Protease

A technology of protease and enzymatic activity, applied in the field of Deinococcus radiodurans protease, which can solve undiscovered problems and achieve the effect of high specificity and good heat resistance

Active Publication Date: 2014-12-17
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, the protease activity and enzyme substrate of PprI have never been found

Method used

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Examples

Experimental program
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Effect test

Embodiment 2

[0037] (1) Protease activity and substrate cleavage sequence specificity of PprI

[0038] The purified PprI protein and its substrate protein DdrO were mixed in reaction buffer (100mM NaCl, 30mM Tris-HCl 8.0, 1mM DTT, 3.0mM MnCl 2 ) for 40 minutes. Through SDS-PAGE electrophoresis and mass spectrometry molecular weight detection, the PprI protein can cut the DdrO enzyme into two segments. The specific sequences of PprI protease cleavage substrates were ELRGKR, ELRGAR, ELAGKR and ELAGAR by point mutation of the amino acid residues near the DdrO cleavage site. In addition, the cleavage position can be obtained between the second and third amino acid residues by mass spectrometry sequencing of the C-terminal of the protein.

[0039](2) The optimum temperature range and temperature resistance of PprI protease activity

[0040] The optimum temperature for the enzymatic cleavage reaction between PprI protease and substrate is between 35-40°C. Protease activity is highest in...

Embodiment 3

[0042] (1) Protease activity and substrate cleavage sequence specificity of PprI

[0043] The purified PprI protein and its substrate protein DdrO were mixed in reaction buffer (200mM NaCl, 20mM Tris-HCl 8.0, 1mM DTT, 5.0mM MnCl 2 ) for 40 minutes. Through SDS-PAGE electrophoresis and mass spectrometry molecular weight detection, the PprI protein can cut the DdrO enzyme into two segments. Through the point mutation of the amino acid residues near the DdrO restriction site, the specific sequences of the PprI protease digestion substrates are ELRGKR, ELRGAR, ELRGER, ELAGKR, ELAGAR and ELAGER. In addition, the cleavage position can be obtained between the second and third amino acid residues by mass spectrometry sequencing of the C-terminal of the protein.

[0044] (2) The optimum temperature range and temperature resistance of the protease activity of PprI

[0045] The optimum temperature for the enzymatic cleavage reaction between PprI protease and substrate is between 35-4...

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Abstract

The invention discloses a protease. The core protein sequence of the protease is represented by SEQIDNO:1; the specific sequence of the substrate of the protease is ELXGXR (wherein X is anyone of essential amino acids), and the cutting position is between a second amino acid residue and a third amino acid residue; and a best temperature of an enzyme digestion reaction is 35-40DEG C, and the protease still has activity at 65DEG C. The protease has the advantages of high specificity of an enzyme digestion sequence, and good heat resistance.

Description

technical field [0001] The present invention relates to a newly discovered Deinococcus radiodurans protease, in particular to the enzymatic cleavage substrate, substrate cleavage specific sequence, active reaction system, optimum temperature and temperature resistance of the protease. Background technique [0002] Protease is a general term for a class of enzymes that hydrolyze protein peptide bonds, and can catalyze the hydrolysis of peptide bonds in proteins. Proteases widely exist in animals, plants and microorganisms. So far, there are more than 100 commercial proteases in the international market. Due to limited animal and plant resources, protease preparations produced in industry mainly come from microorganisms, which are extracted and prepared by using microorganisms such as Bacillus subtilis, yeast, mold, and Escherichia coli. With the deepening of protease research, its industrial application has attracted more and more people's attention. [0003] Protease has ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/52C12R1/01
CPCC12N9/52
Inventor 华跃进王云光王梁燕
Owner ZHEJIANG UNIV
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