3 research outputs found
Modification of pancreatic lipase properties by directed molecular evolution
9 pags, 4 figs, 3 tabsCystic fibrosis is associated with pancreatic insufficiency and acidic intraluminal conditions that limit the action of pancreatic enzyme replacement therapy, especially that of lipase. Directed evolution combined with rational design was used in the aim of improving the performances of the human pancreatic lipase at acidic pH. We set up a method for screening thousands of lipase variants for activity at low pH. A single round of random mutagenesis yielded one lipase variant with an activity at acidic pH enhanced by ∼50 on medium- and long-chain triglycerides. Sequence analysis revealed two substitutions (E179G/N406S) located in specific regions, the hydrophobic groove accommodating the sn-1 chain of the triglyceride (E179G) and the surface loop that is likely to mediate lipase/colipase interaction in the presence of lipids (N406S). Interestingly, these two substitutions shifted the chain-length specificity of lipase toward medium- and long-chain triglycerides. Combination of those two mutations with a promising one at the entrance of the catalytic cavity (K80E) negatively affected the lipase activity at neutral pH but not that at acidic pH. Our results provide a basis for the design of improved lipase at acidic pH and identify for the first time key residues associated with chain-length specificity. © The Author 2010.This work was supported by the French Cystic Fibrosis Foundation and the French Research Medical Foundation (D.Y.C)
Exploring The Active Site Cavity Of Human Pancreatic Lipase
Within the scope of improving the efficiency of pancreatic enzyme replacement
therapy in cystic fibrosis, the feasibility of shifting the pH-activity profile
of pancreatic lipase toward acidic values was investigated by site specific
mutagenesis in different regions of the catalytic cavity. We have shown that
introducing a negative charge close to the catalytic histidine induced a shift of
the pH optimum toward acidic values but strongly reduced the lipase activity. On
the other hand, a negative charge in the entrance of the catalytic cleft gives
rise to a lipase with improved properties and twice more active than the native
enzyme at acidic pH