Amylase Production from Eco-Enzyme Filtrate of Citrus maxima: It’s Purification, Characterization, and Activity

Abstract

Background: Making enzymes from agro waste has been promoted as a sustainable and cost effective solution to meet growing industrial demand. One promising application for the abundant agro residue Citrus maxima peel is the production of eco enzymes and amylases.

Materials and Methods: An eco enzyme was produced through anaerobic fermentation using a ratio of 3:1:10 for Citrus maxima peel, jaggery, and water. Amylase was isolated using a combination of solvent precipitation, ammonium sulphate precipitation, DEAE Sephadex ion exchange chromatography, and Sephadex G 100 gel filtration chromatography. Enzyme characterization was carried out using SDS PAGE, zymogram analysis, and western blot analysis. The effects of substrate concentration, temperature, thermal stability, and pH on enzyme activity were also evaluated.

Results: Purified amylase was obtained with a recovery of 10.14 percent, a fold purification of 8.34, and a specific activity of 16.39 U per mg. An approximate molecular weight of 50 kDa was confirmed, and western blot analysis verified the presence of alpha amylase. The highest enzyme activity was observed at pH 7.4, a temperature of 40 degrees Celsius, and a starch concentration of 1 percent. The enzyme exhibited moderate thermal stability when maintained at 40 degrees Celsius.

Conclusion: The study demonstrated that Citrus maxima peel waste can be utilized as a sustainable and environmentally beneficial substrate for amylase production. The findings highlight its potential as a low cost resource for enzyme production and agro waste valorization within the framework of a circular bioeconomy.