m.

K TEKNOLOGI BIOINDUSTRI
TIN 330 (2-3)

AMINO ACIDS PRODUCTION

Agroindustrial Technology Department

FATETA - IPB
2016

Asam Amino
Asam amino = senyawa organik yang memiliki gugus
fungsional karboksil (-COOH) dan amino ( -NH2)

Macam asam amino

Ada 20 macam asam amino, yang masing-masing ditentukan
oleh jenis gugus R (rantai samping ). Jika gugus R berbeda
maka jenis asam amino berbeda

CURRENT CONDITION
The amino acid business is a multi-billion dollar enterprise. The
major producers of amino acids are based in Japan, the US,
South Korea, China and Europe.
Commercial production of MSG is the largest and
biggest industries all over the world.
Glutamic acid > lysine
Aspartic acid

> methionine

> threonine

Uses Of Amino Acids In Industrial Applications :

Food industry : 65%
Feed Additives : 30%
Pharmaceutical :
5%

>

APPLICATION of AMINO ACIDS in FOOD INDUSTRY

MSG, glysine, alanine : flavor enhancers
Glycine : added to food containing sweeteners
DL-alanine : added to fruit juices to round off the
taste
Tryptophan and histidine act as antioxidants to preserve
milk powder.
L-Cystein is used for fruit juices as an antioxidant and
improves the quality of bread during baking process
(breaks the disulfide bonds of gluten elasticity of dough
higher and helping it to rise during baking).
Aspartame, dipeptide (aspartyl-phenylalanine-methyl
ester) is 200 sweeter than sucrose. Used as low calorie
artificial sweetener.
Essential amino acids or those deficient in plant based
foods like lysine, methonine, threonine, tryptophane
used as cereal supplementation

CHEMICAL INDUSTRY
Used as a precursor for making other products, such as :
- Glycine used to manufacture Glyphosate & Threonine
used for Azthreonam (herbicide)
- Poly methyl glutamate : manufacturing of synthetic leather

- Alanine and lysine for making polymer e.g polyalanine

fibers, lysine isocyanate resins, and polyaspartate, a watersoluble biodegradable polymer that can be used in
disposable diapers and agriculture
- N-acyl derivatives of amino acids used for making
cosmetics (e.g N-acyl derivatives of phenylalanine or
tyrosine) used to add moisturizing properties to lotions and
conditioners) and surface active substances

PHARMACEUTICAL INDUSTRY
Used as medicines, infusions etc
Medicine :
e.g. N-acetyl-L-cysteine is a pharmaceutical drug and
nutritional supplement precursor in the formation of
the antioxidant glutathione in the body

Infusions Application (amino acids mixture)

- The patient cannot take food by mouth
(after surgery)
- There is a problem with amino acids or
proteins absorption
- The patient need greater than normal
amounts of nutrients including proteins (e.g
patients with severe burns).
http://www.mydr.com.au/webroot/cmis/OutXHTML/CM06911.htm

http://www.otsuka.co.id

METHODS FOR PRODUCTION OF AMINO ACIDS

- Extraction
- Chemical Synthesis
- Microbiological Synthesis (Fermentation)

1. EXTRACTION : hydrolysis of proteins to isolate amino

acids like cystein, lysine, tyrosyne, leucine and asparagine,
(source plant, animal) low yield.
2. CHEMICAL SYNTHESIS shorter time.
Result in racemic mixture (DL-mino acids, (but only Lforms are used by cells)).
e.g glycine, DL-alanine, DL-methionine, and DLtryptophane. Suitable method if the amino acid precursor
is supplied at low prices.
Glycine is manufactured industrially by reacting
chloroacetic acid with ammonia. :
ClCH2COOH + 2 NH3 H2NCH2COOH + NH4Cl

3. MICROBIOLOGICAL SYNTHESIS (Fermentation)

Advantage : low cost
a. Direct fermentation : microorganisms use carbon
sources to produce amino acids (e.g glucose, fructose,
alkanes, ethanol, glycerol, molasses, starch, methanol
etc.). Example : glutamic acid
b. Conversion of metabolic intermediates to amino
acids: such as glycine
Asam amino L-serine glycine (Corynebacterium
glynophilum)
a. Use of enzymes (microbial) or immobilized cells to
produce amino acids such as L-alanine, L-aspartic acid,
L-DOPA (dihydroxyphenylalanine), L-tryptophane and
L-tyrosine.

