Mutation of Aspergillus oryzae for improved production of 3, 4-dihydroxy phenyl-L-alanine (L-DOPA) from L-tyrosine

Abstract

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 organism were tested for the production of L-DOPA in submerged fermentation. Among these putative mutant strains, mutant designated SI-12 gave maximum production of L-DOPA (300 mg L-DOPA.g -1 cells). The production of L-DOPA from different carbon source solutions (So= 30 g.l -1 ) by mutant culture was investigated at different nitrogen sources, initial pH and temperature values. At optimum pH (pHo= 5.0), and temperature (t=30ºC), 100% sugars were utilized for production and cell mass formation, corresponding to final L-DOPA product yield of 150 mg.g -1 substrate utilized, and maximum volumetric and specific productivities of 125 mg.l , respectively. There was up to 3-fold enhancement in product formation rate. This enhancement is the highest reported in literature. To explain the kinetic mechanism of L-DOPA formation and thermal inactivation of tyrosinase, the thermodynamic parameters were determined with the application of Arrhenius model: activation enthalpy and entropy for product formation, in case of mutant derivative, were 40 k j/mol and 0.076 k j/mol. K for L-DOPA production and 116 k j/mol and 0.590 k j/mol. K for thermal inactivation, respectively. The respective values for product formation were lower while those for product deactivation were higher than the respective values for the parental culture. Therefore, the mutant strain was thermodynamically more resistant to thermal denaturation.

Biological sources of L-DOPA: An alternative approach

“…There were many biological soures reported for enantiometrically pure L-DOPA ( Table 1). Stemonitis herbicola 50 mg [25] Aspergillus oryzae ( mutant) 1.28 mg/ml [26] Aspergillus oryzae 1.28 mg/ml [27] Aspergillus oryzae UV7( double mutant) 1.28 mg ml [28] Aspergillus oryzae 1.86 mg/ml [29] Aspergillus oryzae UV-7 444 g cells [30] Aspergillus oryzae ME2 (Illite) 1.686 mg/ml [31] Aspergillus oryzae ME2 (Celite) 0.428 mg/ml [32] Aspergillus oryzae (Double mutant) 300 mg [33] Aspergillus oryzae IIB-6 1.34 mg/ml [34] Acremonium retilum 0.89 mg/ml [35] Aspergillus niger 0.365 mg/ml [36] Actinomycetes 28.6% [37] Yeast Yarrowia lipolytica NRRL-143 2.96 mg/ml [38] Egyptian halophilic black yeast 66 ug/ml [39] Bacteria Vibrio tyrosinaticus 4 mg/ml [40] Pseudomonas melanogenum 8 mg/ml [41] E. coli W(ATCC 11105) (p-hydroxyphenyl acetate 3-hydroxylase) 48 mM in reaction mixture [42] Bacillus sp. JPJ 0.497 mg/ml [7] Recombinant The production of L-DOPA from biological sources involves the oxidation of L-tyrosine by enzyme tyrosinase (E.C.1.14.18.1), which is copper containing enzyme widely distributed in plants, animals and microorganisms [13].…”

“…L-DOPA produced by the method was enantiometrically pure as well as it is cost effective. Similarly, biotransformation of L-DOPA from L-tyrosine was carried out using Acremonium reticulum by submerged fermentation process yields more amount of L-DOPA in the broth [24][25][26][27][28][29][30][31][32][33][34][35][36][37]. Production of L-DOPA also reported from yeast species [38,39].…”

Co-Immobilization of Peroxidase and Tyrosinase onto Hybrid Membranes Obtained by the Sol-Gel Method for the Construction of an Optical Biosensor

“…1.14.18.1) is a copper-containing enzyme which catalyzes the oxidation of different phenolic substrates in sequential steps (5,9,22). tyrosinase contains about 13 % carbohydrate residues (10), and peroxidase (hRP) about 18 % (20), which allows an effective covalent immobilization to be conducted by prior activation of these residues.…”

Production of 3,4-dihydroxy L-phenylalanine by a newly isolated Aspergillus niger and parameter significance analysis by Plackett-Burman design

“…In addition, L-dopa is an unstable product in the reaction mixture and is further converted into dopaquinone and the final product melanin [5]. Because of the higher production cost and its greater commercial value, many researchers have investigated on the alternative production of L-dopa [6,7]. Investigations have now centred upon microbiological L-dopa production from Erwinia herbicola and Escherichia coli [8,9].…”