An optimized micro-plate assay for high-throughput screening of recombinant Pichia pastoris strains
来源期刊：中南大学学报(英文版)2012年第11期
论文作者：WANG Xiao-liang(王晓亮) ZHENG Jia(郑甲) WANG Xiao(王筱) CHEN Qin-hua(陈青花) HU Yi-wei(胡祎玮)
文章页码：3046 - 3054
Key words：screening; high-throughput; Pichia pastoris; microplate
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.
SHEN Li(申丽)1,2, WANG Xiao-liang(王晓亮)2, ZHENG Jia(郑甲)2, WANG Xiao(王筱)2, CHEN Qin-hua(陈青花)2, HU Yi-wei(胡祎玮)2, ZHA
(1. Key Laboratory of Biometallurgy of Ministry of Education (Central South University), Changsha 410083, China;
2. School of Minerals Processing and Bioengineering, Central South University, Changsha 410083, China)
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.
Key words:screening; high-throughput; Pichia pastoris; microplate
