High-Throughput Screening of Mutant α-Amylase Libraries for Increased Activity at 129°C
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.
KeywordsSugar Hydrolysis Starch Glycerol Agar
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