Production of L-DOPA by Thermostable Tyrosine Phenol-lyase of a Thermophilic Symbiobacterium Species Overexpressed in Recombinant Escherichia coli

  • Lee, Seung-Goo (Microbial Conversion Resrarch Unit, Applied Microbiology Research Divistion, Korea Research Institute of Bioscience & Biotechnology, KIST) ;
  • Ro, Hyeon-Su (Microbial Conversion Resrarch Unit, Applied Microbiology Research Divistion, Korea Research Institute of Bioscience & Biotechnology, KIST) ;
  • Hong, Seung-Pyo (Microbial Conversion Resrarch Unit, Applied Microbiology Research Divistion, Korea Research Institute of Bioscience & Biotechnology, KIST) ;
  • Kim, Eun-Hwa (Microbial Conversion Resrarch Unit, Applied Microbiology Research Divistion, Korea Research Institute of Bioscience & Biotechnology, KIST) ;
  • Sung, Moon-Hee (Microbial Conversion Resrarch Unit, Applied Microbiology Research Divistion, Korea Research Institute of Bioscience & Biotechnology, KIST)
  • Published : 1996.04.01

Abstract

A thermostable tyrosine phenol-lyase gene of a thermophilic Symbiobacterium species was cloned and overexpressed in Escherichia coli in order to produce the biocatalyst for the synthesis of 3, 4-dihy-droxyphenyl-L-alanine (L-DOPA). The substrates used for the synthetic reaction were pyrocatechol, so-dium pyruvate, and ammonium chloride. The enzyme was stable up to $60^{\circ}C$, and the optimal temperature for the synthesis of L-DOPA was $37^{\circ}C$ . The optimal pH of the reaction was about 8.3. Enzyme activity was highly dependent on the amount of ammonium chloride and the optimal concentration was estimated to be 0.6 M. In the case of pyrocatechol, an inactivation of enzyme activity was observed at con-centrations higher than 0.1 M. Enzyme activity was increased by the presence of ethanol. Under op-timized conditions, L-DOPA production was carried out adding pyrocatechol and sodium pyruvate to the reaction solution intermittently to avoid substrate depletion during the reaction. The concentration of L-DOPA reached 29.8 g/l after 6 h, but the concentration didn t increase further because of the formation of byproducts by a non-enzymatic reaction between L-DOPA and pyruvate.

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