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Asian Journal of Biochemistry
  Year: 2008 | Volume: 3 | Issue: 5 | Page No.: 320-329
DOI: 10.3923/ajb.2008.320.329
 
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Identification of Catalytically Essential Amino Acid Residues and Immobilization of Glutamate Dehydrogenase from Rumex Cotyledons

H.M. El-Shora and M.M. Youssef

Abstract:
The present study was designed to characterize and immobilize the glutamate dehydrogenase (GDH, EC 1.4.1.2) from Rumex dentatus cotyledons. The enzyme from Rumex dentatus was purified with specific activity of 145 U mg-1 protein. The indispensable role of arginine, lysine and tyrosine at the active site of the enzyme was demonstrated through chemical modification by 1,2-cyclohexanedione (CHD), trinitrobenzenesulfonic acid (TNBS) and tetranitromethane (TNM), respectively. The three modifiers were inactivated GDH enzyme with pseudo-first order kinetics and second order-rate constants of 22, 0.70 and 0.5 mM-1 min-1, respectively. Both α-ketoglutarate and NADH offered GDH a protection against the inactivation. These studies suggested the involvement of arginine, lysine and tyrosine residues in the enzyme catalysis. GDH immobilized on gelatin beads via cross-linking with glutaraldehyde. The resulting immobilized enzyme was stored at 4 °C for 10 days without loosing its activity. Km of immobilized enzyme increased while Vmax reduced compared to the 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, respectively. The immobilized enzyme has a long life as compared with the native enzyme.
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How to cite this article:

H.M. El-Shora and M.M. Youssef, 2008. Identification of Catalytically Essential Amino Acid Residues and Immobilization of Glutamate Dehydrogenase from Rumex Cotyledons. Asian Journal of Biochemistry, 3: 320-329.

DOI: 10.3923/ajb.2008.320.329

URL: https://scialert.net/abstract/?doi=ajb.2008.320.329

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