Part:BBa_K3039001
PETase T88A_S121E_D186H_R280A
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.
Different mutations that have been reported in past papers to increase the activity of PETase have been combined into a novel mutant, in order to test if this would result in an overly active mutant. This sequence is the Escherichia coli K12 (E. coli K12) codon optimized DNA of the novel mutant of PETase, with an N-terminal His-tag. The His-tag was added to allow for conformation of expression and subsequent purification.
The native predicted signal peptide (Met1-Ala33) was removed from the WT PETase sequence (Seo et al 2019) and replaced with a start codon (Met), however all mutations are numbered according to the full-length WT sequence. TThe 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.
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 (PETase T88A_S121E_D186H_R280A) is labeled with 2. A clear band is visible with a size of about 30 kDa which is the size of PETase 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 (PETase T88A_S121E_D186H_R280A) is labeled with 2. A clear band is visible with a size of about 30 kDa which is the size of PETase with the His tag attached to it.
Protein Purification
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 50 ml shows protein elution.
Size Exclusion Chromatography (Superdex-200)
Further purification of the enzyme by size exclusion chromatography using a calibrated Superdex-200 column. The large peak at an elution volume of ~90 ml shows the monomeric form of the protein. There is a smaller peak at ~65 ml which could be a higher oligomeric state. This will be investigated further in the future as sometimes larger oligomeric states have been found to be more thermal stable.
Esterase Activity
Activity was measured by spectrophotometrically flowing the hydrolysis of p-nitrophenyl acetate (pNPA) into acetate and p-nitrophenol. This was performed at room temperature in buffer containing 50 mM NaPhosphate buffer pH7.5, 100 mM NaCl. A range of substrate concentrations were tested and a blank used to subtract the auto-hydrolysis of the pNPA. The production of p-nitrophenol was measured at 405 nm.
The esterase activity assay shows the production of p-nitrophenol (A405nm) at different substrate concentrations.
The specific activity of the enzyme at differing substrate concentrations.
Thermal Stability Graphs
Thermal Stability
The thermostability of the enzyme was investigated incubating enzyme samples at a range of temperatures (20 °C - 90 °C) for one hour using the gradient function in a SensOQuest LabCycler (Geneflow) before samples are cooled to 4 °C and assayed for activity using the esterase assay method described previously.
The thermal stability assay shows the production of p-nitrophenol (A405nm) after the pre-incubation of the enzyme at increasing temperatures before the esterase assay was carried out.
The SP1 enzyme shows activity between 20 and 50°C.
Thermal Stability of BBa_K3039001 (SP1) Vs. Wild Type PETase
The % activity of the enzymes compared to the activity at room temperature. WT PETase is most active at 40 °C before immediately falling off to 0% activity at 50 °C. PETase S212E_D186H_R280A (PTS) is also most active at 40 °C but is able to retain ~70 % activity at 50 °C before falling to 0% activity at 60 °C. SP1and SP2 although are not as active at the lower temperatures but SP1 is able to retain ~35 % activity at 50 °C before falling to 0% activity at 60 °C.
Fibre Assay
The PETase assay was carried out using fibres obtained from the washing machine and filter as part of this project. 35 µg of fibres was incubated in 500 µl of enzyme at a range of concentrations (50 to 2000 µM) in 50 mM Na Phosphate buffer pH 7.5 with 50 mM NaCl. The fibres were incubated with the enzyme solution for 76 hours before the reaction was terminated by heating at 80 °C for 15 mins. 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 breakdown of PET fibres harvested from our filter into its constitutive parts with a change in enzyme concentration over a 76 hour period.
BHET Assay
The breakdown of PET fibres harvested from our filter into BHET with a change in enzyme concentration over a 76 hour period.
MHET Assay
The breakdown of PET fibres harvested from our filter into MHET with a change in enzyme concentration over a 76 hour period.
TPA Assay
The breakdown of PET fibres harvested from our filter into TPA with a change in enzyme concentration over a 76 hour period.
Conclusion
The enzyme is over expressed and found to be active in both the esterase assay as well as the being able to break down PET fibres collected as part of the washing machine filter. PETase T88A_S121E_D186H_R280A is not as active at the lower temperatures but is able to retain ~35 % activity at 50 °C before falling to 0% activity at 60 °C. The enzyme had a lower specific activity when compared to the WT PETase (0.5 x).
References
[1] Hyeoncheol Francis Son, In Jin Cho, Seongjoon Joo, Hogyun Seo, Hye-Young Sagong, So Young Choi, Sang Yup Lee, Kyung-Jin Kim; Rational Protein Engineering of Thermo-Stable PETase from Ideonella sakaiensis for Highly Efficient PET Degradation (2019) ACS Catal. 9(4), 3519-3526
Sequences and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 667
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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