Download 5.Amino acids

AMINO ACIDS
Ikeda in 1908, working on the flavouring component in kelps
Discovered GLUTAMIC ACID (L-glutamate) after acid hydrolysis and
fractionation of kelp and neutralization with caustic soda.
These treatments enhance the taste of kelp
Gave rise to the birth of
MONO SODIUM GLUTAMATE (MSG), flavor enhancing compound
It was extracted from soy and wheat now micro-organisms
(Corynebacterium glutamicum) are used
Commercial production of MSG is the largest and biggest industries
world over.
Commercial Production
Glutamic acid > lysine > methionine > threonine > Aspartic acid
The market is growing steadily by about 5–10% per year.
USES OF AMINO ACIDS IN INDUSTRIAL APPLICATIONS
Food industry:
65%
Feed Additives: 30%
Pharmaceutical: 5%
FOOD INDUSTRY
• Flavor enhancers, MSG, glycine, alanine. Tryptophan and histidine
act as antioxidants to preserve milk powder. For fruit juices
cysteine is used as an antioxidant.
• Aspartame, dipeptide (aspartyl-phenylalanine-methyl ester)
produced by combination of asp and Phe is 200 sweeter than
sucrose. Used as low calorie artificial sweetener in soft drink
industry
• Essential amino acidsor those deficient in plant based foods like
lys, met, thr, Trp improves nutritional quality of food and feed
additives (animal). Bread: lysine, soy products or soyabean meal
(pigs/animals): methionine
PHARMACEUTICAL INDUSTRY
Used as medicines, infusions to patients with post operative treatment
CHEMICAL INDUSTRY
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Used as a precursor for production of several cpds
Glycine used to manufacture GLYPHOSATE (a herbicide)
Threonine used for AZTHREONAM (herbicide)
Poly methyl glutamate: manufac. Of synthetic leather
N-acyl derivatives of amino acids used for making cosmetics
METHODS FOR PRODUCTION OF AMINO ACIDS
1.
EXTRACTION: hydrolysis of proteins to isolate amino acids like
cys, tyr, leu
2. CHEMICAL SYNTHESIS: can result in racemic mixture (D and L
amino acids), most applications are for L-form sometime DL or D
maybe required.
3. MICROBIOLOGICAL SYNTHESIS
a. Direct fermentation: MO use carbon sources and produce aa.
Carbon like glu, fructose, alkanes, ethanol, glycerol, molasses,
starch, methanol etc.
b. Conversion of metabolic intermediates to amino acids:
c. Use of enzymes (microbial) or immobilized cells: resting cells,
crude cell extracts, immobilized cells can be used.
STRAIN IMPROVEMENT METHODS FOR AA PRODUCTION
Because of regulatory control of metabolic reactions natural over
production is rare
Regulatory control has to be removed
Mutagenesis and screening for mutants are done
1. Auxotrophic mutants: lack of formation of regulatory end
product (repressor or effector molecule). Intermediates
accumulate and get excreted.
2. Genetic recombination: for overproduction (recombinant
molecules created) or protoplast fusion to develop hybrids
3. Recombinant DNA Technology: gene cloning, gene engineering
4. Functional genomics: whole chromosome sequencing data
L-GLUTAMIC ACID
Corynebacterium glutamicum, is a short, aerobic, Gram-positive
rod capable of growing on a simple mineral salt medium with
glucose, provided that biotin is also added.
Production of L-glutamic acid by C. glutamicum is maximal at a
critical biotin concentration of 0.5 mg g-1 of dry cells, which is
suboptimal for growth
Detergents like Tween-40,
addition of penicillin,
use of Glucose,
Glucose-6P,
CO2,
fatty acid auxotrophic strains, or
addition of ethambutol- inhibiting
synthesis.
arabinogalactan
L-GLUTAMIC ACID
Metabolic pathway
C.glutamicum used glycolysis, PPP and
Citric acid cycle
Krebs cycle is replenished
produced is high in amount
the
Glu
biotin
biotin
Phosphoenol pyruvate
carboxylase
Glutamic acid bacteria have high
activity
of
Glutamate
dehydrogenase and low activity of
a-ketoglutarate dehydrogenase
Microbacterium
Brevibacterium
Arthrobacter
PDH
Isocitrate
dehydrogenase
L-GLUTAMIC ACID
Regulatory control:
Good supply of glucose and efficient conversion of phosphoenol
pyruvate to oxaloacetate
Phosphoenol pyruvate carboxylase and pyruvate carboxylase, pyruvate
dehydrogenase
a-ketoglutarate dehydrogenase (low activity by adding penicillin,
surfactants)
Glutamate dehydrogenase (high activity)
1 mole of glucose should produce 1 mole of glu
In practice, efficiency is 70%
• Glu is synthesized intracellularly
• Carrier mediated processess
• Biotin is essential co factor (for Acetyl CoA carboxylase),
deficiency of biotin affects fatty acid biosynthesis, membrane
formation falters, permeability is affected and intracellular
export of glu is altered
FACTORS INFLUENCING PRODUCTION
1.
2.
3.
4.
Carbon sources
Nitrogen source: ammonia for carbon to glu pH control
Growth factors :biotin
O2 supply: high conc inhibits growth and low O2 leads to lactic
acid production and succinic acid, Afftects Glu production in both
cases
Nutrients
Glucose (12%)
Dissolving tank
38oC
30-35h
Sterlizer
Buffer tank
Inoculum
Sterile air
FERMENTER
Cell separator
Ammonia, pH control (7.8)
(ammonium acetate 0.5%)
Eluted in NaOH
Anion exchanger
Evaporation
Crystallization
100g/L