ABSTRACT- - In this present study, the biotransformation of phenol to L-tyrosine was studied with resting cells of Citrobacter freundii MTCC 2424 containing high tyrosine phenol lyase activity. Different process parameters leading to synthesis of L-tyrosine were optimized. The L-tyrosine formed from biotransformation reactions was detected and quantified by HPLC technique. The maximum L-tyrosine conversion 69% (6.49g/l) was obtained with ammonium chloride 0.25M, phenol 0.1M, and sodium pyruvate 0.2M in borate buffer (0.1M) of pH 8.5 at 35°C temperature for 45min of incubation. Higher phenol concentration was found to be inhibitory for biotransformation due to phenol inactivation of catalyst.
Key-words- Citrobacter freundii MTCC 2424, L-tyrosine, Tyrosine phenol lyase, Biotransformation

INTRODUCTION
L-tyrosine is an aromatic amino acid, one of the building blocks of protein and found in many protein containing food products such as soy products, chicken, turkey, fish, peanuts, almonds, bananas, milk, cheese, yogurt, pumpkin seeds and sesame seeds. A number of studies on human have found tyrosine to be useful during condition of stress, cold, fatigue [1], loss of a loved one such as in death, prolonged work and sleep deprivation [2-3], improvements in cognitive and physical performance [4-6]. Citrobacter freundii, a member of the genus Citrobacter belongs to family Enterobacteriaceae. These are areobic gram negative bacilli, long rod shaped bacteria (1-5 µm in length). Its habitat includes the environment (soil, water, and sewage), food, and the intestinal tracts of animals and humans [7].
Tyrosine phenol lyase (TPL) is an enzyme that catalyzes the synthesis of L-tyrosine. This enzyme has been found to exist in a number of bacteria but some species in particular namely Citrobacter freundii, Escherichia intermedia and Erwinia herbicola have been recognized for high enzyme activity [8]. This enzyme catalyzes the multiple reactions such as a,ß-elimination [9], reversal of a,ß-elimination [10], ß-replacement [11-12], and racemization reactions [13]. These reactions are important for enzymatic synthesis of L-tyrosine [14-16] and its related amino acids including 3,4-dihydroxyphenylalanine or L-DOPA [17], treatments of phenolics in water [18] and for biotransformation of L-serine [15].
L-tyrosine has been used as nutritional supplements and mild antioxidants to alleviate the acute cases of Parkinson’s symptoms [19]. L-tyrosine is required to make several neurotransmitters such as L-DOPA, dopamine, epinephrine and norepinephrine [20-22]. L-phenylalanine can also be converted into L-tyrosine utilizing the enzyme phenylalanine hydroxylase and in turn L-tyrosine is converted to levodopa (L-DOPA) by enzyme tyrosine hydroxylase. This can be further converted into dopamine, epinephrine and norepinephrine. The derivatives of L-tyrosine in body fluids play regulatory roles in functions of hormonal system in the adrenal, thyroid, and pituitary glands. The hormones epinephrine and norepinephrine have therapeutic use such as cardiostimulants in the treatment of acute circulatory insufficiency and hypotension [23]. Considering the importance of L-tyrosine and its use in synthesis of molecules of therapeutic and industrial value the present study entitled “Biotransformation of phenol to L-tyrosine with resting cells of Citrobacter freundii MTCC 2424” was carried with objectives to develop a laboratory scale process for production of therapeutically and industrially important molecule L-tyrosine.

MATERIALS AND METHODS
Microorganism and Maintenance of culture: The culture of C. freundii MTCC 2424 was procured from Department of Biotechnology, Himachal Pradesh University, Shimla, India and used for this study. C. freundii MTCC 2424 was maintained on L-tyrosine agar media containing (%, w/v) meat extract 0.5, yeast extract 0.5, peptone 0.25, L-tyrosine 0.1 and agar 2.0 [24]. The pH of media was maintained at 7.5. The plates were incubated at 30°C for 24 h after inoculation. Periodical sub-culturing was carried out and glycerol stocks of culture were prepared and stored at -40°C.

