Production of Amino Acids

(Glutamate)

Department of Pharmaceutical Technology (Biotechnology)

National Institute of Pharmaceutical Education and Research
SAS Nagar, Punjab
 Background
• Industrial production of amino acids have started with availability of
monosodium glutamate (MSG) in 1909

• Discovered by Dr. Kikunae Ikeda in 1908

• Originally manufactured by extraction from acid hydrolysis of plant

protein

• In late 1950 fermentation technology was established and commercially

exploited for other amino acids

• L-Glutamine fermentation started in late 1960

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Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
 Consumption and production

• Worldwide consumption of amino acids is about 2 million tonnes

• About 1.5 million tonnes was sold in 2001

• 4% annual growth in sale is observed

• The annual demand of amino acids in food and pharmaceutical industry is

4,60,000 tonnes

• The annual worldwide production of L-glutamine is 3,70,000 tonnes

Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555. 3

 Uses
MSG is used commercially as a flavour enhancer.
Although once stereotypically associated with foods in Chinese
restaurants; it is now found in many common food items, particularly
processed foods
Examples include:
 Canned soups
 Pre-prepared stocks
 Common snack foods
 Most fast foods
 Instant meals such as the seasoning mixtures for instant noodles

4
 Table 1 - Major uses of glutamic acid and its derivatives in research
PROTEIN ENGINEERING Peptide synthesis Protein modification Polymer supports

BIOCHEMICAL/CELL BIOLOGY Biochemical experiments Cell biology research

ANALYTICAL APPLICATIONS Analytical standards Diagnostic products and procedures

Table 2 - Analytical uses of glutamic acid and its derivatives

   
Gas Pure glutamic acid Research, medical,
chromatography or derivatives food processing
Affinity Glutamic acids bonded to Separating complex
chromatography polymer and other types proteins/high
molecular weight
materials
Radioisotope Radiolabelled Medical,
tracers liquid or solid pharmaceutical

Table 3 - Medical and pharmaceutical applications

TREATING DISEASE Parenteral nutrition, Prescription dietary

supplements, Congenital metabolic diseases,
Hypertension, Neuroregulators, Ophthalmic
solutions
DIAGNOSING DISEASE Congenital metabolic diseases, Disorders or
malfunction of brain/nervous system
5
 Therapeutic applications of glutamic acid
 ENTERAL NUTRITION
  Crystalline glutamic acid in solution: It is the source of protein precursors for hospitalized patients
unable to eat or eat enough to get or stay healthy.

 PARENTERAL NUTRITION

 Crystalline glutamic acid in solution: Given to the patient through circulatory system via a vein

 PRESCRIPTION DIETARY SUPPLEMENTS

Tablet, capsule or powder protein precursors: For people able to eat but who need very highly
concentrated source to recover from illness or surgery

 CONGENITAL METABOLIC DISEASES


Powder Prescription diets: For newborn babies and others who need a diet devoid of or with highly
reduced content of specific amino acid

 HYPERTENSION (Capsules or injection)

Prescription drugs that reduce blood pressure

 NEURO-REGULATORS (Capsules or injection)

Prescription drugs to stimulate sluggish nerve activity or to dampen neural activity in disorders of the
nervous system
6
 Structure

O O

HO HO
NH2

●Glutamic acid is a dicarboxylic monoamino acid

●Non-essential or dispensible amino acid
 General manufacturing process
 The manufacturing methods of amino acids are-
 Extraction from acid hyrolysates
 Chemical synthesis
 Fermentation
 Enzymatic
 Leucine, proline, tyrosine, cystine are manufactured by extraction, fermentation
and chemical synthesis
 L-Glutamic acid is manufactured world wide using fermentation

 The manufacturing process of an amino acid by fermentation comprises

fermentation, crude isolation and purification processes.
 In the crude isolation process, most impurities contained in the fermentation
broth are removed by combining various technologies

Contd…..
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Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
 Final purification is performed to ensure the required quality for the
intended use

 The final product is obtained as a crystalline powder

 The product is released only after quality tests have verified that the product
meets specific requirements, and the normal functioning of each process
step has been verified

 All manufacturing processes for the production of amino acids for medical
use must comply with current good manufacturing practice requirements

