INTRODUCTION 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

TYPES OF FERMENTATION Batch Fermentation Fed-batch Fermentation Continuous Fermentation Enzymatic Method

BATCH FERMENTATION 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.

FED-BATCH FERMENTATION 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.

CONTINUOUS FERMENTATION 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 BioreactorPlug 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)

ENZYMATIC METHOD

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 substrate2) High substrate yields3) High rate of product production

Biocatalyst Preparation : 1. Low fermentation medium cost2. Short fermentation time3. High enzyme recovery yield

PRODUCTION STRAINS 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

METHODS TO SEPARATE AMINO ACID 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

CENTRIFUGATION

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.

FILTRATION 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.

CRYSTALLISATION 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

ION EXCHANGE 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.

ELECTRODIALYSIS 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.

SOLVENT EXTRACTION 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;

DECOLORISATION 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 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.

PRODUCTION OF AJINOMOTO 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.

AMINO ACID PRODUCTIONS The amino acid produces many products. For example :

Lysine HCl Threonine Aspartate

LYSINE Lysine application

Food & dietary supplement Medicine, cosmetics, chemicals Feed : essential aminoacid for most mammals

Glucose

Oxygen

Ammonia

Minerals &Vitamins

Lysine

CORYNEBACTERIUM GLUTAMICUM

EXAMPLE OF AMINO ACID PRODUCTION

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.

THERONINE 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 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.

PROBLEM IN FERMENTATION OF AMINO ACID 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)

HOW TO OVERCOME THESE PROBLEMS 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.

ADVANTAGES OF AMINO ACID FERMENTATION 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.

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