Concanavalin A carrying reactive beads for yeast invertase purification

A novel polymeric carrier using an immobilized form of Concanavalin A (Con A) was proposed for the purification of invertase from Saccharomyces cerevisiae. For the synthesis of the carrier, cross-linked, spherical poly(P-chloromethylstyrene), (PCMS) beads with an average diameter of 186 pm were obtained by suspension polymerization. The selected ligand, Con A was covalently attached onto the bead surface via a direct chemical reaction between the chloromethyl groups of the support and the primary amine groups of Con A. Invertase was adsorbed onto the Con A-carrying PCMS beads. The desorption of invertase from the carrier was achieved by the use of methyl-alpha-D-mannopyranoside as the counter-ligand. The kinetic parameters of purified invertase (i.e. V-m and Km) were determined according to the Michealis-Menten model and compared with the crude one. The specific activity of purified invertase (V-m = 2061 U/mg protein) was determined as 4.2 times higher than the activity of crude invertase mixture (V-m = 495 U/ mg protein). Km value of the purified invertase (% 1.06, w/w) was found to be approximately half of the Km (% 2.00, w/ w) of the crude one. The activity half-life of purified invertase was found to be approximately 1.4 times higher. The activation energy of purified invertase (3428 cal/mol) was higher than that of the crude one (2829 cal/mol). These results showed that the carrier developed based on a reactive polymer (i.e. PCMS) could be efficiently used for invertase purification. (C) 2004 Elsevier B.V. All rights reserved.