Difference between revisions of "Team:CPU CHINA/Improve"

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         <div id="detail" class="clearfix">
 
         <div id="detail" class="clearfix">
 
             <div class="section" id="section1">
 
             <div class="section" id="section1">
−
                 <h2 id='background'><span>BACKGROUND</span></h2>
+
                 <h2 id='background'>BACKGROUND</h2>
−
                 <p><span> </span><span>As mentioned in our design page, as the most critical enzyme in our multi-enzyme
+
                 <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
 
                         complex, manganese peroxidase plays a very important role. However, during our experiments, we
 
                         found
 
                         found
−
                         that the stability of wild-type MnP (<strong>wtMnP, </span>
+
                         that the stability of wild-type MnP (<strong>wtMnP,  
−
                             <partinfo><a href="http://parts.igem.org/Part:BBa_K500001">BBa_K500001</a></partinfo><span></em>) was not good enough. Therefore, we tried to
+
                             <partinfo><a href="http://parts.igem.org/Part:BBa_K500001">BBa_K500001</a></partinfo></em>) was not good enough. Therefore, we tried to
 
                         enhance
 
                         enhance
−
                         its stability through directed evolution.</span>
+
                         its stability through directed evolution.
 
                 </p>
 
                 </p>
 
             </div>
 
             </div>
 
             <div class="section" id="section2">
 
             <div class="section" id="section2">
−
                 <h2 id='design'><span>DESIGN</span></h2>
+
                 <h2 id='design'>DESIGN</h2>
−
                <p><span> </span><span>We made rational designs based on thermostability. By introducing
+
                </span<p>>We made rational designs based on thermostability. By introducing
−
                     </span><em><span>parameters including</span></em><span> salt bridge, secondary structure, RMSF,
+
                     <em>parameters including</em> salt bridge, secondary structure, RMSF,
 
                         RMSD,
 
                         RMSD,
 
                         protein gyration radius (GYRATE), hydrogen bond number, solvent accessibility surface area
 
                         protein gyration radius (GYRATE), hydrogen bond number, solvent accessibility surface area
 
                         (SASA)
 
                         (SASA)
−
                         and </span><em><span>so forth</span></em><span>, comprehensive analysis of these datas, we
+
                         and <em>so forth</em>, comprehensive analysis of these datas, we
−
                     </span><strong><span>established a single point mutation database</span></strong><span> (File 1) based on
+
                     <strong>established a single point mutation database</strong> (File 1) based on
 
                         FoldX,
 
                         FoldX,
−
                         also we established a </span><strong><span>multifunctional enzyme library</span></strong><span> (File 2)
+
                         also we established a <strong>multifunctional enzyme library</strong> (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.</p>
−
                 <p class="imgdescribe"><strong><span>Table 1 Mutation sites of ten mutants with the smallest ΔΔG according to computational
+
                 <p class="imgdescribe"><strong>Table 1 Mutation sites of ten mutants with the smallest ΔΔG according to computational
−
                             simulation.</span></strong></p>
+
                             simulation.</strong></p>
 
                 <figure>
 
                 <figure>
 
                     <table class="lab">
 
                     <table class="lab">
 
                         <thead>
 
                         <thead>
 
                             <tr>
 
                             <tr>
−
                                 <th><span>Mutant No.</span></th>
+
                                 <th>Mutant No.</th>
−
                                 <th><span>Position of amino acids</span></th>
+
                                 <th>Position of amino acids</th>
−
                                 <th><span>Mutation</span></th>
+
                                 <th>Mutation</th>
−
                                 <th><span>ΔΔG(kcal/mol)</span></th>
+
                                 <th>ΔΔG(kcal/mol)</th>
 
                             </tr>
 
                             </tr>
 
                         </thead>
 
                         </thead>
 
                         <tbody>
 
                         <tbody>
 
                             <tr>
 
                             <tr>
−
                                 <td><strong><span>1#</span></strong></td>
+
                                 <td><strong>1#</strong></td>
−
                                 <td><span>74</span></td>
+
                                 <td>74</td>
−
                                 <td><span>E-P</span></td>
+
                                 <td>E-P</td>
−
                                 <td><span>-2.255</span></td>
+
                                 <td>-2.255</td>
 
