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(SASA) | (SASA) | ||
and </span><em><span>so forth</span></em><span>, comprehensive analysis of these datas, we | and </span><em><span>so forth</span></em><span>, comprehensive analysis of these datas, we | ||
− | </span>< | + | </span><strong><span>established a single point mutation database</span></strong><span> (File 1) based on |
FoldX, | FoldX, | ||
− | also we established a </span>< | + | also we established a </span><strong><span>multifunctional enzyme library</span></strong><span> (File 2) |
based | based | ||
on Rosetta and Funclib for activity analysis. It is expected that manganese peroxidase with | on Rosetta and Funclib for activity analysis. It is expected that manganese peroxidase with | ||
higher | higher | ||
temperature stability can be obtained.</span></p> | temperature stability can be obtained.</span></p> | ||
− | <p>< | + | <p><strong><span>Table 1 Mutation sites of ten mutants with the smallest ΔΔG according to computational |
− | simulation.</span></ | + | simulation.</span></strong></p> |
<figure> | <figure> | ||
<table class="lab"> | <table class="lab"> | ||
Line 203: | Line 203: | ||
<p><span> </span><span>By analyzing the established single mutation library, ten mutants with the | <p><span> </span><span>By analyzing the established single mutation library, ten mutants with the | ||
smallest | smallest | ||
− | ΔΔG were selected for stability verification (</span>< | + | ΔΔG were selected for stability verification (</span><strong><span>Table 1</span></strong><span>). We |
applied | applied | ||
site-directed mutagenesis (<a | site-directed mutagenesis (<a | ||
Line 221: | Line 221: | ||
monitoring the oxidation of 2,6-dimethoxyphenol (2,6-DMP) at 469 nm. After comparison, we found | monitoring the oxidation of 2,6-dimethoxyphenol (2,6-DMP) at 469 nm. After comparison, we found | ||
that | that | ||
− | </span>< | + | </span><strong><span>mutant 2</span><sup><span>#</span></sup><span> performed |
− | outstandingly</span></ | + | outstandingly</span></strong><span>. |
</span></p> | </span></p> | ||
<img src="https://static.igem.org/mediawiki/2021/6/6d/T--CPU_CHINA--Part-improvement--Fig_1.png" | <img src="https://static.igem.org/mediawiki/2021/6/6d/T--CPU_CHINA--Part-improvement--Fig_1.png" | ||
Line 232: | Line 232: | ||
<img src="https://static.igem.org/mediawiki/2021/1/15/T--CPU_CHINA--Part-improvement--Fig_2.png" | <img src="https://static.igem.org/mediawiki/2021/1/15/T--CPU_CHINA--Part-improvement--Fig_2.png" | ||
referrerpolicy="no-referrer"> | referrerpolicy="no-referrer"> | ||
− | <p class="imgdescribe">< | + | <p class="imgdescribe"><strong><span>Fig. 2 Effect of temperature on the stability of mutant |
2</span><sup><span>#</span></sup><span> | 2</span><sup><span>#</span></sup><span> | ||
− | and wtMnP after 6 h incubation.</span></ | + | and wtMnP after 6 h incubation.</span></strong><span> </span><i><span>The initial MnP activity |
before | before | ||
incubation was set as 100%. r.t. refers to room | incubation was set as 100%. r.t. refers to room | ||
Line 240: | Line 240: | ||
</span><sup><span>*</span><span>*</span></sup><span>P < 0.01.</span></i></p> | </span><sup><span>*</span><span>*</span></sup><span>P < 0.01.</span></i></p> | ||
<p><span> </span><span>Firstly, we detected the changes in the stability of mutant | <p><span> </span><span>Firstly, we detected the changes in the stability of mutant | ||
− | 2</span><sup><span>#</span></sup><span> over time at different temperatures (</span>< | + | 2</span><sup><span>#</span></sup><span> over time at different temperatures (</span><strong><span>Fig |
− | 1</span></ | + | 1</span></strong><span>). After 2 h incubation, the enzyme activities of mutant |
2</span><sup><span>#</span></sup><span> incubated at different temperatures reduced to varying | 2</span><sup><span>#</span></sup><span> incubated at different temperatures reduced to varying | ||
degrees. It is worth noting that in the subsequent incubation, </span><strong><span>mutant | degrees. It is worth noting that in the subsequent incubation, </span><strong><span>mutant | ||
