Identification and enhanced activity of the tyrosine phenol-lyase from Mycobacterium tuberculosis.

L-DOPA (3,4-dihydroxyphenyl-L-alanine) is a key drug for the Parkinson's disease. The global demand of L-DOPA is continuously increasing. Tyrosine phenol-lyase (TPL) is the environmentally friendly biocatalyst for L-DOPA biosynthesis. In this study, a novel TPL from Mycobacterium tuberculosis (Mt-TPL) was identified and expressed in Escherichia coli. The characterization of Mt-TPL indicated that the optimal temperature and pH values were 40 °C and 9.0, respectively, and the half-lives (t1/2) at 30 °C were 6.52 h. To further improve its catalytic performance, protein engineering of Mt-TPL was undertaken using a consensus sequence-based strategy. It was found that the I384V mutant exhibited the increase of 40.31 % in the enzymatic activity as compared with the wild-type enzyme. Molecular docking and molecular dynamics (MD) simulations revealed that the I384V mutation changed the local flexibility of enzyme and the orientation of substrate, and shortened the proton transfer distance between the generic acid Y71 and substrate pyrocatechol. Finally, the whole-cell catalysts with wild type and I384V Mt-TPL were used to product the L-DOPA, the L-DOPA production of 95.46 g/L for whole-cells with I384V Mt-TPL was achieved in a 5 L fermenter by the fed-batch, 38.6 % higher than that of the wild type enzyme.