                             </tr>
 
                             </tr>
 
                             <tr>
 
                             <tr>
−
                                 <td><strong><span>2#</span></strong></td>
+
                                 <td><strong>2#</strong></td>
−
                                 <td><span>74</span></td>
+
                                 <td>74</td>
−
                                 <td><span>E-M</span></td>
+
                                 <td>E-M</td>
−
                                 <td><span>-2.059</span></td>
+
                                 <td>-2.059</td>
 
                             </tr>
 
                             </tr>
 
                             <tr>
 
                             <tr>
−
                                 <td><strong><span>3#</span></strong></td>
+
                                 <td><strong>3#</strong></td>
−
                                 <td><span>182</span></td>
+
                                 <td>182</td>
−
                                 <td><span>D-I</span></td>
+
                                 <td>D-I</td>
−
                                 <td><span>-1.711</span></td>
+
                                 <td>-1.711</td>
 
                             </tr>
 
                             </tr>
 
                             <tr>
 
                             <tr>
−
                                 <td><strong><span>4#</span></strong></td>
+
                                 <td><strong>4#</strong></td>
−
                                 <td><span>182</span></td>
+
                                 <td>182</td>
−
                                 <td><span>D-V</span></td>
+
                                 <td>D-V</td>
−
                                 <td><span>-1.637</span></td>
+
                                 <td>-1.637</td>
 
                             </tr>
 
                             </tr>
 
                             <tr>
 
                             <tr>
−
                                 <td><strong><span>5#</span></strong></td>
+
                                 <td><strong>5#</strong></td>
−
                                 <td><span>182</span></td>
+
                                 <td>182</td>
−
                                 <td><span>D-T</span></td>
+
                                 <td>D-T</td>
−
                                 <td><span>-1.544</span></td>
+
                                 <td>-1.544</td>
 
                             </tr>
 
                             </tr>
 
                             <tr>
 
                             <tr>
−
                                 <td><strong><span>6#</span></strong></td>
+
                                 <td><strong>6#</strong></td>
−
                                 <td><span>232</span></td>
+
                                 <td>232</td>
−
                                 <td><span>S-P</span></td>
+
                                 <td>S-P</td>
−
                                 <td><span>-1.306</span></td>
+
                                 <td>-1.306</td>
 
                             </tr>
 
                             </tr>
 
                             <tr>
 
                             <tr>
−
                                 <td><strong><span>7#</span></strong></td>
+
                                 <td><strong>7#</strong></td>
−
                                 <td><span>74</span></td>
+
                                 <td>74</td>
−
                                 <td><span>E-L</span></td>
+
                                 <td>E-L</td>
−
                                 <td><span>-1.239</span></td>
+
                                 <td>-1.239</td>
 
                             </tr>
 
                             </tr>
 
                             <tr>
 
                             <tr>
−
                                 <td><strong><span>8#</span></strong></td>
+
                                 <td><strong>8#</strong></td>
−
                                 <td><span>78</span></td>
+
                                 <td>78</td>
−
                                 <td><span>S-P</span></td>
+
                                 <td>S-P</td>
−
                                 <td><span>-1.173</span></td>
+
                                 <td>-1.173</td>
 
                             </tr>
 
                             </tr>
 
                             <tr>
 
                             <tr>
−
                                 <td><strong><span>9#</span></strong></td>
+
                                 <td><strong>9#</strong></td>
−
                                 <td><span>183</span></td>
+
                                 <td>183</td>
−
                                 <td><span>Q-P</span></td>
+
                                 <td>Q-P</td>
−
                                 <td><span>-1.079</span></td>
+
                                 <td>-1.079</td>
 
                             </tr>
 
                             </tr>
 
                             <tr>
 
                             <tr>
−
                                 <td><strong><span>10#</span></strong></td>
+
                                 <td><strong>10#</strong></td>
−
                                 <td><span>182</span></td>
+
                                 <td>182</td>
−
                                 <td><span>D-C</span></td>
+
                                 <td>D-C</td>
−
                                 <td><span>-0.9288</span></td>
+
                                 <td>-0.9288</td>
 
