liquids to form a reaction system, and starting enzyme catalysis reaction; the mixed reaction system usually needs a period of time in a water bath to reach the set temperature; therefore, the time point at which the enzyme-catalyzed reaction starts is not the time point at which the set catalysis temperature is reached. In the termination reaction stage, whether the enzyme-catalyzed reaction is terminated by heating to boil or by adding an inhibitor of the enzyme, the termination operation takes a certain time, and thus the time point at which the reaction is terminated cannot be accurately determined. To reduce the errors caused by the above factors, the influence of the metering error is often reduced by higher concentrations of substrate and longer enzyme-catalyzed reaction duration (usually controlled at 10-60 min) [ Miyabe R, Takahashi Y, Matsufuji H, Ogihara J, Itou K, Kawaiy, et al.purification and partial catalysis of an X-propyl-dipeptidylaminopeptidase from Lactobacillus gasseri ME-284.Food Science and technology research.2015; 21(3) 445-51.] [ Mejias L, Cerda A, Barrena R, Gea T, SanchezA. microbiological protocols for cellular and xylanase production through soluble-state transfer of geographic from biological. Sustainability.2018; https:// doi.org/10.3390/su10072433.] [ Alhelli AM, Abdul Man MY, Mohammed AS, Mirhosseini H, Suliman E, Shad Z, et al.response surface methodology of an aqueous two-phase system for the purpose of purification of a protease organism from polymeric library (PCA1/TT031) under solid state conversion and situ biological chemistry, International Journal of molecular science, 2016; https:// doi. org/10.3390/ijms17111872 ].

The kinetics of the enzyme reaction have been studied to show that, in the initial stage of the enzyme reaction, the amount of change in the substrate or product increases linearly with the increase in the reaction time, and the catalytic reaction rate of the enzyme is the greatest, and the reaction rate of the enzyme at this time is referred to as the initial catalytic reaction rate. As the reaction time increases, the rate of consumption of the substrate or production of the product decreases and the rate of the catalytic reaction of the enzyme decreases. The factors causing the reduction of the enzyme reaction rate are many, the substrate concentration is continuously reduced along with the continuation of the reaction, the product is continuously increased, the reverse reaction becomes obvious gradually from the beginning to the end, and meanwhile, the factors such as acid, alkali, heat and the like also slowly act to destroy and inactivate the enzyme. Therefore, the only real representation of the catalytic activity of an enzyme is the initial catalytic reaction rate of the enzyme [ Junesiu amber. enzyme engineering Manual. Beijing: china light industry press; 2011: 172.] is a characteristic index that can be used for standardization of enzymatic activity comparisons. The current "two-point method", which measures only the substrate or product concentration at the beginning and end of the reaction, cannot determine whether the substrate concentration is always within the concentration range that keeps the enzyme at the initial catalytic reaction rate during the reaction [ Hu HL, van den Brink J, Gruber BS, Wosten HAB, Gu JD, de Vries RP.improved enzyme production by co-cultivation of Aspergillus niger and Aspergillus oryzae and with other enzymes International biodegradation & Biodegradation.2011; 65(1):248-52.] [ Alberto Batista-Garcia R, Balcazar-Lopez E, Miranda-Miranda E, Sanchez-Reyes A, Curvo-Soto L, Aceves-Zamouio D, et al.Characterisation of lignocelluloses from a modular halophiles string of Aspergillus caesium spent from a transgenic breast tissue transfer. P.S. one.2014; https:// doi.org/10.1371/journal.pone.0105893.] [ Ding ZY, Chen YZ, Xu ZH, Peng L, Xu GH, Gu ZH, et.production and characterization of milk from Pleurotus ferulae injected transfer. annals of microbiology.2014; 64(1) 121-9 ], the measured result is actually the average catalytic rate between two measuring time points, the accuracy and comparability of the enzyme activity detection result are influenced, and one of the reasons of confusion of the enzyme activity standard is also provided.

