Abstract
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 0 hydrophobic amino acids. While EA1 npr showed similar efficiencies while cleaving short peptides with any one of six hydrophobic amino acids at the P1 0 position, thermolysin showed marked preference for leucine. A single amino acid difference in the S1 0 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.