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

Kanzhigitova M. Yessimova A. Narymbayeva Z. Mamytova A. Abubakirova A. Tashmuxamedov M. Tashmukhamedova Sh.S.
December 2025 University of Guilan

Caspian Journal of Environmental Sciences
2025 #23 Issue 5 1271 - 1282 pp.

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 utilization 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. In addition, 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 emphasis 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.

Aspergillus oryzae , Characterization , Enzyme-Linked Immunosorbent Assay (ELISA) , Polygalacturonase , α-amylase

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M. Auezov South Kazakhstan University, Shymkent City, Kazakhstan
Zhumabek Akhmetuly Tashenev University, Shymkent City, Kazakhstan
Zhanibekov University, Shymkent City, Kazakhstan
Tashkent Institute of Chemical Technology, 32, Navoi Street, Tashkent, Uzbekistan
National University of Uzbekistan, named after Mirzo Ulugbek, Tashkent City, Uzbekistan

M. Auezov South Kazakhstan University
Zhumabek Akhmetuly Tashenev University
Zhanibekov University
Tashkent Institute of Chemical Technology
National University of Uzbekistan

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