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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 54
  
• 1000
  COMPATIBLE 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 from iGEM17-UESTC-China

Km value of PETase towards pNPB

This year we did some exploration of the Km value of PETase. As the direct detection of PETase activity to PET is difficult to achieve, we used 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 kind of lipase, it is possible to determine the ability of PETase which hydrolyzes polymerized polyethylene terephthalate to MHET by determining the ability of PETase cleaving ester bonds with pNP-Assay.

Fig. 1 The mechanism of pNPB degradation.

Using this method, we detected PETase Km value under the conditions of pH 7.4, 37 ℃.

Fig. 2 Relationship between PETase and pNPB concentration.

We fitted our detected data by the nonlinear regression, then got the Km value-1.79383 mM from the equation.

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.

Fig. 3 3D model of PETase.

It is reported that the oxyanion hole of the substrate immobilized with hydrogen bonds in PETase is Tyr87 and Met161 [1].

Fig. 4 Active sites of PETase.

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.

Fig. 5 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.

Reference

1.Yoshida, S., et al., A bacterium that degrades and assimilates poly(ethylene terephthalate). Science, 2016. 351(6278): p. 1196-9.

2.Kamal, M.Z., et al., Role of active site rigidity in activity: MD simulation and fluorescence study on a lipase mutant. PLoS One, 2012. 7(4): p. e35188.