Production of amino acid tyrosine by conventional and modern method. And a case study of synthesis of tyrosine my using micro organism and its optimisation study. Clinical significance of tyrosine and Why there is need to produce it artificially?

1. PRODUCTION OF TYROSINE
By-
Abhishek Rajesh Indurkar
(17PBT202)
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2. TYROSINE
Tyrosine (4-hydroxyphenylalanine) is a non-essential
amino acid is synthesised in vivo from L-phenylalanine.
It readily passes the blood-brain barrier. Once in the brain,
it is a precursor for the neurotransmitters dopamine,
norepinephrine and epinephrine, better known as adrenalin.
It is also the precursor for hormones, thyroid, and the
major human pigment, melanin.
It is an important amino acid in many proteins, peptides
and even enkephalins, the body's natural pain reliever.
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3. CLINICAL IMPORTANCE
Tyrosine is used in protein supplements to treat an inherited disorder called
phenylketonuria (PKU). People who have this problem can't process phenylalanine
properly, so as a result they can't make tyrosine. To meet their bodies' need,
supplemental tyrosine is given.
Tyrosine is also given to patients suffering from
1. Depression,
2. Attention deficit disorder (ADD),
3. Attention deficit-hyperactivity disorder (ADHD),
4. The inability to stay awake (narcolepsy).
It is also used for stress, premenstrual syndrome (PMS), Parkinson's disease,
Alzheimer's disease, chronic fatigue syndrome (CFS), alcohol and cocaine
withdrawal, heart disease and stroke.
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4. METHODS FOR SYNTHESIS
Conventional.
Modern.
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5. CONVENTIONAL METHOD
Liver of the rat, guinea pig, rabbit, dog, chicken, and human convert phenylalanine to tyrosine.
The enzyme system (phenylanaline hydroxylase) is found only in the liver.
Rats were stunned and then decapitated to permit the blood to drain. The livers were immediately
removed and homogenised in a Waring blendor with 2 to 3 times their weight of isotonic KCI.
Very low yield obtained.
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6. MODERN METHOD
Use of micro organism for production of L-tyrosine
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MICRO-ORGANISM YIELD
E. coli 2gm/l
Corynebacterium glutamicum 15.4gm/l
Citrobacter freundii 6.49gm/l
Saccharomyces cerevisiae 0.52mg/l

7. MICRO-ORGANISM
Citrobacter freundii MTCC 2424 were used for
Biotransformation of phenol to L-tyrosine.
It is a species of facultative anaerobic gram-negative bacteria of
the Enterobacteriaceae family. The bacteria have a long rod
shape with a typical length of 1–5 μm.
It is a soil organism, but can also be found in water, sewage, food
and in the intestinal tracts of animals and humans.
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8. MEDIA
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C. freundii MTCC 2424 was maintained on L-tyrosine agar media
containing
COMPONENTS COMPONENTS
Meat extract 0.5
Yeast extract 0.5
Peptone 0.25
L-tyrosine 0.1
Agar 0.2
ph 7.5

9. PREPARATION OF SEED AND
PRODUCTION MEDIUM
Seed and production medium used were of
same composition as shown in table.
Sterile seed culture (50ml) was inoculated with
a loopful of a culture and incubated at 25°C in
shaker incubator 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 shaker incubator at 150rpm for 16h.
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COMPONENTS (W/V)
Meat extract 0.5
Yeast extract 0.5
Peptone 0.25
L-tyrosine 0.1
ph 7.5

10. CONT..
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.
Tyrosine phenol lyase (TPL) is an enzyme that catalyses
the synthesis of L-tyrosine.
Ammonia + Pyruvate + Phenol L-tyrosine + water
Biotransformation of phenol to tyrosine was carried out
in 2L lab fermentor.
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11. OPTIMISATION OF VARIOUS PROCESS
PARAMETERS FOR BIOTRANSFORMATION
1. Selection of suitable ammonium salt-
Different ammonium salts (ammonium chloride, ammonium
sulphate, ammonium nitrate, ammonium acetate) were used (1M) in
reaction mixture (500ml) for biotransformation. 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.
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12. CONT..
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Maximum conversion of Phenol to
L-tyrosine was found to be 26%
(1.22g/l) when ammonium chloride
was used . Other ammonium salts
used like ammonium sulfate,
ammonium nitrate and ammonium
acetate showed comparatively a
lower conversion, which was 17%,
13%, and 7% respectively.

13. OPTIMISATION OF VARIOUS PROCESS
PARAMETERS FOR BIOTRANSFORMATION
2. Optimisation 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.
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14. CONT..
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.
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15. OPTIMISATION OF VARIOUS PROCESS
PARAMETERS FOR BIOTRANSFORMATION
3. Optimisation 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
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16. CONT..
Maximum biotransformation
(48%) of phenol to L-tyrosine
was obtained at 0.1M
concentration of phenol with
4.52g/l biosynthesis of L-
tyrosine. However, as the
concentration of phenol was
increased further to 0.25M in
the reaction mixture, the
conversion was reduced to
15%.
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17. OPTIMISATION OF VARIOUS PROCESS
PARAMETERS FOR BIOTRANSFORMATION
4.Optimization of concentration of sodium pyruvate for
Biotransformation-
Sodium pyruvate provides propionic acid(-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.
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18. CONTD..
Maximum biotransformation
(54%) was observed at 0.2M
concentration of sodium
pyruvate with 5.08g/l
biosynthesis of L-tyrosine.
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19. OPTIMISATION OF VARIOUS PROCESS
PARAMETERS FOR BIOTRANSFORMATION
5. Optimisation 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.
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20. CONTD..
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.
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21. OPTIMISATION OF VARIOUS PROCESS
PARAMETERS FOR BIOTRANSFORMATION
6. Optimisation of incubation temperature for Biotransformation
To find out optimum temperature, biotransformation reactions
were performed at different temperatures (25°C to 45°C).
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22. CONTD..
Maximum L-tyrosine
conversion (61%) was
observed at 35°C. L-
tyrosine biosynthesis
observed at 35°C was
5.74g/l.
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23. OPTIMISATION OF VARIOUS PROCESS
PARAMETERS FOR BIOTRANSFORMATION
7. 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.
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24. CONTD..
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
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25. CONCLUSION
The various process parameters were individually optimised to
maximise the biosynthesis of L-tyrosine. Out of different process
parameters optimised, 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.
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26. REFERENCES
National Center for Biotechnology Information. PubChem Compound Database; CID=6057,
https://pubchem.ncbi.nlm.nih.gov/compound/6057 (accessed Nov. 4, 2017).
https://www.webmd.com/vitamins-supplements/ingredientmono-1037-TYROSINE.aspx (accessed Nov. 4,
2017).
Juminaga et al.”Modular Engineering of L-Tyrosine Production in Escherichia coli”. Applied and
Environmental Microbiology p. 89–98.
H Hagino, K Nakayama, H Yoshida (1970) (US 3787287) “A Process for the production of l-tyrosine”.
Gold et al. Microbial Cell Factories (2015)”Metabolic engineering of a tyrosine overproducing yeast
platform using targeted metabolomics”14:73
Kumari V: Biotransformation of Phenol to L-tyrosine with Resting Cells of Citrobacter freundii MTCC
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https://tools.thermofisher.com/content/sfs/manuals/IFU9960.pdf (accessed Nov. 4, 2017).
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Citrobacter freundii isolates in two different time periods". Journal of microbiology, immunology, and
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27. …Thank You…
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