FEMS Microbiology Letters 72 (19~O) 195-200 195, Published by Elsevier

FEMSLE 04198

Production of L-DOPA by Aspergillus terreus

Sharmila Chattopadhyay and Arati Das

Department of Mlcrob~olo~'. Bose Institute. Caleuutt India

Received 20 August 1989 Revision received 11 June 1990

Accepted 26 June 1990

Key words: L-DOPA; Tyrosine conversion: Intermittent feeding

1. SUMMARY

Production of L-DOPA by the activity of tyrosinase from a strain of Aspergillus terreus 104 was investigated. Effects of substrate (L-tyrosine) concentrations, its mode of feeding to Ihe assay mixture and also the effects of t-ascorbic acid and other antioxidants on the production rate of L- DOPA wei¢ studied. The parameters for optimal production of L-DOPA with a maximum conver- zion of 85% of 0.1% L-tyrosine were determined.

2, INTRODUCTION

L-Tyrosine is converted to b3,4-dihydroxy- phenyl-L-alanine (L-DOPA) by a one step oxida- tion reaction catalysed by tyrosinase or tyrosine hydroxylase in living organisms. In view of the pharmaceutical importance of L-DOPA, attempts have been made to find suitable microorganisms capable of producing L-DOPA on a commercial scale. Although microbial synthesis of L-DOPA was first detected from fungal sources in 1969 [1], the literature indicates that such work has mostly

Correspondence to: A, Das, Department of Microbiology, Bose Institute, 93/I A.P.C. Road, Calcutta 700 009 India.

utilized bacteria [2-6]. Comparatively little infor- mation is available on [ungal production of L- DOPA, all from various studies of Aspergillus oryzae [7 9]. In a study undertaken in this labora- tory a strain of A. oryzae AR5 was found to produce L-DOPA from 0.5% L-tyrosine with a maximum conversion of 7570 of the substrate [10]. in an attempt to search for more promising fungal strains fresh screening was carried out and a strain of AspergiXus terreus 104 was isolated with a bet- ter yield of L-DOPA compared to A. oryzae ARS. The present investigation was undertaken in order to determine the parameters for optimum produc- tion of L-DOPA by the selected strain.

3. MATERIALS AND METHODS

3.1. Microorganism Preliminary screening was carried out by inoc-

ulating 140 fungal isolates derived from soil sources onto thc solid screening medium (SM) described below. On the basis of intensity of violet-black coloured zones produced due to tyrosinase activity, two isolates were selected. These were next grown in liquid screening medium for quantitative estimation of L-DOPA production capacity, and Aspergillus terreus 104 was finally selected as the most active producer of t-DOPA.

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196

This strain was maintained in 2% malt-extract (ME) medium at 4¢C.

3.2. Culture media and cultivation Screening medium (SM): For preliminary

screening of fungal isolates with strong tyrosinase activity, the screening medium of Tanaka et al. [4] was used after modification. Its composition was (g/100 ml distilled water): glucose, 2.0; (NH4)2 SO 4, 0.5; KHyPO4; 0.01; MgSOn-7H~O, 0.02; L-tyrosine, 0.2; peptone, 2.0; ferrous lactate, 0.5; afar, 2; pH 6.5. The constituents of other media used for mycelial growth are presented in Table 1.

Liquid cultures were grown in Erlenmeyer flasks and following incubation for 48 h the mycelia were harvested, washed with ice cold water, pressed between filter papers by keeping the material un- der vacuum (100 nun head of mercury) for 2 rain. These wet mycelial samples were weighed and stored at 4 ° C for no longer than 15 rain before further use. All cultures were incubated in static conditions at 28 ° C.

3.3. Assay men,ca The synthesis of L-DOPA was studied using

L-tyrosine as the substrate. An assay mixture con- taining 350 mg of wet mycelia (equivalent to a dry weight of 100 mg 5:2.6 rag), 5 mg of L-tyrosine (unless mentioned otherwise) and 10 mg of L- ascorbic acid in 5 ml of acetate buffer solution was agitated for a reaction period of 60 nfin at 45 ° C. The supernatant was then used for qualita- tive confirmation by TLC and quantitative de- termination by Arnow's method [11] of L-DOPA.

3.4. Analytical methods Chromatography was performed by TLC using

a solvent system of n-butanol:acetic acid:water (2 : 1 : 2) and the spraying reagent was a mixture of equal proportion of 5% FeCI 3 and 10% KaFe(CN) 6 producing violet black spots [12], Quantitative estimation was carried out by spec- trophotometry in a Shimadzu UV-240 spectropho- tometer at 540 nm with reference to a L-DOPA calibration curve.

3. 5. Chemicals Malt extract, yeast extract, L-DOPA, L-tyrosine

and o-tyrosin¢ were purchased from Sigma Chem-

icals Co. (U.S.A.) and ferrous lactate from Fluka Chemika, Switzedand. All other chemicals were of analytical reagent grade and obtained from local suppliers.

