Improving the thermostability and activity of lipoxygenase from Anabaena sp. PCC 7120 by directed evolution and site-directed mutagenesis

A sensitive and reliable high-throughput approach to screen lipoxygenase (LOX) mutants was developed to permit a directed evolution study in an attempt to increase the activity and thermostability of LOX from Anabaena sp. PCC 7120 using error-prone polymerase chain reaction (PCR) and DNA shuffling. The best mutant, named D22, contained one key amino acid change (N305D) and its half-life of inactivation at 50 °C was increased by approximately twofold in comparison to the wild-type enzyme, while specific activity 1.87-fold higher than the wild-type enzyme. The N305D substitution was confirmed to be critical for the thermostability and catalytic activity of LOX by site-saturation mutagenesis. Indeed, the reaction temperature for maximum activity increased from 45 °C to 50 °C, while catalytic efficiency (kcat/Km) was increased by 83%. This study reports for the first time on the directed evolution of the LOX from Anabaena sp. for improving thermostability and catalytic activity, and the mutant is of potential importance in food processing and flavor biosynthesis.