Amino acid Productions

1. Amino acid Productions

2. Amino acid Amino Acids Are Molecules Containing An Amine Group, A Carboxylic Acid Group And A Side-chain That Varies Between Different Amino Acids. The Key Elements Of An Amino Acid Are Carbon , Hydrogen , Oxygen , And Nitrogen An Alpha-amino Acid Has The Generic Formula H2NCHRCOOH, Where R Is An Organic Substituent. Amino Acids Are Critical To Life, And Have Many Functions In Metabolism. One Particularly Important Function Is To Serve As The Building Blocks Of Proteins , Which Are Linear Chains Of Amino Acids. Amino Acids Can Be Linked Together In Varying Sequences To Form A Vast Variety Of Proteins.

3. Essential and Nonessential AAs Histidine Alanine Isoleucine Arginine * Leucine Asparagine Lysine Aspartic acid Methionine Cysteine * Phenylalanine Glutamic acid Threonine Glutamine * Tryptophan Glycine Valine Ornithine * Proline * Selenocysteine * Serine * Taurine * Tyrosine * (*) Essential only in certain cases

4. EAAs • An essential amino acid or indispensable amino acid is an amino acid that cannot be synthesized de novo by the organism being considered, and therefore must be supplied in its diet. The nine amino acids humans cannot synthesize are phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine, and histidine.

5. Non EAAs • Six amino acids are considered conditionally essential in the human diet, meaning their synthesis can be limited under special pathophysiological conditions, such as prematurity in the infant or individuals in severe catabolic distress. These six are arginine, cysteine, glycine, glutamine, proline and tyrosine . • Five amino acids are dispensable in humans, meaning they can be synthesized in the body. These five are alanine, aspartic acid, asparagine, glutamic acid and serine.

6. Amino Acid by Fermentation • NOW A DAYS THREE MAJOR AMINO ACIDS ARE BEING PRODUCED ON LARGE SCALE. GLUTAMIC ACID LYSINE METHIONINE • All PRODUCED BY PROCESS OF FERMENTATION USED TO BE PRODUCED BY CHEMICAL SYNTHESIS

7. Why Produced on a Large Scale? • The amino acid business is a multi-billion dollar enterprise, All twenty amino acids are sold, although each in greatly different quantities • Amino acids are used as animal feed additives (lysine, methionine , threonine ), flavor enhancers (monosodium glutamic, serine, aspartic acid) and as specialty nutrients in the medical field. • Glutamic acid, lysine and methionine account for the majority, by weight, of amino acids sold . • GLUTAMATE IS MEDICALY USED AS A NEUROTRANSMITTER.

8. Glutamic Acid Production • 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 (Corynebacteriumglutamicum) are used for MSG production. • 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.

9. USES OF AMINOACIDS 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 times sweeter than sucrose. Used as low calorie artificial sweetener in soft drink industry • Essential amino acids are 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

10. PHARMACEUTICAL INDUSTRY Used as medicines, infusions to patients with post operative treatment CHEMICAL INDUSTRY • Used as a precursor for production of several cpds • Glycine used to manufacture GLYPHOSATE • Threonine used for AZTHREONAM (herbicide) • Poly methyl glutamate: manufac. Of synthetic leather • N-acyl derivatives of amino acids used for making cosmetics

11. METHODS FOR PRODUCTION OF AMINOACIDS • EXTRACTION: hydrolysis of proteins to isolate amino acids like cys, tyr, leu • CHEMICAL SYNTHESIS: can result in racemic mixture (D and L amino acids), most applications are for L-form sometime DL or D maybe required. • MICROBIOLOGICAL SYNTHESIS • Direct fermentation: MO use carbon sources and produce aa. Carbon like glu, fructose, alkanes, ethanol, glycerol, molasses, starch, methanol etc. • Conversion of metabolic intermediates to amino acids: • Use of enzymes (microbial) or immobilized cells: resting cells, crude cell extracts, immobilized cells can be used.

12. 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 Auxotrophic mutants: lack of formation of regulatory end product (repressor or effector molecule). Intermediates accumulate and get excreted. Genetic recombination: for overproduction (recombinant molecules created) or protoplast fusion to develop hybrids Recombinant DNA Technology: gene cloning, gene engineering Functional genomics: whole chromosome sequencing data

13. 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 arabinogalactan synthesis.

14. 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%

15. 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 alters, permeability is affected and intracellular export of glu is altered FACTORS INFLUENCING PRODUCTION • 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

16. Nutrients Glucose (12%) 38oC 30-35h Dissolving tank Sterlizer Buffer tank FERMENTER Cell separator Anion exchanger Evaporation Crystallization Ammonia, pH control (7.8) (ammonium acetate 0.5%) Inoculum Sterile air Eluted in NaOH 100g/L

19. Bakteri asam laktat juga memproduksi asam format, asetat, propionat, kaproat, kaprilat, kaprat, butirat, dan isovalerat darimetabolisme fermentasi dan transformasi enzimatik asam amino. Protease merupakan enzim yang mendegradasi protein. Protease tergolong enzim hidrolase yang mengkatalisis reaksi degradasi substrat dengan pengolahan air. Pengertian protease menurut Winarno (1985) ada dua yaitu proteinase yang mengkatalisisreaksi hidrolisis molekul protein menjadi fragmen besar polipeptida, dan peptidase yang menghidrolisis fragmen besar polipeptidamenjadi asam amino. Kasein pada yogurt sebagai substrat untuk uji aktivitas protease dengan metode Lowry. Kasein akan terhidrolisis menjadi peptida dan asam amino dengan bantuan air oleh enzim protease.