High-Throughput Screening of Mutant α-Amylase Libraries for Increased Activity at 129°C

Abstract

We describe a high throughput screening setup for measuring α-amylase activity at temperatures far above the boiling point of water. The system consists of a sealed aluminum 384-well assay plate incubated between two preheated aluminum blocks. Samples consisting of starch solution and α-amylase contained in the supernatants of E. coli cultures are rapidly and uniformly heated up to 125°C in less than 30 s. The final temperature of 129°C is reached in less than 1 min. After the high temperature incubation, the α-amylase activity is determined by measuring the concentration of released sugar moieties using p-hydroxy benzoic acid hydrazide. Several thousand clones from mutant libraries generated by error prone PCR techniques can be screened within a week. The described setup could be readily adapted to screen libraries of other enzymatic systems for increased thermoactivity, or to evaluate other properties of enzymes at extreme thermal conditions.