Difference between revisions of "Part:BBa K3853059"

 
 
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<partinfo>BBa_K3853059 short</partinfo>
 
<partinfo>BBa_K3853059 short</partinfo>
  
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<p>Manganese peroxidase (MnP) is the key enzyme in our degrading system. In order to improve its catalyzing ability, we tried rational design. And  according to the computational redesign results, 6 mutants were chosen  and tested, including their relative enzyme activity and the effect of  temperature/pH/organic solvents on them. MnP(S232P) is one of the most promising mutant of MnP. We use <partinfo>BBa_K3853059</partinfo>  to construct the expression system to express and purify the protein.</p>
  
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===Biology===
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===Usage and Biology===
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Manganese peroxidase (MnP), a glycosylated heme enzyme derived from the white-rot fungus <i>Phanerochaete chrysosporium</i>, can oxidize Mn<sup>2+</sup> to Mn<sup>3+</sup> under the action of H<sub>2</sub>O<sub>2</sub>. Mn<sup>3+</sup> can be released outside the enzyme under the action of a chelate such as malonic acid and can oxidise a wide range of phenolic and non-phenolic compounds as a common substrate. The Mn<sup>3+</sup>-malonic acid chelate can be detected at 469 nm by oxidation of 2,6-dimethyloxyphenol (2,6-DMP), which is also the main enzyme activity detection method for MnP. MnP (S232P) is obtained by mutating the serine at position 232 of wild-type MnP (<partinfo>BBa_K3853000</partinfo>) to proline.
  
