Double mutant of Aspergillus oryzae for improved production of L-dopa (3,4-dihydroxyphenyl-L-alanine) from L-tyrosine.

Aspergillus oryzae mutant strain UV-7 was further improved for the production of L-dopa from L-tyrosine using chemical mutation. Different putative mutant strains of the organism were tested for the production of L-dopa in triplicate shake-flask cultures. Among these putative mutants, the strain designated SI-12 gave a maximal production of L-dopa (444+/-14 mg of L-dopa/g of cells). The regulation of L-dopa from different carbon source solutions [initial substrate concentration (S(0))=30 g/l] by the mutant culture was investigated. At an initial pH (pH(0)) of 5.0 and a temperature (T) of 30 degrees C, 100% of sugars were utilized for product and cell mass formation, corresponding to final L-dopa product yield of 189+/-8 mg/g of substrate utilized and maximum volumetric and specific productivities of 145+/-5 mg/h per litre and 155+/-8 mg/h per g of cells respectively. There was up to 3-fold enhancement in product formation rate. This enhancement is, to our knowledge, the highest reported in the literature. To explain the kinetic mechanism of L-dopa formation and its thermal inactivation, the thermodynamic parameters were determined with the application of the Arrhenius model. Activation enthalpy and entropy for product formation, in the case of the mutant derivative, were 40 kJ/mol and 0.076 kJ.mol(-1).K(-1) for its production and 116 kJ/mol and 0.590 kJ.mol(-1).K(-1) for thermal inactivation respectively. The respective values for product formation and product de-activation were lower than the respective values for the parental culture. Therefore the mutant strain was thermodynamically more resistant to thermal denaturation during the product-formation process.