To learn and understand the principles and methods of the 3,5-dinitrosalicylic acid (DNS) method for the determination of cellulase activity, and to understand the action characteristics of cellulase.
Principle
The basic principle of cellulase activity measurement is that cellulase is a multi-component enzyme, including three main components: 0 enzyme, Cx enzyme and glucosidase. The G enzyme hydrolyzes natural cellulose to amorphous cellulose, the Cx enzyme hydrolyzes amorphous cellulose to oligosaccharides, and the glucosidase enzyme hydrolyzes oligosaccharides to glucose. Cellulase hydrolyzes cellulose to produce cellobiose, glucose and other reducing sugars that can reduce 3,5-dinitrosalicylic acid (DNS) under alkaline conditions to produce brown-red amino compounds with maximum light absorption at 540 nm, and the amount of reducing sugars is proportional to the color strength of the reaction solution within a certain range, so that the cellulase enzyme can be used to determine its activity by measuring the amount of reducing sugars produced using the colorimetric method. The activity of cellulase can be determined by measuring the amount of reducing sugar produced using colorimetric method.
Operation method
Determination of cellulase activity
Principle
The basic principle of cellulase activity measurement is that cellulase is a multi-component enzyme, including three main components: 0 enzyme, Cx enzyme and glucosidase. The G enzyme hydrolyzes natural cellulose to amorphous cellulose, the Cx enzyme hydrolyzes amorphous cellulose to oligosaccharides, and the glucosidase enzyme hydrolyzes oligosaccharides to glucose. Cellulase hydrolyzes cellulose to produce cellobiose, glucose and other reducing sugars that can reduce 3,5-dinitrosalicylic acid (DNS) under alkaline conditions to produce brown-red amino compounds with maximum light absorption at 540 nm, and the amount of reducing sugars is proportional to the color strength of the reaction solution within a certain range, so that the cellulase enzyme can be used to determine its activity by measuring the amount of reducing sugars produced using the colorimetric method. The activity of cellulase can be determined by measuring the amount of reducing sugar produced using colorimetric method.
Materials and Instruments
Materials: Xinhua quantitative filter paper, skimmed cotton, sodium stearylmethylcellulose, salicylic acid Xie. Move The basic procedure for the determination of cellulase activity can be divided into the following steps:1. Preparation of glucose standard curve: Take 8 washed and dried 20 mL stoppered graduated test tubes, number them and add standard glucose solution and evaporated water according to Table 50-1 to make a series of glucose solutions of different concentrations. After shaking well, add 1.5 mL of DNS solution to each test tube, shake well and boil for 5 min, remove and cool, then reconstitute to 20 mL with evaporated water and mix well. Under the wavelength of 540 nm, use the No.1 tube as a blank control, adjust the zero point, determine the optical density value of other tubes and record the results. The glucose standard curve was plotted on the coordinate paper with the glucose content (mg) as the horizontal coordinate and the corresponding optical density value as the vertical coordinate.Table 50-1 Glucose Standard Curve Spiking Chart （1） Preparation of a glucose standard curve (Table 50-2):Table 50-2 Standard Curve Preparation Spiking Table Caveat 1. When preparing DNS solution, add NaOH solution containing DNS into hot water solution containing potassium sodium tartrate, pour slowly and stir while pouring to prevent being scalded. 2. The concentration of cellulase solution can be adjusted according to the vitality of different enzyme preparations. If the enzyme vigor is high, the enzyme concentration can be smaller; on the contrary, if the enzyme vigor is low, the enzyme concentration should be larger.3 . When measuring the enzyme activity, the substrate such as filter paper strips and skimmed cotton must be fully immersed in the reaction solution. For more product details, please visit Aladdin Scientific website.
Equipments: Visible spectrophotometer, thermostatic water bath, boiling water bath, electric stove, scissors, analytical balance, thermostatic drying oven, refrigerator, test-tube racks, rubber-tipped burette, 20 mL stoppered graduated test tubes, 0.5 mL, 2 mL pipettes or pipettes with liquid dispenser, 100 mL, 1,000 mL volumetric flasks, 50 mL, 100 mL, 500 mL measuring cylinders, 100 mL, 500 mL, 1,000 mL beakers. 100 mL, 500 mL, 1,000 mL beaker.
