The Inducted Expression Of D-amino Acid Oxidase And The Study Of Enzyme Engineering

The use of lactose as inducer substitute IPTG for expression of D-amino acid oxidase gene(daao) in the recombinant Escherichia coliBL21(DE3)/pWY. The influences such as lactose concentration, cell density, the time of induction, the induction temperature on expression of the recombinant protein were investigated in shaking flask. Under the condition of the optical density at 600nm reached about 1.0, lactose concentration was 10g per liter, induced 6h at 28℃, the DAAO enzyme activity reached up to 2400U per liter. In 5L fermentor, and inducted with lactose as inducer in 23℃,25℃,28℃,32℃,37℃for expression to establish the optimum inducted temperature according to the DAAO enzyme activity in different temperature, added two times lactos,and under the condition of lactose concentration was 1%, inducted about 22h at 28℃, the DAAO enzyme activity reached up to 3784.32U/L, cell density(OD₆₀₀) reached to 27.44.The recombinant His-tagged DAAO was one-step purified by affinity resi-n-EPI-30-ARG-IDA-Co²⁺. The purification was ten folds and the activity yield was 38.35%. The DAAO protein was analyzed by SDS-PAGE to get more pure DAAO.The immobilized DAAO on Eupergit C exhibited an enhancement of stability against thermal and pH inactivation as an increaseing in the appareal Michaelisconstant (Km). The activity yield of immobilized DAAO was about 4 2.44%.