Cloning, expression, and characterization of an adenylate cyclase from Arthrobacter sp. CGMCC 3584

The cya gene encoding adenylate cyclase was cloned from Arthrobacter sp. CGMCC 3584 by thermal asymmetric interlaced PCR for the first time. It exhibited an open reading frame containing 1,125 bp and encoding 374 amino acids. Amino acid sequence analysis showed that this enzyme was a class III adenylate cyclase. Expression of the cya gene was carried out in Escherichia coli Rosetta, and purification was performed via Ni²⁺-NTA agarose gel column. SDS-PAGE indicated that the molecular mass of the recombinant adenylate cyclase was 45 kDa. The Vmax and Km were determined to be 5.06 μmol/min/mg and 7.56 mM, respectively. The optimum pH and temperature were 8.0 and 35 °C. Several divalent metal ions were found to activate the enzyme to different extents, and the maximal specific activity reached 3.04 μmol/min/mg when 50 mM Mg ²⁺ was added. This was the first report of the cloning of an adenylate cyclase gene from Arthrobacter sp.