Protein engineering by random mutagenesis and structure-guided consensus of Geobacillus stearothermophilus Lipase T6 for enhanced stability in methanol

Appl Environ Microbiol. 2014 Feb;80(4):1515-27. doi: 10.1128/AEM.03371-13. Epub 2013 Dec 20.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Enzyme Inhibitors / metabolism*
  • Enzyme Stability
  • Geobacillus stearothermophilus / enzymology*
  • Geobacillus stearothermophilus / genetics
  • Lipase / chemistry
  • Lipase / genetics*
  • Lipase / metabolism*
  • Methanol / metabolism*
  • Models, Molecular
  • Molecular Dynamics Simulation
  • Mutagenesis*
  • Mutant Proteins / chemistry
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism
  • Protein Engineering / methods*
  • Solvents


  • Enzyme Inhibitors
  • Mutant Proteins
  • Solvents
  • Lipase
  • Methanol