Abstract
BackgroundThe turkey pancreatic lipase (TPL) was purified from delipidated pancreases. Some biochemical properties and kinetic studies were determined using emulsified system and monomolecular film techniques. Those studies have shown that despite the accumulation of free fatty acids at the olive oil/water interface, TPL continues to hydrolyse efficiently the olive oil and the TC4 in the absence of colipase and bile salts, contrary to most classical digestive lipases which denaturate rapidly under the same conditions. The aim of the present study was to express TPL in the methylotrophic yeast Pichia pastoris in order to get a large amount of this enzyme exhibiting interesting biochemical properties, to purify and characterize the recombinant enzyme.ResultsThe recombinant TPL was secreted into the culture medium and the expression level reached about 15 mg/l after 4 days of culture. Using Q-PCR, the number of expression cassette integrated on Pichia genomic DNA was estimated to 5. The purified rTPL, with molecular mass of 50 kDa, has a specific activity of 5300 U/mg on emulsified olive oil and 9500 U/mg on tributyrin. The optimal temperature and pH of rTPL were 37°C and pH 8.5. The stability, reaction kinetics and effects of calcium ions and bile salts were also determined.ConclusionsOur results show that the expressed TPL have the same properties as the native TPL previously purified. This result allows us the use of the recombinant enzyme to investigate the TPL structure-function relationships.
Highlights
The turkey pancreatic lipase (TPL) was purified from delipidated pancreases
Lipases are defined as triacylglycerol acylhydrolases (E.C.3.1.1.3) that catalyze the hydrolysis of fats and oils at the oil-water interface to glycerol and free fatty acids
We report the expression of the TPL gene in Pichia pastoris, the recombinant enzyme was purified and its properties are compared to the native one
Summary
Some biochemical properties and kinetic studies were determined using emulsified system and monomolecular film techniques. Those studies have shown that despite the accumulation of free fatty acids at the olive oil/water interface, TPL continues to hydrolyse efficiently the olive oil and the TC4 in the absence of colipase and bile salts, contrary to most classical digestive lipases which denaturate rapidly under the same conditions. The resolution of the three-dimensional structure of human pancreatic lipase (HPL) [2] has shown that the single polypeptide chain (449 amino acids) is fold into two domains: a large N-terminal. Colipase binds to the C-terminal domain of HPL and exposes the hydrophobic tips of its fingers at the opposite side of its lipase-binding site [5]. The open lid and the extremities of the colipase fingers, as well as the b9 loop, form an impressive continuous hydrophobic plateau extending over more than 50 A°2, which might be to interact strongly with a lipid/water interface [6]
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