Difference between revisions of "Part:BBa K3039004"

Latest revision as of 11:12, 18 October 2021

MHETase W397A

Usage and Biology

The enzymes PETase and MHETase were first discovered in Ideonella sakaiensis in 2016 by a group of researchers in Japan. These enzymes were found to degrade polyethylene terephthalate (PET) into its monomers, terephthalic acid (TPA) and ethylene glycol (EG). PETase degrades PET into Mono-(2-hydroxyethyl)terephthalic acid (MHET), Bis(2-Hydroxyethyl) terephthalate (BHET) and TPA, the main product being MHET. MHET is further degraded by MHETase into TPA and EG. We are aiming to use mutants of these enzymes to degrade the microfibres that are coming off clothing during washing cycles.

This sequence is the Escherichia coli K12 (E. coli K12) codon optimized DNA of the W397A mutant MHETase, with an N-terminal His-tag. The His-tag was added to allow for conformation of expression and subsequent purification. These mutations have been reported in the literature to increase the activity of MHETase (Gottfried et al 2019).

The native predicted signal peptide (Met1-Ala19) was removed from the WT MHETase sequence (Palm et al 2019) and replaced with a start codon (Met), however all mutations are numbered according to the full-length WT sequence. The amino acid sequence was submitted to Twist Bioscience who codon optimised the sequence for E. coli, ensuring that there were no forbidden restriction sites, to allow for potential cloning into alternative BioBrick plasmids. The resulting CDS was synthesised and cloned, by Twist, into pET28. This added a 21 AA His-tag and thrombin cleavage site to the N-terminal of the protein, a T7 promoter and T7 terminator.

Characterisation

In order to characterise our part and determine the rate of its activity and prove its functionality we have run a series of experiments. After transforming the Arctic Express, Rosetta Gami and BL21 DE3 strains of E. coli with our plasmid we induced the expression of the enzymes using IPTG. In order to confirm that the enzyme expression has been successful we ran a western blot which showed the presence of the enzyme in the soluble fractions of the sonicated cells. Afterwards the enzyme was purified and used in assays to show its functionality and determine the rate of its activity.

Western blot of the soluble fraction of Arctic Express strain showing expression of all mutants. The PageRuler Plus prestained protein ladder was used and labeled with the corresponding sizes. The negative control is labeled with 1. This part (MHETase W397A) is labeled with 6. A clear band is visible with a size of about 65 kDa which is the size of MHETase with the His tag attached to it.

Conclusions

Unfortunately while we were able to express this enzyme in E.coli Arctic Express cells in the soluble fraction we were unable to express enough to characterise the enzyme.

References

[1] Gottfried J. Palm, Lukas Reisky, Dominique Böttcher, Henrik Müller, Emil A. P. Michels, Miriam C. Walczak, Leona Berndt, Manfred S. Weiss, Uwe T. Bornscheuer & Gert Weber; Structure of the plastic-degrading Ideonella sakaiensis MHETase bound to a substrate (2019) Nat. Commun. 10(1717)

New characterization data added by ASTWS-China 2021

1 Plasmid Construction

We designed our functional parts and cloning into pET-21a(+) backbone plasmid chemical synthesized by GenScript. To confirm the correctness of the plasmid, we used BamHI and EcoRI restriction enzymes to digest the plasmids. The gel electrophoresis results (Figure 1) showed that the MHETase (1821bp) genes were constructed as expected. In addition, we confirmed the results by sequencing the entire plasmid.

Figure 1 Nucleic acid gel electrophoresis results of OmpR234, PETase and MHETase.

2 Protein expression test

SDS-PAGE electrophoresis was used to check the expression of MHETase proteins. As shown in Figure 2, compared to the blank control, the lane contained MHETase(42 kDa) protein indicated that these three proteins have been successfully expressed.

Figure 2 Protein SDS-PAGE electrophoresis results of OmpR234, PETase and MHETase.

3 Enzyme Activity Test of MHETase

We measured the concentration of TPA (MHET degradation product) by HPLC to analyze the degradation activity of MHETase. The result is shown in Figure 3-A, and the relationship between the concentration of product TPA and MHET is shown in Figure 3-B. In the case of sufficient MHETase, the concentration of the product TPA increases proportionally with the concentration of the substrate. It also further explained the degradation activity of MHETase.

Figure 3 (A) HPLC results of MHETase-ST, (B) Relationship between the concentration of product TPA and MHET

Sequences and Features