Purification and Comparative Insight into Physico-chemical and Kinetic Properties of Fatty Acid Esterases Novel Isoforms from Pearl Millet and their Effect on Shelf Life

Abstract

The current study focused on isolating and characterising fatty acid esterase from pearl millet, specifically comparing enzymes derived from F1 seeds and F2 grains. Purification was achieved through ammonium sulphate precipitation, dialysis, and gel filtration chromatography. Separation on a Sephadex G-75 column revealed two distinct isoforms, termed Fatty Acid Esterase I and II. In F1 seeds, these isoforms were purified to 10.05 and 8.34-fold levels with activity recoveries of 13.3% and 8.6%, respectively, whereas in F2 grains, purification reached 4.65- and 5.39-fold levels with recoveries of 6.71% and 9.11%. Both isoforms exhibited optimal function within a pH range of 7.8-8.0. Molecular weight determinations showed that fatty acid esterase I measured 69.18 kDa in F1 seeds and 66.09 kDa in F2 grains, while fatty acid esterase II had molecular masses of 23.98 kDa and 22.90 kDa, respectively. The optimal temperature for the activity of both esterases from both sources was 45 °C. Thermostability tests indicated that Esterase II from F1 seeds and Esterase I from F2 grains were more heat-resistant than their counterparts. Kinetic analyses revealed Vmax/Km values of 10.85 and 13.19 units mL⁻¹ µM⁻¹ for esterase I from F1 seeds and F2 grains, while esterase II displayed a higher Vmax/Km value in F2 grains (8.45 units mL⁻¹ µM⁻¹) compared to F1. Therefore, the development of fat acidity and specific fat acidity in stored flour strongly correlated with lipid content. Increase in fat content was directly proportional to an increase in Specific fat acidity and fat acidity in both genotypes of pearl millet. Development of fat acidity was higher in high-temperature storage flour as compared to low-temperature stored flour. So, Low temperature increased the shelf life of pearl millet flour. F2 grains contained lipid content and fat acidity, and SFA were higher compared to F1 seeds of HHB-94. So, fatty acidity and SFA directly depend on fat content. Lower temperatures slow down these kinetics, thereby prolonging shelf life. Thus, Rancidity was higher in F2 grains due to higher lipid content. Both isoforms from the two genotypes were inhibited by ascorbic acid, suggesting that the vitamin C could suppress in situ lipid hydrolysis, thereby extending the shelf life of millet flour.