2</span><sup><span>#</span></sup><span> enzyme activity at 37℃ was | 2</span><sup><span>#</span></sup><span> enzyme activity at 37℃ was | ||
− | improved.</span></strong><span> Compared with wtMnP, </span>< | + | improved.</span></strong><span> Compared with wtMnP, </span><strong><span>the stability of mutant |
2</span><sup><span>#</span></sup><span> at r.t., 50℃, and 60℃ has been significantly | 2</span><sup><span>#</span></sup><span> at r.t., 50℃, and 60℃ has been significantly | ||
improved | improved | ||
− | (Fig 2).</span></ | + | (Fig 2).</span></strong></p> |
<img src="https://static.igem.org/mediawiki/2021/c/c5/T--CPU_CHINA--Part-improvement--Fig_3.png" | <img src="https://static.igem.org/mediawiki/2021/c/c5/T--CPU_CHINA--Part-improvement--Fig_3.png" | ||
referrerpolicy="no-referrer"> | referrerpolicy="no-referrer"> | ||
− | <p class="imgdescribe">< | + | <p class="imgdescribe"><strong><span>Fig. 3 Effect of pH on the stability of mutant 2</span><sup><span>#</span></sup><span> and |
wtMnP | wtMnP | ||
− | after 12 h incubation.</span></ | + | after 12 h incubation.</span></strong><span> </span><i><span>The initial MnP activity before |
incubation was set as 100%. </span><sup><span>**</span></sup><span>P < 0.01.</span></i> | incubation was set as 100%. </span><sup><span>**</span></sup><span>P < 0.01.</span></i> | ||
</p> | </p> | ||
Line 260: | Line 260: | ||
<p><span> After incubation, the stability of mutant 2</span><sup><span>#</span></sup><span> and wtMnP | <p><span> After incubation, the stability of mutant 2</span><sup><span>#</span></sup><span> and wtMnP | ||
under | under | ||
− | different pH condition displayed similar tendencies (</span>< | + | different pH condition displayed similar tendencies (</span><strong><span>Fig 3</span></strong><span>). At |
pH | pH | ||
= 4, the stability between the two enzyme showed a significant difference as mutant | = 4, the stability between the two enzyme showed a significant difference as mutant | ||
Line 266: | Line 266: | ||
<p><span> </span><span>All in all, we screened single point mutations of manganese peroxidase. Mutant | <p><span> </span><span>All in all, we screened single point mutations of manganese peroxidase. Mutant | ||
2</span><sup><span>#</span></sup><span> outcompetes other screened mutants, displayed a | 2</span><sup><span>#</span></sup><span> outcompetes other screened mutants, displayed a | ||
− | </span>< | + | </span><strong><span>significant improvement regarding thermostability</span></strong><span>, and was |
basically | basically | ||
consistent with wtMnP in other aspects. In conclusion, mutant | consistent with wtMnP in other aspects. In conclusion, mutant | ||
− | 2</span><sup><span>#</span></sup><span> is more </span>< | + | 2</span><sup><span>#</span></sup><span> is more </span><strong><span>suitable</span></strong><span> for |
use in | use in | ||
− | </span>< | + | </span><strong><span>higher temperature environments</span></strong><span> than wtMnP, while its applications |
under other physiochemical conditions will not be impaired.</span></p> | under other physiochemical conditions will not be impaired.</span></p> | ||
</div> | </div> |
Revision as of 17:32, 19 October 2021
![](https://static.igem.org/mediawiki/2021/c/c4/T--CPU_CHINA--loading.gif)
![](https://static.igem.org/mediawiki/2021/c/cf/T--CPU_CHINA--Parts--background.png)
IMPROVEMENT
![](https://static.igem.org/mediawiki/2021/b/b2/T--CPU_CHINA--experimentalize.png)
BACKGROUND
As mentioned in our design page, as the most critical enzyme in our multi-enzyme
complex, manganese peroxidase plays a very important role. However, during our experiments, we
found
that the stability of wild-type MnP (wtMnP,
DESIGN
We made rational designs based on thermostability. By introducing parameters including salt bridge, secondary structure, RMSF, RMSD, protein gyration radius (GYRATE), hydrogen bond number, solvent accessibility surface area (SASA) and so forth, comprehensive analysis of these datas, we established a single point mutation database (File 1) based on FoldX, also we established a multifunctional enzyme library (File 2) based on Rosetta and Funclib for activity analysis. It is expected that manganese peroxidase with higher temperature stability can be obtained.
