Bacillus subtilis as a heterologous host for production and secretion of a hyperthermostable archaeal subtilisin-like protease

Pernisine is a subtilisin-like protease from the hyperthermophilic archaeon Aeropyrum pernix . Its ability to degrade resistant protein substrates such as prion proteins makes this protease attractive for industrial applications. Here, we used Bacillus subtilis as a heterologous host for expression and secretion prepropernisine, a precursor of pernisine. The native secretion signal peptide of prepropernisine was replaced with a signal peptide of native B. subtilis preprosubtilisin E to achieve efficient secretion. Among the four promoters tested, xylose-inducible promoter yielded the highest level of propernisine production, with a titer of 175 mg per l of culture. The produced propernisine was purified from the culture medium using a combination of ultrafiltration, anion-exchange chromatography, and heat treatment. Approximately 115 mg of propernisine with 80% purity was obtained per liter of culture, representing a 10-fold higher yield than previously achieved with other heterologous hosts. Activated pernisine obtained from B. subtilis exhibited remarkably high thermostability, with optimal activity at temperatures above 90 °C and across a pH range of 7 to 9. In addition, it retained approximately 60% of initial activity after autoclaving and efficiently degraded collagen and meat. The stability and performance of pernisine from B. subtilis were nearly identical to that of pernisine produced in Escherichia coli . Overall, these findings demonstrate that B. subtilis is a promising host for further optimization of cultivation conditions and scale-up to controlled bioreactor cultivation, to achieve production of pernisine at industrial level. Key points • Bacillus subtilis efficiently produced and secreted recombinant propernisine. • Propernisine was readily purified from the culture medium. • Activated pernisine exhibited high thermostability and efficient meat hydrolysis.