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Industrial Production of L-Lysine by Fermentation

Lysine is an essential amino acid that is used in the biosynthesis of proteins. Lysine is required for the nutrition of animals and humans. Lysine is useful as medicament, chemical agent, food material (food industry) and feed additives (animal food). It's demand has been steadily increasing in recent years. Several thousand tones of L-lysine are annually produced worldwide, almost by microbial fermentation.
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Industrial Production of L-Lysine by Fermentation

  1. 1. Production of Amino Acids (Lysine) by Fermentation Technology By: Harshita Mishra Jyoti Dangwal Kuldeep Sharma Devashish Somani
  2. 2. Introduction What are amino acids? Amino acids are building blocks of proteins. Amino acids are a group of organic compounds containing two functional groups amino and carboxyl
  3. 3. • The amino group (-NH2) is basic while the carboxyl group (-COOH) is acidic in nature. • with a side-chain (R group) specific to each amino acid is also present • all the 20 amino acids need not be taken in the diet. Based on the nutritional requirements amino acids are grouped into two classes essential and nonessential
  4. 4. Facts and History • French chemists isolated a compound in asparagus that was named asparagine, the first amino acid to be discovered. • Cystine was discovered in1810 • Glycine and leucine were discovered in 1820. • The last of the 20 common amino acids to be discovered was threonine in 1935 by William Cumming Rose. • who also determined the essential amino acids
  5. 5. Essential amino acids • The amino acids which cannot be synthesized by the body and need to be supplied through the diet are called essential amino acids. • They are required for proper growth and maintenance of the individual. The ten amino acids listed below are essential for humans: Arginine, Valine, Histidine, lsoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan.
  6. 6. There’s a catch • The two amino acids arginine and histidine can be synthesized by adults and not by growing children, hence these are considered as semi-essential amino acids. Thus, 8 amino acids are absolutely essential while 2 are semi-essential.
  7. 7. Non essential amino acids • The body can synthesize 10 amino acids to meet the biological needs, hence they need not be consumed in the diet. These are- glycine, alanine, serine, cystein e, aspartate,a sparagni e, glutamate,glutamine, tyrosine and proline. • Some microorganisms are capable of producing certain amino acids such as lysine, glutamic acid and tryptophan.
  8. 8. • There are 20 naturally occurring amino acids , which are required for the synthesis of variety of proteins besides other biological functions • However, these 20 amino acids need not to be taken in diet . • Based on nutrition requirements amino acids are grouped into 2 classes :essential & non essential.
  9. 9. Types of amino acids…… Essential amino acids: the amino acids which cannot be synthesized by the body and therefore, need to be supplied through the diet . Non Essential amino acids : the amino acids which can be synthesized by the body and therefore, need not to be supplied through the diet.
  10. 10. History…. In 1889 , first it was isolated from casein. Lysin was commercially introduced as a feed around 1956. In 1978, first fermented L- lysin was produced by japanese company “kyowa Hokko Kogyo”. Recent method of lysin production is based on fementation of carbohydrates.
  11. 11. Lysine production plant of the BASF AG located in Gunsan, South Korea with an annualcapacity of about 100 000 tons. Copyright BASF AG—The chemical company (2003).
  12. 12. Three amino acids which are produced at large scale includes, L-lysine L-glutamic acid DL- methionine , arginine, phenylalanine. So, lysine is one of amongst them which covering more then 90% of total world amino acid production Synthesis of lysine 80% by Fermentation , 20% by chemical synthesis . Two routes -DAP pathway - α -aminoadipate pathway
  13. 13. DAP pathway
  14. 14. α -aminoadipate pathway
