A method for determining alpha-amylase activity

Abstract

The present application belongs to the technical field of enzyme activity determination, and aims at the problems of low sensitivity, non-stoichiometry, poor substrate solubility and standard curve intercept deviating from the origin in the existing amylase activity determination technology, and provides a determination method of alpha-amylase activity, wherein starch or hydroxypropyl starch is used as a substrate to prepare a substrate solution, different concentrations of glucose standard solutions prepared by using the substrate solution as a solvent are taken, fresh prepared MBTH reagent is added under alkaline conditions, ammonium ferric sulfate reagent is added, color development reaction is carried out, absorbance value is determined, and a standard corresponding relationship curve is drawn according to the relationship between each group of glucose concentrations and the determined absorbance values; the substrate solution is taken, amylase is added, enzyme hydrolysis reaction is carried out, and absorbance value is determined; and amylase activity is calculated. Compared with the MBTH method of the prior art, the method is more sensitive and accurate, and the color development product is more stable.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of enzyme activity determination, and particularly relates to a method for determining alpha-amylase activity. BACKGROUND

[0002] Amylase is a polysaccharide hydrolase capable of degrading starch and other polysaccharides containing alpha-1, 4-glucosidic bonds. Amylase is widely used in medicine, medical diagnosis, food, feed, bioenergy, etc., and enzyme activity is a key parameter thereof. The enzyme activity is generally determined by a method for determining the initial speed of enzyme reaction. According to WS / T349-2011 Reference Method for Determination of Alpha-amylase Catalytic Activity Concentration, the activity unit of amylase can be defined as the amount of 1 micromole of reducing end group equivalent to glucose produced per milliliter of reaction solution per minute under the condition of 37 DEG C and optimal pH. The measured enzyme activity includes the total enzyme activity of endo- and exo-starch glycosidic bonds.

[0003] At present, the amylase activity determination method still has the following problems:

[0004] Substrate problem. Currently marketed starches include corn starch, potato starch, soluble starch, etc. Corn starch and potato starch have poor solubility and need to be heated to dissolve. However, the solubilization process is accompanied by a substantial increase in viscosity, and the high viscosity results in extremely low mass transfer rate between the enzyme and the substrate, and the enzyme reaction process is a non-zero order reaction, making it difficult to ensure the accuracy of the amylase enzyme activity determination method. The commercially available soluble starch also needs to be heated to dissolve, and although its viscosity has been greatly reduced, precipitation will occur after cooling. Therefore, in WS / T349-2011 Reference Method for Determination of Alpha-amylase Catalytic Activity Concentration, a synthetic substrate 4, 6-ethylene (G1)-4-nitrobenzene (G7)-alpha-(1->4)-D-maltose heptasaccharide (EPS) is used to determine enzyme activity. However, this substrate is extremely expensive and is a non-natural substrate, and its apparent enzyme activity is relatively low, so it cannot be used as a routine test method for amylase production and application enterprises.

[0005] The DNS method is currently used to determine the activity of amylase (GB / T 36861-2018), which has relatively low sensitivity. Low sensitivity of the determination method has two effects on the determination of enzyme activity. On the one hand, a larger substrate enzymolysis rate is required to measure the concentration of the reducing end group of the enzymolysis product, and the initial speed of enzymolysis cannot be measured. On the other hand, a higher enzyme concentration is required, and the impurity concentration in the enzyme solution is also correspondingly higher, and their activation or inhibition of amylase will be more obvious. In addition, the intercept of the glucose concentration standard curve of the DNS method is large, but the origin of the coordinate axis, and different dilution multiples of amylase will directly affect the accuracy of the determination of amylase activity. The problem that the DNS method does not comply with stoichiometry has been reported early (Mccleary B V, Mcgeough P. A Comparison of Polysaccharide Substrates and Reducing Sugar Methods for the Measurement of endo-1,4-β-Xylanase [J]. Applied Biochemistry & Biotechnology, 2015, 177(5): 1152-1163).

[0006] Chinese patent document with application number 202010619456.1 discloses a method for detecting the activity of α-amylase in breast milk. Fresh breast milk sample is mixed with saturated ammonium sulfate solution, shaken thoroughly, centrifuged to obtain clear liquid, and then the activity of α-amylase in breast milk is determined by iodine-starch colorimetry. The disadvantages of this method are that the starch solution is unstable and prone to sedimentation, which greatly interferes with the results, and it is low in efficiency and time-consuming.

[0007] The MBTH method (Zhang Y Q, Wang Z P, Xu J, Xue C H, and Jan Z C. Study on the determination of xylose content by MBTH method [J]. Food Science and Technology, 2010, 35(04): 247-250. DOI: 10.13684 / j.cnki.spkj.2010.04.027.) can be used to determine the activity of xylanase, lysozyme and β-glucanase. The inventors found that the chromogenic product is not stable enough when using the MBTH method to determine the activity of α-amylase, which leads to a significant change in the slope of the glucose standard curve over time. In addition, in the prior art, the calculation of enzyme activity is based on the glucose concentration standard curve, and the substrate in the enzyme solution also has a reducing end group, both of which participate in the MBTH reaction. Therefore, when optimizing the glucose concentration determination method, the effect of the substrate on glucose determination must be considered. The above problems will directly affect the accuracy, repeatability and stability of the determination of α-amylase activity. SUMMARY

[0008] In view of the problems of the existing starch amylase activity determination technology, such as difficult to dissolve and high viscosity of the substrate, low sensitivity of DNS method, deviation of the standard curve intercept from the coordinate origin and instability of the chromogenic product, the application provides a kind of determination method of alpha-amylase activity, by improving the existing substrate dissolution method and modifying the substrate structure, the substrate is more easily dissolved and has lower viscosity, by improving the MBTH reagent, detection wavelength, drawing method of glucose standard curve, ammonium ferric sulfate reagent, the obtained standard curve intercept is closer to the coordinate origin, the chromogenic product is more stable and the substrate in the enzyme solution does not interfere with the determination of glucose, so as to improve the sensitivity and accuracy of the detection and the repeatability of the experiment.

[0009] The purpose of the application is achieved by the following technical solutions:

[0010] The application provides a kind of determination method of alpha-amylase activity, comprising the following steps:

[0011] (1) preparation of substrate solution:

[0012] The substrate solution is prepared by alkali dissolution method, which is to disperse starch or hydroxypropyl starch into strong alkali solution, mix thoroughly until the starch is dissolved, adjust the pH value of the hydroxypropyl starch solution to the measured value with an acidic solution, and then dilute to volume with a buffer solution of the measured pH value to obtain the substrate solution, wherein the acidic solution is a conjugate acid solution;

[0013] Alternatively, the substrate solution is prepared by heating method, which is to disperse hydroxypropyl starch (HPS) into a buffer solution of the measured pH value and heat to dissolve the substrate at 70-100 DEG C;

[0014] The measured pH value is the pH value for determining the activity of starch amylase, and the molar substitution degree of the hydroxypropyl starch is 0-0.5;

[0015] The same buffer solution is used to prepare the starch amylase solution, that is, the buffer solution used for preparing the starch amylase solution and the buffer solution used for preparing the substrate solution are completely the same in terms of type, concentration and pH value; preferably, a buffer solution with good stability is selected to shorten the operation time as much as possible, so that the type, concentration and pH value of the buffer solution are completely the same at the time of preparing the starch amylase solution and the substrate solution, thereby eliminating the possible influence on the detection results.

[0016] The viscosity of the solution obtained by dissolving starch by alkali solution method is much lower than that by heating. The starch solution will precipitate due to aging phenomenon if left for a long time, so it is better to prepare it immediately before use. The aging phenomenon of hydroxypropyl starch is not obvious and will not affect the determination. Compared with the preparation of substrate by heating method, alkali solution method has its own advantages. If this method is applied to automatic high-throughput analysis, alkali solution method has more advantages because of short dissolution time and no need for heating and waiting. The heating method is relatively simple and suitable for manual operation in the laboratory.

[0017] HPS is a non-ionic starch derivative, and its glycosidic bond has a spatial structure closer to starch than malt oligosaccharide and carboxymethyl starch. If the degree of substitution is too low, the viscosity is high. If the degree of substitution is too high, too much hydroxypropyl will seriously affect the spatial structure of α-1, 4 glycosidic bond in starch, which will directly affect the affinity of the substrate and the enzyme. As preferred, the molar degree of substitution of HPS is 0.05-0.5.

