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

作　　者：申丽 王晓亮 郑甲 王筱 陈青花 胡祎玮 赵伟 

机构地区：[1]Key Laboratory of Biometallurgy of Ministry of Education(Central South University) [2]School of Minerals Processing and Bioengineering,Central South University

出　　处：《Journal of Central South University》2012年第11期3046-3054,共9页中南大学学报（英文版）

基　　金：Project(31000350)supported by the National Natural Science Foundation of China

摘　　要：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 50μL of diluted enzyme sample and 50 μL substrate, incubating at 45 ℃ for exactly 5 min in micro-plate, mixing with 100 μL 3,5-dinitrosalicylic acid （DNS） reagent, maintaining at boiling point for 15 rain, 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 200μL 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.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.

关 键 词：SCREENING HIGH-THROUGHPUT Pichia pastoris MICROPLATE 

分 类 号：TQ926[轻工技术与工程—发酵工程]