Abstract:

Objective: To obtain high-purity glutamate dehydrogenase (GDH) from duck liver and characterize this enzyme. Methods: Crude GDH solution was obtained from duck liver after acetone defatting, addition of MnCl2 for impurity precipitation, ammonium sulfate salting-out and DEAE-Sepharose ion exchange and Sephacryl S-200 gel permeation chromatographic fractionation. Purity identification and relative molecular mass determination were conducted using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results: A GDH enzyme of electrophoretic homogeneity was obtained, with a purification fold of 60.93, an activity recovery of 11.02% and a specific activity of 24.37 U/mg. This enzyme had a relative molecular mass of 371.41 and a subunit relative molecular mass of 61.60. It was deduced that this enzyme was composed of the same six subunits. Its apparent Km towards NADH was 53.19μmol/L, and the optimal reaction pH and temperature 10.0 and 35 ℃, respectively. This enzyme had excellent stability at around pH 8.0 and at a temperature below 40 ℃. Zn2+, Li+ and Cu2+ had significant inhibition on this enzyme. Conclusion: A high-purity GDH enzyme has been successfully prepared. This enzyme greatly deserves to be developed and utilized.

Key words: duck liver, glutamate dehydrogenase (GDH), purification, characterization