INTRODUCTION:

Though L-methionine is one of the essential sulfur containing amino acids for human nutrition, but human beings are not capable of producing this amino acid in their bodies [1]. Vegetable diets are also deficient in L-methionine [2]. Considering the fact, several attempts have been made to commercialize the L methionine production. Among the various methods for production of different L-amino acids, e.g., chemical synthesis, protein hydrolysis and microbial fermentation methods, the later now dominates industrial production of optically active amino acids [3]. Thus, selection of suitable microorganism is foremost important aspect for this method. Several efforts have been made to improve these techniques. Wild type of microorganisms produce little amounts of amino acids which needed for their own cellular growth and metabolism [4].

The over production of L-amino acids required in the industrial scale need to release the product mediated feed-back inhibition and repression by environmental manipulations of fermentation methods or genetic alteration of microorganisms involved [5]. Most of the genetic manipulations have been made by mutagenesis followed by selection of suitable over producing either auxotrophic or regulatory mutants [6]. In addition to this, protoplast fusion is also widely used now a days to develop high yielding strains which is resistant to different agents like product analogue(s) ,antibiotics etc. Kaneko and Sekiguchi (1979), Akamatsu and Sekiguchi (1982) first used the protoplast fusion method in bacteria using Bacillus and Brevibacterium in Japan [3,4]. Tosaka et al. (1983) first applied this technique for L-lysine and L-threonine fermentation using Brevibacterium lactofermentum [5].In this present study an effort has been made to develop multiple L-methionine analogue-resistant high L-methionine yielding strain of Corynebacterium glutamicum by induced mutation.

MATERIALS AND METHODS:

Selection of microorganism:

A regulatory mutant Corynebacterium glutamicum X1 (accumulated only 0.6 mg /ml L-methionine) developed in our laboratory from its parent strain Corynebacterium glutamicum (basically a L-glutamic acid producing bacterium which does not accumulate L-methionine) which was isolated from North Bengal soil was subjected for mutational study.

Chemical and Physical mutagensis:

To develop a high L-methionine yielding strain, the above mentioned regulatory strain was subjected to mutational treatments using Ethyl Methane Sulfonate (EMS) and UV irradiations as Chemical and Physical mutagens respictively as follows:

Exposure to EMS:

1 ml cell suspension (containing 3x10⁸ cells) was added to 9ml EMS solution of different concentrations (221.8 mmol /ml, 186.3 mmol /ml 76.9 mmol /ml respectively) and was incubated (10,20,30,40 and 60 minutes respectively).From each sample , 1 ml cell suspension was then plated on CD agar medium and kept at 30⁰C for 48 hours[6].

Treatment with UV irradiation:

2 ml cell suspension (containing 3x10⁸cells/ml) was taken in a petridish (5 cm diameter) and expose it to UV irradiation, using Hanovia germicidal lamp (15 Watt) from a distance of 12 cm for different periods of time (1-9 minutes). The UV treated cells were plated in similar ways as mentioned above [6].

Development of multiple L-methionine resistant strain : Multiple L-methionine analogue-resistant strain was develop by adding different L-methionine analogue ( 20-100 mg/ml) to the growth medium (namely: α-Methyl methionine , DL-ethionine ,D-methionine sulphate and DL-norleucine[7,8].

Physical conditions for growth:

The fermentation was carried out using medium volume, 30 ml ; initial pH 7 ; shaker speed , 200 rpm ; age of inoculum, 48 hours ; cell density , 3 x 10⁸ cells / ml at 30⁰C[9].

Protoplast preparation, fusion and regeneration:

Two superior strains (namely, Corynebacterium glutamicum X164 which is high L-methionine yielding and Corynebacterium glutamicum X124 which is a multiple analogue resistant strain) were selected for protoplast fusion. The cells were harvested in 100 ml growth medium composed of : glucose , 20 gm / L ; peptone , 10 gm / L ; yeast extract ; 10 gm /L ; NaCl , 2.5 gm / L ;MgSO₄.7H₂O , 0.25 gm / L ;MnSO₄.4H₂O , 0.1 gm / L ; K₂HPO₄ , 1.0 gm / L ,KH₂PO₄ , 1.0 gm / L and biotin, 2 µg / ml in 250 ml Erlenmeyer conical flask at 30⁰C for 24 hours. Then the cell suspensions were centrifuged separately at 10,000 rpm for 10 minutes. The pellets were collected and transferred aseptically to a protoplasting medium composed of: sucrose, 0.5 M, maleate buffer (pH 6.5), 0.02 M; MgCl₂.H₂O, 20 mM and lysozyme, 100 µg / ml. After protoplast fusion (observed under phase contrast microscope), protoplast were fused in a medium containing the same composition similar to the protoplasting medium along with polyethane glycol (30%), dimethyl sulfide (15%) and CaCl₂ , 10 mM . The suspension was shaking at 50 rpm on a rotary shaker with incubator at 30⁰C for 10 minutes and then it was diluted 10 fold with protoplast medium buffer (pH 6.5) .The suspension was then centrifuged for 5 minutes at 25,000 rpm at 5^oC using a cold centrifuge apparatus (EPLX3761) . The pellet was collected and plated for colony formation for 48 hours at 30⁰C. The colonies were transferred to agar (2%) slants containing the same growth medium.

