Novel extremophilic metalloproteases for consumer product application

Abstract

Proteases are one of the largest classes of commercial enzymes worldwide, with applications in a range of industries, including detergents, tanning and dairy. This project aims to develop novel metalloproteases for use in the detergents industry.

Metagenomic libraries were screened for likely protease candidates, predicting biochemical characteristics based on their primary sequence. Candidate DNA was then synthesised, codon-optimised for expression in Bacillus subtilis. A protease deficient B. subtilis strain was engineered for this purpose through CRISPR-MAD7. This included a system for overexpression of the foldase prsA, that has been shown to increase secretion of heterologous proteins. Proteases were expressed in this B. subtilis chassis downstream of a signal peptide library to optimise secretion. Five protease candidates were successfully expressed and secreted, and of these, four had optimal activities in the desired ranges of pH and temperature.

Of these candidates, one was purified and further characterised. This protease was optimally active at 40 °C, pH 9.0 and was stable across a pH range of 5.0 – 10.0. It was found to be composed of an N-terminal propeptide, a catalytic domain and a C-terminal domain. The C-terminal domain, although inactive by itself, proved to be crucial for achieving full protease activity. This protease displayed a non-specific enzymatic profile, showing particular effectiveness in tackling blood and gravy stains, indicating its potential for use in detergent applications. Furthermore, the enzyme exhibited remarkable resilience in the presence of sequestrants, suggesting its suitability for various detergent formulations. In addition, comparative analyses against the commercial proteases Neutrase® and Carnival Evity™ 16 L highlighted the protease’s potential advantages in terms of salt tolerance and its activity in hard water. These encouraging initial findings warrant further investigation into the practical applications of this protease in the detergent industry.