Proteomic analysis and optimized production of Alkalihalobacillus patagoniensis PAT 05^T extracellular proteases

Extracellular proteolytic extracts from the haloalkalitolerant strain Alkalihalobacillus patagoniensis PAT 05^T have proved highly efficient to reduce wool felting, as part of an ecofriendly treatment suitable for organic wool. In the present study, we identified the extracellular proteases produced by PAT 05^T and we optimized its growth conditions for protease production through statistical methods. A total of 191 proteins were identified in PAT 05^T culture supernatants through mass spectrometry analysis. Three of the 6 detected extracellular proteases belonged to the serine-endopeptidase family S8 (EC 3.4.21); two of them showed 86.3 and 67.9% identity with an alkaline protease from Bacillus alcalophilus and another one showed 50.4% identity with Bacillopeptidase F. The other 3 proteases exhibited 55.3, 49.4 and 61.1% identity with D-alanyl-D-alanine carboxypeptidase DacF, D-alanyl-D-alanine carboxypeptidase DacC and endopeptidase LytE, respectively. Using a Fractional Factorial Design followed by a Central Composite Design optimization, a twofold increase in protease production was reached. NaCl concentration was the most influential factor on protease production. The usefulness of PAT 05^T extracellular proteolytic extracts to reduce wool felting was possible associated with the activity of the serine-endopeptidases closely related to highly alkaline keratinolytic proteases. The other identified proteases could cooperate, improving protein hydrolysis. This study provided valuable information for the exploitation of PAT 05^T proteases which have potential for the valorization of organic wool as well as for other industrial applications.