9
 Manufacturing of L-Gln
 It is essential to the outcome of the fermentation process to maintain a clean
and sterile fermentation tank
 Compared with wild-type strains, L-Gln-producing strains are weak and are
compromised in a contaminated environment
 Furthermore, it is important to maintain the tank under positive pressure by
aeration during fermentation to prevent contamination by other
microorganisms and external materials
 The fermentation medium consists of glucose as a carbon source, ammonia
as a nitrogen source, a small amount of minerals and vitamins as growth
factors
 Control factors during fermentation are pH, temperature and dissolved
oxygen

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Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
Schematic representation of glutamic acid production
11
 Strains producing glutamic acid

 Most exploited is Corynebacterium glutamicum

 Other genera of Corynebacterium is also used

 Brevibacterium sp., Microbacterium sp., Arthrobacter sp. are also

used
 All glutamic acid producers require biotin for their activity

 All the strains show little activity of -ketoglutarate dehydrogenase

 Increased activity of glutamate dehydrogenase

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Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
Biosynthesis of glutamic acid

Glutamic Acid

13
 Conditions of manufacture
 Carbon sources: glucose, sucrose, maltose, xylose, sugarcane and
sugarbeet, molasses, strach, hydrolysates
 Nitrogen sources: ammonium salts, ammonia, urea

 Growth factors: biotin, L-cystiene

 Oxygen supply:

 Optimal production occurs at Kd of 0.0000035 moles of O2/

atm.min.ml
 Lower oxygen content causes excretion of lactate

 Higher oxygen content inhibits α-ketoglutarate production

Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.

14
Culture medium

15
 Conditions during fermentation
• pH is set at 8.5 and automatically maintained at 7.8 during
the course of fermentation

• Temperature is set at 380C during the fermentation process

• Feeding of glucose is done until the end of fermentation (160

g/L)

• Aeration is controlled such that CO2 in exhaust is not more

than 4.5%

Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.

16
Overview of fermentation

17
 Fermented broth

Centifugation

Collect supernatant

Concentration Ion exchange treatment

Direct Crystallization Resin preparation

Column packing

Separation of fluid
(acidified to pH 3.2, with 1 N HCl. Storage at 20°C for 48 h)

Crystallization

Downstream Processing
K. Madhavan Nampoothiri et al; Revista de
microbiologta.1999; 30. 18
 Preparation of resin
 Spherical particles of cation exchange resin, Amberlite is used

 The resin is washed thoroughly two times with 4 N HCI

After two washes with distilled water, the resin is then washed with
2 N NaOH until the filtrate was alkaline

The resulting material (sodium salt of the resin) is suspended in 3-times its
volume of 1 N NaOH and heated over a steam bath for 2 h with occasional
mixing

The supernatant fluid was decanted after 30 minutes of settling and

replaced with fresh hot 1 N NaOH

The procedure was repeated two times. The resin was filtered and washed
with distilled water to make it free of alkali

 The resin was finally stored as the moist sodium salt

19
 Flow diagram of the isolation process.

20
Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
 Effect of temperature on solubility of glutamate

• The solubility of L-Gln is barely affected by temperature as shown by the flat

solubility curve.
• Consequently, cooling crystallization is not applicable for harvesting L-Gln.

21
Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
 Crystallization
 Purification of amino acids by crystallization is an effective means to produce
polymorphism, two crystal forms can be used

 After crystallization of amino acid in the one form, the crystals are dissolved, and
then recrystallized in the other form

 In this manner, it is possible to remove impurities based on their different

affinities for the two crystal forms

 Unfortunately, L-Gln occurs only as one crystal form

 Therefore, to use crystallization for purification, there is no way other than the
inefficient simple repetitive crystallization of the one crystal form of
L-Gln

 By changing the pH to the isoelectric point (3.2) and by the subsequent cooling
of the eluent, glutamic acid was crystallized out.
22
 Simplified production flow chart of the L-Gln manufacturing
process

23
Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
 On-line optimization
 Higher glutamate concentration could be achieved by constantly
controlling dissolved oxygen concentration (DO) at a lower level; however,
by-product lactate also severely accumulated

 Activities of glutamate and lactate dehydrogenases changed during the

the fermentation

 The entire metabolic network flux analysis showed that the lactate
overproduction was because the metabolic flux in TCA cycle was too low to
balance the glucose glycolysis rate

 As a result, the respiratory quotient (RQ) adaptive control based “balanced

metabolic control” (BMC) strategy was proposed and used to regulate the
TCA metabolic flux rate at an appropriate level to achieve the metabolic
balance among glycolysis, glutamate synthesis, and TCA metabolic flux

