Modification of pancreatic lipase properties by directed molecular evolution.
Cystic 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 approximately 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.
INRA, UMR 1260 "Nutriments Lipidiques et Prévention des Maladies Métaboliques", 27 Bd Jean Moulin, Marseille F-13385, France.
Damien Yann
DY
Colin
Paule
P
Deprez-Beauclair
Noella
N
Silva
Lourdes
L
Infantes
Brigitte
B
Kerfelec
D019943:Amino Acid Substitution; D001483:Base Sequence; D003550:Cystic Fibrosis / Q000188:drug therapy*; D017931:DNA Primers / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010843:Pichia; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D008049:Lipase; C518354:PNLIP protein, human