Production, purification and biochemical characterization of lactate oxidase wild type and double mutant

Abstract

Mutations N212S and S178N of Lactate Oxidase (LOX) from Aeorcoccus viridanswere prepared by site directed mutagenesis and confirmed by sequencing. Once these two mutations were created, the mutated protein was expressed, purified and characterized along with the Wild Type (WT). A protocol was established for purifying the Double Mutant (DM) and the WT, consisting ofan ammonium sulfide precipitation before two subsequent FPLC steps,the former one with a hydrophobic interaction column (HIC), and a proper MonoQ ion exchange column. After obtaining an adequate level of purification confirmed by an electrophoresis gel, a basic biochemical characterization was performed. The specific activity, Vmax and Km values of the WT and the Double Mutant were calculated with SIGMAPLOT 9.0 software. Comparisons between each other showed a considerable loss of activity by the double mutant. After comparing the biochemicalparameters between WT and DM, the preliminary conclusion is that all though the mutantstill shows some activity, it might be failing to accomplishat least part ofthe two-stepreactionthat the WT does, due to a steric change in the substrate entrance.