Biosynthesis of Amino Acids

HMF /
Pentosa
Fosfat

Jalur Embden-Meyerhof Parnas (EMP) atau Glikolisis

Heksokinase
Glu-6 fosfat isomerase
Fosfofrukto kinase
Frukosa difosfat Aldolase

2 NAD+ + 2

+ 2 H+

(PEP)

Jalur Heksosamonofosfat (HMF) = Pentosa Fosfat =

Fosfoglukonat
Glukosa-6-fosfat dehidrogense

Glikolisis

Siklus Asam Sitrat (TCA Cycle = Siklus Krebs)

Synthesis of the various amino acids in a family

frequently requires many separate enzymatically catalyzed
steps starting from the parent amino acid
Sumber : 210.36.18.48/gxujingpin/dwwswx/ppt/6.ppt

GLUTAMIC ACID PRODUCTION

Glutamic acid and its ions and
salts, called glutamates, are flavor
enhancing compounds which
provide an umami (savory) taste to
food.
Monosodium glutamate (MSG), is
widely used as additive in the food
industry
Bacteria : Corynebacterium glutamicum
Brevibacterium sp.
Microbacterium sp.

Glutamate Producer
Various glutamate-producing bacteria have been isolated
and classified as Arthrobacter, Brevibacterium, or as
members of other genera, but recent work has shown that
almost all of these strains belong to the genus
Corynebacterium.

Wild-type cultures produced up to 10 g/l glutamic acid.

Yields were quickly improved by process engineering and
by developing over-producing mutants. Yields are now in
excess of 100 g per liter. It means that a 100,000 liter
bioreactor yields about 10,000 kg of glutamate

Metabolic pathway

C. glutamicum used

glycolysis, PPP (pentose

phosphate pathway)
and Citric acid cycle

Glutamic acid
bacteria have high
activity of
Glutamate
dehydrogenase and
low activity of aketoglutarate
dehydrogenase)
imtech.wikispaces.com/file/view/5.Amino+acids.pptx

GLUTAMIC ACID PRODUCTION

Glutamate producers requirements :
1. overproduce glutamate in excess of their normal
metabolic needs .
2. excrete it into culture broth (Glutamate is the most
abundant free amino acid in bacterial cytoplasm)
by altering permeability of bacterial cell membrane
For completing these two requirements:
use bacteria which lack of -ketoglutaric acid
dehydrogenase enzyme
use of minimum conc. of biotin, addition of penicillin
or surfactants altered cell wall increase
permeability of the bacterial cell membrane results
in over production of glutamic acid

Biotin
cofactor of acetyl-CoA carboxylase that converts
Acetyl- CoA + CO2 Malonyl-CoA (FA biosynthesis !)
(deficiency of biotin affects fatty acid biosynthesis
membrane formation permeability is affected
intracellular export of glutamate is altered
Penicillin & Surfactants
Alter the permeability of the bacterial cell wall and thus
allowing more of the glutamic acid to excrete.
Penicillin inhibits the linking of the amino acid side chains
of peptidoglycan units which have role in the permeability
of the outer membrane.
Surfactant e.g. polyoxylene-sorbitan monostearate
(tween 60) and tween 40 (-monopalmitate) have actions
similar to biotin

Process Control Parameters:

Production is carried out by submerged culture methodfed batch fermenter . Aerobic process, high aeration
required pH 6-8; Temperature should be controlled at 3032 0C
Carbon Source : Cane molasses, beet molasses, and
tapioca/corn hydrolisate are mainly used as the carbon
source because of their low cost.
Example composition of a medium :
Glucose, corn steep liquor , enzymatic casein
hydrolisate , K2HPO4 , MgSO4.7H2O, Urea
Nitrogen Source : the process involves intermittent
addition of urea, ammonia or ammonium salts which
serves as source of ammonium ions

Media Composition of Glutamic Acid Fermentation

By Brevibacterium divaricatum

The process is carefully

controlled at each step such
that cell density, nutrient
composition, temperature,
pH, aeration, agitation rates
and sugar flow rates
Fed-batch :
Sugar is fed into the
bioreactor as the
fermentation proceeds up to
about 36 h

Production tank of about

50,000-500,000 L

MSG and Glutamic Acid

Production

http://www.vedaninternational.com/products/product1.htm

NaOH

The crystals of l-glutamic acid

hydrochloride were separated from the
liquid by filtration and redissolved in water.