Viable counting of protoplast (Reversion of protoplast): Protoplasts were diluted with 10 ml of dilution fluid and plated into petridish (diameter 5cm) containing agar medium allowed to grow at 30⁰C for 48 hours and subjected for subsequent fermentation trials[10].

Composition of basal salt medium for L-methionine production:

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 [10,11] .

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 n-butanol: 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 [11]. All the chemicals used in this study were analytical grade (AR) grade and obtained from E. Merck. 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 [12].

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 AND DISCUSSION:

The wild type Corynebacterium glutamicum X1 was not capable to accumulate L-methionine in the fermentation broth in excess amount than necessary for its own survival (accumulated only 0.6 mg / ml after 72 hours of incubation). In addition to that, this strain responded to feed-back inhibition and repression also. The main problem of screening was the selection of strains after mutagenic treatments. Accumultion of different L-amino acids by the different mutants of Corynebacterium glutamicum developed on exposure to different concentration of EMS for 15 minutes and their nutritional requirements is depicted in table1.

Table 1: Relation between nutritional requirements of the mutant strains of Corynebacterium glutamicum with EMS and their L-amino acid accumulation
EMS			Extracellular L-amino acid pattern							r
Concentration (mmol / ml)	Exposure Time (min)	Total Number of auxotroph	L-methionine			Other L-amino acids			Requirements of vitamins
Concentration (mmol / ml)	Exposure Time (min)	Total Number of auxotroph	Similar to parents	More than parents	Less than parents	L-glutamic acid	L-lysine	L-threonine	Requirements of vitamins
221.8	10	26	18	-	03	03	02	-	08	3
	20	76	25	11	19	11	06	04	23	2

	30	55	19	06	07	13	07	03	31	2
	40	58	09	04	-	21	17	07	26	6
	50	44	18	04	07	10	04	01	16	2
	60	33	21	-	-	10	02	-	19	2
186.3	10	28	14	02	01	06	02	03	11	2
	20	65	22	05	06	17	11	04	33	4
	30	43	07	02	11	18	05	-	21	7
	40	39	16	05	05	11	02	-	11	2
	50	31	16	01	01	13	-	-	07	1
	⁶⁰	⁶⁴	32	11	06	09	04	02	17	3
76.9	10	42	21	17	-	04	-	-	23	4
	20	56	13	28	02	08	04	01	41	3
	30	71	24	36	07	02	02	-	37	2
	40	49	22	11	02	04	06	04	27	3
	50	55	11	18	07	08	08	03	36	3
	60	48	07	16	04	10	07	04	26	6
r is the frequency of spontaneous reversion X 10⁶

Among the two mutagens used in this present study, UV irradiation showed sharp killing effect on bacteria (Fig.1 and Table 2).

*Table 2: Relation between vitamin requirements and L-amino acid accumulation by the mutants obtained by exposure of Corynebacterium* glutamicum X184 to UV irradiation**
Periods of exposure to UV irradiation (minutes)	Total number of auxotroph	Extracellular L- amino acid pattern
		L-methionine			Other L- amino acids			Requirements of vitamins	r
		Similar to parents	More than parents	Less than parents	L-glutamic acid	L-lysine	L-threonine	Requirements of vitamins	r
1	116	39	08	10	29	19	11	44	4
2	92	58	17	-	17	-	-	64	1
3	88	36	21	07	11	04	09	31	1
4	64	32	19	-	11	01	01	24	6
5	42	20	11	-	09	02	-	22	2
6	33	11	11	04	03	03	01	16	7
7	11	04	03	01	02	02	-	03	3
8	07	01	02	-	04	-	-	05	2
9		-	-	-	-	-	-	-	-
r is the frequency of spontaneous reversion X 10⁶