Estimation of cell mass: Cells of C. freundii MTCC 2424 were harvested by centrifuging the broth at 10,000 rpm for 10 min in a refrigerated centrifuge (4°C) and known amount of wet cell pellet was placed in oven at 80°C for overnight and corresponding absorbance of cell slurry was measured at 600 nm in a spectrophotometer. The known dried cell weight corresponding to their optical cell density was recorded and a standard graph was plotted between dry cell weight and A600. The cell mass in terms of dry cell weight (dcw) was measured from standard curve.

Tyrosine Phenol Lyase (TPL) assay: The a,ß-elimination reaction was used for assay of enzyme. TPL converts tyrosine to phenol, pyruvate, and ammonia. The amount of liberated ammonia was measured via spectrophotometer. Since TPL was found to be intracellular in nature, the resting cells suspended in borate buffer (0.1M, pH 8.5) were used for enzyme assay. Activity of TPL from whole cell of C. freundii MTCC 2424 was expressed in terms of units (U).

Estimation of Ammonia: Ammonia released from hydrolysis of L-tyrosine was estimated by Berthelot color reaction [25] for assay of enzyme activity.

HPLC analysis of L-tyrosine: The L-tyrosine formed from biotransformation reactions was detected and quantified by HPLC equipped with a reverse phase column and UV spectrophotometer. The reaction mixture was centrifuged (10000rpm for 10min) to remove all suspended particles. The supernatant was filtered through 0.20µ filters and 10µl of samples were loaded on HPLC column. The column was eluted with 0.01M ammonium acetate buffer (pH 3.5) at a flow rate of 1ml/min and the detection was done at 280nm. The amount of L-tyrosine formed was calculated from standard curve.

Preparation of seed and production culture: Seed and production medium used were of same composition containing (%, w/v) meat extract 0.5, yeast extract 0.5, peptone 0.25, L-tyrosine 0.1 and pH 7.5 [24]. Sterile seed culture (50ml) was inoculated with a loopful of a culture grown an agar plates and incubated at 25°C in a temperature controlled incubator shaker at 150rpm for 4h. The exponential phase cell mass (4h old) was used as inoculum (6%, v/v) for 100ml sterile production medium and flasks were incubated at 25°C in a temperature controlled incubator shaker at 150rpm for 16h. After 16h, the broth was centrifuged at 10,000rpm for 10min and cells pellet was washed three times with borate buffer (0.1M, pH 8.5). The washed pellet was suspended in 10ml borate buffer. The resting cells (30 OD, 0.48mg/ml dcw) were used as catalyst for biotransformation reactions.

Optimization of various process parameters for biotransformation of phenol to L-tyrosine with resting cells of C. freundii MTCC 2424 in a Fermenter:Biotransformation studies were carried out in a 2l laboratory fermenter. The bioconversion percentage was calculated on the basis of phenol supplied and L-tyrosine formed (w/w).

Selection of suitable ammonium salt for Biotransformation : The different ammonium salts (ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium acetate) were used (1M) in reaction mixture (500ml) for biotransformation and amount of L-tyrosine formed was analyzed by HPLC technique. The reaction was carried out with resting cells of C. freundii MTCC 2424 in borate buffer (0.1M, pH 8.5), containing known amount of call mass (48mg, dcw), 0.05M phenol, 0.1M sodium pyruvate at 30°C (100rpm) for 30min. The reaction was stopped by taking 2ml of reaction mixture with 1ml of 1.0N HCl, centrifuged to recover the clear supernatant, filtered and injected into HPLC.

