Team:SZU-China/Measurement

In order to detect the enzymatic activity of cellulase CL34 and IARI-SP-2, we used DNS oxidation method to extract cellulase from bacteria. We optimized the protocol to suit our experiments.

Improvement

We replaced the buffer from the acetic acid-sodium acetate (pH = 5) and the Tris-HCl (pH = 8.8) to PBS (pH = 7). And we increased the reaction system from 150μl to 1.4ml, changed the reaction container from 1ml ep test tube to 15ml test tube.

Experimental program

1.Reagents and solutions

Glucose solution 10.0mg/ml
1g anhydrous glucose solid, add water to make up to 100ml
PBS
10 times dilution
Carboxymethyl Cellulose Sodium solution 0.8%
0.8g Carboxymethyl Cellulose Sodium, add 80ml PBS, heat and stir to dissolve, stop heating and continue stirring for 30mins, then add PBS to make the volume to 100ml. Shake well before using it.
Enzyme solution
DNS solution
18.2g of seignette salt, dissolve in 50ml of distilled water and heat, add 0.03g of 3,5-Dinitrosalicylic acid, 2.1g of NaOH and 0.5g of phenol to the hot solution, stir to dissolve, then cool and dilute with distilled water to 100ml and store in a brown bottle.

2.Protocol

Cell disruption
(1)Take 10ml of the bacterial solution in a 15 ml ep tube, add it to liquid nitrogen and cool it until the bacterial solution freezes. Then immediately add it to 37℃ warm water to melt. After the melt is complete, put it in liquid nitrogen. Repeat the operation 5 times in a row. Pay attention to the state of the ep tube during repeated freezing and thawing to prevent it from cracking.
(2)After freezing and thawing, paut it in a centrifuge and spin at 10000 speed for 1 min.
(3)After centrifugation, carefully pipette the supernatant into a new tube, and discard the bottom sediment. And store the supernatant in the refrigerator at -20℃.

Preparation of standard curve
(1)Prepare blank sample: 0.4ml PBS buffer solution, 0.4ml DNS, heating in a boiling water bath for 5 minutes, cooling the water bath to room temperature, adding water to make the volume up to 4ml
(2)Prepare glucose standard solution: take 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7ml glucose solution respectively. The buffer solution is adjusted to 10ml as standard solution.
(3)Take 0.4 ml of the above standard solution, add 0.4 ml of water and 0.4 ml of DNS respectively. (Electromagnetic) oscillate for 3 seconds, heat in a boiling water bath for 10 minutes, and cool the water bath to room temperature. Add water and make up to 4ml.
(4)The y-axis of od540 is the glucose concentration, and the absorbance OD is the x-axis.

Enzyme activity determination
(1)Equilibrate the carboxymethyl cellulose sodium solution and enzyme solution in a 37°C water bath for 10 minutes.
(2)Take 0.4ml of the above enzyme solution in a test tube and heat it with boiling water for 10 minutes. Then add 0.4ml of the above carboxymethyl cellulose sodium solution. Electromagnetic shock for 3s to mix. Incubate at 37°C for 30 min to prepare saccharification liquid. Add 0.4ml DNS solution to the saccharification solution and heat it with boiling water for 10 minutes. Then the water bath was cooled to room temperature. Add water to the volume to 4ml, and then (electromagnetic) oscillate. Measure od540 and mark it as AB.
(3)Take 0.4ml of the above enzyme solution and test tube, add 0.4ml of the above carboxymethyl cellulose sodium solution. Electromagnetic shock for 3s to mix. Incubate at 37°C for 30 min to prepare saccharification liquid. Add 0.4ml DNS solution to the saccharification solution and heat it with boiling water for 10 minutes. After cooling the water bath to room temperature, add water to make the volume up to 4ml, and then (electromagnetic) shake. Measure od540 and record it as AD.
(4)According to the definition of enzyme activity: unit cellulase activity concentration (U/ml) is equal to the amount of 1μg reducing sugar produced per minute in 1ml reaction system under the action of enzyme. Calculate enzyme activity.

Experimental results

We drew the standard curve of the glucose solution according to the protocol, and drew the enzyme activity temperature curves for the two enzymes we used as follows.

Before changing the buffer system, the enzyme activity we measured was very small. It is 0.1 U/ml level of enzyme activity. When the reaction vessel is a 1ml ep tube, we cannot detect the enzyme activity for the same enzyme solution. After changing the test tube, considerable enzyme activity was measured. Among them, at the optimum temperature, the enzyme activity concentration of CL34 is 29.65138U/ml, and the enzyme activity concentration of IARI-SP-2 is 28.6964U/ml.