Part:BBa_K258006

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.

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.

Lipase action of TliA

The Km and Vmax values of TliA where determined by Warwick iGem 2019 using a p-nitrophenol octanoate assay. This was to determine 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 monitored using colorimetry at 400nm at a pH of 8. A more detailed protocol can be found on the Warwick 2019 iGem page. The data gathered was compiled into a linear plot using the Michaelis Menten equation. The data is as follows:

From this 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).

Absorption spectra of TliA

The full absorption spectra of TliA was measured using a light spectrophotomer, the results are as follows:

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 651
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]