A method for determining dextranase activity and its application

Abstract

The present invention belongs to the technical field of enzyme activity determination. Aiming at the problems of low sensitivity and deviation of the intercept of the standard curve from the origin of the coordinate axis in the existing dextranase activity determination technology, the present invention discloses a method for determining the activity of dextranase and its application, which specifically includes the following steps: a. Refining of substrate; b. Enzyme activity determination: preparing a standard corresponding relationship curve between the glucose absorbance value measured by the MBTH method and its corresponding concentration; using the MBTH method to detect the absorbance value equivalent to glucose produced after the dextran to be tested hydrolyzes dextran; estimating the activity of dextranase; c. Determination of enzyme activity in toothpaste and mouthwash. The enzyme activity determination method of the present invention has high sensitivity, and the intercepts of the used glucose standard curve and the corresponding enzyme concentration curve are close to zero, and the dilution multiple of the sample will not affect the accuracy of the enzyme activity determination.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme activity determination, and specifically relates to a method for determining dextranase activity and an application thereof. Background Art

[0002] Dextranase, also known as α-glucanase, is an enzyme that specifically hydrolyzes the α-1,6 glycosidic bond in dextran molecules. Its hydrolysis types include endo-type and exo-type. The main hydrolysis product of endo-dextranase (Endodextranase EC 3.2.1.11, α-1,6-glucan 6-glucanohydrolase) is isomaltooligosaccharide; the main hydrolysis product of exo-dextranase (Exodextranase EC 3.2.1.70, glucan 1,6-α-glycosidase) is glucose. Dextran is a component of dental plaque biofilm. Dextranase can destroy dental plaque biofilm by degrading dextran, thereby achieving the purpose of preventing dental caries. Therefore, dextranase is often added to toothpaste or mouthwash as an anti-caries ingredient.

[0003] Enzyme activity is an important indicator for measuring enzyme activity. The activity unit of dextranase can be defined as the amount of glucose equivalent to 1 μmol produced per minute in each milliliter of reaction solution under certain enzymatic conditions. At present, the main method for determining the activity of dextranase is the DNS method (Chang Guowei, Li Zhide, Huang Zengwei, et al. Study on the enzymatic characteristics and thermal inactivation kinetics of dextranase [J]. Chinese Condiment, 2021, 46(5); Zhao Yahua. Biochemistry and Molecular Biology Experimental Technology Tutorial [M]. Beijing: Higher Education Press, 2005: 69-70). The DNS method calculates the activity of dextranase by measuring the rate of increase of reducing sugar concentration during the enzymatic hydrolysis of dextranase. However, the sensitivity of the DNS method is relatively low. On the one hand, a larger substrate enzymatic hydrolysis rate is required to measure the concentration of the reducing end group of the enzymatic hydrolysis product, and the initial enzymatic hydrolysis rate cannot be measured. Especially for dextranase with a larger Km value, it is difficult to obtain the enzymatic hydrolysis rate of the zero-order reaction, so it is impossible to use the enzymatic hydrolysis time period (such as 30 minutes of enzymatic hydrolysis) to calculate the initial enzymatic hydrolysis rate; on the other hand, a higher enzyme concentration is required, and the concentration of impurities in the enzyme solution (such as from toothpaste and mouthwash) is also correspondingly higher, and their activation or inhibition effect on dextranase will be more obvious. In addition, the problem that the DNS method does not conform to stoichiometry has been reported (Mccleary BV, McGeough PA 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). Moreover, the deviation of the glucose standard curve measured by the DNS method from the origin of the coordinate axis has been described in the above-mentioned article by Chang Guowei et al. So far, there is no standard document on the method for determining the activity of dextranase.

[0004] The MBTH method can be used to determine the activity of xylanase, lysozyme and β-glucanase. Taking the determination of β-glucanase activity as an example, the MBTH reagent used is a mixture of MBTH solution and DTT solution. This method is simpler, faster, more accurate and cheaper than the turbidimetric method; its sensitivity is significantly higher than the DNS method and the potassium ferrocyanide method. The inventor tried to use the MBTH method for the determination of dextranase activity and found that the following problems exist in the application of this method: (1) Commercially available dextran products are not suitable for enzyme activity determination, because ① medium and low molecular weight dextran (such as dextran 20, 40, 70) causes the background value of the determination to be too high, and its absorbance value may even exceed the upper limit of the instrument measurement; ② pharmaceutical grade and standard grade dextran with large molecular weight (molecular weight ≥ 70kDa) has a background value suitable for enzyme activity determination, but its price is extremely expensive (price > 250 yuan / g), resulting in excessively high detection costs; ③ ordinary grade dextran available on the market, although There are products with large molecular weight, but their molecular weight distribution is too wide, which still makes the background value of the measurement too high; ④ The raw materials of the dextran described in ① and ② above are the same as those in ③, and there is also the problem of too high background value; (2) The solubility of large molecular weight dextran in water is low, and it is difficult to prepare a substrate solution with a concentration suitable for enzyme activity determination; (3) Although the intercept of the obtained glucose standard curve is smaller and better than the DNS method, it still deviates from the origin of the coordinate axis, which will affect the accuracy of the enzyme activity determination due to different dilution multiples of the enzyme solution; (4) The color product is not stable enough, resulting in the slope of the glucose standard curve changing over time.

[0005] In addition, when measuring the dextranase activity in toothpaste and mouthwash, the inventor found that the composition in toothpaste is extremely complicated, and there are viscous components such as xanthan gum, cellulose gum and water-insoluble components such as calcium carbonate and calcium pyrophosphate. If the precision and accuracy of the measurement are not affected, if they are removed, dextranase may be adsorbed on these insolubles and removed simultaneously, which also affects the accuracy of the test result. And in mouthwash, the inventor found that due to the use of the protective agent of enzyme, the determination of enzyme activity will also be affected to a certain extent. Therefore, the method of removing insolubles and protective agent is also crucial for the accuracy of the dextranase activity measurement. Summary of the invention

[0006] Aiming at the problems of low sensitivity, deviation of the intercept of the standard curve from the origin of the coordinate axis, obvious interference of impurities in toothpaste and mouthwash on enzyme activity determination in the existing dextranase activity determination technology, and excessively high substrate background value, deviation of the intercept of the standard curve from the origin of the coordinate axis, and unstable color development product in the existing MBTH method for dextranase activity determination, the present invention provides a method for determining dextranase activity and its application, and designs a means for fully automated high-throughput enzyme activity determination, which is achieved by the following means: 1). Providing a simple and inexpensive dextran concentrate 1). The invention discloses a method for preparing a dextranase from toothpaste and mouthwash; 2). The invention discloses a method for preparing a dextranase from toothpaste and mouthwash; 3). The invention discloses a method for preparing a dextranase from toothpaste and mouthwash; 4). The invention discloses a method for preparing a dextranase from toothpaste and mouthwash; 5). The invention discloses a method for preparing a dextranase from toothpaste and mouthwash; 6). The invention discloses a method for preparing a dextranase from toothpaste and mouthwash; 7). The invention discloses a method for preparing a dextranase from toothpaste and mouthwash; 8). The invention discloses a method for preparing a dextranase from toothpaste and mouthwash; 9). The invention discloses a method for preparing a dextranase from toothpaste and mouthwash; 10). The invention discloses a method for preparing a dextranase from toothpaste and mouthwash; 11). The invention discloses a method for preparing a dextranase from toothpaste and mouthwash; 12). The invention discloses a method for preparing a dextranase from toothpaste and mouthwash; 13). The invention discloses a method for preparing a dextranase from toothpaste and mouthwash; 14). The invention discloses a method for preparing a dextranase from toothpaste and mouthwash; 15). The invention discloses a method for preparing a dextranase from toothpaste and mouthwash; 16). The invention discloses a method for preparing a dextranase from toothpaste and mouthwash; 17). The invention discloses a method for preparing a dextranase from toothpaste and mouthwash; 18). The invention discloses a method for preparing a dextranase from toothpaste and mouthwash; 19). The invention discloses a method for preparing a dextranase from toothpaste and mouthwash; 20). The invention discloses a method for dissolving a substrate suitable for enzymatic hydrolysis; 30). The invention discloses a method for extracting dextran from toothpaste and mouth

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] (I) Method for determining dextranase activity

[0009] (1) Refined dextran:

[0010] The present invention uses dextran as a substrate. If the weight average molecular weight of dextran is ≥70 kDa and the minimum molecular weight is

[0011] >10kDa, it can be used directly as a purified substrate, omitting the following purification steps. The method for purifying the substrate is as follows:

[0012] a. The dextran is crushed to prepare a dextran powder, preferably the substrate is ground until all pass through a 60 mesh sieve or more;

[0013] b. Disperse the dextran powder in a 35%-70% ethanol solution, stir thoroughly, centrifuge, and discard the supernatant. Repeat this step several times to remove small molecules in the dextran powder;

[0014] Preferably, the concentration of the ethanol solution is 60-70%, the oligosaccharides are more easily dissolved, and the resulting precipitate contains high molecular weight substrates;

[0015] Preferably, the mass (g): volume (mL) of the substrate and the ethanol solution is 1:(130-170), more preferably 1:(140-160);

[0016] Preferably, the stirring time is 3-5h;

[0017] Preferably, the centrifugation is performed at 2500-4000 RCF, and the centrifugation time is 5-20 min, more preferably 10-15 min.

