amino acid

1.  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.

2.  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

3.  Batch Fermentation
 Fed-batch Fermentation
 Continuous Fermentation
 Enzymatic Method

4.  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.

5.  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.

7.  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.

9.  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

10.  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)

13.  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.

14.  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

15.  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

16.  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

17. 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

18.  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

19.  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.

20.  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.

21.  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

22.  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

23.  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.

24.  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.

26.  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;

27.  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.

29.  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.

30.  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

31.  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.

32.  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.

33.  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.

34.  The amino acid produces many products.
 For example :
 Lysine HCl
 Threonine
 Aspartate

35.  Lysine application
 Food & dietary supplement
 Medicine, cosmetics, chemicals
 Feed : essential aminoacid for most mammals

36. Glucose
Oxygen
Ammonia
Minerals &
Vitamins
Lysine

38.  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.

41.  Application of theronine
 Vitamins
 supplements

43.  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.

44.  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.

45.  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.

47.  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.

48.  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)

49.  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.

50.  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.