 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 & Lysine

Vitamins
 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.