재조합 대장균에서 과발현된 Citrobacter freundii KCTC2006 유래의 β-tyrosinase를 이용한 3,4-dihydroxyphenyl-L-alanine의 생산

Abstract

By using the β-tyrosinase of Citrobacter freundii KCTC2006, which was cloned and overexpressed in Escherichia coli, 3,4-dihydroxy phenyl-L-alanine (L-DOPA) was synthesized efficiently from pyrocatechol, sodium pyruvate, and ammonium acetate. Optimal temperature and pH for the reaction were determined to be about 18°C and 8.5, respectively. The effects of substrate concentrations were also examined at different concentrations of ammonium acetate, sodium pyruvate, and pyrocatechol. Ammonium acetate and sodium pyruvate increased the reaction rate until the concentrations reached to 300 mM and 50 mM, respectively. Although pyrocatechol showed the optimal concentration at 20 mM, it was controlled between 20 mM and 50 mM to avoid the depletion of substrate during the enzymatic synthesis. Meanwhile the synthetic rate was improved about 20% when ethanol was included in the reaction solution. Based on above results, a reaction medium for the production of L-DOPA was prepared and incubated with 1 unit/ml of β-tyrosinase. Pyrocatechol and sodium pyruvate was added to the reaction solution intermittently to avoid the substrate depletion during the enzymatic reaction. After 24 hour of reaction, 31.6 g/l of L-DOPA was accumulated in the reaction solution as soluble and precipitated ones and the conversion yield was about 85.2%.