Revision as of 20:06, 21 October 2019

BHETase 2

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. The enzymes would be secreted into a filter that captures the microfibres.

Degradation of PET by PETase also results in the formation of BHET. Therefore for complete degradation of PET a further BHETase enzyme is required. This sequence is the Escherichia coli K12 (E. coli K12) codon optimized DNA of the R411A_S419G_F424N mutant MHETase, with an attached His tag. The His tag was attached in order to more easily identify the enzymes. The wild type MHETase does not show BHET degrading activity. These mutations have been reported in past papers to give MHETase the ability to degrade BHET.

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, BsaI or SapI, to allow for potential TypeIIS assembly. The resulting CDS was synthesised and cloned, by Twist, into pET28. This added a 63 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.

Expression in E.coli

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 R411A_S419G_F424N) is labeled with 8. 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.

Western blot of the soluble fraction of Rosetta Gami 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 R411A_S419G_F424N) is labeled with 8. 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.

Purification graphs

Nickel Affinity Chromatography

Nickel affinity column trace taken during initial purification of the enzyme. The light blue line shows the change in imidazole concentration with increasing volume run through the column and the purple line shows the corresponding change in A280 of eluent from the column. The peak at 32 ml shows BHETase 1 elution.

Nickel affinity trace showing the elution of the protein from the column with an increase in Imidazole concentration

We were unable to further purify this enzyme using size exclusion chromatography. This could have been due to degradation of the protein sample

BHET Assay Graphs

BHETase 2 Assay

The concentration of the metabolites BHET, MHET and TPA in the assay solution after 24 hours with a change in enzyme concentration.

BHET Standard Curve

MHET Standard Curve

TPA Standard Curve

Conclusion

Enter conclusion

Sequence and Features