Effect of vitamin B-complex, L-amino acids and complex nutrients
on L-methionine fermentation by a multiple analogue resistant
mutant
Corynebacterium glutamicum X300
1.Subhadeep Ganguly
2.Kunja Bihari Satapathy *
Post-Graduate Department of Botany, Utkal University, Vani Vihar
Bhubaneswar-751004, Odisha2
*Address for correspondence : Dr Kunja Bihari Satapathy , Post-Graduate Department of Botany,
Utkal University, Vani Vihar
Bhubaneswar-751004, Odisha
Communicating Email :res_biol@rediffmail.com
Abstract
An experimental study was conducted to evaluate the effect of vitamin B-complex(vitamin B12, folic acid, nicotinic
acid, riboflavin, pyridoxine-HCl, thiamine-HCl, inositol,calcium pantothanate and para amino benzoic acid), Lamino acids L-glycine,L-alanine,L-Valine,L-leucine, L-isoleucine, L-phenylalanine, L-tyrosine, L-trpytophan, Lserine, L-proline, L-threonine, L-cystine, L-asparagine, L-glutamine, L-lysine, L-histidine, L-arginine, L-aspartate,
L-glutamic acid, 4-hydroxyproline, 5-hydroxylysine, 6N methyllysine, γ-carboxyglutamate, desmosine and
selenocystine) and complex nutrients(paddy soak liquor, wheat bran extract, rice bran extract, malt extract, peptone,
beef extract, corn steep liquor, meat extract and yeast extract) on L-methionine fermentation by a multiple analogue
resistant mutant Corynebacterium glutamicum X300.
[ Key words:vitamin, amino acids, complex nutrients, Corynebacterium glutamicum X300]
INTRODUCTION
Successful production of L-methionine largely depends on judicial selection of suitable nutritional ingredients added
to the production medium [1]. Medium composition exerts pivotal impacts on microbial growth and production of
different metabolites like carbon,nitrogen,minerals,vitamins and amino acids played a vital role on microbial growth
and different enzyme activities.Kase and Nakayama (1974) examined the effect of different amino acids on the
production of o-acetyl-L-homoserine , an intermediate of L-methionine biosynthesis[2]. Ganguly and Banik also
investigated the effect of Vitamin B-complex, L-amino acids and complex nutrients on L-glutamic acid fermentation
by a mutant Micrococcus glutamicus AB100[3-5].Thus ,in the present investigation , we were intended to examine the
effect of vitamin B-complex (vitamin B12, folic acid, nicotinic acid, riboflavin, pyridoxine-HCl, thiamine-HCl,
inositol,calcium pantothanate and para amino benzoic acid),L-amino acids(L-glycine,L-alanine,L-Valine,L-leucine,
L-isoleucine, L-phenylalanine, L-tyrosine, L-trpytophan, L-serine, L-proline, L-threonine, L-cystine, L-asparagine,
L-glutamine, L-lysine, L-histidine, L-arginine, L-aspartate, L-glutamic acid, 4-hydroxyproline, 5-hydroxylysine, 6N
methyllysine, γ-carboxyglutamate, desmosine and selenocystine) and complex nutrients (paddy soak liquor, wheat
bran extract, rice bran extract, malt extract, peptone, beef extract, corn steep liquor, meat extract and yeast extract)
on L-methionine fermentation by the mutant Corynebacterium glutamicum X300.
MATERIALS AND METHODS
Microorganism: A multiple analogue resistant mutant Corynebacterium glutamicumX300 developed in our
laboratory from Corynebacterium glutamicumX1 was used throughout the study [6].
Composition of basal salt medium for L-methionine fermentation: L methionine production was carried out
using the following basal salt medium (per litre): glucose, 60 g; (NH4)2SO4, 1.5 g ; K2HPO4, 1.4 g;
MgSO4·7H2O, 0.9 g; FeSO4·7H2O, 0.01 g ;biotin, 60μg [7,8].
Optimum cultural conditions: Volume of medium ,25 ml; initial pH ,7.0; shaker’s speed ,150 rpm; age of
inoculum ,48 hours; optimum cell density ,4.0X10 8 cells/ml; temperature280C and period of incubation ,72 hours[9].