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===Usage===
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<p>We mutated the serine at position 232 of wild-type MnP to proline through single-point mutation in order to improve the stability of wild-type MnP. We use <partinfo>BBa_K3853059</partinfo>  to construct the expression system to express and purify the protein.</p>
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===Characterization===
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<p><b>1. Identification</b></p>
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After receiving the synthetic plasmid, we electrotransformed it into <i>Pichia pastoris</i>, and selected monoclonal colonies for colony PCR to verify the successful transformation.
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    <figure style="display: flex; justify-content: center; align-items: center;">
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        <img src="https://2021.igem.org/wiki/images/d/dd/T--CPU_CHINA--gel_fig_4.PNG" width="70%" style="float:center;">
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<p style="text-align:center"><b>Fig. 1 Agarose gel electrophoresis of PCR products of monoclonal colonies of MnP (S232P).</b> <i>lane 4 for MnP (S232P)</i></p>
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<p><b>2. Proof of the expression</b></p>
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After the expressed protein was re-dissolved by ammonium sulfate precipitation, it was verified by running gel, and the target protein band was observed by SDS-PAGE (<b>Fig. 2</b>).
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        <img src="https://2021.igem.org/wiki/images/3/37/T--CPU_CHINA--PART_FIG1.png" width="70%" style="float:center;">
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<p style="text-align:center"><b>Fig. 2 SDS-PAGE of MnP (S232P)</b>. <i>lane 4 for MnP (S232P)</i></p>
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<p><b>3. Enzyme Activity</b></p>
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<p>MnP activity of MnP (S232P) was measured by monitoring the oxidation of 2,6-dimethyloxyphenol (2,6-DMP) at 469 nm<sup>[1]</sup>. H<sub>2</sub>O<sub>2</sub> concentration were determined using ε<sub>240</sub> = 43.6 M<sup>-1</sup> cm<sup>-1</sup>.The reaction mixtures contained 0.4 mM MnSO<sub>4</sub>, 50 mM sodium malonate (pH 4.5), and 1 mM 2, 6-DMP. For a 96-well plate, 140 μl of the above reaction mixtures and 20 μl enzyme solution were mixed uniformly in advance and then 40 μl 0.1 mM H<sub>2</sub>O<sub>2</sub> were added to initiate reaction. The concentration of 2, 6-DMP's oxidation products, 2, 2', 6, 6'-tetramethoxydibenzo-1, 1'-diquinone, were determined using ε<sub>469</sub> = 49.6 mM<sup>-1</sup> cm<sup>-1</sup>. One unit (U) of MnP activity is defined as the amount of enzyme required to convert 1 μM 2, 6-DMP to 2, 2', 6, 6'-tetramethoxydibenzo-1, 1'-diquinone in 1 minute.</p>
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<p>As shown in <b>Fig. 3A</b>, the absorbance of the reaction system with MnP (S232P) continued to rise within 1 min, while the absorbance of the control group (without enzyme) did not change. Through UV-visible spectrum of the reaction system after 1 min, the characteristic absorption at 469 nm was observed (<b>Fig. 3B</b>).</p>
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<html>
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    <figure style="display: flex; justify-content: center; align-items: center;">
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        <img src="https://2021.igem.org/wiki/images/4/48/T--CPU_CHINA--BBa_K3853016_fig_3.png" width="70%" style="float:center;">
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        <figcaption>
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<p style="text-align:center"><b>Fig. 3 The detection of 2, 2', 6, 6'-tetramethoxydibenzo-1, 1'-diquinone.</b> <i>Mutant 6<sup>#</sup> refers to MnP (S232P). Control group refers to the reaction system without enzyme. <b>A:</b> The absorbance change at 469 nm in the reaction system within 1 min. <b>B:</b> UV-visible spectrum of the reaction system after 1 min.</i></p>
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<p><b>4. Thermostability</b></p>
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<p>To evaluate thermal stability, the purified MnP (S232P) were incubated in 20 mM sodium malonate buffer (pH 5.5) with 100 mM NaCl at different temperature for 6 h (<b>Fig. 4</b>) and the residual enzyme activity were measured and calculated every 2 h. The relative enzyme activity under different temperatures were calculated with the following equation: </p>
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    <figure style="display: flex; justify-content: center; align-items: center;">
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        <img src="https://2021.igem.org/wiki/images/c/ca/T--CPU_CHINA--BBa_K3853008_fig_B.png" width="60%" style="float:center;">
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<p>As shown in <b>Fig. 4</b>, relative enzyme activity of MnP (S232P) under different incubation temperatures displayed distinct characteristics. When the temperature reached  to 37℃, enzyme activity slightly decreased within 2 h, while a sharp decline of enzyme activity could be observed while temperature exceeded 60℃, but both above gradually stabilized in the following 4 hours. </p>
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    <figure style="display: flex; justify-content: center; align-items: center;">
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        <img src="https://2021.igem.org/wiki/images/7/70/T--CPU_CHINA--BBa_K3853016_fig_4.png" width="70%" style="float:center;">
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<p style="text-align:center"><b>Fig. 4 Thermal stability of MnP (S232P).</b> <i>The initial MnP activity before incubation was set as 100%.</i></p>
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<p>Compared to the MnP without any mutation, MnP (S232P)'s thermostability had significantly decreased as it exhibited poorer relative enzyme activity at 37℃ and 50℃ after 6 h incubation (<b>Fig. 5</b>).</p>
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<html>
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    <figure style="display: flex; justify-content: center; align-items: center;">
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        <img src="https://2021.igem.org/wiki/images/3/37/T--CPU_CHINA--BBa_K3853016_fig_5.png" width="70%" style="float:center;">
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        <figcaption>
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<p style="text-align:center"><b>Fig. 5 Effect of temperature on the stability of MnP (S232P) and MnP after 6 h incubation.</b> <i>The initial MnP activity before incubation was set as 100%. Mutant 6<sup>#</sup> refers to MnP (S232P). <sup>*</sup>P < 0.05, <sup>**</sup>P < 0.01.</i></p>
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<p><b>5. pH stability</b></p>
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<p>To evaluate pH stability, the purified MnP (S232P) were incubated in 20 mM sodium malonate buffer with 100 mM NaCl under pH 3-7 for 12 h at room temperature. The relative enzyme activity at different pH conditions were calculated with the following equation:</p>
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    <figure style="display: flex; justify-content: center; align-items: center;">
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        <img src="https://2021.igem.org/wiki/images/f/fa/T--CPU_CHINA--BBa_K3853008_fig_C.png" width="60%" style="float:center;">
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        <figcaption>
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<p>Compared to the MnP without any mutation, MnP (S232P) exhibited poorer performance at low pH range (pH 3-6) (<b>Fig. 6</b>), which may caused by the decrease in the number of hydrogen bonds after mutating the serine at position 232 to proline, as the former could form a large number of hydrogen bonds with surrounding amino acid residues before the mutation(<b>Fig. 7</b>). </p>
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    <figure style="display: flex; justify-content: center; align-items: center;">
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        <img src="https://2021.igem.org/wiki/images/5/55/T--CPU_CHINA--BBa_K3853016_fig_6.png" width="70%" style="float:center;">
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        <figcaption>
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        </p>
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<p style="text-align:center"><b>Fig. 6 Effect of pH on the stability of MnP (S232P) and MnP after 12 h incubation.</b> <i>The initial MnP activity before incubation was set as 100%. Mutant 6<sup>#</sup> refers to MnP (S232P). <sup>*</sup>P <0.05, <sup>**</sup>P < 0.01.</i></p>
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<p style="text-align:center"><b>Fig. 7 Position of Ser232 in wild-type MnP.</b> <i>The residue in wheat color refers to 232S.  The dashed line indicates the hydrogen bond formed by serine at position 232 and the surrounding amino acid residues. For serine, the red dashed line indicates the hydrogen bond acceptor, while the blue dashed line indicates the hydrogen bond donor. Green ball indicates Ca<sup>2+</sup> and purple ball indicates Mn<sup>2+</sup>.</i></p>
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 22:47, 21 October 2021


