• 한국미생물·생명공학회지
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• 제24권1호
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• pp.44-49
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• 1996

By using the ${\beta}$-tyrosinase of Citrobacter freundii KCT2006, 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$^{\circ}C$ and 8.5, respectively. The effects of substrate concentrations were also examined at different concentrations of ammonium acetate, sodium pyruvate, and pyrocatechol. Ammoniumacetate and sodium pyruvate increased the reaction rate until the concentrations reached to 300mM and 50mM, respectively. Although pyrocatechol showed the optimal concentration at 20mM, it was controlled between 20mM and 50mM 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 productin of L-DOPA was prepared and incubated with 1 unit/ml of ${\beta}$-tyrosinase. Pyrocatechol and sodium pyruvate was added to the reaction solutin intermittently to avoid the substrate depletion during the enzymatic reaction. After 24 hour of reaction, 31.6g/l of L-DOPA was accumulated in the reaction solution as soluble and precipitated ones and the conversion yield was about 85.2%.

• 한국미생물·생명공학회지
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• 제24권2호
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• pp.222-226
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• 1996

The enzymatic synthesis of 3, 4-dihydroxyphenyl-L-alanine (L-DOPA) was examined for the effects of the reaction additives such as sodium borate, alcohol, and organic solvents. The enzyme used was tyrosine phenol-lyase of Citrobacter freundii KCTC 2006 produced in Escherichia coli. The amounts of tyrosine phenol-lyase and pyridoxal-5-phosphate were optimized to 2.0 units/ml and 0.1 mM, respectively, for the synthetic reaction. Sodium borate, a substance that forms a complex with pyrocatechol, reduced the enzyme deactivation by pyrocatechol although it seriously inhibited the enzyme activity. Among the organic solvents tested, dimethylsulfoxide, dimethylformamide, and alcohol increased the productivity of the L-DOPA synthesis. In a reaction system with 5% methanol, L-DOPA concentration increased up to 210 mM after 24 hours, and 77.1% of which was separated as precipitates. The L-DOPA was purified to 99.96% by solubilizing and recrystallyzing the precipitates.