Purification and characterisation of a novel protease from Cordyceps sinensis and determination of the cleavage site motifs using oriented peptide library mixtures

A novel protease, from the edible fungus Cordyceps sinensis, was purified and characterised. Its cleavage site motifs were determined by oriented peptide library mixtures and validated by synthetic peptides and natural proteins. otease was purified to homogeneity using anion-exchange chromatography, sieve chromatography, native PAGE and reversion phase chromatography. Its molecular weight, estimated by SDS–PAGE, was approximately 43 kDa. The results of MS-MS, MALDI-TOF MS and de novo sequencing demonstrated that it was a completely new protease. d oriented peptide library mixtures to determine cleavage site motifs. Cleavage requires lysine at P1 and proline or lysine at P3′. P2 and P1′ also show some preference. A series of synthetic peptides and natural proteins were used to validate the substrate specificity. The protease has special substrate preferences different from other proteases. It also has excellent biochemical properties, which make it able to withstand harsh conditions and suitable for industrialisation and commercialisation.