Different types, Biosynthesis methods, Isolation, Strain Development, media components, applications.

1. TRYPTOPHAN
B H A G Y A S H R I K E S A R W A N I
M T B / 2 0 / 1 0 1 2
A M I T Y U N I V E R S I T Y , N O I D A

2. • T
ryptophan is an essential amino acid.
• Chemical name B-indole-alanine and L-B-3-
indolylalanine, is also known as L-pancreatic
protein amino acid.
• It is widely used in medicine & as a supplement in
animal feed.

3. C11H12N2O2 -
Molecular weight: 204.22 g/mol -
Non-polar aromatic essential amino acid -
- White or slightly yellow crystalline or
crystalline powder.
- Slightly bitter taste.
- Odourless
Properti
es
Soluble in water -
Melting point: 290.5 °C -
pka: 2.38 (carboxyl), 9.39 (amino) -
Encoded by the codon UGG -
- Precursor to the co-enzymes NAD &
NADP.
- Precursor to serotonin, melatonin,
tryptamine, quinolinic acid, kynurenic
acid and vitamin B3.

4. 4
Milk Proteins
Enzyma
tic
Hydroly
sis
Free & peptide-bound Tryptophan
Precursor of
Biomolecules
Serotonin, Melatonin,
Tryptamine, Niacin, etc
Bioactive
Peptides
Antioxidant,
Antidiabetic, etc.
Psychoactive
Agents
Stress, Depression,
Cognition, etc.
• Tryptophan was first isolated by
Hopkins & Cole in 1901 through
hydrolysis of casein, a protein found in
milk.
• From 600 grams of crude casein, 4 -
8 grams of tryptophan was obtained.
I S O L A T I O N

5. I.
II. D - TRYPTOPHAN
L - TRYPTOPHAN
II.
L-Tryptophan can only be utilised in the structure or enzymes proteins while D-Tryptophan is normally present in
naturally occurring peptides.
STEREOISOMERS OF TRYPTOPHAN

6. L - TRYPTOPHAN SYNTHESIS METHOD
Microbial Fermentation
Chemical Synthesis Method
Conversion Method
This method has been gradually
eliminated due to the existence of
complex processes, complex product
composition and other reasons.
Enzyme conversion method and microbial
conversion method has been achieved in
industrialisation. Plants and microorganisms
commonly synthesise tryptophan from shikimic
acid or anthranilate.
Industrial production is biosynthetic & based on
fermentation of serine & indole using either wild-type or
genetically modified bacteria such as B.
amyloliquefaciens, B. subtilis, C. glutamicum or E. coli.
The conversion is catalysed by the enzyme tryptophan
synthase. L-Trptophan synthesis includes three parts:
central metabolic pathway, aromatic amino acid
common pathway , and L-Tryptophan branch pathway.

7. TRYPTOPHAN BIOSYNTHESIS

8. EDIA
Generally, Lysogeny broth (LB) media is used for generating and propagating recombinant strains with pH adjusted to 7.0.
Liquid LB contains:
1% (w/v) tryptone,
0.5% (w/v) yeast extract, and
1% (w/v) NaCl.
If solid LB plates are required, XY % (w/v) agar is added.
The shake-flask fermentation medium is composed of glucose (30 g/L), MgSO4·7H2O (0.5 g/L), KH2PO4 (3 g/L),
K2HPO4 (12 g/L), (NH4)2SO4 (4 g/L), yeast extract (1 g/L), monosodium citrate (2 g/L), and FeSO4·7H2O (0.1 mg/mL).
Ampicillin and tetracycline is used at a concentration of 100 mg/L when indicated.
M

9. i. In a baffled flask seed media is first inoculated with a single colony strain generated in LB media and cultivated at 36°C with
shaking at 200rpm for 12 hours.
ii. Inoculum from this culture is aseptically added to a 5L fed-batch seed fermenter which contains 3L seed medium. It is
cultivated at 36°Cfot 16 hours.
iii.The culture grown in the fed-batch seed fermenter is inoculated aseptically into 18L fermentation medium in a 30L fed-batch
fermenter. The temperature is maintained at 36°C and the pH to 7.0 with 25% ammonium hydroxide during the cultivation
period, and the level of dissolved oxygen (DO) is controlled at 20 and 30%. Glucose is added to the fermenter according to
the DO feedback strategy.
ULTURE
C

10. TRAIN IMPROVEMENT
S
Significant efforts have been made for the improvement of strain for L-tryptophan
production which mainly relies on mutagenesis and screening procedures, with the
expectation of obtaining strains with deleted competing pathways and released feedback
regulations in the biosynthetic pathways.

11. APPLICATIONS
L-Tryptophan is
widely used as
Leavening
agent, Oxidant
& Anti-
Oxidant in food
production.
L-Tryptophan is
widely used as
nutritional
supplement in
beverages.
L-Tryptophan is
widely used as
nutritional
supplement in
Agriculture/
Animal Feed.
L-Tryptophan is
widely used as
amino acid
whitening
agents in
cosmetics.
L-Tryptophan is
used as
sedatives and
psychotropic
drug.

12. T K U
Y
H N
A O !