High performance microbiological transformation of L-tyrosine to L-dopa by Yarrowia lipolytica NRRL-143

Abstract

Background: The 3,4-dihydroxy phenyl L-alanine (L-dopa) is a drug of choice for Parkinson's disease, controlling changes in energy metabolism enzymes of the myocardium following neurogenic injury. Aspergillus oryzae is commonly used for L-dopa production; however, potential improvements in ease of handling, growth rate and environmental impact have led to an interest in exploiting alternative yeasts. The two important elements required for L-dopa production are intracellular tyrosinases (thus pre-grown yeast cells are required for the transformation of L-tyrosine to L-dopa) and L-ascorbate, which acts as a reducing agent.

Results: Pre-grown cells of Yarrowia lipolytica NRRL-143 were used for the microbiological transformation of L-tyrosine to L-dopa. Different diatomite concentrations (0.5-3.0 mg/ml) were added to the acidic (pH 3.5) reaction mixture. Maximum L-dopa biosynthesis (2.96 mg/ml L-dopa from 2.68 mg/ml L-tyrosine) was obtained when 2.0 mg/ml diatomite was added 15 min after the start of the reaction. After optimizing reaction time (30 min), and yeast cell concentration (2.5 mg/ml), an overall 12.5 fold higher L-dopa production rate was observed when compared to the control. Significant enhancements in Yp/s, Qs and qs over the control were observed.

Conclusion: Diatomite (2.0 mg/ml) addition 15 min after reaction commencement improved microbiological transformation of L-tyrosine to L-dopa (3.48 mg/ml; p < or = 0.05) by Y. lipolytica NRRL-143. A 35% higher substrate conversion rate was achieved when compared to the control.

Figure 1

The effect of different diatomite concentrations on L-dopa production by Y. lipolytica NRRL-143 (L-tyrosine consumed -∘-, L-Dopa produced -×-). A total of 3.5 mg/ml L-tyrosine, 3.0 mg/ml cell biomass and varying diatomite amounts were added at the start of the biochemical reaction. The total reaction time was 50 min at 50°C.

Figure 2

The effect of time of addition of diatomite on L-dopa production by Y. lipolytica NRRL-143 (L-tyrosine consumed -∘-, L-Dopa produced -×-). A total of 2.0 mg/ml diatomite was added to 3.5 mg/ml L-tyrosine and 3.0 mg/ml cell biomass. The total reaction time was 50 min at 50°C.

Figure 3

Time course of L-dopa production and L-tyrosine consumption by Y. lipolytica NRRL-143 (L-tyrosine consumed -∘-, L-Dopa produced -×-). a. Control (3.5 mg/ml L-tyrosine and 3.0 mg/ml cell biomass) b. Test (2.0 mg/ml diatomite added 15 min after the start of reaction to 3.5 mg/ml L-tyrosine and 3.0 mg/ml cell biomass). The total reaction time was 50 min at 50°C.

Figure 4

The effect of various levels of cell biomass (Y. lipolytica NRRL-143) on L-dopa production (L-tyrosine consumed -∘-, L-Dopa produced -×-). a. Control (3.5 mg/ml L-tyrosine only). b. Test (3.5 mg/ml L-tyrosine and 2.0 mg/ml diatomite added 15 min after the start of reaction), The total reaction time was 50 min at 50°C.