[0018] c. Dehydrating the precipitate obtained in step b step by step with ethanol solutions of increasing concentrations. Preferably, the precipitate and 80-100% ethanol are mixed in a mass (g): volume (mL) ratio of 1: (20-25). Then, 80-100% ethanol is added again for dispersion. This can be repeated several times, and then centrifuged, the supernatant is discarded, anhydrous ethanol is added to the precipitate, centrifuged, and the supernatant is discarded;

[0019] Preferably, 90%-100% ethanol is added; preferably, the volume of ethanol solution added each time is the same; preferably, the operation of adding ethanol solution for dispersion is repeated 2-5 times, more preferably 3 times; preferably, the next repetitive operation is performed after sufficient mixing for 10 minutes; preferably, centrifugation is performed at 2500-4000RCF, more preferably at 3000-4000RCF; preferably, the centrifugation time is 5-20 minutes each time, more preferably 10-15 minutes.

[0020] d. adding acetone to the precipitate obtained in step c, allowing it to stand after ultrasonic treatment, and performing reduced pressure rotary evaporation under high vacuum conditions to obtain a dried refined substrate, wherein the high vacuum degree refers to a pressure ≤-0.089 MPa; preferably, the mass (g) of the precipitate and the volume (mL) of acetone is 1:(20-100), more preferably 1:(50-60); preferably, the ultrasonic time is 1-5 min, more preferably 2-3 min; preferably, the standing time is 1-2 h; preferably, the pressure range of the high vacuum degree is -0.09 MPa--0.098 MPa, and the rotary evaporation temperature is maintained in the range of 25-40 ° C.

[0021] Compared with dialysis or ultrafiltration to remove small molecule substrates, this substrate purification method does not consume a large amount of ultrapure water like dialysis or ultrafiltration, nor does it take a long time (more than three days) like dialysis. It can be completed in just one day, greatly reducing costs.

[0022] Dextran has strong hydrophilicity. The dextran precipitate obtained by alcohol precipitation contains a large amount of ethanol and a small amount of water. If it is dried at normal pressure or a conventional vacuum drying oven, the ethanol in the precipitate will gradually decrease as the drying process progresses. At this time, the water in the precipitate will instantly interact with the dextran molecules to form a film or block, causing incomplete drying. In order to solve the above problems, first, the method of increasing the ethanol concentration step by step is used to dehydrate step by step, and finally immerse in acetone for further dehydration; then, under high vacuum conditions, the sample is locally gelled by rotary evaporation to avoid ethanol removal. Therefore, step-by-step dehydration and high vacuum rotary evaporation are the key to obtaining dextran refined samples. The dextran refined sample obtained in this way is uniform, loose, and fine powder, which is far superior to the prior art. Compared with freeze-drying method for drying substrates, this method of substrate drying is more time-saving and more cost-saving.

[0023] (2) Preparation of substrate solution and dextranase solution:

[0024] Add dextran or a refined substrate having a weight average molecular weight ≥ 70 kDa and a minimum molecular weight > 10 kDa to a strong alkaline solution having a concentration of 0.1-1 mol / L, mix thoroughly to dissolve it, add acid to adjust the pH to the pH value to be measured, and dilute to a volume with a buffer solution having the pH value to be measured, thereby obtaining a substrate solution;

[0025] The dextranase solution is prepared with the same buffer solution; specifically, the same buffer solution means that the buffer solution used to prepare the dextranase solution and the buffer solution used to prepare the substrate solution are exactly the same in type, concentration and pH value; it is best to use a buffer solution with good stability and shorten the operation time as much as possible to ensure that the type, concentration and pH value of the buffer solution are exactly the same when preparing the dextranase solution and the substrate solution, so as to eliminate possible influence on the test results.

[0026] Preferably, the strong base is NaOH or KOH, and the acid for adjusting the pH is the conjugate acid for preparing the buffer solution. After the strong base is dissolved and the conjugate acid is added to adjust the pH, the ionic strength in the system is the same as the ionic strength of the buffer solution used for constant volume.

[0027] Preferably, the buffer solution is a buffer solution of the optimum pH for measuring the activity of dextranase; more preferably, it is a succinic acid-sodium succinate buffer solution or an acetic acid-sodium acetate buffer solution.

[0028] The existing method of dissolving the substrate is to dissolve it in hot water (same as heating and dissolving). The viscosity of the dextran solution after dissolution is relatively high, which is not conducive to the enzymatic hydrolysis reaction. The prepared substrate solution of the present invention has a lower viscosity and is more conducive to the enzymatic hydrolysis reaction. The slope of the obtained standard curve is larger, and the sensitivity of detecting the reducing end group is higher.

[0029] Preferably, the substrate solution is prepared and used immediately. The slope of the standard curve obtained by storing the dextran solution prepared at room temperature at 4°C and -18°C is significantly reduced, and the intercept is significantly increased. Therefore, the dextran solution should be prepared and used immediately, and should not be stored for a long time.

[0030] (3) Draw the standard corresponding relationship curve between glucose absorbance value and glucose concentration:

[0031] a. Using the substrate solution prepared in step (2), prepare a series of glucose standard solutions with a concentration gradient, wherein the concentration of the substrate in the series of glucose standard solutions with a concentration gradient is the same and the glucose concentration range is 0-0.5mmol / L;

[0032] b. adding an alkaline reagent to the series of glucose standard solutions with a concentration gradient to obtain an alkaline glucose standard solution, wherein the concentration of the alkaline reagent in the alkaline glucose standard solution is the same, and then adding a freshly prepared MBTH reagent to the alkaline glucose standard solution, wherein the initial concentration range of MBTH is 0.3-4.5 mmol / L, and the initial concentration range of DTT is 0-1.6 mmol / L, reacting at 50-100° C. for 5-180 min, and then adding an acidic iron reagent, the reaction system becomes acidic, and the H + The concentration is 0.02-0.3 mol / L, Fe 3+ The initial concentration range is 1-6mmol / L, a color reaction occurs, and after the color development is stable, the absorbance value is measured at a wavelength of 580-680nm. A standard corresponding relationship curve is drawn based on the relationship between each group of glucose concentrations and the measured absorbance values;

[0033] The MBTH reagent is prepared from MBTH and DTT, or only MBTH; the acidic iron reagent is prepared from soluble Fe 3+ It is prepared from salt and strong acid;

[0034] “The initial concentration range of MBTH is 0.3-4.5 mmol / L, and the initial concentration range of DTT is 0-1.6 mmol / L”. It can be understood that the concentration of MBTH reagent here describes the initial concentration at the moment of adding MBTH reagent and before reaction.

[0035] “Fe 3+ The initial concentration range is 1-6mmol / L". It can be understood that Fe 3+ The concentration describes the initial concentration at the moment the acidic iron reagent is added, before the reaction.

[0036] Unreacted MBTH reagent and Fe 3+ The reaction needs to occur in an acidic environment. +The concentration describes the H concentration after the addition of the acidic iron reagent and the completion of the acid-base neutralization reaction. + concentration.

[0037] Preferably, the soluble Fe in the acidic iron reagent 3+ The salt is at least one of ferric nitrate, ferric chloride and ammonium ferric sulfate, and the strong acid is at least one of aminosulfonic acid, sulfuric acid and hydrochloric acid.

[0038] Preferably, after adding the freshly prepared MBTH reagent to the alkaline glucose standard solution, the initial concentration of MBTH in the system is in the range of 0.95-3.6 mmol / L, the initial concentration of DTT is 0 mmol / L, and after adding the acidic iron reagent, the Fe 3+ The initial concentration range is 3-5mmol / L. Compared with the MBTH reagent containing DTT (the DTT concentration in the determination method of β-glucanase activity is 1mg / mL), the slope of the standard curve obtained without DTT is larger, the sensitivity of detecting the reducing end group is higher, and the intercept is closer to 0.

[0039] Preferably, the acidic iron reagent is added while hot, cooled to room temperature, and the absorbance value is measured after the color development stabilizes.

[0040] Preferably, the color is allowed to stand at room temperature for 50-180 min, and the absorbance is measured at 590 nm-650 nm during the color development time, more preferably at 590 nm-610 nm.

