AbstractThe aim of the present study was the characterisation of three true subtilisins and one phylogenetically intermediate subtilisin from halotolerant and halophilic microorganisms. Considering the currently growing enzyme market for efficient and novel biocatalysts, data mining is a promising source for novel, as yet uncharacterised enzymes, especially from halophilic or halotolerantBacillaceae, which offer great potential to meet industrial needs. Both halophilic bacteriaPontibacillus marinusDSM 16465TandAlkalibacillus haloalkaliphilusDSM 5271Tand both halotolerant bacteriaMetabacillus indicusDSM 16189 andLitchfieldia alkalitellurisDSM 16976Tserved as a source for the four new subtilisins SPPM, SPAH, SPMI and SPLA. The protease genes were cloned and expressed inBacillus subtilisDB104. Purification to apparent homogeneity was achieved by ethanol precipitation, desalting and ion-exchange chromatography. Enzyme activity could be observed between pH 5.0–12.0 with an optimum for SPPM, SPMI and SPLA around pH 9.0 and for SPAH at pH 10.0. The optimal temperature for SPMI and SPLA was 70 °C and for SPPM and SPAH 55 °C and 50 °C, respectively. All proteases showed high stability towards 5% (w/v) SDS and were active even at NaCl concentrations of 5 M. The four proteases demonstrate potential for future biotechnological applications.Key points• Halophilic and halotolerant Bacillaceae are a valuable source of new subtilisins.• Four new subtilisins were biochemically characterised in detail.• The four proteases show potential for future biotechnological applications.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00253-023-12553-w.