Part:BBa_K3853014
MnP (E74M)
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
2. Proof of the expression
3. Enzyme Activity
MnP activity of MnP (E74M) was measured by monitoring the oxidation of 2,6-dimethyloxyphenol (2,6-DMP) at 469 nm. 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 (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. 3B).
Fig. 3 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 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 (E74M) under different incubation temperature 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. 4 Thermal stability of MnP (E74M).<b> The initial MnP activity before incubation was set as 100%. <p><b>5. pH stability
6. Organic solvents stability
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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