title: Improved activity and thermostability of Bacillus pumilus lipase by directed evolution.
abstract: To improve enzymatic activity of Bacillus pumilus lipases, DNA shuffling was applied to two lipase genes from local B. pumilus isolates. Using a high-throughput activity assay, the mutant with highest activity was selected. This chimeric mutant (L3-3), carrying two crossover positions and three point mutations, has a specific activity 6.4 and 8.2 times higher than the two parent enzymes. The mutant also is more tolerant to various detergents and organic solvents, and has a 9 times longer half-life at 50 °C. Homology modeling of mutant L3-3, based on the highly homologous B. subtilis lipase A, shows that the increased thermostability is likely due to structural rigidification and reduced surface hydrophobicity. Increased specific activity may result from the location of mutations close to the active site. Together, our results show that it is possible to evolve, by DNA shuffling, B. pumilus lipase variants with improved applicability as biocatalysts, even if the two parent enzymes are highly similar.
authors: Department of Molecular Biology and Genetics, Gebze Institute of Technology (GIT), 41400 Kocaeli, Türkiye. nakbulut@gyte.edu.tr
authors: Nagihan
authors: N
authors: Akbulut
authors: Merve
authors: M
authors: Tuzlakoğlu Öztürk
authors: Tjaard
authors: T
authors: Pijning
authors: Saliha
authors: S
authors: İşsever Öztürk
authors: Füsun
authors: F
authors: Gümüşel
mesh_terms: D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D032621:DNA Shuffling / Q000379:methods; D019020:Directed Molecular Evolution / Q000379:methods*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006358:Hot Temperature; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein
chemical_list: D011994:Recombinant Proteins; D008049:Lipase