Creating an Efficient Methanol-Stable Biocatalyst by Protein and Immobilization Engineering Steps towards Efficient Biosynthesis of Biodiesel

ChemSusChem. 2016 Nov 23;9(22):3161-3170. doi: 10.1002/cssc.201601158. Epub 2016 Oct 25.


Two ternary sol-gel matrices, an octyltriethoxysilane-based aliphatic matrix and a phenyltriethoxysilane (PTEOS)-based aromatic matrix, were used to immobilize a methanol-stable variant of lipase from Geobacillus stearothermophilus T6 for the synthesis of biodiesel from waste oil. Superior thermal stability of the mutant versus the wildtype in methanol was confirmed by intrinsic protein fluorescence measurements. The influence of skim milk and soluble E. coli lysate proteins as bulking and stabilizing agents in conjunction with sol-gel entrapment were investigated. E. coli lysate proteins were better stabilizing agents of the purified lipase mutant than skim milk, as evidenced by reverse engineering of the aromatic-based system. This was also shown for commercial Candida antarctica lipase B (CaLB) and Thermomyces lanuginosus lipase (TLL). Uniform, dense, and nonaggregated particles imaged by scanning electron microscopy and a small particle size of 13 μm pertaining to the system comprising PTEOS and E. coli lysate proteins correlated well with high esterification activity. Combining protein and immobilization engineering resulted in a durable biocatalyst with efficient recycling ability and high biodiesel conversion rates.

Keywords: biodiesel; enzymes; immobilization; protein engineering; sol-gel processes.

MeSH terms

  • Animals
  • Biocatalysis*
  • Biofuels*
  • Enzyme Stability / drug effects
  • Enzymes, Immobilized / chemistry*
  • Enzymes, Immobilized / genetics
  • Enzymes, Immobilized / metabolism*
  • Escherichia coli Proteins / chemistry
  • Geobacillus stearothermophilus / enzymology
  • Hydrolysis
  • Lipase / chemistry*
  • Lipase / genetics
  • Lipase / metabolism*
  • Methanol / pharmacology*
  • Milk / chemistry
  • Models, Molecular
  • Protein Conformation
  • Protein Engineering
  • Temperature


  • Biofuels
  • Enzymes, Immobilized
  • Escherichia coli Proteins
  • Lipase
  • Methanol