Addition of vitamin B-complex to the synthetic medium : Initially, the basal medium contained only biotin as a
member of vitamin B-complex. Different members of vitamin B-complex namely vitamin B12, folic acid, thiamineHCl, riboflavin, nicotinic acid, pyridoxine-HCl, inositol, biotin, Calcium pantothanate, paraaminobenzoic acid and
biotin were added separately to the medium at varying concentrations (0.2-0.8 µg/ml) [10].
Addition of amino acids : The effects of different amino acids at varying concentrations (0.5-3.0 mg/ml)
on L-methionine accumulation by this mutant were studied [4].
Preparation and addition of complex
nutrients :
(a) Preparation of rice bran and wheat bran extract : 40 gm of each material was taken into 250 ml of warm
distilled water separately. The suspension was kept at 28oC for 48h. The extracts were filtered separately through
cotton and evaporated to dryness under vaccum to recover solid content.
(b) Preparation of corn steep liquor : About 150 gm of corn was taken into 300 ml distilled water and kept it for
24h at 28oC. The extract was filtered through cotton and evaporated to dryness under vaccum to recover solid
content.
(c) Preparation of paddy soak liquor : 100 gm paddy was added to 250 ml of distilled water and kept it at 28oC
for 24h. The extract was filtered through cotton and evaporated to dryness under vaccum to recover solid content.
(d) Preparation of soybean extract : 50 gm of soybean was poured into 250 ml of distilled water and allowed to
swallow for 24h at 28oC. The soaked water was extracted thoroughly and filtered through cotton. It was then dried
for determination of solid content.
All the above mentioned complex nutrients were added to the synthetic medium according to them solid content in a
sterile condition, but peptone, yeast extract, beef extract, malt extract and tryptone were added directly to the
synthetic medium[5].
Analysis of L-methionine: Descending paper chromatography was employed for detection of L-methionine in
culture broth and was run for 18 hours on Whatman No.1 Chromatographic paper . Solvent system used include nbutanol: acetic acid : water (2:1:1). The spot was visualized by spraying with a solution of 0.2 % ninhydrin in
acetone and quantitative estimation of L-methionine in the suspension was done using colorimetric method
[7]
. All
the chemicals used in this study were analytical grade (AR) grade and obtained from E mark .Borosil glass goods
and triple distilled water used throughout the study .
