Purification and Characterization of Hordolisin, a Subtilisin-like Serine Endoprotease from Barley

A protease was purified from green barley (Hordeum vulgare L.) malt with an apparent molecular mass of 74 kD and a pI of 6.9. Activity was assayed using an internally quenched fluorogenic peptide substrate, and the inhibitor profile indicated that the catalytic site contained a serine residue. This was confirmed by labelling with [14C]-diisopropyl fluorophosphate. The N-terminal amino acid sequence of the purified protease was homologous to plant subtilisin-like serine endoproteases, such as cucumisin. The barley protease (trivial name hordolisin) had a pH optimum of 6 and was stable up to 60 °C. The substrate specificity of hordolisin was determined from P4 to P3′ using a series of internally quenched fluorogenic peptide substrates. Hordolisin was similar to Savinase and subtilisin BPN′, except that Ala was better accepted at P1, and Arg was preferred at P1′. Hordolisin does not appear to be important in the degradation of the hordein storage proteins during barley grain germination.