so of ci. Rath, E. Se Va 1, No. (209; 196 386
Shi Sree haters
OPTIMIZING THE CONDITIONS OF L-GLUTAMIC ACID
RODUCTION BY CORYNE BACTERIUM GLUTAMICUM
ARUN SASI, A, KALIDOSS, M, RAVIKUMAR, A.CHANTHRU,
TL BHAKYARAJ AND N. YOGANANTH
PG. and Research Department of Microbiology, Collegeof Arts and Scence,
Puduhkett, Tam Nad, India
Key ood: L-Gatn cid Corea gatemicam Bin, Wavy, Optimist
[Abetnt- Amino aed sich htc acid were yet hana nique physi and chemi propertas
[have many aplication e goa medical and pharracetcal fel Coryebacertm gutaicur me
Procuce, age smoune of Loghtams ace hn ther organisms Calture collected form MTCC sti of
[rerbol tehsogy. To optimiae the production of Lamu ald by ehanging the carbon source he
‘Kamene lnone plocose ete, manaold, mannoe, sascha sucrose. Temporamue 30. 3- and
Ent were ached in 95,65, 710,78 respect oun eee se
Aincywilact aan sdcional ad eheapar carbon source. hen
esmcetation cf 200 L100
of 1g is hed, Llane
‘ns produced at marian of 30 p/m sets incase ct scarbon source and empectue of 37°C or
Frodoction When oun of 100. wou ade 0 ohey the predation wes mui of 275 n/m ith
Ecnperntare of 3>C and pol 85
INTRODUCTION
GGutamic acid (GA) is one ofthe amino acids that
Synthesized in human body. Glutamle 2c often
‘Sats inside all kinds of animal and plans. [thas a
Unique physical, chemical of pharmaceutical elds.
(Aida et, 1986). Glutamic acid reagents and
derivatives are associated vith protein synthesis
techniques and research in cell biology and
[iochemstry Amino acids compounds destined to
become druge polymers and other chemical
products, Glutamic acid a intermediate is
hlathone one kind ofiver drag. GA happens tobe
Se ofthe main components the ell well of Gram=
pasitivehaciera. GA is one othe major amino acids
Erplan protein and GA plays «role ofthe major
ntrogen storage for plants GA has two carbonic
[roupe hich ean fom chelates with many’ eet
alone, The respectively glutamic acd was used as
prescription drugs, that reduced the bod pressure.
(Runhwimer et 1970).
Coryoebaclerum phtamicum are widely used for
the production & L-Glutamme acd. (Kinorbitae
1957) They ae plemorphie, asparogevens, Gram
postive bacteria, widely dstoued innature. in lst
‘con of Bergey's manal of ystematic Bacteriology
(1969) Corynabacera are involve in 15 section as
regular, sporing. Gram positive ods together
achiverse collection of 22 genera (Alda eal. (1988)
(Qakao ot 1972) this study tried to detect four
things i Include, optimieing the temperature, pH,
tnd molet important nutrient content (Demein et
1198), Use cheaper medium inode whey obtained
form cheese industry ean be used forthe production
‘of L-Ghutamic ac (Peter S-Weaisch ea. 199). (Del
Sgunay ef af 2002), And Biotin actas additional
Carbon soinrce for L-glutamic acié production
(Gletman ot 1992).
MATERIAL AND METHODS
Corynebacterium glutamicum Is used for the
production of L-Gtstamic acd. The eulture was
Eollected from IMTECH. Corynebacterium
glutamicum has the MTCC number of 26 The calare
{Sin the frowze dried form, The revived culture
Allowed to grown in Nutrient broth medium and
‘neubated at 30°C for 48 hour. Inoculum was added
Jn SOmL of Nutrient broth and then allowed the
bacteria to grown,
Tn all the experiments, Corynebacterium
slutamicum was used. Cols were groven in
———6. Suara.
Trlenmayer Flacs on a totaryshacker (150m) at
{9°C The following basal medium was used (L)
INH) 50, 3g. Urea 5g, KU,PO, 285 kHPO,3H.O
28g, MgS0,7 1,0 0.25¢, FeS0,7H,0, 0.01,
MaS0,4H,0 001g, COcl,2H,0, 0.415, ZnSO,
7140, has gH,BO,001g, Coe AH,0 007, Cac
GH,0 0.03%, Nici, SH,0 0.0im, Namo 20,24,
(im Suga Sg Biotin’ Ig pH (changed) pH-7D.
