An optimized micro-plate assay for high-throughput screening of recombinant Pichia pastoris strains

Abstract

A simple optimized microplate-based method to assay endo-1,4-β-mannosidase activity was described as an improved high-throughput screening method. A series of experimental conditions were optimized. It is revealed that the optimum measurement procedure is as follows: adding 50 μL of diluted enzyme sample and 50 μL substrate, incubating at 45 °C for exactly 5 min in micro-plate, mixing with 100 μL 3,5-dinitrosalicylic acid (DNS) reagent, maintaining at boiling point for 15 min, cooling down to room temperature before determining the ABS value at 540 nm using an ELISA micro-plate reader. The reaction volume of the optimized microplate-assay is reduced to 200 μL from 2 500 μL used in the standard β-mannanase macro-assay. The optimized micro-assay is significantly more sensitive in all of the 643 candidates during endo-1,4-β-mannosidase screening. Statistical analyses show that the sensitivity of the optimized micro-method is significantly greater than that of the macro-assay. The optimized method is convenient, fast, and cheap for high throughput enzyme screening.