Method for starting and improving enzyme activity of Deinococcus radiodurans protease ppri
A technology of Deinococcus radiodurans and protease, applied in microorganism-based methods, biochemical equipment and methods, hydrolytic enzymes, etc., can solve problems such as undiscovered, and achieve the effect of improving enzyme cleavage activity and shortening reaction time
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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 (150mMNaCl, 20mM Tris-HCl8.0, 1mMDTT, 10mMMgCl 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 recA Gene promoter in reaction buffer (150mMNaCl, 20mM Tris-HCl8.0, 1mMDTT, 10mMMgCl 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 digestion efficien...
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 (150mMNaCl, 20mM Tris-HCl8.0, 1mMDTT, 10mMMgCl 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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