CN102796806A - Method for determining cellulase activity of sample - Google Patents

Abstract

The present invention proposes a method for determining the cellulase activity of a sample. The method comprises: in a reaction vessel, mixing a sample to be tested with a cellulase substrate; placing the reaction vessel under conditions suitable for the action of the cellulase for a first predetermined time; adding a chromogenic substrate and placing the reaction vessel under conditions suitable for color development for a second predetermined period of time; determining the glucose content in the reaction vessel; and based on the glucose content, determining the Cellulase activity. Using this method, the cellulase activity of the sample can be effectively determined.

Description

Method for Determining Cellulase Activity of a Sample

technical field

The invention relates to the field of biochemical industry, and more specifically, the invention relates to a method for sampling cellulase activity.

Background technique

Cellulose degradation is closely related to the activity of related microbial cellulase. At present, the most common method for measuring cellulase activity in the world is "Method for Determination of Cellulase Activity" published by T.K.Ghose on Pure & Appli.Chem in 1987, which was promulgated by the International Union of Purification and Applied Chemistry (IUPAC). authoritative method, which is incorporated herein by reference. The basic principle is that reducing sugars such as cellobiose and glucose produced by cellulase hydrolysis of cellulose can reduce 3,5-dinitrosalicylic acid (DNS) under alkaline conditions to generate brown-red amino compounds. There is a maximum light absorption at a wavelength of 540nm, and the amount of reducing sugar is proportional to the color intensity of the reaction solution within a certain range. The activity of cellulase can be determined by measuring the amount of reducing sugar produced by colorimetry. Among them, the activity of endoglucanase can be detected by carboxymethylcellulose sodium (CMC) enzyme activity assay method, and the whole enzyme (including endoglucanase, exoglucanase and β- Glucosidase) activity can be detected by filter paper (equivalent to cellulose) enzyme activity assay.

However, current methods for determining cellulase activity still need to be improved.

Contents of the invention

The present invention has been accomplished based on the following findings of the inventors:

The current international standard system for the determination of CMC enzyme activity contains 0.5ml enzyme, 0.5ml 2% by weight CMC, 3ml DNS and 20ml distilled water. The international standard system of filter paper enzyme activity assay method includes 0.5ml enzyme, 1ml PH4.8 0.05M sodium acetate-acetate buffer solution (in this paper, sometimes referred to as "acetate buffer solution"), 1cm × 6cm filter paper, 3ml DNS and 20ml of distilled water. For the convenience of operation, some adjustments are usually made on the basis of the above-mentioned international standard methods in specific research or technical inventions. For example, with regard to the assay method of CMC enzyme activity, some studies have used a 120 μl system in a 96-well plate, which includes 30 μl enzyme, 30 μl 2% CMC and 60 μl DNS. Regarding the assay method of filter paper enzyme activity, some studies have used a 96-well plate 200μl system, which includes 20μl enzyme, 40μl 0.05M acetate buffer, 7mm diameter filter paper disk and 120μl DNS. After DNS color development, suck out 36μl + 160μl distilled water , measured A ₅₄₀ value. However, when large-scale, high-throughput cellulase activity assays are required, the traditional reaction system is time-consuming and laborious.

The present invention aims at solving one of the above technical problems at least to a certain extent or at least providing a useful commercial choice. Therefore, an object of the present invention is to propose a method capable of effectively determining the cellulase activity of a sample.

The method for determining the cellulase activity of a sample according to an embodiment of the present invention includes: mixing the sample to be tested with a cellulase substrate in a reaction vessel; placing the reaction vessel under conditions suitable for the action of cellulase, for a first predetermined time; adding a chromogenic substrate to the reaction vessel, and placing the reaction vessel under conditions suitable for color development for a second predetermined time; determining the glucose content in the reaction vessel; Based on the glucose content, the cellulase activity of the sample is determined. Using the method according to the embodiment of the present invention, the cellulase activity of the sample can be effectively determined. According to the embodiment of the present invention, the method for determining the cellulase activity of the sample according to the embodiment of the present invention is consistent with the result of measuring the enzyme activity of the same commercial enzyme with the same concentration using the international standard method (IU). Compared with other existing methods, the method for determining cellulase activity of a sample according to the embodiment of the present invention can simultaneously measure CMC enzyme activity and filter paper enzyme activity, and the measured enzyme activity value is consistent with the international standard method (IU) measurement results, It is reliable and stable. According to the method for determining the cellulase activity of a sample according to the embodiment of the present invention, the reaction and color development are carried out in the same container, such as the same 96-well plate, without changing the reaction container, the operation is simple, and the amount of determination samples and reagents is small , The determination speed is fast, and a large number of samples can be determined at the same time to achieve high throughput.