                             </tr>
 
                             </tr>
 
                         </tbody>
 
                         </tbody>
 
                     </table>
 
                     </table>
 
                 </figure>
 
                 </figure>
−
                 <p><span> </span><span>By analyzing the established single mutation library, ten mutants with the
+
                 <p> By analyzing the established single mutation library, ten mutants with the
 
                         smallest
 
                         smallest
−
                         ΔΔG were selected for stability verification (</span><strong><span>Table 1</span></strong><span>). We
+
                         ΔΔG were selected for stability verification (<strong>Table 1</strong>). We
 
                         applied
 
                         applied
 
                         site-directed mutagenesis (<a
 
                         site-directed mutagenesis (<a
Line 218: Line 218:
 
                         construct our Manganese
 
                         construct our Manganese
 
                         peroxidase
 
                         peroxidase
−
                         mutants.</span></p>
+
                         mutants.</p>
 
             </div>
 
             </div>
 
             <div class="section" id="section3">
 
             <div class="section" id="section3">
−
                 <h2 id='result'><span>RESULT</span></h2>
+
                 <h2 id='result'>RESULT</h2>
−
                 <p><span> </span><span>Finally, mutants 1</span><sup><span>#</span></sup><span>,
+
                 <p> Finally, mutants 1<sup>#</sup>,
−
                         2</span><sup><span>#</span></sup><span>, 5</span><sup><span>#</span></sup><span>,
+
                         2<sup>#</sup>, 5<sup>#</sup>,
−
                         6</span><sup><span>#</span></sup><span>, 7</span><sup><span>#</span></sup><span>, and
+
                         6<sup>#</sup>, 7<sup>#</sup>, and
−
                         8</span><sup><span>#</span></sup><span> were successfully expressed in </span><em><span>Pichia
+
                         8<sup>#</sup> were successfully expressed in <em>Pichia
−
                             pastoris</span></em><span>. The enzyme activity of the mutants and wtMnP was compared by
+
                             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. After comparison, we found
 
                         monitoring the oxidation of 2, 6-dimethoxyphenol (2, 6-DMP) at 469 nm. After comparison, we found
 
                         that
 
                         that
−
                     </span><strong><span>mutant 2</span><sup><span>#</span></sup><span> performed
+
                     <strong>mutant 2<sup>#</sup> performed
−
                             outstandingly</span></strong><span>.
+
                             outstandingly</strong>.
−
                     </span></p>
+
                     </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"
 
                     referrerpolicy="no-referrer">
 
                     referrerpolicy="no-referrer">
−
                 <p class="imgdescribe"><strong><span>Fig. 1 Thermostability of mutant
+
                 <p class="imgdescribe"><strong>Fig. 1 Thermostability of mutant
−
                             2</span><sup><span>#</span></sup><span>.</span></strong><span> </span><em class="reference"><span>The initial
+
                             2<sup>#</sup>.</strong> <em class="reference">The initial
 
                             MnP
 
                             MnP
−
                             activity before incubation was set as 100%</span></em><span>.</span></p>
+
                             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"
 