Estimation of Dry Cell Weight (DCW): The cell paste was obtained from the fermentation broth by centrifugation
and dried in a dried at 1000C until constant cell weight was obtained [11].
Statistical analysis: All the data were expressed as mean±SEM.Data were analyzed using One Way
ANOVA followed by Dunett’s post hoc multiple comparison test using a± soft-ware Prism 4.0.
RESULTS
Table 1: Effect of vitamin B-complex on the L-methionine fermentation
Vitamin (s)
Vitamin B12
Folic acid
Nicotinic acid
Riboflavin
Pyridoxine-HCl
Thiamine-HCl
Inositol
Calcium Pantothanate
Para
Amino
Acid(PABA)
Benzoic
Concentration(s)
[µg/ml]
0.0(control)
0.2
0.4
0.6
0.8
0.0(control)
0.2
0.4
0.6
0.8
0.0(control)
0.2
0.4
0.6
0.8
0.0(control)
0.2
0.4
0.6
0.8
o.o(control)
0.2
0.4
0.6
0.8
o.o(control)
0.2
0.4
0.6
0.8
o.o(control)
0.2
0.4
0.6
0.8
o.o(control)
0.2
0.4
0.6
0.8
o.o(control)
L-methionine (mg/ml)
Dry cell weight(mg/ml)
52.1±1.661
52.6±1.312
*53.1±1.091
*53.1±0.883
*53.1±1.613
52.1±1.113
52.4±0.971
52.5±0.991
*52.9±1.871
*53.1±1.901
52.1±0.913
52.4±1.361
52.7±1.661
52.7±1.099
52.7±1.788
52.1±0.913
52.4±1.871
52.5±1.993
52.5±0.991
52.5±1.683
52.1±1.113
52.5±0.867
52.6±1.181
52.9±1.901
52.9±0.988
52.1±1.688
52.4±1.965
52.5±1.971
52.7±1.553
*53.1±1.911
52.1±0.871
52.3±1.661
52.5±1.873
52.8±1.119
*53.2±1.065
52.1±1.776
52.6±0.991
52.9±1.881
52.9±1.913
52.9±0.897
52.1±1.683
28.5±0.661
28.7±0.913
28.9±0.791
28.9±1.613
28.9±0.882
28.5±0.761
28.6±0,883
28.6±0.661
28.8±0.913
28.9±0.881
28.5±1.881
28.6±0.917
28.7±0.832
28.7±1.991
28.7±1.311
28.5±0.882
28.6±1.111
28.6±0.668
28.6±0.312
28.6±0.912
28.5±0.875
28.6±1.118
28.6±0.993
28.8±0.598
28.8±0,661
28.5±0.881
28.6±0.913
28.6±1.165
28.6±1.887
28.9±0.991
28.5±0.687
28.6±1.191
28.6±0.991
28.8±0.883
28.9±1.917
28.5±1.683
28.6±0.991
28.8±1.268
28.8±0.913
28.8±0.992
28.5±1.116
0.2
0.4
0.6
0.8
52.4±1.993
52.7±1.687
*53.0±1.991
*53.0±0.968
28.6±1.881
28.8±1.883
28.9±0.993
28.9±1.168
(Values were expressed as mean±SEM, where n=6,*p<0.05 when compared to control.)
Table 2: Effect of L-amino acids on the L-methionine fermentation
L-amino acid (s)
Nature of amino Concentration(s)
L-methionine
acid(s)
[mg/ml]
(mg/ml)
1.L-Glysine
Nonpolar aliphatic 0.0(control)
52.1±1.683
amino acid
0.5
52.7±1.661
1.0
*53.1±1.992
1.5
53.4±1.773
2.0
53.4±1.918
2.5
53.4±0.971
3.0
53.4±1.439
2.L-Alanine
0.0(control)
52.1±1.663
0.5
52.4±2.136
1.0
52.4±1.914
1.5
52.4±1.773
2.0
52.4±1.922
2.5
52.4±1.992
3.0
52.4±1.683
3.L-Valine
0.0(control)
52.1±1.871
0.5
52.5±2.136
1.0
52.9±1.882
1.5
52.9±0.972
2.0
52.9±1.113
2.5
52.9±1.651
4.L-leucine
3.0
52.9±1.892
0.0(control)
52.1±2.221
0.5