  15. 15. UPSTREAM PROCESSING
  16. 16. 1. Fermentation Process  Submerged Fermentation  Aerobic Fermentation 2. Mode of Operation  Batch Process  Fed-Batch Process 3. Fermenter Type  Stirred Tank Reactors  Air – Lift Bioreactors
  17. 17. 4. Microorganism Corynebactrium glutamicum (ATCC 13287) • Gram positive bacterium • Soil Bacterium • Non-Motile • Rod shaped • Non – spore producing • Non-pathogenic bacterium Electron micrograph of C. glutamicum (kromer et al., 2006).
  18. 18. • Homoserine auxotrophs strains • Genetically Engineered strains I. Overproducing genes  dapA (Dihydrodipicolinate Synthase)  lysC (Aspartate Kinase)  dpaB (Dihydrodipicolinate Reducatse) II. Lysine excreting mutants  lysE (lysine export carrier gene)
  19. 19. 5. Fermentation Media  Carbon Source : Cane Molasses  Nitrogen Source: Corn steep liquor / Soybean meal  Minerals and Salts: KH2PO4/K2HPO4, CaCO3  Trace Elements: Corn steep liquor  Antifoaming Agents: PEG-2000, Silicone based oils 6. Process Parameters • Optimum pH : 7.2 • Optimum Temperature: 35-37 °C • Time: 100 hours production cycle
  20. 20. 7. Fermentation Process I. Seed Inoculum preparation The typical medium composition includes; Glucose 100g/L Urea 5 g/L Biotin 0.01 g/L Leucine 0.4 g/L MgSO4 2.85 g/L KH2PO4/K2HPO4 0.5 g/L MnSO4 0.016 g/L CaCO3 20 g/L (NH4)2SO4 46 g/L II. Main Fermenter (upto 500 m3 volume)
  21. 21. DOWNSTREAM PROCESSING
  22. 22. PROCESS FLOW CHART
  23. 23. Product Recovery • The fermentation broth is sent to an ultrafiltration system for the removal of cell debris and other suspended solids. Subsequently, the liquor from ultrafiltration is fed to ion-exchange columns, where L-lysine is selectively adsorbed. The adsorbed L-lysine is eluted from the ion-exchange resins by washing with an aqueous ammonia solution.
  24. 24. The lysine has an amino group on its side chain, its isoelectric point is at pH=9.7. At pH=1 the lysine possesses two positive, at pH=5.6 two positive and one negative, at pH=9.7 one positive and one negative and at pH=11 one negative charge.
  25. 25. Product Concentration & Drying • The L-lysine eluted from the ion-exchange columns is mixed with mother liquor from the product filtration step and concentrated by evaporation. The concentrated lysine solution is acidified with hydrochloric acid, and free L-lysine is converted to L- lysine HCl. • The L-lysine HCl solution is then sent to the crystallizer, and lysine salt is filtered. The mother liquor is recycled to the evaporator and the wet cake is conveyed to dryers. Final dry L-lysine-HCl (98.5 wt.%) is obtained and sent to a packaging line before being stored in bags.
  26. 26. CRYSTALLIZER
  27. 27. SPRAY DRYER
  28. 28. • As the drum rotates, it is partially submerged in the feed slurry. • Vacuum draws liquid through the filter medium (cloth) on the drum surface which retains the solids. • The vacuum pulls air (or gas) through the cake and continues to remove moisture as the drum rotates. • If required, the cake can be washed to remove impurities or to extract more product. Additional drying of the cake follows washing.
  29. 29. • Finally, the cake is discharged from the drum to a conveyor or chute to the next process step. • The filtrate and air pulled through the medium flow through internal filtrate pipes and pass though the rotary valve and into the filtrate receiver. • The liquid stream is separated from the vapor stream in the receiver. • Liquid filtrate is then pumped to the next step in the process. • Vacuum is applied using a liquid ring vacuum pump or other means.
  30. 30. Regulation of Lysine production • The pathway leading to lysine biosynthesis is initiated with the conversion of aspartate to aspartyl-P via the enzyme aspartate-kinase (AK). • The phosphorylated aspartate is then converted to aspartyl-semialdehyde (ASA) that can converted to homoserine by homoserine dehydrogenase (HSD) or to diaminopimelic acid (DAP) by a series of five enzymatic conversions, and hence to lysine. • Lysine feedback inhibits the activity of aspartate kinase
  31. 31. THANK YOU

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