Chemical modification and blocking ratio assessment of Taq DNA polymerases using a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-based assay

Mainstreaming modification approaches of commercial hot-start Taq DNA polymerases include antibody modification, nucleic acid adaptor modification, and chemical modification, among which the chemical modification possesses merits of lower cost and higher efficiency excluding animal-derived matters compared with the former two. In chemical modification, the overall modification ratio of lysine residues is a key parameter to indicate the blocking ratio of modified enzyme without compromising its stability. In this study, 2,4,6-trinitrobenzenesulfonic acid (TNBS)-based assay was applied for chemically modified Taq DNA polymerase activity blocking ratio for the first time. The results showed that at the modification ratio of about 50%, the enzyme activity was completely blocked. This detection method's features were easy-operation and time-saving for the screening of optimized anhydride modified Taq DNA polymerase in multiple incubation conditions. Notably, this new protocol provided a practical reference for identification of novel chemical compounds that can modify DNA polymerases, thereby facilitating the development of advanced enzymes.