52.4±1.981
1.0
52.7±1.411
1.5
*53.1±0.883
2.0
*53.1±1.612
2.5
*53.1±1.913
3.0
*53.1±1.118
5.L-Isoleucine
o.o(control)
52.1±0.991
0.5
52.4±1.611
1.0
52.7±1.562
1.5
*53.0±1.381
2.0
*53.0±2.168
2.5
*53.0±1.221
3.0
*53.0±1.919
6.L-phenylalanine
Aromatic
amino 0.0(control)
52.1±1.843
acids
0.5
52.5±1.556
1.0
52.7±1.771
1.5
52.9±1.832
2.0
53.3±1.961
2.5
53.6±1.772
3.0
53.6±1.991
Dry cell weight (mg
/ml)
28.5±0.668
28.8±0.776
*29.0±0.762
*29.2±0.913
*29.2±1.002
*29.2±0.836
*29.2±0.991
28.5±1.116
28.6±0.871
28.6±0.932
28.6±0.661
28.6±0.591
28.6±0.771
28.6±0.991
28.5±0.874
28.6±0.812
28.8±0.882
28.8±0.669
28.8±0.812
28.8±0.782
28.8±0.892
28.5±0.771
28.9±0.872
**29.2±0.861
**29.4±0.992
**29.4±0.831
**29.4±0.812
**29.4±0.971
28.5±0.861
28.9±0.781
*29.1±0.882
*29.3±0.885
*29.3±0.912
*29.3±0.692
*29.3±0.881
28.5±0.871
28.7±0.791
*29.0±0.912
*29.2±0.872
**29.6±0.923
**29.7±0.891
**29.7±0.772
7.L-Tyrosin
8. L-Tryptophan
9.L-serine
10.L-proline
11.L-threonine
12.L-cystine
13.L-asparagine
14.L-glutamine
Polar
uncharged
amino acids
0.0(control)
52.1±1.731
28.5±0.774
0.5
1.0
1.5
2.0
2.5
3.0
0.0(control)
0.5
1.0
1.5
2.0
2.5
3.0
0.0(control)
0.5
1.0
1.5
2.0
2.5
3.0
0.0(control)
0.5
1.0
1.5
2.0
2.5
3.0
0.0(control)
0.5
1.0
1.5
2.0
2.5
3.0
0.0(control)
0.5
1.0
1.5
2.0
2.5
3.0
0.0(control)
0.5
1.0
1.5
2.0
2.5
3.0
0.0(control)
0.5
1.0
52.3±1.991
52.5±0.833
52.8±2.771
52.8±1.913
52.8±1.773
52.8±1.683
52.1±1.871
52.4±1.911
52.7±0.913
52.9±1.683
52.9±1.871
52.9±1.771
52.9±0.993
52.1±1.871
52.3±1.683
52.3±1.821
52.3±1.842
52.3±1.901
52.3±1.672
52.3±1.991
52.1±1.832
52.4±1.554
52.4±1.483
52.4±0.991
52.4±1.117
52.4±1.665
52.4±1.572
52.1±1.383
52.4±1.991
52.4±1.882
52.4±1.602
52.4±1.882
52.4±1.281
52.4±1.008
52.1±1.592
52.4±1.661
52.5±1.749
52.7±1.927
53.0±1.891
53.0±1.892
53.0±1.992
52.1±1.901
52.3±2.832
52.4±1.942
52.8±1.991
52.8±1.882
52.8±1.792
52.8±1.882
52.1±1.902
52.3±1.722
52.6±1.902
28.5±0.991
28.6±0.771
28.8±0.913
28.8±0.692
28.8±0.719
28.8±0.913
28.5±0.683
28.6±0.661
28.8±0.762
28.8±0.881
28.8±0.669
28.8±0.713
28.8±0.783
28.5±0.881
28.6±0.991
28.6±0.771
28.6±0.881
28.6±0.712
28.6±0.771
28.6±0.891
28.5±0.771
28.6±0.781
28.6±0.991
28.6±0.683
28.6±0.813
28.6±0.832
28.6±0.782
28.5±0.771
28.6±0.662
28.6±0.683
28.6±0.881
28.6±0.892
28.6±0.852
28.6±0.912
28.5±0.771
28.6±0.875
28.6±0.590
28.7±0.812
28.7±0.881
28.7±0.912
28.7±0.712
28.5±0.669
28.6±0.891
28.6±0.882
28.8±0.773
28.8±0.782
28.8±0.771
28.8±0.912
28.5±0.772
28.6±0.792
28.8±0.832
15.L-lysine
Positively charged
(R )L-amino acids
16.L-histidine
17.L-arginine
18.L-aspartate
19.L-glutamate
20.
