Production of Amino Acids(Glutamate)
Department of Pharmaceutical Technology (Biotechnology)National Institute of Pharmaceutical Education and Research
SAS Nagar, Punjab
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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
Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
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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.
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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
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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
Gaschromatography
Pure glutamic acidor derivatives
Research, medical,food processing
Affinitychromatography
Glutamic acids bonded to polymer and other types
Separating complexproteins/high
molecular weightmaterials
Radioisotopetracers
Radiolabelledliquid or solid
Medical,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
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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
Therapeutic applications of glutamic acid
Structure
HO HO
O O
NH2
●Glutamic acid is a dicarboxylic monoamino acid●Non-essential or dispensible amino acid
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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…..
Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
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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
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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
Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
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Schematic representation of glutamic acid production
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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
Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
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Glutamic Acid
Biosynthesis of glutamic acid
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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.
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Culture medium
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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.
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Overview of fermentation
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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.
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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
Preparation of resin
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Flow diagram of the isolation process.
Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
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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.
Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
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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.
Crystallization
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Simplified production flow chart of the L-Gln manufacturing process
Kusumoto I., Journal of Nutrition. 2001; 131:2552-2555.
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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
Xiao J et al; Bioprocess Biosystem Engineering. 2006; 29(2);109–117.
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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.
Analytical methods
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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.
On-line control system
Xiao J et al; Bioprocess Biosystem Engineering. 2006;29(2):109–117.
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• 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%
Observations after setting a particular DO level
Contd….. Xiao J et al; Bioprocess Biosystem Engineering. 2006;29(2):109–117.
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•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…..
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Effect of different DO levels on glucose consumptions during aerobic conditions
Open circle: DO = 10%
Open triangle: DO = 50%
Xiao J et al; Bioprocess Biosystem Engineering. 2006;29(2):109–117.
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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…..
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● 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…..
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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%
Effect of DO Levels on various parameters
Xiao J et al; Bioprocess Biosystem Engineering. 2006;29(2):109–117.
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• 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
Fermentation and recovery of L-glutamic acid from cassava starch hydrolysate
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●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.
Batch process for Casava starch hydrolysate
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● 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
Contd…..K. Madhavan Nampoothiri et al; Revista de microbiologia.1999;30
Fed-batch process for Casava starch hydrolysate
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●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)
K. Madhavan Nampoothiri et al; Revista de microbiologia. 1999;30.
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Growth and glutamic acid production based on the hydrolysate having different DE values
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Consumption of reducing sugars by Brevibacterium sp. at different DE values
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Yields of L-glutamic acid at different DE value
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Percentage conversion at different DE values
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.
A= 15-20% DEB= 30-35% DEC= 45-50% DED= 60-65% DEE= 85-90% DE
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Comparison of bacterial growth and L-glutamic acid production in batch and fed-batch process
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● 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.
Recovery of glutamic acid
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Glutamic acid recovered at different elution volumes through ion-exchange resin column
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