4. RESULTS A N D DISCUSSION

4. L Selection of working strain After preliminary screening of 140 fungal soil

isolates, two isolates (Aspergillus terrens 104 and A. niger AR 107) were observed as the most active producers of L-DOPA, and by quantitative estima- tion of their L-DOPA production capacity A. ter- reus 104 was solected as the working strain for farther experiments.

A. terreus 104 was grown in different liquid media (Table 1) and from the second day on- wards, myceha were harvested daily for quantita- tive estimation of their tyrosinase activity and production of L-DOPA from L-tyrosine. Results indicated (Table 2) that the enzyme was most activc ia tli~ ,,~:~,~clla of 72 i~ oid t:,'ture~, irrespec- tive of the medium composition. However, MI was the most fa~urable medium and therefore used for all succeeding experiments.

4.Z Effect of substrate specificity on the ~roduetien of L-DOPA

To study the specificity of Lqyrosine and i~. tyrosine on L~DOPA production, reactions were carried out with both these substrates and conver-

Table 1 Composition of different cultivalion media (rag/t00 lift)

MI MII Mill MIV MV Malt exlract Yeast exuaet Olm:os¢ Cornstccp liquor Peptone NH4CI KH:~PO4 MBSO~, 7HzO NaNO3 K~HPO4 KCI FeSO 4

~00 500 500 250

1000 t000 11300 2000 100

t00 30O 3O0 2t~

3000

S0 300 100 50

I

197

Table 2

Effect of medium composition and mycelial age on the L-DOPA production

Error limits correspond to one slandard deviation calculated on the basis of five replicate samples. All biomass quantities given are wet weights, determined as described in MATERIALS AND METHODS.

Culture Media Period (h) MI MII Mill MIV MV

L-DOPA Biomass L-DOPA Biomass L-DOPA niomass :-DOPA Biomass L-DOPA Biomass (mg/g (g/I) (mg/g (g/ll (mg/g (g/I) (mg/g (g/l) (mg/g (g/I) biomass) bicmass) biomass) biomass) hiomass)

24 4.3±0.18 2.8+C.2 3.5:t:0.15 2.0±0.14 2.8±0.16 2.0±0.14 1.4±0.2 1.0±O.I 1.1+0.17 1.1+0.14 48 7.8+0.17 4.0±0.18 5,7±0,18 3.8±0,2 5.0±0.16 2,8±0.16 3.4±0. i7 1.8±0.14 2.1±0.14 1.6±0.12 72 10.0±0.17 7.0±0.09 8.8±0.18 5.0+0.16 7.3±0.2 4.O±0.17 4.3±0.11 2.5±0.09 2.84-O.16 2.8±0.2 96 9.2+0.2 7.8±0.2 7.1±0.1g 5.9±0,11 5.7±0.18 5.02.0.16 2.8±0.2 3.2±0.12 1.7±0.18 3.5±O.15

s ion rate was found to be h igher in presence of L-tyrosine (Table 3). L-Tyrosine was used as the subs t ra te in the assay mix ture of all succeeding exper iments .

4.3. Effect of reaction period and substrate con- centration on the production of t.~r 9PA

_~iffe.rC'nt. c ' ~ . ~ ! ' t r ~ t ~ n S Of T .|~.rt~¢irt~ ~v~r~ ii~erl

in the assay mix ture and observat iona *~ete m a d e at 15 rain intervals (Fig . 1). O p t i m a l concen t ra t ion of the substrate was found to be 1.0 m g / m l when a m a x i m u m convers ion of 70% (10 m g g - ~ ) was observed o n 60 rain incubat ion . In subsequent exper iments the react ion was therefore carr ied out for 60 rain wi th 1.0 m g / m l of subs t ra te concentra- t ion.

4.4, Effect of ascorbic acid and other antioxidants on the production of t-DOPA

L-Aseorbic acid is general ly added to the assay mix ture to prevent fur ther bydroxyla t ion of L-

Table 3

Effect of substrate specificity on tbe I.-DOPA production

Error limits got'respond to one standard deviation determined on the basis of five replicate samples. Biomass quantities are wet weights,

Substrate Concentration L-DOPA (mg/ml) (mg/g biomass)

t.-Tyrosine I 10,0±O.17 2 7.5 ± 0.2

~Tyrosine 1 5.1 +0,17 2 4.0 ~r 0.Is

D O P A [1]. In o rde r to check efficacy of o ther chemicals as ant ioxidants , a n u m b e r of chemicals were used replacing L-ascorhic acid in the assay mixture (Table 4). It can be seen that L -DOPA synthesis was ra,>st effective in presence of ascorbic acid followed by araboascorbic acid, whereas o ther add; l ives were less effective in this respect. It is well k n o w n that ascorbic acid catalys,~s ~yrosinase

i0( FIg,I

9(

6(

~ 6t

15 30 45 60

Reocti0n period[min)

Fig. I. Effect of substrat¢ concentrations (Q [3). 0.5: • • , 1.0; o • o, 2.O; O O, 4.0 mg /m l ) and reaction period on L-DOPA production. Each value represents mean±S.D, of five observarions and coefficient variability is less than 2% in all cases. Biomass is expressed as g wet weight.

determined as described in MATERIALS AND MYrHODS.

