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 0.02–13.53 ppm per mg protein indicating that amylases are Ca2+ dependent. Molecular weight analysis revealed 12 to 147 kDa. The UB, O1, and H2 amylases with appropriate molecular masses of 64, 49, and 25 kDa 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 12 weeks. 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.
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Data availability
All data included in this study are available upon request by contact with the corresponding author. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al. 2022) partner repository. The datasets generated and analyzed during the current study are available in PRIDE database (PRIDE—Proteomics Identification Database (ebi.ac.uk) via ProteomeXchange (www.proteomexchange.org) repository with accession number or identifier PXD033432 and PXD033433.
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This research was supported by the research capability enhancement program through graduate student scholarship, Faculty of Science, Khon Kaen University.
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Suthathip Phetlum performed the experiments and wrote the first draft of the manuscript. Chamaiporn Champasri acquired the funding, prepared materials and methods, wrote the final draft, revised and edited the manuscript. All authors read and approved the final manuscript.
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Experiments performed under the ethical principles for use and care of animals in science are approved by the Institutional Animal Care and Use Committee of Khon Kaen University (IACUC KKU). All experimental protocols and the care and use of experimental animals complied with animal welfare laws of Thailand, and guidelines and policies approved by ThaiIACUC (permit reference number U1-04,584-d2559). Fish collection and digestive tract preparation were done to minimize fish suffering.
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Phetlum, S., Champasri, C. Purification and characterization of amylases from three freshwater fish species providing new insight application as enzyme molecular markers for zymography. Fish Physiol Biochem 49, 1257–1276 (2023). https://doi.org/10.1007/s10695-023-01255-9
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DOI: https://doi.org/10.1007/s10695-023-01255-9