In addition, according to an embodiment of the present invention, the above-mentioned method for determining the cellulase activity of a sample may also have the following additional technical features:

In one embodiment of the present invention, the reaction vessel is a 96-well plate.

In one embodiment of the present invention, the cellulase substrate is at least one selected from filter paper and a solution containing sodium carboxymethylcellulose.

In one embodiment of the present invention, the size of the filter paper is 6×10 square millimeters, and the solution containing sodium carboxymethyl cellulose contains 2% by weight of sodium carboxymethyl cellulose and 0.05M acetate buffer, the pH of which is is 4.8.

In one embodiment of the present invention, the cellulase substrate is a solution containing sodium carboxymethylcellulose, and the reaction vessel is a 96-well plate, wherein, in the reaction vessel, the sample to be tested is mixed with the cellulase substrate It further includes: firstly adding 50 microliters of the sample to be tested into at least one well of the 96-well plate, and then adding 50 microliters of the solution containing sodium carboxymethylcellulose.

In one embodiment of the present invention, the reaction vessel is a 96-well plate, and the cellulase substrate is filter paper, wherein, in the reaction vessel, mixing the sample to be tested with the cellulase substrate further includes: Add 50 microliters of the sample to be tested and 50 microliters of pH4.8, 0.05M acetate buffer in order to at least one well; and immerse the filter paper into the resulting mixed solution.

In one embodiment of the present invention, the reaction container is a 96-well plate, and placing the reaction container under conditions suitable for the action of cellulase for a first predetermined time further includes: placing the 96-well plate at 50 degrees Celsius, maintaining 20-100 minutes.

In one embodiment of the present invention, the chromogenic substrate is a solution containing 3,5-dinitrosalicylic acid.

In one embodiment of the present invention, the solution containing 3,5-dinitrosalicylic acid contains 1.4% by weight of 3,5-dinitrosalicylic acid, 0.28% by weight of phenol, 0.07% by weight of Sodium sulfite, 28% by weight sodium potassium tartrate and 1.4% by weight sodium hydroxide.

In one embodiment of the present invention, the reaction container is a 96-well plate, wherein, placing the reaction container under conditions suitable for color development for a second predetermined time further includes: placing the 96-well plate at 80-110 degrees Celsius , hold for 5 minutes.

In one embodiment of the present invention, determining the glucose content in the reaction vessel is determined by measuring the absorbance value at a wavelength of 540 nm and using a glucose standard curve.

In one embodiment of the present invention, a glucose standard curve is determined using a series of glucose solutions of known concentrations.

The method for determining the cellulase activity of a sample according to an embodiment of the present invention may have at least one of the following advantages:

1. The method for determining the cellulase activity of a sample according to the embodiment of the present invention can be applied to the determination of cellulose CMC enzyme activity and filter paper enzyme activity at the same time.

2. According to the method for determining the cellulase activity of the sample according to the embodiment of the present invention, a 96-well plate can be used as the reaction vessel, thus, a blank group can be set, and the blank group and the experimental group can be on the same 96-well plate. Operations were all performed in the same 96-well plate. There is no need to replace the reaction container, the operation is simple, the amount of samples and reagents used is small, the measurement speed is fast, and a large number of samples can be measured at the same time to achieve high throughput.

3. According to the method for determining the cellulase activity of the sample according to the embodiment of the present invention, the amount of enzyme and substrate added is appropriate, and the enzyme activity of the filter paper is measured on the same 96-well plate without dilution, and errors caused during the experimental operation Smaller, can meet the experimental conditions of most laboratories.

4. The method for determining the cellulase activity of a sample according to the embodiment of the present invention is easy to operate. The measurement speed is fast (30 minutes for CMC enzyme activity, 60 minutes for filter paper enzyme activity), and is suitable for the determination of a large number of samples, which can achieve high throughput.

5. The measured value of the method for determining the cellulase activity of the sample according to the embodiment of the present invention is consistent with the international standard, the measurement is accurate, the error is small, and the reproducibility is good.

Additional aspects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Description of drawings

The above and/or additional aspects and advantages of the present invention will become apparent and comprehensible from the description of the embodiments in conjunction with the following drawings, wherein:

Figure 1 shows the standard curve adopted in the calculation of the national standard cellulase filter paper enzyme activity;

Figure 2 shows the standard curve used in the calculation of the national standard cellulase CMC enzyme activity; and

Fig. 3 shows the standard curve used in the calculation of cellulase activity according to the embodiment of the present invention.

Detailed ways

Embodiments of the present invention are described in detail below. The embodiments described below by referring to the figures are exemplary and are intended to explain the present invention and should not be construed as limiting the present invention.

