title: N-terminal domain of turkey pancreatic lipase is active on long chain triacylglycerols and stabilized by colipase.
abstract: The gene encoding the TPL N-terminal domain (N-TPL), fused with a His6-tag, was cloned and expressed in Pichia pastoris, under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. The recombinant protein was successfully expressed and secreted with an expression level of 5 mg/l of culture medium after 2 days of culture. The N-TPL was purified through a one-step Ni-NTA affinity column with a purification factor of approximately 23-fold. The purified N-TPL, with a molecular mass of 35 kDa, had a specific activity of 70 U/mg on tributyrin. Surprisingly, this domain was able to hydrolyse long chain TG with a specific activity of 11 U/mg using olive oil as substrate. This result was confirmed by TLC analysis showing that the N-TPL was able to hydrolyse insoluble substrates as olive oil. N-TPL was unstable at temperatures over 37°C and lost 70% of its activity at acid pH, after 5 min of incubation. The N-TPL exhibited non linear kinetics, indicating its rapid denaturation at the tributyrin-water interface. Colipase increased the N-TPL stability at the lipid-water interface, so the TPL N-terminal domain probably formed functional interactions with colipase despite the absence of the C-terminal domain.
authors: Laboratory of Biochemistry, National Engineering School of Sfax (ENIS), University of Sfax, Sfax, Tunisia.
authors: Madiha
authors: M
authors: Bou Ali
authors: Aida
authors: A
authors: Karray
authors: Youssef
authors: Y
authors: Gargouri
authors: Yassine
authors: Y
authors: Ben Ali
mesh_terms: D000818:Animals; D002999:Clone Cells; D003089:Colipases / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008049:Lipase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism; D010179:Pancreas / Q000201:enzymology*; D010843:Pichia / Q000378:metabolism; D011485:Protein Binding; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors; D014170:Transformation, Genetic; D014280:Triglycerides / Q000378:metabolism*; D014304:Triolein / Q000378:metabolism; D014422:Turkeys / Q000378:metabolism*
chemical_list: D003089:Colipases; D018076:DNA, Complementary; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D014280:Triglycerides; D014304:Triolein; D006639:Histidine; D008049:Lipase; C005830:tributyrin
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