24
Xiao J et al; Bioprocess Biosystem Engineering. 2006; 29(2);109–117.
 Analytical methods

 The concentrations of cells, glucose, glutamate, and lactate are measured during
the course of fermentation

 The CO2 and O2 concentrations (partial pressure) in the inlet and exhaust gas were
on-line measured by a gas analyzer

 The collected on-line data were smoothly filtered, and then oxygen uptake rate
(OUR) and CO2 evolution rate (CER) were on-line calculated

 Respiratory quotient (RQ) was determined by its definition (RQ = CER/OUR) using

the on-line measured OUR and CER data.

Xiao J et al; Bioprocess Biosystem Engineering. 2006;29(2):109–117. 25

 On-line control system

Based on the RQ set-points and the measured RQ, the PC on-line regulated
the agitation rate of the fermentor (AGT) with the equation below

• k represented the current control instant

•RQset was the RQ set-point which might be subject to changes during the
control
•KC and τI were proportional and integral constants of the feedback
controller, respectively
•KC and τI were determined by observing the RQ response to a step change
in the input (agitation rate) during a certain period of the glutamate
production phase.
26
Xiao J et al; Bioprocess Biosystem Engineering. 2006;29(2):109–117.
 Observations after setting a particular DO level
• Glutamate and lactate formation pattern strongly depended on the DO control
level

• A higher glutamate production rate could be achieved when the DO was

controlled at a lower level of 10% and the final glutamate concentration reached
about 91.5 g/L at 34 h

• Final glutamate concentration stopped at a lower level of 72.7 g/L (30 h) when

controlling DO at 50%

• On the other hand, lactate severely accumulated up to 28 g/L when DO was

controlled at a lower level of 10%

• Almost no lactate accumulation occurred when controlling DO at 50%

Contd…..
Xiao J et al; Bioprocess Biosystem Engineering. 2006;29(2):109–117. 27
•These results suggested that the enzymatic activities of GDH and LDH under lower and higher
DO level might be quite different

•Generally, it is considered that the anaerobic condition is extremely harmful to glutamate

production

•To verify the above speculation, an experiment under extremely low DO level was conducted.
In the fermentation, DO was initially controlled at 30%, and the agitation rate was manually
reduced to bring DO down to 0% instantly at 12 h

•Then, the same agitation rate was kept for the next 6 h.

•During this period, the fermentation could be considered as implemented under anaerobic
condition, glutamate production stopped and lactate overflowed

•At 18 hr, the automatic control of DO was resumed to quickly bring DO back to 30%, a partial
recovery of glutamate production was observed

•However, the final glutamate concentration ended at a very low level of about 45 g/L

•The results indicated that the occurrence of anaerobic condition even for a short period
would be both fatal and irretrievable to glutamate fermentation
Contd…..
28
Effect of different DO levels on glucose consumptions during aerobic
conditions

Open triangle: DO = 50%

Open circle: DO = 10%

Xiao J et al; Bioprocess Biosystem Engineering. 2006;29(2):109–117. 29

 Balanced metabolic control
● In glutamate fermentation glycolysis rate should balance
with glutamate synthesis, lactate formation and TCA
metabolic flux
● Severe lactate accumulation at lower DO control (DO = 10%)
was due to the carbon metabolic balance rather than the
higher LDH activity by the following facts-
1) The changing patterns of glycolysis rate (r1) at different DO levels were
almost the same

2) No significant differences in LDH activities were shown under different DO level

3) The metabolic flux of TCA significantly decreased with the decrease in DO

control level

Contd…..
30
● Under the lower DO level, even though GDH activity was higher, the
higher glutamate synthesis rate (r6) still could not completely balance
with the glycolysis rate (r1), as TCA cycle was almost closed completely
and the TCA metabolic flux (r4) was very low

● Under this circumstance, lactate had to be overflowed or excreted (r5)

into the broth to achieve the entire intracellular carbon balance

● On the other hand, under the higher DO level, GDH activity was
relatively low, but the TCA cycle was nearly open for a complete
oxidation
● Under this condition, lactate was not necessarily overflowed, as the
glutamate synthesis rate (r6) plus the higher TCA metabolic flux (r4)
were big enough to balance with the glycolysis rate (r1), even though
LDH exhibited almost equivalent activity as compared with that of the
lower DO case

