Purification and characterization of amylases from three freshwater fish species providing new insight application as enzyme molecular markers for zymography.

Abstract

Purification of amylases from digestive tracts of three freshwater fish species with Q-Sepharose Fast Flow and Sephacryl S-200 columns displayed two isoforms of amylases from Osteochilus hasselti (O1, O2) and three isoforms of those from both Hampala dispar (UB, H1, H2) and Puntioplites proctozystron (P1, P2, P3). The optimum pH values displayed at 7.0 and 8.0, while the optimum temperatures revealed at 40 and 50°C. Almost isoenzyme activities were activated by NaCl and CaCl2, whereas EDTA and SDS strongly inhibited all enzymatic activities. Verification with an atomic absorption spectrophotometry exhibited the presence of Ca2+ ions in the range of 0.02-13.53ppm per mg protein indicating that amylases are Ca2+ dependent. Molecular weight analysis revealed 12 to 147kDa. The UB, O1, and H2 amylases with appropriate molecular masses of 64, 49, and 25kDa validated with LC-MS/MS were selected. Three certain enzymes revealed high stability in a sample buffer after five cycles of freeze-thawing process upon storage at - 20°C for 12weeks. No protein degradation was observed on polyacrylamide gel, and the enzymes still displayed sharp and clear bands on zymograms. The result suggested that the purified fish amylases, which expressed high activities and stabilities, were potentially used as enzyme molecular weight markers for zymography.