[0018] The specific dissolution phenomenon is shown in Table 1.

[0019] (2) Draw the standard corresponding relationship curve of glucose absorbance value and glucose concentration:

[0020] a. Use the substrate solution prepared in step (1) to prepare a series of glucose standard solutions with concentration gradient, and the concentration of the substrate in the series of glucose standard solutions is the same, and the glucose concentration range is 0-0.3 mmol / L

[0021] b. Add an alkaline reagent to the series of glucose standard solutions with concentration gradient to obtain an alkaline glucose standard solution, the concentration of the alkaline reagent in the alkaline glucose standard solution is the same, then add freshly prepared MBTH reagent, the OH - concentration in the system is 0.01-0.2 mol / L, the concentration range of MBTH is 0.3-4.5 mmol / L, and the concentration range of DTT is 0-1.6 mmol / L, and the reaction is carried out at 50-90℃ for 5-180 min; then add an acidic iron reagent, the H + concentration in the system is 0.02-0.3 mol / L, and the initial concentration range of Fe 3+ is 1-6 mmol / L, and the color reaction is carried out, and the absorbance value is measured at 580-680 nm after color development is stable, and the standard corresponding relationship curve is drawn according to the relationship between each group of glucose concentration and the measured absorbance value;

[0022] The MBTH reagent is an aqueous solution prepared from MBTH and DTT, or an aqueous solution prepared only from MBTH; and the acidic iron reagent is an aqueous solution prepared from a soluble Fe 3+ salt and a strong acid;

[0023] The concentration range of MBTH is 0.3-4.5 mmol / L, and the concentration range of DTT is 0-1.6 mmol / L. It can be understood that the concentration of MBTH reagent described here is the initial concentration at the moment of adding the MBTH reagent before reaction.

[0024] The concentration range of Fe 3+ is 1-6 mmol / L. It can be understood that the concentration of Fe 3+ described here is the initial concentration at the moment of adding the acidic iron reagent before reaction.

[0025] The unreacted MBTH reagent and Fe 3+ need to react in an acidic environment. The aforementioned H + concentration describes the H + concentration after neutralization of the acid and base after adding the acidic iron reagent.

[0026] (3) Enzymatic reaction of amylase:

[0027] The substrate solution prepared in step (1) is added to the amylase solution to ensure that the concentration of the substrate in the obtained solution is the same as that in the glucose standard solution in step (2), and then the enzymatic reaction is carried out. The alkaline reagent is added to terminate the enzymatic reaction, and the alkaline enzymatic solution is obtained. The concentration of the alkaline reagent in the alkaline enzymatic solution is the same as that in the alkaline glucose standard solution in step (2).

[0028] (4) Preparation of alkaline enzymatic blank solution:

[0029] Specifically, it is the method of step (3), the difference is that the alkaline reagent is added to the substrate solution first, and then the amylase solution is added, thereby obtaining the alkaline enzymatic blank solution.

[0030] (5) Determination of alkaline enzymatic solution and alkaline enzymatic blank solution:

[0031] According to the method of step (2), the MBTH reaction is carried out to determine the alkaline enzymatic solution and the alkaline enzymatic blank solution, thereby obtaining the concentration of the reducing end group produced by enzymatic reaction, and further obtaining the amylase activity.

[0032] Table 1 Solubility and gel state of starch and HPS solution at room temperature

[0033]

[0034]

[0035] The addition of substrate during the experiment can improve the accuracy of the standard curve, thereby improving the accuracy of the determination of enzyme activity. Starch is the natural substrate of amylase, but its poor solubility affects the determination of enzyme activity. The substrate described in the present application is hydroxypropyl starch, which is a non-ionic starch derivative, and its glycosidic bond has a spatial structure closer to starch. Its degree of substitution is too low, and hydroxypropyl starch needs a higher OH - concentration (0.4 mol / L) to dissolve, and the high concentration of salt produced by adjusting the pH of the enzyme to the optimum pH will affect the determination of amylase activity; a too high degree of substitution will directly affect the affinity of the substrate and the enzyme. Further preferably, the molar degree of substitution of the hydroxypropyl starch is 0.05-0.5; the OH - concentration is preferably 0.1-0.2 mol / L.

[0036] As a preferred, in the step (1), the concentration of the substrate solution is 1-20 mg / mL, more preferably 5-15 mg / mL.

[0037] As a preferred, in the step (1), the pH value of the substrate solution is within a certain range of the optimum pH value of the measured amylase, more preferably the optimum pH ± 1, and further preferably the optimum pH value, and the pH is acidic or alkaline.

[0038] As a preferred, in the step (1), the buffer solution is an acetate buffer solution, a citrate buffer solution, a phosphate buffer solution, a borate buffer solution or a succinate buffer solution, more preferably a sodium acetate buffer solution, a sodium citrate buffer solution, a sodium phosphate buffer solution, a sodium borate buffer solution or a sodium succinate buffer solution.

[0039] As a preferred, the specific preparation method of the substrate solution in the step (1) is: dispersing hydroxypropyl starch into an alkaline solution, then freezing, adding an acid solution to adjust the pH value to be measured after the substrate is completely dissolved, and then supplementing with a buffer solution of the required pH to the required volume.

[0040] As a preferred, except as otherwise specified, the same kind and concentration of reagent for adjusting the pH value in different steps of the present application is preferred to maximize the factors that may cause errors. The alkaline reagent can be selected from sodium hydroxide and potassium hydroxide, preferably sodium hydroxide.

[0041] As a preferred, in the step (2), after adding MBTH to the alkaline glucose standard solution, the OH - concentration in the system is in the range of 0.1-0.2 mol / L, the concentration of MBTH is in the range of 0.95-3.6 mmol / L, and the concentration of DTT is 0 mmol / L;

[0042] After adding the acidic iron reagent, the H+ Fe2+ initial concentration ranges from 3 to 5 mmol / L. 3+

[0043] When DTT is not contained in the MBTH reagent, the slope of the standard curve of the reducing sugar is larger and more stable, the sensitivity of detecting the reducing sugar is higher, and the intercept is closer to 0, which is more consistent with the Lambert-Beer law.

[0044] The MBTH reagent is prepared in advance and is effective within one day. In some embodiments of the present application, the concentration of MBTH in the prepared MBTH is 5.5-7.5 mmol / L, and the concentration of DTT is 0-5 mmol / L. In the prepared acidic iron reagent, the concentrations of ammonium ferric sulfate and strong acid H + are 6-15 mmol / L and 0.35-0.55 mol / L, respectively. The concentration of the prepared basic reagent is 0.3-0.6 mol / L. As a preferred, the volume ratio of the amount of glucose standard solution: basic reagent: MBTH reagent: acidic iron reagent is 1: (0.8-1.2): (0.8-1.2): (1.6-2.4).

[0045] As a preferred, in the step (2), the reaction temperature of the MBTH reagent with the glucose or the enzymatic hydrolysate or the enzymatic hydrolysis blank solution is 75-85℃, and the reaction time is 9-17 min, more preferably 78-82℃, and 10-15 min. In some embodiments provided by the present application, the reaction temperature of the MBTH reagent with the glucose or the enzymatic hydrolysate or the enzymatic hydrolysis blank solution is 80℃, and the reaction time is 13 min. When the reaction is carried out for 13 min, the absorbance value of the reaction solution is the largest and remains unchanged with the extension of the reaction time.

[0046] As a preferred, the soluble Fe 3+ salt is at least one of ferric nitrate, ferric chloride, and ammonium ferric sulfate, and the strong acid is preferably hydrochloric acid, sulfuric acid, or sulfonic acid, and more preferably the acidic iron reagent is prepared from an ammonium ferric sulfate and an amino sulfonic acid solution.

[0047] As a preferred, after the step (2) of adding the acidic iron reagent, the solution is naturally cooled to room temperature, and the absorbance value is determined after the color development is stable. As a preferred, the color development time is 50-180 min, and more preferably the color development time is 60-120 min.