Reagents:
(1) Glucose standard solution with concentration of 1 mg - mL-1: Dry glucose at 105°C in a constant temperature drying oven until constant weight, weigh 100 mg accurately in a 100 mL beaker, dissolve it with a small amount of distilled water, and then transfer it into a 100 mL volumetric flask and dilute it with distilled water until it reaches 100 mL, and then mix it thoroughly. Store in the refrigerator at 4°C (available for 12~15 d).
(2) 3,5-Z1 nitrosalicylic acid (DNS) solution: accurately weigh 6.3 g of DNS in a 500 mL beaker, dissolve it in a small amount of evaporated water, add 2 mol - L_1NaOH solution 262 mL, and then add it to 500 mL of potassium sodium tartrate (C) solution, which contains 185 g of DNS.
4
HtO
6
KNa - 4 H
2
O, relative density 282.22) in hot aqueous solution, add 5 g of crystallized phenol (C
6
H
5 g crystallized phenol (C 6 H
OH, relative density 94.11) and 5 g of anhydrous sodium sulfite (Na
2
SO
3 , relative density 126.04), stirred and dissolved.
, relative density 126.04), stirred to dissolve, cooled and transferred into a 1 000 mL volumetric flask, and then diluted to 1 000 mL with evaporated water, and mixed thoroughly. Store in a brown bottle at room temperature for 1 week before use.
(3) 0.05 mol・L_1 pH 4.5 citrate buffer:
Liquid A (0.1 mol・L "1 citric acid solution): accurately weigh C
6
H
8
O
7
-'s 0 (relative density of 210. 14) 21.014 g in a 500 mL beaker, with a small amount of distilled water dissolved, transferred to 1 000 mL volumetric flask and fixed to 1 000 mL with distilled water, mix well. 4 ° C refrigerator to keep spare.
Liquid B (0.1 mol - L "1 sodium citrate solution): weigh accurately Na
0.1 mol - L "1 sodium citrate solution: accurately weigh Na
Liquid B (0.1 mol - L "1 sodium citrate solution): accurately weigh Na
6
H
5
O
7
- 2 H
2
O (relative density 294.12) 29.412 g in a 500 mL beaker, dissolved with a small amount of distilled water, transferred to a 1 000 mL volumetric flask, and then diluted to 1 000 mL with distilled water, and mixed thoroughly. Store in the refrigerator at 4°C. Take 27.12 mL of liquid A and 22.88 mL of liquid B, mix well, then transfer them into a 100 mL volumetric flask, and then dilute to 100 mL with evaporated water, mix well, that is, the citric acid buffer solution of 0.05 mol・L^pH 4.5. Keep it in the refrigerator at 4 笆 for the determination of the enzyme activity on filter paper.
(4) 0.05 mol - L 'pH 5.0 citrate buffer: take 20.5 mL of the above liquid A, 29.5 mL of liquid B, mix well and transfer to 100 mL volumetric flask with distilled water to 100 m", mix well. The solution is 0.05 mol - L 1 pH 5.0 citrate buffer. Store in the refrigerator at 4°C for the determination of G-enzyme activity.
(5) 0.51% sodium cunning methyl cellulose (CMC) solution: weigh 0.51 g CMC in a 100 mL beaker, add 0.05 mol - L l pH 5.0 citrate buffer, heat to dissolve and transfer to a 100 mL volumetric flask, and 0.05 mol - L ~ l pH 5.0 citrate buffer to 100 mL, shake well before use. Store in the refrigerator at 4°C for the determination of Cx enzyme activity.
(6) 0.5% salicylic acid solution: Accurately weigh 0.5 g of salicylic acid in a 100 mL beaker. Dissolve with a small amount of 0.05 mol - L'1 pH 4.5 citrate buffer, transfer to a 100 mL volumetric flask, and then dilute to 100 mL with 0.05 mol - L'1 pH 4.5 citrate buffer, mix well. Store in the refrigerator at 4°C for the determination of glucosidase activity.
(7) Preparation of cellulase solution: Weigh 500 mg of cellulase preparation accurately in a 100 mL beaker, dissolve it with a little amount of spiked water - transfer it to a 100 mL volumetric flask, and then make it into 100 mL by constant volume of spiked water, and then keep it in a refrigerator at 4°C for spare use.
