Potato polyphenol oxidase preparation and property experiments.

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< "color: rgb (70,135,221); "> I, experimental purposes

1. Learn how to prepare enzymes fromcells.

2. To understand the role of polyphenol oxidase and the effects of various factors on its effects.

II, Experimental Principle

Polyphenol oxidase is a copper-containing enzyme with a maximum pH of 6 to 7. Reactions catalyzed by polyphenol oxidases, such as phthalates as a substrate, can be oxidized to form phthalates. The redox reaction, catalyzed by polyphenol oxidase, can be identified by changes in the color of the solution, which is common in nature, such as peeled potatoes and fruits turning brown as a result of the enzyme's action.

the most appropriate substrate for polyphenol oxidase is phthalates (child tea phenols). Interphenols and paraphenols have similar structures to phthalates, and they can also be oxidized into a variety of colored substances.

ase is a biocatalyst whose catalytic activity is susceptible to a variety of factors, such as temperature, pH, substrate species, substrate concentration, enzyme concentration, as well as inhibitors andprotein denaturation agents will change their biocatalytic activity.

III, experimental equipment

1.

2 . Knife, gauze, < a href> "> funnel, other glassware

3.. Centrifuges

4.refrigerator

5.. Constant temperaturewater bath

4, material andreagent

1.potato

2.0.1mol/L NaF solution: 4.2g of sodium fluoride dissolved in 1000L.

3.0.01mol/L phthalates solution: 1.1g phthalates dissolved in 1000mL water, with a pH of 6.0 for thin NaOH-regulated solutions to prevent their own oxidation. When the solution turns brown, it should be rearranged. The newly made solution should be stored in a brown bottle.

4.pH6.8 phosphate buffer

5. 5. 5% tricloacetic acid solution

6. Thiophenyl

7.01mol/L interphenol solution: 0.11g interphenol dissolved in 100mL water.

8.0.01mol/L solution: 0.11 g of paraphenol dissolved in 100mL water.

9. Solid ammonium sulfate

10.0.8% HCl: 19.2mL thick HCl water diluted to 1000mL.

11.0.2% and 0.3% (V/V) lactic acid solutions.

12. 0.5% sodium carbonate solution

13.0.01% sodium carbonate solution

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); 1. Preparation of polyphenol oxidase

Together with each of the three groups, 150g potatoes (new potatoes can not be peeled), cut and put into a homogenizer, add 150mNaF solution, homogenously use four layers of gauze.

each group measured 50mL filter liquid in centrifugal tubes, centrifuged at 3500 rpm 5 to 10min, took the night, added solid ammonium sulfate 16g, dissolved, placed at 4 degrees C 30min, centrifuged at 3500 rpm 15min, poured out the liquid, precipitated with 15mL pH6.8 phosphate buffer dissolved, that is, coarse enzyme solution, containing potato polyphenol oxidase.

catalytic effect of polyphenol oxidase

each reagent is added to table 1 to observe the reaction phenomenon and record and analyze the cause.

1 Catalytic effect of polyphenol oxidase