Molecular Cloning, Heterologous Expression, And Characterization Of Tyrosine Decarboxylase From Enterococcus Faecium R615Z1 And Enterococcus Faecalis R612Z1

Enterococcus faecium and Enterococcus faecalis are two tyramine-producing bacteria and were both concernd to have ability to produce tyamine and phenylethylamine. In our study, molecular cloning, heterologous expression, and characterization of tyrosine decarboxylase from Enterococcus faecium R615Z1 and Enterococcus faecalis R612Z1 commonly found in Nanjing traditional food-water boiled salted duck were studied.Two bacteria were identified to be Enterococcus faecium and Enterococcus faecalis respectively. And PCR method was used to amplified the tyrosine decarboxylase (TDC) genes, alignment were tested. Enterococcus faecium R612Z1 TDC gene has 1921 bp bases, while the Enterococcus faecalis R615Z1 TDC gene has 2006 bp bases. By compared these two gene basic groups and amino acid alignment, high similarity had been found. And PLP binding sites were found in the alignment, which means these two enzymes are PLP-dependent decarboxylases.PCR products from chapter two were used to construct express plasmids, and SDS-PAGE method was used to identify the purified enzymes, and finally the activity of both enzymes were tested, reaults showed that both of the enzymes' weights are approximately 70 kDa, and tyrosine and phenylalanine can be catalyzed by both enzymes.Values of Km and Vm for both enzymes were tested and the optimal pH and temperature of the enzymes were also found. Results comes to Kmt=0.021 mol-L-1, Vmaxt=2.088 mol-L-1min-1, optimum temperature 25?, and optimum pH 6.0 for 612TDC; and Kmp=0.019 mol-L-1, Vmaxp=0.314 mol·L-1min-1, optimum temperature 25?, and optimum pH 5.5 for 615TDC. With the increase of NaCl, tyramine production merely changed, on the contrary, phenyl ethylamine production decreased seriously.