title: Protein engineering by random mutagenesis and structure-guided consensus of Geobacillus stearothermophilus Lipase T6 for enhanced stability in methanol.
abstract: The abilities of enzymes to catalyze reactions in nonnatural environments of organic solvents have opened new opportunities for enzyme-based industrial processes. However, the main drawback of such processes is that most enzymes have a limited stability in polar organic solvents. In this study, we employed protein engineering methods to generate a lipase for enhanced stability in methanol, which is important for biodiesel production. Two protein engineering approaches, random mutagenesis (error-prone PCR) and structure-guided consensus, were applied in parallel on an unexplored lipase gene from Geobacillus stearothermophilus T6. A high-throughput colorimetric screening assay was used to evaluate lipase activity after an incubation period in high methanol concentrations. Both protein engineering approaches were successful in producing variants with elevated half-life values in 70% methanol. The best variant of the random mutagenesis library, Q185L, exhibited 23-fold-improved stability, yet its methanolysis activity was decreased by one-half compared to the wild type. The best variant from the consensus library, H86Y/A269T, exhibited 66-fold-improved stability in methanol along with elevated thermostability (+4.3°C) and a 2-fold-higher fatty acid methyl ester yield from soybean oil. Based on in silico modeling, we suggest that the Q185L substitution facilitates a closed lid conformation that limits access for both the methanol and substrate excess into the active site. The enhanced stability of H86Y/A269T was a result of formation of new hydrogen bonds. These improved characteristics make this variant a potential biocatalyst for biodiesel production.
authors.affiliation: Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa, Israel.
authors.forename: Adi
authors.initials: A
authors.lastname: Dror
authors.forename: Einav
authors.initials: E
authors.lastname: Shemesh
authors.forename: Natali
authors.initials: N
authors.lastname: Dayan
authors.forename: Ayelet
authors.initials: A
authors.lastname: Fishman
mesh_terms: D019943:Amino Acid Substitution; D004791:Enzyme Inhibitors / Q000378:metabolism*; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000432:Methanol / Q000378:metabolism*; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016296:Mutagenesis; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D012997:Solvents
chemical_list: D004791:Enzyme Inhibitors; D050505:Mutant Proteins; D012997:Solvents; D008049:Lipase; D000432:Methanol