(Clvstan olsenen et a 1950) Different sugar
can be used. They ate dextrose lactone, Glucose,
{cos Mannie, Maneose Sarh Sucrose, at
ancanration 5% Biotin concentration of 200 yg.
‘Tablet Botin 00H)
and 100 pg/L was used (Table 1 and Fig.1)
{Corian hoishen ea. 1950; Daly, 200,
Kimara eal. 1997), In addition to production
medium use whey bas the prexiuction medium.
100 mL production medium in 250ml. conical
sask with different biotin concentration \08
prapored and sterilized, Dilferent sugars like
Dentose, fructose, Glucose, Lactose, mannitol,
TPonnoae and sare and sucrose was used 25% of
Iovculum was added and incubated at °C and
5° for 48 hours. {Table 2). Fermentation medium
‘dusted pH55, 65,7. and 75. n addon to this
‘they also used os production medium. (Table 3 &
Fig 2) without biotin also weed 2s production
radia Table 4)
SNe Caton Sours Temperature pH
cme) | a saa
7 Davos we 16 180 a
ut 1 40 130 20
2. Fecteee 2 a ‘uo 0 ve
x 83 200 m0 Ee
a Glucose #0 100 7 18. m
” 108 2 138 0
4 Lacove » 10 mf ws 180
we 2 = 0 109
5 Mani » 0 rr] 108 09
> 8 1 18 2
6 Mena x 74 Ms 108 128
= Be 168 i 1S
2 Sarch = rat 8 104 rs
” 16 a 0 182
8 Suc » 100 10 ns as
7 m7 1% ra a
“Table 2 Biotin (1001)
SN Coben Sues Temperawre pH
ea
a =m
are a Py ms 2
2 Faetose 0 03 68 18
are 83 corn) 198
2 Chace 2 2% 20 98 BL
y im 2 Ww ma
4 eee > waa 2 iat mz
z La x0 22 soa
5 Marit x = wo 10 Bl
a & Ww 120 7
6 Mannose x” s2 200 138 180
re at Bo mu ro
sort 0 108 = 10 1908;
y 0 Bi ct 83.
8 Sucre » sas waka ins
we a7 2263 20Optimising the Conditions of -Glutare Aid Reduction by Cory Bates Gitamiaum 187
ear Conor of KGa Ac Rottion by Corye actertum Gstamicam 187
Tables. Whey only
SNe Cabos Source Tempera J
ee oe
ie) Whey oF 10 275 735,
me ims
2 bin FY 10s
200 9. 2 ss
3 ets » 109
= vs as
135 0 rr
1 mu
ms us
pt x use ws mas 105
SS iis
“Tae Without stn
SX Caren Suse Tenens
=
Tenperare
wy 70 75,
rs = TE 5 3 1a
a re 240 2 ra
2 acne a os 03m 16
a mM 03 BD ims
hos. = no mi ime
” Fe] m2 aa 1m2
4 Lactose » 3 10 im 120
a a4 3 ns 10
5 Manstt » 23 oe 1084 12
y se ime as 12
i » 03 a3. 1103 1082
a m3 os 1203 tesa
7 Sarch x» Se 2 ss 1053
¥ 2 93 a 1082
% Scere x 105 1 15 rs
7 ins 20 20 to
Aller fermentation the total aminoacid content
tyasdetermines by Ninhgdrin method of Moene std
Stein 1948 with glutamic aid has standard. The
calture ites were contrifuged and employed for
fstimation of amino acid 2mL of 30% TCA was
sulle 010 ml of cultured filtrate and cenit
for 20 minates.2 mL of2% Ninhyerin was alded in
2omLov supernatant and heated at OT in weter bath
for? minute. The intensity of blue colour then
developed wa read at 570 nn
RESULTS AND DISCUSSION
# temperature, pH, and nuteient
_smeentaton forthe production of Lami ac
‘The effect ofcorbon source like Deslese, tricone,
slucose,lactoe, mannitol, mannose, starch and
‘Sucrse for Lglatamic and procter. The medium,
With pH of 55,65, 7,75 and adaltional natin
supplement was biotin, il was used ata
concentration of 100 ug/L ‘and 200 pg/L
"spectively. Some cause biotin was not used, Whey
as alo used asthe production medium, When
‘lots of 200 ig/ Ls sed the L-Clutam acid wos
rosuced at maximum of 300 g/mL with ates
‘atbon source and! temperatte of 37°C of pit 6S,
Was suitable forbest production.
