Abstract
Enzymes are attractive tools for synthetic applications. To be viable for industrial use, enzymes need sufficient stability towards the desired reaction conditions such as high substrate and cosolvent concentration, non-neutral pH and elevated temperatures. Thermal stability is an attractive feature not only because it allows for protein purification by thermal treatment and higher process temperatures but also due to the associated higher stability against other destabilising factors. Therefore, high-throughput screening (HTS) methods are desirable for the identification of thermostable biocatalysts by discovery from nature or by protein engineering but current methods have low throughput and require time-demanding purification of protein samples. We found that nanoscale differential scanning fluorimetry (nanoDSF) is a valuable tool to rapidly and reliably determine melting points of native proteins. To avoid intrinsic problems posed by crude protein extracts, hypotonic extraction of overexpressed protein from bacterial host cells resulted in higher sample quality and accurate manual determination of several hundred melting temperatures per day. We have probed the use of nanoDSF for HTS of a phylogenetically diverse aldolase library to identify novel thermostable enzymes from metagenomic sources and for the rapid measurements of variants from saturation mutagenesis. The feasibility of nanoDSF for the screening of synthetic reaction conditions was proved by studies of cosolvent tolerance, which showed protein melting temperature to decrease linearly with increasing cosolvent concentration for all combinations of six enzymes and eight water-miscible cosolvents investigated, and of substrate affinity, which showed stabilisation of hexokinase by sugars in the absence of ATP cofactor. ENZYMES: Alcohol dehydrogenase (NADP+ ) (EC 1.1.1.2), transketolase (EC 2.2.1.1), hexokinase (EC 2.7.1.1), 2-deoxyribose-5-phosphate aldolase (EC 4.1.2.4), fructose-6-phosphate aldolase (EC 4.1.2.n).