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+ | <img class="background" src="https://static.igem.org/mediawiki/2021/c/cf/T--CPU_CHINA--Parts--background.png" alt=""> | ||
+ | <h1 id="title">IMPROVEMENT</h1> | ||
+ | <svg width="100%" height="100%" viewBox="0 0 1920 600" version="1.1" | ||
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+ | <div class="catalogue-title">IMPROVEMENT</div> | ||
+ | <ul class="catalogue"> | ||
+ | <li class="num"> | ||
+ | <a href="#section1" title="BACKGROUND">BACKGROUND</a> | ||
+ | </li> | ||
− | < | + | <li class="num"> |
− | < | + | <a href="#section2" title="DESIGN">DESIGN</a> |
− | + | </li> | |
− | < | + | <li class="num"> |
− | <p> | + | <a href="#section3" title="RESULT">RESULT</a> |
− | + | </li> | |
− | <p> | + | </ul> |
− | + | </nav> | |
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− | </ | + | </div> |
− | + | <div id="detail" class="clearfix"> | |
− | + | <div class="section" id="section1"> | |
− | < | + | <h2 id='background'>BACKGROUND</h2> |
− | <div class=" | + | <p> 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 |
− | <p> | + | that the stability of wild-type MnP (<strong>wtMnP, |
− | + | <partinfo><a href="http://parts.igem.org/Part:BBa_K500001">BBa_K500001</a></partinfo> | |
− | <p> | + | </strong>) was |
− | < | + | not good enough. Therefore, we tried to |
− | <a href=" | + | enhance |
− | + | its stability through directed evolution. | |
− | </a> | + | </p> |
− | </ | + | </div> |
− | + | <div class="section" id="section2"> | |
− | </ | + | <h2 id='design'>DESIGN</h2> |
− | </div> | + | <p>We made rational designs based on thermostability. By introducing |
+ | <em>parameters including</em> salt bridge, secondary structure, RMSF, | ||
+ | RMSD, | ||
+ | protein gyration radius (GYRATE), hydrogen bond number, solvent accessibility surface area | ||
+ | (SASA) | ||
+ | and <em>so forth</em>, comprehensive analysis of these datas, we | ||
+ | <strong>established a single point mutation database</strong> (<a href="https://static.igem.org/mediawiki/2021/2/27/T--CPU_CHINA--improverment_Average_protein.txt"><strong> File 1</strong></a> ) based on | ||
+ | FoldX, | ||
+ | also we established a <strong>multifunctional enzyme library</strong> (<a href="https://static.igem.org/mediawiki/2021/c/c5/T--CPU_CHINA--improverment_multifunctional_enzyme_library.pdf"><strong>File 2</strong></a>) | ||
+ | based | ||
+ | on Rosetta and Funclib for activity analysis. It is expected that manganese peroxidase with | ||
+ | higher | ||
+ | temperature stability can be obtained. | ||
+ | </p> | ||
+ | <p class="imgdescribe"><strong>Table 1 Mutation sites of ten mutants with the smallest ΔΔG according to | ||
+ | computational | ||
+ | simulation.</strong></p> | ||
+ | <figure> | ||
+ | <table class="lab"> | ||
+ | <thead> | ||
+ | <tr> | ||
+ | <th>Mutant No.</th> | ||
+ | <th>Position of amino acids</th> | ||
+ | <th>Mutation</th> | ||
+ | <th>ΔΔG(kcal/mol)</th> | ||
+ | </tr> | ||
+ | </thead> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td><strong>1#</strong></td> | ||
+ | <td>74</td> | ||
+ | <td>E-P</td> | ||
+ | <td>-2.255</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>2#</strong></td> | ||
+ | <td>74</td> | ||
+ | <td>E-M</td> | ||
+ | <td>-2.059</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>3#</strong></td> | ||
+ | <td>182</td> | ||
+ | <td>D-I</td> | ||
+ | <td>-1.711</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>4#</strong></td> | ||
+ | <td>182</td> | ||
+ | <td>D-V</td> | ||
+ | <td>-1.637</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>5#</strong></td> | ||
+ | <td>182</td> | ||
+ | <td>D-T</td> | ||
+ | <td>-1.544</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>6#</strong></td> | ||
+ | <td>232</td> | ||
+ | <td>S-P</td> | ||
+ | <td>-1.306</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>7#</strong></td> | ||
+ | <td>74</td> | ||
+ | <td>E-L</td> | ||
+ | <td>-1.239</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>8#</strong></td> | ||
+ | <td>78</td> | ||
+ | <td>S-P</td> | ||
+ | <td>-1.173</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>9#</strong></td> | ||
+ | <td>183</td> | ||
+ | <td>Q-P</td> | ||
+ | <td>-1.079</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>10#</strong></td> | ||
+ | <td>182</td> | ||
+ | <td>D-C</td> | ||
+ | <td>-0.9288</td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | </figure> | ||
+ | <p> By analyzing the established single mutation library, ten mutants with the | ||
+ | smallest | ||
+ | ΔΔG were selected for stability verification (<strong>Table 1</strong>). We | ||
+ | applied | ||
+ | site-directed mutagenesis (<a | ||
+ | href="https://static.igem.org/mediawiki/2021/c/c0/T--CPU_CHINA--Part--improvement--Primer_sequence_designed_for_MnP_mutant_construction.pdf" | ||
+ | target="_blank" rel="noopener noreferrer">click here to see primer sequence</a>) to | ||
