High-Throughput Feasible Screening Tool for Determining Enzyme Stabilities against Organic Solvents Directly from...

Biotransformations in organic chemistry frequently suffer from limitations caused by low water-solubility of substrates and product inhibition. Both, usually are addressed by the addition of organic cosolvents, which often accompanies at the expense of enzyme stability. A common method for measuring enzyme stability is to determine the melting temperature (T_m ) of the enzyme. However, current methods are limited to the application of purified enzymes. Herein, for the first time, an easy and fast (<1 h) high-throughput feasible method to determine enzyme stabilities directly from crude extracts is reported. In pure buffer, the T_m value measured in the crude extract was identical to that obtained for the purified enzyme. Through the addition of different organic compounds, the T_m values in the crude extract differed by up to 2.4 °C from that of the purified enzymes due to the presence of the host-cell proteins. Thus, the measurement of enzyme stabilities in crude extracts appears to represent conditions in whole-cell catalysts even better. The applied nano differential scanning fluorimetry technology is further proven to be suitable for whole-cell catalysts with two overexpressed enzymes; thus representing a tool for the rapid screening of natural and mutant enzyme libraries in terms of process stability for challenging biotransformations.