title: Aromatic amino acid mutagenesis at the substrate binding pocket of Yarrowia lipolytica lipase Lip2 affects its activity and thermostability.
abstract: The lipase2 from Yarrowia lipolytica (YLLip2) is a yeast lipase exhibiting high homologous to filamentous fungal lipase family. Though its crystal structure has been resolved, its structure-function relationship has rarely been reported. By contrast, there are two amino acid residues (V94 and I100) with significant difference in the substrate binding pocket of YLLip2; they were subjected to site-directed mutagenesis (SDM) to introduce aromatic amino acid mutations. Two mutants (V94W and I100F) were created. The enzymatic properties of the mutant lipases were detected and compared with the wild-type. The activities of mutant enzymes dropped to some extent towards p-nitrophenyl palmitate (pNPC16) and their optimum temperature was 35°C, which was 5°C lower than that of the wild-type. However, the thermostability of I100F increased 22.44% after incubation for 1 h at 40°C and its optimum substrate shifted from p-nitrophenyl laurate (pNPC12) to p-nitrophenyl caprate (pNPC10). The above results demonstrated that the two substituted amino acid residuals have close relationship with such enzymatic properties as thermostability and substrate selectivity.
authors.affiliation: Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, No. 1037, Luoyu Road, Wuhan 430074, China.
authors.forename: Guilong
authors.initials: G
authors.lastname: Wang
authors.affiliation: Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, No. 1037, Luoyu Road, Wuhan 430074, China.
authors.forename: Zimin
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, No. 1037, Luoyu Road, Wuhan 430074, China.
authors.forename: Li
authors.initials: L
authors.lastname: Xu
authors.affiliation: Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, No. 1037, Luoyu Road, Wuhan 430074, China.
authors.forename: Yunjun
authors.identifier: 0000-0001-6643-597X
authors.initials: Y
authors.lastname: Yan
mesh_terms: D024322:Amino Acids, Aromatic / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics*; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs / Q000235:genetics*; D055550:Protein Stability; D013329:Structure-Activity Relationship; D013696:Temperature; D025062:Yarrowia / Q000235:genetics*
chemical_list: D024322:Amino Acids, Aromatic; D005656:Fungal Proteins; C408971:LIP2 protein, Yarrowia lipolytica; D008049:Lipase