The terms "first" and "second" used herein are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features. Thus, a feature defined as "first" and "second" may explicitly or implicitly include one or more of these features. In the description of the present invention, "plurality" means two or more, unless otherwise specifically defined.

The present invention has been accomplished based on the following findings of the inventors:

The current international standard system for the determination of CMC enzyme activity contains 0.5ml enzyme, 0.5ml 2% by weight CMC, 3ml DNS and 20ml distilled water. The international standard system of filter paper enzyme activity assay method includes 0.5ml enzyme, 1ml pH4.8 0.05M sodium acetate-acetate buffer solution (in this paper, sometimes referred to as "acetate buffer solution"), 1cm × 6cm filter paper, 3ml DNS and 20ml of distilled water. For the convenience of operation, some adjustments are usually made on the basis of the above-mentioned international standard methods in specific research or technical inventions. For example, with regard to the assay method of CMC enzyme activity, some studies have used a 120 μl system in a 96-well plate, which includes 30 μl enzyme, 30 μl 2% CMC and 60 μl DNS. Regarding the assay method of enzyme activity on filter paper, some studies have used a 200 μl system in a 96-well plate, which includes 20 μl enzyme, 40 μl 0.05M acetate buffer, a filter paper disc with a diameter of 7 mm, and 120 μl DNS. , measured A ₅₄₀ value. However, when large-scale, high-throughput cellulase activity assays are required, the traditional reaction system is time-consuming and laborious.

According to an embodiment of the present invention, the present invention proposes a method for determining the cellulase activity of a sample. According to an embodiment of the present invention, the method includes the following steps:

First, in a reaction vessel, the sample to be tested is mixed with the cellulase substrate. According to the embodiments of the present invention, the type of the reaction vessel for determining the enzyme activity is not particularly limited, and can be determined according to needs. According to an embodiment of the present invention, the reaction vessel that can be used is a 96-well plate. Therefore, it can meet the needs of high-throughput determination, and can conveniently use PCR instrument as the temperature control equipment for enzymolysis reaction and color reaction, and can use microplate reader as the equipment for detecting absorbance, so as to effectively improve the determination of fiber The efficiency of the enzyme activity method. Thus, a blank group can be set, and the blank group and the experimental group can be on the same 96-well plate, and the entire operation is performed on the same 96-well plate. There is no need to replace the reaction container, the operation is simple, the amount of samples and reagents used is small, the measurement speed is fast, and a large number of samples can be measured at the same time to achieve high throughput.

According to the embodiments of the present invention, the types of cellulase substrates that can be used are not particularly limited, as long as they contain substances that can be degraded by cellulase. In one embodiment of the present invention, the cellulase substrate may be at least one selected from filter paper and a solution containing sodium carboxymethylcellulose. Thus, the filter paper enzyme activity and CMC enzyme activity can be determined. In one embodiment of the present invention, the size of the filter paper is 6×10 square millimeters, and the solution containing sodium carboxymethyl cellulose contains 2% by weight of sodium carboxymethyl cellulose and 0.05M acetate buffer, the pH of which is is 4.8. Thus, the efficiency of determining the enzyme activity of the filter paper and the enzyme activity of the CMC can be further improved. Depending on the type of cellulase substrate used, the method of mixing the sample to be tested with the cellulase substrate can be adjusted. In one embodiment of the present invention, the cellulase substrate is a solution containing sodium carboxymethylcellulose, wherein, in the reaction vessel, mixing the sample to be tested with the cellulase substrate further includes: In at least one well of the orifice plate, first add 50 microliters of the sample to be tested, and then add 50 microliters of the solution containing sodium carboxymethylcellulose. In one embodiment of the present invention, the cellulase substrate is filter paper, wherein, in the reaction vessel, mixing the sample to be tested with the cellulase substrate further includes: in at least one well of a 96-well plate, Add 50 microliters of the sample to be tested and 50 microliters of pH4.8, 0.05M acetate buffer in sequence; and immerse the filter paper into the resulting mixed solution. The inventors surprisingly found that by adopting the mixing method according to the embodiment of the present invention, the efficiency of determining cellulase activity can be effectively improved. The existing 96-well plate 120 μl system measures CMC enzyme activity, and the substrate addition amount is 30 μl 2% by weight of CMC, because of its high viscosity, it is easy to cause errors. The existing 96-well plate 200μl system measures the enzyme activity of the filter paper in proportion to the national standard. The measurement process needs to be carried out on two 96-well plates, that is, the solution after DNS color development needs to be transferred to another 96-well plate for dilution. And the reaction volume of the enzyme and the substrate is (20μl enzyme + 40μl buffer), this system can not only cause operational errors, but also after adding filter paper, the contact between the enzyme and the substrate will be insufficient due to the water absorption of the filter paper, and the final result will be imprecise. According to the method for determining the cellulase activity of the sample according to the embodiment of the present invention, the amount of enzyme and substrate added is more appropriate, and the enzyme activity of the filter paper is measured on the same 96-well plate without dilution, and the error caused during the experimental operation is even smaller. Small enough to meet the experimental conditions of most laboratories.

