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Enzyme activity starting and improving method of Deinococcus radiodurans protease PprI

A technology of Deinococcus radiodurans and protease, applied in the direction of microorganism-based methods, biochemical equipment and methods, hydrolytic enzymes, etc., can solve undiscovered problems, achieve the effect of improving enzyme cutting activity and shortening reaction time

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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  • Enzyme activity starting and improving method of Deinococcus radiodurans protease PprI
  • Enzyme activity starting and improving method of Deinococcus radiodurans protease PprI
  • Enzyme activity starting and improving method of Deinococcus radiodurans protease PprI

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] (1) The protease activity of PprI depends on the metal ion Mn 2+

[0031] When PprI protein performs protease activity, it needs metal ion Mn 2+ The presence. in Mn 2+ The activity is optimal when the final concentration is 2mM / L.

[0032] (2) Restriction substrate gene wxya The promoter can promote the efficiency of PprI digestion

[0033] Digest substrate protein DdrO and substrate gene promoter in reaction buffer (150mM NaCl, 20mM Tris-HCl 8.0, 1mM DTT, 10mM MgCl 2 ) for 40 minutes. Next, the purified PprI protein was added to the reaction solution. Then, add MnCl at a final concentration of 2.0 mM 2 Start the reaction. After 40 minutes, the reaction was terminated and detected by SDS-PAGE electrophoresis. Experiments have shown that the interaction between the substrate protein and its own promoter can greatly promote the efficiency of PprI digestion.

Embodiment 2

[0035] (1) The protease activity of PprI depends on the metal ion Mn 2+

[0036] When PprI protein performs protease activity, it needs metal ion Mn 2+ The presence. in Mn 2+ The activity is optimal when the final concentration is 2mM / L. Other divalent ions such as Ni 2+ ,Zn 2+ etc., the final concentration is greater than 0.25mM / L, all have inhibitory effect on protease activity.

[0037] (2) recA Gene promoter can promote PprI digestion efficiency

[0038] Digestion of substrate protein DdrO with recAGene promoter in reaction buffer (150mM NaCl, 20mM Tris-HCl 8.0, 1mM DTT, 10mM MgCl 2 ) for 40 minutes. Next, the purified PprI protein was added to the reaction solution. Then, add MnCl at a final concentration of 2.0 mM 2 Start the reaction. After 40 minutes, the reaction was terminated and detected by SDS-PAGE electrophoresis. Experiments have shown that substrate proteins and recA The interaction between gene promoters can greatly promote the digestio...

Embodiment 3

[0040] (1) The protease activity of PprI depends on the metal ion Mn 2+

[0041] When PprI protein performs protease activity, it needs metal ion Mn 2+ The presence. in Mn 2+ The activity still exists when the final concentration is 5mM / L. Other divalent ions such as Fe 2+ ,Cu 2+ etc., the final concentration is greater than 0.25mM / L, all have inhibitory effect on protease activity.

[0042] (2) Non-specific DNA cannot promote the digestion efficiency of PprI

[0043] Digest substrate protein DdrO and non-specific DNA in reaction buffer (150mM NaCl, 20mM Tris-HCl 8.0, 1mM DTT, 10mM MgCl 2 ) for 40 minutes. Next, the purified PprI protein was added to the reaction solution. Then, add MnCl at a final concentration of 2.0 mM 2 Start the reaction. After 40 minutes, the reaction was terminated and detected by SDS-PAGE electrophoresis. Experiments have shown that non-specific DNA cannot promote the efficiency of PprI digestion.

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Abstract

The invention discloses an enzyme activity starting and improving method of protease PprI. The enzyme activity starting of the protease PprI needs 2.0-5.0mM of Mn<2+> ions. An enzyme digestion reaction buffer solution contains 150-250mM OF NaCl, 10-50Mm of Tris-HcL 8.0, 0.1mM of DTT and 2.0-5.0mM of MnCl2. The combination of gene dr2574 and dr2340 promoter fragments with a substrate DdrO improves the enzyme digestion activity of the protease PprI, shortens the reaction time, and has positive contribution to future utilization of the enzyme.

Description

technical field [0001] The invention relates to a newly discovered Deinococcus radioresistant protease PprI, which relates to the initiating effect of metal ions on enzyme activity and the promoting effect of gene promoter on enzyme cutting efficiency. 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 been widely used in fur, leather,...

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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