Biosynthesis of L-DOPA by Aspergillus oryzae

Ikram-ul-Haq *, Sikander Ali, M.A. Qadeer

Biotechnology Research Laboratories, Department of Botany, Government College, Lahore, Pakistan

Received 22 May 2001; received in revised form 21 February 2002; accepted 7 March 2002

Abstract

The present investigation deals with the biosynthesis of L-DOPA by parental (GCB-6) and mutant (UV-7) strains of Aspergillus

oryzae. There was a marked difference between the mycelial morphology and pellet type of parental and UV-irradiated mutant

culture. The mutant strain of A. oryzae UV-6 exhibited pellet-like mycelial morphology and improved tyrosinase activity. Mould

mycelium was used for biochemical conversion of L-tyrosine to L-DOPA because tyrosinase is an intracellular enzyme. The mutant

was found to yield 3.72 fold higher production of L-DOPA than the parental strain. The mutant strain is stable and D-glc-resistant.

The comparison of kinetic parameters was also done which showed the greater ability of the mutant to yield L-DOPA (i.e., Yp=x

40:00 � 0:01d mg/mg with parent and 182:86 � 0:02a mg/mg in case of mutant). When cultures grown for various incubation pe-

riods, were monitored for Qp, Qs and qp, there was significant enhancement (p < 0:0025–0.005) in these variables by the mutant

strain of A. oryzae UV-7 over GCB-6 on all the rates. L-DOPA (3,4-dihydroxy phenyl L-alanine) is a drug of choice in the treatment

of Parkinson’s disease and myocardium following neurogenic injury. � 2002 Elsevier Science Ltd. All rights reserved.

Keywords: L-DOPA; Aspergillus oryzae; Biosynthesis; Fermentation; Amino acid studies; Parkinson’s disease; Myocardium; Neurogenic injury

1. Introduction

L-DOPA (3,4-dihydroxy phenyl L-alanine) is a usefuldrug in the treatment of Parkinson’s disease and myo-cardium following neurogenic injury (Raju et al., 1993).It occurs naturally in beans of Vicia faba and seeds ofMucana pruriens. Its production has been described by anumber of workers (Sih et al., 1969; Haq et al., 1998;Fling and Paul, 2001). L-DOPA is produced from L-tyrosine by a one-step oxidation reaction by submergedfermentation (Haneda et al., 1973). The optimisation ofcultural conditions is necessary for the successful fer-mentation process. The key enzyme responsible forbiosynthesis of L-DOPA is ‘tyrosinase’ (Rosazza et al.,1995; Ali and Haq, 2000). Tyrosinases are widely dis-tributed and highly purified enzymes, derived from mi-crobial (Aspergillus, Rhizopus and Neurospora spp.) andplant sources (Agaricus and Vicia spp.). However, in mi-croorganisms tyrosinase activity is generally very weakand L-tyrosine and L-DOPA are rapidly decomposed toother metabolites. Thus, stoichiometric formation of

LL-DOPA is difficult to achieve (Kumagai et al., 1969;James and Fling, 2001). The mycelial activity ofAspergillus oryzae or A. flavus catalysing L-tyrosine toLL-DOPA was observed in the acidic range below pH 5.0(Singh, 1999; Haq et al., 2000).

In the present study, the goal was to increase thebiomass of A. oryzae and consequently the productionof L-DOPA, using a shake flask technique. Tyrosinaseis an intracellular enzyme. So, mould mycelium wasused for the biochemical conversion of L-tyrosine toLL-DOPA. The wild-type culture of A. oryzaeGCB-6 wasimproved after ultraviolet irradiation to increase theproduction of L-DOPA in the reaction mixture.

2. Methods

2.1. Organism

A. oryzae strain GCB-6 was used for the presentstudy. This wild-type culture of A. oryzae was takenfrom Biotechnology Research Laboratories, Depart-ment of Botany, Government College, Lahore. It wasmaintained on potato dextrose agar medium and storedat 4 �C in a refrigerator.

Bioresource Technology 85 (2002) 25–29

* Corresponding author.

E-mail address: ikrhaq@yahoo.com (Ikram-ul-Haq).

0960-8524/02/$ - see front matter � 2002 Elsevier Science Ltd. All rights reserved.

