CHARACTERIZATION OF HETEROLOGOUSLY PRODUCED CARBONIC-ANHYDRASE FROM METHANOSARCINA-THERMOPHILA

The gene encoding carbonic anhydrase from Methanosarcina thermophila w as hyperexpressed in Escherichia coli, and the heterologously produced enzyme was purified 14-fold to apparent homogeneity. The enzyme purif ied from E. coli has properties (specific activity, inhibitor sensitiv ity, and thermostability) similar to those of the authentic enzyme iso lated from M. thermophila; however, a discrepancy in molecular mass su ggests that the carbonic anhydrase is posttranslationally modified in either E. coli or M. thermophila. Both the authentic and heterologousl y produced enzymes were stable to heating at 55 degrees C for 15 min b ut were inactivated at higher temperatures, No esterase activity was d etected with p-nitrophenylacetate as the substrate, Plasma emission sp ectroscopy revealed approximately 0.6 Zn per subunit. As judged from t he 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 a re regulated in response to the growth substrate, with protein levels higher in acetate than in methanol- or trimethylamine-grown cells.