First, the principle and method
PPO catalyzes the oxidation of various phenols and O2 to hydrazine. In this experiment, catechol was used as a substrate. In a reaction system of 0.2 M disodium hydrogen phosphate-0.1 M citrate buffer pH 5.8, PPO catalyzed the formation of brown quinone in catechol, in a spectrophotometer 410 nm. The OD value of the reaction system is changed, and the enzyme activity of the PPO is determined by the change in the reading of the OD value.
The method comprises the following steps: adding 0.05 ml of a 0.05 M catechol solution to a 0.2 M sodium hydrogen phosphate-0.1 M citrate buffer tube containing 4 ml of pH 5.8, preheating in a constant temperature water bath at 30 ° C, and adding 0.05 ml of the enzyme solution. The reaction was carried out for 5 minutes, and the absorbance was read at 410 nm of a spectrophotometer. Under the above conditions, an AOD increase of 0.01 OD per minute is defined as an enzyme activity unit (U).
Second, instruments and reagents
(1) Sample:
Polyphenol oxidase crude enzyme solution
Ammonium sulfate precipitation component
DE-52 elution component
Dextran gel elution component
(2) Substrate:
Catechol: O.01M catechol solution
(3) Reaction system:
0.2M sodium dihydrogenate-0.1M citrate buffer pH 5.8
(4) Vessels and instruments:
Test tube, constant temperature water bath, etc.
Spectrophotometer
Third, the specific activity of the enzyme protein
Specific activity = enzyme unit (U) / mg protein
Fourth, experimental results and analysis
1. Relative concentration (OD590) and relative enzyme activity (OD410) of the eluted fraction of Sephadex G-200
Result list
Test tube number protein concentration PPO enzyme live tube number protein concentration PPO enzyme activity
Blank 0 0 3 0.16 0.09
Crude enzyme solution 0.20 0.14 4 0.03 0.03
1 0.00 0.01 5 0.03 0.01
2 0.13 0.09 6 0.02 0.02
Analysis: 2 and 3 tubes contain most of the desired protein and are stored for further purification.
2. Relative concentration (OD590) and relative enzyme activity (OD410) of the eluted fraction of DE-52
Result list
Test tube number protein concentration PPO enzyme live tube number protein concentration PPO enzyme activity
Blank 0 0 3 0.03 0.05
Crude enzyme solution 0.22 0.16 4 0.09 0.05
1 0.00 0.00 5 0.07 0.02
2 0.03 0.02 6 0.03 0.00
Analysis: The activity of the enzyme is not necessarily proportional to the protein content, and the 3 and 4 tubes contain more protein.
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