[0048] As a preferred, the wavelength for determining the absorbance value is 580-655 nm, and more preferably the wavelength for determining the absorbance value is 590-610 nm, and preferably an enzyme label instrument is used to determine the absorbance. In some embodiments provided by the present application, the color development time is 60-120 min, and the absorbance value is determined by using an enzyme label instrument at a wavelength of 590-610 nm. When the determination wavelength is 590 nm, the intercept is close to 0, and the R 2 ​More close to 1. The standard curve slope obtained is higher when the determination wavelength is 610 nm. As preferred, the shaking operation in the enzyme reader before determination is more stable.

[0049] Step (3), the enzymolysis time is preferably 1-60 min, more preferably 3-40 min, and more preferably 10-30 min. As long as the enzymolysis reaction occurs, the enzyme activity can be determined by the method of the present application. A longer enzymolysis time is beneficial to large batch determination, but it cannot be too long to avoid the decrease of enzyme activity in the enzymolysis process. As preferred, the enzymolysis reaction is carried out at 37℃ for 10-30 min.

[0050] Step (5), since the method has high sensitivity and the enzymolysis process is a zero-order reaction, the enzymolysis speed in the determination time is the same, so the generation amount of the product in the enzymolysis time period can be used to calculate the enzymolysis rate, thereby obtaining the enzyme activity.

[0051] The present application has the following beneficial effects:

[0052] 1. The present application provides a preparation method of a natural substrate of amylase. Compared with the starch that cannot be dissolved in a buffer solution and the starch solution prepared by a heating method with high viscosity, the starch solution prepared by the preparation method has the advantages of complete dissolution and significantly reduced viscosity, and is suitable for amylase activity determination.

[0053] 2. The present application provides a water-soluble substrate of amylase, which is a hydroxypropyl starch with a molar substitution degree of 0.05-0.5. The advantages are that it is completely dissolved in a buffer solution, the preparation process is simple, the solution has low viscosity, and the low substitution degree does not affect the combination with amylase, and is suitable for amylase activity determination.

[0054] 3. Compared with the prior art MBTH method, the time stability of the present application is greatly improved. The chromogenic product of the enzymolysis solution tested by the MBTH method remains stable within 180 minutes, which improves the stability and repeatability of the determination.

[0055] 4. Compared with the prior art MBTH method, the accuracy of the present application is greatly improved. The specific measures are as follows: a standard curve of substrate for simulating enzymolysis reaction is used for enzyme activity calculation; a DTT-free MBTH reagent is used; and the absorbance value is determined at 580-680 nm. The enzyme concentration curve obtained is linear, the intercept of the equation is close to 0, and the accuracy of enzyme activity determination is not affected by different dilution multiples of the enzyme solution. BRIEF DESCRIPTION OF DRAWINGS

[0056] Figure 1The figures show the solubility of different starches under heating and non-heating conditions. Samples 1, 2, 3, 4, and 5 in the figure represent potato starch (pharmacographa grade), corn starch (reagent grade), corn starch (pharmaceutical grade), potato starch (powder), and soluble starch, respectively. The buffer solution used was pH 6.9 0.05M phosphate buffer. Left: The state of the five starches after mixing with the buffer solution; none were completely dissolved. Middle: The state of the samples in the left figure after heating; none showed precipitation. Right: After the samples in the middle figure were left to stand overnight, all except sample 4 showed no precipitation.

[0057] Figure 2 This shows the solubility of different starches under alkaline conditions. Samples 1, 2, 3, 4, and 5 in the figure are shown below. Figure 1 As described in the left figure: 0.2M NaOH solution was added to the starch samples, followed by the addition of acid to neutralize the added alkali, and then buffer solution was added to make up the volume. No precipitation was formed in any of them. After standing for one day, except for sample No. 4 which was still in a dissolved state, all the others had precipitation to varying degrees.

[0058] Figure 3 The figures show the background values ​​of different starch solutions after the colorimetric reaction. Samples 1, 2, 3, 4, and 5 in the figure are shown below. Figure 1 The absorbance value of sample No. 5 was the highest, but this did not affect the measurement.

[0059] Figure 4 The standard curves for glucose determination are shown for the prior art DNS method and the present invention MBTH method. The standard curve for glucose obtained by the MBTH method has a larger slope, that is, the MBTH method is more sensitive.

[0060] Figure 5 The solubility of HPS samples with different degrees of substitution (MS values ​​of 0, 0.029, 0.072, 0.052, 0.075, 0.414, 1.597, 1.003, and 1.865) in water at room temperature is shown, with an HPS concentration of 10 mg / mL. MS samples were soluble upon heating to a temperature below 0.075°C, and soluble at room temperature above 0.4°C.

[0061] Figure 6 This invention relates to the effect of DTT concentration in the MBTH reagent on the slope of the glucose standard curve.

[0062] Figure 7 This is the MBTH glucose standard curve at 590 nm, as presented in this invention.

[0063] Figure 8 The figures show the reaction kinetics curve of amylase using the MBTH method of this invention (left figure, zero-order reaction) and the reaction rate curve of enzyme mass concentration (right figure, which is linear). Detailed Implementation

[0064] The technical solutions in the present application will be described clearly and completely below in combination with the embodiments in the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the protection scope of the present application.

[0065] The main instruments and reagents used in the following embodiments are as follows:

[0066] Main instruments: water bath constant temperature oscillator, SHZ-82, Changzhou Zhiluorui Technology Co., Ltd.; FE28 type pH meter, Mettler-Toledo Instruments Co., Ltd.; Bio-rad 680 enzyme label instrument, Bole Life Science Products (Shanghai); UV-2600 ultraviolet visible spectrophotometer, SHIMADZU; AL204 type electronic balance, Mettler-Toledo Instruments Co., Ltd.

[0067] Main reagents: soluble starch, purchased from Sinopharm Chemical Reagent Co., Ltd.; glucose standard was purchased from Sigma Aldrich Trading Co., Ltd.; hydroxypropyl starch with a molar substitution degree of 0.05-2.0 was self-made in the laboratory (the preparation method is prior art); amylase, Wuhan Xinhua Yang Biological Co., Ltd.; MBTH and DTT reagents were from Sigma Aldrich Trading Co., Ltd.; sulfamic acid, FeNH4(SO4)2·12H2O, Fe(NO3)3·9H2O and FeCl3 were purchased from Sinopharm Chemical Reagent Co., Ltd.; other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd., and other reagents were all analytical pure. Distilled water was used in the experimental process. The enzyme activity in the following embodiments and comparative examples was calculated according to the following formula:

[0068]

[0069] In the formula: U-amylase activity, U;

[0070] A-absorbance value of the enzyme hydrolysis solution;

[0071] A0-absorbance value of the enzyme hydrolysis blank solution;

[0072] b-intercept of the glucose standard curve;

[0073] k-slope of the glucose standard curve, mL / μmoL;

[0074] t-hydrolysis time, min.

[0075] Example 1

[0076] 1. Preparation of substrate solution

[0077] Hydroxypropyl starch with a molar substitution of 0.05 was dispersed in a PBS buffer solution with a pH of 6.9 and a concentration of 0.05 mol / L, and was dissolved by heating at 80°C for 1.5 h to prepare a 6 mg / mL hydroxypropyl starch solution.

[0078] 2. Amylase activity determination

[0079] Step (1): A series of glucose standard solutions with different concentrations were prepared in a sodium phosphate monobasic-sodium phosphate dibasic buffer solution with a pH of 6.9 and a concentration of 0.05 mol / L. The concentration of glucose was in the range of 0-100 μg / mL (equivalent to 0-0.3 mmol / L). 0.5 mL of each glucose standard solution was taken and mixed with 0.5 mL of the above hydroxypropyl starch solution, 1 mL of 0.5 mol / L NaOH solution, and 1 mL of a freshly prepared MBTH reagent composed of 3 mg / mL (12.8 mmol / L) MBTH and 1 mg / mL (6.5 mmol / L) DTT. The concentration of OH - in the initial reaction system was 0.17 mol / L, the concentration of MBTH was 2.14 mmol / L, and the mixture was heated at 80°C for 13 min. Then, 2 mL of an acidic iron reagent composed of 5 g / L (10 mmol / L) (FeNH4(SO4)2)·12H2O and 0.4 mol / L sulfamic acid was added. The concentration of H + in the initial reaction system was 0.06 mol / L, and the molar concentration of Fe 3+ was 4 mmol / L. The mixture was rapidly cooled to room temperature in cold water at 4-10°C and was colored for 60 min. Then, 200 μL of the mixture was taken and mixed well in a micro-cuvette of an enzyme-labeled plate. The absorbance value was measured at a wavelength of 590 nm using an enzyme-labeled instrument. Three parallel samples were prepared for each concentration. A standard curve was prepared according to the relationship between the concentration of glucose and the measured absorbance value of glucose.

