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High throughput screening method of aminopeptidase and high-yield strain thereof

A screening method, aminopeptidase technology, applied in the fields of microbial breeding, enzyme engineering and fermentation engineering, can solve the problems of long cycle, heavy workload, high cost, etc., and achieve less medium and reagents, fast speed, and reduced labor. Effect

Inactive Publication Date: 2013-03-27
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to overcome the problems of large workload, long period and high cost in conventional methods for screening novel aminopeptidases and their high-yield strains, the present invention provides a method for miniaturized culture based on deep-well plates and micro-detection based on microplate readers. High Throughput Screening Methods

Method used

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  • High throughput screening method of aminopeptidase and high-yield strain thereof
  • High throughput screening method of aminopeptidase and high-yield strain thereof
  • High throughput screening method of aminopeptidase and high-yield strain thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Comparison of the maximum absorption wavelength of the enzyme plate method and the conventional p-nitroaniline method.

[0038] Prepare 400 μmol / L p-nitroaniline, respectively use microplate plate and microplate reader method, cuvette and ultraviolet-visible spectrophotometer, and scan continuously from 280nm to 480nm. To plot the absorbance curve for p-nitroaniline, see figure 2 and image 3 . Depend on figure 2 and image 3 It can be seen that the maximum absorption peak of the ELISA method is consistent with that of the conventional p-nitroaniline method, both at about 380 nm, and the absorbance is also consistent.

Embodiment 2

[0040] High-throughput assay of methionine aminopeptidase enzyme activity.

[0041] Add 40 μL of ammonia-ammonium chloride buffer (0.05mol / L, pH8.0) and 20 μL of methionine p-nitroaniline substrate solution (6mmol / L) into the microwells of the microplate, and then add 20 μL of the crude enzyme solution to be tested that has been appropriately diluted, and 20 μL of distilled water for the blank control instead of the crude enzyme solution. Cover the lid and react in a water bath at 60°C for 10 min, and immediately add 120 μL of 50% acetic acid solution to terminate the reaction. Measure the absorbance with a microplate reader preferably at 380nm, and calculate the enzyme activity of the crude enzyme solution according to the standard curve and the dilution factor of the crude enzyme solution. Definition of enzyme activity: at a temperature of 60°C, the amount of enzyme needed to hydrolyze methionine-p-nitroaniline to produce 1 μmol of p-nitroaniline per minute is defined as on...

Embodiment 3

[0045] Comparison of enzyme activity determination results by ELISA plate method and conventional p-nitroaniline method.

[0046] Prepare p-nitroaniline standard solutions with concentrations of 150 μmol / L, 200 μmol / L, 250 μmol / L, 300 μmol / L, 350 μmol / L and 400 μmol / L respectively, with 0 content as the control, at the wavelength of 380 nm, use enzyme-labeled The content of p-nitroaniline was determined by plate method and conventional p-nitroaniline method. Take the absorbance as the abscissa, and the p-nitroaniline concentration as the ordinate, draw a standard curve, and the standard curves of the two methods are as follows: Figure 4 , Figure 5 . Depend on Figure 4 , Figure 5 As shown, the standard curve of p-nitroaniline by the microtiter plate method satisfies the regression correlation coefficient above 0.999, and the corresponding p-nitroaniline concentration at the two endpoints of the regression line is 20.720-55.252 μg / mL, which is consistent with the convent...

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Abstract

The invention discloses a high throughput screening method of aminopeptidase and a high-yield strain thereof, relates to a high throughput screening method of aminopeptidase and a high-yield microorganism bacterial strain thereof. The method comprises the following steps of: vaccinating a single colony in the hole of a deep-hole plate I on a medium containing a seed liquid, performing shaking cultivation, vaccinating the seed liquid in the hole of the deep-hole plate I correspondingly into the hole of a deep-hole plate II equipped with a fermentation medium, performing shaking cultivation, and centrifuging the deep-hole plate II to obtain the supernatant crude enzyme; adding a buffer solution and a chromogenic substrate solution or fluorogenic substrate solution in the micropore of an elisa plate, adding the dilute crude enzyme to be tested, carrying out water-bath reaction for 10-30 minutes at 30-70 DEG C, adding a 30-50% acetic acid solution immediately after the water-bath reaction and terminating the reaction; and measuring the absorbance or fluorescence intensity of the reaction liquid by a microplate reader, and calculating the enzyme activity according the standard curve and the dilution ratio of the crude enzyme. According to the invention, the goals of high throughput cultivation, high throughput preparation of crude enzyme and the high throughput test of the enzyme activity are achieved.

Description

technical field [0001] The invention relates to a high-throughput screening method for aminopeptidase and high-yield microbial strains thereof, and belongs to the technical fields of microbial breeding, enzyme engineering and fermentation engineering. Background technique [0002] Aminopeptidase (aminopeptidase) is a class of exopeptidases in proteolytic enzymes. They can hydrolyze amino acids from the N-terminal sequence of proteins and polypeptide chains. The catalytic mechanism can be expressed by the following formula: [0003] [0004] In organisms, aminopeptidase not only maintains the homeostasis of cells, plays an important role in cell maturation, growth and defense, but also participates in the activation of antibiotics as a transcriptional regulator, a specific site recombination factor and a toxin receptor with transshipment. There are many kinds of aminopeptidases, and different aminopeptidases have different hydrolysis abilities to N-terminal amino acid res...

Claims

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Application Information

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IPC IPC(8): C12N1/02C12Q1/37C12Q1/04G01N21/64G01N21/31
Inventor 姜文侠杨玉兰刘琦杨萍陈宁
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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