Innovative Effect of Illite on Improved Microbiological Conversion of L-Tyrosine to 3,4 Dihydroxy Phenyl L-Alanine (L-DOPA) by Aspergillus oryzae ME₂ Under Acidic Reaction Conditions

Abstract

In the present investigation, the previous ultraviolet irradiated mutant strain of Aspergillus oryzae UV-7 was further improved in terms of 3,4 dihydroxy phenyl L-alanine (L-DOPA) activity after chemical mutagenesis through 1-methyl 3-nitro 1-nitroso guanidine (MNNG = 250–1500 μg/ml) treatment (0–30 min). Among several mutant variants, the one that produced a larger amount of L-DOPA from L-tyrosine was designated to as ME₂ and it was made 2-deoxy-D-glucose-resistant by growing it at various concentrations of 2 dg (0.01–0.025 %, w/v) in Vogel’s agar medium. Relatively better production of L-DOPA (> 0.60 mg/ml) was obtained when 2.0% (w/v) glucose was used as a carbon source in the mycelium production medium and the tyrosinase activity increased constitutively (1.08 mg/ml), which resulted in a greater production of L-DOPA. At optimum pH₀ (pH 6.0) and reaction time (60 min), more than 65% sugar was utilized for cell mass formation. The maximum conversion of L-tyrosine to L-DOPA (0.428 mg/ml) was achieved 60 min after the biochemical reaction. Mould mycelium was used for microbiological conversion of L-tyrosine to L-DOPA because tyrosinases, β-carboxylases, and tyrosine hydroxylases are intracellular enzymes. The effect of illite (1.0 × 10⁶–6.0 × 10⁶ M) on biochemical conversion of L-tyrosine to L-DOPA by Aspergillus oryzae ME₂ was also carried out. Best results of L-DOPA biosynthesis were observed when the concentration of illite was 3.5 × 10⁻⁶ M (1.686 mg/ml L-DOPA produced with 1.525 mg/ml consumption of L-tyrosine). It was noted that the addition of illite not only increased enzyme activity but also enhanced the permeability of cell membrane to facilitate the secretion of enzymes into the reaction broth. The comparison of kinetic parameters showed the ability of mutant to yield L-DOPA (i.e., Y_p/x 7.360 ± 0.04 mg/mg). When the culture grown on various illite concentrations was monitored for Q_p, Q_s, and q_p, there was significant enhancement (p < 0.025) in these variables over the control, which indicate that the study can be commercially applicable on stirred and magnetic rotary drums. Overall, there was up to 3-fold (Q_p = 0.290 mg/L-DOPA produced/ml/h) enhancement in the product formation rate, which is highly encouraging (HS, LSD 0.456).