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Method for high-flux assay of phytase activity in transgenic phytase crop seed

A technology for converting phytase and phytase, which is applied in the direction of biochemical equipment and methods, and microbial measurement/inspection, can solve the problems of large error, low stability, and high coefficient of variation of detection methods, and achieve good accuracy, High stability and good repeatability

Inactive Publication Date: 2012-02-15
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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Problems solved by technology

[0006] The Chinese invention patent with the publication number CN 101914508A (invention name: a phytase high-throughput screening method based on a 96-well plate) discloses a high-throughput method for measuring phytase activity. On the basis of ammonium vanadate method for determining phytase activity, a method for determining phytase activity by microplate method was established. Although this method can detect a large number of samples at a time and has the advantage of high-throughput detection, there are errors in this detection method. Large, low stability, high coefficient of variation and other defects need to be improved

Method used

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  • Method for high-flux assay of phytase activity in transgenic phytase crop seed
  • Method for high-flux assay of phytase activity in transgenic phytase crop seed
  • Method for high-flux assay of phytase activity in transgenic phytase crop seed

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Embodiment 1

[0048] Embodiment 1 The establishment of the method for the high-throughput determination of phytase enzyme activity in the phytase crop seeds of the present invention or the feed added with the phytase crop seeds

[0049] 1. Standard curve drawing

[0050] Dilute the phosphorus standard solution with acetic acid buffer to 1.5625mmol / L, 2.5000mmol / L, 3.1250mmol / L, 5.0000mmol / L and 6.2500mmol / L solutions. The reaction was carried out according to the operation steps in Table 1, and then the absorbance was measured at a wavelength of 415 nm after standing at room temperature for 10 minutes. Draw a standard curve with the phosphorus content as the abscissa and the absorbance as the ordinate.

[0051] 2. Preparation of sample solution

[0052] 1. Sample grinding

[0053] Put the sample in the grinding tube (transformed phytase corn or feed containing the phytase corn), add steel balls, place it in the grinder, set the speed at 1400 strokes / min, 30 sec each time, and conduct at ...

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Abstract

The invention discloses a method for high-flux assay of phytase activity in a transgenic phytase crop seed. The method comprises the following steps: (1) building a phosphorus standard curve based on a phosphorus content as a horizontal coordinate and a light absorption value as a vertical coordinate; (2) leaching out phytase in a sample to be assayed so as to obtain leach liquor of phytase; (3) carrying out enzyme activity assay reaction of phytase on the leach liquor of phytase in a microplate with 96 holes; and (4) assaying the light absorption value of a reaction solution, calculating the phosphorus concentrations of various samples to be assayed according to a linear regression equation corresponding to the phosphorus standard curve, calculating the phytase activities of the samples to be assayed according to an enzyme activity calculation formula. Accuracy and sensitivity testes show that the assayed result obtained by using the method has good accuracy, small error, high stability and good repeatability, and coefficient of variation of the assayed result obtained by using the method is far lower than that obtained by the existing microplate method. The assay method disclosed by has the advantages of high flux, high assay efficiency, low cost, high accuracy and the like, and is easy to operate.

Description

technical field [0001] The invention relates to a method for measuring phytase activity, in particular to a method for quickly and high-throughput detection of phytase activity in phytase-transformed crop seeds or feed added with phytase-transformed crop seeds, which belongs to transgenic Field of determination of phytase activity in crop seeds. Background technique [0002] From 1996 to 2010, the planting area of ​​genetically modified crops in the world increased by 87 times in 15 years, reaching 148 million hectares by 2010, and growing at a rate of more than 10% every year. So far, a total of 59 countries have approved the cultivation or import of biotech crops for food and feed, including 29 countries that have approved the localization of biotech crops, and 75% of the world's population lives in these 59 countries. [0003] Among the genetically modified plants developed so far, genetically modified corn, soybean, cotton, rapeseed and potato are all related to feed an...

Claims

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Application Information

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IPC IPC(8): C12Q1/44
Inventor 初晓宇杨文竹伍宁丰陈茹梅范云六姚斌
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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