Abstract
2 Abstract: Glutamate dehydrogenase (GDH, EC 1.4.1.2) was purified from Rumex dentatus cotyledons with specific activity of 145 U mg protein. The indispensable role of arginine, lysine and tyrosine at the active -1 site o f the enzyme was demonstrated through chemical modification by 1,2-cyclohexanedione (C HD), trinitrobenzenesulfonic aci d (TNBS) and tetranitromethane (TNM), respectively. The t hree modifiers inactivated GDH enzyme wi th pseud o-first order ki netics and second order-rate co nstants of 22, 0.70 and 0.5 mM min , respectively. Both a- ketoglutarate and NADH offered GDH a pro tection agai nst the -1 -1 inactivation. These studies s uggested the i nvolvement of arginine, lysine and tyrosine res idues in t he enzyme catalysis. GDH was immobilized on gelatin beads via c ross-linking with g lutaraldehyde. m The resulting immobilized e nzyme was stored at 4 ° C f or 10 days without loosing its activity. K of max immobilized enzyme increased while V reduced compared to t he free one. The immobilization of GDH resulted in a shift of pH optimum from 7.5 to 8.0. The optimum temperatures of the free and immobilized enzyme were 45 °C and 60 °C, re spectively. The immobilized enzyme has a long life a s c ompared with the native enzyme.
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