[0041] Preferably, the absorbance value is measured using an ELISA reader after shaking the plate.

[0042] (4) Dextranase enzymatic reaction:

[0043] The substrate solution prepared in step (2) is added to the dextranase solution to ensure that the concentration of the substrate is the same as the concentration of the substrate in the glucose standard solution in step (2), and then an enzymatic hydrolysis reaction is carried out, and then an alkaline reagent is added to terminate the enzymatic hydrolysis reaction to obtain an alkaline enzymatic hydrolyzate, wherein the concentration of the alkaline reagent in the alkaline enzymatic hydrolyzate is the same as the concentration of the alkaline reagent in the alkaline glucose standard solution in step (2).

[0044] Preferably, the substrate concentration in the enzymatic reaction system is in the range of 0.5-5 mg / mL; the enzymatic reaction temperature is 25-60°C, and the reaction time is 5-60 min; the reaction is performed at 37°C for 30 min as an example, and the dextranase concentration in the enzymatic reaction system is preferably in the range of 0.032-16.5 mU / mL.

[0045] (5) Preparation of alkaline enzymatic blank solution:

[0046] Specifically, referring to the method of step (4), the difference is that before the substrate solution and the dextranase solution are mixed, an alkaline reagent is first added to the dextranase solution and then the substrate solution is added, thereby obtaining an alkaline enzymatic blank solution; this step is carried out according to the setting method of the blank control group commonly used in the laboratory, and the amounts of the substrate solution, the alkaline reagent, and the dextranase solution conform to the proportional relationship of step (4), and preferably the amounts of the substrate solution, the alkaline reagent, and the dextranase solution are the same as those in step (4).

[0047] (6) Determination of alkaline enzymatic solution and alkaline enzymatic blank solution:

[0048] The MBTH reaction is carried out according to the method of step (3), and the absorbance value after the reaction of the alkaline enzymatic solution obtained in step (4) and the alkaline enzymatic blank solution obtained in step (5) is measured, so as to obtain the concentration of reducing end groups generated by enzymatic hydrolysis, and then obtain the dextranase activity.

[0049] (ii) The present invention also provides the above method for determining the activity of dextranase, and its application in determining the activity of dextranase in toothpaste and mouthwash.

[0050] 1. Determination of dextranase activity in toothpaste:

[0051] First, it is necessary to fully extract the dextranase in the toothpaste, accurately weigh a certain amount of toothpaste, transfer it to a tissue homogenizer with a buffer solution, grind it finely, transfer the mixed solution to a centrifuge tube, centrifuge it under a temperature condition that ensures that the enzyme is not inactivated, adjust the concentration and pH value of the dextranase in the supernatant with a buffer solution, make up the volume, and prepare a solution with a toothpaste concentration of 0.1-100 mg / mL as the enzyme solution to be tested. The determination method refers to Part (I). The spiked recovery rate of enzyme extraction in the sample is 100%-120%, proving that the sample treatment method can fully extract dextranase. The purpose of using the tissue homogenizer is to fully desorb the dextranase present in the stabilizer in the toothpaste.

[0052] Preferably, the amount of buffer solution added is 0.01-10 L of buffer solution for every 1 g of toothpaste; preferably, centrifugation is performed at 3000-4000 RCF for 10-15 min; preferably, centrifugation is performed at 4°C; preferably, 0.1-10 g of toothpaste is accurately weighed to prepare a toothpaste solution with a concentration of 0.1-100 mg / mL.

[0053] 2. Determination of dextranase activity in mouthwash:

[0054] Accurately weigh a certain amount of mouthwash, add a large amount of buffer solution at one time, and prepare a mouthwash solution with a concentration of 0.1-100 mg / mL. The mouthwash solution can be used as a dextranase solution for enzyme activity determination. The determination method refers to Part (I). The purpose of adding the buffer solution at one time is to destroy the protected state of dextranase in the mouthwash and completely release and dissolve it in the buffer solution.

[0055] Preferably, the enzymatic hydrolysis reaction and MBTH reaction in step (3), step (4) and step (5) can be continuously performed at the same temperature in the metal bath to achieve high-throughput determination. This method does not require heating of the metal bath after the enzymatic hydrolysis reaction is completed, and the MBTH solution can be directly added for reaction. After the color development is completed, the metal bath does not need to be cooled before enzymatic hydrolysis can be performed again. The enzyme activity can be continuously determined, which avoids cumbersome operating procedures and can achieve fully automated sample detection.

[0056] The present invention has the following beneficial effects:

[0057] 1. The substrate purification method of the present invention is simple, rapid and efficient, and its preparation cost is much lower than that of membrane separation methods such as ultrafiltration and dialysis. It efficiently solves the problem of high substrate background value and greatly reduces the detection cost.

[0058] 2. The method of the present invention has high sensitivity and can accurately measure the initial enzymatic hydrolysis velocity of dextranase with extremely low enzyme concentration (the present invention takes enzymatic hydrolysis at 37° C. for 30 min as an example, and the limit of quantification is 0.032 mU / mL). Since the dilution multiple of the enzyme solution to be tested is high, impurities in the enzyme solution, such as impurities in toothpaste and mouthwash, have little interference with the enzyme activity determination, which is much better than the DNS method (the DNS method takes enzymatic hydrolysis at 37° C. for 30 min as an example, and the limit of quantification is 0.105-0.213 mU / mL in the four DNS methods in the comparative example). Therefore, it is particularly suitable for products with low enzyme content.

[0059] 3. Compared with the existing MBTH method (method for determining β-glucanase activity) applied to the determination of dextranase activity, the method of the present invention has a standard curve intercept that is closer to the origin of the coordinate axis and a more stable color product, thereby improving the sensitivity and accuracy of the detection, and providing a better method for the determination of dextranase activity.

[0060] 4. The method of the present invention is simple to operate, has rapid determination, and can be applied to high-throughput analysis of large-scale determinations, while also being able to realize a fully automated factory operation mode;

[0061] 5. The present invention is a method for determining the activity of dextranase, which is not only suitable for enzyme preparation manufacturers, but also can be applied to toothpaste and mouthwash containing dextranase, thus providing a method for quality control of anti-caries products containing dextranase such as toothpaste and mouthwash. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 The background values ​​of the dextran before and after the MBTH reaction were compared using the method of Examples 1-4 as the substrate (commercially available ordinary grade dextran). Nos. 1-6 were commercially available dextran and dextran production raw materials from different manufacturers, and their models were 70 kDa, 500 kDa, 200 kDa, 100 kDa, 70 kDa and pharmaceutical grade dextran production raw materials, respectively. The concentrations were all 10 mg / mL. The background value was determined by ELISA plate. The results are as follows:

[0063] Sample No. Before substrate purification After substrate purification 1 1.71 1.19 2 2.88 0.687 3 2.97 0.735 4 3.00 0.359 5 3.00 0.960 6 3.44 0.451

[0064] Figure 2 The left picture shows the substrate obtained after alcohol precipitation with 60% ethanol, and the substrate obtained by direct drying after centrifugation is a hard gel solid. The middle picture and the right picture show the substrate obtained after 60% ethanol precipitation and 70% ethanol precipitation of the present invention, respectively, which are loose solids. The loose solids are conducive to being easy to dissolve when preparing solutions as enzymatic hydrolysis substrates.

[0065] Figure 3 The dissolution conditions of substrate solutions of the same concentration obtained by different preparation methods are shown in Figure 1. a is direct dissolution at room temperature, and the solution contains a large amount of insoluble matter; b is dissolution heated at 80°C for 1 hour, and the solution is slightly milky white; c is the substrate dissolution method of the present invention, first adding alkali to dissolve, and then adjusting the pH with conjugate acid, and the solution is clearer and more transparent than b.

[0066] Figure 4 The standard curves of enzyme mass concentration and absorbance for the dissolution method of adding conjugate acid after alkali dissolution and the dissolution method of heating at 80°C for 1h of the present invention are shown. When measured by ELISA plate, the absorbance value corresponding to the present invention at the same enzyme concentration is larger, indicating that conventional heating to dissolve the substrate makes the substrate gelatinized with a slightly milky white color, increases the viscosity of the substrate solution, is not conducive to the enzymatic hydrolysis reaction, and leads to a low absorbance value, indicating that the substrate dissolution method of the present invention is more conducive to the enzymatic hydrolysis reaction.

[0067] Figure 5 The effects of substrate preparation time and storage method on the slope (upper figure) and intercept (lower figure) of the glucose standard curve were determined by ELISA plate. The slope of the glucose standard curve prepared immediately before use was the highest and the intercept was closer to 0.