Next, the reaction vessel is placed under conditions suitable for the action of cellulase for a first predetermined time, so that the cellulase contained in the sample can play a role, and the cellulase substrate is decomposed to obtain glucose. According to the embodiments of the present invention, the conditions for performing the enzymatic hydrolysis reaction are not particularly limited. According to the embodiment of the present invention, the enzymatic hydrolysis reaction can be carried out at 50 degrees Celsius. In one embodiment of the present invention, placing the reaction vessel under conditions suitable for the action of cellulase for the first predetermined time further comprises: placing the 96-well plate at 50 degrees Celsius for 20-100 minutes. According to an embodiment of the present invention, different reaction times can be used for different types of cellulase substrates, for example, for the determination of CMC enzyme activity, it can be kept at 50 degrees Celsius for 30 minutes, and for the determination of filter paper enzyme activity, it can be used at 60 minutes at 50°C. Therefore, the method for determining the cellulase activity of a sample according to the embodiment of the present invention is easy to operate. The measurement speed is fast (30 minutes for CMC enzyme activity, 60 minutes for filter paper enzyme activity), and is suitable for the determination of a large number of samples, which can achieve high throughput.

Next, adding a chromogenic substrate into the reaction vessel, and placing the reaction vessel under conditions suitable for color development for a second predetermined period of time, so that the cellulase decomposition product can act on the chromogenic substrate , so that the concentration change of glucose in the reaction vessel can be determined by measuring the degree of color development, for example, by measuring the absorbance, and then the cellulase activity of the sample can be determined. According to an embodiment of the present invention, the type of chromogenic substrate that can be used is not particularly limited. According to an embodiment of the present invention, in one embodiment of the present invention, the chromogenic substrate contains 3,5- A solution of dinitrosalicylic acid. In one embodiment of the present invention, the solution containing 3,5-dinitrosalicylic acid contains 1.4% by weight of 3,5-dinitrosalicylic acid, 0.28% by weight of phenol, 0.07% by weight of Sodium sulfite, 28% by weight sodium potassium tartrate and 1.4% by weight sodium hydroxide. Thereby, the efficiency of determining cellulase activity can be further improved. In one embodiment of the present invention, the reaction container is a 96-well plate, wherein, placing the reaction container under conditions suitable for color development for a second predetermined time further includes: placing the 96-well plate At 80-110 degrees Celsius, keep for 5 minutes.

Next, the glucose content in the reaction vessel was determined. According to the embodiments of the present invention, the method for determining the glucose content produced in the reaction vessel is not particularly limited. In one embodiment of the present invention, determining the glucose content in the reaction vessel is determined by measuring the absorbance value at a wavelength of 540nm and using a glucose standard curve. Thus, the efficiency of determining the glucose content can be further improved, thereby further improving the efficiency of determining the cellulase activity.

Wherein, according to an embodiment of the present invention, the glucose standard curve can be determined according to the following method:

Prepare 1mg/ml anhydrous glucose solution (glucose is dried at 105°C for 3-4 hours in advance), take 0, 20, 40, 60, 80, 100 μl of the anhydrous glucose solution and add them to a 96-well plate, correspondingly add 100 μl of distilled water , 80, 60, 40, 20, 0 μl; then add 100 μl of the prepared DNS reagent, boil in water for 5 minutes and then cool in an ice bath; finally transfer to a microtiter plate and mix well, and measure the corresponding OD values at 540nm. Then draw a standard curve with the absorbance value A as the abscissa and the glucose content as the ordinate.

Finally, based on the glucose content, the cellulase activity of the sample was determined. Using the method according to the embodiment of the present invention, the cellulase activity of the sample can be effectively determined. According to an embodiment of the present invention, after obtaining the glucose content in the reaction vessel, the cellulase activity of the sample can be quantified according to the following formula:

$<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 1</span>}<span\; class="notranslate">\; )</span>\mathrm{<span\; class="notranslate">\; FPU</span>}<span\; class="notranslate">\; /</span>\mathrm{<span\; class="notranslate">\; ml</span>}<span\; class="notranslate">\; =</span>\mathrm{<span\; class="notranslate">\; Ymg</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 5.55</span>}\mathrm{<span\; class="notranslate">\; \&mu;mole</span>}<span\; class="notranslate">\; /</span>\mathrm{<span\; class="notranslate">\; mg</span>}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; (</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; 60</span>}\mathrm{<span\; class="notranslate">\; min</span>}}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; (</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; Xml</span>}}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; ;</span>$

