Heterologous expression and characterization of a thermostable MprT metalloprotease from thermophilic Bacillus subtilis BSP in dual host systems

Thermophilic bacteria represent a valuable source of robust enzymes for industrial applications, yet their high growth temperatures often limit large-scale cultivation. In this study, the MprT gene encoding a thermostable metalloprotease was cloned from the thermophilic Bacillus subtilis BSP and heterologously expressed in both Escherichia coli BL21(DE3) and the protease-deficient B. subtilis 1A751. The full-length MprT open reading frame (1638 bp), including its native signal and pro-peptide sequences, was amplified by PCR and inserted into the pET29b vector under the control of the T7 promoter for expression in E. coli. The same gene was subsequently cloned into the integration vectors pDR111 (IPTG-inducible hyper-spank promoter) and pSG1154 (xylose-inducible promoter) for chromosomal integration and extracellular secretion in B. subtilis 1A751. Recombinant constructs were verified by restriction digestion, Sanger sequencing, and SDS-PAGE, which confirmed production of a 36 kDa polypeptide. Functional expression was demonstrated on casein-supplemented agar plates containing appropriate inducers, with clear hydrolytic halos appearing after incubation at 60 °C. The purified recombinant MprT protease displayed optimal activity at 50 °C and pH 7-8, retained 82% of its initial activity after 30 min at 60 °C, and remained stable across a broad pH range (4-11). Activity was strongly stimulated by Ca²⁺, Fe²⁺, and Mn²⁺ ions and was completely abolished by EDTA, consistent with its classification as a metalloprotease. Compared with previous reports on thermolysin-like proteases, this work provides 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 preserving the enzyme's native thermostability and alkaline tolerance. These properties position the recombinant MprT as a promising candidate for detergent formulations and waste-to-biofuel processes conducted under harsh industrial conditions.