Industrial processing of amino acid slide
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Industrial processing of amino acid slide Industrial processing of amino acid slide Presentation Transcript

  •  Amino acids have always played an important role in the biology of life, in biochemistry and in (industrial) chemistry.  amino acids are the building blocks of proteins and they play an essential role in the reguiation of the metabolism of living organisms.  Large scale chemical and microbial production processes have been commercialised for a number of essential amino acids.  current interest in developing peptide-derived chemotherapeutics has heightened the importance of rare and non-proteinogenic pure amino acids.
  •  amino acids are versatile chiral (optically active) building blocks for a whole range of fine chemicals.  Amino acids are, therefore, important as nutrients (food and feed), as seasoning, flavourings and starting material for pharmaceuticals, cosmetics and other chemicals.  Amino acid can be produced by :  Chemical synthesis  Isolation from natural materials  Fermentation  Chemo-enzyme methods
  •  Batch Fermentation  Fed-batch Fermentation  Continuous Fermentation  Enzymatic Method
  •  Widely use in the production of amino acid  Fermentation is a closed culture system which contains an initial, limited amount of nutrient.  A short adaptation time is usually necessary (lag phase) before cells enter the logarithmic growth phase (exponential phase).  Nutrients soon become limited and they enter the stationary phase in which growth has (almost) ceased.  In amino acid fermentations, production of the amino acid normally starts in the early logarithmic phase and continues through the stationary phase.
  •  For economical reasons the fermentation time should be as short as possible with a high yield of the amino acid at the end.  A second reason not to continue the fermentation in the late stationary phase is the appearance of contaminant-products  The lag phase can be shortened by using a higher concentration of seed inoculum.  The seed is produced by growing the production strain in flasks and smaller fermenters.
  •  Batch fermentations which are fed continuously, or intermittently, with medium without the removal of fluid.  In this way the volume of the culture increases with time.  The residual substrate concentration may be maintained at a very low level.  This may result in a removal of catabolite repressive effects and avoidance of toxic effects of medium components  Oxygen balance.  The feed rate of the carbon source (mostly glucose) can be used to regulate cell growth rate and oxygen limitation,especially when oxygen demand is high in the exponential growth phase.
  •  In continuous fermentation, an open system is set up.  Sterile nutrient solution is added to the bioreactor continuously and an equivalent amount of converted nutrient solution with microorganisms is simultaneously removed from the system.  Two basic types of continuous fermentations can be distinguished:  Homogeneously Mixed Bioreactor  Plug Flow Reactor
  •  Advantages :  higher productivity, operation for a very long period of time, and lower installation and maintenance costs  Disadvantages :  chance of contamination by other microorganisms during the long fermentation runs (sometimes several weeks).  occurrence of variants of the parent production strain by back mutation or loss of genetic elements (plasmids)
  •  An amino acid precursor is converted to the target amino acid using 1 or 2 enzymes.  Allows the conversion to a specific amino acid without microbial growth, thus eliminating the long process from glucose.  Raw materials for the enzymatic step are supplied by chemical synthesis  The enzyme itself is either in isolated or whole cell form which is prepared by microbial fermentation.
  •  Bioprocess keys : enzymatic production of amino acid Bioreactor : 1) low unit cost of substrate 2) High substrate yields 3) High rate of product production Biocatalyst Preparation : 1. Low fermentation medium cost 2. Short fermentation time 3. High enzyme recovery yield
  •  Amino acid fermentation is closely connected with screening or selection of suitable putative production organisms.  The selection of organism based on :  Non-pathogenicity  Wide spectrum of assimilable carbon source  Rapid growth on cheap carbon and nitrogen sources  High ability to metabolize carbon sources  Resistance to bacteriophage attack
  •  Production strains can be divided into 3 type of strains :  Wild type strain  Mutant strain  Genetically modified strain Wild type strain  Capable to produce specific amino acid under defined conditions Mutant Strain  Feedback regulations are bypassed by partially starving them of their requirements or by genetic removal of metabolic control
  • Genetically modified Strain  Biosynthetic capacity of cells making specific amino acids is improve by amplifying genes coding for rate-limiting enzymes  Improvement involve strains capable to produce amino acid at higher yields  They also produce lower by-product because they dominate costs for downstream procesing
  •  Specific method is require to separate the amino acid produced from its contaminant products  There are 8 methods :  Centrifugation  Filtration  Crystallisation  Ion exchange  Electrodialysis  Solvent extraction  Decolorisation  Evaporation
  •  Common method used in industry  Can be operate semi-continuous or continuous basis  Large scale tests have to performed to choose a suitable centrifuge  Poor centrifugation can be improved by adding flocculation agent  This agent will neutralize the anionic charges on the surface of microbial cells.
  •  Also widely use in industrial  Based on a few factors :  Properties of the filtrate  Nature of the solid particles  Adequate pressure to obtain adequate flow rate  Negative effects of antifoaming agents on filtration  Filtration can be improved by using filteraids  Filteraids improved the porosity of a resulting filter cake leading to a faster flow rates.
  •  Method to recover amino acid  Because of the amphoteric character of amino acid, their solubility are greatly influenced by the pH of a solution  Temperature also influence the solubility of amino acid and their salts  Thus, lowering the temperature can be used to obtain the required product  Precipitation of amino acid with salts are commonly used
  •  Used for the extraction and purification of amino acids form the fermentation broth  Strongly affected by pH of the solutions and the present of contaminant ions  There are two types of ion exchange resins  Cation exchange resins  Anion exchange resins  Cation exchange resins  Bind with positively charged amino acids
  •  Anion exchange resins  Bind with negatively charged amino acid  Anion exchange resins are generally lower in their exchange capacity and durability than cation exchange resins  ion exchange as a tool for separation is only used when other steps fail, because of its tedious operation, small capacity and high costs.