Optimization of concentration of ammonium chloride for Biotransformation: The concentration of most suitable ammonium salt (ammonium chloride) was determined for biotransformation reaction by varying its concentration from 0.001M to 1.25M. The reactions with resting cells of C. freundii MTCC 2424 were carried out in borate buffer (0.1M, pH 8.5), containing known amount of cell mass (48mg, dcw), 0.05M phenol, 0.1M sodium pyruvate at 30°C (100rpm) for 30 min.

Optimization of concentration of phenol on its Biotransformation to L-tyrosine: The varying concentrations (0.05M to 0.25M) of phenol were used for biotransformation reaction along with 0.25M ammonium chloride and 0.1M sodium pyruvate at 30°C. The rest of reaction conditions were maintained same and L-tyrosine synthesized was analyzed by HPLC technique.

Optimization of concentration of sodium pyruvate for Biotransformation: Sodium pyruvate provides (-CH2-CH-COOH) to tyrosine. Varying concentrations (0.1M to 0.5M) of sodium pyruvate were used along with 0.1M phenol. The rest of reaction conditions were maintained same.

Optimization of pH of borate buffer for Biotransformation: Reactions were carried out at various pH (7.5 to 9.5) of borate buffer to study its effect on biotransformation.

Optimization of incubation temperature for Biotransformation: To find out optimum temperature, biotransformation reactions were performed at different temperatures (25°C to 45°C).

Optimization of incubation time for Biotransformation: Biotransformation reactions were performed under previously described conditions for 75 min and samples were withdrawn at regular intervals of 15min. In each sample L-tyrosine synthesized was analyzed by HPLC technique.

RESULTS
Colonies of C. freundii MTCC 2424 were observed on inoculated L-tyrosine agar media after incubation at 30°C for 24 h (Fig. 1). Biotransformation studies were carried out in 2l laboratory fermenter (Fig. 2). Maximum conversion of Phenol to L-tyrosine was found to be 26% (1.22g/l) when ammonium chloride was used (Fig. 3). Other ammonium salts used like ammonium sulfate, ammonium nitrate and ammonium acetate showed comparatively a lower conversion, which was 17%, 13%, and 7% respectively.

   

Fig. 1: Colonies of C. freundii MTCC 2424 cells   Fig. 2: Fermenter used for biotransformation

   

Fig. 3: Effect of different ammonium salts on Biotransformation   Fig. 4: Effect of ammonium chloride concentrations on biotransformation

   

Fig. 5: Effect of phenol concentrations on biotransformation   Fig. 6: Effect of sodium pyruvate concentrations on biotransformation

   

Fig. 7: Effect of buffer pH on biotransformation   Fig. 8: Effect of incubation temperature on biotransformation

Resting cells of C. freundii MTCC 2424 were showed maximum conversion (39%) at 0.25M concentration of ammonium chloride. The maximum L-tyrosine biosynthesis was recorded to be 1.84g/l (Fig. 4). Maximum biotransformation (48%) of phenol to L-tyrosine was obtained at 0.1M concentration of phenol with 4.52g/l biosynthesis of L-tyrosine (Fig. 5). However, as the concentration of phenol was increased further to 0.25M in the reaction mixture, the conversion was reduced to 15%. Maximum biotransformation (54%) was observed at 0.2M concentration of sodium pyruvate with 5.08g/l biosynthesis of L-tyrosine (Fig. 6). Maximum L-tyrosine conversion (55%) was observed with borate buffer (0.1M) at pH 8.5 with 5.18g/l accumulation of L-tyrosine in reaction mixture (Fig. 7). Maximum L-tyrosine conversion (61%) was observed at 35°C. L-tyrosine biosynthesis observed at 35°C was 5.74g/l (Fig. 8). L-tyrosine production was found to increase initially with increasing incubation time (69% at 45min) and then attained constant value as the reaction proceeds. Maximum L-tyrosine biosynthesis (6.49g/l) was observed at 45min of incubation (Fig. 9).

