Part:BBa_K2013002
pelB and PETase
This part contains the sequence of coding pelB signal peptide and PETase, Extracellular PETase can hydrolyze PET to MHET and TPA. This enzyme is an important step for us, which creates a new step hydrolyzing PET .PETase comes from a bacterium called Ideonella sakaiensis 201-F6 that Japanese scientists newly discovered.According to literature,The activity of the PETase protein against the PET film is 120, 5.5, and 88 times as high as that of TfH, LCC, and FsC that are close to PETase respectively.Therefore,PETase may be a promising enzyme that achieve effective degradation of PET.PETase in this bacterium is a secreted protein with a signal peptide. Adding 5 aspartate sequence behind the PelB signal peptide can enhance the secretion of secreted protein.we did codon optimization before synthesizing it to encode the target product successfully in E. coli.
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 54
- 1000COMPATIBLE WITH RFC[1000]
Experimental Validation
This part is validated through Four ways: amplification, PCR, Enzyme cutting and Sequence.
Amplification
Enzyme:KOD
Primer-F:5′- GAATTCGCGGCCGCTTCTAGATGAAGTACCTGCTGCCGACCG-3′
Primer-R:5′- TCGTACCGCGAATTGCAGCTAATAATACTAGTAGCG-3′
Results
PCR
Enzyme:Taq
Primer-F:5′-CCACCTGACGTCTAAGAAAC-3′
Primer-R:5′-GTATTACCGCCTTTGAGTGA-3′
Results
Double digestion
After the assembly ,the plasmid was transferred into the Competent E. coli top10. After culturing overnight in LB,we minipreped the plasmid for double digestion .The first cutting procedure was performed with EcoRI and EcoRV restriction endonuclease. The second cutting procedure was performed with PstI and NcoI restriction endonuclease.The plasmid was cutted in a 25μL system at 37 ℃ for 1 hours. The Electrophoresis was performed on a 1％ Agarose glu.
Results
Expression of enzymes
SDS-PAGE
The extracellular expression level of PETase was tested from the supernatant fractions collected from E. coli culture after 4h incubation by SDS-PAFE analysis. Surprisingly, we found that our targeted band represents the dominant band in the agarose gel. The results might indicate that the extracellular expression level reached at maximum at 4h. With prolonged incubation time, host cells produced more extracellular proteins and might interfere with the function of PETase.
M: protein marker;Line1:piGEM2016-001;Line2:piGEM2016-002;
Line3:piGEM2016-003;Line4:piGEM2016-005;Line5:piGEM2016-006;
Line6:piGEM2016-007; Line7:pUC57; Line8: E. coli BL21 (DE3)
characterization
Km of PETase on pNPB
This year we have some exploration of the Km value of PETase. As the direct detection of PETase activity to PET is difficult to achieve, we use a relatively simple enzyme activity detection method: pNP-Assay [1]. pNP-Assay is that,based on para-Nitrophenylbutyrate(pNPB) as a substrate, lipase can hydrolysis pNPB to produce a color of p-nitrophenol(pNP). And the pNP has a specific absorption peak at 405 nm. Because PETase is a lipase, it is possible to determine the ability of PETase to cleave ester bonds by the pNP-Assay, which can determine the ability of PETase to hydrolyze polymerized polyethylene terephthalate into MHET.
By this method, we detect PETase Km value under the conditions of pH 7.4, 37 ℃.
The nonlinear regression fitting is performed using the detected data, and we obtain a Km value of 1.79383 mM.
3D Model of PETase
Since the structure of PETase is unknown, we try to predict the 3D structure of PETase by using the Q-Site Finder in the protein 3D structure prediction website ROBETTA.
Figure 1.PETase 3D structure
It is reported that the oxyanion hole of the substrate immobilized with hydrogen bonds in PETase is Tyr87 and Met161 [1].
Furthermore，according to Catalytic mechanism of ester hydrolysis by B. subtilis lipase, Ser and His are directly involved in catalytic reaction, acting as nucleophilic attacking group and general acid-base catalytic elements respectively. Asp acts as activator of His and helps in stabilization of positive charged developed on His during the course of reaction. Oxyanion hole stabilizes the negative charge developed onto the tetrahedral intermediates.
Figure 3. Catalytic mechanism of ester hydrolysis by B. subtilis lipase[2]
According to the catalytic mechanism, the distances between the amide group His and Met, Tyr and the Hydroxyl group O of Ser should not be too large, otherwise the PETase enzyme will be unable to perform its function.
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