[0068] Figure 6It is a comparison diagram of the glucose standard curve between the method of the present invention and the DNS method (DNS-1, DNS-2, DNS-3, DNS-4) of the prior art, which is measured by cuvette, wherein DNS-1 is the DNS method for measuring the activity of dextranase in the literature (Chang Guowei, Li Zhide, Huang Zengwei, et al. Study on the enzymatic characteristics and thermal inactivation kinetics of dextranase [J]. Chinese Condiments, 2021, 46(5):84-88); DNS-2 is the DNS method for measuring the activity of alkaline pectinase in the literature (Xing Mingxia, Wang Pengbo, Xu Wenting, et al. Optimization of the determination method of alkaline pectinase specific activity by kinetic method [J]. Food Industry Science and Technology, 2020, 41(23):223-228); DNS-3 is the national standard of the People's Republic of China (Spectrophotometric method for determination of xylanase activity of feed additives GB / T 23874-2009, Spectrophotometric method for determination of β-mannanase activity of feed additives GB / T 36861-2018) DNS method for determining xylanase and β-mannanase activities; DNS-4 is the DNS method for determining cellulase activity in the National Standard of the People's Republic of China (Analysis Method for Cellulase Activity of Forestry Biomass Raw Materials GB / T35808-2018); compared with the glucose standard curves determined by the four existing DNS methods, the method of the present invention not only has a larger slope but also passes through the origin, and the method has higher sensitivity and accuracy.

[0069] Figure 7 After adding the freshly prepared MBTH reagent to the alkaline glucose standard solution, OH in the reaction system - Effect of concentration on the slope (upper figure) and intercept (lower figure) of the glucose standard curve, measured by ELISA plate, OH - The glucose standard curve can be drawn in the range of 0.01-0.2 mol / L to determine the dextranase activity, among which OH - The slope is the largest and the intercept is closest to 0 when the concentration is in the range of 0.1-0.2 mol / L.

[0070] Figure 8 The invention shows the influence of MBTH concentration in the reaction system on the slope (upper figure) and intercept (lower figure) of the glucose standard curve after adding the freshly prepared MBTH reagent to the alkaline glucose standard solution. The glucose standard curve can be drawn when the MBTH concentration is in the range of 0.3-4.5 mmol / L, and the dextranase activity is determined by ELISA plate. The slope is the largest and the intercept is closest to 0 when the MBTH concentration is in the range of 0.95-3.6 mmol / L, and the time stability is the best.

[0071] Fig. 9The figure shows the effect of the DTT concentration in the MBTH reagent of the present invention on the slope (upper figure) and intercept (lower figure) of the glucose standard curve. The glucose standard curve can be drawn when the DTT concentration is in the range of 0-1.6 mmol / L using an ELISA plate to measure the dextranase activity. When the DTT concentration is 0, the slope is the largest and the intercept is closest to 0.

[0072] Fig.10 Compared with the prior art β-glucanase activity determination method using MBTH method (DTT concentration is 1 mg / mL), the slope of the glucose standard curve drawn by the method of the present invention is larger and the intercept is closer to 0.

[0073] Fig.11 The Fe in the reaction system after adding the acidic iron reagent of the present invention 3+ The effect of molar concentration on the slope (upper figure) and intercept (lower figure) of the glucose standard curve was determined by ELISA plate. 3+ The molar concentration of Fe can be used to draw a glucose standard curve in the range of 1-6 mmol / L to determine the dextranase activity. 3+ The slope is largest and the intercept is closest to 0 when the molar concentration is in the range of 3-5mmol / L, and the time stability is the best.

[0074] Fig.12 The slope (upper figure) and intercept (lower figure) of the glucose standard curve of different soluble iron salts change with time. All three soluble iron salts can be used for the dextranase activity determination.

[0075] Fig.13 In the present invention, after adding the freshly prepared MBTH reagent to the alkaline glucose standard solution, OH - When the concentration is 0.07 and 0.1 mol / L, the acidic iron reagent is added, and the remaining H + The effect of the concentration of H on the slope (upper figure) and intercept (lower figure) of the glucose standard curve was determined by ELISA plate. + The glucose standard curve can be drawn in the range of 0.02-0.3 mol / L to determine the dextranase activity. + When the concentration reaches 0.06 mol / L, the intercept is basically stable, and the slope is + The concentration remains stable in the range of 0.06-0.1mol / L.

[0076] Fig.14 This is the influence of the slope (upper figure) and intercept (lower figure) of the vibrating plate and the non-vibrating plate before measuring the absorbance value of the ELISA plate of the present invention. Compared with the non-vibrating plate, the slope measured after the vibrating plate is larger, the intercept is closer to 0, and the time stability is better.

[0077] Fig.15 The figure shows the influence of the wavelength of the absorbance value determination of the ELISA plate of the present invention on the slope (upper figure) and intercept (lower figure) of the glucose standard curve. When the wavelength is 590nm and 610nm, the intercept is close to 0, which is better than the wavelength of 650nm used in the determination method of β-glucanase activity.

[0078] Fig.16 The toothpaste is diluted, ground, centrifuged (centrifuged at 4000RCF for 10 min at 4°C), and the supernatant is taken to obtain the solution. Samples 1, 2, and 3 in the figure (corresponding to the samples in Example 3) are toothpaste products from different companies. The concentrations in the left figure are 0.2 mg / mL, 2 mg / mL, and 20 mg / mL, respectively. The solutions are clear and transparent and can be used for determining enzyme activity by the MBTH method (see Example 3), but cannot be used for determining enzyme activity by the DNS method; the concentrations in the right figure are 2 mg / mL, 20 mg / mL, and 200 mg / mL, respectively. Solution No. 1 is clear and transparent, solution No. 2 is not completely dissolved, and solution No. 3 is an insoluble emulsion. Only under this concentration condition can it be used for determining enzyme activity by the DNS method (see Comparative Example 4). If other extraction methods are used, such as filtration, the filter pores will be directly blocked, and multiple centrifugation will increase the operating error. At the same time, the extraction time is increased, and it is difficult to ensure that the enzyme will not be inactivated when extracted from the protective matrix. The present invention is still applicable to samples with low enzyme content or low enzyme activity.

[0079] Fig.17 This is the enzyme mass concentration reaction rate curve obtained for the enzymatic hydrolysis reaction and MBTH reaction at 55°C for 30 min under metal bath conditions. The curve is linear and can be used for fully automatic high-throughput determination using a discharge gun.

[0080] Fig.18 The MBTH method of the present invention uses an ELISA plate to measure the reaction kinetics curve of dextranase (the upper figure is a zero-order reaction) and the enzyme mass concentration reaction rate curve (the lower figure is a linear curve), and the accuracy of the measurement will not be affected by different dilution multiples. DETAILED DESCRIPTION

[0081] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0082] The main instruments and reagents used in the following examples are as follows:

[0083] Main instruments: water bath constant temperature oscillator, SHZ-82 model, Changzhou Zhiborui Co., Ltd.; FE2 laboratory pH meter, Mettler-Toledo Instrument Co., Ltd.; Bio-rad 680 microplate reader, Bio-Rad Life Sciences Products (Shanghai) Co., Ltd.; UV-2600 ultraviolet visible spectrophotometer, SHIMADZU; AL204 electronic balance, Mettler-Toledo Instrument Co., Ltd.; CT14RD centrifuge, Shanghai Tianmei Biochemical Instrument Equipment Engineering Co., Ltd.

[0084] Main reagents: Glucose was purchased from Sinopharm Chemical Reagent Co., Ltd.; dextran 70 kDa was purchased from Sinopharm Chemical Reagent Co., Ltd.; dextran 70 kDa, 100 kDa, 200 kDa, and 500 kDa were purchased from Xi'an Xinlu Biotechnology Optimization Company; the production raw materials of pharmaceutical-grade dextran were purchased from Shaanxi Institute of Microbiology; dextranase was purchased from Sino Biotechnology Development Jiangsu Co., Ltd.; toothpaste was purchased from Hangzhou Baixi Brand Management Co., Ltd., Colgate-Palmolive (China) Co., Ltd., Kangbo Daily Chemical Group Co., Ltd., Hangzhou Nami Zhikang Technology Co., Ltd., Shanghai Chuanmei Industrial Co., Ltd., and Lion Co., Ltd. Co., Ltd., Weimeizi Industrial (Guangdong) Co., Ltd.; mouthwash was purchased from Hangzhou Baixi Brand Management Co., Ltd., Kangbo Daily Chemical Group Co., Ltd., and Lion Corporation of Japan; 3-methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate (MBTH) was from Sigma-Aldrich Trading Co., Ltd.; DL-dithiothreitol (DTT) was from Sigma-Aldrich Trading Co., Ltd.; aminosulfonic acid, FeNH4(SO4)2·12H2O, Fe(NO3)3·9H2O, and FeCl3 were purchased from Sinopharm Chemical Reagent Co., Ltd.; other reagents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0085] The enzyme activity was calculated according to the following formula:

[0086]

[0087] Where:

[0088] U-dextranase activity, μmoL / (mL·min);

[0089] A-absorbance value of enzymatic solution;

[0090] A0-absorbance value of enzymatic blank solution;

[0091] b-intercept of glucose standard curve;

[0092] Slope of k-glucose standard curve, mL / μmoL;

[0093] t-enzymatic hydrolysis time, min.

