Thermostable MprT metalloprotease from *Bacillus subtilis* BSP successfully overexpressed in dual host systems, retaining high activity at 60 °C.

Industrial enzyme applications, particularly in detergents and waste treatment, demand robust enzymes that maintain activity under harsh conditions like high temperatures and extreme pH. While thermophilic bacteria are a rich source of such enzymes, their high cultivation temperatures pose challenges for large-scale production. Developing efficient heterologous expression systems for these enzymes in mesophilic hosts is crucial to enable cost-effective, high-yield manufacturing while preserving their native stability and catalytic properties. This study addresses the gap in producing highly stable enzymes like metalloproteases from thermophiles in more industrially viable hosts.

Researchers cloned the MprT gene (1638 bp), encoding a thermostable metalloprotease from thermophilic Bacillus subtilis BSP, into two host systems. For E. coli expression, the gene was inserted into the pET29b vector under a T7 promoter. For extracellular secretion in protease-deficient B. subtilis 1A751, it was cloned into integration vectors pDR111 (IPTG-inducible) and pSG1154 (xylose-inducible). Recombinant constructs were verified by restriction digestion, Sanger sequencing, and SDS-PAGE, confirming a 36 kDa polypeptide. Functional expression was assessed on casein-supplemented agar plates, observing hydrolytic halos after incubation at 60 °C.

The purified recombinant MprT protease demonstrated optimal activity at 50 °C and a pH range of 7-8. Notably, the enzyme exhibited remarkable thermostability, retaining 82% of its initial activity after 30 min of incubation at 60 °C. It also maintained stability across a broad pH range, from pH 4 to pH 11. The metalloprotease activity was significantly stimulated by the presence of Ca²⁺, Fe²⁺, and Mn²⁺ ions, but was completely abolished by the chelating agent EDTA, confirming its classification. > This work represents the first successful functional overexpression of the BSP-derived MprT gene in a protease-deficient B. subtilis host, enabling efficient extracellular production at moderate cultivation temperatures while fully preserving the enzyme's native thermostability and alkaline tolerance.