title: Engineering lipase A from mesophilic Bacillus subtilis for activity at low temperatures.
abstract: Loops or unordered regions of a protein are structurally dynamic and are strongly implicated in activity, stability and proteolytic susceptibility of proteins. Diminished activity of proteins at lower temperatures is considered to be due to compromised dynamics of the protein at lower temperatures. To evolve an active mesophilic lipase (Bacillus subtilis) at low temperatures, we subjected all the loop residues (n = 88) to site saturation mutagenesis (SSM). Based on a three-level screening protocol, we identified 14 substitutions, among 16,000 mutant population, which contributed to a substantial increase in activity at 5 °C. Based on the preliminary activity of recombinants at several temperatures, 5 substitutions among the 14 were found to be beneficial. A recombinant of these five mutations, named as 5CR, exhibited 7-fold higher catalytic efficiency than wild-type (WT) lipase at 10 °C. All the mutants, individually and in a recombinant (5CR), were characterized by substrate-binding parameters, melting temperatures and secondary structure. 5CR was similar to WT in substrate preferences and showed a significant improvement in activity at both lower and higher temperatures compared with the WT. To establish the contribution of mutations on the dynamics of the protein, we performed 100-ns molecular dynamics (MD) simulations on the WT and mutant lipase at 10 and 37 °C. The root mean square fluctuations (RMSFs) indeed showed that the mutations enhance the protein dynamics locally in the loop region having a catalytic residue, which may help in improved activities at lower temperatures.
authors.affiliation: Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Uppal Road, Hyderabad 500007, India.
authors.forename: Virender
authors.initials: V
authors.lastname: Kumar
authors.forename: Poornima
authors.initials: P
authors.lastname: Yedavalli
authors.forename: Vishal
authors.initials: V
authors.lastname: Gupta
authors.forename: Nalam Madhusudhana
authors.initials: NM
authors.lastname: Rao
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003080:Cold Temperature; D004795:Enzyme Stability; D008049:Lipase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D011487:Protein Conformation
chemical_list: D001426:Bacterial Proteins; D008049:Lipase
keywords: Bacillus subtilis; MD simulation; cold activity; lipase; loops; site saturation mutagenesis