[0094] The method of embodiment 1-4:

[0095] Step (1), the substrate is crushed so that all the substrate passes through an 80-mesh sieve, 1 g of the substrate is taken out and stirred in 150 mL of 60% ethanol for 3 h, centrifuged at 4000 RCF (6400 rpm) for 15 min, the supernatant is discarded, 25 mL of 95% ethanol is added, and the mixture is thoroughly mixed. After 10 min, the same volume of 95% ethanol is added again and this step is repeated 3 times, centrifuged at 3000 RCF (5600 rpm) for 15 min, the supernatant is discarded, 25 mL of anhydrous ethanol is added again, centrifuged at 3000 RCF (5600 rpm) for 15 min, the supernatant is discarded, 50 mL of acetone is added to the precipitate, ultrasonicated for 2 min, allowed to stand for 1.5 h, and evaporated at 35° C. under a high vacuum pressure of -0.098 MPa until the sample is dry to obtain a refined substrate.

[0096] Step (2), adding 4 mL of 0.4 mol / L NaOH solution to 0.1 g of the treated refined substrate, stirring to dissolve the substrate, adding 4 mL of 0.2 mol / L succinic acid solution, mixing, continuing to add 0.1 mol / L succinic acid solution to the substrate solution to adjust the pH to 5.5, adding 0.1 mol / L succinic acid-sodium succinate buffer solution with a pH of 5.5 to the fixed volume, preparing a dextran solution with a concentration of 10 mg / mL, the solution is prepared and used immediately; adding succinic acid-sodium succinate buffer solution with a pH of 5.5 to 0.1 g of dextranase to the fixed volume, preparing a dextranase solution.

[0097] Step (3), take the dextran solution in step (2) and 0.1mol / L, pH 5.5 succinic acid-sodium succinate buffer solution and mix them in equal volumes to prepare a glucose standard solution with a concentration of 0-100μg / mL (0-0.5mmol / L). Add 1mL of 0.5mol / L NaOH solution to 1mL of the above glucose standard solutions of different concentrations, mix well, and then add 1mL of freshly prepared 1.5mg / mL (6.4mmol / L) MBTH solution. At this time, OH in the reaction system is 1.5mg / mL (6.4mmol / L). - The concentration of is 0.17 mol / L, and the initial concentration of MBTH is 2.1 mmol / L. After heating in a water bath at 80°C for 13 min, 2 mL of ferric nitrate reagent (composed of ferric nitrate with a final concentration of 10 mmol / L, 0.05 mol / L aminosulfonic acid, and 0.45 mol / L hydrochloric acid) is added while hot. At this time, the system becomes acidic, and H + The concentration is 0.1 mol / L, Fe 3+The initial concentration was 4 mmol / L. After cooling to room temperature, the color developed for 50 minutes. 200 μL was taken and placed on an ELISA plate. After shaking the plate, the absorbance of glucose was determined at a wavelength of 590 nm using an ELISA reader. Three parallel samples were made for each concentration. A standard correspondence curve was drawn based on the relationship between the concentration of each group of glucose solutions and the corresponding glucose absorbance values ​​measured.

[0098] Step (4), adding 0.5 mL of the dextranase solution to be tested (using 0.1 mol / L, pH 5.5 succinic acid-sodium succinate buffer solution) into a test tube and preheating it in a water bath shaker for 5 min, adding 0.5 mL of the 10 mg / mL dextran substrate solution in step (2) preheated to the corresponding temperature for enzymolysis reaction, the enzymolysis time is 30 min, quickly adding 1 mL of 0.5 mol / L NaOH solution, and immediately mixing thoroughly to terminate the enzyme reaction to obtain an alkaline enzymolysis solution.

[0099] Step (5), add 0.5 mL of dextran solution to the test tube and preheat in a water bath shaker for 5 min, then continue to keep warm for 30 min, quickly add 1 mL of 0.5 mol / L NaOH solution to the test tube, and immediately mix thoroughly, add 0.5 mL of the same preheated dextran solution as in step (4), mix thoroughly, and obtain an enzymatic blank.

[0100] Step (6), add 1 mL of the same MBTH solution as in step (3) to the alkaline enzymatic hydrolyzate in step (4) and the enzymatic hydrolyzate blank in step (5), heat in a water bath at 80°C for 13 min, add 2 mL of the same ferric nitrate reagent as in step (3) while hot, cool at room temperature for 50 min, take 200 μL of each onto an ELISA plate, vibrate the plate, and measure the absorbance at a wavelength of 590 nm.

[0101] Step (7), the absorbance value measured in step (6) is substituted into the standard corresponding relationship curve in step (3), and the reducing sugar concentration of the alkaline enzymatic solution and the enzymatic blank equivalent to the glucose concentration is obtained, so as to calculate the increase of the reducing sugar concentration per unit time, and then calculate the dextranase activity. The activity unit of dextranase can be defined as the amount of glucose equivalent to 1 μmol produced per minute in each milliliter of reaction solution under the above conditions as one unit of enzyme activity.

[0102] Embodiment 1:

[0103] The steps (1) to (7) of the method of Example 1-4 were used, and enzymatic hydrolysis was carried out at 55°C.

[0104] Table 1 Dextranase activity assay results in Example 1

[0105]

[0106] Embodiment 2:

[0107] The steps (1) to (7) of the method of Example 1-4 were used, and enzymatic hydrolysis was carried out at 37°C.

[0108] Table 2 Dextranase activity assay results in Example 2

[0109]

[0110] Examples 1-2 show that dextranase activity can be accurately measured within the range of 37-55°C.

[0111] Embodiment 3:

[0112] Three commercially available toothpastes with high, medium and low dextranase contents were selected, and the enzyme activity was determined using the method of the present invention.

[0113] Accurately weigh 0.1 g, 0.2 g, and 2 g of toothpaste with high, medium, and low dextranase content, respectively, add 10 mL of buffer solution, crush with a tissue homogenizer, centrifuge at 4°C, 4000 RCF (6400 rpm) for 10 min, discard the lower precipitate, take the supernatant for later use, repeat the above steps three times, and prepare toothpaste solutions with concentrations of 0.4 mg / mL, 4 mg / mL, and 40 mg / mL with the supernatant.

[0114] The steps (1) to (7) of the method of Example 1-4 were used, and enzymatic hydrolysis was carried out at 37°C.

[0115] Table 3 Dextranase activity determination results in Example 3 toothpaste

[0116]

[0117] Example 3 shows that for commercially available toothpaste, no matter whether the amount of dextranase contained is high or low, the present invention can measure it under the condition of human body temperature of 37°C.

[0118] Embodiment 4:

[0119] Accurately weigh 0.1 g of mouthwash, add 0.1 mol / L succinic acid-sodium succinate buffer solution with a pH of 5.5 at one time, stir thoroughly, and prepare a mouthwash solution with a concentration of 0.4 mg / mL.

[0120] The steps (1) to (7) of the method of Example 1-4 were used, and enzymatic hydrolysis was carried out at 37°C.

[0121] Table 4 Dextranase activity determination results in Example 4 mouthwash

[0122]

[0123] Example 4 shows that for commercially available mouthwash, the present invention can accurately measure the dextranase activity under the condition of human body temperature of 37°C.

[0124] Embodiment 5:

[0125] Step (1), the substrate is crushed so that all the substrate passes through a 60-mesh sieve, 1 g of the substrate is taken out and stirred in 170 mL of 35% ethanol for 5 h, centrifuged at 2500 RCF (5100 rpm) for 20 min, the supernatant is discarded, 25 mL of 80% ethanol is added, and the mixture is thoroughly mixed. After 10 min, the same volume of 80% ethanol is added again and this step is repeated 5 times, centrifuged at 4000 RCF (6400 rpm) for 10 min, the supernatant is discarded, 25 mL of anhydrous ethanol is added again, centrifuged at 4000 RCF (6400 rpm) for 20 min, the supernatant is discarded, 100 mL of acetone is added to the precipitate, ultrasonicated for 1 min, allowed to stand for 1 h, and evaporated at 40° C. under a high vacuum pressure of -0.093 MPa until the sample is dry to obtain a refined substrate.

