title: Substitution of Asp189 residue alters the activity and thermostability of Geobacillus sp. NTU 03 lipase.
abstract: Error-prone PCR was used to create more thermoactive and/or thermostable variants of thermoalkalophilic lipases. A variant of the α6 helix (lid domain), with an 189E to V substitution at residue 189, lost its thermostability but exhibited higher activity than that of its wild-type predecessor (r03Lip). Site-saturation mutagenesis was used to explore the sequence-function relationship. Five other mutants also lost thermostability (20-40%) but exhibited enhanced thermoactivity (6.3-79-fold). The mutant E189I showed the highest activity retaining 50% activity after maintaining it at 65 °C for 24 h. In comparison to r03Lip, the mutant E189I had a higher affinity for p-nitrophenyl palmitate and p-nitrophenyl stearate (61 and 56% decreased Km) and catalytic efficiency (42-fold and 18-fold increased kcat/Km). The mutant lipase retained its tolerance to n-hexane, but had an improved transesterification activity. The results suggest that residue Glu189 plays a significant role in the thermostability and activity of this thermoalkalophilic lipase.
authors.affiliation: Department of Biochemical Science and Technology, College of Life Science, National Taiwan University, 1 Roosevelt Rd. Sec. 4, Taipei 106, Taiwan, ROC. d93b47106@ntu.edu.tw
authors.forename: Tsung-Wei
authors.initials: TW
authors.lastname: Shih
authors.forename: Tzu-Ming
authors.initials: TM
authors.lastname: Pan
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D056506:Geobacillus / Q000201:enzymology*; D006358:Hot Temperature; D007700:Kinetics; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D010168:Palmitates / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D011487:Protein Conformation; D055550:Protein Stability; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial; D050505:Mutant Proteins; D010168:Palmitates; C050015:4-nitrophenyl palmitate; D008049:Lipase