(164q) A Novel Thermophilic Enzyme for DNA Based Error Correction | AIChE

(164q) A Novel Thermophilic Enzyme for DNA Based Error Correction


Sabnis, R. - Presenter, Texas A&M University
Sun, Q., Texas A&M University
de Figueiredo, P., Texas A&M Health Science Center
The ability to synthesize accurate DNA is important for advancements in synthetic biology and biotechnology. Contrary to DNA replication in vivo, in vitro DNA synthesis lacks DNA repair mechanism and pathways to detect and repair mismatches. Enzymatic error correction has emerged as a cost effective and convenient method for error mitigation that are embedded in synthetically generated DNA fragments. Several commercial enzymes have shown to reduce error rate of synthetic genes in vitro. However, due to limitations related to enzyme kinetics and thermal stability, the error correction efficiencies are inadequate for downstream applications. Prior research has reported that despite environments more favorable to mutations, the mutation frequency in hyperthermophiles is lower than their mesophilic bacteria counterparts. We hypothesize that enzymes obtained from thermophiles will demonstrate higher robustness and better error correction efficiencies. We identified, expressed, purified, and characterized one error correction enzyme from thermophilic archaeon Thermococcus Barophilus. We tested our hypothesis by comparing the enzyme activity and found that the thermophilic enzyme has better performance as compared to the commercially available error correction enzyme in detecting and correcting G/C mismatches. Furthermore, we carried out kinetic and activity assay of the purified enzyme. We observed that the thermophilic enzymes exhibit lower unwanted exonuclease activity that is responsible for error free DNA template degradation.