Method for determining activity of endotrypsin A

A technology of protease and yeast, which is applied in the field of fermentation engineering, can solve the problems of few application reports of resonance light scattering technology and restrict the development of pure draft beer, etc., and achieve the effects of less interference, low detection limit and good sensitivity

Inactive Publication Date: 2012-07-18
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are not many reports on the application of resonance light scattering technology in enzyme catalysis, and there are no reports on the dete

Method used

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  • Method for determining activity of endotrypsin A
  • Method for determining activity of endotrypsin A
  • Method for determining activity of endotrypsin A

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Embodiment 1: Determination of protease A secretion amount of yeast

[0028] Heat 0.25g of casein with 60mL 6mol / L HCl, dissolve it by ultrasonic, add Gly-HCl buffer solution to make the final concentration 0.1mol / L, adjust the pH to 3 with saturated NaOH solution, adjust the volume to 250mL, and filter with medium-speed filter paper , which is the casein solution. Dissolve 408.48g of trichloroacetic acid in distilled water, set the volume to 1L, and filter to obtain a 2.5mol / L trichloroacetic acid solution. 150g of yeast paste with 300mLNa 2 HPO 4 -KH 2 PO 4 Buffer solution (pH 6.5, ionic strength 1 / 15mol / L) was autolyzed at room temperature for 24 hours, ground the solution with quartz sand in an ice bath, filtered, centrifuged, and the supernatant was taken as crude enzyme solution.

[0029] Add 0.1mL of crude enzyme solution to a 10mL small test tube, then add 1mL of 0.1% casein solution (pH=3), mix well, react at 40°C for 20min, add 2mL of 2.5mol / L trichloroac...

Embodiment 2

[0035] Embodiment 2: Determination of protease A content in pure draft beer

[0036] Heat 0.25g of casein with 60mL 6mol / L HCl, dissolve it by ultrasonic, add Gly-HCl buffer solution to make the final concentration 0.1mol / L, adjust the pH to 3 with saturated NaOH solution, adjust the volume to 250mL, and filter with medium-speed filter paper , which is the casein solution. Dissolve 408.48g of trichloroacetic acid in distilled water, set the volume to 1L, and filter to obtain a 2.5mol / L trichloroacetic acid solution. 100mL of pure draft beer was salted out in ammonium sulfate at 4°C (50% saturation) for 2h, centrifuged at 8000r / m for 20min, the precipitate was dissolved in water and the volume was adjusted to 50mL, and the supernatant obtained by centrifugation was the crude enzyme solution.

[0037]Add 0.1mL of crude enzyme solution to a 10mL small test tube, then add 1mL of 0.1% casein solution (pH=3), mix well, react at 40°C for 20min, add 2mL of 2.5mol / L trichloroacetic ac...

Embodiment 3

[0043] Embodiment 3: Determination of protease A content in fermented liquid

[0044] Heat 0.25g of casein with 60mL 6mol / L HCl, dissolve it by ultrasonic, add Gly-HCl buffer solution to make the final concentration 0.1mol / L, adjust the pH to 3 with saturated NaOH solution, adjust the volume to 250mL, and filter with medium-speed filter paper , which is the casein solution. Dissolve 408.48g of trichloroacetic acid in distilled water, set the volume to 1L, and filter to obtain a 2.5mol / L trichloroacetic acid solution. 100mL of fermentation broth was salted out in ammonium sulfate at 4°C (50% saturation) for 2h, centrifuged at 8000r / m for 20min, the precipitate was dissolved in water and the volume was adjusted to 100mL, and the supernatant obtained by centrifugation was the crude enzyme solution.

[0045] Add 0.1mL of crude enzyme solution to a 10mL small test tube, then add 1mL of 0.1% casein solution (pH=3), mix well, react at 40°C for 20min, add 2mL of 2.5mol / L trichloroace...

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Abstract

The invention discloses a method for determining the activity of endotrypsin A, belonging to the field of fermentation engineering. The invention relates to a novel method for determining the activity of the endotrypsin A by using a resonance light scattering technology. According to the method, trichloroacetic acid and trace amount of protein can form associated-particles; according to the principle that the concentration of micro particles and the resonance scattering light intensity form a a proportional relationship under certain conditions, the trichloroacetic acid is associated with protein in a solution before and after the endotrypsin A reacts with casein protein; the light intensity of the protein is determined by using a fluorospectro photometer; and the activity of the endotrypsin A is obtained according to the degradation amount of casein. The method has the advantages of low detection limit, favorable sensitivity, high stability, wide linear range, little influence from a chaff interference, quickness, simpleness and convenience, low detection cost and greater potential; and the requirements for determining the activity of trace amount of endotrypsin A in fermentation broth and beer can be met and reliable base for deeply researching the endotrypsin A is provided.

Description

technical field [0001] A method for measuring the activity of yeast protease A belongs to the field of fermentation engineering. Background technique [0002] Yeast protease A (EC3.4.23.6) is an aspartic acid family protease encoded by the PEP4 gene. The protease A secreted to the outside of the cell can reduce the foam stability of pure draft beer by degrading the foam active protein in the fermentation broth and finished beer. How to accurately determine the very small amount of protease A in pure draft beer has always been a problem that researchers have been concerned about. [0003] The existing protease A detection methods can be roughly divided into two categories: the detection using natural substrates and the detection using synthetic substrates. Natural substrates can use casein, azo casein, denatured hemoglobin, unfolded myoglobin, insulin A chain and B chain, ribonuclease, etc., and are generally detected by UV spectrophotometry, Lowry method or Bradford method...

Claims

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Application Information

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IPC IPC(8): G01N21/64
Inventor 李崎刘春凤宋群李永仙郑飞云王金晶顾国贤
Owner JIANGNAN UNIV
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