Purification and characterization of a thermostable alkaline protease produced by Yarrowia lipolytica

purification of the extracellular prote ase produced by yarrowia lipolytica was realized in fo ur steps: ammonium sulfate precipitation, anionic exchange (2x) and gel filtration. the enzyme showed m olecular weight of 61.5 kda (sds-page) and optimum activity at 52.4°c at ph 10-11. the thermal stability was modified in presence of ca2+ (10 mm) providing an in crease of 73, 6 and 11% at 40, 50 and 60°c respectively. the thermodynamic parameters (enthalpy and entropy) indicate that the stability of the enzyme is not provided by non-covalent linkages. furthermore the ion ca2+ is important for thermodynamic stabilization of the enzymatic structure. the proteolytic activity was inhibited by pmsf; suggesting that the enzyme can be classify in the serine protease family. the results of thermodynamic stability allow classifying the protease studied as thermostable. the importance of the ca2+ on the thermostability was corroborated; this is the first report on thermal stability and thermodynamic properties of proteases produced by y. lipolytica.