Table 3: Selection of multiple analog resistant strain
Analogue(s)	Mutant strain(s)	Concentration(s) of analog(s)[mg/ml]
Analogue(s)	Mutant strain(s)	20	40	60	80	100
α-Methyl methionine	*Corynebacterium* *glutamicum* X216	+	+	+	+	+
α-Methyl methionine	*Corynebacterium* *glutamicum* X224	+	+	+	+	+
DL-ethionine	*Corynebacterium* *glutamicum* X224	+	+	-	-	_
DL-ethionine	*Corynebacterium* *glutamicum* X224	+	+	+	+	+
D-methionine sulphate	*Corynebacterium* *glutamicum* X224	+	+	+	_	_
D-methionine sulphate	*Corynebacterium* *glutamicum* X224	+	+	+	+	+
DL-norleucine	*Corynebacterium* *glutamicum* X224	+	+	+	_	_
DL-norleucine	*Corynebacterium* *glutamicum* X224	+	+	+	+	+

This led to a great problem to obtain high L-methionine yielding mutant. In our present study, another important section is the development of a multiple L-methionine analogue-resistant strain development. The selection procedure was based on the principle that the mutants which grew well on medium containing different L-methionine analogues (shown in Table 3).

The third section of our study is to develop L-methionine yielding strain of Corynebacterium glutamicum by protoplast fusion . The conditions for appropriate fusion were examined in this study.Appearence of spherical cell and their disruption at hypotonic solution was used as the indices of protoplast study. Cells suspended into hypotonic solution exhibited plasmolysis after 3 hours.The soft agar concentrations also showed marked effect on colony formation and thus, viable count of the strain (Table 4).Maximum colony formation was obtained with 0.5 % soft agar concentration . Colonies appeared after 48 hours of incubation at 30⁰C, but their count was increased in the next week.

Fig 4: Effect of agar concentration on the viable count
Soft agar concentration (%)	Viable count/ml
0.1	**6X10⁴±2X10³
0.2 ^©	8X10⁶±6X10³
0.3	11X10⁶±2X10⁴
0.4	*10X10⁷±7X10³
●0.5	*16X10⁷±3X10³
0.6	**11X10⁹±6X10³
0.7	**9X10⁸±1X10³
0.8	*11X10⁷±4X10⁴
0.9	*7X10⁷±3X10³
(Values were expressed as Mean±SEM ,where n=6, p<0.05 and *p<0.01 when compared to control(©).● stands for maximum production.)

For Protoplast fusion, mainly different concentrations of polyethylene glycol or PEG(10-80%), incubation period (with PEG),pH and additives (namely sodium succinate,sucrose,MgSO₄.7H₂O,EDTA and K₂HPO₄) were examined one by one (Tables 5-12) as mentioned below:

Table 5: Effect of PEG
Concentration of PEG(%)	Fusion frequency
10	**11X10³±4X10³
20	**6X10⁴±6X10²
●30^©	16X10⁷±3X10³
40	*11X10⁶±1X10³
50	*7X10⁶±1X10³
60	**16X10³±3X10²
Values were expressed as Mean±SEM ,where n=6,p<0.05 and *p<0.01 when compared to control(©).● stands for maximum production)

Table 6:Incubation period with PEG
Time (minutes)	Fusion frequency
10	13 X 10⁷±3X10³
●20	*11X10⁸±7X10²
30^©	*16X10⁷±4X10²
40	*9X10⁶±1X10²
Values were expressed as Mean±SEM ,where n=6,p<0.05 and *p<0.01 when compared to control(©).● stands for maximum production)

Table 7:ffect of pH
pH	Fusion frequency
6.0	**13 X 10⁷±4X10³
●6.5	*17X10⁹±3X10⁴
7.0^©	11X10⁸±1X10³
7.5	**9X10⁶±1X10³
Values were expressed as Mean±SEM ,where n=6,p<0.05 and *p<0.01 when compared to control(©).● stands for maximum production)
Table 8 : Effect of Sodium succinate
Sodium succinate(M)		Fusion frequency
0.10		**7X10⁶±2X10³
0.15		*11X10⁸±1X10³
0.20		*17X10⁸±1X10³
●0.25^©		17X10⁹±4X10²
0.30		*11X10⁸±7X10³
0.35		**16X10⁴±1X10²
Values were expressed as Mean±SEM ,where n=6,p<0.05 and *p<0.01 when compared to control(©).● stands for maximum production)
Table 9: Effect of Sucrose
Sucrose(M)		Fusion frequency
0.10		*17X10⁸±4X10³
0.15		13X10⁹±7X10⁴
●0.20		*27X10¹⁰±6X10⁴
0.25^©		17X10⁹±2X10²
0.30		17X10⁹±4X10³
0.35		*11X10⁸±2X10³
Values were expressed as Mean±SEM ,where n=6,p<0.05 and *p<0.01 when compared to control(©).● stands for maximum production)