In addition, no mature device and method for on-line detection of enzyme activity exists so far, and timely feedback control cannot be implemented in research, production, separation, purification and application of enzyme.

Therefore, if the operation can be simplified, the metering precision is improved, the detection time is greatly shortened, the initial catalytic reaction rate of the enzyme is measured, and a more accurate detection result is obtained in time, the method is not only beneficial to establishing a general standard of the enzyme activity measuring method, but also lays a foundation for developing an online enzyme activity detecting system.

Disclosure of Invention

The invention aims to provide a rapid enzyme activity determination method based on real-time absorbance determination in a constant-temperature catalytic reaction process.

The invention provides a method for measuring enzyme activity based on a spectrophotometry, which comprises the following steps:

(1) the constant temperature catalytic reaction process of the enzyme and the detection of the absorbance value are carried out in the same time and space, namely: carrying out real-time measurement on the absorbance value of the reaction system while carrying out constant-temperature catalytic reaction on the enzyme to be measured;

(2) drawing a variation curve of the absorbance value along with time; if the change curve of the absorbance value along with the time is a straight line with invariable slope, the absorbance values measured at two time points are selected randomly to calculate the enzyme activity of the enzyme to be measured; if the variation curve of the absorbance value along with the time comprises a straight line part, selecting the absorbance values measured at any two time points in the straight line part to calculate the enzyme activity of the enzyme to be measured; if the change curve of the absorbance value along with the time is not a straight line with a constant slope and does not contain a straight line part, changing or optimizing the measuring condition, and repeating the step (1) until the change curve of the absorbance value along with the time is a straight line with a constant slope or contains a straight line part; all of the above straight-line portions have at least three absorbance values and a slope different from zero.

The method can pass through R of a linear part²Value judging the accuracy of the enzyme activity determination value, wherein R²The defined range of values is determined by the degree of accuracy of the assay result desired in actual practice. To distinguish the current "two-point method", the method established by the present invention is called "multipoint method".

Further, in the step (1), the real-time measurement of the absorbance values is to measure the absorbance values of at least 3 time points, the absorbance values of the first two time points can be used for calculating a detection value of the enzyme activity, and the last absorbance value can be used for verifying whether the change of the absorbance values of at least 3 time points along with time is a straight line verification value.

Further, in the step (2), the straight line or the straight line portion should be obtained under the condition that the enzyme to be tested is maintained at the initial catalytic reaction rate.

Further, the absorbance value corresponding to said straight line or said straight line portion in the step (2) should be measured in the case where the substrate concentration in the reaction system is not lower than the lowest concentration at which said enzyme to be measured maintains the initial catalytic reaction rate; in step (2), the absorbance value corresponding to the straight line or the straight line part is determined within the maximum time length range for keeping the enzyme to be detected at the initial catalytic reaction rate in the reaction process.

Wherein the minimum concentration of the substrate capable of maintaining the initial catalytic reaction rate of the enzyme to be tested and the maximum duration of time capable of maintaining the initial catalytic reaction rate of the enzyme to be tested are determined from the absorbance value versus time curve.

Further, in step (2), the changing or optimizing of the assay conditions may specifically be diluting the enzyme solution to be tested and/or increasing the substrate concentration.

In the step (1), the catalytic reaction of the enzyme to be detected is carried out, specifically, the catalytic reaction can be carried out in a container (such as a cuvette of a spectrophotometer) which can transmit ultraviolet light and/or visible light in a device capable of measuring absorbance, and the temperature of an enzyme catalytic reaction system in the container is accurately controlled through a medium (such as circulating water bath); and (3) carrying out constant-temperature catalytic reaction on the enzyme to be detected and simultaneously measuring the absorbance of the catalytic system in real time.

