Purification, characterization and investigation of the applications of protease enzyme from strawberry

The aim of this study was purification, characterization, immobilization and investigating some applications of the protease isolated from strawberry (Fragaria ananassa) fruit. The molecular weight of protease, that was 395.6-fold purified by using Sepharose-4B-L-tyrosine-p-amino benzoic acid affinity chromatography, was determined to be 65.8 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Optimum pH and temperature for the pure enzyme were 6 and 40 °C, respectively. The pure protease was stable at a wide range of temperature and pH. Co2+ ions stimulated protease activity very strongly. Cu2+, Hg2+, Cd2+ and Mn2+ ions significantly inhibited protease activity. While 2-propanol completely inhibited the enzyme, the enzyme maintained its activity better in the presence of ethanol and methanol. The strawberry protease showed the highest specificity towards hemoglobin among all the natural and modified substrates tested. The specificity of the enzyme towards synthetic substrates was also investigated and it was concluded that it has broad substrate specificity. Inhibition studies indicated that this purified protease was highly-likely a serine protease and its activity was significantly affected by the presence of metal ions. Furthermore, after the immobilization of crude strawberry protease in Ca-alginate beads, the use of crude protease as a detergent additive and milk coagulating agent was tested. During immobilization of the enzyme in Ca-alginate beads, the alginate concentration was kept constant at 2% and the CaCl2 solution concentration at 3%. Co2+ ions (1 mM) immobilized together with the crude enzyme were found to increase the enzyme activity by 8.4 fold. The immobilization yield was determined to be 65% for the beads containing only the crude enzyme and 69% for the beads containing crude enzyme and Co2+ ions together. As a result of the immobilization, the optimum temperature value of the enzyme did not change and the optimum pH value changed from 6 to 7. After immobilization, there was a slight decrease in Vmax and a significant increase in Km of the crude enzyme. While the reusability of the strawberry protease was at an average level, the storage stability was found to be high. In terms of all the parameters tested, the stability of the strawberry protease increased as a result of immobilization. The application studies showed that strawberry protease can be considered as a good detergent additive but a weak milk clotting enzyme alternative.