Identification and characterization of a second butyrate kinase from Clostridium acetobutylicum ATCC 824

A gene encoding a new butyrate kinase isozyme (BKII) was identified from the C. acetobutylicum ATCC 824 DNA database. The enzyme was expressed in Escherichia coli, purified, and characterized. The purified enzyme exhibited a subunit molecular mass of 43 kDa by SDS-PAGE, and a native molecular mass of 80 kDa by gel filtration suggesting it functions as a dimer. In the butyryl phosphate-forming direction the optimal pH of BKII was 8.5. The enzyme had a Km of 0.62 M and a turn over rate of 2.2 x 10(5)/sec (Vmax of 165 units/mg). The presence of a mRNA encoding the BKII was demonstrated using a reverse transcription PCR reaction. The expression of the BKII in Clostridium acetobutylicum ATCC 824 was further examined by Western blot analysis using a polyclonal antibody prepared against recombinant BKII.