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INTRODUCTION
Proteins,
indispensable
a
for
class
of
life.
complex
They
are
organic
molecules,
bio-polymers
of
are
simpler
compounds known as am:l.no acids. Amino acids possess amino group
attached
to
the
carbon atom lying next to the carboxyl
oC. -
group. Due to the presence of at least one asymmetric carbon atom
amino acids, except glycine, exist in two isomeric forms, d and 1
of which only the 1-forms are found in proteins.
discovered in 1806 and 1810 are asparagine and
amino acids
cystine,
The 1st two
respectively.
There are
23 amino acids which may be
seen to occur frequently in protein hydrolyzates.
Besides these
there are more than 170 amino acids known to occur in living
systems (Meister, 1965).
Animals including man are unable to synthesize some amino
acids called "essential amino acids" from simple ingredients. These
essential amino acids are isoleucine,
phenylalanine,
threonine,
leucine,
lysine,
tryptophan and valine.
methionine,
All these amino
acids must be present in requisite quantities in the ingested food
materials.
The absence or shortage of any one of these amino
acids in the food
may cause malnutrition and protein deficiency
(Karlson, 1969). Population in developing countries like India, are
mainly dependent on vegetable diet. It is known that the vegetable
proteins are deficient in essential amino acid content.
For this
reason, sole dependence on vegetable diet leads to the appearance
of
symptoms
of
protein
deficiency.
An
inadequate
supply
of
balanced protein is the main reason for high rate of sickness and
death in developing countries (Fontanel, 1972). It is reported that
nausea,
dizziness
deficiency.
and
Methionine
hypersensitivity
deficiency
results
observed
in
in
hepatic
lysine
and
renal
damage. Apart from their use as food supplement amino acids are
also
used
in
pharmaceutics,
herbicide)
and
industry.
importance
as
therapeutic
cosmetic,
Amino
agents,
agriculture
acids,
taste
( ani_mal
now-a-days,
improver
of
feed,
are
of
foods,
2
flavouring
agent
and
ditterent
laboratory
reagents besides their
nutritional importance (Table 1) .
Table 1. Amino acid application ( Plachy, 1989)
Amino acid
Area of
application
Application
Lysine, Methionine,
Feedstuffs
Fortification of
Threonine.
Feedstuffs.
Lysine, Methionine,
Food
Food additives,
Threonine, Glutamic
Industry
seasioning
acid, Glycine, Alanine,
Sweeteners.
Asparagine,
Pheny !alanine.
Lysine, Methionine,
Medical
Therapeutic
1hreonine, Arginine,
field
application
Tryptophan, Other
inf'usion
essential and non
solutions, synthesis
essential amino acids.
ot peptides and
vitamins.
Poly- L-alanine,
Chemical
Surface active
poly- {?> -glutamic
Industry
agents, detergents
acid etc.
polymers,
synthetic fibres
and leather.
Mixture ot essential amino acids are used in treatment of
nephrosis,
ulcers,
early stages ot liver cirrhosis,
drug
toxicity,
anaemia,
during
gastric and duodenal
convalescence
from
3
infectious diseases,
before and a Her surgery
(Dulaney,
1967).
Japanese people add amino acids as condiments tor improvement ot'
taste ot tood. 1he organoleptic property of L-glutamic acid is used
widely
for
improving
the
taste
of
"Kombu'
a
traditional
preparation made from Laminaria. L-glutamate is marketed for its
characteristic
flavour
in
the
trade name
"Ajinomoto".
Lysine
derivative have been made to improve the flavour in alcoholic and
non-alcoholic
beverages.
Lysine
adipate
and
lysine
succinate
enhance the food taste (Hause and Todd, 1962). The characteristic
taste of
Emmentaler cheese is said to be imparted by certain
amino acids like threonine, ci.. -aminobutyric acid, glycine, alanine
and proline. In United States primary dietary use of DL ·methionine
is used for supplementation of chick feed. Mono hydrochloride of
lysine, arginine,
glutamic acid and histidine are used {Miyake et
al, 1971) as meat improving agent. Lysine is used as a supplement
for bread and other food stuffs. It is the limiting essential amino
acid
required
lysine
is
in
being
vaccine (Dorval
the
diet
used
by
~ ~
of pol:1.HTJ {David,
industry
to
1987).
stabilize
the
Recently,
poliovirus
1990). 1hus it is obvious that if the amino
acids are to be used as nutritional supplements or as organoleptic
agents these are to be produced commercially and to be marketed
at a cheaper rate. For this, several methods have been adopted
to produce essential amino acids in a large scale. These include
chemical
synthesis,
extraction,
enzymatic
synthesis
and
fermentation ; of which fermentative method is popular because it
is cheaper,
easier and the amino acids obtained from microbial
fermentation
are
exclusively
in
the
biologically active 1-form.
Several methods are used to produce amino acids (Table 2 ) •
Table 2. Methods of amino acid preparation (Plachy. 1989).