[0126] Step (2), adding 4 mL of 0.1 mol / L KOH solution to 0.1 g of the treated refined substrate, stirring to dissolve the substrate, adding 4 mL of 0.1 mol / L acetic acid solution, mixing, continuing to add 0.05 mol / L acetic acid solution to the substrate solution to adjust the pH to 5.5, adding 0.05 mol / L acetic acid-sodium acetate buffer solution with a pH of 5.5 to the fixed volume, preparing a dextran solution with a concentration of 2 mg / mL, and the solution is prepared and used immediately; adding succinic acid-sodium succinate buffer solution with a pH of 5.5 to 0.1 g of dextranase to the fixed volume, preparing a dextranase solution with a concentration of 0.2 μg / mL.

[0127] Step (3), take the dextran solution in step (2) and mix it with 0.05mol / L acetic acid-sodium acetate buffer solution with a pH of 5.5 in equal volumes to prepare a glucose standard solution with a concentration of 0-100μg / mL (0-0.5mmol / L). Add 1mL of 0.06mol / L NaOH solution to 1mL of the above glucose standard solutions of different concentrations, mix well, and then add 1mL of freshly prepared 3.2mg / mL (13.5mmol / L) MBTH solution. At this time, OH in the reaction system - The concentration of is 0.02 mol / L, and the initial concentration of MBTH is 4.5 mmol / L. After heating in a 100°C water bath for 5 min, 2 mL of ammonium ferric sulfate reagent (composed of ammonium ferric sulfate with a final concentration of 2.5 mmol / L and 0.1 mol / L aminosulfonic acid) is added while hot. At this time, the system becomes acidic, and H + The concentration is 0.03 mol / L, Fe 3+The initial concentration was 1 mmol / L. After cooling to room temperature, the color developed for 70 minutes. 200 μL was taken and placed on an ELISA plate. After shaking the plate, the absorbance of glucose was determined at a wavelength of 610 nm using an ELISA reader. Three parallel samples were made for each concentration. A standard correspondence curve was drawn based on the relationship between the concentration of each group of glucose solutions and the corresponding glucose absorbance values ​​measured.

[0128] Step (4), adding 0.5 mL of the dextranase solution to be tested (using 0.05 mol / L, pH 5.5 acetic acid-sodium acetate buffer solution) into a test tube and preheating it in a 30°C water bath shaker for 5 min, adding 0.5 mL of the 0.1 mg / mL dextran substrate solution in step (2) preheated to the corresponding temperature for enzymolysis reaction, the enzymolysis time is 20 min, quickly adding 1 mL of 0.06 mol / L NaOH solution, and immediately mixing thoroughly to terminate the enzyme reaction to obtain an alkaline enzymolysis solution.

[0129] Step (5), add 0.5 mL of dextran solution to the test tube and preheat in a water bath shaker for 5 min, then continue to keep warm for 20 min, quickly add 1 mL of 0.06 mol / L NaOH solution to the test tube, and immediately mix thoroughly, add 0.5 mL of the same preheated dextran solution as in step (4), mix thoroughly, and obtain an enzymatic blank.

[0130] Step (6), add 1 mL of the same MBTH solution as in step (3) to the alkaline enzymatic hydrolyzate in step (4) and the enzymatic hydrolyzate blank in step (5), heat in a water bath at 100°C for 5 minutes, add 2 mL of the same ferric nitrate reagent as in step (3) while hot, cool at room temperature for 70 minutes, take 200 μL of each onto an ELISA plate, vibrate the plate and measure the absorbance at a wavelength of 610 nm.

[0131] Step (7), the absorbance value measured in step (6) is substituted into the standard corresponding relationship curve in step (3), and the reducing sugar concentration of the alkaline enzymatic solution and the enzymatic blank equivalent to the glucose concentration is obtained, so as to calculate the increase of the reducing sugar concentration per unit time, and then calculate the dextranase activity. The activity unit of dextranase can be defined as the amount of glucose equivalent to 1 μmol produced per minute in each milliliter of reaction solution under the above conditions as one unit of enzyme activity.

[0132] Table 5 Dextranase activity assay results in Example 5

[0133] Parallel Sample 1 2 Enzyme concentration in the reaction system (μg / mL) 0.10 0.10 The difference in absorbance between the enzymatic solution and the blank solution 0.151 0.152 Concentration of reducing end groups produced by enzymatic hydrolysis (μmol / mL) 0.0864 0.0871 Enzyme activity (U / mg) 43.2 43.5

[0134] Embodiment 6:

[0135] Step (1), the substrate is crushed so that all the substrate passes through a 60-mesh sieve, 1 g of the substrate is taken out and stirred in 130 mL of 70% ethanol for 3 h, centrifuged at 2500 RCF (5100 rpm) for 10 min, the supernatant is discarded, 20 mL of anhydrous ethanol is added, and the mixture is thoroughly mixed. After 10 min, the same volume of anhydrous ethanol is added again and this step is repeated twice, centrifuged at 4000 RCF (6400 rpm) for 5 min, the supernatant is discarded, 20 mL of anhydrous ethanol is added again, centrifuged at 4000 RCF (6400 rpm) for 5 min, the supernatant is discarded, 40 mL of acetone is added to the precipitate, ultrasonicated for 2 min, allowed to stand for 2 h, and evaporated at 25° C. under a high vacuum pressure of -0.09 MPa until the sample is dry to obtain a refined substrate.

[0136] Step (2), add 1 mL of 1 mol / L NaOH solution to 0.1 g of the treated refined substrate, stir to dissolve the substrate, add 1 mL of 1 mol / L acetic acid solution, then add water to dilute to 20 mL, mix, continue to add 0.1 mol / L acetic acid solution to the substrate solution to adjust the pH to 5.5, add 0.1 mol / L acetic acid-sodium acetate buffer solution with a pH of 5.5 to make up the volume, prepare a dextran solution with a concentration of 10 mg / mL, and the solution is prepared and used immediately; add succinic acid-sodium succinate buffer solution with a pH of 5.5 to 0.3 g of dextranase to make up the volume, and prepare a dextranase solution with a concentration of 0.3 μg / mL.

[0137] Step (3), take the dextran solution in step (2) and mix it with 0.1mol / L acetic acid-sodium acetate buffer solution with pH 5.5 in equal volumes to prepare a glucose standard solution with a concentration of 0-100μg / mL (0-0.5mmol / L). Add 1mL of 0.6mol / L KOH solution to 1mL of the above glucose standard solutions of different concentrations, mix well, and then add 1mL of freshly prepared 0.2mg / mL (0.9mmol / L) MBTH solution. At this time, OH in the reaction system - The concentration of 0.2 mol / L and the initial concentration of MBTH were 0.3 mmol / L. After heating in a 50°C water bath for 120 min, 2 mL of ammonium ferric sulfate reagent (composed of ferric chloride with a final concentration of 15 mmol / L and 0.42 mol / L sulfuric acid) was added while hot. At this time, the system became acidic and H + The concentration is 0.05 mol / L, Fe 3+The initial concentration was 6 mmol / L. After cooling to room temperature, the color developed for 180 minutes. 200 μL was taken and placed on an ELISA plate. After shaking the plate, the absorbance of glucose was determined at a wavelength of 590 nm using an ELISA reader. Three parallel samples were made for each concentration. A standard correspondence curve was drawn based on the relationship between the concentration of each group of glucose solutions and the corresponding glucose absorbance values ​​measured.

[0138] Step (4), adding 0.5 mL of the dextranase solution to be tested (using 0.1 mol / L, pH 5.5 acetic acid-sodium acetate buffer solution) into a test tube and preheating it in a 60° C. water bath shaker for 5 min, adding 0.5 mL of the 10 mg / mL dextran substrate solution in step (2) preheated to the corresponding temperature for enzymolysis reaction, the enzymolysis time is 30 min, quickly adding 1 mL of 0.6 mol / L KOH solution, and immediately mixing thoroughly to terminate the enzyme reaction to obtain an alkaline enzymolysis solution.

[0139] Step (5), add 0.5 mL of dextran solution to the test tube and preheat in a water bath shaker for 5 min, then continue to keep warm for 30 min, quickly add 1 mL of 0.06 mol / L KOH solution to the test tube, and immediately mix thoroughly, add 0.5 mL of the same preheated dextran solution as in step (4), mix thoroughly, and obtain an enzymatic blank.

[0140] Step (6), add 1 mL of the same MBTH solution as in step (3) to the alkaline enzymatic hydrolyzate in step (4) and the enzymatic hydrolyzate blank in step (5), heat in a 50°C water bath for 120 min, add 2 mL of the same ferric chloride reagent as in step (3) while hot, cool at room temperature for 180 min, take 200 μL of each onto an ELISA plate, vibrate the plate, and measure the absorbance at a wavelength of 590 nm.

