Difference between revisions of "Part:BBa K3039005"

Latest revision as of 02:32, 22 October 2019

BHETase 1

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 S416A_F424N 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 give MHETase the ability to degrade BHET (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 Type IIS 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.

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 S416A_F424N) is labeled with 7. A clear band is visible with a size of about 65 kDa which is the size of BHETase1 with the His tag attached to it.

The western blot demonstrated that we had successfully expressed our BHETase 1 enzyme and that it was in the soluble fraction. Therefore we were able to over-express and purify this enzyme for use in vitro assays.

Purification from E. coli

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.

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

BHETase 1 activity assay

The assay enzyme activity reactions (100 µl) with 100 µg/ml substrate in 40 mM Na Phosphate buffer pH 7.5 with 50 mM NaCl and 20 % (v/v) DMSO were incubated at 25 °C with the enzyme at 3 different concentrations (250, 500 and 1000 µM) and the reaction incubated for 24 hours at 30 °C. The reaction is terminated by heating at 80 °C for 15 mins. BHET was purchased pure form Sigma Aldrich. The reaction was then analysed by HPLC. Samples (10 µl) was analysed using a high-performance liquid chromatography system (HPLC, Agilent 1200) using an Eclipse Plus C18 column (Agilent, UK). The mobile phase was 99.9 % Water with 0.1 % Formic Acid at a flow rate of 0.8 ml min-1 and the effluent monitored at 240 nm. The typical elution condition was 10 min with 20% - 80% acetonitrile. The amounts of products (BHET, MHET and TPA) were calculated by comparison to a standard curve. All samples were analysed in triplicate and the data averaged and standard errors calculated.

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

The assay shows the decrease in BHET concentration during the 24 hour BHETase assay. This BHET is being converted into MHET and a small amount of TPA as can be seen by the appearance of these compounds. We can also see from these assays that an increase in protein concentration corresponds to an increase in the amount of BHET used up as well as as increase in the amount of MHET and TPA produced.

Conclusion

As part of this years Exeter iGEM project we have been able to express in the soluble fraction BHETase 1 and purify it using a Nickel affinity column. This purified enzyme was then used in BHETase assay. As the enzyme concentration used in the assay increased so did the amount of BHET hydrolysed as well as increase in the hydrolysis products MHET and TPA. This BHETase enzyme can be used to degrade BHET in our filter set up and that will be tested in the future.

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)

Sequences and Features