Properties of Immobilized Fumarase by Radiation-Polymerization and Its Utilization for Continuous Production of L-Malate

Abstract

Some properties of fumarase which was immobilized by radiopolymerization at a low temperature were investigated. The michaelis constant, K_m for L-malate increased by 4.6 times after the immobilization, but for fumarate it showed a value of 20 times as much high as that of native one. The equilibrium constant, K_eq was not so much changed by the immobilization. The optimum temperature of immobilized fumarase for L-malate was apt to go down. Thermal stability of immobilized fumarase became higher than that of native one. Immobilized fumarase also showed considerable activity under acidic area. The immobilized fumarase particles (13.6g wet) were packed in a column (9×250mm). L-Malate was continuously produced when 40-1000 mM fumarate (pH 7.5) were feeded into the column. The conversion rate of 1000mM fumarate to L-malate was estimated about 50% at the flow rate of 0.47hr_-1 (S.V.) at 25°C. In this flow test, apparent Km increased at the low flow rates. Higher concentration of the substrate declined the conversion rate, that would be due to shrinkage of the enzyme beads. In this column, it was observed that the initial activity reduced to about 80% after 120 days continuous operation in day and night at the flow rate of 0.47hr^-1 at 25°C.