biotechnology ppt b.pharm 6th semester

1. Study Of Production Of Glutamic Acid.
Submitted To- Dr. Sandip Pawar
Submitted By- 1735, 1733, 1736, 1740, 1737,
1739, 1743, 1724, 1738
B. Pharm 6th Semester
BIOTECHNOLOGY ASSIGNMENT

2. INTRoduction
 Glutamic Acid is a non essential α-Amino acid used in biosynthesis of proteins.
HISTORY:
 Glutamic Acid was discovered by Ritthausen in 1866.
 In 1908, Prof. Ikeda found that material enhancing the taste of sea weed soup
came from its sodium salt (Monosodium Glutamate or MSG).
 Before 1956, obtained mainly by hydrolysis of gluten.
 After 1956, Japanese investigators reported possibility of its production by
microbiological means.
 Thereafter its production began on an industrial scale.
CHEMICAL NAMES:
 2-aminopentanedioic Acid
 1-aminopropane-1,3-dicarboxylic acid
 α-Aminoglutaric acid
COMMON NAME: Glutaminic Acid

3. STRUCTURE-
* *
L-glutamic Acid
D- glutamic acid
Amino group; exists
as -NH3
+
Carboxylic group;
exists as –COO-
• Aliphatic Amino Acid
•It is polar negatively charged (at Physiological pH)
•L- glutamic acid is naturally found and used by cells.
•Isoelectric point at pH- 3.22
•Molecular weight- 147.14
•Boiling Point- 333.8oC
• It is non essential in humans i.e. we need not take it from
external source. It can be synthesized by body.
Both contain Side
chain carboxylic
Acid

4. PRODUCTION OF GLUTAMIC ACID
It can be produced by a number of ways-
 Hydrolysis of protein rich food material like like wheat glutin
 Cleavage of Pyrrolidone carboxylic acid found in stiffens
mollases.
 One step fermentation process requiring single
microorganisms.
 2 step fermentation process involving α-keto glutaric acid and
its conversion by another enzyme or an enzyme process.
 A method still under study is the Immobilization of C.
glutamicum and mixed culture of C.
glutamicum and Pseudomonas reptilivora for L-glutamic acid
production using submerged fermentation. 2% sodium alginate
immobilized beads were used for production and reusability of
immobilized cells and for production of high yields.

5. • Microbe used- Micrococcus No. 541.
•Media Requirements-
o Microbe being a Biotin Auxotroph, requires 0.5-5 μg/lt biotin.
o Carbon Source: Glucose, Fructose, Sucrose, Maltose, Xylose, or Hydrolyzed
Starch.
o Nitrogen Source: At least one of Urea, Ammonia, Ammonium salts, peptones,
corn steep liquor, hydrolyzed casein, Meat extract, digested soyabean meals or
fish meals.
o Ph: 6-9 (optimum- 7 to 8.5)
o Ammonium ion is essential for growth.
o Temperature: 27-30o C
o Proper Aeration essential.
• PROCESS- (Takes Place in a single step)
One step fermentation process requiring single microbes.
Nutrient
media
Strain of
Cepahalosporium. Eg- C.
salmosynnematum, C.
diospyri, etc.
fermentation
Production Of L-
Glutamic Acid

6. TWO step fermentation process
Carbon
source in
an
optimum
nutient
medium
Step 1 Step 2
L- glutamic acid
Via enzymatic
conversion or by
microbes
using
K. citrophila; or
P. fluorescens
 Other processes of α-keto glutarate production include:
• In microbes, α-KG is mainly synthesized by oxidative decarboxylation of
isocitrate by isocitrate dehydrogenase via TCA, and then is partly consumed to
synthesized glutamate catalyzed by glutamate dehydrogenase.
• Chemical synthesis process. However its low yield and application of toxic
chemical and solvents increase the cost and limit its wider application.
 Enzymatic Methods for conversion of α-keto glutarate to L- glutamic acid
include-
• Biological catalytic system (with reversibly oxidized nicotinic acid containing co
enz)
Micrbial
Fermentatio
n

