Difference between revisions of "Part:BBa K258006"

Latest revision as of 23:14, 21 October 2019

Thermostable lipase (TliA) of Pseudomonas fluorescens SIK W1

TliA has four glycinerich repeats (GGXGXD) in its C-terminus, which appear in many ABC transporter-secreted proteins.Export of fusion proteins with the whole TliA through the ABC transporter was evident on the basis of lipase enzymatic activity. Upon supplementation of E. coli with ABC transporter, EGF-TliA was excreted into the culture supernatant.The thermostable lipase (TliA) of P. fluorescens SIK W1 is comprised of 476 amino acids and has the characteristic C-terminal signal sequence recognized by the ABC transporter. Whole TliA(the largest LARD-lipase ABC transporter recognition domain) were attached to C-termini of model proteins and enabled the export of the model proteins such as GFP and EGF which has 3 disulfide bonds in E. coli supplemented with ABC transporter. Activity domain (residues 1–268) and secretion/chaperon domain (residues 279–476). In our experiment, we observed that TliA fused proteins were excreted to supernatant culture succesfully by detecting lipase activity with tributyrin and spectrophotometric detection with the substrate p-nitrophenyl phosphate at 420 nm. At the experiments, Tlia fused proteins were excreted ten-fold more with ABC transporter PrtDEF of Erwinia chrysanthemi.

Figure 1. Tributyrin. Lipolytic pathway for tributyrin degradation into butyric acid.

In our Wound Dressing project, EGF is one of tke key proteins although EGF has three disulfide bridges. Thus, exportation of EGF from E.coli was actually difficult. Happily with Jung Hoon Ahn's support, EGF was achieved to be exported in E. coli with TliA and also LARD1 into supernatant media, showing the possibility that the protein having disulfide bonds can be exported.

Warwick iGEM 2019 - Lipase action of TliA

Figure 2. pNPO Assay.The p-Nitrophenol octanoate (pNPO) substrate undergoes lipolysis via TliA enzyme action to become p-Nitrophenol (pNP), causing increased absorption of light at a wavelength of 400nm.

The Warwick iGEM 2019 characterised Pseudomonas fluorescens (SIK W1) Thermostable lipase (TliA) activity by measuring change in light absorbance at a wavelength of 400nm, which directly correlated to the concentration of p-Nitrophenol (pNP), caused by lipolysis of p-Nitrophenol octanoate (pNPO) substrate. The data from the pNPO lipolysis assay defined the enzyme activity and was processed into kinetic data for TliA. The assay determined the efficency of TliA as a means to breakdown triglycerides. A solution of TliA was mixed in a cuvette containing different concentrations of p-nitrophenol octanoate and was then monitored using colorimetry at 400nm at a pH of 8. The data gathered was compiled into a linear plot using the Michaelis Menten equation.

Figure 3. Kinetic Graph. TliA Kinetics Characterisation via Lineweaver-Burk Plot. Thermostable lipase A (TilA - BBa_K258006) has a KM of 0.256mM and a Vmax of 0.00375 mM/min, where the Kcat is 0.0972 mM / min / mg.

From Figure 3 graph we extrapolated the Km of TliA to be 0.256mM, Vmax to be 0.00486 mM/min and kcat to be 0.0972 mM/min/mg. Giving TliA an enzyme efficency of 0.380 at pH 8 (25C).

Figure 4. TliA Absorbance spectrum.Absorbance of TliA protein at a range of wavelengths.