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Preparation method of polyvinylpyrrolidone modified wheat esterase

A technology of polyvinylpyrrolidone and wheat esterase, which is applied in the direction of enzyme stabilization and the like, can solve the problems of harsh storage conditions and difficult to obtain materials, and achieves the effects of easy source, increased activity and simple modification process.

Inactive Publication Date: 2015-11-25
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The animal cholinesterase inhibition method has the advantages of rapidity, simplicity, and sensitivity, but it is derived from animals, and it is not easy to obtain materials and the storage conditions are harsh.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] 1) Add 0.07mol / L NaCl solution to wheat flour, the volume ratio of material to liquid is 1:2 (m:v), shake and extract at 29°C for 120min, then filter through double-layer gauze, and the filtrate obtained is at 6000r / min , centrifuge at 4°C for 10min, collect the supernatant; then salt out with 20%-70% ammonium sulfate, centrifuge at 10,000r / min at 4°C for 15min to collect the precipitate, use a dialysis bag with a molecular weight cut-off of 8,000-14,000Da, put the dialysis bag dialyzed into distilled water to remove ammonium sulfate. It is the prepared wheat esterase.

[0024] 2) Sodium periodate and polyvinylpyrrolidone (2:1, m:m) were dissolved in 40 mL of distilled water, and after stirring for 20 hours at 25°C in the dark, the resulting product was dissolved in distilled water with a dialysis bag with a molecular weight cut-off of 8,000 to 14,000 Da. Dialyzed for 20 hours to obtain an oxidized solution of polyvinylpyrrolidone.

[0025] 3) Mix the prepared wheat e...

Embodiment 2

[0028] 1) Add 0.15 mol / L NaCl solution to wheat flour, the volume ratio of material to liquid is 1:6 (m:v), shake and extract at 40°C for 40 minutes, and then filter through double-layer gauze. / min, centrifuge at 4°C for 5min, collect the supernatant; then salt out with 20%-70% ammonium sulfate, centrifuge at 10,000r / min at 4°C for 19min to collect the precipitate, and use a dialysis bag with a molecular weight cut-off of 8,000-14,000Da to dialyze the precipitate The bag was put into distilled water and dialyzed to remove ammonium sulfate. It is the prepared wheat esterase.

[0029] 2) Sodium periodate and polyvinylpyrrolidone (4:1, m:m) were dissolved in 45 mL of distilled water, and after stirring for 35 hours in the dark at 25°C, the resulting product was dissolved in distilled water with a dialysis bag with a molecular weight cut-off of 8,000-14,000 Da. Dialyzed in medium for 19 hours to obtain an oxidized solution of polyvinylpyrrolidone.

[0030] 3) Mix the prepared w...

Embodiment 3

[0033] 1) Add 0.07mol / L NaCl solution to wheat flour, the volume ratio of material to liquid is 1:3 (m:v), shake and extract at 30°C for 60min, then filter through double-layer gauze, and the filtrate obtained is at 30000r / min , centrifuged at 4°C for 7 minutes, and collected the supernatant; then salted out with 20%-70% ammonium sulfate, centrifuged at 10,000 r / min at 4°C for 16 minutes to collect the precipitate, and used a dialysis bag with a molecular weight cut-off of 8,000 to 14,000 Da, and put the dialysis bag into dialyzed into distilled water to remove ammonium sulfate. It is the prepared wheat esterase.

[0034] 2) Sodium periodate and polyvinylpyrrolidone (2.5:1, m:m) were dissolved in 90 mL of distilled water, and after stirring for 30 hours in the dark at 25°C, the resulting product was dissolved in distilled water with a dialysis bag with a molecular weight cut-off of 8,000-14,000 Da. Dialyzed in medium for 19 hours to obtain an oxidized solution of polyvinylpyr...

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Abstract

The invention relates to a preparation method of polyvinylpyrrolidone modified wheat esterase, and belongs to the field of modified use of wheat esterase. The wheat esterase can be used for rapid detection of organophosphorus and carbamates pesticides. The method comprises the steps as follows: after polyvinylpyrrolidone is oxidized, the wheat esterase is modified, so that the specific activity of the wheat esterase can be increased to 130%-150% of the proenzyme activity, heat stability of the wheat esterase is improved, appetency to substrate acetic acid-1-naphthaline ester is enhanced, and the detection cost for rapidly detecting organophosphorus and carbamates pesticides with an enzymatic inhibition method is saved.

Description

technical field [0001] The invention relates to a preparation method for modifying wheat esterase with polyvinylpyrrolidone, which belongs to the field of modification and utilization of wheat esterase, in particular to a method for modifying wheat esterase and improving the specific activity of the enzyme. Background technique [0002] Organophosphorus and carbamate pesticides are widely used and are commonly used in my country. Therefore, when used improperly, it often leads to residues of agricultural products, which affects food safety and quality. [0003] At present, the detection methods of organophosphorus and carbamate pesticides include chromatography, enzyme-linked immunoassay, and enzyme inhibition. Chromatography requires special, expensive equipment and detection costs are high. Enzyme-linked immunoassay has the advantages of high sensitivity, strong specificity, low detection limit, simple and quick operation, but has certain limitations. Because of strong s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/96
Inventor 陆利霞熊晓辉陈铭游京晶
Owner NANJING TECH UNIV
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