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.
Highlights
Lipases are defined as triacylglycerol acylhydrolases (E.C.3.1.1.3) that catalyze the fats and oils hydrolysis at the oilwater interface to glycerol and free fatty acids.Our understanding of the lipase action mode has made much progress as a result of the 3D structure resolutions of more than twenty lipases over the past few years [1].The three-dimensional (3-D) structure of the human pancreatic lipase (HPL) consists of two functional domains [2]
Generation of Recombinant Clones Expressing N-turkey pancreatic lipase (TPL) The P.pastoris X33 strain was transformed by electroporation using plasmid DNA linearized by the BspHI restriction enzyme
In this paper, we investigated the TPL N-terminal domain expression in Pichia pastoris to study the effects of the C-terminal deletion effects on the N-terminal domain TPL (N-TPL) activity, stability and some other biochemical properties
Summary
Lipases are defined as triacylglycerol acylhydrolases (E.C.3.1.1.3) that catalyze the fats and oils hydrolysis at the oilwater interface to glycerol and free fatty acids. The three-dimensional (3-D) structure of the human pancreatic lipase (HPL) consists of two functional domains [2]. The Nterminal domain belongs to the a/b hydrolase fold family of proteins [3] and contains the active site which involves a catalytic triad analogous to that present in serine proteases. HPL requires a small protein cofactor, colipase, for the enzyme to be able to bind water/triglyceride interface in presence of bile salt. 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 [4]
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