7. INDUSTRIAL MICROBIOLOGY
Microbes are typically grown on a large scale to produce products or to carry
out chemical transformations.
• Glutamic acid is produced via fermentation process.
• Wild strains of certain microbes are used for Glutamic acid production.
• These organisms are gram positive and non motile.
• Classic methods are used to select for high yielding microbial variants.
Source; Vyas.S.P; Dixit.V.K; Pharmaceutical Biotechnology; ed-10th; pp-
211
Microbial
Production Of
L-glutamic
acid from
α-Keto
glutarate

8.  Microbes used include-
 Corynebacterium Spps (like C. glutamicum, C. lilum)…….…(most preferred)
 Brevibacterium spps (like B.divericartum, B. alanicum)
 Microbacterium Spps. (M. flavumvar, A. glutamicum)
 Arthrobacter (A. globiformis, A. aminofaciens).
+ NH2
Glutarate
• They lack or have very low amounts of enzyme α-keto glutarate, formed by removal
of CO2 from isocitrate formed in TCA cycle. Since α-keto glutarate is not
dehydrogenated, it is available to form glutarate by reacting with ammonia.

9.  Properties Of useful Industrial microbe include-
 Produces spores or are easily inoculated.
 Grows rapidly on a large scale in inexpensive medium
 Produces desired product quickly
 Should not be pathogenic
 Amenable to genetic manipulation.
 Conditions Of Fermentation-
 Medium Composition-
 Glucose: 10% (Hydrocarbons can also be used)
 Corn Steep Liquor: 0.25%
 Enzymatic Casein Hydrosylate: 0.25%
 KHPO (Dipotassium Phosphate): 0.1%
 MgSO4 : 7%
 H2O: 0.25%
 Urea: 0.5%
 Optimum Temperature: 30- 35oC
 High degree of Aeration.

10. BIOCHEMICAL BASIS FOR GLUTAMIC ACID PRODUCTION-
 When Biotin limiting - Glutamic acid production
 When Biotin in excess- Lactic Acid excretion instead of glutamic acid
 Therefore, optimum Biotin level - 0.5 mg per g of dry cells.
 Isocitrate- succinate part of TCA cycle is needed for growth. As after the
growth phase, glutamic acid production is optimal.
 Increase in Cell Permeability (altered Permeability) facilitates outward
diffusion of glutamic acid, thus increasing its production. It is one of the
major factor. This increase can be achieved by-
 Biotin deficiency of medium
 Treatment with fatty acid derivatives
 Ensuring oleic Acid deficiency in mutant bacterias requiring Oleic Acid.
(Cells treated with above 3 factors have cell membrane abnormal saturated:
unsaturated fatty acid ratio, thus leading to destroyed permeability barrier
and glutamic acid accumulates in the medium.)
 Addition of Pencillin during growth of glutamic acid bacteria.
(Prevents cell wall formation, thus enabling use of molasses which are rich in
biotin for glutamic acid production.)

11. INDUSTRIAL PRODUCTION OF GLUTAMIC ACID
 Manufacturing Process includes-
Fermentatio
n
Crude
Isolation
Purification
FERMENTATION
Batch
Fermentation
Fed Batch
Fermentation
Continuous
Fermentation

12. Batch Fermentation
• Widely used in most amino
acid production
• Closed culture system
• Contains intial limited
nutrient amount
• Lag phase required before
cells enter log phase
(Exponential Phase)
• Nutrients soon become
ceased and enter stationery
phase in which growth has
almost ceased.
• Glutamic Acid production
normally starts in early log
phase and continues through
5 steps-

13.  Short fermentation time with high yield
of glutamic acid should be aimed.
 Fermentation should not be continued in
late stationery phase as contaminants
appear.
 Lag phase can be shortened using a high
seed inoculum concetration.
 Seed inoculum is produced by growing
production strain in flasks and small
fermentors.

14. Fed- Batch Fermentation
 Continuously fed Batch Fermentations
 So, volume of cultures increases with
time.
 Preferred over batch culture.
 Residual substrate concentration may
be mantained at very low level.
 May result in removal of catabolite
repressive effects and avoidance of
toxic effects of medium components.
 Oxygen balance
 Feed rate of glucose regulates growth
rate and oxygen limitation, esp. when
oxygen demand high in exponential
growth phase.

15. Continuous Fermentation
 Open system is set up.
 Sterile nutrient solution added to
bioreactor continuously.
 Equivalent amount of converted
nutrient solution with microorganisms
is simultaneously removed from the
system.