PAOX1-α-factor-his-tag-MnP(S232P)-AOX1 Terminator

Manganese peroxidase (MnP) is the key enzyme in our degrading system. In order to improve its catalyzing ability, we tried rational design. And according to the computational redesign results, 6 mutants were chosen and tested, including their relative enzyme activity and the effect of temperature/pH/organic solvents on them. MnP(S232P) is one of the most promising mutant of MnP. We use BBa_K3853059 to construct the expression system to express and purify the protein.

Biology

Manganese peroxidase (MnP), a glycosylated heme enzyme derived from the white-rot fungus Phanerochaete chrysosporium, can oxidize Mn2+ to Mn3+ under the action of H2O2. Mn3+ can be released outside the enzyme under the action of a chelate such as malonic acid and can oxidise a wide range of phenolic and non-phenolic compounds as a common substrate. The Mn3+-malonic acid chelate can be detected at 469 nm by oxidation of 2,6-dimethyloxyphenol (2,6-DMP), which is also the main enzyme activity detection method for MnP. MnP (S232P) is obtained by mutating the serine at position 232 of wild-type MnP (BBa_K3853000) to proline.

Usage

We mutated the serine at position 232 of wild-type MnP to proline through single-point mutation in order to improve the stability of wild-type MnP. We use BBa_K3853059 to construct the expression system to express and purify the protein.

Characterization

1. Identification

After receiving the synthetic plasmid, we electrotransformed it into Pichia pastoris, and selected monoclonal colonies for colony PCR to verify the successful transformation.

Fig. 1 Agarose gel electrophoresis of PCR products of monoclonal colonies of MnP (S232P). lane 4 for MnP (S232P)

2. Proof of the expression

After the expressed protein was re-dissolved by ammonium sulfate precipitation, it was verified by running gel, and the target protein band was observed by SDS-PAGE (Fig. 2).