Contd…..
31
 Effect of DO Levels on various parameters

Glutamate concentration: (filled circle) DO 10%, (filled triangle) DO 50%; lactate concentration:
(open circle) DO 10%, (open triangle) DO 50%. b GDH activity: (filled circle) DO 10%, (open circle)
DO 50%; LDH activity: (filled triangle) DO 10%, (open triangle) DO 50%. c Cells concentration:
(filled circle) DO 10%, (filled triangle) DO 50%. d Glucose concentration: (filled circle) DO 10%,
(filled triangle) DO 50%

Xiao J et al; Bioprocess Biosystem Engineering. 2006;29(2):109–117. 32

33
 Fermentation and recovery of L-glutamic acid
from cassava starch hydrolysate

• Carried by K. Nampoothiri and Ashok Pandey, Biotechnology Division,

Regional Research Laboratory, CSIR, Trivandrum

• Brevibacterium sp. was used

• Initial studies were carried out in shake flasks, which showed that even
though the yield was high with 85-90 DE (Dextrose Equivalent value), the
maximum conversion yield (~34%) was obtained by using only partially
digested starch hydrolysate, i.e. 45-50 DE

K. Madhavan Nampoothiri et al;Revista de microbiologia. 1999;30 34

 Batch process for Casava starch hydrolysate

● Cassava starch hydrolysate (85-90 DE) was diluted to 5% initial sugar concentration
and was supplemented with 1 ml mineral solution, 100 µl corn steep liquor and one
drop of Tween 80 in 100 ml starch hydrolysate (pH 7.2)

● Fermentation was carried out with a working volume of 2.5 L in a 5 L fermenter

● Dissolved oxygen was maintained at 60% of air saturated medium

K. Madhavan Nampoothiri et al; Revista de microbiologia.1999;30. 35

Fed-batch process for Casava starch hydrolysate

● Fed-batch process was also carried out in the fermenter

● The initial concentration of reducing sugars in the medium was 5%, and at the
stages, where the concentration fell to 2%, starch hydrolyzate solution containing
10% reducing sugars, was added to bring the sugar concentration of fermenting
medium as 5%

● Fermentation conditions were the same as for batch process

K. Madhavan Nampoothiri et al; Revista de microbiologia.1999;30 Contd….. 36

● Fermentations were carried out in batch mode in a 5 L fermenter, using
suitably diluted cassava starch hydrolysate, using a 85-90 DE value hydrolysate

● Media supplemented with nutrients resulted in an accumulation of 21 g/L

glutamic acid with a fairly high (66.3%) conversation yield of glucose to
glutamic acid (based on glucose consumed and on 81.74% theoretical
conversion rate)

● The bioreactor conditions most conducive for maximum production were pH

7.5, temperature 30°C and an agitation of 180 rpm

● When fermentation was conducted in fed-batch mode by keeping the residual

reducing sugar concentration at 5% w/v, 25.0 g/L of glutamate was obtained
after 40 h fermentation (16% more the batch mode)

37
K. Madhavan Nampoothiri et al; Revista de microbiologia. 1999;30.
Growth and glutamic acid production based on the
hydrolysate having different DE values

38
Consumption of reducing sugars by Brevibacterium sp. at
different DE values

39
Yields of L-glutamic acid at different DE value

40
 Percentage conversion at different DE values

A= 15-20% DE
B= 30-35% DE
C= 45-50% DE
D= 60-65% DE
E= 85-90% DE

With 85-90 DE hydrolysate, the conversion was lowest (~27%). Thus, if conversion
factor has to be considered as a major criterion, a low DE value hydrolysate, i.e. 45-50
DE would be sufficient for L-glutamic acid production.

K. Madhavan Nampoothiri et al; Revista de microbiologia. 1999;30. 41

Comparison of bacterial growth and L-glutamic acid production
in batch and fed-batch process

42
 Recovery of glutamic acid

● The fermented broth contained various impurities such as bacterial cells,

macromolecules, pigments, inorganic substances, organic substances etc., which
were removed by filtration and centrifugation

● Glutamic acid was purified from cation exchange resin

K. Madhavan Nampoothiri et al; Revista de microbiologia.1999;30.

43
Glutamic acid recovered at different elution volumes
through ion-exchange resin column

44
45
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47
Thank
You !!!