4-hydroxyproline
Non-standard amino
acid
1.5
2.0
2.5
3.0
0.0(control)
52.6±1.662
52.6±0.992
52.6±1.612
52.6±1.984
52.1±1.002
28.8±0.888
28.8±0.921
28.8±0.845
28.8±0.771
28.5±0.682
0.5
1.0
1.5
2.0
2.5
3.0
0.0(control)
0.5
1.0
1.5
2.0
2.5
3.0
0.0(control)
0.5
1.0
1.5
2.0
2.5
3.0
0.0(control)
0.5
1.0
1.5
2.0
2.5
3.0
0.0(control)
0.5
1.0
1.5
2.0
2.5
3.0
0.0(control)
52.3±1.912
52.7±1.883
*53.1±1.991
*53.1±1.892
*53.1±1.882
*53.1±1.902
52.1±1.832
52.4±1.990
52.4±1.771
52.4±1.203
52.4±1.006
52.4±1.913
52.4±1.221
52.1±1.991
52.3±1.832
52.3±1.991
52.3±1.668
52.3±1.612
52.3±0.932
52.3±1.112
52.1±0.991
52.4±1.992
52.7±1.212
*53.2±0.881
*53.2±1.112
*53.2±0.872
*53.2±1.981
52.1±1.081
52.4±1.112
52.5±2.081
52.7±1.008
52.7±0.931
52.7±0.912
52.7±1.113
52.1±1.089
28.5±0.817
28.7±0.885
*29.0±0.893
*29.0±0.891
*29.0±0.913
*29.0±0.761
28.5±0.892
28.6±0.661
28.6±0.890
28.6±0.769
28.6±0.991
28.6±0.862
28.6±0.791
28.5±0.992
28.6±0.963
28.6±0.832
28.6±0.918
28.6±0.871
28.6±0.662
28.6±0.782
28.5±0.590
28.6±0.917
28.8±0.892
*29.0±0.782
*29.0±0.881
29.0±0.692
29.0±0.771
28.5±0.869
28.6±0.669
28.7±0.943
28.7±0.881
28.7±0.762
28.7±0.881
28.7±0.590
28.5±0.881
0.5
52.3±1.097
28.5±0.872
1.0
52.6±1.007
28.6±0.881
1.5
*53.1±1.991
28.8±0.991
2.0
*53.1±1.321
28.8±0.993
2.5
*53.1±1.009
28.8±0.892
3.0
*53.1±1.887
28.8±0.771
Other non-standard amino acids studied namely, 5-hydroxylysine,6N-methylysine,γ-carboxyglutamate,desmosine
and selenocystine showed no effect on L-methionine by this mutant. (Values were expressed as mean±SEM, where
n=6,*p<0.05 ,**p<0.01when compared to control.)
Table 3: Effect of complex nutrients on the L-methionine fermentation
Complex nutrient(s)
Concentration(s)
L-methionine (mg/ml)
1.Paddy soak liquor
0.0(control)
52.1±1.991
1.0
52.7±1.612
2.0
53.0±0.981
3.0
52.8±0.991
4.0
52.4±0.832
5.0
52.2±1.613
2. Wheat bran extract
0.0(control)
52.1±0.963
1.0
52.4±0.882
2.0
52.2±0.991
3.0
51.9±0.962
4.0
51.6±0.893
5.0
*51.1±0.911
3.Rice bran extract
0.0(control)
52.1±1.671
1.0
52.6±0.993
2.0
52.2±0.791
3.0
51.8±1.992
4.0
51.3±1.111
5.0
*51.0±0.883
4.Malt extract
0.0(control)
52.1±0.912
1.0
52.4±0.992
2.0
52.8±1.116
3.0
*53.1±0.831
4.0
*53.4±0.991
5.0
*53.1±1.683
5.Peptone
0.0(control)
52.1±0.992
1.0
52.4±0.871
2.0
52.7±0.891
3.0
52.9±0.991
4.0
52.6±0.865
5.0
52.1±0.958
6.Beaf extract
0.0
52.1±1.331
1.0
52.4±0.983
2.0
52.8±0.762
3.0
*53.4±0.814
4.0
*53.9±0.972
5.0
*53.6±1.683
7.Corn steep liquor
0.0
52.1±0.991
1.0
52.4±1.892
2.0
52.8±0.992
3.0
*53.4±0.831
4.0
*53.9±1.682
5.0
*53.7±0.991
8.Meat extract
0.0(control)
52.1±1.771
1.0
52.5±0.954
2.0
53.0±0.882
3.0
*53.6±0.862
4.0
*53.4±0.948
5.0
*53.1±0.992
9.Yeast extract
0.0(control)
52.1±0.996
1.0
52.4±0.831
2.0
52.7±0.972
Dry cell weight (mg/ml)
28.5±0.662
28.7±0.991
28.8±0.683
28.7±0.662
28.6±0.591
28.5±0.836
28.5±0.771
28.6±0.913
28.5±0.683
28.4±0.661
28.2±0.917
28.0±0.772
28.5±0.662
28.8±0.591
28.5±0.662
28.3±0.688
*28.0±0.661
*27.9±0.613
28.5±0.531
28.7±0.662
28.9±0.913
*29.0±0.663
*29.1±0.683
*29.0±0.791
28.5±0.591
28.6±0.771
28.7±0.769
28.8±0.613
28.7±0.661
28.5±0.692
28.5±0.615
28.7±0.661
28.9±0.592
*29.2±0.615
*29.4±0.992
*29.3±0.661
28.5±0.692
28.7±0.653
28.9±0.773
*29.2±0.792
*29.6±0.661
*29.4±0.661
28.5±0.971
28.7±0.992
28.9±0.681
*29.3±0.662
*29.2±0.683
*29.1±0.912
28.5±0.661
28.7±0.691
28.9±0.662
3.0
4.0
5.0
*53.1±1.983
*53.6±0.991
*53.3±0.884
*29.1±0.713
*29.4±0.913
*29.2±0.991
(Values were expressed as mean±SEM, where n=6,*p<0.05 when compared to control.)
