Application of 4-sulfonated calix [n] arene/CTAB complex system in improving activity of polyphenol oxidase

[0023] (1) Preparation of crude enzyme solution

[0024] Pineapple crude extract: peel the pineapple and cut into pieces, weigh 24.3158g, add 24mL of ethylenediaminetetraacetic acid disodium (EDTA·2Na)-containing phosphate buffer solution (40mM, pH6.0), squeeze it with a juicer The juice is 3-5 minutes, and then filtered with gauze. The filtrate is centrifuged in a high-speed refrigerated centrifuge with a rotating speed of 10000 rpm and a temperature of 4°C for 25 minutes, and the supernatant obtained after separation is stored at -20°C.

[0025] Crude apple extract: Wash the apples in clean water, drain them, and freeze them at -20°C for 12 hours. Cut 58.82g of the frozen apple, add 100mL of phosphate buffer solution (0.2M pH=6.5, containing 0.25% (v/v) TritonX-100 and 2% (w/w) polyvinylpyrrolidone), mix and beat for 1 min, Then it was filtered with gauze, the filtrate was centrifuged in a high-speed refrigerated centrifuge with a rotating speed of 10000 rpm and a temperature of 4°C for 10 minutes, and the supernatant obtained by separation was stored at -20°C.

[0026] (2) Preparation of substrate solution

[0027] Catechol:

[0028] Prepare 300 mM catechol solution in 100 mM phosphate buffer solution at pH 7.0.

[0029] (3) Preparation of surfactant mother liquor

[0030] Prepare a 2mg/mL CTAB solution in water.

[0031] (4) Preparation of calixarene mother liquor

[0032] Prepare 4mg/mLSC[n] solution in water.

[0033] (5) Enzyme activity determination (UV spectrophotometer)

[0034] Polyphenol oxidase

[0035] Using catechol as the substrate, the enzyme activity of polyphenol oxidase in the crude pineapple extract was tested. In the test system, the concentration of catechol is 150mM, and the test wavelength is 420nm. Plot the absorbance value (A) at 420nm with time (t), calculate the slope of the linear part of the figure, and combine the molar absorption coefficient (1100M -1 cm -1 ), the initial reaction rate ν(μM·s -1 ). The enzyme activity without any additives is defined as 100%.

[0036] Example 1

[0037] The effect of SC[4]/CTAB compound system on the activity of polyphenol oxidase in crude pineapple extract:

[0038] The experiment was divided into two groups: group 1 mixed a certain amount of SC[4] mother liquor and pineapple crude extract, group 2 mixed a certain amount of CTAB mother liquor, SC[4] mother liquor and pineapple crude extract, and equilibrated at 30℃ for 1h . The obtained mixed solution is added to the catechol solution to carry out a catalytic reaction. After the reaction is completed, the absorbance value of the reaction solution is detected at 420 nm over time. In the total reaction system, the concentration of SC[4] in group 1 was 0, 0.02 mg/mL, 0.06 mg/mL, 0.08 mg/mL, 0.1 mg/mL, and the concentration of catechol was 150 mM. In group 2, the concentration of CTAB is 0.1mg/mL, the concentration of SC[4] is 0, 0.02mg/mL, 0.06mg/mL, 0.08mg/mL, 0.1mg/mL, and the concentration of catechol is 150mM . Plot the absorbance value (A) at 420nm with time (t), calculate the slope of the linear part of the figure, and combine the molar absorption coefficient (1100M -1 cm -1 ), the initial reaction rate ν(μM·s -1 ). The enzyme activity without any additives is defined as 100%.

[0039] Example 2

[0040] The effect of SC[6]/CTAB compound system on the activity of polyphenol oxidase in crude pineapple extract:

[0041] The experiment is divided into two groups: group 1 mixes a certain amount of SC[6] mother liquor and pineapple crude extract; group 2 mixes a certain amount of CTAB mother liquor, SC[6] mother liquor and pineapple crude extract, and balance at 30°C 1h. The obtained mixed solution is added to the catechol solution to carry out a catalytic reaction. After the reaction is completed, the absorbance value of the reaction solution is detected at 420 nm over time. In the total reaction system, in group 1, the concentration of SC[6] was 0, 0.02mg/mL, 0.06mg/mL, 0.08mg/mL, 0.1mg/mL, and the concentration of catechol was 150mM. In group 2, the concentration of CTAB is 0.1mg/mL, the concentration of SC[6] is 0, 0.02mg/mL, 0.06mg/mL, 0.08mg/mL, 0.1mg/mL, and the concentration of catechol is 150mM . Plot the absorbance value (A) at 420nm with time (t), calculate the slope of the linear part of the figure, and combine the molar absorption coefficient (1100M -1 cm -1 ), the initial reaction rate ν(μM·s -1 ). The enzyme activity without any additives is defined as 100%.