title: Characterization of heterologously produced carbonic anhydrase from Methanosarcina thermophila.
abstract: The gene encoding carbonic anhydrase from Methanosarcina thermophila was hyperexpressed in Escherichia coli, and the heterologously produced enzyme was purified 14-fold to apparent homogeneity. The enzyme purified from E. coli has properties (specific activity, inhibitor sensitivity, and thermostability) similar to those of the authentic enzyme isolated from M. thermophila; however, a discrepancy in molecular mass suggests that the carbonic anhydrase is posttranslationally modified in either E. coli or M. thermophila. Both the authentic and heterologously produced enzymes were stable to heating at 55 degrees C for 15 min but were inactivated at higher temperatures. No esterase activity was detected with p-nitrophenylacetate as the substrate. Plasma emission spectroscopy revealed approximately 0.6 Zn per subunit. As judged from the estimated native molecular mass, the enzyme is either a trimer or a tetramer. Western blot (immunoblot) analysis of cell extract proteins from M. thermophila indicates that the levels of carbonic anhydrase are regulated in response to the growth substrate, with protein levels higher in acetate than in methanol- or trimethylamine-grown cells.
authors.affiliation: Department of Biochemistry and Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg, 24061-0305, USA.
authors.forename: B E
authors.initials: BE
authors.lastname: Alber
authors.forename: J G
authors.initials: JG
authors.lastname: Ferry
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002256:Carbonic Anhydrases / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D017020:Methanosarcina / Q000201:enzymology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000302:isolation & purification; D015032:Zinc / Q000032:analysis
chemical_list: D011994:Recombinant Proteins; D002256:Carbonic Anhydrases; D015032:Zinc