PURIFICATION AND CHARACTERIZATION OF TWO INVERTASES FROM MUTANT STRAIN OF SACCHAROMYCES CEREVISIAE

Abstract

In the present study, a mutant strain of Saccharomyces cerevisae EMS-42 was used for the biosynthesis of invertase (E.C.3.2.1.26). Both types of invertases i.e., extracellular and intracellular invertase are present in S. cerevisae. An extracellular invertase was purified to homogeneity by two step purification i.e., ammonium sulfate precipitation and DEAE-Sephadex A-50. The enzyme was present in the supernatant of 85% saturation being glycoprotein in nature. DEAE column chromatography eluted enzyme as single active fraction at 0.2 M NaCI. The enzyme was purified by 15 fold with recovery of 38%. The molecular mass of 110 kDa was determined after SDS-PAGE. The carbohydrate content was found to be 48%. The intracellular invertase contains both forms of glycosylated (large) and non-glycosylated (small). The similar above procedure was applied for glycosylated intracellular invertase (L-form) while three steps for non-glycosylated invertase (S-form). The L-form was purified by 19 fold with recovery of 32%. Like extracellular invertase, the molecular weight was (110 kDa) for L-form. Ammonium sulfate precipitation separated the enzyme (S-form) as insoluble fraction. The enzyme was eluted at 0.3 M NaCl using DEAE-Sephadex. A single band of molecular weight (55 kDa) was estimated after Sephadex G-50 with purification (16 fold) and recovery of 17%. Both types of invertases were isolated as monomeric protein. The optimum pH, temperature, MnCl2 and the values of the Km and Vmax for non-glycosylated and glycosylated were found to be as 5, 50 and 60ºC, (109 and 111%), (1.2 mM and 909 U/ml/min, 1.8 mM and 1429 U/ml/min), respectively.