[0080] Step (2): 0.2 mL of the hydroxypropyl starch solution prepared in step (1) was taken and added to a test tube. The test tube was preheated in a water bath shaker at 37°C for 2 min. Then, 0.2 mL of a preheated amylase solution (α-amylase sample enzyme, 1 μg / mL, dissolved in a sodium phosphate monobasic-sodium phosphate dibasic buffer solution with a pH of 6.9 and a concentration of 0.05 mol / L) was added. After 30 min, 0.4 mL of 0.5 mol / L NaOH solution was rapidly added to terminate the enzymatic reaction, and an alkaline enzymatic hydrolysate was obtained.

[0081] Take 0.2 ml of the hydroxypropyl starch solution prepared in step (1) into a test tube, preheat in a 37°C water bath shaker for 2 min, then continue to incubate for 30 min (same as the enzyme digestion time), then quickly add 0.4 mL of 0.5 mol / L NaOH solution and mix quickly, then add 0.2 ml of 1 μg / mL preheated starch enzyme solution at the same temperature, thus obtaining the alkaline enzyme digestion blank solution.

[0082] Step (3), determination of alkaline enzyme digestion solution and alkaline enzyme digestion blank solution: according to the method of step (1), MBTH reaction was carried out to determine the alkaline enzyme digestion solution and the alkaline enzyme digestion blank solution, so as to obtain the concentration of the reducing end group produced by enzyme digestion, and further obtain the activity of amylase.

[0083] Table 2 Experimental data of amylase activity in Example 1

[0084]

[0085]

[0086] Example 2

[0087] 1. Preparation of substrate solution

[0088] The hydroxypropyl starch with a molar substitution degree of 0.05 was dispersed in a pH 6.9 0.05 mol / L PBS buffer solution, heated at 80°C for 1.5 h to dissolve it, and prepared into a 6 mg / mL hydroxypropyl starch solution.

[0089] 2. Determination of amylase activity

[0090] Step (1), a series of glucose standard solutions with concentration gradient were prepared in a pH 6.9 0.05 mol / L sodium dihydrogen phosphate-sodium hydrogen phosphate buffer solution, and the concentration of glucose was in the range of 0-100 μg / mL (equivalent to 0-0.3 mmol / L). 0.5 mL of glucose standard solution was taken, 0.5 mL of the above hydroxypropyl starch solution was added, 1 mL of 0.5 mol / L NaOH solution was added, mixed, and then 1 mL of freshly prepared MBTH reagent composed of 3 mg / mL (12.8 mmol / L) MBTH and 1 mg / mL (6.5 mmol / L) DTT was added. At this time, the concentration of OH - in the initial reaction system was 0.17 mol / L, the concentration of MBTH was 2.14 mmol / L, and it was heated at 80°C for 13 min. Then 2 mL of acidic iron reagent composed of 5 g / L (10 mmol / L) (FeNH4(SO4)2)·12H2O and 0.4 mol / L sulfamic acid was added. At this time, the concentration of H +concentration of 0.06 mol / L, the molar concentration of Fe 3+ The molar concentration of Fe was 4 mmol / L, and the color was developed by rapidly cooling to room temperature in 4-10°C cold water for 60 min, fully mixing, taking 200 μL into the micro-wells of the enzyme label plate, and measuring the absorbance value at a wavelength of 630 nm after shaking the plate using an enzyme label instrument. Three parallel samples were prepared for each concentration. The standard corresponding relationship curve was drawn according to the relationship between the glucose concentration of each group and the measured glucose absorbance value.

[0091] Step (2), 0.2 ml of the hydroxypropyl starch solution prepared in step (1) was taken and added to a test tube, preheated in a 37°C water bath shaker for 2 min, then 0.2 mL of preheated amylase solution (α-amylase sample enzyme, 1 μg / mL, in a pH 6.9 0.05 mol / L sodium dihydrogen phosphate-sodium hydrogen phosphate buffer solution) at the same temperature was added, and after 30 min, 0.4 mL of 0.5 mol / L NaOH solution was rapidly added and mixed to terminate the enzymolysis reaction, to obtain an alkaline enzymolysis solution;

[0092] 0.2 ml of the hydroxypropyl starch solution prepared in step (1) was taken and added to a test tube, preheated in a 37°C water bath shaker for 2 min, and then continuously incubated for 30 min (the same as the enzymolysis time), 0.4 mL of 0.5 mol / L NaOH solution was rapidly added and mixed, and then 0.2 ml of preheated amylase solution at the same temperature with a concentration of 1 μg / mL was added, to obtain an alkaline enzymolysis blank solution.

[0093] Step (3) determination of the alkaline enzymolysis solution and the alkaline enzymolysis blank solution: the MBTH reaction was carried out according to the method of step (1), and the alkaline enzymolysis solution and the alkaline enzymolysis blank solution were determined, so as to obtain the concentration of the reducing end group produced by enzymolysis, and further obtain the amylase activity.

[0094] Table 3 experimental data of amylase activity in example 2

[0095] Parallel sample 1 2 Difference in absorbance between enzymatic solution and enzymatic blank solution 0.401 0.395 Concentration of reducing end groups generated by enzymatic digestion (pmol / mL) 0.148 0.145 Enzymatic activity (U / mg) 9.84 9.69

[0096] Example 3

[0097] 1. Preparation of substrate solution

[0098] The soluble starch of Sinopharm was dispersed in a pH 6.9 0.05 mol / L PBS buffer solution, heated at 80°C for 1.5 h to dissolve, and prepared into a 6 mg / mL soluble starch solution.

[0099] 2. Determination of amylase activity

[0100] Step (1), a series of glucose standard solutions with different concentrations were prepared in a pH 6.9 0.05 mol / L sodium phosphate buffer solution as the solvent, the concentration of glucose was in the range of 0-100 μg / mL (equivalent to 0-0.3 mmol / L), 0.5 mL of each glucose standard solution was taken, 0.5 mL of the above starch solution was added, 1 mL of 0.5 mol / L NaOH solution was added, mixed, and then 1 mL of freshly prepared 1.5 mg / mL (6.4 mmol / L) MBTH solution was added. At this time, the concentration of OH - in the initial reaction system was 0.17 mol / L, the concentration of MBTH was 2.14 mmol / L, and it was heated in a water bath at 80°C for 13 min, then 2 mL of an acidic iron reagent composed of 5 g / L (10 mmol / L) (FeNH4(SO4)2)·12H2O and 0.4 mol / L sulfamic acid was added. At this time, the concentration of H + in the initial reaction system was 0.06 mol / L, and the molar concentration of Fe 3+ was 4 mmol / L. It was quickly cooled to room temperature in 4-10°C cold water and developed color for 60 min. After mixing thoroughly, 200 μL was taken into a micro-well of an enzyme-labeled plate, and the absorbance value was measured at a wavelength of 590 nm using an enzyme-labeled instrument after shaking the plate. Three parallel samples were prepared for each concentration. The standard corresponding relationship curve was drawn according to the relationship between the glucose concentration and the measured glucose absorbance value.

[0101] Step (2), 0.2 ml of the starch solution prepared in step (1) was taken and added to a test tube, which was preheated in a 37°C water bath shaker for 2 min, then 0.2 mL of preheated starch enzyme solution (α-amylase sample enzyme, 1 μg / mL, in a pH 6.9 0.05 mol / L sodium phosphate buffer solution as the solvent) at the same temperature was added. After 30 min, 0.4 mL of 0.5 mol / L NaOH solution was quickly added and mixed to terminate the enzymatic hydrolysis reaction, obtaining an alkaline enzymatic hydrolysis solution.

