Variant of the Thermomyces lanuginosus lipase with improved kinetic stability: a candidate for enzyme replacement therapy.
Lipases with high kinetic stability and enzymatic efficiency in the human gastro-intestinal tract may help against exocrine pancreatic insufficiency. Here we mimic gastric conditions to study how bile salts and pH affect the stability and activity of Thermomyces lanuginosus lipase (TlL) and its stabler variant StL using spectroscopy, calorimetry and gel electrophoresis. Both enzymes resist trypsin digestion with and without bile salts. Bile salts activate native TlL and StL equally well, bind weakly to denatured TlL and StL at lower pH and precipitate native TlL and StL at pH 4. StL refolds more efficiently than TlL from gastric pH in bile salts, regaining activity when refolding from pH as low as 1.8 and above while TlL cannot go below pH 2.6. StL also unfolds 10-40 fold more slowly in the denaturant guanidinium chloride and the anionic surfactant SDS. We ascribe StL's superior performance to general alterations in its electrostatic potential which makes it more acid-resistant. These superior properties make StL a good candidate for pancreatic enzyme replacement therapy.
Interdisciplinary Nanoscience Centre, Center for Insoluble Protein Structures (inSPIN), Department of Molecular Biology and Genetics, University of Aarhus, Gustav Wieds Vej 14, DK, 8000 Aarhus C, Denmark.
H
H
Wang
J
J
Hagedorn
A
A
Svendsen
K
K
Borch
D E
DE
Otzen
D001203:Ascomycota / Q000201:enzymology*; D001647:Bile Acids and Salts / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D056947:Enzyme Replacement Therapy; D010188:Exocrine Pancreatic Insufficiency / Q000201:enzymology / Q000628:therapy*; D005765:Gastrointestinal Agents / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D017510:Protein Folding
D001647:Bile Acids and Salts; D005765:Gastrointestinal Agents; D050505:Mutant Proteins; D008049:Lipase