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...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.展开更多
基金Project(31000350)supported by the National Natural Science Foundation of China
文摘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.