‘Then dextrose shows maxamm proccion of 23
ug/L temperasure of SAC and pH of 65 when
_shicose shows manimum production of 274 g/mL,
At temperature of 27°C and pH 65, Mannose sous
‘maximum production of 236 ug/ml at a
femperature of 37°C and pH of 65) Slarch shows
‘maximum production of 231 ug/ml ate a
femperature of 37°C and pH 6.5, Sucrose shoves
‘maximm production of 23.2 ug/ml at a pH
femperature of 37°C and pH 6.5. Manmitel shows
maximum production of 149 g/mL at a
lemperature of 3°C and pH of 5. Fractve shows
maximum production of 138-8 iyg/mt at a
temperature of 37°C and pH 6 5. Whey sbowe
‘maxlinum production of 2775 g/L with Blt of
100 ng/L and temperature of 3™C and pH of 65
‘When without Biotin, dextrose shows shaximam
“ip A Producion Meda Withand WiikoutCalice B
production of 240 ug/ml a a temperate of 3
tnd pl of 5.
|L-latsmic acid produced at axdemum amcun at
‘temperature of 370C and Biotin concentration of
100uL and a pH of 6S and maximom amoun! w:
REFERENCES
Aida, K/Chitata,K, Nakayama, K. Tabisaa
"Yamade, H. 1985. Bjiechnleny of A
Pda Elser Vo} 21-87
Via of Spt Batley 1989 (
Iams od Wiking S220 2
CChnitin Hosseten and Reinhard Kramer, 19%0
mate seretion isCo
wn games [of Baeridogy 3400-3
DanunceL,Dammadery M, Racha, M. Pizzo,
200, A amily of Corynebacterium Eco bustle
‘ects fr gene dosing, coro gene exes
aa promis probing Brain Re
ela 3, Clg Dy Bacher, MF, Engi, Gay
vac Aan George [L198 Imgrtanea
Proephoenol pyrzvate carboxylase of
Conpoehacienim giotamiccum during the
triggered glutamic acid production
21
‘emai Lapis, P, Engen, M. ane Compe].
998" Fletblty of the met abolisiy of
ceri glutamicum 262 oa ard
ip socks eds Marcel Been 488-91.
sang, B, Daler, T, Mavi, Ty Wintertllr,C and
Bac, A199 Revs] by cat the lod aeate
fea arise iain of atari aid producto
Dy Coryrebaceram giatamicim ppl Met
797-288
Fig 2A, Whey With and Wihoxt Caltre
Gutmann, o, Hosehen, Cand Kraet R 1992 Caer
adit glutamic aectetion by Coryrabocteriur
Slatin under Sain kittie. ace. pry
petty
Jeter, Bakers, Noen and Burns 4.1997
“Giiamie sys of Corynebacterium:
iSnot ese far gitate shane gli
by teagan ta Mirabgy 280-9
2, and Kawabara, G. 1997. Glutamate
Production of “Corynebacterium becieri,
Temas kaka havo, its 274
‘ina, 5, Uda, Sand shimone, 1957
hes cl ermenetan Pap
vac renga 1 Gh
ton
phystlgy anc median, FEMS Morbid Re 13
Nehametsu, T. and Kimura, B 2003. Metaboli
tin. Ae Bch
iy peels
Testing cally of Coryoebscteriam
otal. Bre os BS,
enki Hr sara, Woe Mead Nog K
‘A atation on Caryneecerum ation
axene causes sareeplabiity fo
Tenpersture sogutive growth and L-plulamate
production, [arto Re. 38576
pate Wedsen H. Red, C, Retna. D, Penge P
‘Mockel, B, Peteraim, & and S:bm. 1.1999
Sharawigrsakon of phospiathel pyiots
Gasboryimose. gene (rom Cofymebacerium
siutaecnm aaa significance of penzysce fo
Srovthand amine 2 prodcin eM. Microb
Beton 4:90-37.
te