[0141] Step (7), the absorbance value measured in step (6) is substituted into the standard corresponding relationship curve in step (3), and the reducing sugar concentration of the alkaline enzymatic solution and the enzymatic blank equivalent to the glucose concentration is obtained, so as to calculate the increase of the reducing sugar concentration per unit time, and then calculate the dextranase activity. The activity unit of dextranase can be defined as the amount of glucose equivalent to 1 μmol produced per minute in each milliliter of reaction solution under the above conditions as one unit of enzyme activity.

[0142] Table 6 Dextranase activity assay results of Example 6

[0143] Parallel Sample 1 2 Enzyme concentration in the reaction system (μg / mL) 0.15 0.15 The difference in absorbance between the enzymatic solution and the blank solution 0.141 0.142 Concentration of reducing end groups produced by enzymatic hydrolysis (μmol / mL) 0.255 0.257 Enzyme activity (U / mg) 56.7 57.1

[0144] Embodiment 7:

[0145] Step (1), the substrate is crushed so that all the substrate passes through a 100-mesh sieve, and 1 g of the substrate is taken out and placed in 160 mL of 70%

[0146] The mixture was stirred in ethanol for 4 h, centrifuged at 4000RCF (6400rpm) for 5 min, the supernatant was discarded, 25 mL of 90% ethanol was added, and the mixture was thoroughly mixed. After 10 min, the same volume of 90% ethanol was added again and this step was repeated 5 times. The mixture was centrifuged at 4000RCF (6400rpm) for 10 min, the supernatant was discarded, 25 mL of anhydrous ethanol was added again, and the mixture was centrifuged at 4000RCF (6400rpm) for 10 min. The supernatant was discarded, 60 mL of acetone was added to the precipitate, and the mixture was ultrasonicated for 5 min. The mixture was allowed to stand for 2 h, and the sample was evaporated at 30°C under a high vacuum pressure of -0.095 MPa until the sample was dry to obtain a refined substrate.

[0147] Step (2), adding 4 mL of 0.4 mol / L NaOH solution to 0.1 g of the treated refined substrate, stirring to dissolve the substrate, adding 4 mL of 0.2 mol / L succinic acid solution, mixing, continuing to add 0.1 mol / L succinic acid solution to the substrate solution to adjust the pH to 5.5, adding 0.1 mol / L succinic acid-sodium succinate buffer solution with a pH of 5.5 to the fixed volume, preparing a dextran solution with a concentration of 4 mg / mL, and the solution is prepared and used immediately; adding succinic acid-sodium succinate buffer solution with a pH of 5.5 to 0.1 g of dextranase to the fixed volume, preparing a dextranase solution with a concentration of 0.1 μg / mL.

[0148] Step (3), take the dextran solution in step (2) and 0.1mol / L, pH 5.5 succinic acid-sodium succinate buffer solution and mix them in equal volumes to prepare a glucose standard solution with a concentration of 0-100μg / mL (0-0.5mmol / L). Add 1mL of 0.5mol / L NaOH solution to 1mL of the above glucose standard solutions of different concentrations, mix well, and then add 1mL of freshly prepared 1.8mg / mL (10.8mmol / L) MBTH solution. At this time, OH in the reaction system - The concentration of is 0.17 mol / L, and the initial concentration of MBTH is 3.6 mmol / L. After heating in a water bath at 80°C for 13 min, 2 mL of ammonium ferric sulfate reagent (composed of ammonium ferric sulfate with a final concentration of 7.5 mmol / L and 0.55 mol / L hydrochloric acid) is added while hot. At this time, the system becomes acidic, and H + The concentration is 0.1 mol / L, Fe 3+The initial concentration is 3mmol / L. After cooling to room temperature, the color develops for 50 minutes. Take 200μL of it and put it on the ELISA plate. After shaking the plate, use an ELISA reader to measure the absorbance of glucose at a wavelength of 590nm. Make three parallel samples for each concentration, and draw a standard correspondence curve based on the relationship between the concentration of each group of glucose solutions and the corresponding glucose absorbance values ​​measured.

[0149] Step (4), adding 0.5 mL of the dextranase solution to be tested (using 0.1 mol / L, pH 5.5 succinic acid-sodium succinate buffer solution) into a test tube and preheating it in a 37°C water bath shaker for 5 min, adding 0.5 mL of the 5 mg / mL dextran substrate solution in step (2) preheated to the corresponding temperature for enzymolysis reaction, the enzymolysis time is 60 min, quickly adding 1 mL of 0.5 mol / L NaOH solution, and immediately mixing thoroughly to terminate the enzyme reaction to obtain an alkaline enzymolysis solution.

[0150] Step (5), add 0.5 mL of dextran solution to the test tube and preheat in a water bath shaker for 5 min, then continue to keep warm for 60 min, quickly add 1 mL of 0.5 mol / L NaOH solution to the test tube, and immediately mix thoroughly, add 0.5 mL of the same preheated dextran solution as in step (4), mix thoroughly, and obtain an enzymatic blank.

[0151] Step (6), add 1 mL of the same MBTH solution as in step (3) to the alkaline enzymatic hydrolyzate in step (4) and the enzymatic hydrolyzate blank in step (5), heat in a water bath at 80°C for 13 min, add 2 mL of the same ferric nitrate reagent as in step (3) while hot, cool at room temperature for 50 min, take 200 μL of each onto an ELISA plate, vibrate the plate, and measure the absorbance at a wavelength of 590 nm.

[0152] Step (7), the absorbance value measured in step (6) is substituted into the standard corresponding relationship curve in step (3), and the reducing sugar concentration of the alkaline enzymatic solution and the enzymatic blank equivalent to the glucose concentration is obtained, so as to calculate the increase of the reducing sugar concentration per unit time, and then calculate the dextranase activity. The activity unit of dextranase can be defined as the amount of glucose equivalent to 1 μmol produced per minute in each milliliter of reaction solution under the above conditions as one unit of enzyme activity.

[0153] Table 7 Dextranase activity assay results of Example 7

[0154] Parallel Sample 1 2 Enzyme concentration in the reaction system (μg / mL) 0.05 0.05 The difference in absorbance between the enzymatic solution and the blank solution 0.463 0.465 Concentration of reducing end groups produced by enzymatic hydrolysis (μmol / mL) 0.139 0.140 Enzyme activity (U / mg) 46.4 46.6

[0155] Comparative Example 1 (compared to Example 1):

[0156] The activity of β-glucanase was determined by enzymatic hydrolysis at 55°C.

[0157] Table 8 Dextranase activity test results of comparative example 1

[0158]

[0159] By comparing Example 1 with Comparative Example 1, it can be seen that when the DTT concentration is 0 and the measurement wavelength is 590 nm, the slope of the glucose standard curve is the largest and the intercept is closest to 0 when the plate is vibrated to reduce the surface tension before the measurement, and the time stability is good (see Figure 7 , Figure 8 , Fig. 9 ), therefore, the determination method of the present invention has higher accuracy.

[0160] DNS method used in Comparative Example 2-4:

[0161] DNS-1: Chang Guowei, Li Zhide, Huang Zengwei, et al. Study on enzymatic properties and thermal inactivation kinetics of dextranase[J]. Chinese Condiments, 2021, 46(5): 84-88;

[0162] DNS-2: Xing Mingxia, Wang Pengbo, Xu Wenting, et al. Optimization of the determination method of alkaline pectinase specific activity by kinetic method[J]. Food Industry Science and Technology, 2020, 41(23): 223-228, 235;

[0163] DNS-3: GB / T 23874-2009 Determination of xylanase activity in feed additives by spectrophotometry, GB / T36861-2018 Determination of β-mannanase activity in feed additives by spectrophotometry;

[0164] DNS-4: GB / T 35808-2018 Analysis method for forestry biomass raw materials - Determination of cellulase activity.

[0165] Table 9 Four DNS methods

[0166]

[0167] Comparative Example 2 (compared to Example 1):

[0168] The methods of DNS-1, DNS-2, DNS-3 and DNS-4 were used, and the substrate treatment, solution preparation and enzymatic hydrolysis process were carried out at 55° C. according to the example method.

[0169] Table 10 Dextranase activity test results of comparative example 2

[0170]

[0171] Comparative Example 3 (compared to Example 2):

[0172] The methods of DNS-1, DNS-2, DNS-3 and DNS-4 were used, and the substrate treatment, solution preparation and enzymatic hydrolysis process were carried out at 37° C. according to the example method.

