title: Highly efficient enzymatic preparation of c-di-AMP using the diadenylate cyclase DisA from Bacillus thuringiensis.
abstract: 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 100mM CHES (pH 9.5) containing 2μM btDisA, 10mM ATP, and 10mM MgCl2 was incubated at 50°C for 4h, a high conversion rate of c-di-AMP was obtained. Coupling this process with HPLC purification and lyophilization yielded 100mg of highly pure c-di-AMP that was harvested in white powder form from a 50mL 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.
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China.
authors.forename: Cao
authors.initials: C
authors.lastname: Zheng
authors.forename: Jieping
authors.initials: J
authors.lastname: Wang
authors.forename: Yunchao
authors.initials: Y
authors.lastname: Luo
authors.forename: Yang
authors.initials: Y
authors.lastname: Fu
authors.forename: Jianmei
authors.initials: J
authors.lastname: Su
authors.forename: Jin
authors.initials: J
authors.lastname: He
mesh_terms: D001413:Bacillus thuringiensis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D015226:Dinucleoside Phosphates / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D005612:Freeze Drying; D007218:Industrial Microbiology / Q000379:methods*; D019761:Phosphorus-Oxygen Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D015226:Dinucleoside Phosphates; C528998:cyclic diadenosine phosphate; D019761:Phosphorus-Oxygen Lyases