Purification and characterisation of two extracellular acid proteinases from Monascus pilosus

Genus Monascus is one of the most important microorganisms in the fermentation industry in Asia. However, only a little attention has been paid to the proteinases produced by this fungus and their role in the fermentation process. The main objective of this study was to purify and characterise acid proteinases produced by Monascus pilosus. Two acid proteinases (MpiAP1 and MpiAP2) were purified to homogeneity. Both purified enzymes, MpiAP1 and MpiAP2, were monomeric structures with molecular masses of around 43 and 58 kDa, respectively. The former was an acidic non-glycoprotein, whereas the latter was an acidic glycoprotein with 27% carbohydrate content. Although amino-terminal amino acid sequence analysis of both enzymes (MpiAP1 and MpiAP2) of 20 amino acid length showed over 90% similarity, their amino-terminal amino acids were different from each other. Both enzymes were optimally active at 55 °C and at pH 2.5–3.0 against casein or human haemoglobin. The T1/2 values of MpiAP1 and MpiAP2 were 65 and 70 °C, respectively. Both of the enzymes were completely inhibited by pepstatin A, and markedly by SDS. MoO3 also showed a partial inhibition of both enzymes. Milk casein and haemoglobin were good substrates for these enzymes. Eleven cleavages were detected using the oxidised insulin B-chain as a peptide for the proteolytic specificity test of MpiAP1, while seven cleavages were detected for MpiAP2.