C-PHYCOCYANIN FROM Spirulina platensis AS A SUBSTRATE CANDIDATE FOR PROTEASE ACTIVITY ASSAY OF PANCREATIN: ISOLATION, CHARACTERIZATION AND KINETIC ASSAY

Abstract

Several substrates have been used for the determination of protease activity of pancreatin. However, there are still weaknesses of substrates such as insolubility, preparation, and a number of reagents used. Therefore, the alternative finding for the substrate for protease activity assay of pancreatin is still openly investigated. This study was aimed to isolate and characterize C-phycocyanin (CPC) from Spirulina platensis and its application as a substrate in protease activity assay. The research includes isolation, purification and characterization of CPC, the interaction of CPC with pancreatin, determining the relationship between initial rate of reaction and protease activity and study of pancreatin protease kinetics. From the experimental results, the ATPS-D fraction with a CPC content of 138.5 ± 0.06 μg/mL and a CPC purity of 0.74 ± 0.00 was used as a substrate solution. The results of the interaction of CPC with pancreatin showed a decrease in CPC content in the incubation time used. The relationship between the initial rate of reaction and protease activity (5 – 30 IU/mL) showed a linear curve, especially at an incubation time of 5 -30 minutes with R2 > 0.9. Enzyme kinetics study showed that the Vmax and Km values for reaction incubation time of 10, 20, 30 minutes were 2.59 g/mL/minute and 91.66 g/mL; 1.47 g/mL/minute and 70.86 g/mL; 1.31 g/mL/minute and 79.22 g/mL/minute, respectively. In conclusion, CPC can be used as a new substrate candidate for the determination of pancreatin protease activity.