[0102] 0.2 ml of the starch solution prepared in step (1) was taken and added to a test tube, which was preheated in a 37°C water bath shaker for 2 min, then 0.2 mL of preheated starch enzyme solution (α-amylase sample enzyme, 1 μg / mL, in a pH 6.9 0.05 mol / L sodium phosphate buffer solution as the solvent) at the same temperature was added. After 30 min, 0.4 mL of 0.5 mol / L NaOH solution was quickly added and mixed to terminate the enzymatic hydrolysis reaction, obtaining an alkaline enzymatic hydrolysis solution.

[0103] Step (3) determination of alkaline enzymatic hydrolysis solution and alkaline enzymatic hydrolysis blank solution: according to the method of step (1), MBTH reaction was carried out to determine the alkaline enzymatic hydrolysis solution and the alkaline enzymatic hydrolysis blank solution, thereby obtaining the concentration of the reducing end group produced by enzymatic hydrolysis, and further obtaining the amylase activity.

[0104] Table 4 Experimental data of amylase activity of Example 3

[0105] Parallel sample 1 2 Difference in absorbance between enzymatic solution and enzymatic blank solution 1.990 1.920 Concentration of reducing end groups generated by enzymatic digestion (pmol / mL) 0.630 0.608 Enzymatic activity (U / mg) 41.986 40.518

[0106] Example 4

[0107] 1. Preparation of substrate solution

[0108] To the soluble starch of Sinopharm, 40% of 0.1 mol / L NaOH solution was added to dissolve, then 1 mol / L HCl was added to neutralize the added NaOH, and water was added to make up the volume to 50% of the total volume, to prepare a 12 mg / mL substrate solution, then 0.1 mol / L disodium hydrogen phosphate or sodium dihydrogen phosphate was used to adjust the pH to 6.9, and then 0.1 mol / L PBS buffer solution at pH 6.9 was added to the required total volume, to prepare a 6 mg / mL starch solution.

[0109] 2. Determination of amylase activity

[0110] Step (1), a series of glucose standard solutions with concentration gradient were prepared in pH 6.9 0.05 mol / L sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution, the concentration of glucose was in the range of 0-100 μg / mL (equivalent to 0-0.3 mmol / L), 0.5 mL of glucose standard solution was taken respectively, 0.5 mL of the above starch solution was added, 1 mL of 0.5 mol / L NaOH solution was added, mixed well, then 1 mL of freshly prepared 1.5 mg / mL (6.4 mmol / L) MBTH solution was added, at this time the concentration of OH in the initial reaction system was 0.17 mol / L, the concentration of MBTH was 2.14 mmol / L, and it was heated in 80°C water bath for 13 min, then 2 mL of acid iron reagent composed of 5 g / L (10 mmol / L) (FeNH4(SO4)2)·12H2O and 0.4 mol / L sulfamic acid was added, at this time the concentration of H in the initial reaction system was 0.06 mol / L, and the molar concentration of Fe was 4 mmol / L, it was quickly cooled to room temperature in 4-10°C cold water, and color development was carried out for 60 min, then 200 μL was taken into the micro-wells of the enzyme-labeled plate, mixed well, and the absorbance value was measured at wavelength 590 nm using an enzyme-labeled instrument after shaking the plate. Three parallel samples were prepared for each concentration. The standard corresponding relationship curve was drawn according to the relationship between the glucose concentration of each group and the measured glucose absorbance value. - + 3+

[0111] ​​​Step (2) Take 0.2 ml of the starch solution prepared in step (1) and add to a test tube, preheat in a 37°C water bath shaker for 2 min, then add 0.2 mL of the preheated starch enzyme solution (α-amylase sample enzyme, 1 μg / mL, in a pH 6.9 0.05 mol / L sodium dihydrogen phosphate-sodium hydrogen phosphate buffer solution) at the same temperature, after 30 min, quickly add 0.4 mL of 0.5 mol / L NaOH solution and mix well to terminate the enzymatic reaction, to obtain an alkaline enzymatic hydrolysis solution;

[0112] Take 0.2 ml of the starch solution prepared in step (1) and add to a test tube, preheat in a 37°C water bath shaker for 2 min, then continue to incubate for 30 min (same as the enzymatic hydrolysis time), quickly add 0.4 mL of 0.5 mol / L NaOH solution and mix well, then add 0.2 ml of the preheated starch enzyme solution at the same temperature (1 μg / mL), to obtain an alkaline enzymatic hydrolysis blank solution.

[0113] Step (3) Determination of the alkaline enzymatic hydrolysis solution and the alkaline enzymatic hydrolysis blank solution: perform MBTH reaction according to the method of step (1), determine the alkaline enzymatic hydrolysis solution and the alkaline enzymatic hydrolysis blank solution, to obtain the concentration of the reducing end groups produced by enzymatic hydrolysis, and further obtain the amylase activity.

[0114] Table 5 Experimental data of amylase activity in Example 4

[0115] Parallel sample 1 2 Difference in absorbance between enzymatic solution and enzymatic blank solution 2.042 2.210 Concentration of reducing end groups generated by enzymatic digestion (pmol / mL) 0.646 0.699 Enzymatic activity (U / mg) 43.077 46.589

[0116] Example 5

[0117] 1. Preparation of substrate solution: add 0.1 mol / L NaOH solution to the total volume of 40% to dissolve the powder level of potato starch, then add 1 mol / L HCl to neutralize the added NaOH, add water to make up the volume to 50% of the total volume, prepare a 12 mg / mL substrate solution, then adjust the pH to 6.9 with 0.1 mol / L sodium phosphate or sodium dihydrogen phosphate, and then add a pH 6.9 0.1 mol / L PBS buffer solution to the required total volume to prepare a 6 mg / mL starch solution.

[0118] 2. Determination of amylase activity

[0119] Step (1), a series of glucose standard solutions with different concentrations were prepared in a pH 6.9 0.05 mol / L sodium phosphate buffer solution as the solvent, the concentration of glucose was in the range of 0-100 μg / mL (equivalent to 0-0.3 mmol / L), 0.5 mL of each glucose standard solution was taken, 0.5 mL of the above starch solution was added, 1 mL of 0.5 mol / L NaOH solution was added, mixed, and then 1 mL of freshly prepared 1.5 mg / mL (6.4 mmol / L) MBTH solution was added. At this time, the concentration of OH - in the initial reaction system was 0.17 mol / L, the concentration of MBTH was 2.14 mmol / L, and it was heated in a water bath at 80°C for 13 min. Then 2 mL of an acidic iron reagent composed of 5 g / L (10 mmol / L) (FeNH4(SO4)2)·12H2O and 0.4 mol / L sulfamic acid was added. At this time, the concentration of H + in the initial reaction system was 0.06 mol / L, and the molar concentration of Fe 3+ was 4 mmol / L. It was quickly cooled to room temperature in 4-10°C cold water and developed color for 60 min. After mixing thoroughly, 200 μL was taken into a micro-well of an enzyme-labeled plate, and the absorbance value was measured at a wavelength of 590 nm using an enzyme-labeled instrument after shaking the plate. Three parallel samples were prepared for each concentration. The standard corresponding relationship curve was drawn according to the relationship between the glucose concentration and the measured glucose absorbance value.

[0120] Step (2), 0.2 ml of the starch solution prepared in step (1) was taken and added to a test tube, which was preheated in a 37°C water bath shaker for 2 min, and then 0.2 mL of preheated starch enzyme solution (α-amylase sample enzyme, 1 μg / mL, in a pH 6.9 0.05 mol / L sodium phosphate buffer solution as the solvent) at the same temperature was added. After 30 min, 0.4 mL of 0.5 mol / L NaOH solution was quickly added to terminate the enzymatic hydrolysis reaction, and an alkaline enzymatic hydrolysate was obtained.

[0121] 0.2 ml of the starch solution prepared in step (1) was taken and added to a test tube, which was preheated in a 37°C water bath shaker for 2 min, and then 0.2 mL of preheated starch enzyme solution (α-amylase sample enzyme, 1 μg / mL, in a pH 6.9 0.05 mol / L sodium phosphate buffer solution as the solvent) at the same temperature was added. After 30 min, 0.4 mL of 0.5 mol / L NaOH solution was quickly added to terminate the enzymatic hydrolysis reaction, and an alkaline enzymatic hydrolysate was obtained.