Method
Amino acids
Extraction from protein
Cysteine, leucine, tyrosine, DL-alanine,
hydrolyzates
asparagine, phenylalanine, glycine,
4
Method
Amino acids
methi onine, threonine, tryptophan,
valine.
Microbiological
Lysine, L-alanine.
methods
Enzymic method
Aspartic acid, tryptophan
Precursor method
Lysine, arginine.
Fermentation
Phenylalanine,
histidine,
glutamic acid,
isoleucine,
glutamine,
leucine,
proline,
threonine, valine.
An
individual
liquors
extensive
ami.no
of
acids
food
literature
from
exists
simple
processing
on
the
production
precursors or
industries,
of
from
waste
supplemented
with
appropriate nitrogen sources and cofactors. Recently, amino acids
are isolated from farm wastes ( Tsuruoka, 1987). Production of Lamino acids by fermentation is now being used in industrial scale
and this potentiality of microbes have been exploited commercially
in countries like Japan and USA (Dulaney, 1967 J • Japan is a main
producer of amino acids ( lable 3). Among other countries amino
acids
Union.
are
produced
commercially
in
Czechoslovakia
and Soviet
5
Table 3 • Amino acid market in Japan (Plachy, 1989).
Amino acid
1
Annual production
p nee
. 2
Producer
Glutamic acid
10000 - 100000
5-10
Ajinomoto, Kyowa
Methionine
10000 - 100000
5-10
Degussa, Ajinomoto,
Rhone - Poulene
Lysine
5-10
10000 - 100000
Ajinomoto, Kyowa,
Toray.
Isoleucine
100- 1000
Ajinomoto, Tanabe,
100-500
Kyowa.
100- 1000
Threonine
50-100
Ajinomoto, Kyowa
Aspartic acid
10D- 1000
5-10
Ajinomoto, Tanabe
Glycine
100- 1000
5-10
Ajinomoto, Kyowa.
Pheny !alanine
100-1000
100-500
Ajinomoto, Kyowa.
Tryptophan
100-1000
·lOD-500
Ajinomoto, 1anabe
Valine
100-1000
10-50
Ajinomoto, Tanabe
50
10-50
Ajinomoto, Kyowa,
Tyrosine
1anabe.
1. Annual production in tons;
2. Price in $/Kg.
In Japan,
Kinoshita et
( 1957) for the first time showed the
~
cheap way of amino acid production by microbial fermentation. In
microbial
fermentation
microorganism
excrete
large
quantities
of
amino acid in the fermentation medium from which they could be
isolated,
purified and marketed at a
product obtained
through
reasonably
low
cost.
The
fermentation needs no resolution before
biological consumption because amino acids are produced exclusively
in 1 - torms.
microorganism
Kinoshita ( 1959) isolated a glutamic acid producing
Micrococcus
Corynebacterium glutamicum
then
Kinoshita
( 1959)
glutamicus
later
on
reidentified
(Nakayama and Kinoshita,
made
an
extensive
study
on
as
1966). Since
amino
acid
6
production by screening thousands of micro-organisms and reported
that amino acid production is not restricted to any particular group
of microorganism.
Production
of
important
amino
acids
through
genetically
modified bacterial strain and to improve the yield is one ot the
aims of modern
biotechnological research.
It
is known that the
vegetable proteins are deficient in essential amino acid content. It
can be thought, therefore, that in the coming years a large scale
microbial
production
of
amino
acids
will
be necessary
as
food
supplement to avoid malnutrition in developing countries (Albanese,
1967).
The objective of the present study was to isolate mutant
strains
that
would
produce
threonine,
to
optimise
the
cultural
conditions for threonine production and to improve the threonine
yield
further
by
isolation
of
mutants
resistant
to
threonine
analogues.
The problem was investigated through the following steps
(..'"'
1.
Revival of lyophilized culture of the threonine producing strain\ S{':--""
M56 (L-methionine auxotroph).
2.
Confirmation
of
the nature of amino acid
selected strain by
produced by
the
paper chromatography and microbiological
assay.
3.
Taxonomic identification of the strain.
4.
Mutagenic treatment of the strain with N-methyl-N •-nitroN-nitrosoguanidine
and
selection
of
double
auzotrophs in order to improve the threonine yield.
and
triple
,
7
5.
Selection ot' potent threonine producing auxotrophic mutants.
6.
Standardization of optimal conditions for threonine production by
the selected single, double and triple auxotrophic mutants.
7.
Improvement
of
the
threonine
yield
by
isolation of
mutants
resistant to threonine analogue and lysine analogue.
8.
Study ol the activity and regulation of aspartokinase, the key
enzyme pertaining to threonine accumulation.
9.
Standardization of method for the product recovery and study
of the homogeneity of the isolated product.
A review
of literature pertaining
to
threonine
and
acid
following
pages
perspective.
some
to
other
analyse
amjno
the
microbial
is
production
presented
programme
in
the
in
of
the
proper