Table 1 Mutation sites of ten mutants with the smallest ΔΔG according to computational simulation.
Mutant No. | Position of amino acids | Mutation | ΔΔG(kcal/mol) |
---|---|---|---|
1# | 74 | E-P | -2.255 |
2# | 74 | E-M | -2.059 |
3# | 182 | D-I | -1.711 |
4# | 182 | D-V | -1.637 |
5# | 182 | D-T | -1.544 |
6# | 232 | S-P | -1.306 |
7# | 74 | E-L | -1.239 |
8# | 78 | S-P | -1.173 |
9# | 183 | Q-P | -1.079 |
10# | 182 | D-C | -0.9288 |
By analyzing the established single mutation library, ten mutants with the smallest ΔΔG were selected for stability verification (Table 1). We applied site-directed mutagenesis ( click here to see primer sequence) to construct our Manganese peroxidase mutants.
RESULT
Finally, mutants 1#, 2#, 5#, 6#, 7#, and 8# were successfully expressed in Pichia pastoris. The enzyme activity of the mutants and wtMnP was compared by monitoring the oxidation of 2,6-dimethoxyphenol (2,6-DMP) at 469 nm. After comparison, we found that mutant 2# performed outstandingly.
![](https://static.igem.org/mediawiki/2021/6/6d/T--CPU_CHINA--Part-improvement--Fig_1.png)
Fig. 1 Thermostability of mutant 2#. The initial MnP activity before incubation was set as 100%.
![](https://static.igem.org/mediawiki/2021/1/15/T--CPU_CHINA--Part-improvement--Fig_2.png)
Fig. 2 Effect of temperature on the stability of mutant 2# and wtMnP after 6 h incubation. The initial MnP activity before incubation was set as 100%. r.t. refers to room temperature. *P < 0.05, **P < 0.01.
Firstly, we detected the changes in the stability of mutant 2# over time at different temperatures (Fig 1). After 2 h incubation, the enzyme activities of mutant 2# incubated at different temperatures reduced to varying degrees. It is worth noting that in the subsequent incubation, mutant 2# enzyme activity at 37℃ was improved. Compared with wtMnP, the stability of mutant 2# at r.t., 50℃, and 60℃ has been significantly improved (Fig 2).
![](https://static.igem.org/mediawiki/2021/c/c5/T--CPU_CHINA--Part-improvement--Fig_3.png)
Fig. 3 Effect of pH on the stability of mutant 2# and wtMnP after 12 h incubation. The initial MnP activity before incubation was set as 100%. **P < 0.01.
Considering that manganese peroxidase may be applied under various complex environments in reality, we subsequently tested its pH stability.
After incubation, the stability of mutant 2# and wtMnP under different pH condition displayed similar tendencies (Fig 3). At pH = 4, the stability between the two enzyme showed a significant difference as mutant 2# demonstrates an improved activity.
All in all, we screened single point mutations of manganese peroxidase. Mutant 2# outcompetes other screened mutants, displayed a significant improvement regarding thermostability, and was basically consistent with wtMnP in other aspects. In conclusion, mutant 2# is more suitable for use in higher temperature environments than wtMnP, while its applications under other physiochemical conditions will not be impaired.