Part:BBa_K3853057

P_AOX1-α-factor-his-tag-MnP(E74M)-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(E74M) is one of the most promising mutant of MnP. We use BBa_K3853057 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 (E74M) is obtained by mutating the glutamate at position 74 of wild-type MnP (BBa_K3853000) to methionine.

Usage

We mutated the glutamate at position 74 of wild-type MnP to methionine through single-point mutation in order to improve the stability of wild-type MnP. We use BBa_K3853057 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 (E74M). lane 2 for MnP(E74M)

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(E74M). lane 2 for MnP(E74M)

For assaying the mRNA expression of MnP (E74M), qRT-PCRs were performed. We did data analysis using a variation of the Livak method. To determine the relative expression of MnP (E74M) vs. reference gene ACT1, total RNA was prepared from an equal volum of yeast solution. In Fig. 3, MnP (E74M) reached its peak to a fold difference of 0.11 after 2% methanol inducing for 36 h. The C_T values for the MnP (E74M) and the reference gene ACT1 were then used to calculate the fold difference with the following equation:

Fig. 3 qRT-PCR results of MnP (E74M) using the relative quantitative method. Microbial samples were taken every 24 hours and ACT1 was applied as the reference gene. This result indicated that the MnP (E74M) expression reached the maximum after 36 h of fermentation in buffered complex methanol medium (BMMY) medium.

3. Enzyme Activity

MnP activity of MnP (E74M) 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. 4A, the absorbance of the reaction system with MnP (E74M) 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. 4B).

Fig. 4 The detection of 2, 2', 6, 6'-tetramethoxydibenzo-1, 1'-diquinone. Mutant 2^# refers to MnP (E74M). 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 (E74M) were incubated in 20 mM sodium malonate buffer (pH 5.5) with 100 mM NaCl at different temperatures for 6 h (Fig. 5) 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. 5, relative enzyme activity of MnP (E74M) under different incubation temperatures displayed distinct characteristics. It exhibited good stability when the temperature wasn't high (below 60°C), and the relative enzyme activities after 6 h incubation at room temperature, 37℃ and 50℃ were 106.1%, 97.26% and 83.19%, respectively. When the temperature exceeded 60℃, a sharp decline of enzyme activity within 2 h could be observed, and it gradually decreased in the following 4 hours.

Fig. 5 Thermal stability of MnP (E74M). The initial MnP activity before incubation was set as 100%.

Compared to the MnP without any mutation, MnP (E74M)'s thermostability showed the same trend and it had significantly improved as it exhibited higher relative enzyme activity at almost all temperature gradient we set after 6 h incubation (Fig. 6).

Fig. 6 Effect of temperature on the stability of MnP (E74M) and MnP after 6 h incubation. The initial MnP activity before incubation was set as 100%. Mutant 2^# refers to MnP (E74M). ^*P < 0.05, ^**P < 0.01.

5. pH stability

To evaluate pH stability, the purified MnP (E74M) 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 (E74M) exhibited higher relative enzyme activity at pH 4 and there was no significant difference under other pH conditions (Fig. 7). This may be due to the fact that after mutating the glutamate at position 74 to methionine, the latter retained hydrogen bond with S66 and A136 (Fig. 8). These result may indicated that the hydrogen bond with S66 and A136 is important for MnP's pH stability as our other mutant exhibited poorer pH stability.

Fig. 7 Effect of pH on the stability of MnP (E74M) and MnP after 12 h incubation. The initial MnP activity before incubation was set as 100%. Mutant 2^# refers to MnP (E74M). ^**P < 0.01.

Fig. 8 Position of Glu74 in wild-type MnP. The residue in wheat color refers to 74E. The dashed line indicates the hydrogen bond formed by glutamate at position 74 and the surrounding amino acid residues. For glutamate, 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⁺.

References

[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).

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]