Difference between revisions of "Part:BBa K3853014"

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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).

4. Thermostability

5. pH stability

6. Organic solvents stability

Sequence and Features