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Method for detecting activity of fatty oxygenase

A lipoxygenase and activity technology, applied in the field of detecting lipoxygenase activity, can solve the problems of high price, inconvenient operation and the like

Inactive Publication Date: 2009-04-29
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The radiation intensity of the isotope labeling method can track the progress of the reaction, but it is expensive and inconvenient to operate

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Determination of lipoxygenase enzyme activity in soybean

[0021] 1) Soybean is pulverized and leached 9 times with petroleum ether to obtain defatted soybean powder (40-mesh sieve). Add water to defatted soy flour (the ratio of solid to liquid is 1:10 (w / v)) and adjust the pH to 4.5 with 1mol / L hydrochloric acid, stir for 60 minutes, and filter; add solid ammonium sulfate to the filtrate to 40% saturation, centrifuge for 20 minutes, discard Remove the precipitate; add solid ammonium sulfate to the supernatant to 60% saturation, centrifuge for 20 min, and dissolve the precipitate with a borate buffer solution with a pH of 9.0 and 0.2 mol / L to obtain a lipoxygenase enzyme solution.

[0022] 2) Disperse 0.1mL of Tween20 in 10mL of 0.2mol / L borate buffer, add 0.1mL of linoleic acid drop by drop under shaking, mix thoroughly, add 1mol / L of hydrochloric acid to adjust the pH to 9.0, and then dissolve the above buffer Dilute to 500mL and use as a substrate.

[002...

Embodiment 2

[0029] Example 2 Determination of lipoxygenase enzyme activity in tomato

[0030] 1) Tomatoes are pulverized and added with water (the ratio of solid to liquid is 1:10 (w / v)) and adjusted to pH 4.5 with 1mol / L hydrochloric acid, stirred for 60 minutes, filtered; solid ammonium sulfate is added to the filtrate until 40% saturated, and centrifuged for 20 minutes , discard the precipitate; solid ammonium sulfate was added to the supernatant to 60% saturation, centrifuged for 20 min, and the precipitate was dissolved with pH 9.0, 0.2 mol / L borate buffer to obtain lipoxygenase enzyme solution.

[0031] 2) Disperse 1.0mL Tween20 in 10mL1.5mol / L borate buffer, add 1.0mL linoleic acid drop by drop under shaking, mix well, add 1mol / L hydrochloric acid to adjust the pH to 9.0, and then add the above buffer Dilute to 500mL and use as a substrate.

[0032] 3) The polyacrylate prepolymer of 0.5g is formulated into a solution with 10g of n-butanol in a mass fraction ratio of 1:2 under ligh...

Embodiment 3

[0038] Example 3 Determination of lipoxygenase enzyme activity in potatoes

[0039]1) After crushing the potatoes, add water (the ratio of solid to liquid is 1:10 (w / v)) and adjust the pH to 4.5 with 1mol / L hydrochloric acid, stir for 60 minutes, and filter; add solid ammonium sulfate to the filtrate to 40% saturation, and centrifuge for 20 minutes , discard the precipitate; solid ammonium sulfate was added to the supernatant to 60% saturation, centrifuged for 20 min, and the precipitate was dissolved with pH 9.0, 0.2 mol / L borate buffer to obtain lipoxygenase enzyme solution.

[0040] 2) Disperse 0.5mL of Tween20 in 10mL of 1.0mol / L borate buffer, add 0.5mL of linoleic acid drop by drop under shaking, mix thoroughly, add 1mol / L of hydrochloric acid to adjust the pH to 9.0, and then dissolve the above buffer Dilute to 500mL and use as a substrate.

[0041] 3) the polyacrylate prepolymer of 0.5g is formulated into a solution with 10g of n-butanol in a mass fraction ratio of 1:...

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PUM

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Abstract

The invention relates to a method for detecting activity of lipoxygenase. The method comprises the following steps: keeping lipoxygenase standard sample solution with known enzyme activity and substrate solution in a water bath tank at a constant temperature of between 10 and 40 DEG C for 10 minutes; placing 60 milliliters of the substrate solution in an electrolyzer; adding 1 milliliter of the lipoxygenase standard sample solution in the electrolyzer filled with the substrate solution; placing the water bath tank at a temperature of between 10 and 40 DEG C to react for 3 minutes; using a spiral platinum wire as an auxiliary electrode, a saturated calomel (SCE) electrode as a reference electrode, and a horse radish peroxidase modified electrode as a working electrode; beginning measuring current values of the electrodes when the voltage is between -0.4 and -0.1 volt; drawing concentration of current values obtained from standard samples with different concentrations as a standard curve; and taking a lipoxygenase sample to be detected, repeating fore processes, contrasting the obtained value with the standard curve, and learning enzyme activity. The result shows that the method has the characteristics of quick response speed, no relation to turbidity of the system, and on-line measurement.

Description

Technical field: [0001] The invention relates to a method for detecting the activity of lipoxygenase, in particular to a method for detecting the activity of lipoxygenase with a horseradish peroxidase electrode. Background technique: [0002] So far, the publicly reported methods for the determination of lipoxygenase activity include oxygen electrode method, volume pressure method, spectrophotometry, Fe(CNS)3 chromogenic method, thiobarbituric acid chromogenic method, isotope labeling method, etc. , the determination principles and determination parameters of various methods are different, and have their own advantages and disadvantages. The oxygen electrode method has high sensitivity, and the measurement parameters have nothing to do with the turbidity of the reaction system, but the temperature and air tightness of the reaction system need to be strictly controlled. The measurement parameters of the pressure measurement method have nothing to do with the turbidity of the...

Claims

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Application Information

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IPC IPC(8): G01N27/416G01N27/327
Inventor 高海燕刘仁许芳萍顾伟
Owner JIANGNAN UNIV
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