Fig. 9: Effect of incubation time on biotransformation

DISCUSSION
On the basis of our results, it was evident that intact cells of C. freundii MTCC 2424 prepared from culture broth cultivated for 16h contained high enzymatic activity. Enzymatic synthesis of L-tyrosine was first demonstrated by Yamada and Kumagai [17]. Ammonium chloride was found to be best salt for biotransformation. Ammonium chloride (4M) was used as a source of ammonium ion for L-tyrosine production [26]. The 2M ammonium chloride was found to be optimum for biotransformation of phenol to L-tyrosine in a simulated waste water containing phenolics by a recombinant thermotolerant TPL of thermophillic Symbiobacterium sp. SMH-1 [18]. Many scientists were also reported ammonium acetate as a source of ammonium ions for L-tyrosine production [27-28]. Inhibition of TPL activity with phenol and its derivatives was studied [14]. They observed 83% inhibition in TPL activity at 1.0mM concentration of phenol and its derivatives in reaction mixture. It was observed that phenol concentration in reaction mixture should be as low as possible to avoid inhibition and inactivation of catalyst [29]. In addition, phenol, which can partially destroy cell walls and denature proteins, was often infused at a minimum concentration to avoid inhibition and inactivation of catalyst [30-31]. Optimum sodium pyruvate concentration was found to be 0.2M for biotransformation. Same concentration of sodium pyruvate (0.2M) was used in reaction mixture for L-tyrosine synthesis by E. herbicola ATCC 21433 [32]. Production processes using solubilized, intact cells of E. herbicola achieved a maximum concentration of 60.5g/l L-tyrosine from 20g/l sodium pyruvate added twice, 50g/l ammonium acetate and a phenol concentration maintained at 10g/l throughout reaction [27]. Concentration of about 0.11M (20g/l) L-tyrosine was reported with addition of 0.30M (33g/l) sodium pyruvate, 0.65M (50g/l) ammonium acetate and 0.13M (12.4g/l) phenol after 2h [28].
In the present investigation, optimum buffer pH and temperature for biotransformation was found to be 8.5 and 35°C respectively. The incubation time for biotransformation was found to increase initially and then attained a constant valve as the reaction proceeds. This might be due to reason that as the reaction proceeds, the enzyme got saturated with substrate and the reaction rate becomes constant with time. L-tyrosine (14.5g/l) synthesis from ammonium acetate (0.65M), phenol (0.1M) and sodium pyruvate (0.18M) was observed with intact cells of E. herbicola and optimum pH for reaction was around 8.0 and optimum temperature range was from 30°C to 37°C [33].

CONCLUSIONS
The present investigation attempts to find out the optimum reaction conditions for synthesis of L-tyrosine with resting cells of C. freundii MTCC 2424. The various process parameters were individually optimized to maximize the biosynthesis of L-tyrosine. Out of different process parameters optimized, ammonium chloride 0.25M, phenol 0.1M, sodium pyruvate 0.2M, buffer 0.1M, pH 8.5, reaction temperature 35°C and incubation time 45min were found to be optimum for maximum production of L-tyrosine. Higher phenol concentration was found to be inhibitory due to phenol inactivation of catalyst. Employing whole cells as biocatalyst for L-tyrosine synthesis offers clear advantages over in vitro enzymatic conversion because microbial synthesis proceeds under environmentally beneficial and non toxic conditions. In view of recent advances, it is clear that microbial fermentation is becoming a very viable alternative option for L-tyrosine production.

ACKNOWLEDGMENT
I would like to give my sincere thanks and regards to Dr. Wamik Azmi, Assistant Professor, HPU Shimla, India for their valuable support and guidance.

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How to cite this article: Kumari V: Biotransformation of Phenol to L-tyrosine with Resting Cells of Citrobacter freundii MTCC 2424. Int. J. Life. Sci. Scienti. Res., 2017; 3(5):1339-1344. DOI:10.21276/ijlssr.2017.3.5.12 Source of Financial Support:Nil, Conflict of interest: Nil