Table 4

Effect of L-ascorbic and other antioxidants or~ L-DOPA plo- duclion Error l~mits correspond to 1 S.D., calculaled on the basis of five replicate sample~. Biomass quanlities are wet weights.

Antioxidants Concentration I.-DOPA (mg/ml) (mg/g biomass)

:1: S.D.

L-Ascorbic acid 1 5.8 ± 0.17 2 t0.04-0,17 3 7.4+0.16

Araboascorbic acid I 5A ±0.17 2 7.0 :t: 0.11 3 6.0±0.17

Citric acid 1 3.0 + 0,'1 2 4.0+0.18 3 3.1 ±0.18

Hydroquinone 1 4.75:0.2 2 5.8+0.17 3 5.4+0,16

Resor¢inol l 4.45:0.09 2 6,0-1-0.17 3 6.0+ 0,2

Pyrocalechol l 4.5 ± 0A 1 2 5.8 + 0.17 3 5.5 +0,14

or tyrosine hydroxylase activity [13], Evidently the acceleration of the production of L-DOPA could be due to accelerating action of ascorbic acid. Araboaseorbic acid, being a s te~oisomer of aseorbie acid, obviously hydroxylates t - tyrosine to L-DOPA. But on account of its different structural configuration it is possibly less active than ascorbic acid in hydroxylating reaction leading to some- what reduced production of L-DOPA. A similarly effective role of araboascorbic acid and L-aseorbie acid was also observed in L-DOPA formation by

Vibrio tyrosinaticus [2]. On the other hand araboascorbi¢ acid was found to be superior to L-aseorbic acid in studies with Fseudomonas melanogenum [3]. These are evidently examples of strain specificity. Low yields of L-DOPA in pres- ence of phenolic compounds e.g. hydroquinone, resoreinol and pyrocatechol could be due to their functions as competi tors with tyrosine in hydrox- ylating reactions [14]. In the presence of citric acid the yield of L-DOPA was very much reduced. This acid possibly suppressed hydroxylating action by chelating metal ions for hydroxylating reaction. leading to poor yield of I_-DOPA.

4.5. Mode of L-O,rosine addition on the production of L-DOPA

The amount of L-DOPA produced was found to he dependent on the mode of feeding of the substrale, L-tyrosine to tile assay mixture (Table 5). 70% conversion was observed when the entire substrate was added in a single batch at the begin- ning of the reaction. In order to study the in- fluence of mode of substrate feeding to the assay mixture, experiments were carr ied out where the ent ire substrate (5 m e / 5 ml) was divided into equal parts (2, 4 or 5 fractions) and these were fed to the assay mixture in powder form at specific intervals s tar t ing from the beginning (Tab/e. 5). It was noted that compared to single feeding inter- mit tent feeding of the substrate in different batches improved the yield of L-DOPA in all cases. How- ever, maximum conversion of 85~ was observed when feeding was carr ied out in four batches dur ing the same reaction period of 60 rain in eomparisc, a to the single feeding or two-batch- feedings,

Table 5

Method or I.-tyrosine feeding to the reaction mixture

Error limits correspond to 1 S,D. calculated on the basis or five replicate sampb,~, Bi0mass quantities are wet weights.

Number of l.-Tyrosine feeding t°A~rbic I..DOPA Conversion substrale feeds Tolal Feeding acid tmg/ml) (mg/g biomaas) (~)

amount tmg/ml) iatorvals (min)

Single feed t ,0 - 2~0 t0,04- O.I 7 70 Two feeds 1,0 30 2.0 10,7 +0,16 ' 75 Four feeds 1.0 15 2.0 12,14-0,16 85 Five feeds L0 12 2~0 it.7 +0.11 82

In view of al l these obse rva t i ons it c a n b e

r e a s o n a b l y s t a t ed tha t A. terreus s t r a i n 104 pos- sesses an a p p r e c i a b l e c a p a c i t y to p r o d u c e L- D O P A ,

A C K N O W L E D G E M E N T S

T h e Ind i an Co u n c i l o f M e d i c a l R e s e a r c h , In- d ia , is a c k n o w l e d g e d for a f inanc ia l con t r i bu t i on . T h a n k s a rc due to Prof . D .P . C ~ a k r a b o r t y . D e p t . o f C h e m i s t r y for his sugges t ions a n d d iscuss ion .

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190

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