[0122] Step (3) determination of alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysis blank solution: MBTH reaction was carried out according to the method of step (1), and the alkaline enzymatic hydrolysate and the alkaline enzymatic hydrolysis blank solution were determined, so as to obtain the concentration of the reducing end group produced by enzymatic hydrolysis, and further obtain the amylase activity.

[0123] Table 6 Experimental data of amylase activity of Example 5

[0124] Parallel sample 1 2 Difference in absorbance between enzymatic solution and enzymatic blank solution 0.994 0.926 Concentration of reducing end groups generated by enzymatic digestion (pmol / mL) 0.276 0.257 Enzymatic activity (U / mg) 18.384 17.142

[0125] Example 6

[0126] 1. Preparation of substrate solution

[0127] To the pharmacopoeia grade potato starch, 40% of 0.1 mol / L NaOH solution was added to dissolve, then 1 mol / L HCl was added to neutralize the added NaOH, water was added to make up the volume to 50% of the total volume, and a 12 mg / mL substrate solution was prepared, then 0.1 mol / L disodium hydrogen phosphate or sodium dihydrogen phosphate was used to adjust the pH to 6.9, and then 0.1 mol / L PBS buffer solution at pH 6.9 was added to the required total volume to prepare a 6 mg / mL starch solution.

[0128] 2. Determination of amylase activity

[0129] Step (1), a series of glucose standard solutions with concentration gradient were prepared in 0.05 mol / L sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution at pH 6.9, and the concentration of glucose was in the range of 0-100 μg / mL (equivalent to 0-0.3 mmol / L), 0.5 mL of glucose standard solution was taken respectively, 0.5 mL of the above starch solution was added, 1 mL of 0.5 mol / L NaOH solution was added, mixed well, and then 1 mL of freshly prepared 1.5 mg / mL (6.4 mmol / L) MBTH solution was added. At this time, the concentration of OH - in the initial reaction system was 0.17 mol / L, the concentration of MBTH was 2.14 mmol / L, and it was heated in a water bath at 80°C for 13 min, then 2 mL of acidic iron reagent composed of 5 g / L (10 mmol / L) (FeNH4(SO4)2)·12H2O and 0.4 mol / L sulfamic acid was added. At this time, the concentration of H + in the initial reaction system was 0.06 mol / L, and the molar concentration of Fe 3+ was 4 mmol / L, and it was quickly cooled to room temperature in 4-10°C cold water for color development for 60 min, mixed well, 200 μL was taken into the micro-wells of the enzyme-labeled plate, and after shaking, the absorbance value was measured at a wavelength of 590 nm using an enzyme-labeled instrument. Three parallel samples were made for each concentration. The standard correspondence curve was drawn according to the relationship between the glucose concentration of each group and the measured glucose absorbance value.

[0130] Step (2) Take 0.2 ml of the starch solution prepared in step (1) and add to a test tube, preheat in a water bath shaker at 37°C for 2 min, then add 0.2 mL of the preheated amylase solution (α-amylase sample enzyme, 1 μg / mL, in a pH 6.9 0.05 mol / L sodium dihydrogen phosphate-sodium hydrogen phosphate buffer solution) at the same temperature, after 30 min, quickly add 0.4 mL of 0.5 mol / L NaOH solution and mix well to terminate the enzymatic hydrolysis reaction, to obtain an alkaline enzymatic hydrolysate;

[0131] Take 0.2 ml of the starch solution prepared in step (1) and add to a test tube, preheat in a water bath shaker at 37°C for 2 min, then continue to incubate for 30 min (same as the enzymatic hydrolysis time), quickly add 0.4 mL of 0.5 mol / L NaOH solution and mix well, then add 0.2 ml of the preheated amylase solution (1 μg / mL) at the same temperature, to obtain an alkaline enzymatic hydrolysis blank solution.

[0132] Step (3) Determination of alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysis blank solution: according to the method of step (1), perform MBTH reaction to determine the alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysis blank solution, to obtain the concentration of the reducing end group produced by enzymatic hydrolysis, and further obtain the amylase activity.

[0133] Table 7 Experimental data of amylase activity in Example 6

[0134] Parallel sample 1 2 Difference in absorbance between enzymatic solution and enzymatic blank solution 1.156 1.144 Concentration of reducing end groups generated by enzymatic digestion (pmol / mL) 0.320 0.317 Enzymatic activity (U / mg) 21.343 21.133

[0135] Example 7

[0136] 1. Preparation of substrate solution

[0137] To the reagent-grade corn starch, add a total volume of 40% 0.1 mol / L NaOH solution to dissolve, then add 1 mol / L HCl to neutralize the added NaOH, add water to make up the volume to a total volume of 50%, prepare a 12 mg / mL substrate solution, then adjust the pH to 6.9 with 0.1 mol / L sodium phosphate or sodium dihydrogen phosphate, and then add a pH 6.9 0.1 mol / L PBS buffer solution to the desired total volume to prepare a 6 mg / mL starch solution.

[0138] 2. Determination of amylase activity

[0139] Step (1), a series of glucose standard solutions with different concentrations were prepared in a pH 6.9 0.05 mol / L sodium phosphate buffer solution as the solvent, the concentration of glucose was in the range of 0-100 μg / mL (equivalent to 0-0.3 mmol / L), 0.5 mL of each glucose standard solution was taken, 0.5 mL of the above starch solution was added, 1 mL of 0.5 mol / L NaOH solution was added, mixed, and then 1 mL of freshly prepared 1.5 mg / mL (6.4 mmol / L) MBTH solution was added. At this time, the concentration of OH - in the initial reaction system was 0.17 mol / L, the concentration of MBTH was 2.14 mmol / L, and it was heated in a water bath at 80°C for 13 min. Then 2 mL of an acidic iron reagent composed of 5 g / L (10 mmol / L) (FeNH4(SO4)2)·12H2O and 0.4 mol / L sulfamic acid was added. At this time, the concentration of H + in the initial reaction system was 0.06 mol / L, and the molar concentration of Fe 3+ was 4 mmol / L. It was quickly cooled to room temperature in 4-10°C cold water and developed color for 60 min. After mixing thoroughly, 200 μL was taken into a micro-well of an enzyme-labeled plate, and the absorbance value was measured at a wavelength of 590 nm using an enzyme-labeled instrument after shaking the plate. Three parallel samples were prepared for each concentration. The standard corresponding relationship curve was drawn according to the relationship between the glucose concentration and the measured glucose absorbance value.

[0140] Step (2), 0.2 ml of the starch solution prepared in step (1) was taken and added to a test tube, which was preheated in a 37°C water bath shaker for 2 min, and then 0.2 mL of preheated starch enzyme solution (α-amylase sample enzyme, 1 μg / mL, in a pH 6.9 0.05 mol / L sodium phosphate buffer solution as the solvent) at the same temperature was added. After 30 min, 0.4 mL of 0.5 mol / L NaOH solution was quickly added and mixed to terminate the enzymatic hydrolysis reaction, obtaining an alkaline enzymatic hydrolysis solution.

[0141] 0.2 ml of the starch solution prepared in step (1) was taken and added to a test tube, which was preheated in a 37°C water bath shaker for 2 min, and then 0.2 mL of preheated starch enzyme solution (α-amylase sample enzyme, 1 μg / mL, in a pH 6.9 0.05 mol / L sodium phosphate buffer solution as the solvent) at the same temperature was added. After 30 min, 0.4 mL of 0.5 mol / L NaOH solution was quickly added and mixed to terminate the enzymatic hydrolysis reaction, obtaining an alkaline enzymatic hydrolysis solution.

[0142] Step (3) determination of alkaline enzymatic hydrolysis solution and alkaline enzymatic hydrolysis blank solution: according to the method of step (1), MBTH reaction was carried out to determine the alkaline enzymatic hydrolysis solution and the alkaline enzymatic hydrolysis blank solution, so as to obtain the concentration of the reducing end group produced by enzymatic hydrolysis, and further obtain the amylase activity.