  •  Based on the principle that charged particles move towards the electrodes in the electric field.  A mixture of the required amino acid and contaminant salts can be separated at a pH where the amino acid has a net zero charge (at the IEP).  The salt ions are captured by the ion exchange membranes that are present.  The applications are limited to desalting amino acid solutions.
  •  has only limited applications.  The distribution coefficients of amino acids between organic solvent and water phases are generally small.  Some possibilities based on alteration of amino acid  cyclisation of L-glutamic acid and extraction with alkyl and aromatic alcohols  conversion of contaminant organic acids (like acetic acid) to the ester form and extraction of the ester  extraction of basic amino acids (like L-lysine) from aqueous solution with water immiscible solvents containing higher fatty acids;
  •  performed to get rid of the coloured impurities in the fermentation broth.  based on the fact that amino acids (especially the non-aromatic amino acids) do not adsorb onto activated charcoal.  Although the treatment is very effective, some of the amino acid is lost during this step.  Alternative ways :  addition of cationic surfactants, high molecular synthetic coagulants or some phenolic compounds  washing of crystals with weakly alkaline water as in the case of glutamic acid.
  •  Evaporation of the amino acid containing solution is a quick but commercially unattractive way (high energy costs) to obtain amino acids from solution.  used when the total amount of contaminant products is very low, since these compounds are not removed and appear in a concentrated form in the product.
  •  Use natural product such as sugar cane  Then, the sugar cane is squeezed to make molasses  The glutamic acid is produced through the fermentation process
  •  The heat sterilize raw material and other nutrient are put in the tank.  The microorganism producing glutamic acid is added to the fermentation broth  The microorganism reacts with sugar to produce glutamic acid.  Then, the fermentation broth is acidified and the glutamic acid is crystallized.
  •  The glutamic acid crystal cake is then separated from the acidified fermentation broth.  The glutamic acid crystal cake is added to the sodium hydroxide solution and converted into monosodium glutamate.  The monosodium glutamate is more soluble in water, less likely absorb moisture and has strong umami taste.  The monosodium glutamate is cleaned by using active carbon.  Active carbon has many micro holes on their surface. The impurities is absorb onto the surface of active carbon.
  •  The clean monosodium glutamate solution is concentrated by heating and the monosodium glutamate crystal is formed.  The crystal produce are dried with a hot air in a closed system.  Then, the crystal is packed in the packaging and ready to be sold.
  •  The amino acid produces many products.  For example :  Lysine HCl  Threonine  Aspartate
  •  Lysine application  Food & dietary supplement  Medicine, cosmetics, chemicals  Feed : essential aminoacid for most mammals
  • Glucose Oxygen Ammonia Minerals & Vitamins Lysine
  •  The pathway leading to lysine (also threonine, isoleucine, methione) biosynthesis is initiated with the conversion of aspartate to aspartyl-P via the enzyme aspartokinase (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.
  •  Application of theronine  Vitamins  supplements
  •  The regulation of threonine biosynthesis in E. coli is more complex than that in C. glutamicum.  Corynebacterium, E. coli has three aspartate kinases, AKI, AKII and AKIII.  Two (AKI and AKII) are multidomain proteins that also have homoserine dehydrogenase activity responsible for the third step of the pathway.  AKI is feedback inhibited by threonine and its synthesis is repressed by a combination of threonine and isoleucine.  The synthesis of AKII is repressed by methionine.  AKIII is feedback inhibited and repressed by lysine.
  •  The second step of the pathway is catalyzed by aspartate semialdehyde dehydrogenase (ASD).  The last two enzymes, homoserine kinase (HK; thrB) and threonine synthase (TS; thrC) are coexpressed along with AKI (thrA) as part of the thrABC operon.  This operon is controlled by transcriptional attenuation.
  •  Aspartate is a vitamin-like substance called an amino acid.  Aspartates are used to increase absorption of the minerals.  reduce brain damage caused by cirrhosis of the liver.
  •  Aspartic acid is made by the enzyme aspartate ammonia lyase (aspartase) that carries out the following reaction in presence of ammonium fumarate  -OOCCH=CHCOO- + NH4 + -OOCCH2CH(NH3+)COOO  Once immobilized, the cells are quite stable retaining aspartase activity for well over 600 days even at 37°C.  The process is carried out at pH 8.5 with ammonium fumarate as the substrate.  Immobilized Pseudomonas dacunhae cells can convert aspartate to alanine using the pyridoxalphosphate dependent aspartate β- carboxylase.
  •  contamination of the culture by other microorganisms during fermentation.  bad fermentation reproducibility due to differences in raw material.  back mutation or loss of genetic material of the production strain.  infection of the culture by bacterial viruses (phages)
  •  make use of fresh starting material (inoculum) for each run.  adsorption onto the bacterial cell followed by introduction of genetic material into the bacterium.  isolation of phage resistant strains.  construction of a strain in such a way that it is energetically advantageous to overproduce the required amino acid, thus keeping the construct in the cell.
  •  normally the production strain is constructed in such a way that overproduction of the desired amino acid is obtained and no, or only minor concentrations of, unwanted contaminants appear.  optical resolution steps are not necessary (as in the case of most chemical-processes) since only the L-form is synthesised.  the required amino acid can be relatively easily separated from cells and protein impurities.