PII: S0960-8524 (02 )00060-3

2.2. Improvement of strain after UV-irradiation

The A. oryzae strain GCB-6 was improved after UV-irradiation. The dose of ultraviolet irradiation was givenat a rate of 1:2 � 106 J/m2/s. Conidia from 3 to 5 daysold cultures were harvested in phosphate buffer con-taining (g/l); K2HPO4 3.5, KH2PO4 1.5 at pH 7.2. Theconidial suspension was exposed to UV-irradiation fordifferent time intervals (15–120 min), aseptically. Theirradiated conidial suspension was poured on agar–maltextract with tyrosine plates (Roy and Das, 1978). Themutant strain of A. oryzae UV-3, a hyper producer ofLL-DOPA obtained after UV-irradiation was used formycelial mutation. One hundred ml of Vogel mediumcontaining (g/l); trisodium citrate 2.5, NH4NO3 2.0,KH2PO4 5.0, (NH4)2SO4 4.0, MgSO4 � 7H2O 0.2, pep-tone 2.0, yeast extract 1.0 at pH 5.5 with 15–20glass beads (2 mm2, diameter) in a 1 l cotton woolplugged conical flask was sterilized at 15 lbs/in.2 (121 �C)for 15 min. A small quantity of conidia from the slant(3–5 days old) was aseptically transferred with an in-oculating needle to the flask. The flask was incubated at30 �C in an incubator shaker at 200 rpm for 24 h. Theinoculum was kept homogeneous and optical densitywas maintained at 1.0 with the help of a photoelectriccolorimeter, using 530 nm filter. Five ml of the vege-tative inoculum was taken in a petriplate and thenUV-treatment was given from 15 to 25 min intervals,following the method of Pontecarvo et al. (1969). Themutant cultures were incubated at 30 �C for 3–4 daysfor maximum sporulation. There was a marked dif-ference between the mycelial morphology and pellettype of parental and UV-irradiated mutant cultures.The mutant strain of A. oryzae UV-6 showed pellet-like mycelial morphology and improved tyrosinase ac-tivity.

2.3. Cultivation of mycelium

A submerged culture method (Raju et al., 1993) wasemployed for cultivation of mycelium. Conidial inocu-lum was prepared in 10 ml of Monoxal O.T. (Dioctylester of sodium sulphosuccinic acid). Twenty-five ml ofcultivation medium containing (% w/v); glucose 2.0,polypeptone 1.0, NH4Cl 0.3, KH2PO4 0.3, MgSO4 �7H2O 0.02, yeast extract 1.0 at pH 5.0 was takenin 250 ml shake flasks. The medium was autoclavedat 15-lbs/in.2 pressure (121 �C) for 15 min and seededwith 1 ml conidial suspension. The flasks were thenincubated in a rotary incubator shaker (200 rpm) at 30 �Cfor 48 h. The mycelium was harvested by filteringthrough a funnel and washed free of adhering me-dium with ice-cold water. The mycelium was dried infilter paper folds and it was stored at 5 �C till furtheruse.

2.4. Reaction procedure

The reaction for L-DOPA production from L-tyro-sine was carried out in a suspension of intact mycelia.The mycelia were suspended in reaction mixture (Han-eda et al., 1973). Fifteen ml of acetate buffer (pH 3.5; 50mM) containing (mg/ml); L-tyrosine 2.5 ml, L-ascorbicacid 5 ml and intact mycelia 75 ml were taken in a 250ml Erlenmeyer flask. The reaction was carried out aero-bically at 50 �C for 60 min in a hot plate with magneticstirrer. The sample was withdrawn, centrifuged (5000/g)and the supernatant was kept under dark for furtherinvestigation.

2.5. Assay methods

L-DOPA and L-tyrosine were determined colorimet-rically according to the method of Arnow (1937). Fordetermination of L-DOPA, 1 ml of the supernatant wastaken and in it 1.0 ml of 0.5 N HCl along with 1.0 ml ofnitrite molybdate reagent was added. A yellow colourappeared. Then 1.0 ml of 1.0 N NaOH was added,which gave a red colouration and the total volumewas made up to 5 ml. The colour intensity was readby photoelectric colorimeter (Model: AE-II, ERMA,Japan) using a green Wratten filter of 530 nm and theamount of L-DOPA was determined from Arnow’s stan-dard curve of L-DOPA (Arnow, 1937). For L-tyrosine,1 ml of supernatant from the reaction mixture was takenand to it 1 ml of mercuric sulphate reagent was added.Placed in a boiling water bath for 10 min. After which itwas cooled and 1 ml of nitrite reagent added. Totalvolume was made up to 5 ml with distilled water. It wascompared in a colorimeter and the amount of L-tyrosinewas determined from Arnow’s standard curve of L-tyrosine (Arnow, 1937).

