Isolation and partial characterisation of milk-clotting aspartic protease from Streblus asper

A rennin-like milk-clotting protease from the twigs of Streblus asper was purified by a factor of 65 times with 36% recovery using ethanol precipitation, ion-exchange and size-exclusion chromatographic techniques. The enzyme was found to be monomeric in nature having a molecular mass of 55kDa. The enzyme acts optimally at 55 degrees C and was stable in the temperature range of 30-40 degrees C. Easy enzyme inactivation by moderate heating, makes this protease extract potentially useful for cheese production. The purified enzyme is an acid protease with an optimum pH of 5.5 and it retained 96% of its residual activity between pH 5.0 and 6.0. Pepstatin A inhibited the proteinase activity, whereas iodoacetamide, phenylmethyl sulphonyl fluoride, beta-mercaptoethanol and ethylenediaminetetraacetic acid had no significant inhibitory effect suggesting the presence of aspartic acid residue at the active site. The milk-clotting aspartic protease showed predominant alpha-helical conformation in phosphate buffer as evidenced from circular dichroic spectroscopy.