Directed evolution of Thermomyces lanuginosus lipase to enhance methanol tolerance for efficient production of biodiesel from waste grease
Directed evolution of Thermomyces lanuginosus lipase to enhance methanol tolerance for efficient production of biodiesel from waste grease
- Bioresource Technology, 245, p.1491-1497, 2017 .
Engineering a methanol tolerant lipase is of great importance in biodiesel production. Here, the first semi-rational method for directed enzyme evolution to enhance methanol tolerance by targeting high B-factor residues for iterative saturation mutagenesis (ISM)is reported. The best double mutant, TLL-S105C/D27R, retained 71 percent of its original activity after incubation in methanol, showing 30 percent greater methanol tolerance than TLL. TLL-S105C/D27R also displayed 27 percent higher activity over TLL. Structure modelling suggested that the increased stability of TLL-S105C/D27R was caused by the formation of a new hydrogen bond which stabilized the protein structure. E. coli (TLL-S105C/D27R)-catalyzed biotransformation of waste grease produced biodiesel in 81 percent yield in 8h, showing improvement over the 67 percent yield for E. coli (TLL), while retaining 92 percent productivity after 4 cycles of biotransformation of waste grease. The engineered TLL mutant shows high potential for commercial biodiesel production
B-FACTOR
BIODIESEL
DIRECTED EVOLUTION
METHANOL TOLERANCE
THERMOMYCES LANUGINOSUS LIPASE
Engineering a methanol tolerant lipase is of great importance in biodiesel production. Here, the first semi-rational method for directed enzyme evolution to enhance methanol tolerance by targeting high B-factor residues for iterative saturation mutagenesis (ISM)is reported. The best double mutant, TLL-S105C/D27R, retained 71 percent of its original activity after incubation in methanol, showing 30 percent greater methanol tolerance than TLL. TLL-S105C/D27R also displayed 27 percent higher activity over TLL. Structure modelling suggested that the increased stability of TLL-S105C/D27R was caused by the formation of a new hydrogen bond which stabilized the protein structure. E. coli (TLL-S105C/D27R)-catalyzed biotransformation of waste grease produced biodiesel in 81 percent yield in 8h, showing improvement over the 67 percent yield for E. coli (TLL), while retaining 92 percent productivity after 4 cycles of biotransformation of waste grease. The engineered TLL mutant shows high potential for commercial biodiesel production
B-FACTOR
BIODIESEL
DIRECTED EVOLUTION
METHANOL TOLERANCE
THERMOMYCES LANUGINOSUS LIPASE