+ | construct our Manganese | ||
+ | peroxidase | ||
+ | mutants.</p> | ||
+ | </div> | ||
+ | <div class="section" id="section3"> | ||
+ | <h2 id='result'>RESULT</h2> | ||
+ | <p>Finally, mutants 1<sup>#</sup>, | ||
+ | 2<sup>#</sup>, 5<sup>#</sup>, | ||
+ | 6<sup>#</sup>, 7<sup>#</sup>, and | ||
+ | 8<sup>#</sup> were successfully expressed in <em>Pichia | ||
+ | pastoris</em>. The enzyme activity of the mutants and wtMnP was compared by | ||
+ | monitoring the oxidation of 2, 6-dimethoxyphenol (2, 6-DMP) at 469 nm<sup>[1]</sup>. After | ||
+ | comparison, we found | ||
+ | that | ||
+ | <strong>mutant 2<sup>#</sup>(<a href="http://parts.igem.org/Part:BBa_K3853014">BBa_K3853014</a>) performed | ||
+ | outstandingly</strong>. | ||
+ | </p> | ||
+ | <img src="https://static.igem.org/mediawiki/2021/6/6d/T--CPU_CHINA--Part-improvement--Fig_1.png" | ||
+ | referrerpolicy="no-referrer"> | ||
+ | <p class="imgdescribe"><strong>Fig. 1 Thermostability of mutant | ||
+ | 2<sup>#</sup>.</strong> <em class="reference">The initial | ||
+ | MnP | ||
+ | activity before incubation was set as 100%</em>.</p> | ||
+ | <img src="https://static.igem.org/mediawiki/2021/1/15/T--CPU_CHINA--Part-improvement--Fig_2.png" | ||
+ | referrerpolicy="no-referrer"> | ||
+ | <p class="imgdescribe"><strong>Fig. 2 Effect of temperature on the stability of mutant | ||
+ | 2<sup>#</sup> | ||
+ | and wtMnP after 6 h incubation.</strong><em class="reference">The initial MnP activity | ||
+ | before | ||
+ | incubation was set as 100%. r.t. refers to room | ||
+ | temperature. <sup>*</sup>P < 0.05, | ||
+ | <sup>**</sup>P < 0.01.</em></p> | ||
+ | <p> Firstly, we detected the changes in the stability of mutant | ||
+ | 2<sup>#</sup> over time at different temperatures (<strong>Fig | ||
+ | 1</strong>). After 2 h incubation, the enzyme activities of mutant | ||
+ | 2<sup>#</sup> incubated at different temperatures reduced to varying | ||
+ | degrees. It is worth noting that in the subsequent incubation, <strong>mutant | ||
+ | 2<sup>#</sup> enzyme activity at 37℃ was | ||
+ | improved.</strong> Compared with wtMnP, <strong>the stability of mutant | ||
+ | 2<sup>#</sup> at r.t., 50℃, and 60℃ has been significantly | ||
+ | improved | ||
+ | (Fig 2).</strong></p> | ||
+ | <img src="https://static.igem.org/mediawiki/2021/c/c5/T--CPU_CHINA--Part-improvement--Fig_3.png" | ||
+ | referrerpolicy="no-referrer"> | ||
+ | <p class="imgdescribe"><strong>Fig. 3 Effect of pH on the stability of mutant 2<sup>#</sup> and | ||
+ | wtMnP | ||
+ | after 12 h incubation.</strong> <em class="reference">The initial MnP activity before | ||
+ | incubation was set as 100%. <sup>**</sup>P < 0.01.</em> | ||
+ | </p> | ||
+ | <p> Considering that manganese peroxidase may be applied under various complex | ||
+ | environments in reality, we subsequently tested its pH stability. </p> | ||
+ | <p> After incubation, the stability of mutant 2<sup>#</sup> and wtMnP | ||
+ | under | ||
+ | different pH condition displayed similar tendencies (<strong>Fig 3</strong>). At | ||
+ | pH | ||
+ | = 4, the stability between the two enzyme showed a significant difference as mutant | ||
+ | 2<sup>#</sup> demonstrates an improved activity. </p> | ||
+ | <p> All in all, we screened single point mutations of manganese peroxidase. Mutant | ||
+ | 2<sup>#</sup> outcompetes other screened mutants, displayed a | ||
+ | <strong>significant improvement regarding thermostability</strong>, and was | ||
+ | basically | ||
+ | consistent with wtMnP in other aspects. In conclusion, mutant | ||
+ | 2<sup>#</sup> is more <strong>suitable</strong> for | ||
+ | use in | ||
+ | <strong>higher temperature environments</strong> than wtMnP, while its applications | ||
+ | under other physiochemical conditions will not be impaired. | ||
+ | </p> | ||
+ | <h4><strong><span>Reference</span></strong></h4> | ||
+ | <p class="reference">[1] Wariishi, H., Valli, K. & Gold, M. H. Manganese(II) oxidation by manganese | ||
+ | peroxidase from the basidiomycete Phanerochaete chrysosporium. Kinetic mechanism and role of | ||
+ | chelators. <em>The Journal of biological chemistry</em> <strong>267</strong>, 23688-23695 (1992). | ||
+ | </p> | ||
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Latest revision as of 21:06, 21 October 2021
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![](https://static.igem.org/mediawiki/2021/c/cf/T--CPU_CHINA--Parts--background.png)
IMPROVEMENT
![](https://static.igem.org/mediawiki/2021/3/38/T--CPU_CHINA--flying.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[1]. After comparison, we found that mutant 2#(BBa_K3853014) 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.
Reference
[1] Wariishi, H., Valli, K. & Gold, M. H. Manganese(II) oxidation by manganese peroxidase from the basidiomycete Phanerochaete chrysosporium. Kinetic mechanism and role of chelators. The Journal of biological chemistry 267, 23688-23695 (1992).