Isolation, Purification and Partial Characterization of Intracellular Invertase from Palm Wine Yeast (Saccharomyces sp.)

Invertase belongs to the class of enzymes called glycosidase. The enzyme is responsible for the catalytic hydrolysis of sucrose to release monosaccharides known as invert sugars. The aim of this study was the isolation, purification, and physicochemical properties of intracellular invertase from palm wine yeast (Saccharomyces sp.) as an alternative enzyme in several industrial applications. The yeast was harvested from the palm wine through flocculation and the intracellular invertase was isolated from the yeast cell by mechanical grinding using acid washed sand. The intracellular invertase was purified using combination of ion-exchange chromatography on DEAE-trisacryl and gel filtration on Sephacryl S-300. The kinetics and other physicochemical properties of the purified enzyme were determined. The two-step purification scheme employed gave a final yield of 168% and a purification fold of 3.0. Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) of the intracellular invertase gave six subunits with the molecular mass of 73.5 ± 2.1 kDa, 52.3 ± 8.1 kDa, 48.6 ± 3.0 kDa, 37.4 ± 4.8 kDa, 26.5 ± 3.6, and 24 ± 4.3 kDa, respectively, while non-denaturing PAGE revealed the presence of single entity invertase with the molecular mass of 267 kDa. Also, native molecular mass estimated on calibrated Sephacryl S-300 was 266 ± 28 kDa, revealing that the purified palm wine Saccharomyces invertase (PWSInv) is heterohexameric in nature. The Km and Vmax of the purified invertase were 30.8 ± 3.2 mM and 9672 ± 169.0 units/mg protein, respectively, leading to catalytic efficiency, kcat/Km of 1.43 × 106 M-1 s-1. The optimum temperature and pH were 60 °C and 3.0, respectively. The activation energy (Ea) of the intracellular invertase for the hydrolysis of sucrose was estimated to be 280.42 kJ/mol. The study established the presence of intracellular invertase from palm wine yeast and investigated some properties and characteristics of the purified invertase, which could be exploited for several biotechnological and industrial processes.