2. Determination of enzyme activity of filter paper: Take four washed and dried 20 mL stoppered graduated test tubes, number them and add 0.5 mL of enzyme solution and 1.5 mL of 0.05 mol - L !pH 4.5 citrate buffer to each of them, and add 1.5 mL of DNS solution into test tube No. 1 to passivate the enzyme activity, which will serve as a blank control, and be used for adjusting the zero in the colorimetric analysis. Preheat the four test tubes in a 50°C water bath for 5~10 min, then add 50 mg of filter paper (Xinhua quantitative filter paper, about 1 cm X 6 cm) into each tube, keep warm in the 50°C water bath for 1 h, then take out and immediately add 1.5 mL of DNS solution into each of the test tubes No.2, No.3, and No.4 to terminate the reaction of the enzyme, after shaking well and then boil the water bath for 5 min, then take out and cool down, and then make it volume with evaporated pyrule water to 20 mL, mix it well. Mix well. The zero point was adjusted with the No.1 tube solution as blank control, and the enzyme was measured at 540 nm.The optical density of No.2, No.3 and No.4 test tubes was measured at 540 nm and the results were recorded.According to the average value of the optical density of 3 replicates, the corresponding glucose content was calculated on the standard curve, and the activity of the enzyme was calculated according to the following formula (U - g_1 - min-1). Under the above conditions, the amount of enzyme required to produce 1 卩 mol of glucose from the substrate per hour is defined as one unit of enzyme activity (U).3. Determination of G enzyme activity: Dilute 5 mg-mL "1 of the original enzyme solution 10-15 times and use skimmed cotton as the substrate for the determination of G enzyme activity.Four washed and dried 20 mL stoppered graduated test tubes were numbered and filled with 50 mg of cotton wool, 1.5 mL of 0.05 mol-IZ1 pH 5.0 citrate buffer, and 1.5 mL of DNS solution was added to test tube No. 1 to passivate the enzyme activity as a blank control and for adjusting the zero in the colorimetric analysis. Preheat the 4 test tubes at 45°C for 5~10 min, then add 0.5 mL of diluted enzyme solution into each tube, keep warm at 45°C for 24 h. Immediately after removing the tubes, add 1.5 mL of DNS solution into each of test tubes No.2, No.3, and No.4 to terminate the enzyme reaction, shaking well and then boil for 5 min, then remove the tubes and cool down, and then dilute the enzyme solution to 20 mL with evaporated water, and then mix well. Take the No.1 test tube solution as a blank control to adjust the zero point, and measure the optical density of the No.2, 3 and 4 test tubes under the wavelength of 540 nm and record the results.Based on the average of the three replicates of optical density, the corresponding glucose content was plotted on the standard curve, and the enzyme activity (U - g 1 - min-^o) was calculated according to the following formula The amount of enzyme required to produce 1 Mmol of glucose from the substrate under the above conditions for 24 h is defined as a unit of enzyme activity (U).4. Determination of Cx enzyme activity: 5 mg-mL-1 of the original enzyme solution was diluted 5-fold for the determination of Cx enzyme activity, and CMC was used as the substrate.Four washed and dried 20 mL stoppered test tubes were numbered and added with 1.5 mL of 0.51% CMC citrate buffer, and 1.5 mL of DNS solution was added to test tube 1 to passivate the enzyme activity, which served as a blank control and was used for adjusting the zero during the colorimetric analysis. Preheat all 4 test tubes at 50°C for 5~10 min, add 0.5 mL of diluted enzyme solution into each tube, keep warm at 50°C for 30 min, immediately add 1.5 mL of DNS solution into each of test tubes No.2.3.4 to terminate the enzyme reaction, shake well and boil for 5 min, remove from the test tubes and cool down, and then make it into 20 mL of evaporated memory water, and then mix it well. Take No.1 tube as a blank control to adjust the zero point, and measure the optical density of No.2, 3 and 4 tubes at 540 nm and record the results.Based on the average value of the optical density of three replicates, the corresponding glucose content was found on the standard curve, and