New robust subtilisins from halotolerant and halophilic Bacillaceae

The 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 halotolerant Bacillaceae, which offer great potential to meet industrial needs. Both halophilic bacteria Pontibacillus marinus DSM 16465(T) and Alkalibacillus haloalkaliphilus DSM 5271(T) and both halotolerant bacteria Metabacillus indicus DSM 16189 and Litchfieldia alkalitelluris DSM 16976(T) served as a source for the four new subtilisins SPPM, SPAH, SPMI and SPLA. The protease genes were cloned and expressed in Bacillus subtilis DB104. 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 degrees C and for SPPM and SPAH 55 degrees C and 50 degrees 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.

Automated summary

The aim of this study was to characterize four new subtilisins from halotolerant and halophilic microorganisms. The proteases showed activity over a wide pH range and exhibited high stability towards SDS and high concentrations of NaCl. These enzymes demonstrate potential for future biotechnological applications.