Design of Halotolerant Bovine Dnase Guided By Phylogenetic and Domain Structure Studies of Bacterial Dnases

Analysis of sequence databases suggested that non - specific DNA binding domain is characteristic to many halophilic or halotolerant bacterial DNase species and it could serve as a mechanism to maintain enzyme activity at high ionic strengths.

This hypothesis was tested on one secreted bacterial DNase from halophilic bacterium Thioalkalivibrio sp., which was experimentally shown to maintain activity at almost saturated solution of sodium chloride. The enzyme is composed from two domains: N-terminal domain which exhibits DNase activity and C-terminal domain comprising duplicate HhH motifs, which natural functions are recognition and interaction with DNA.

 We have experimentally proved, that the C-terminal domain in Thioalkalivibrio sp. DNase is responsible for enzyme halophilicity. Based on these findings we designed two variants of bovine DNase I C-terminal fusions: one with fused C-terminal domain of DNase from halophilic bacterium Thioalkalivibrio sp. , the second one with fused homologous domain from ComEA protein of B. subtilis. These fusions were showed to be more salt tolerant than wild type DNase and subsequently superior to it at least in several practical applications