Table 10: Effect of MgSO₄.7H₂O
MgSO₄.7H₂O(M)	Fusion frequency
0.005	11X10⁹±3X10³
●0.01^©	27X10¹⁰±1X10³
0.02	12X10¹⁰±1X10³
0.03	*17X10⁹±1X10³
0.04	**11X10⁷±3X10³
Values were expressed as Mean±SEM ,where n=6,p<0.05 and *p<0.01 when compared to control(©).● stands for maximum production)

Table 11: Effect of EDTA
EDTA(mM)	Fusion frequency
0.1	**11x10⁴±6X10²
0.2	**19X10⁶±4X10²
0.3	**16X10⁸±1X10²
0.4	**11X10⁹±4X10²
●0.5	*12X10¹¹±6X10³
0.7^©	27X10¹⁰±2X10³
0.8	**11X10⁸±3X10³
Values were expressed as Mean±SEM ,where n=6,p<0.05 and *p<0.01 when compared to control(©).● stands for maximum production)

Table 12: Effect of K₂HPO₄
K₂HPO₄	Fusion frequency
0.05	**21x10⁸±2X10²
0.10	**17X10⁹±6X10³
0.20^©	12X10¹¹±3X10³
0.30	*12X10¹²±3X10³
0.40	*32X10¹²±4X10²
0.50	21X10¹¹±6X10³
0.60	**19X10⁹±1X10³
Values were expressed as Mean±SEM ,where n=6,p<0.05 and *p<0.01 when compared to control(©).● stands for maximum production)

In this purpose, high L-methionine yielding strain (Corynebacterium glutamicum X168) and multiple L- methionine analogue-resistant strain Corynebacterium glutamicum X124 was subjected to protoplast fusion to develop a multi-analogue resistant , high L-methionine yielding strain Corynebacterium glutamicum (Fig2) .

Fig1: Phse contrast microscopic representation of protoplasting between Corynebacterium glutamicum X168 and X 124.

Corynebacterium glutamicum X300 was derived in this process which accumulated significantly (**p<0.01) higher amount of L-methionine (9.6 mg/ml) in the fermentation broth than Corynebacterium glutamicum X1(which accumulated only 0.6 mg/ml of L-methionine. This strain was recommended for further studies.

DISCUSSION:

Microbial fermentation and its success depend on the potential of the microbial strain used. So far as the reviews were concerned, no wild type microorganism has been isolated till now which accumulated large amount of L-methionine in the fermentation broth. Thus, a genetically altered mutant must be produced to improve its fermentative accumulation [6]. Development of high L-amino acid yielding mutants using mutagenesis has been studied extensively [6].Different amino acid analogues can be used which can acts as feed-back inhibitors without altering other cellular functions of the microorganisms [13-15]. L-methionine analogue-resistant mutants possess altered and deregulated enzyme systems which does not show feed-back inhibition and repression and thus able to accumulate L-methionine in the fermentation broth in absence of analogues [15].Thus microbial production of L-methionine was first carried out in Japan in 1970s using Corynebacterium glutamicum [16].Japanese Scientists introduced several screening methods to develop new strains of Corynebacterium glutamicum. Among the L-methionine analogues, ethionine is most commonly used [7]. In our present investigation, Corynebacterium glutamicum X300 was derived via random mutagenesis. In this process, not only L-methionine production pathway, but several other biochemical pathways appear. So, Corynebacterium glutamicum X300 was developed in order to produce L-methionine in a larger quantity,but the development of other mutants capable of producing other L-amino acids could not avoided in this process as claimed by Kumar et al .(2003) [17].One of the major advantages of this process is that the unrequested mutations which led to accumulate other biochemical metabolites than the desired one ,can be avoided .In Gram +ve bacteria , protoplast fusion can be done more efficiently, may obtain up to 80% transformants. In this present study, an efficient result was obtained.

CONCLUSION:

Industrial production of L-methionine by fermentation in large quantity can be achieved by using the newly developed mutant Corynebacterium glutamicum X300 which accumulated comparatively larger quantity (9.6 mg/ml)of L-methionine in the fermentation broth than the wild strain Corynebacterium glutamicum X1 which accumulated only 0.6 mg/ml L-methionine in the broth.However, to further improve the production by this mutant , optimization of different Physico-Chemical parameters is require which is under process.

ACKNOWLEDGEMENT:

We express our cordial gratitude to the Department of Chemical Engineering, University of Calcutta for their kind co-operation without we could not able to finish the work.

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