The fermentation last 35-45 hours

With NaOH/KOH, and this solution was

stored to allow l-glutamic acid to crystallize
out MSG

Optimizing Production
Glutamic acid production is highest when biotin is
limiting. When biotin is optimal, growth is luxuriant and
lactic acid, not glutamic acid, is excreted as by product

Cell membranes in which the saturated to unsaturated

fatty acid ratio is abnormal, therefore the permeability
barrier is destroyed and glutamic acid accumulates in the
medium.

Effect of Biotin on Glutamic Acid Production

Glutamic
Acid (g/Lt)

Cells Growth

Glutamate Production

biotin

Dry Weight Cells

(g/l)

Key factors in controlling the production of Lglutamic acid :

The presence of biotin in the range of 5 to 10
Ug/L, which is optimal for the excretion of Lglutamic acid through cell walls
A sufficient supply of oxygen to reduce the
accumulation of lactic acid and succinic acid as
by-products (Corynebacterium bersifat aerobik)

Alternative of Downstream Process : RECOVERY AND

PURIFICATION :
Filtration for removal of microbial cell mass in broth
Concentration of the filtered broth (evaporator)
Adsorption ion exchange resins
Concentration (evaporator)

Crystallization
Recrystallization to obtain L- glutamic acid in purified form
+ NaOH MSG.

Produsen MSG di Indonesia :

1. PT. Sasa Inti
Probolinggo, Jawa Timur
2. PT. Miwon Indonesia
Gresik, Jawa Timur
3. PT. Ajinex International
Mojokerto, Jawa Timur
4. PT. Ajinomoto Indonesia
Mojokerto, East Java
5. PT. Palur Raya
Semarang, Jawa Tengah
Persatuan Pabrik Monosodium Glutamate dan Glutamic
Acid Indonesia (PPMI) - Kebon Jeruk, Jakarta Barat

LYSINE PRODUCTION
essential amino acid

Lysine is not synthesized in animals, hence it must be

ingested as lysine or lysine-containing proteins.
In plants and bacteria, it is synthesized from aspartic
acid (aspartate)

Lysine Production
Lysine can be produced by :
chemical synthesis, extracting from protein hydrolyzate,
enzymatic method & fermentation method
Among these methods, fermentation is the most
economical and practical means of producing lysine
biological form of lysine (L-lysine) is produced.
Application :
L-lysine is useful as medicament, chemical agent, food
material (food industry) and feed additive (animal food).

Biosintesis Asam Amino

Glikolisis
HMF /
Pentosa Fosfat

Siklus Krebs

FERMENTASI LISIN
Aspartat

aspartokinase

Aspartil fosfatase
Aspartat semi aldehid

Dihidropikolinat
sintetase

Mutan
auksotrofik
tidak mempunyai
enzim homoserin
dehidrogenase,
=
tapi tetap
memerlukan
threonin dan
metionin untuk
pertumbuhannya
Homoserin
dehidrogenase

Homoserin

Penghambatan
Sinergis/harmonis

Lisin

Threonin

Metionin
Isoleusin

Fermentasi Lisin
Threonin ditambahkan pada batas konsentrasi untuk
pertumbuhan, sehingga tidak terjadi penghambatan
umpan balik sinergis/harmonis lisin diproduksi secara
berlebihan.
Contoh mikroba : Corynebacterium glutamicum &
Brevibacterium flavum

http://www.vedaninternational.com/products/product1.htm

Downstream Process
Separation/extraction methods (ultrafiltration or
centrifugation, separation or ion exchange extraction,
crystallization, drying) and sold as a powder.
Alternatively, spray dried pellets or liquid fermentation
broth can be used as animal feed supplement.
One common use of L-Lysine
is for the treatment of herpes/
cold sores.

