Abstract:

The aim was to establish a convenient and high throughput method for screening Pichia pastoris with high-level recombinant glucose oxidase (GOD). Recombinant P. pastoris clones were cultured on the MM plate and induced by methanol for 2 days, then the plate were covered with color solution. After 30 min reaction at room temperature, measure brown circle area around the colony, then calculate correlation coefficient between the color area and GOD activity. Brown circle appeared clearly when the sample GOD activity higher than 0.0005 U. This new screening method showed high sensitivity, good stability and repeatability, and considerable reliability compared with the traditional screening method. The correlation coefficient between color area and GOD activity reached up to 0.63 when GOD activities lower than 0.1 U/mL. A rapid and high throughput method for screening of P. pastoris with recombinant glucose oxidase was established. At the same time, it may provide a reference for screening of P. pastoris clones with other recombinant enzyme.