$<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 2</span>}<span\; class="notranslate">\; )</span>\mathrm{<span\; class="notranslate">\; CMC</span>}<span\; class="notranslate">\; /</span>\mathrm{<span\; class="notranslate">\; ml</span>}<span\; class="notranslate">\; =</span>\mathrm{<span\; class="notranslate">\; Ymg</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 5.55</span>}\mathrm{<span\; class="notranslate">\; \&mu;mole</span>}<span\; class="notranslate">\; /</span>\mathrm{<span\; class="notranslate">\; mg</span>}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; (</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; 30</span>}\mathrm{<span\; class="notranslate">\; min</span>}}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; (</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; Xml</span>}}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; .</span>$

Wherein, when the cellulase substrate is filter paper, formula (1) is applicable. In formula (1), FPU/ml is the enzyme activity of the tested enzyme at the dilution of preparation. Ymg is the glucose mg number corresponding to the sample absorbance value A ₅₄₀ in the standard curve equation. 5.55μmole/mg is the number of μmoles in 1mg of glucose. 60min is the reaction time. X ml is the volume of enzyme solution added when measuring cellulase, ie 0.5ml.

Wherein, when the cellulase substrate is a solution containing sodium carboxymethylcellulose, formula (2) is applicable. In formula (2), CMC/ml is the enzyme activity of the tested enzyme at the dilution of preparation. Ymg is the glucose mg number corresponding to the sample absorbance value A ₅₄₀ in the standard curve equation. 5.55μmole/mg is the number of μmoles in 1mg of glucose. 30min is the reaction time. X ml is the volume of enzyme solution added when measuring cellulase, ie 0.5ml.

According to the embodiment of the present invention, the method for determining the cellulase activity of the sample according to the embodiment of the present invention is consistent with the result of measuring the enzyme activity of the same commercial enzyme with the same concentration using the international standard method (IU). Compared with other existing methods, the method for determining cellulase activity of a sample according to the embodiment of the present invention can simultaneously measure CMC enzyme activity and filter paper enzyme activity, and the measured enzyme activity value is consistent with the international standard method (IU) measurement results, It is reliable and stable. According to the method for determining the cellulase activity of a sample according to the embodiment of the present invention, the reaction and color development are carried out in the same container, such as the same 96-well plate, without changing the reaction container, the operation is simple, and the amount of determination samples and reagents is small , The determination speed is fast, and a large number of samples can be determined at the same time to achieve high throughput.

The present invention is explained below through specific examples. It should be noted that the following examples are only illustrative and do not limit the present invention in any way. In addition, all instruments, materials, etc. used in the following examples are commercially available, and operations that are not clearly indicated in the following examples can be performed by conventional operating methods for those skilled in the art.

general method

Experimental materials: Cellulase is a commercial enzyme in the laboratory; 2% by weight of CMC (prepared with pH 4.8, 0.05M acetate buffer) as a substrate; DNS formula is 1.4% by weight of 3,5-dinitrosalicyl Acid, 0.28% by weight of phenol, 0.07% by weight of sodium sulfite, 28% by weight of potassium sodium tartrate (Rochelle salt) and 1.4% by weight of sodium hydroxide, prepared one week before the experiment and stored in the dark. The detection wavelength is 540nm.

I. Preparation of standard curve

Prepare 1 mg/ml anhydrous glucose solution, dry the glucose at 105°C for 3-4 hours in advance, take 0, 20, 40, 60, 80, 100 μl of anhydrous glucose solution and add them to a 96-well plate, correspondingly add distilled water 100, 80 μl , 60, 40, 20, 0 μl; then add 100 μl of the prepared DNS reagent, boil in water for 5 minutes and then cool in an ice bath; finally transfer to a microtiter plate and mix well, and measure its corresponding OD value at 540nm. Then draw a standard curve with the absorbance value A as the abscissa and the glucose content as the ordinate. The results are shown in Figure 3. Fig. 3 shows the standard curve used in the calculation of cellulase activity in the method for determining the cellulase activity of a sample according to the present invention.

II. CMC enzyme activity

experimental method:

1. Prepare 0.05 mg/ml enzyme solution to be tested, and then add 50 μl to 12 wells of a 96-well plate, of which 6 wells are used as the experimental group, and the other 6 wells are used as the control group.

2. Add 100 μl DNS to each well of the control group, and then put the 96-well plate into a PCR instrument at 50°C for 10 minutes to inactivate the enzyme in the blank control group.

