Selection of Suitable Maintenance Medium and Determination of Auxotrophic Nature of the Multiple Analogue Resistant Mutant Micrococcus glutamicus X300 for L-methionine Fermentation

 

Subhadeep Ganguly, Kunja Bihari Satapathy*

Post-Graduate Department of Botany, Utkal University, Vani Vihar, Bhubaneswar-751004, Odisha

*Corresponding Author E-mail: res_biol@rediffmail.com

 

 

ABSTRACT:

The present study was conducted to select a suitable maintenance medium for the maintenance of the multiple analogue resistant mutant and also to determination of its auxotrophic requirements. Among the three medium studied, the strain maintained its constant rate of production of L-methionine for many generations(studied up to 12 months) with  glucose, 20 gm/L; (NH4)2SO4; 1.6 gm /L; NaCl, 2.5gm/L; MgSO4.7H2O,0.25 gm /L; MnSO4.4H2O, 0.1 gm/L; K2HPO4, 1.0 gm/L; KH2PO4,1.0 gm/L; biotin, 2µg/ml; thiamine-HCl, 2µg/ml and agar 2% as a solidifying agent which can prevent its degeneration. This strain thus showed double auxotrophic requirements for both biotin and thiamine-HCl.

     

KEYWORDS: Degeneration, strain, auxotrophic, biotin, thiamine-HCl.

 


 

INTRODUCTION:

Though the L-methionine is an essential amino acid in human nutrition, but human beings are incapable of producing it in their body1. Vegetable diets are also deficient in L-methionine2. Considering the serious matter, several attempts have been made to commercialize the production of L-methionine. Among this methods, chemical synthesis and enzymatic production are very common3,4. Chemical synthesis produce a DL- mixture of the amino acid from which separation of L-methionine from the medium is very difficult5. But when Kinoshita et al.(1957) isolated Micrococcus glutamicus (later renamed Corynebacterium glutamicum), a L-glutamic acid producer, a new mild stone has been set in the field of industrial microbiology6. Since then several attempts have been made to isolate different microbial strains, which is cheaper as well as easier process. Several mutational treatments have been used to develop high yielding strains7,8. But one of the draw-backs of this mutant is the tendency to degenerate7. Thus, to prevent such degeneration, selection of suitable maintenance medium for maintaining the culture is one of the most essential aspects while working with such mutant strain.

 

In the present study we were intended to select a suitable maintenance medium for the maintenance of the multiple analogue-resistant mutant Corynebacterium glutamicum X300.

 

METHODS AND MATERIALS:

Microorganism: A multiple analogue resistant mutant Corynebacterium glutamicum X300 developed in our laboratory from Corynebacterium glutamicumX1 was used throughout the study.

 

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, 60μg9,10.

 

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 method9. All the chemicals used in this study were of analytical grade (AR) grade and obtained from E mark. Borosil glass goods and triple distilled water used throughout the study.


 

Table 1: Selection of maintenance medium for the mutant  Corynebacterium glutamicum X300

Medium

Month(s)

 

1

1

2

3

4

5

6

7

8

9

10

11

12

9.6©

±0.683

*9.0

±0.668

**8.3

±0.813

**6.4

±0.613

**4.2

±0.719

**2.8

±0.832

**2.1

±0.661

**0.3

±0.661

-

-

-

-

2

9.6©

±0.791

9.6

±0.613

9.3

±0.681

*9.0±0.662

**8.4

±0.811

**8.1

±0.641

**7.6

±0.871

**7.1

±0.832

**6.6

±0.571

**6.1

±0.613

**5.4

±0.642

**5.0

±0.661

3

9.6©

±0.661

9.6

±0.661

9.6

±0.668

9.6±0.663

9.6

±0.681

9.6±0.668

9.6

±0.683

9.6

±0.731

9.6

±0.882

9.6

±0.661

9.6

±0.771

9.6

±0.681

(Values were expressed as mean ± SEM , where n=6, p<0.05 ,p<0.01 when compared to control. © stands for control.)

 


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 obtained11.

 

Composition of the synthetic media: The following media were examined to select the suitable one for maintenance of the strain and determination of the auxotrophic nature of the strain:

Medium 1: glucose, 20 gm/L; (NH4)2SO4; 1.6 gm /L; NaCl, 2.5gm/L ; MgSO4.7H2O,0.25 gm /L; MnSO4.4H2O, 0.1 gm/L; K2HPO4, 1.0 gm/L; KH2PO4,1.0 gm/L and agar 2% as a solidifying agent.

