New method for measuring activity of protease

A protease and activity technology, applied in the field of bioengineering, can solve the problems of large sample demand and long time required, and achieve the effect of small sample demand, short time consumption and fast speed.

Pending Publication Date: 2019-09-27
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
View PDF4 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the process of measuring enzyme activity by the national standard method, steps such as diluting the sample, hydrolyzing the substrate, terminating the reaction, centrifuging the reaction solution to obtain the supernatant, and developing the color of folin phenol are required. The time required for the determination of enzyme activity for a single sample is long and the sample requirements large amount

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • New method for measuring activity of protease
  • New method for measuring activity of protease
  • New method for measuring activity of protease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: the drafting of the standard curve of mixed substrate assay protease enzymatic activity

[0030] 1) Prepare different concentrations of p-nitroaniline solutions with DMSO.

[0031] 2) Composition of the reaction system: 80 μL of phosphate buffer (pH=7.5), 20 μL of p-nitroaniline solution, and 100 μL of phosphate buffer (pH=7.5).

[0032] 3) Take tube 0 without p-nitroaniline as blank, at OD 410 The absorbance was measured respectively.

[0033] 4) Take the absorbance A as the ordinate, and the final concentration C of p-nitroaniline as the abscissa, draw a standard curve (the curve should pass through the zero point). Calculate the concentration of p-nitroaniline when the absorbance is 1 according to the drawing, which is the absorbance constant K value. standard curve as figure 1 shown.

Embodiment 2

[0034] Embodiment 2: Utilize oligopeptide composition to measure the enzymatic activity of various proteases

[0035] 1. Using oligopeptide composition to measure the enzyme activity of 1398 neutral protease

[0036] 1) Prepare a mixed substrate solution of 20 oligopeptide compounds with DMSO, wherein the concentration of each oligopeptide compound is 4mM;

[0037] 2) Reaction system composition: 80 μL phosphate buffer (pH=7.5), 20 μL mixed substrate solution, 100 μL diluted enzyme solution (3-4 U / mL) of the sample to be tested;

[0038] 3) Mix well, let stand at 40°C for 10 minutes, measure OD 410 Absorbance value at time;

[0039] 4) Calculate the enzyme activity in the enzyme solution to be tested, and express the result with one decimal place.

[0040] 2. Determination of enzyme activity of alkaline protease derived from Bacillus clausii using oligopeptide composition

[0041] 1) Prepare a mixed substrate solution of 20 oligopeptide compounds with DMSO, wherein the con...

Embodiment 3

[0062] Embodiment 3: trypsin activity assay

[0063] Trypsin is a protease with significant specificity. It has a high cutting efficiency for the peptide bond formed by arginine (R) and lysine (K). Using this enzyme to react with 20 kinds of oligopeptide compounds respectively, you can To verify the feasibility of using oligopeptide composition to reflect protease substrate cleavage specificity.

[0064] Use mixed substrates and 20 kinds of oligopeptide compounds to react with trypsin, the specific operation is as follows:

[0065] 1) Prepare 4 mM AAPX substrate solution with DMSO (X represents any one of 20 common amino acids); reaction system composition: 80 μL phosphate buffer (pH=7.5), 20 μL AAPX substrate solution, 100 μL diluted Enzyme solution (3-4U / mL);

[0066] 2) Prepare a mixed substrate solution of 20 oligopeptide compounds with DMSO, wherein the concentration of each oligopeptide compound is 4 mM; the composition of the reaction system: 80 μL phosphate buffer (p...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention provides a new method for measuring the activity of protease. The actual hydrolysis ability of the protease can be evaluated more comprehensively and accurately. The method mainly comprises the following steps: taking a composition consisting of 20 oligopeptide compounds as a substrate, wherein the oligopeptide compounds have a general formula of Suc-Ala-Ala-Pro-X-pNA, and X represents any one of 20 types of common amino acids; synthesizing the 20 oligopeptide compounds, preparing a reaction system consisting of a mixed substrate solution, a buffer solution and protease liquid, wherein a pNA monomer which is generated by hydrolyzing a peptide bond between amino acid X and paranitroaniline pNA with the protase has an absorbing peak at the position of OD410; and mixing the 20 oligopeptide compounds to serve as the substrate, measuring the OD410 value after hydrolyzing the mixed substrate with the protease, and then measuring a light absorption value of OD410 after reaction to obtain the maximum enzyme activity of the protease.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a new method for measuring protease activity. Background technique [0002] Among numerous enzyme preparations, protease occupies a very important position because of its wide application in industry. Proteases can catalyze the peptide bonds in protein macromolecules and hydrolyze them into amino acids, peptides, and peptones, so they are also called peptidases. Due to the hydrolysis effect of protease, it is widely used in leather industry, detergent, medicine, food and feed processing and other industries. Using a protease of subtilisin in the detergent greatly improves the washing effect. [0003] Protease is one of the most important and abundant enzyme systems in the biological industry. Protease has a wide range of sources and is widely distributed in animal offal, microorganisms, plant stems, leaves, and fruits. Among them, protease derived from bacteria account...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12Q1/37
CPCC12Q1/37
Inventor 路福平刘逸寒李玉曹雪李家霖刘夫锋张会图王洪彬彭冲
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap