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

Abstract

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.

Pseudomonas fluorescensStrain R124 Encodes Three Different MIO Enzymes

“…To characterize the enzymes, unfolding events of the proteins were measured with a nanoDSF device by monitoring the intrinsic tryptophan fluorescence at emission wavelengths of λ =330 and 350 nm. Because the melting temperature ( T m ) of a lipase determined by differential scanning calorimetry (DSC) was identical to that obtained by nanoDSF, this technique proved to be a straightforward, simple, and highly correlative method to traditional techniques.…”

Bridges to Stability: Engineering Disulfide Bonds Towards Enhanced Lipase Biodiesel Synthesis

“…Recombinant Geobacillus stearothermophilus T6 lipase (EMBL, AF429311.1) fused to a His‐tag was expressed in Escherichia coli SHuffle cells C3030 (NEB, Massachusetts, USA) as previously described for BL21 – . Thermomyces lanuginosus lipase (TLL) was purchased from Sigma‐Aldrich (Rehovot, Israel) as a soluble solution, while tyrosinase from Bacillus megaterium (TyrBm) was kindly donated by Deri et al .…”

“…The denaturation temperature was measured with the nanoDSF instrument as previously described . Aggregation temperature in buffer or methanol was determined simultaneously under the same temperature gradient with light scattering measurement.…”

Immobilization engineering – How to design advanced sol–gel systems for biocatalysis?

“…Lipases represent one of the most frequently used enzyme classes due to their ability to catalyze a wide range of reactions such as esterification, transesterification, aminolysis, polymerizationin a mild and selective manner without external cofactors. 5 Lipase-catalyzed reactions cover a wide range of applications ranging from producing biofuels, 6,7 fragrances, 8 and food ingredients 9 to the enantioselective synthesis of enantiopure active pharmaceutical ingredients (API). 10 Kinetic resolution (KR) of racemic mixtures is one of the most important applications of lipases.…”

“…16 The process proved to be an easy and effective way to immobilize many different types of enzymes such as lipases. 7,17 Sol-gel immobilization often enhances the thermostability, mechanical resistance, solvent tolerance and storage stability of enzymes. 17 The sol-gel process is initiated by the hydrolysis of tetraalkoxysilanes [Si(OR) 4 ] followed by polycondensation in the presence of the enzyme to be trapped producing a dense silica gel polymeric network.…”