3. In the experimental group and the control group, 50 μl of 2% by weight CMC (prepared with pH 4.8, 0.05M acetate buffer) was added respectively, and the 96-well plate was placed in a PCR instrument at 50°C for 30 minutes for enzymatic hydrolysis.

4. Add 100 μl DNS to the experimental group, and perform a color reaction at 99.9°C for 5 minutes in a PCR instrument.

5. After the temperature dropped to room temperature, measure A _{540 of} 100 μl of the solution in the 96-well plate with a microplate reader.

6. Calculate the glucose content according to the glucose standard curve (see Figure 3).

III. Filter paper enzyme activity

experimental method:

1. Prepare 0.5 mg/ml enzyme solution to be tested, and then add 50 μl to 12 wells of a 96-well plate, of which 6 wells are used as the experimental group, and the other 6 wells are used as the control group.

2. Add 100 μl DNS to each well of the control group, and then put the 96-well plate into a PCR instrument at 50°C for 10 minutes to inactivate the enzyme in the blank control group.

3. In the experimental group and the control group, 50 μl of pH 4.8, 0.05M acetic acid buffer solution were added, and 6×10mm ² filter paper was added and placed in a PCR instrument at 50°C for 60 minutes for reaction.

4. Add 100 μl DNS to the experimental group, and develop the color in the PCR instrument at 99.9°C for 5 minutes.

5. After the temperature drops to room temperature, transfer 100 μl of the solution in the 96-well plate to a microplate plate, and measure A ₅₄₀ with a microplate reader.

6. Calculate the glucose content according to the glucose standard curve (see Figure 3).

IV. Enzyme activity quantification

After obtaining the glucose content in the reaction vessel, the cellulase activity of the sample can be quantified according to the following formula:

$<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 1</span>}<span\; class="notranslate">\; )</span>\mathrm{<span\; class="notranslate">\; FPU</span>}<span\; class="notranslate">\; /</span>\mathrm{<span\; class="notranslate">\; ml</span>}<span\; class="notranslate">\; =</span>\mathrm{<span\; class="notranslate">\; Ymg</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 5.55</span>}\mathrm{<span\; class="notranslate">\; \&mu;mole</span>}<span\; class="notranslate">\; /</span>\mathrm{<span\; class="notranslate">\; mg</span>}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; (</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; 60</span>}\mathrm{<span\; class="notranslate">\; min</span>}}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; (</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; Xml</span>}}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; ;</span>$

$<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 2</span>}<span\; class="notranslate">\; )</span>\mathrm{<span\; class="notranslate">\; CMC</span>}<span\; class="notranslate">\; /</span>\mathrm{<span\; class="notranslate">\; ml</span>}<span\; class="notranslate">\; =</span>\mathrm{<span\; class="notranslate">\; Ymg</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 5.55</span>}\mathrm{<span\; class="notranslate">\; \&mu;mole</span>}<span\; class="notranslate">\; /</span>\mathrm{<span\; class="notranslate">\; mg</span>}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; (</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; 30</span>}\mathrm{<span\; class="notranslate">\; min</span>}}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; (</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; Xml</span>}}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; .</span>$

Wherein, when the cellulase substrate is filter paper, formula (1) is applicable. In formula (1), FPU/ml is the enzyme activity of the tested enzyme at the dilution of preparation. Ymg is the glucose mg number corresponding to the sample absorbance value A ₅₄₀ in the standard curve equation. 5.55μmole/mg is the number of μmoles in 1mg of glucose. 60min is the reaction time. X ml is the volume of enzyme solution added when measuring cellulase, ie 0.5ml.

Wherein, when the cellulase substrate is a solution containing sodium carboxymethylcellulose, formula (2) is applicable. In formula (2), CMC/ml is the enzyme activity of the tested enzyme at the dilution of preparation. Ymg is the glucose mg number corresponding to the sample absorbance value A ₅₄₀ in the standard curve equation. 5.55μmole/mg is the number of μmoles in 1mg of glucose. 30min is the reaction time. X ml is the volume of enzyme solution added when measuring cellulase, ie 0.5ml.

V. National standard method

(1) Determination of cellulase filter paper enzyme activity by international standards

1. Method:

(1) Add 1.0ml 0.05M, pH4.8 sodium citrate into a 25ml test tube.

(2) Add 0.5ml of enzyme solution (dilute the enzyme with buffer to prepare the enzyme solution, at least 2 dilutions of the enzyme solution need to be prepared. The concentrations of the 2 dilutions of the enzyme solution are greater than and less than the catalytic substrate. 2.0 mg glucose).

(3) Heat to 50°C, add substrate filter paper (1.0×6.0cm).

(4) React for 60 minutes at 50°C.

(5) Add 3.0ml DNS and mix well. Place the test tubes in the test tube rack.