L-Methionine Production

Amino acid needed by the body but not manufactured

naturally by it, L-Methionine can be acquired through
proper diet and supplements.
Comercially, methionine produce by chemical reaction
which produce racemic mixture of acetylated DL
Methionine separated by immobilized aminoacylase
(using DEAE-Sephadex) deacylated of L-methionine
L-methionine

Chemical Synthesis of DL-Methionine

MMP : methyl mercaptan-propionaldehyde

http://www.allaboutfeed.net/managment/markets-trade/the-complex-process-of-manufacturing-methionine-12677.html

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid
acylase for the resolution of racemic mixtures of chemically
synthesized amino acids.
Amino acid acylase catalyses the deacetylation of the L form of
the N-acetyl amino acids leaving unaltered the N-acetyl-d
amino acid, that can be easily separated, racemized and
recycled.
Some of the methods of enzyme immobilization used for this
purpose :
- ionic binding to DEAE-sephadex
- entrapped as microdroplets of its aqueous solution into fibres
of cellulose triacetate .
- immobilized on macroporous beads made of flexiglass-like
material

DL-R-CH-COOH + H2O L-R-CH-COOH +

D-R-CH-COOH
NHCOR
NH2
NHCOR
Aminoacylase
(EC35114)
Racemization

A chemically synthesized acyl-DL-amino acid is asymmetrically

hydrolyzed by aminoacylase to give L-amino acid and unhydrolyzed
acylDamino acid. Both compounds are easily separated by the
difference of their solubilities.
Acyl-D-amino acid is racemized, and reused for the resolution

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical
industries and is needed for the production of aspartame (aspartic
acid + phenylalanine; as low -calorific sweetener )

The aspartase enzyme has been immobilized using the whole cells
of Escherichia coli onto e.g. phenolformal-dehyde resin, for
adsorbing aspartase

Fumaric acid
(chemical)

Aspartic acid

aspartase

Enzymatic production of L-aspartate and L-alanine

Use fumaric acid as precursor with enzyme :

E1 : aspartate ammonia lyase (aspartase catalyses
addition of ammonia to the double bond of
fumaric acid) from E. coli,
E2 : aspartate -decarboxylase from Pseudomonas
dacunhae

http://en.bioamid.com/catalogue/farm/l-asparaginic_acid.html

Aspartame
- artificial sweetener used in reduced calorie foods
200 times sweeter than sugar
- chemical name N-L-aspartyl-L-phenylalanine-l-methyl ester
(APM)
- has a sweet taste without the bitter chemical or metallic
aftertaste
Production :
phenylalanine is modified by a reaction with methanol and
then combined with a slightly modified aspartic acid which
eventually forms aspartame.

http://www.madehow.com/Volume-3/Aspartame.html

BAHAYA ASPARTAM
Telah digunakan pada banyak jenis produk makanan
dan minuman di seluruh dunia. Terutama digunakan
di minuman soda dan permen.
Dalam keadaan normal, fenilalanin diubah menjadi
tirosin dan dibuang dari tubuh. Gangguan dalam
proses ini menyebabkan fenilalanin terakumulasi
dalam darah dan dapat meracuni otak serta
menyebabkan keterbelakangan mental. Penyakit ini
dinamakan phenylketonuria (PKU).

Aturan Penggunaan Aspartam

Kep. Ka. BPPOM
Wajib mencantumkan peringatan Fenilketonuria:
mengandung fenilalanin, yang ditulis dan terlihat
jelas pada label jika makanan atau minuman atau
sediaan menggunakan pemanis buatan aspartam.
Pada label kemasan harus dicantumkan :
Produk ini mengandung aspartam ,sehingga tidak
disarankan untuk penderita phenylketonuria

References
http://www.holisticmed.net/aspartame/aminoacid.pdf
Appl Microbiol Biotechnol (2005) 69: 18
DOI 10.1007/s00253-005-0155-y
MINI-REVIEW
Wolfgang Leuchtenberger . Klaus Huthmacher .
Karlheinz Drauz
Biotechnological production of amino acids and derivatives:
current status and prospects
Received: 13 June 2005 / Revised: 22 August 2005 / Accepted: 29 August 2005 /
Published online: 30 September 2005
# Springer-Verlag 2005
http://faculty.kfupm.edu.sa/CHEM/belali/CHEM%20456/Chapter%209/Chapter%209%20Industrial%20Fermentation.pdf
Etc. Etc.