Elisa-based enzyme isolation, purification, and characterization of polygalacturonase and α-amylase from Aspergillus oryzae

Document Type : Research Paper

Authors

1 M. Auezov South Kazakhstan University, Shymkent City, Kazakhstan

2 Zhumabek Akhmetuly Tashenev University, Shymkent City, Kazakhstan

3 Zhanibekov University, Shymkent City, Kazakhstan

4 Tashkent Institute of Chemical Technology, Tashkent, 32, Navoi Street, Uzbekistan

5 National University of Uzbekistan, named after Mirzo Ulugbek, Tashkent City, Uzbekistan

10.22124/cjes.2025.8916

Abstract

The α-amylase (α-A) and polygalacturonase (PG) enzymes produced by Aspergillus oryzae are the primary subjects of this research, which also aims to isolate, purify, and characterize them. Biological material utilisation was conducted by all applicable ethical norms. To achieve highly pure enzymes with improved activity, the purification process included ammonium sulphate precipitation, dialysis, and size-exclusion chromatography employing Toyopearl HW-65 resin. We used SDS-PAGE and gel filtration to find the molecular weights of α-amylase and polygalacturonase. We tested the enzyme activity under different pH, temperature, and substrate conditions. According to Michaelis-Menten kinetics, substrate-specific activity was found through a kinetic study. α-amylase showed optimal activity at pH 6.8, and polygalacturonase at pH 4.8. Enzyme-linked immunosorbent assays (ELISAs) demonstrated the presence and sensitive quantification of these enzymes. To emphasise their regulatory roles, researchers examined how divalent cations and inhibitors affected enzyme activity. By examining the catalytic properties and operational stability of these enzymes, the study highlights their potential industrial applications in food processing, biotechnology, and diagnostics.

Keywords

References

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Caspian Journal of Environmental Sciences
Volume 23, Issue 5
Special issue: Biological Researches and Envireonment, Guest Editor: Prof. Hamed Mousavi-Sabet, University of Guilan, Iran
December 2025
Pages 1271-1282

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