Difference between revisions of "Part:BBa K3576001"
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| + | [[File: PETase-Fig2.png|500px|thumb|center|Figure 2 (A)The mechanism of pNPB degradation; (B) OD405 of pNPB hydrolysis by overexpressed PETase.]] | ||
Revision as of 08:07, 28 July 2022
PETase expression system
It is the key part that is responsible for expressing PETase. The PETase can hydrolyze PET to MHET. In order to ensure that PETase can be fully in contact with extracellular PET. The sequence of coding pelB signal peptide was added before PETase.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 97
Illegal NgoMIV site found at 123 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 267
Results
1. Protein expression test
SDS-PAGE electrophoresis was used to check the expression of PETase protein. As shown in Figure 1, compared to the blank control, the lane contained PETase (40 KDa) protein indicated that this protein has been successfully expressed.
2. Enzyme Activity Test of PETase
The enzyme activity of PETase was performed by p-NP assay which is a common way to quantify hydrolytic activity. We selected p-Nitrophenylbutyrate (pNPB) as the substrate, which can be hydrolyzed to p-nitrophenol (pNP) (Figure 2-A). The concentration of pNP can be measured by the characteristic absorption at 405 nm. As shown in Figure 2-B, with the extension of reaction time, the OD405 value of p-NP gradually increased, which indicates that the degradation activity of the PETase.