[0173] Table 11 Dextranase activity test results of comparative example 3

[0174]

[0175]

[0176] Comparison of the experimental data of Comparative Examples 2-3 with those of Examples 1-2 shows that when dextranase is measured at different enzyme concentrations in the range of 37-55°C using the present invention, the relative standard deviation of the enzyme activity data is small, different experimental results will not be produced due to different prepared enzyme concentrations during the measurement, and the amount of enzyme used during the measurement is small, which is more cost-effective.

[0177] Comparative Example 4 (compared to Example 3):

[0178] Three commercially available toothpastes with high, medium and low dextranase contents were selected, and the enzyme activity was determined using the method of the present invention.

[0179] Accurately weigh 1g, 2g, and 20g of toothpaste with high, medium, and low dextranase content, respectively, add 10mL of buffer solution, crush with a tissue homogenizer, centrifuge at 4°C, 4000RCF (6400rpm) for 10min, discard the lower layer of precipitate, take the supernatant for later use, repeat the above steps three times, and prepare the test solution with concentrations of 4mg / mL, 40mg / mL, and 400mg / mL with the supernatant.

[0180] The methods of DNS-1, DNS-2, DNS-3 and DNS-4 were used, and the substrate treatment, solution preparation and enzymatic hydrolysis process were carried out at 37° C. according to the example method.

[0181] Table 12 Dextranase activity determination results in comparative example 4 toothpaste

[0182]

[0183] The comparison of the experimental data of Comparative Example 4 and Example 3 shows that for toothpaste with low dextranase content on the market, the present invention can accurately determine the enzyme activity, while the DNS method cannot obtain an enzyme solution that can be used for enzyme activity determination due to its low sensitivity (see Appendix Fig.10 ); Compared with the DNS method, the present invention can use a small amount of sample for determination, and will not increase the concentration of the toothpaste matrix in the extracted enzyme solution due to the large amount of sample and interfere with the determination result.

[0184] Of course, the above description is not a limitation of the present invention, and the present invention is not limited to the above operations. All changes, modifications, additions or substitutions made within the essential scope of the present invention should fall within the protection scope of the present invention.

Claims

1. A method for determining dextranase activity, characterized in that: The following steps are involved: (1) Refined substrate: a. crushing the dextran to prepare dextran powder; b. Disperse the dextran powder in a 35%-70% ethanol solution, stir thoroughly, centrifuge, and discard the supernatant. Repeat this step several times to remove small molecules in the dextran powder; c. The precipitate obtained in step b is dehydrated stepwise using an ethanol solution with increasing concentrations, wherein the concentration of the ethanol solution used is 80%-100%; d. adding acetone to the precipitate obtained in step c, allowing to stand after ultrasonic treatment, and performing vacuum rotary evaporation under high vacuum conditions to obtain a dried refined substrate, wherein the high vacuum degree refers to a pressure ≤-0.089MPa; If the weight average molecular weight of dextran is ≥70 kDa and the minimum molecular weight is greater than 10 kDa, it can be used directly as a refined substrate and the above-mentioned refining step can be omitted; (2) preparing a substrate solution and a dextranase solution: adding the refined substrate of step (1) to a strong alkaline solution having a concentration of 0.1-1 mol / L, mixing thoroughly to dissolve it, adding an acid to adjust the pH to a pH value to be measured, and fixing the volume with a buffer solution having a pH value to be measured, thereby obtaining a substrate solution; and using the same buffer solution to prepare a dextranase solution; (3) Draw the standard corresponding relationship curve between glucose absorbance value and glucose concentration: Using the substrate solution prepared in step (2), a series of glucose standard solutions with concentration gradients are prepared, wherein the substrate concentrations in the series of glucose standard solutions with concentration gradients are the same, and the glucose concentration range is 0-0.5 mmol / L; An alkaline reagent is added to the series of glucose standard solutions with a concentration gradient to obtain an alkaline glucose standard solution, wherein the concentration of the alkaline reagent in the alkaline glucose standard solution is the same, a freshly prepared MBTH reagent is added, the OH- concentration in the system is in the range of 0.02-0.2 mol / L, the initial concentration range of MBTH is 0.3-4.5 mmol / L, the initial concentration range of DTT is 0-1.6 mmol / L, the reaction is carried out at 50-100° C. for 5-180 min, and then an acidic iron reagent is added, the reaction system becomes acidic, and the H + The concentration is 0.02-0.3 mol / L, Fe 3+ The initial concentration range is 1-6mmol / L, a color reaction occurs, and after the color development is stable, the absorbance value is measured at a wavelength of 580-680nm. A standard corresponding relationship curve is drawn based on the relationship between each group of glucose concentrations and the measured absorbance values; The MBTH reagent is an aqueous solution prepared by MBTH and DTT, or an aqueous solution prepared by MBTH alone; the acidic iron reagent is a soluble Fe 3+ Aqueous solution of salt and strong acid; (4) dextranase enzymatic hydrolysis reaction: adding the substrate solution prepared in step (2) to the dextranase solution, ensuring that the concentration of the substrate in the resulting solution is the same as the concentration of the substrate in the glucose standard solution in step (2), then performing an enzymatic hydrolysis reaction, and then adding an alkaline reagent to terminate the enzymatic hydrolysis reaction to obtain an alkaline enzymatic hydrolyzate, wherein the concentration of the alkaline reagent in the alkaline enzymatic hydrolyzate is the same as the concentration of the alkaline reagent in the alkaline glucose standard solution in step (2); (5) preparing an alkaline enzymatic hydrolysis blank solution; (6) Determination of alkaline enzymatic hydrolysis solution and alkaline enzymatic hydrolysis blank solution: MBTH reaction is carried out according to the method of step (3), and the absorbance values ​​of the alkaline enzymatic hydrolysis solution obtained in step (4) and the alkaline enzymatic hydrolysis blank solution obtained in step (5) are determined to obtain the concentration of reducing end groups generated by enzymatic hydrolysis, and then the dextranase activity is obtained.

2. A method for measuring dextranase activity according to claim 1, characterized in that, The step (1) is centrifuged at 2500-4000 RCF for 5-20 min; in step b, 60%-70% ethanol is added; in step c, 90%-100% ethanol is added; in step d, the high vacuum pressure range is -0.09MPa--0.098MPa, and the rotary evaporation temperature is maintained in the range of 25-40°C.

3. A method for measuring dextranase activity according to claim 1, characterized in that, The strong alkali solution in step (2) and the alkaline reagent in step (3) are NaOH solution or KOH solution.

4. A method for measuring dextranase activity according to claim 1, characterized in that, The buffer solution of the pH value to be measured is a succinic acid-sodium succinate buffer solution or an acetic acid-sodium acetate buffer solution.

5. A method for measuring dextranase activity according to claim 1, characterized in that, The soluble Fe in the acidic iron reagent in step (3) 3+ The salt is at least one of ferric nitrate, ferric chloride and ammonium ferric sulfate, and the strong acid is at least one of aminosulfonic acid, sulfuric acid and hydrochloric acid.

6. A method for measuring dextranase activity according to claim 1, characterized in that: In the step (3), after adding the freshly prepared MBTH reagent to the alkaline glucose standard solution, the OH- concentration in the system is in the range of 0.1-0.2 mol / L, the initial concentration of MBTH is in the range of 0.95-3.6 mmol / L, and the initial concentration of DTT is 0 mmol / L; After adding the acidic iron reagent, H + The concentration range is 0.06-0.1 mol / L, Fe 3+ The initial concentration range is 3-5mmol / L.

7. A method for measuring dextranase activity according to claim 1, characterized in that: The reaction time of glucose and MBTH in step (3) is 5-120 min.

8. A method for measuring dextranase activity according to claim 1, characterized in that: The color development time after adding the acidic iron reagent in step (3) is 50-180 minutes, and the absorbance value is measured at 590-650nm during the color development time.

9. A method for determining the activity of dextranase in toothpaste, characterized in that: First, the sample is treated so that the dextranase contained in it is within the range that can be quantitatively determined, and then the enzyme activity thereof is determined according to the method of claim 1. The specific method is as follows: The precisely weighed toothpaste sample is transferred to a tissue homogenizer with a buffer solution and finely ground. The mixture is transferred to a centrifuge tube and centrifuged under a temperature condition that ensures that the enzyme is not inactivated. The supernatant is fixed to a volume to prepare a solution with a toothpaste concentration of 0.1-100 mg / mL as the enzyme solution to be tested.

10. A method for determining the activity of dextranase in mouthwash, characterized in that: First, the sample is treated so that the dextranase contained in it is within the range that can be quantitatively determined, and then the enzyme activity thereof is determined according to the method of claim 1. The specific method is as follows: Accurately weigh a certain amount of mouthwash, add a large amount of buffer solution at one time, and prepare a mouthwash solution with a concentration of 0.1-100 mg / mL. The mouthwash solution can be used as a dextranase solution for enzyme activity determination.

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