Engineering of Bacillus lipase by directed evolution for enhanced thermal stability: effect of isoleucine to threonine mutation at protein surface.
A lip gene from a Bacillus isolate was cloned and expressed in E. coli. By thermal denaturation analysis, T(1/2) of lipase was observed to be 7 min at 50°C with less than 10% activity after 1 h incubation at 50°C. To expand the functionality of cloned lipase, attempts have been made to create thermostable variants of lip gene. A lipase variant with an isoleucine to threonine amino acid substitution at the protein surface was isolated that demonstrated higher thermostability than its wild type predecessor. To explore the structure-function relationship, the lip gene product of wild type (WT) and mutant was characterized in detail. The mutation enhanced the specific activity of enzyme by 2-folds when compared with WT. The mutant enzyme showed enhanced T(1/2) of 21 min at 50°C. The kinetic parameters of the mutant enzyme were significantly altered. The mutant enzyme displayed higher affinity for substrate (decreased K ( m )) in comparison to the wild type. The k (cat) and catalytic efficiency (k (cat)/K ( m )) of mutant were also enhanced by two and five times, respectively, as compared with the WT. The mutation resides on the part of helix which is exposed to the solvent and away from the catalytic triad. The replacement of a solvent exposed hydrophobic residue (Ile) in WT with a hydrophilic residue (Thr) in mutant might impart thermostability to the protein structure.
Khurana
Jyoti
J
Department of Biotechnology, Panjab University, Chandigarh, India.
Singh
Ranvir
R
Kaur
Jagdeep
J
D019943:Amino Acid Substitution; D000818:Animals; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D007532:Isoleucine / Q000235:genetics*; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D013379:Substrate Specificity; D013696:Temperature; D013912:Threonine / Q000235:genetics*
D007532:Isoleucine; D013912:Threonine; D008049:Lipase