title: Overexpression in a non-native halophilic host and biotechnological potential of NAD+-dependent glutamate dehydrogenase from Halobacterium salinarum strain NRC-36014.
abstract: Enzymes produced by halophilic archaea are generally heat resistant and organic solvent tolerant, and accordingly important for biocatalytic applications in 'green chemistry', frequently requiring a low-water environment. NAD(+)-dependent glutamate dehydrogenase from an extremely halophilic archaeon Halobacterium salinarum strain NRC-36014 was selected to explore the biotechnological potential of this enzyme and genetically engineered derivatives. Over-expression in a halophilic host Haloferax volcanii provided a soluble, active recombinant enzyme, not achievable in mesophilic Escherichia coli, and an efficient purification procedure was developed. pH and salt dependence, thermostability, organic solvent stability and kinetic parameters were explored. The enzyme is active up to 90 °C and fully stable up to 70 °C. It shows good tolerance of various miscible organic solvents. High concentrations of salt may be substituted with 30 % DMSO or betaine with good stability and activity. The robustness of this enzyme under a wide range of conditions offers a promising scaffold for protein engineering.
authors.affiliation: Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland.
authors.forename: Nayla
authors.initials: N
authors.lastname: Munawar
authors.forename: Paul C
authors.initials: PC
authors.lastname: Engel
mesh_terms: D019843:Archaeal Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D001622:Betaine / Q000737:chemistry; D001709:Biotechnology; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D004795:Enzyme Stability / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005969:Glutamate Dehydrogenase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D019617:Halobacterium salinarum / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D019843:Archaeal Proteins; D011994:Recombinant Proteins; D001622:Betaine; D005969:Glutamate Dehydrogenase; C061421:glutamate dehydrogenase (NAD(P)+); D004121:Dimethyl Sulfoxide