A highly stable Zn(II) dependent phospholipase C obtained by consensus design

VAL DIEGO; DI NARDO LUISINA; MARCHISIO FIORELA; PAOLETTI LUCIANA; PEIRÚ SALVADOR; AGUIRRE, ANDRES; CASTELLI, M EUGENIA; ABRIATA LUCIANO; MENZELLA, HUGO; RASIA, RODOLFO M.

Mendoza

Congreso; SAIB 2022; 2022

SAIB

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 to address critical environmental and industrial challenges is commonly pursued in protein engineering studies. In particular, the enzymatic removal of phospholipids from crude vegetable oils, or enzymatic oil degumming, by phospholipases is a key enabling technology to obtain cleaner and more efficient methods. Nonetheless, advanced enzymes are needed for their widespread adoption. In this work, in order to thermally stabilize a bacterial phospholipase C by consensus sequence design, several sequenced homologs from different sources, selecting a subset of examples for expression and characterization were retrieved and analyzed. As a result, a non-natural consensus sequence, called ChPLC, showed the highest stability and activity among those tested. Comparison of activity and stability parameters between this stabilized mutant with other natural variants bearing similar mutations allowed the characterization of the sites most likely to be responsible for the enhancement. In addition, an enzymatic degumming process based on ChPLC was developed. This new degumming process does not require modifications in current soybean oil factories, avoiding upfront investments, and is four times faster than the enzymatic processes available in the market. A techno-economic analysis predicts a ~60 % cost reduction for a base-case scenario, establishing the largely expected conditions to promote the global adoption of enzymatic oil degumming by refineries of all sizes.