title: Overproduction of Thermus sp. YS 8-13 manganese catalase in Escherichia coli production of soluble apoenzyme and in vitro formation of active holoenzyme.
abstract: Overproduction of Thermus sp. YS 8-13 manganese catalase in Escherichia coli BL21(DE3) was accomplished by introducing a derivative of pET-23a(+) containing a copy of the coding gene into the multicloning site. E. coli BL21(DE3)/pETMNCAT produced abundant quantities of manganese catalase as insoluble inclusion bodies. Regeneration of active catalase was achieved by denaturation in guanidine hydrochloride and subsequent dialysis in the presence of manganese ion. When the E. coli chaperone genes GroEL, GroES, DnaK, DnaJ and GrpE were coexpressed with manganese catalase, a significant fraction of the overproduced protein was partitioned into the soluble fraction. However, almost all of the soluble enzyme was isolated in a manganese-deficient apo form which could subsequently be converted into active holoenzyme by incubation with manganese ion at high temperatures. Further experiments on this apo catalase suggested that the structure of this protein was virtually identical to the active holoenzyme.
authors.affiliation: Department of Biotechnology, Faculty of Engineering, Tottori University, Japan. mizobata@bio.tottori-u.ac.jp
authors.forename: T
authors.initials: T
authors.lastname: Mizobata
authors.forename: M
authors.initials: M
authors.lastname: Kagawa
authors.forename: N
authors.initials: N
authors.lastname: Murakoshi
authors.forename: E
authors.initials: E
authors.lastname: Kusaka
authors.forename: K
authors.initials: K
authors.lastname: Kameo
authors.forename: Y
authors.initials: Y
authors.lastname: Kawata
authors.forename: J
authors.initials: J
authors.lastname: Nagai
mesh_terms: D001051:Apoenzymes / Q000096:biosynthesis*; D002374:Catalase / Q000096:biosynthesis*; D002460:Cell Line; D002850:Chromatography, Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics*; D020035:Holoenzymes / Q000096:biosynthesis*; D018832:Molecular Chaperones / Q000096:biosynthesis; D013824:Thermus / Q000201:enzymology*
chemical_list: D001051:Apoenzymes; D020035:Holoenzymes; D018832:Molecular Chaperones; D002374:Catalase