title: Knowledge-guided laboratory evolution of protein thermolability.
abstract: In rare but nevertheless important cases it is of practical interest to decrease the thermostability of an enzyme, that is, to increase thermolability in a controlled manner. In the present model study, this unconventional goal has been reached by applying directed evolution to the lipase from Pseudomonas aeruginosa (PAL). By utilizing the B-factor iterative test (B-FIT), previously developed to increase the thermostability of enzymes, it was possible to reduce the T(15)(50) value from 71.6 degrees C in the case of wild type (WT-PAL) to 35.6 degrees C (best mutant) without affecting the catalytic profile in terms of substrate acceptance or enantioselectivity at room temperature. Accordingly, saturation mutagenesis was performed at sites in PAL, which on the basis of its X-ray structure, have the lowest B-factors indicative of high rigidity. Focused mutations were introduced which can be expected to decrease rigidity, the ensuing increased flexibility leading to higher thermolability without changing the actual catalytic profile.
authors.affiliation: Max-Planck-Institut für Kohlenforschung, Mülheim, Germany. reetz@mpi-muelheim.mpg.de
authors.forename: Manfred T
authors.initials: MT
authors.lastname: Reetz
authors.forename: Pankaj
authors.initials: P
authors.lastname: Soni
authors.forename: Layla
authors.initials: L
authors.lastname: Fernández
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D019020:Directed Molecular Evolution / Q000379:methods*; D004795:Enzyme Stability / Q000235:genetics*; D015723:Gene Library; D008049:Lipase / Q000737:chemistry* / Q000235:genetics*; D008958:Models, Molecular; D016296:Mutagenesis; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D008049:Lipase