posted on 2024-01-11, 16:06authored byDiego
S. Val, Luisina Di Nardo, Fiorela Marchisio, Salvador Peiru, María Eugenia Castelli, Luciano Andrés Abriata, Hugo G. Menzella, Rodolfo M. Rasia
Proteins’
extraordinary performance in recognition and catalysis
has led to their use in a range of applications. However, 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 homologues 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 the stability
parameters of this stabilized mutant and other natural variants bearing
similar mutations allows 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 harsh
oil degumming conditions, making it suitable for industrial applications.