DISCUSSION
Microorganisms change their enzyme activities in response to their growth medium composition and thus ,medium
composition should carefully maintained.In our present investigation, Among different vitamins,L- amino acids and
complex nutrients (readily available in our country)examined ,all the vitamins, major amino acids and complex
nutrients showed positive impacts on L-methionine fermentation by this mutant.Kase and Nakayama(1975) claimed
that 100µg/L biotin is essential for a altered strain of Corynebacterium glutamicum[8].Banik and Majumdar
(1975)incorporated 1mg/L biotin in the medium for L-methionine biosynthesis by Micrococcus glutamicus[12].Tani
et al.(1988) used 200 µg/L thiamine-HCl and 2 µg/L biotin for L-methionine biosynthesis by a methylotrophic yeast
Candida boidinii No.2201 [13].
CONCLUSION
From this present study, it can tentatively concluded that the production of L-methionine by the mutant
Corynebacterium glutamicum X300 can be increased by incorporating Vitami µg/ml; n B-complex, L-amino acid
and complex nutrients in the synthetic medium as recommended in the following block:
Vitamin B12,0.4µg/ml; folic acid, 0.84µg/ml; nicotinic acid,0.4 µg/ml; riboflavin, 0.4 µg/ml; pyridoxine-HCl, 0.6
µg/ml; µg/ml; thiamine-HCl,0.8 µg/ml; inositol, 0.8 µg/ml; calcium pantothanate,0.4 µg/ml; paraaminobenzoic
acid,0.6 µg/ml;L-glycine, 1.5 mg/ml ;L-alanine, 0.5 mg/ml; L-valine, 1.0 mg/ml; L-leucine, 1.5 mg/ml; Lisoleucine, 1.5 mg/ml; L-phenylalanine, 2.5 mg/ml; L-tyrocine, 2.0 mg/ml; L-tryptophan, 1.5 mg/ml; L-serine, 0.5
mg/ml; L-proline, 0.5 mg/ml; L-threonine,1.0 mg/ml; L-cystine, 2.0 mg/ml; L-asparagine, 1.5 mg/ml; L-glutamine,
1.0 mg/ml; L-lysine, 1.0 mg/ml; L-histidine, 0.5 mg/ml; L-arginine, 0.5 mg/ml; L-aspartate, 1.5 mg/ml; L-glutamate,
1.5 mg/ml; 4-hydroxyproline, 1.5 mg/ml; paddy soak liquor, 2.0%; wheat bran extract, 1.0%; rice bran extract,
1.0%; malt extract , 4.0%; peptone, 3.0 %; beef extract, 4.0%; corn steep liquor, 4.0; malt extract , 3.0% and yeast
extract, 4.0%.
ACKNOWLRDGEMENT
We express our cordial thanks to Prof.(Dr) Ajit Kumar Banik, Professor , Department of Chemical Engineering ,
University of Calcutta for his outstanding contribution and cooperation without which we could not able to finish the
work.
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