2.6. Kinetic parametric studies and statistical analyses

The kinetic parameters were studied according to theprocedures of Pirt (1975). The statistical analyses (US-statistica, version-4) were based on Duncan’s multi-ple range and ANOVA-II design tests (Snedecor andCochran, 1980).

3. Results and discussion

The data for the screening of UV-irradiated mutantstrains of A. oryzae for the production of L-DOPA inshake flasks are shown in Table 1. All the fermentationswere carried out at 30 �C temperature with 160 rpm,agitation rate. The production of L-DOPA by mutantcultures ranged from 0.08 to 1.28 mg/ml. Of all the

26 Ikram-ul-Haq et al. / Bioresource Technology 85 (2002) 25–29

mutant cultures examined, the strain UV-7 gave thehighest yield of L-DOPA. It might be due to the fact thatUV-irradiation had altered the actual structure of DNAby photolysis i.e., formation of pyrimidine dimers. Thestructural change in DNA is related to the activity of theenzyme tyrosinase. Thymidine–thymidine dimers pro-mote mycelial growth in the form of round pellets andsubsequently enzyme activity, which result in the greaterexcretion of L-DOPA from the mycelial cells. Only in afew research reports with similar findings have beenobserved (Kumagai et al., 1969). In the present study,the mutant strain of A. oryzae UV-7 was found to yield3.72-fold higher production of L-DOPA as compared tothe parent culture.

A comparative study of L-DOPA production andkinetic parameters of wild-type culture of A. oryzae andthe more optimal UV-irradiated mutant isolates is pre-sented in Table 2. The specific growth rate (l), cell mass(x) and product yield coefficients (Yp=x, Qp and qp) ofUV-7 showed higher values as compared to wild-typeculture as well as other mutant strains of A. oryzae. TheYp=s (product/substrate utilized g/g) in case of UV-7 was4.2 fold better than GCB-6. On the basis of standarddeviation among the three replicates of flasks, the valueswith letters differ significantly at p < 0:05. The uptake oftyrosine was less effective when A. oryzae was grownunder submerged culture conditions. Although completeuptake of substrate from the media was observed at the

lowest concentrations employed (0.5 and 1.0 mg/mg),this decreased to only 50% at 1.5 mg/ml and to about20% at the highest concentration employed (3.0 mg/ml).The work is not substantiated with Sarin et al. (1980)and Singh (1999) who reported higher product forma-tion at higher concentrations of substrate. However,biomass formation was not influenced in their case (i.e.,Yx=s ¼ 0:0042 mg/mg).

Biosynthesis of L-DOPA by parental (GCB-6) andmutant (UV-7) strains of A. oryzae using the shake flasktechnique is shown in Table 3. The cultures were incu-bated for different time intervals (24–96 h). At 24 h ofincubation, the production of L-DOPA was low (0.21mg/ml in case of the parental strain and 0.16 mg/ml incase of mutant strain of A. oryzae). The maximumproduction of L-DOPA by the parental GCB-6 was 0.36mg/ml, whereas 1.34 mg/ml L-DOPA was obtained bymutant UV-7, 48 h after incubation. The overall valuesof growth yield coefficient Yx=s (mg/mg) of the mutantculture were generally higher than the parent strain.Reduction in the formation of L-DOPA occurred whenthe incubation period was increased beyond 48 h. At 96h of incubation period, the production of L-DOPA be-came very low, for both the parental (0.18 mg/ml) aswell as the mutant strain (0.92 mg/ml). The decrease inLL-DOPA production with the increase in incubationperiod might be due to the overgrowth of fungi and alsoan age factor. Enzyme activity is directly related with the

