Kinetic basis of celite (CM 2:1) addition on the biosynthesis of 3,4-dihydroxyphenyl-L-alanine (L-DOPA) by Aspergillus oryzae ME2 using L-tyrosine as a basal substrate

Celite is a unique variety of 2:1 clay mineral and is referred to as hydrous mica because it is believed to be the weathering product of mica. The present study describe the effect of celite on microbiological conversion of l-tyrosine to 3,4-dihydroxyphenyl-l-alanine (L-DOPA) by Aspergillus oryzae ME2. At optimum pH (pH(o)=6), and temperature (t = 30 degrees C), 100% sugars were utilized for cell mass formation. Mould mycelium was used for biochemical conversion of L-tyrosine to L-DOPA because tyrosinases, beta-carboxylases and tyrosine hydroxylases are intracellular enzymes. The maximum conversion of L-tyrosine to L-DOPA (0.428 mg/ml) was achieved after 60 min of reaction. To further enhance the production of L-DOPA, different concentration of celite were added to the reaction mixture. Best results were observed when the concentration of celite was 3.5 mu M (1.686 mg L-DOPA produced/ml with 1.525 mg L-tyrosine consumed/ml). There was up to 3-fold enhancement in the product formation rate. This enhancement is the highest so far reported. Celite not only increased enzyme activity but also enhanced the permeability of the cell membrane to facilitate the secretion of enzymes in to the reaction broth. The comparison of kinetic parameters showed the ability of the mutant to yield L-DOPA (i.e., Y-p/x 7.360 +/- 0.04 mg/mg). When the culture grown on various celite levels was monitored for Q(p), Q(s) and q(p), there was significant enhancement (P < 0.025) in these variables over the control, which indicates that the study can be commercially applicable (HS, LSD 0.456).