Enhanced Extracellular Expression of a Ca- and Mg-Dependent Hyperthermostable Protease EA1 in Systematic Screening of Optimal Signal Peptides.

Proteases have been widely applied in various industries, including tanning, silk, feed, medicine, food, and environmental protection. Herein, the protease EA1 (GenBank accession no. U25630.1) was successfully expressed in and demonstrated to function as a Ca- and Mg-dependent hyperthermostable neutral protease. At 80 °C, its half-life () in the presence of 10 mM Mg and Ca was 50.4-fold longer than that in their absence (7.4 min), which can be explained by structural analysis. Compared with the currently available commercial proteases, protease EA1 has obvious advantages in heat resistance. The largest peptide library was used to enhance the extracellular expression of protease EA1 constructing and screening 244 signal peptides (SPs). Eleven SPs with high yields of protease EA1 were identified from 5000 clones using a high-throughput assay. Specifically, the enzyme activity of protease produced by the strain (217.6 U/mL) containing the SP XynD was 5.2-fold higher than that of the strain with the initial SP. In brief, the protease is a potential candidate for future use in the high-temperature industry.