                 <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"><strong><span>Fig. 2 Effect of temperature on the stability of mutant
+
                 <p class="imgdescribe"><strong>Fig. 2 Effect of temperature on the stability of mutant
−
                             2</span><sup><span>#</span></sup><span>
+
                             2<sup>#</sup>
−
                             and wtMnP after 6 h incubation.</span></strong><em class="reference"><span>The initial MnP activity
+
                             and wtMnP after 6 h incubation.</strong><em class="reference">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
−
                             temperature. </span><sup><span>*</span></sup><span>P &lt; 0.05,
+
                             temperature. <sup>*</sup>P &lt; 0.05,
−
                         </span><sup><span>*</span><span>*</span></sup><span>P &lt; 0.01.</span></em></p>
+
                         <sup>**</sup>P &lt; 0.01.</em></p>
−
                 <p><span> </span><span>Firstly, we detected the changes in the stability of mutant
+
                 <p> Firstly, we detected the changes in the stability of mutant
−
                         2</span><sup><span>#</span></sup><span> over time at different temperatures (</span><strong><span>Fig
+
                         2<sup>#</sup> over time at different temperatures (<strong>Fig
−
                             1</span></strong><span>). After 2 h incubation, the enzyme activities of mutant
+
                             1</strong>). After 2 h incubation, the enzyme activities of mutant
−
                         2</span><sup><span>#</span></sup><span> incubated at different temperatures reduced to varying
+
                         2<sup>#</sup> 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, <strong>mutant
−
                             2</span><sup><span>#</span></sup><span> enzyme activity at 37℃ was
+
                             2<sup>#</sup> enzyme activity at 37℃ was
−
                             improved.</span></strong><span> Compared with wtMnP, </span><strong><span>the stability of mutant
+
                             improved.</strong> Compared with wtMnP, <strong>the stability of mutant
−
                             2</span><sup><span>#</span></sup><span> at r.t., 50℃, and 60℃ has been significantly
+
                             2<sup>#</sup> at r.t., 50℃, and 60℃ has been significantly
 
                             improved
 
                             improved
−
                             (Fig 2).</span></strong></p>
+
                             (Fig 2).</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"><strong><span>Fig. 3 Effect of pH on the stability of mutant 2</span><sup><span>#</span></sup><span> and
+
                 <p class="imgdescribe"><strong>Fig. 3 Effect of pH on the stability of mutant 2<sup>#</sup> and
 
                             wtMnP
 
                             wtMnP
−
                             after 12 h incubation.</span></strong><span> </span><em class="reference"><span>The initial MnP activity before
+
                             after 12 h incubation.</strong> <em class="reference">The initial MnP activity before
−
                             incubation was set as 100%. </span><sup><span>**</span></sup><span>P &lt; 0.01.</span></em>
+
                             incubation was set as 100%. <sup>**</sup>P &lt; 0.01.</em>
 
                 </p>
 
                 </p>
−
                 <p><span> </span><span>Considering that manganese peroxidase may be applied under various complex
+
                 <p> Considering that manganese peroxidase may be applied under various complex
−
                         environments in reality, we subsequently tested its pH stability. </span></p>
+
                         environments in reality, we subsequently tested its pH stability. </p>
−
                 <p><span> After incubation, the stability of mutant 2</span><sup><span>#</span></sup><span> and wtMnP
+
                 <p> After incubation, the stability of mutant 2<sup>#</sup> and wtMnP
 
                         under
 
                         under
−
                         different pH condition displayed similar tendencies (</span><strong><span>Fig 3</span></strong><span>). At
+
                         different pH condition displayed similar tendencies (<strong>Fig 3</strong>). 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
−
                         2</span><sup><span>#</span></sup><span> demonstrates an improved activity. </span></p>
+
                         2<sup>#</sup> demonstrates an improved activity. </p>
−
                 <p><span> </span><span>All in all, we screened single point mutations of manganese peroxidase. Mutant
+
                 <p> 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<sup>#</sup> outcompetes other screened mutants, displayed a
−
                     </span><strong><span>significant improvement regarding thermostability</span></strong><span>, and was
+
                     <strong>significant improvement regarding thermostability</strong>, 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><strong><span>suitable</span></strong><span> for
+
                         2<sup>#</sup> is more <strong>suitable</strong> for
 
                         use in
 
                         use in
−
                     </span><strong><span>higher temperature environments</span></strong><span> than wtMnP, while its applications
+
                     <strong>higher temperature environments</strong> than wtMnP, while its applications
−
                         under other physiochemical conditions will not be impaired.</span></p>
+
                         under other physiochemical conditions will not be impaired.</p>
 
             </div>
 
             </div>
 
         </div>
 
         </div>

Revision as of 18:46, 19 October 2021

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, BBa_K500001) was not good enough. Therefore, we tried to enhance its stability through directed evolution.

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.

Fig. 1 Thermostability of mutant 2#. The initial MnP activity before incubation was set as 100%.

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).

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.