원문정보
초록
영어
In this study, we describe a method for parallel generation and screening of transaminases through the protocols of PCR-based cell-free protein synthesis. Cloned nucleotide sequences of ω-aminotransferases and putative genes were PCR-amplified and directly expressed into corresponding enzymes in a cell-free protein synthesis system. For the subsequent colorimetric analysis ,we used 2,4-dinitrophenylhydrazine (2,4-DNPH). 2,4-DNPH formed yellow precipitates of 2,4-dinitrophenylhydrazone upon the reaction with ketone group of pyruvate, which served as an amine acceptor during the aminotransfer reactions. Our results demonstrate that combination of the cell-free protein synthesis techniques with properly designed assay methods can accelerate the procedures for the screening of required enzymes from the pools of nucleotide
sequences.