In order to avoid an error caused by the fact that the temperature of the catalytic system does not reach the set temperature in the initial reaction stage, in step (1) of the method, the temperature of the enzyme solution of the enzyme to be detected and the temperature of the test solution for enzyme activity measurement are made to be the same as the catalytic reaction temperature in advance, for example, the enzyme solution is placed in a water bath with the same catalytic reaction temperature and is subjected to sufficient water bath constant temperature.

In the method, the enzyme to be tested may be an enzyme capable of detecting a change in the concentration of its substrate or product spectrophotometrically.

Further, the enzyme to be tested may be an enzyme that catalyzes a substrate including dissolved oxygen and an enzyme that catalyzes a substrate not including dissolved oxygen.

Further, the enzyme that catalyzes a substrate including dissolved oxygen may be an oxidase.

Still further, the oxidase is a laccase.

The term "substrate" as used herein means: the enzyme-acting substance can form a product by the action of enzyme.

"dissolved oxygen" in the present invention is molecular oxygen dissolved in an aqueous solution. The content of dissolved oxygen in the aqueous solution is related to the partial pressure of oxygen in the air and the temperature of the solution.

In a specific embodiment of the present invention, the enzyme to be detected is specifically any one of the following:

(A) catalytic substrates include oxygen-dissolving enzymes such as: laccase or glucose oxidase;

(B) the catalytic substrate does not include oxygen-dissolving enzymes such as α -chymotrypsin, aminopeptidase, alcohol dehydrogenase, horseradish peroxidase, manganese peroxidase or catalase.

In addition, the following uses of the method provided by the invention also belong to the protection scope of the invention: comparing the activities of different enzymes or different enzyme preparations.

The invention enables the constant temperature catalytic reaction of the enzyme and the detection of the absorbance to be carried out at the same time and space, realizes the accurate control of the temperature of the enzyme catalytic reaction and the accurate measurement of the duration of the catalytic reaction in the detection process, and greatly reduces the error caused by the measurement. Because the concentration of the product (or the substrate) and the change of the reaction rate can be monitored in real time while the enzyme is in the constant-temperature catalytic reaction, on one hand, the error of the measurement result caused by the consumption of the substrate in the reaction process can be avoided, the enzyme activity can be calculated through the initial catalytic reaction rate, and the measurement result is accurate. On the other hand, since the initial catalytic reaction rate is an important characteristic of the enzyme, the comparison of the activities of different enzymes and preparations thereof is more convenient. On the basis, the invention not only obviously shortens the time required by detection, but also greatly reduces the concentrations of the substrate and the enzyme solution to be detected, and saves the reagent cost. The invention does not need to terminate the catalytic reaction, greatly simplifies the operation procedure and further shortens the total time consumed in the whole detection process.

Drawings

FIG. 1 is a schematic view of a water bath thermostatic cuvette holder. 1 is a cuvette; and 2 is an external circulation pipeline.

FIG. 2 shows the change of the absorbance with time during the enzyme activity detection of α -chymotrypsin.

FIG. 3 is a graph showing the change of absorbance with time in the process of detecting the enzyme activity of aminopeptidase.

FIG. 4 is a graph showing the change of absorbance with time in the process of detecting the enzyme activity of alcohol dehydrogenase.

FIG. 5 shows the change of absorbance with time during the detection of horseradish peroxidase activity.

FIG. 6 is a graph showing the change of absorbance with time in the enzyme activity detection process of manganese peroxidase.

FIG. 7 shows the change of the dissolved oxygen concentration in the enzyme catalysis system for enzyme activity detection of laccase at three different temperatures with time.

FIG. 8 shows the change of dissolved oxygen concentration in enzyme activity detection systems with different laccase concentrations.

FIG. 9 shows the change of absorbance value with time during the laccase activity detection process. Wherein (a) is before diluting the enzyme solution to be tested; (b) after diluting the enzyme solution to be tested.

FIG. 10 shows the change of absorbance with time during the detection of glucose oxidase enzyme activity.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.