title: Hydrophobic substitution of surface residues affects lipase stability in organic solvents.
abstract: A novel lipase has been recently isolated from a local Pseudomonas sp. (GQ243724). In the present study, we have tried to increase the organic solvent stability of this lipase using site-directed mutagenesis. Eight variants N219L, N219I, N219P, N219A, N219R, N219D, S251L, and S251K were designed to change the surface hydrophobicity of this enzyme with respect to the wild-type. Among these variants, the stability of N219L and N219I significantly increased in the presence of all tested organic solvents, whereas two mutants (N219R and N219D) significantly exhibited decreased stabilities in all the organic solvent studied, suggesting that improvement of hydrophobic patches on the enzyme surface enhances the stability in organic media. Furthermore, replacing Ser²⁵¹ with hydrophobic residues on the enzyme surface dramatically diminished its stability in the tested condition. In spite of the distance of the mutated sites from the active site, the values of k(cat) and K(m) were affected. Finally, structural analysis of the wild-type and mutated variants was carried out in the presence and absence of some organic solvents using circular dichroism and fluorescence spectroscopy.
authors.affiliation: Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran.
authors.forename: Maryam
authors.initials: M
authors.lastname: Monsef Shokri
authors.forename: Shahin
authors.initials: S
authors.lastname: Ahmadian
authors.forename: Neda
authors.initials: N
authors.lastname: Akbari
authors.forename: Khosro
authors.initials: K
authors.lastname: Khajeh
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D020134:Catalytic Domain; D002942:Circular Dichroism; D004795:Enzyme Stability; D057927:Hydrophobic and Hydrophilic Interactions; D008049:Lipase / Q000737:chemistry / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009930:Organic Chemicals / Q000737:chemistry; D011487:Protein Conformation; D012997:Solvents / Q000737:chemistry
chemical_list: D009930:Organic Chemicals; D012997:Solvents; D008049:Lipase