 

Medium 2: glucose, 20 gm/L; (NH4)2SO4; 1.6 gm /L; NaCl, 2.5gm/L ; MgSO4.7H2O,0.25 gm /L; MnSO4.4H2O, 0.1 gm/L; K2HPO4, 1.0 gm/L; KH2PO4,1.0 gm/L; thiamine-HCl, 2µg/ml and agar 2% as a solidifying agent.

 

Medium 3: glucose, 20 gm/L; (NH4)2SO4; 1.6 gm /L; NaCl, 2.5gm/L ; MgSO4.7H2O,0.25 gm /L; MnSO4.4H2O, 0.1 gm/L; K2HPO4, 1.0 gm/L; KH2PO4,1.0 gm/L; biotin , 2µg/ml; thiamine-HCl, 2µg/ml and agar 2% as a solidifying agent.

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:

The production of L-methionine by the strain on subsequent fermentation trials has been depicted in the table 1.

 

Among the three mentioned medium studied in table 1, Production of L-methionine can be constantly maintained by using the strain maintained in the medium 3.The strain showed double auxotrophic nature to both biotin and thiamine-HCl.

 

DISCUSSION:

Strain degeneration is a great problem for a mutant strain. Strain degeneration in microorganism is characterized by loss of secondary metabolites production capacities. Strain degeneration can be prevented by selection of suitable maintenance medium and providing specific nutrients to which this particular strain shows its auxotrophic nature12.

 

CONCLUSION:

From this study, it can be tentatively concluded that, the newly developed mutant is a double auxotroph to both biotin and thiamine-HCl. The constant rate of L-methionine production can be maintained by using the maintenance medium composed of glucose, 20 gm/L; (NH4)2SO4; 1.6 gm /L; NaCl, 2.5gm/L; MgSO4.7H2O, 0.25 gm /L; MnSO4.4H2O, 0.1 gm/L; K2HPO4, 1.0 gm/L; KH2PO4,1.0 gm/L; biotin, 2µg/ml; thiamine-HCl, 2µg/ml and agar 2% as a solidifying agent which can prevent its degeneration.

 

ACKNOWLEDGEMENT:

We express our sincere gratitude 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.

 

REFERENCES:

1.       Kumar D and Gomes J, Methionine production by germentation, Biotechnol. Advances, 23, 2005, 41-61.

2.       Rose WC, The nutritive significance of amino acids, Physiol Rev., 18, 1938, 109-136.

3.       Mannsfeld SP, Pfeiffer A, Tanner H and Liebertanz E, Continuous process for manufacture of methionine, Us Patent 04069251 (1978).

4.       Leuchtenberger W, Amino acids-technical production and uses. In: Rehm HJ, Reed G, Phuler A and Stadler P(eds.), Products of primary metabolism. Biotechnology, Vol.6, Weinheim : VCH; 1996, p.492.

5.       Fong CV, Goldgraben GR, Konz J, Walker P and Zank NS, Condensation process for DL-methionine production.In: Goldfrab AS(ed.), Organic chemicals manufacturing hazards. Ann Arbor: Ann Arbor publisher; 1981, p.115-194.

6.       Kinoshita S, Udaka S, Shimono M, Amino acid fermentation :1. Production of L-glutamic acid by various microorganisms, J.Gen.Appl.Microbiol., 3, 1957, 193-205.

7.       Banik AK and Majumder SK, Studies on methionine fermentation: Part I. Selection of mutants of Micrococcus glutamicus and optimum conditions for methionine production, Indian J. Exp. Biol, 12, 1974, 363-365.

8.       S. Ganguly and A.K Banik, Induced mutation and selection of high yielding strain of Micrococcus glutamicus for gluconic acid production, J. Indian Chem. Soc., 87, 2010, 717-721.

9.       Iwata M, Made M and Ishiwa H, Protoplast fusion of Lactobacillus fermentum. Appl. Environ. Microbiol., 52, 1986, 392-393.

10.     Kase H and Nakayama K, L-Methionine production by Methionine Analogue resistant mutants of Corynebacterium glutamicum. Agric. Biochem., 39, 1975, 153-160.

11.     Roy SK, Mishra AK and Nanda G, Extracellular production of L-methionine. Currt Sci., 52, 1984, 1296-1297.

12.     Okafor N, Umesh C, Ibenegbu C, Obizoba I and Nnam M, Improvement of garri quality by the inoculation of microorganisms into cassava mash. Int. J. Food Microbiol., 40, 1998, 43-49.

 

 

 

 

Received on 25.09.2013       Modified on 10.11.2013

Accepted on 14.11.2013      © RJPT All right reserved

Research J. Pharm. and Tech. 6(12): Dec. 2013; Page 1319-1320