Severin Wedde, Dr. Christian Kleusch, Dr. Daniel Bakonyi & Prof. Dr. Harald Gröger
ChemBioChem 2017 vol: 18(24) pp: 2399–2403 doi: 10.1002/cbic.201700526
Abstract
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 (Tm) 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 Tm value measured in the crude extract was identical to that obtained for the purified enzyme. Through the addition of different organic compounds, the Tm 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.
Topics: Prometheus – nanoDSF, Proteins, Publications