(6) Accurately heat in a boiling water bath for 5 minutes. Then cool in a cold water bath.

(7) Add 20ml of deionized water or distilled water. Invert several times to mix thoroughly.

(8) After the filter pulp is precipitated, measure the absorbance at 540 nm with a blank tube as a control.

2. Blank tube: 1.5ml buffer, 3.0ml DNS, 5min boiling water bath, 20ml water.

3. Enzyme blank (no substrate): 1.0ml buffer, 0.5ml enzyme, 3.0ml DNS, boil for 5 minutes, 20ml water.

The absorbance value A ₅₄₀ of the final sample is the value after subtracting the blank.

Standard curve preparation: 0.5ml glucose standard solution (glucose solutions with different concentration gradients are required), 1.0ml buffer solution, 3.0ml DNS, 5min boiling water bath, 20ml water. The absorbance was measured against a blank tube. Among them, Figure 1 shows the standard curve used in the calculation of the enzyme activity of the national standard cellulase filter paper.

calculate:

$\mathrm{<span\; class="notranslate">\; FPU</span>}<span\; class="notranslate">\; /</span>\mathrm{<span\; class="notranslate">\; ml</span>}<span\; class="notranslate">\; =</span>\mathrm{<span\; class="notranslate">\; Ymg</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 5.55</span>}\mathrm{<span\; class="notranslate">\; \&mu;mole</span>}<span\; class="notranslate">\; /</span>\mathrm{<span\; class="notranslate">\; mg</span>}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; (</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; 60</span>}\mathrm{<span\; class="notranslate">\; min</span>}}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; (</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; Xml</span>}}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; ,</span>$

Where: FPU/ml is the enzyme activity of the tested enzyme at the dilution of preparation. Ymg is the glucose mg number corresponding to the sample absorbance value A ₅₄₀ in the standard curve equation. 5.55μmole/mg is the number of μmoles in 1mg of glucose. 60min is the reaction time. X ml is the volume of enzyme solution added when measuring cellulase, ie 0.5ml.

(2) Determination of cellulase CMC enzyme activity by international standards

1. Method:

(1) Add 0.5ml of enzyme solution into a 25ml test tube (dilute the enzyme with buffer to prepare the enzyme solution, at least 2 dilutions of the enzyme solution need to be prepared. The concentrations of the 2 dilutions of the enzyme solution are greater than and less than After catalyzing the substrate, 0.5 mg of glucose is generated).

(2) Heat to 50°C.

(3) Add 0.5ml CMC solution (use pH4.8, 0.05M sodium citrate buffer to prepare 2% CMC solution). After mixing well, react at 50°C for 30 minutes.

(4) Add 3.0ml DNS and mix well. Place the test tubes in the test tube rack.

(5) Accurately heat in a boiling water bath for 5 minutes. Then cool in a cold water bath.

(6) Add 20ml of deionized water or distilled water. Invert several times to mix thoroughly.

(7) Measure the absorbance at 540nm with a blank tube as a control.

(8) Calculate the glucose content according to the glucose standard curve, and then convert it into enzyme activity.

2. Blank tube: 0.5ml substrate (2% CMC solution), 30min, 50°C, 3.0ml DNS, 0.5ml buffer, mixed boiling water bath for 5.0min, 20ml H ₂ O

3. Enzyme blank: 0.5ml substrate (2% CMC solution), 30min, 50°C, 3.0ml DNS, 0.5ml buffer, boiling water bath for 5.0min, 20ml H ₂ O

The absorbance value A ₅₄₀ of the final sample is the value after subtracting the blank.

4. Standard curve: 0.5ml substrate, 30min, 50℃, 3.0ml DNS, 0.5ml glucose standard solution (glucose standard solution with different concentration gradient is required), 5min hot water bath, 20ml water. The absorbance was measured against a blank tube. Among them, Figure 2 shows the standard curve used in the calculation of the national standard cellulase CMC enzyme activity.

calculate:

$\mathrm{<span\; class="notranslate">\; CMC</span>}<span\; class="notranslate">\; /</span>\mathrm{<span\; class="notranslate">\; ml</span>}<span\; class="notranslate">\; =</span>\mathrm{<span\; class="notranslate">\; Ymg</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 5.55</span>}\mathrm{<span\; class="notranslate">\; \&mu;mole</span>}<span\; class="notranslate">\; /</span>\mathrm{<span\; class="notranslate">\; mg</span>}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; (</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; 30</span>}\mathrm{<span\; class="notranslate">\; min</span>}}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; (</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; Xml</span>}}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; .</span>$

Where: CMC/ml is the enzyme activity of the tested enzyme at the dilution of preparation. Ymg is the glucose mg number corresponding to the sample absorbance value A ₅₄₀ in the standard curve equation. 5.55μmole/mg is the number of μmoles in 1mg of glucose. 30min is the reaction time. Xml is the volume of enzyme solution added when measuring cellulase, ie 0.5ml.

