Open AccessProperties of the thermostable glutamate dehydrogenase of the mesophilic anaerobe Peptostreptoccus asaccharolyticus purified by a novel method after over‐expression in an Escherichia coli host
Author(s) -
Carrigan John B.,
Coughlan Suzie,
Engel Paul C.
Publication year - 2005
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1016/j.femsle.2005.01.026
Subject(s) - thermostability , glutamate dehydrogenase , oxidoreductase , nad+ kinase , enzyme kinetics , enzyme , escherichia coli , biology , biochemistry , dehydrogenase , mesophile , microbiology and biotechnology , glutamate receptor , bacteria , active site , genetics , receptor , gene
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 °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 k cat / K m 405 times greater than for NADP + . In the reverse direction of reaction, the k cat / K m value for NADH is almost 1000‐fold greater than for NADPH.