amino acid

Production of Amino Acids(Glutamate)

Department of Pharmaceutical Technology (Biotechnology)National Institute of Pharmaceutical Education and Research

SAS Nagar, Punjab

2

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.

3

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


4

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

5

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

6

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

8

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…..


9

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

10

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


11

Schematic representation of glutamic acid production

12

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


13

Glutamic Acid

Biosynthesis of glutamic acid

14

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


15

Culture medium

16

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%


17

Overview of fermentation

18

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.

19

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

20

Flow diagram of the isolation process.


21

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.


22

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

23

Simplified production flow chart of the L-Gln manufacturing process


24

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.

25

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

26

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


27

• 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.

28

•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…..

29

Effect of different DO levels on glucose consumptions during aerobic conditions

Open circle: DO = 10%

Open triangle: DO = 50%


30

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…..

31

● 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…..

32

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


34

• 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

35

●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

36

● 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

37

●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.

38

Growth and glutamic acid production based on the hydrolysate having different DE values

39

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

40

Yields of L-glutamic acid at different DE value

41

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

42

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

43

● 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

44

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

46

Suppliers

Raj Enterprises BUSINESS PROFILE Manufacturing and supplying food ingredients such as xanthan gum, sodium gluconate, silicon di- oxide, potassium sorbate, monosodium glutamate, ascorbic acid use as a chemical preservatives for sauces and homemade products.

CONTACT DETAILSStreet Address: 236/238, 4th Floor, Samuel Street City: Mumbai State: Maharashtra PIN: 400 003 Country: IndiaPhone: +(91)-(22)-23402742 Fax: +(91)-(22)-66315361

Eurofine Chemicals

BUSINESS PROFILEManufacturers and exporters of zinc stearate, zinc cyanide, sodium acetate, mono sodium glutamate, magnesium turnings etc.

CONTACT DETAILSStreet Address: 212, Maker Bhavan No. 3, 21, New Marine Lines City: Mumbai State: Maharashtra PIN: 400 009 Country: IndiaPhone: +(91)-(22)-23455101/23475416/22074206 Fax: +(91)-(22)-23475049

47

P.D. UdyogBUSINESS PROFILEManufacturer of citric acid, dried yeast and mono-sodium-glutamate.CONTACT DETAILSStreet Address: No. 7, 1st Floor, G. S. Market, Patnoolpet O. T. Pet Cross City: Bangalore State: Karnataka PIN: 560 053 Country: IndiaPhone: +(91)-(80)-23381195 Website: http://www.indiamart.com/company/1049219/

Dudley & Brother Private Limited, Kolkata BUSINESS PROFILESellers of various types of mono sodium glutamate.CONTACT DETAILSStreet Address: 13, Ganesh Chandra Avenue City: Kolkata State: West Bengal PIN: 700 013 Country: IndiaPhone: +(91)-(33)-22368899/22367322 Fax: +(91)-(33)-22252714

R. M. Chemicals

BUSINESS PROFILESuppliers of all kinds of mono sodium glutamate, citric acid, ascorbic acid etc.CONTACT DETAILSStreet Address: 47, Nattu Pilliar, Koil Street City: Chennai State: Tamil Nadu PIN: 600 001 Country: IndiaPhone: +(91)-(44)-25221743

Thank Thank You !!!You !!!

amino acid

Documents

derivatives glutamic

major uses of glutamic

pharmaceutical applications

annual demand of amino

journal of nutrition

pharmaceutical industry

disease parenteral nutrition

annual worldwide production