Table 2

Comparative study of L-DOPA production and kinetic parameters of parental strain of A. oryzae and its best UV-irradiated mutant strains

Kinetic parameters LL-DOPA

Parent (GCB-6) UV-3 UV-6 UV-7

l (h�1) 0.0067� 0.02d 0.0154� 0.02c 0.0170� 0.02b 0.0267� 0.02a

Yx=s (mg/mg) 0.0087� 0.01a 0.0087� 0.02a 0.0086� 0.01ab 0.0081� 0.03b

Qs (mg/ml/h) 0.0192� 0.02b 0.0220� 0.01a 0.0196� 0.02b 0.0179� 0.03c

Qs (mg/mg/h) 0.0062� 0.02b 0.0071� 0.01a 0.0062� 0.01b 0.0058� 0.03bc

Yp=x (mg/mg) 40.000� 0.01d 92.500� 0.01c 102.50� 0.01b 182.86� 0.02a

Yp=s (mg/mg) 0.3478� 0.02d 0.6981� 0.02c 0.8723� 0.02b 1.4883� 0.02a

Qp (mg/ml/h) 0.0066� 0.01c 0.0150� 0.02bc 0.0165� 0.02b 0.0266� 0.02a

qp (mg/mg/h) 0.0021� 0.01c 0.0050� 0.01b 0.0055� 0.02b 0.0086� 0.02a

Table 1

Screening of UV-irradiated mutant strains of A. oryzae for the production of L-DOPA in shake flasks

Mutant strains of A. oryzae LL-Tyrosine used (mg/ml) Dry cell mass (mg/ml) LL-DOPA produced (mg/ml) Mycelial morphology

Parent (GCB-6) 0.92 0.008 0.32 Fine round pellets

UV-1 0.95 0.004 0.16 Gelatinous mass

UV-2 1.21 0.005 0.25 Gelatinous mass

UV-3 1.06 0.008 0.74 Small pellets

UV-4 1.35 0.011 0.30 Gelatinous mass

UV-5 0.87 0.010 0.08 Gelatinous mass

UV-6 0.94 0.008 0.82 Small pellets

UV-7 0.86 0.007 1.28 Intermediate pellets

UV-8 1.42 0.009 0.10 Gelatinous mass

UV-9 1.68 0.014 0.26 Gelatinous mass

UV-10 0.98 0.012 0.18 Gelatinous mass

Incubation temperature 30 �C, pH 5.5, agitation 160 rpm.

Ikram-ul-Haq et al. / Bioresource Technology 85 (2002) 25–29 27

age and development of fungi (Ali and Haq, 2000).Similar findings have also been reported by Raju et al.(1993). Haneda et al. (1973) used A. oryzae for theconversion of L-tyrosine to L-DOPA and obtainedmaximum production (0.86 mg/ml), 72 h after inocula-tion. Thus our finding (1.34 mg/ml L-DOPA) is moreencouraging and economically significant due to greaterproduction and decrease in incubation period. The

consumption of L-tyrosine was 0.86 and 0.75 mg/ml byparent (GCB-6) and mutant (UV-7), respectively.

The comparative time course and kinetic parametersi.e., volumetric rates (Qp, Qs and Qx in mg/ml/h) andspecific rate constants (qp and qs in mg/mg/h) on theproduction of L-DOPA by parent (GCB-6) and mutant(UV-7) are depicted in Tables 4 and 5. Maximumgrowth in terms of overall specific growth and produc-

Table 3

Biosynthesis of L-DOPA by parental (GCB-6) and mutant (UV-7) strains of A. oryzae using shake flask technique

Incubation

period (h)

LL-DOPA produced (mg/ml) LL-Tyrosine used (mg/ml) % L-DOPA� Growth yield coefficient, Yx=s (mg/mg)

