Cyclic 3',5'-diadenosine monophosphate (c-di-AMP) is a newly recognized bacterial nucleotide second messenger molecule. In addition, it has been shown to be a potential vaccine adjuvant. Although multiple methods are available for c-di-AMP synthesis, the yields are low and the purification procedures are laborious. Here, we report an enzymatic method for more efficient and economical c-di-AMP synthesis using a diadenylate cyclase DisA from Bacillus thuringiensis BMB 171 (btDisA). After overexpression and purification of btDisA, the enzyme-catalyzed reaction conditions were further investigated. Under the optimum conditions, in which 100 mM CHES (pH 9.5) containing 2 mu M btDisA, 10 mM ATP, and 10 mM MgCl2 was incubated at 50 degrees C for 4 h, a high conversion rate of c-di-AMP was obtained. Coupling this process with HPLC purification and lyophilization yielded 100 mg of highly pure c-di-AMP that was harvested in white powder form from a 50 mL enzyme-catalyzed reaction system. The protocol is not only directly applicable for preparing abundant amounts of c-di-AMP for extensive biochemical and immunological use, but can also be scaled up to meet the requirements for medical applications. (C) 2013 Elsevier Inc. All rights reserved.