Impact of Novel Purified Fatty Acid Esterases on the Biochemical Properties and Shelf Life of Finger Millet

Abstract

This study focused on isolating fatty acid esterase from finger millet flour and examining its physicochemical and kinetic characteristics. The enzyme was partially purified from fresh flour of the GPU 28 finger millet variety using ammonium sulphate precipitation and dialysis, followed by Size Exclusion chromatography. Gel filtration on Sephadex G-75 separated the enzyme into two novel isoforms, named Fatty acid esterase I and II. These isoforms were purified 4.33 and 9.83-fold, with recoveries of 18.35% and 14.48% of the initial enzyme activity, respectively. Both isoforms exhibited optimal activity at pH 8.0 and 45 °C. Their molecular weights, as determined by gel filtration, were 112.2 kDa for isoform I and 19.95 kDa for isoform II. Fatty acid esterase I showed greater thermal stability compared to isoform II. The catalytic efficiency (Vmax/Km) values were 13.74 units mL⁻¹ µM⁻¹ for isoform I and 12.11 units mL⁻¹ µM⁻¹ for isoform II. Both isoforms were inhibited by ascorbic acid. The enzyme displayed a preference for substrates with short-chain fatty acids, especially p-nitrophenyl butyrate. The low Km values for p-nitrophenyl butyrate indicate a strong substrate affinity, which may contribute to the rapid lipid hydrolysis observed in stored pearl millet flour. These properties suggest the enzyme’s potential in producing low molecular weight esters, enhancing flour shelf life in the food industry, and use in chemical applications. Increase in fat content was directly proportional to an increase in Specific fat acidity and fat acidity in finger millet flour. Use of the in vitro inhibitory response of fatty acid esterase by ascorbic acid may be explored in arresting in situ hydrolysis of lipids in flour for increasing the shelf life of millet flour. Thus, the Development of fat acidity and specific fat acidity in stored flour strongly correlated with lipid content. Additionally, no specific studies exist in the literature on the shelf-life kinetics of finger millet flour. Recent research has instead focused on pearl millet flour shelf-life kinetics, providing essential quantitative kinetic data for predicting shelf life and optimizing storage conditions. Research is required to advance novel processing interventions that suppress both hydrolytic and oxidative rancidity pathways in millet flour, thereby achieving superior storage stability.