Parent Mutant Parent Mutant Parent Mutant Parent Mutant

24 0.21 0.16 0.48 0.26 43.75 61.54 0.013� 0.002b 0.015� 0.002c

36 0.34 0.68 0.62 0.48 54.84 80.42 0.013� 0.001b 0.019� 0.002a

48 0.36 1.34 0.86 0.74 41.86 87.55 0.014� 0.001a 0.019� 0.002a

60 0.32 1.26 1.10 0.80 29.09 70.52 0.011� 0.001c 0.017� 0.002b

72 0.29 1.12 1.39 0.86 20.86 62.50 0.009� 0.001d 0.015� 0.001c

84 0.24 1.06 1.78 0.96 13.48 50.02 0.008� 0.001e 0.013� 0.001d

96 0.18 0.92 1.96 1.38 9.18 37.24 0.008� 0.001e 0.011� 0.002e

LL-Tyrosine added 2.5 mg/ml, incubation temperature 30 �C, agitation intensity 160 rpm, *on the basis of L-tyrosine used. Yx=s ¼ Dry cell mass (mg/

ml)/substrate utilized (mg/ml), � indicates the standard deviation among the three parallel replicates, the values with different letters differ signifi-

cantly at p < 0:008.

Table 4

Comparative study of L-DOPA production and kinetic parameters (volumetric rates) by using parental (GCB-6) and mutant (UV-7) strains of

A. oryzae

Incubation

period (h)

Volumetric rates (mg/ml/h)

Qp Qs Qx

Parent Mutant Parent Mutant Parent Mutant

24 0.008� 0.002b 0.007� 0.001g 0.020� 0.002b 0.011� 0.002e 0.0025� 0.001a 0.0017� 0.0001a

36 0.009� 0.001a 0.019� 0.001c 0.017� 0.002e 0.018� 0.002c 0.0022� 0.001b 0.0025� 0.0001b

48 0.007� 0.001c 0.028� 0.001a 0.018� 0.002d 0.021� 0.001b 0.002� 0.002c 0.0029� 0.0002a

60 0.005� 0.001d 0.021� 0.002b 0.018� 0.002d 0.021� 0.001b 0.0017� 0.001d 0.0023� 0.0002c

72 0.004� 0.003e 0.012� 0.002c 0.019� 0.002c 0.022� 0.001a 0.0017� 0.0002d 0.0018� 0.0002d

84 0.003� 0.001f 0.011� 0.001d 0.021� 0.002a 0.022� 0.002a 0.0017� 0.0001d 0.0015� 0.0003f

96 0.002� 0.001g 0.014� 0.001d 0.020� 0.003b 0.022� 0.001a 0.0001� 0.0002e 0.0017� 0.0001e

Qp ¼ Slope of product (mg/ml)/time of fermentation (h), Qs ¼ slope of substrate utilized (mg/ml)/time of fermentation (h), Qx ¼ slope of cell mass

formation (mg/ml)/time of fermentation (h), � indicates the standard deviation of three parallel sets of replicates, within the column values with

different letters differ significantly at p < 0:005.

Table 5

Comparative study of L-DOPA production and kinetic parameters (specific rate constants) by both parental (GCB-6) and mutant (UV-7) strains of

A. oryzae

Incubation

period (h)

Specific rate constants (mg/mg/h)

qp qs

Parent Mutant Parent Mutant

24 0.00184� 0.0001c 0.01107� 0.0005g 0.00420� 0.0001d 0.00773� 0.0002f

36 0.00321� 0.0002b 0.01283� 0.003de 0.00585� 0.0002b 0.00905� 0.0001e

48 0.00270� 0.0001d 0.03740� 0.002a 0.00645� 0.0002a 0.02065� 0.005a

60 0.00170� 0.0001e 0.02646� 0.002b 0.00586� 0.0004b 0.01680� 0.005b

72 0.00117� 0.00005e 0.01742� 0.0005c 0.00560� 0.0002b 0.01334� 0.002c

84 0.00068� 0.00002f 0.01338� 0.002d 0.00509� 0.0005c 0.01212� 0.003d

96 0.00335� 0.0001a 0.00881� 0.0005f 0.00364� 0.0003e 0.01322� 0.0001cd

qp ¼ Specific L-DOPA production rate ðmg=mg=hÞ ¼ ðl � Yp=sÞ, qs ¼ specific substrate uptake rate ðmg=mg=hÞ ¼ ðl � Yx=sÞ. The values differ sig-

nificantly at p < 0:0025 while � indicates the standard deviation among the replicates.