[0143] Table 8 Experimental data of amylase activity of Example 7

[0144] Parallel sample 1 2 Difference in absorbance between enzymatic solution and enzymatic blank solution 1.096 1.174 Concentration of reducing end groups generated by enzymatic digestion (pmol / mL) 0.304 0.325 Enzymatic activity (U / mg) 20.247 21.671

[0145] Example 8

[0146] 1. Preparation of substrate solution

[0147] To the medicinal grade corn starch, add 40% of 0.1 mol / L NaOH solution to dissolve, then add 1 mol / L HCl to neutralize the added NaOH, add water to make up the volume to 50% of the total volume, prepare a 12 mg / mL substrate solution, then use 0.1 mol / L disodium hydrogen phosphate or sodium dihydrogen phosphate to adjust the pH to 6.9, and then add 0.1 mol / L PBS buffer solution at pH 6.9 to the required total volume to prepare a 6 mg / mL starch solution.

[0148] 2. Determination of amylase activity

[0149] Step (1), prepare a series of glucose standard solutions with concentration gradient in 0-100 μg / mL (equivalent to 0-0.3 mmol / L) in 0.05 mol / L sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution at pH 6.9, take 0.5 mL of the glucose standard solution, add 0.5 mL of the above starch solution, add 1 mL of 0.5 mol / L NaOH solution, mix well, then add 1 mL of freshly prepared 1.5 mg / mL (6.4 mmol / L) MBTH solution. At this time, the concentration of OH in the initial reaction system is 0.17 mol / L, the concentration of MBTH is 2.14 mmol / L, heat in a water bath at 80°C for 13 min, then add 2 mL of acid iron reagent composed of 5 g / L (10 mmol / L) (FeNH4(SO4)2)·12H2O and 0.4 mol / L sulfamic acid. At this time, the concentration of H in the initial reaction system is 0.06 mol / L, and the molar concentration of Fe is 4 mmol / L. Rapidly cool to room temperature in 4-10°C cold water, develop color for 60 min, mix well, take 200 μL into the micro-wells of the enzyme-labeled plate, shake the plate, then use an enzyme-labeled instrument to measure the absorbance value at a wavelength of 590 nm. Three parallel samples are prepared for each concentration. According to the relationship between the glucose concentration of each group and the measured glucose absorbance value, a standard correspondence curve is drawn. - + 3+

[0150] ​​​Step (2) : Take 0.2 ml of the starch solution prepared in step (1) and add it to a test tube. Preheat it in a 37°C water bath shaker for 2 min, then add 0.2 mL of the amylase solution (α-amylase sample enzyme, 1 μg / mL, dissolved in a pH 6.9 0.05 mol / L sodium dihydrogen phosphate-sodium hydrogen phosphate buffer solution) preheated to the same temperature. After 30 min, quickly add 0.4 mL of 0.5 mol / L NaOH solution and mix well to terminate the enzymatic reaction, obtaining an alkaline enzymatic hydrolysis solution.

[0151] Take 0.2 ml of the starch solution prepared in step (1) and add it to a test tube. Preheat it in a 37°C water bath shaker for 2 min, then continue to incubate for 30 min (same as the enzymatic hydrolysis time). Quickly add 0.4 mL of 0.5 mol / L NaOH solution and mix well, then add 0.2 ml of the amylase solution (1 μg / mL) preheated to the same temperature, thus obtaining an alkaline enzymatic hydrolysis blank solution.

[0152] Step (3) : Measure the alkaline enzymatic hydrolysis solution and the alkaline enzymatic hydrolysis blank solution: Perform the MBTH reaction according to the method of step (1) to measure the alkaline enzymatic hydrolysis solution and the alkaline enzymatic hydrolysis blank solution, thus obtaining the concentration of the reducing end groups produced by the enzymatic hydrolysis, and further obtaining the amylase activity.

[0153] Table 9 Experimental data of amylase activity in Example 8

[0154] Parallel sample 1 2 Difference in absorbance between enzymatic solution and enzymatic blank solution 1.008 1.075 Concentration of reducing end groups generated by enzymatic digestion (pmol / mL) 0.280 0.298 Enzymatic activity (U / mg) Parallel sample Difference in absorbance between enzymatic solution and enzymatic blank solution Concentration of reducing end groups generated by enzymatic digestion (pmol / mL) Enzymatic activity (U / mg) 18.640 19.863

[0155] Example 9

[0156] 1. Prepare a 6 mg / mL HPS solution

[0157] Add a pH 6.9 0.05 mol / L PBS buffer solution to the HPS with a degree of substitution of 0.05, heat it at 80°C for 1.5 h to dissolve it, and prepare a 6 mg / mL HPS solution.

[0158] 2. Measure the amylase activity

[0159] Step (1), a series of glucose standard solutions with different concentrations were prepared in a pH 6.9 0.05 mol / L sodium dihydrogen phosphate-sodium hydrogen phosphate buffer solution as the solvent, and the concentration of glucose was in the range of 0-100 μg / mL (equivalent to 0-0.3 mmol / L). 0.5 mL of each glucose standard solution was taken, 0.5 mL of the above hydroxypropyl starch solution was added, 1 mL of 0.5 mol / L NaOH solution was added, mixed, and then 1 mL of freshly prepared MBTH reagent composed of 3 mg / mL (12.8 mmol / L) MBTH and 1 mg / mL (6.5 mmol / L) DTT in equal volume was added. At this time, the concentration of OH - in the initial reaction system was 0.17 mol / L, the concentration of MBTH was 2.14 mmol / L, and the mixture was heated at 80°C for 13 min. Then 2 mL of acidic iron reagent composed of 5 g / L (10 mmol / L) (FeNH4(SO4)2)·12H2O and 0.4 mol / L sulfamic acid was added. At this time, the concentration of H + in the initial reaction system was 0.06 mol / L, and the molar concentration of Fe 3+ was 4 mmol / L. The mixture was rapidly cooled to room temperature in 4-10°C cold water and developed color for 60 min. After mixing thoroughly, 200 μL was taken into a micro-well of an enzyme-labeled plate, and the absorbance value was measured at a wavelength of 590 nm using an enzyme-labeled instrument after shaking the plate. Three parallel samples were prepared for each concentration. The standard corresponding relationship curve was drawn according to the relationship between the concentration of glucose in each group and the measured absorbance value of glucose.

[0160] Step (2), 0.2 ml of the hydroxypropyl starch solution prepared in step (1) was taken and added to a test tube, which was preheated at 37°C in a water bath shaker for 2 min. Then 0.2 mL of preheated amylase solution (α-amylase sample enzyme, 1 μg / mL, in a pH 6.9 0.05 mol / L sodium dihydrogen phosphate-sodium hydrogen phosphate buffer solution as the solvent) at the same temperature was added. After 30 min, 0.4 mL of 0.5 mol / L NaOH solution was rapidly added to terminate the enzymatic hydrolysis reaction, and an alkaline enzymatic hydrolysis solution was obtained.

[0161] 0.2 ml of the hydroxypropyl starch solution prepared in step (1) was taken and added to a test tube, which was preheated at 37°C in a water bath shaker for 2 min. Then 0.2 mL of preheated amylase solution (α-amylase sample enzyme, 1 μg / mL, in a pH 6.9 0.05 mol / L sodium dihydrogen phosphate-sodium hydrogen phosphate buffer solution as the solvent) at the same temperature was added. After 30 min, 0.4 mL of 0.5 mol / L NaOH solution was rapidly added to terminate the enzymatic hydrolysis reaction, and an alkaline enzymatic hydrolysis solution was obtained.

[0162] Step (3) determination of alkaline enzyme solution and alkaline enzyme blank solution: according to the method of step (1), the MBTH reaction was carried out, and the alkaline enzyme solution and the alkaline enzyme blank solution were determined, so as to obtain the concentration of the reducing end group produced by enzyme hydrolysis, and then the amylase activity was obtained.

[0163] Table 10 Amylase activity calculated according to different enzyme hydrolysis time of Example 9

[0164]

[0165]

[0166] Comparative Example 1

[0167] 1. Preparation of substrate solution

[0168] The hydroxypropyl starch with a molar substitution degree of 0.57 was dispersed in a pH 6.9 0.05 mol / L PBS buffer solution, heated at 80°C for 1.5 h to dissolve it, and prepared into a 6 mg / mL hydroxypropyl starch solution.