16. PROCESS OF INDUSTRIAL GLUTAMIC ACID PRODUCTION
Raw materials and nutrient supplements like nitrogen source,
inorganic raw material, phosphate, corn steep liquor etc are put
in fermentor tank
Inoculum
Culture is
grown in shake
flask
Transferred
to first
seed tank
Transferred
to second
seed tank
Serves as
Inoculum For
Production
Tank
Natural product such as cane sugar or corn used as carbon source
Sugar Cane squeezed to make molasses (source in Europe and
north America) or corn syrup (USA) made out of corn

17. They are then sterilized in fermentor by steam
When temperature of medium cools down to 30oC, microbe
(Corynebacterium glutamicum) added to fermentation broth in
proper inoculum size.
Microbe incubated for 36 to 48 hours in proper temperature,
pressure and pH conditions.
Microbe reacts with sugar to produce glutamic acid
Then fermentation broth is then acidified.
Glutamic acid is crystallized

18. Downstream Processing
(Seperation and purification of glutamic acid from its contaminant products)
Centrifugatio
n Evaporation Decolorisation
Solvent
Extraction
Electrodialysis
Ion
Exchang
e
Filtration
Crystallisatio
n
• After fermentation, filtration of cells done using Rotary Vaccum
Filter, improved using filter aids (like Kiesselgurh)
• Many Seperation and Purification techniques are employed
•Chromatographic
method
• Glutamic acid binds to
anion exchange resin
(pH of solution higher
than ion exchange
potential)
• Used as it has
amphoteric
character,
solubility
influenced by pH,
temp; shows
minima at
isoelectric point;
COMMONLY USED METHODS

19. Process
Glutamic Acid crystals added to NaOH to make MSG (more water
soluble , less likely to absorb moisture, strong umami taste)
MSG is cleaned by using active carbon, which has many micro
holes on its surface.
Clean MSG is concentrated by heating and MSG crystals are
formed
MSG Crystals produced are dried with hot air in a closed system.
MSG Crystals are then packaged.
These packaged crystals are then ready to be sold.
L-glutamic acid crystals
MSG
CRYSTALS

20. Schematic REPResentation

21. Food Production-
Its sodium Salt (MSG) is mainly used as-
• Food Additive
• Flavor Enhancer.
• Nutritional Supplement
Beverage
• Used in soft drinks and wine as flavor enhancer.
Memory Enhancer
• Important in brain metabolism hence its various
analogues are used in treating various neuropathic
diseases.
Cosmetics
• For Hair loss as hair restorer.
• In preventing ageing.
APPLICATION OF GLUTAMIC ACID

22.  Agriculture/ Animal Feed.
 As an additive nutritional supplement in feed.
 Use as Polymer
 Poly glutamic Acid is a naturally occouring anionic polymerthat is biodegradable,
edible, and non toxic towards human and environment.
 Other Industries
 Used as Thickener
 As Cryoprotectant
 As bitterness relieving agent.
 Curable biological adhesive
 Heavy metal absorber.

23. Adverse effects Of MSg
MSG symptom complex which
includes Headache, Flushing,
Sweating, Facial pressure or
tightness, Numbness, tingling or
burning in the face, neck and other
areas, Rapid, fluttering heartbeats
(heart palpitations), Chest pain,
Nausea, Weakness.
.
Excess of everything is bad…So, FDA
declared MSG Safe for limited usage,
and enlists several side effects linked
to increased MSG consumption
Nestle’s mislabelled claim
“No Added MSG” and excess
lead amount led to ban on
Maggi in 2015. Ban was later
withdrawn and Nestle has
since then removed the
‘said claim’ from its label.

24. REFEREnces
 Vyas.S.P; Dixit.V.K; Pharmaceutical Biotechnology; ed-10th; pp-209- 213.
 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147103/;
 https://www.slideshare.net/NOMIKhanS/glutamic-acid-fermentation;
 https://www.slideshare.net/EssamYahya2/industrial-production-of-chemical-
acids-glutamic-acid;
 http://www.jocpr.com/articles/overproduction-of-ketoglutarate-by-the-
corynebacteriumglutamate.pdf
 https://www.mayoclinic.org/healthy-lifestyle/nutrition-and-healthy-
eating/expert-answers/monosodium-glutamate/faq-20058196