28 Ikram-ul-Haq et al. / Bioresource Technology 85 (2002) 25–29

tion rate was significantly different (p < 0:005 andp < 0:0120, respectively) during growth of GCB-6 andUV-7, 24, 48, 72 and 96 h after incubation. When cul-tures grown for various incubation periods were moni-tored for Qp, Qs and qp, there was significant overallenhancement (p < 0:0025–0.005) in these variables bymutant strain of A. oryzae UV-7 over GCB-6 for all therates. Other workers have also described the hyper-producability of mutants over the isolated cultures ofA. oryzae (Sarin et al., 1980).

3.1. Conclusion

For each strain of fungus, the incubation period is animportant factor to be optimised. It is also suggested thatmutation can raise the status of microorganisms to hy-perproduce the actual product required. There was amarked difference between the mycelial morphology andpellet type of parental and UV-irradiated mutant cul-tures. The mutant strain of A. oryzae UV-7 exhibitedpellet-like mycelial morphology and enhanced tyrosinaseactivity. The mutant strain is stable and D-glc-resistant.The product i.e., L-DOPA is economical and is a highyield product. By optimising more fermentation condi-tions, such as the effect of nitrogen and phosphate sourceson the mould growth, this mutant strain could have greatpotential for commercial production of L-DOPA.

References

Ali, S., Haq, I., 2000. Studies on the microbial transformation of

L-tyrosine to L-DOPA by mutant strains of Aspergillus oryzae.

Pak. J. Pl. Sci 45 (2), 123–129.

Arnow, L.E., 1937. Colorimetric determination of the components of

4-dihydroxy phenyl alanine-tyrosine mixtures. J. Biochem 88 (1),

531.

Fling, G., Paul, J., 2001. Radiation induced production of L-3,4

dihydroxy phenylalanine by Aspergillus oryzae. J. Anal. Chem 67,

185–187.

Haneda, K., Watanabe, S., Takeda, P., 1973. Production of L-DOPA

from L-Tyrosine by microorganisms. J. Ferment. Technol. 51 (6),

398–406.

Haq, I., Iqbal, F., Qadeer, M.A., 1998. Production of L-DOPA from

L-tyrosine by Aspergillus oryzae. Biologia 44 (1 & 2), 31–39.

Haq, I., Ali, S., Manzoor, S., Qadeer, M.A., 2000. Optimization of

cultural conditions for the production of L-DOPA from L-tyrosine

by Aspergillus oryzae. Pak. J. Botany 32 (2), 255–258.

James, P., Fling, G., 2001. Tyrosinase activity of different fungal

strains. Biochem. Biophys 112, 98–103.

Kumagai, L.S., Fling, M.S., Sarin, A.N., 1969. Mushroom tyrosinases.

J. Biol. Chem 68, 178–185.

Pirt, S.J., 1975. In: Principles of Cell Cultivation. Blackwells Scientific,

London, pp. 114–117.

Pontecarvo, J., Roper, J.A., Hemmous, L.M., Buftan, W., 1969. Basic

techniques of UV-irradiation. Adv. Genet 5, 141–183.

Raju, B.G.S., Rao, G.H., Ayyanna, C., 1993. In: Bioconversion of

L-tyrosine to L-DOPA using Aspergillus oryzae. CBS Publishers,

Visakhapatnam India, pp. 106–110.

Rosazza, K.C., Paul, G.M., Wolf, S., 1995. The microbial synthesis of

L-DOPA. Biotechnol. Lett 16 (3), 210–215.

Roy, D., Das, R.M., 1978. Mutation of microorganisms to complete

change in physiological sequences. Amn. Tech. Chem 159 (12),

172–183.

Sarin, A.N., Bedi, K.L., Chopra, C.L., Atal, C.K., 1980. Biocon-

version of L-tyrosine to L-DOPA. Ind. J. Microbiol 20, 122–

124.

Sih, C.J., Foss, P., Rosazza, J., Lembagar, M., 1969. Microbial

synthesis of 4-dihydroxy phenylalanine. J. Amn. Chem. Soc 91, 62–

64.

Singh, H., 1999. Mechanism of oxidation of L-tyrosine by fungal

tyrosinases. J. Chem 172, 83.

Snedecor, G.W., Cochran, W.G., 1980. In: Statistical methods,

seventh ed. Iowa State University, USA, pp. 32–43.

Ikram-ul-Haq et al. / Bioresource Technology 85 (2002) 25–29 29