the Cx enzyme activity (U - gf・mirT]) was calculated according to the following formula. Under the above conditions, the amount of enzyme required to produce 1 卩 mol of glucose from the substrate per hour is defined as one enzyme activity unit (U).5.0 Determination of glucoamylase activity: Take 4 washed and dried 20 mL graduated test tubes, add 1.5 mL of 0.5% salicylic acid and citric acid buffer to each of them after numbering, and add 1.5 mL of DNS solution to test tube No. 1 to passivate the enzyme activity, which will serve as a blank control for adjusting the zero during the colorimetric analysis. Preheat the four test tubes in a 50°C water bath for 5~10 min, add 0.5 mL of enzyme solution to each tube and keep warm at 50°C for 30 min. Immediately after removing the tubes, add 1.5 mL of DNS solution to each of test tubes No.2, No.3 and No.4 to terminate the enzyme reaction. Shake well and boil for 5 min, remove from the test tubes and cool down, and then reconstitute the enzyme solution in evaporated water to 20 mL, and then mix well. Take the No.1 test tube as a blank control to adjust the zero point, and measure the optical density of No.2, 3 and 4 test tubes at 540 nm and record the results.According to the average value of optical density of 3 replicates, the corresponding glucose content was found on the standard curve, and the /? - Glucose shikimate enzyme activity (U - gT - min^). The amount of enzyme required to produce 1 Mmol of glucose from the substrate per hour under the above conditions is defined as one unit of enzyme activity (U).6 . Calculation of results： Tube number Reagent 1 2 3 4 5 6 7 8 Glucose standard solution/mL 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Convulsive water/ mL 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 Glucose content/mg 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
According to the values in the table, the glucose standard curve was plotted on the coordinate paper with the glucose content (mg) as the horizontal coordinate and the corresponding optical density value as the vertical coordinate.(2) Calculate the enzyme activity of filter paper and fill in Table 50-3:Table 50-3: Measurement Data of Filter Paper Acyl Activity Item Tube number 1 2 3 4 5 6 7 8 Glucose content/mg 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Optical density (ODWnni) 0
Filter paper enzyme activity (U - gf・min-l) =Glucose content (mg) X Total volume of enzyme solution fixed (mL) X 5.56 /molVolume of enzyme solution added to reaction solution (mL) X Sample weight (g) X Time (h)Glucose content (mg) X 100 mL X 5. 56 ptmol0. 5 mL X 0. 5 gX 1 hWhere: 5. 56-1 mg The amount of glucose (1,000/180 = 5. 56) plants mol.(3) C] Calculate the results of enzyme activity measurement in Table 50-4;TABLE 50-4 G Data Sheet for Determination of Enzyme Activity Item Tube number Mean value of three tubes 1 2 3 4 Optical density (OD raging) 0 Glucose content/mg 0
G Enzyme activity (U - g 1 - min~1) =Glucose content (mg) X total volume of enzyme solution fixed (mL) X dilution times X 5. 56 卩 mol X 24 h - Amount of enzyme solution added to the reaction solution (mL) X sample weight (g) X time (h)-Glucose content (mg) X 100 mL X dilution times X 5. 56 卩 mol X 24 h0. 5 mL X 0. 5 g X 24 hWhere: 24 - 24 h in the definition of enzyme activity.(4) Calculate the Cx enzyme activity in Table 50-5:Table 50-5G Data sheet for determination of enzyme activity Item Tube number Average of three tubes 1 2 3 4 Optical Density (OD5Wum) 0 Glucose content/mg 0
Cx Enzyme activity (u・ gf - min^) =Glucose content (mg) X total volume of enzyme solution fixed (mL) X dilution factor X 5. 56 /imolVolume of enzyme solution added to reaction solution (mL) X Sample weight (g) X Time (h)-Glucose content (mg) X 100 mL X 5 (times) 乂 5. 56 〃mol0. 5 mL X 0. 5 g X 0. 5 h(5) Calculate the results of the 0-glucose xylase enzyme activity and fill in Table 50-6:Table 50-6 . Data table for the determination of 0-glucosidase activityItem Tube number Average of three tubes 1 2 3 4 Optical density (OD540nm) 0 Glucose content/mg 0
Parent glucose xylase activity (U - g [= 0. 5 mL X 0. 5 gX 0. 5 h) Item Tube number Average of three tubes 1 2 3 4 Optical density (OD5.,0nra) 0 Glucose content/mg 0