Improved seed crude enzyme solution extracting method

A technology of seed crude enzyme and extraction method, which is applied in the preparation of test samples and the measurement of color/spectral properties, etc. Improved repeatability and cost savings of assays

Inactive Publication Date: 2015-07-22
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are a large number of glands in the cotyledons of cotton seeds, and a large amount of phenolic substances secreted by the glands seriously affect the accuracy of enzyme activity determination
The existing crude enzyme liquid extraction method is not accurate and reproducible in the determination of enzyme activity, and even the results cannot be detected. Secondly, the volume of the reaction system in the detection process is relatively large, and the cost of drugs is high, resulting in high detection costs.

Method used

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Examples

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Effect test

Embodiment 1

[0027] Example 1. Improved glutamate synthase activity assay method

[0028] Preparation of modified glutamic acid synthetase extract (50mM phosphate buffer solution with pH=7.2, 5% v / v glycerol, 2% w / v polyvinylpyrrolidone): measure the original extract solution with 50mM phosphate buffer solution with pH=7.2 Liquid 95ml, glycerin 5ml and 0.2g polyvinylpyrrolidone are mixed evenly. The above extract was stored at 4°C; glycerol was the volume percentage concentration, and polyvinylpyrrolidone was the mass concentration (that is, 2 g of polyvinylpyrrolidone was added to 1 L of the improved extract).

[0029] Sampling: Select cotton plants with consistent growth, when 50% of the 1st and 2nd fruiting nodes of cotton 6-8 fruiting branches bloom, put a sign to mark the white flowers on the 1st and 2nd fruiting nodes of the cotton middle fruiting branches. 10 days, 17 days, 24 days, 31 days, 38 days, 45 days after flowering, 6-8 cotton bolls with consistent growth and development w...

Embodiment 2

[0040] Embodiment 2. The assay method of improved phosphoenolpyruvate carboxylase enzyme activity

[0041] Prepare phosphoenolpyruvate carboxylase modified extract (300mM sucrose, 50mM pH=7.0Hepes-Tris, 8mM EDTA, 2mM PMSF, 4mM DTT, 5% v / v glycerol, 2% w / v polyethylene Pyrrolidone): Measure 95ml of the original extract (300mM sucrose, 50mM pH=7.0Hepes-Tris, 8mM EDTA, 2mM PMSF, 4mM DTT), 5ml of glycerin and 0.2g of polyvinylpyrrolidone and mix well. The above extract was stored at 4°C; glycerol was the volume percentage concentration, and polyvinylpyrrolidone was the mass concentration (that is, 2 g of polyvinylpyrrolidone was added to 1 L of the improved extract).

[0042]Sampling: Select cotton plants with consistent growth, when 50% of the 1st and 2nd fruiting nodes of cotton 6-8 fruiting branches bloom, put a sign to mark the white flowers on the 1st and 2nd fruiting nodes of the cotton middle fruiting branches. 10 days, 17 days, 24 days, 31 days, 38 days, 45 days after flo...

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Abstract

The invention discloses an improved seed crude enzyme solution extracting method. The improved seed crude enzyme solution extracting method comprises the following steps: taking seeds in a growing process and pre-cooled improved extracting solution to fully grind to obtain homogenate; adding petroleum ether or diethyl ether to the homogenate, uniformly mixing, leaving the mixture to stand, centrifuging the mixture and separating the mixture to obtain a seed crude enzyme solution; adding glycerinum and polyvinylpyrrolidone to the original extracting solution to obtain the improved extracting solution. According to the improved seed crude enzyme solution extracting method, an oil body, an enzyme preparation and impurities are separated completely; in an activity determination process, the standard error of a determination result is reduced, so that a reaction system can be shrunk in multiples, and the determination cost is reduced; by utilizing the improved seed crude enzyme solution extracting method, the accuracy and the repeatability of enzyme activity determination are remarkably improved.

Description

technical field [0001] The invention relates to an improved extraction method of seed crude enzyme liquid, in particular to an extraction method of crude enzyme liquid in the process of oil and protein formation in the cotton seed development process. Background technique [0002] Cotton is an important raw material for the textile industry in my country. At the same time, cotton seeds, as an important source of oil and protein, have broad application prospects in biodiesel, new materials, and ruminant feed. The processing of biodiesel is to add oil to fossil diesel at a ratio of 10%-20% through esterification, cracking, and bio-enzyme hydrolysis. The oil content and fatty acid quality determine the cost of biodiesel processing. The protein content of degreased and detoxified cottonseed can reach 40%, and the level of protein content determines the value of ruminant feed. [0003] Studying the activities of key enzymes in the synthesis of proteins and oils is the fundamenta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28G01N21/31
Inventor 陈兵林杨洪坤周治国孟亚利赵文青王友华王珊珊
Owner NANJING AGRICULTURAL UNIVERSITY
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