A thermostable phospholipase C obtained by consensus design

Abstract

Proteins extraordinary performance in recognition and catalysis have led their use in a range of applications. But proteins obtained from natural sources are oftentimes not suitable for direct use in industrial or diagnostic setups. Natural proteins, evolved to optimally perform a task in physiological conditions, usually lack the stability required to be used in harsher conditions. Therefore, the alteration of the stability of proteins is commonly pursued in protein engineering studies. Here we achieved a substantial thermal stabilization of a bacterial Zn(II) dependent phospholipase C by consensus sequence design. We retrieved and analyzed sequenced homologs from different sources, selecting a subset of examples for expression and characterization. A non-natural consensus sequence showed the highest stability and activity among those tested. Comparison of activity and stability parameters of this stabilized mutant and other natural variants bearing similar mutations allow us to pinpoint the sites most likely to be responsible for the enhancement. Point mutations in these sites alter the unfolding process of the consensus sequence. We show that the stabilized version of the protein retains full activity even in the harsh oil degumming conditions, making it suitable for industrial applications.