Efficient preparation of c-di-AMP at gram-scale using an immobilized Vibrio cholerae dinucleotide cyclase DncV

•Vibrio cholerae dinucleotide cyclase DncV can efficiently synthesize c-di-AMP from ATP with no product inhibition in vitro.•The method includes four steps: DncV immobilization, in situ enzymatic reaction, purification using SP207 resin, and desiccation.•Up to 1 g c-di-AMP of purity ≥98% was obtained by using ∼19 mg immobilized DncV for reaction and 400 g SP207 resin for purification.•The immobilized DncV on Ni-Chelating resin is reusable and applied two times without obvious decrease in the enzyme activity.•The method can be easily developed for industry to prepare c-di-AMP on a large scale.