Thermostable neutral metalloprotease from <i>Geobacillus</i> sp. <scp>EA1</scp> does not share thermolysin's preference for substrates with leucine at the <scp>P1</scp> ′ position

Cleavage preference determines suitability of proteases for different applications. The cleavage preference of a thermophilic neutral metalloprotease (npr) from Geobacillus sp. EA1 was characterised using mass spectrometry, confirming its similarity to thermolysin in preferring P1' hydrophobic amino acids. While EA1 npr showed similar efficiencies while cleaving short peptides with any one of six hydrophobic amino acids at the P1' position, thermolysin showed marked preference for leucine. A single amino acid difference in the S1' pocket of these enzymes underlies this difference. A variant of EA1 npr (F133L) had intermediate preference, suggesting that approximately half the difference is attributable to this single amino acid change. Broader preference and higher efficiency make EA1 npr a superior candidate for quick digestion of varied substrates. Impact statement This investigation supported two new, useful conclusions. It characterised for the first time the substrate preference of EA1, a commercially significant protease. In the course of comparison to the very well-studied thermolysin, a hitherto unknown subtlety of thermolysin's substrate preference (marked preference for leucine) was found.