title: Properties of the thermostable glutamate dehydrogenase of the mesophilic anaerobe Peptostreptoccus asaccharolyticus purified by a novel method after over-expression in an Escherichia coli host.
abstract: Peptostreptococcus asaccharolyticus glutamate dehydrogenase (L-glutamate: NAD+ oxidoreductase (deaminating); EC 1.4.1.2) overexpressed in Escherichia coli has been purified by two new methods. Enzyme made by the first method showed remarkable thermophilicity, with a temperature optimum of 60 degrees C, and also thermostability, which suggested the second, simpler method, incorporating a heat step. This produced 94 mg of homogeneous protein per litre culture medium. The basic kinetic parameters for P. asaccharolyticus glutamate dehydrogenase with all substrates are revealed at pH 7.0. The enzyme is highly specific for NAD+, with values for kcat/Km 405 times greater than for NADP+. In the reverse direction of reaction, the kcat/Km value for NADH is almost 1000-fold greater than for NADPH.
authors.affiliation: Department of Biochemistry, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: John B
authors.initials: JB
authors.lastname: Carrigan
authors.forename: Suzie
authors.initials: S
authors.lastname: Coughlan
authors.forename: Paul C
authors.initials: PC
authors.lastname: Engel
mesh_terms: D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D005969:Glutamate Dehydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010462:Peptostreptococcus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D009243:NAD; D009249:NADP; D005969:Glutamate Dehydrogenase