Fig. 2 SDS-PAGE of MnP (S232P). lane 4 for MnP (S232P)

3. Enzyme Activity

MnP activity of MnP (S232P) was measured by monitoring the oxidation of 2,6-dimethyloxyphenol (2,6-DMP) at 469 nm[1]. H2O2 concentration were determined using ε240 = 43.6 M-1 cm-1.The reaction mixtures contained 0.4 mM MnSO4, 50 mM sodium malonate (pH 4.5), and 1 mM 2, 6-DMP. For a 96-well plate, 140 μl of the above reaction mixtures and 20 μl enzyme solution were mixed uniformly in advance and then 40 μl 0.1 mM H2O2 were added to initiate reaction. The concentration of 2, 6-DMP's oxidation products, 2, 2', 6, 6'-tetramethoxydibenzo-1, 1'-diquinone, were determined using ε469 = 49.6 mM-1 cm-1. One unit (U) of MnP activity is defined as the amount of enzyme required to convert 1 μM 2, 6-DMP to 2, 2', 6, 6'-tetramethoxydibenzo-1, 1'-diquinone in 1 minute.

As shown in Fig. 3A, the absorbance of the reaction system with MnP (S232P) continued to rise within 1 min, while the absorbance of the control group (without enzyme) did not change. Through UV-visible spectrum of the reaction system after 1 min, the characteristic absorption at 469 nm was observed (Fig. 3B).

Fig. 3 The detection of 2, 2', 6, 6'-tetramethoxydibenzo-1, 1'-diquinone. Mutant 6# refers to MnP (S232P). Control group refers to the reaction system without enzyme. A: The absorbance change at 469 nm in the reaction system within 1 min. B: UV-visible spectrum of the reaction system after 1 min.

4. Thermostability

To evaluate thermal stability, the purified MnP (S232P) were incubated in 20 mM sodium malonate buffer (pH 5.5) with 100 mM NaCl at different temperature for 6 h (Fig. 4) and the residual enzyme activity were measured and calculated every 2 h. The relative enzyme activity under different temperatures were calculated with the following equation:

As shown in Fig. 4, relative enzyme activity of MnP (S232P) under different incubation temperatures displayed distinct characteristics. When the temperature reached to 37℃, enzyme activity slightly decreased within 2 h, while a sharp decline of enzyme activity could be observed while temperature exceeded 60℃, but both above gradually stabilized in the following 4 hours.

Fig. 4 Thermal stability of MnP (S232P). The initial MnP activity before incubation was set as 100%.

Compared to the MnP without any mutation, MnP (S232P)'s thermostability had significantly decreased as it exhibited poorer relative enzyme activity at 37℃ and 50℃ after 6 h incubation (Fig. 5).

Fig. 5 Effect of temperature on the stability of MnP (S232P) and MnP after 6 h incubation. The initial MnP activity before incubation was set as 100%. Mutant 6# refers to MnP (S232P). *P < 0.05, **P < 0.01.


5. pH stability

To evaluate pH stability, the purified MnP (S232P) were incubated in 20 mM sodium malonate buffer with 100 mM NaCl under pH 3-7 for 12 h at room temperature. The relative enzyme activity at different pH conditions were calculated with the following equation:

Compared to the MnP without any mutation, MnP (S232P) exhibited poorer performance at low pH range (pH 3-6) (Fig. 6), which may caused by the decrease in the number of hydrogen bonds after mutating the serine at position 232 to proline, as the former could form a large number of hydrogen bonds with surrounding amino acid residues before the mutation(Fig. 7).

Fig. 6 Effect of pH on the stability of MnP (S232P) and MnP after 12 h incubation. The initial MnP activity before incubation was set as 100%. Mutant 6# refers to MnP (S232P). *P <0.05, **P < 0.01.

Fig. 7 Position of Ser232 in wild-type MnP. The residue in wheat color refers to 232S. The dashed line indicates the hydrogen bond formed by serine at position 232 and the surrounding amino acid residues. For serine, the red dashed line indicates the hydrogen bond acceptor, while the blue dashed line indicates the hydrogen bond donor. Green ball indicates Ca2+ and purple ball indicates Mn2+.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1111
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]