[0169] 2. Determination of amylase activity

[0170] Step (1), a series of concentration gradient glucose standard solutions were prepared with pH 6.9 0.05 mol / L sodium dihydrogen phosphate-sodium hydrogen phosphate buffer solution as solvent, and the concentration of glucose was in the range of 0-100 μg / mL (equivalent to 0-0.3 mmol / L). 0.5 mL of glucose standard solution was taken, 0.5 mL of the above hydroxypropyl starch solution was added, 1 mL of 0.5 mol / L NaOH solution was added, and it was mixed, then 1 mL of freshly prepared MBTH reagent composed of 3 mg / mL (12.8 mmol / L) MBTH and 1 mg / mL (6.5 mmol / L) DTT was added. At this time, the concentration of OH - in the initial reaction system was 0.17 mol / L, the concentration of MBTH was 2.14 mmol / L, and it was heated at 80°C water bath for 13 min. Then 2 mL of acidic iron reagent composed of 5 g / L (10 mmol / L) (FeNH4(SO4)2)·12H2O and 0.4 mol / L sulfamic acid was added. At this time, the concentration of H + in the initial reaction system was 0.06 mol / L, and the molar concentration of Fe 3+ was 4 mmol / L. It was quickly cooled to room temperature in 4-10°C cold water and developed color for 60 min. After fully mixing, 200 μL was taken into the micro-wells of the enzyme-labeled plate, and the absorbance value was measured at a wavelength of 590 nm using an enzyme-labeled instrument after shaking the plate. Three parallel samples were made for each concentration. The standard corresponding relationship curve was drawn according to the relationship between each group of glucose concentration and the measured glucose absorbance value.

[0171] Step (2), take 0.2ml of the hydroxypropyl starch solution prepared in step (1) into a test tube, preheat in a water bath shaker at 37℃ for 2min, then add 0.2mL of preheated to the same temperature amylase solution (α-amylase sample enzyme, 1μg / mL, with pH 6.9 0.05mol / L sodium dihydrogen phosphate-sodium hydrogen phosphate buffer solution as solvent), after 30min, quickly add 0.4mL 0.5mol / L NaOH solution and mix quickly to terminate the enzymatic hydrolysis reaction, to obtain the alkaline enzymatic hydrolysate;

[0172] Take 0.2ml of the hydroxypropyl starch solution prepared in step (1) into a test tube, preheat in a water bath shaker at 37℃ for 2min, then continue to incubate for 30min (same as the enzymatic hydrolysis time), quickly add 0.4mL 0.5mol / L NaOH solution and mix quickly, then add 0.2ml of preheated to the same temperature amylase solution with a concentration of 1μg / mL, to obtain the alkaline enzymatic hydrolysis blank solution.

[0173] Step (3) determination of alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysis blank solution: according to the method of step (1), carry out MBTH reaction, determine the alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysis blank solution, to obtain the concentration of the reducing end group produced by enzymatic hydrolysis, and further obtain the amylase activity.

[0174] Table 11 experimental data of amylase activity of comparative example 1

[0175]

[0176]

[0177] This comparative example is a comparative example of example 1, and the determination parameters of this comparative example and example 1 are completely the same when the amylase activity is determined by the MBTH method of the present application, only the hydroxypropyl starch with a degree of substitution of 0.57 is used as the amylase substrate, and the measured amylase activity is significantly reduced, the average of the amylase activity measured twice is reduced from 8.96 (example 1) to 5.32, which indicates that the affinity of hydroxypropyl starch with a too high degree of substitution (MS>0.5) to amylase is very low, and it is not suitable to be used as an amylase substrate.

[0178] The above-described examples only describe the preferred embodiments of the present application, and do not limit the scope of the present application, and various modifications and improvements to the technical solutions of the present application made by those skilled in the art without departing from the design spirit of the present application shall fall within the protection scope determined by the claims of the present application.

Claims

1. A method for determining the activity of an α-amylase, characterized in that, The method comprises the following steps: (1) preparing a substrate solution and an α-amylase solution: dispersing starch or hydroxypropyl starch into a strong alkali solution, mixing well until the substrate is dissolved, then adding acid to neutralize the strong alkali, adjusting the pH to the pH to be measured, and then adding a buffer solution of the pH to be measured to volume; the pH to be measured is the pH value for measuring the activity of the α-amylase; the molar substitution degree of the hydroxypropyl starch is 0.05-0.5; The α-amylase solution is prepared with the same buffer solution. (2) drawing a standard curve of the absorbance value of glucose versus the concentration of glucose: a. using the substrate solution prepared in step (1), preparing a series of glucose standard solutions with gradient concentrations, wherein the concentrations of the substrate in the series of glucose standard solutions are the same, and the range of the concentration of glucose is 0-0.3 mmol / L; b. Adding an alkaline reagent to the series of glucose standard solutions to obtain an alkaline glucose standard solution, the concentration of the alkaline reagent in the alkaline glucose standard solution being the same, then adding a freshly prepared MBTH reagent, the OH - concentration being 0.01-0.2 mol / L, the concentration of MBTH being 0.3-4.5 mmol / L, and the reaction being carried out at 50-90°C for 5-180 min, then adding an acidic iron reagent, the H + concentration being 0.02-0.3 mol / L, and the initial concentration of Fe 3+ being 1-6 mmol / L, to carry out a color reaction, and measuring the absorbance value at 580-680 nm after the color is stable, and drawing a standard corresponding relationship curve according to the relationship between each group of glucose concentrations and the measured absorbance values. The MBTH reagent is an aqueous solution prepared only from MBTH; the acidic iron reagent is an aqueous solution prepared from a soluble Fe 3+ salt and a strong acid. (3) enzyme hydrolysis reaction of the α-amylase: adding the substrate solution prepared in step (1) to the α-amylase solution to ensure that the concentration of the substrate in the obtained solution is the same as that in the glucose standard solution in step (2), then performing enzyme hydrolysis reaction, adding an alkaline reagent to terminate the enzyme hydrolysis reaction, and obtaining an alkaline enzyme hydrolysis solution, wherein the concentration of the alkaline reagent in the alkaline enzyme hydrolysis solution is the same as that in the alkaline glucose standard solution in step (2); (4) preparing an alkaline enzyme hydrolysis blank solution; (5) measuring the alkaline enzyme hydrolysis solution and the alkaline enzyme hydrolysis blank solution: performing MBTH reaction according to the method in step (2), measuring the alkaline enzyme hydrolysis solution and the alkaline enzyme hydrolysis blank solution, thereby obtaining the concentration of the reducing end group produced by the enzyme hydrolysis, and further obtaining the activity of the α-amylase.

2. The method for determining the activity of α-amylase according to claim 1, characterized in that, The starch in step (1) is potato starch, corn starch or soluble starch.

3. The method for determining the activity of α-amylase according to claim 1, characterized in that, The strong alkali is NaOH or KOH.

4. The method for determining alpha-amylase activity according to claim 1, characterized in that: In step (2), the composition of the acidic iron reagent can include soluble Fe 3+ The salt is at least one of ferric nitrate, ferric chloride, and ferric ammonium sulfate, and the strong acid is at least one of sulfamic acid, hydrochloric acid, and sulfuric acid.

5. The method for determining alpha-amylase activity according to claim 1, characterized in that: After the step (2), OH - The concentration of MBTH is in the range of 0.95-3.6 mmol / L. After adding the acidic iron reagent, the H + The concentration of Fe 3+ The initial concentration of Fe is in the range of 3-5 mmol / L.

6. The method for determining alpha-amylase activity according to claim 1, characterized in that: The reaction temperature of the glucose standard solution in step (2) with the MBTH reagent is 75-85°C, and the reaction time is 9-17 min.

7. The method for determining alpha-amylase activity according to claim 1, characterized in that: After adding the acidic iron reagent in step (2), the system is naturally cooled to room temperature, and the absorbance value is measured after the coloration is stable.

8. The method for determining alpha-amylase activity according to claim 1, characterized in that: The wavelength for measuring the absorbance value in step (2) is 590-680 nm.

9. The method for determining alpha-amylase activity according to claim 1, characterized in that: The coloration time after adding the acidic iron reagent in step (2) is 50-180 min, and the absorbance value is measured within the coloration time, and the wavelength for measuring the absorbance value is 590-610 nm.

Images