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 |
| |
Authors: | Carrigan John B Coughlan Suzie Engel Paul C |
| |
Institution: | Department of Biochemistry, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland. |
| |
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. |
| |
Keywords: | Glutamate dehydrogenase Thermostability Thermophilicity Coenzyme specificity Purification Overexpression Peptostreptococcus |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|