Example 1

The commercial enzyme CELLMLASE "ONOZUKA" R-10CMC enzyme activity was measured using the method of the present invention and international standards (see the aforementioned general method for specific steps). The measurement results are shown in Table 1 and Table 4. It can be seen from Tables 1 and 4 that the method of the present invention (200 μl system) and the international standard system have the same variance and no significant difference in the measured values of enzyme activity (see Tables 2, 3 and 5, 6 for statistical analysis results). The results show that the two sets of data have the same consistency, and the method can accurately measure the CMC enzyme activity, and the repeatability and stability are good.

Table 1 Comparison of CMC enzyme activity assay results of two methods

Table 2 Statistical test results of the data in Table 1 1

Table 3 Statistical test results of the data in Table 1 2

Table 4 Comparison of enzyme activity data of commercial enzyme filter paper measured by this method and international standards

Table 5 Statistical test results of table 4 data 1

Table 6 Table 4 Data Statistical Test Results 2

Table 2, 3 and 5, 6 F test, T-test results prove that the method of the present invention and the international standard system filter paper enzymatic assay value variance is homogeneous and difference is not significant. Illustrate that two groups of data have equivalent consistency, adopt the method of the present invention to accurately measure CMC enzymatic activity and filter paper enzymatic activity.

In the description of this specification, descriptions referring to the terms "one embodiment", "some embodiments", "example", "specific examples", or "some examples" mean that specific features described in connection with the embodiment or example , structure, material or characteristic is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be construed as limitations to the present invention. Variations, modifications, substitutions, and modifications to the above-described embodiments are possible within the scope of the present invention.

Claims (10)

1. A method for determining sample cellulase activity, comprising: In the reaction vessel, the sample to be tested is mixed with the cellulase substrate; subjecting the reaction vessel to conditions suitable for the action of the cellulase for a first predetermined period of time; adding a chromogenic substrate to the reaction vessel, and subjecting the reaction vessel to conditions suitable for color development for a second predetermined period of time; determining the glucose content in the reaction vessel; and Based on the glucose content, the cellulase activity of the sample is determined. 2. The method according to claim 1, wherein the reaction vessel is a 96-well plate. 3. The method according to claim 2, characterized in that, the cellulase substrate is at least one selected from filter paper and a solution containing sodium carboxymethyl cellulose. 4. The method of claim 3, wherein, The size of the filter paper is 6×10 square millimeters, The solution containing sodium carboxymethyl cellulose contains 2% by weight sodium carboxymethyl cellulose and 0.05M acetic acid buffer, and its pH value is 4.8. 5. method according to claim 4, is characterized in that, described cellulase substrate is the solution containing sodium carboxymethyl cellulose, Wherein, in the reaction vessel, mixing the sample to be tested with the cellulase substrate further includes: In at least one well of the 96-well plate, first add 50 microliters of the sample to be tested, and then add 50 microliters of the solution containing sodium carboxymethylcellulose. 6. method according to claim 4, is characterized in that, described cellulase substrate is filter paper, Wherein, in the reaction vessel, mixing the sample to be tested with the cellulase substrate further includes: In at least one well of a 96-well plate, sequentially add 50 microliters of the sample to be tested and 50 microliters of pH4.8, 0.05M acetate buffer; and The filter paper was immersed in the resulting mixed solution. 7. The method according to claim 2, wherein placing the reaction vessel under conditions suitable for the action of cellulase for a first predetermined time further comprises: The 96-well plate was placed at 50°C for 20-100 minutes. 8. The method according to claim 1, wherein the chromogenic substrate is a solution containing 3,5-dinitrosalicylic acid. 9. The method according to claim 8, wherein the solution containing 3,5-dinitrosalicylic acid contains 1.4% by weight of 3,5-dinitrosalicylic acid, 0.28% by weight of Phenol, 0.07% by weight sodium sulfite, 28% by weight sodium potassium tartrate and 1.4% by weight sodium hydroxide. 10. The method according to claim 9, wherein the reaction vessel is a 96-orifice plate, wherein, Exposing the reaction vessel to conditions suitable for color development for a second predetermined time further comprises: Place the 96-well plate at 80-110 degrees Celsius for 5 minutes, Optionally, determining the glucose content in the reaction vessel is determined by measuring the absorbance at a wavelength of 540 nm and using a glucose standard curve, Optionally, the glucose standard curve is determined using a series of glucose solutions of known concentrations.

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