L-Iysine, one of the well known essential amino acid is in great demand as medicament and as an additive to animal feed or human food-stuff. The importance of L-lysine as an essential amino acid in the nutrition of human beings has made it a desirable supplement of the diet in recent years. This is more prominent in underdeveloped and over-populated areas or the world, where the chief staples have been found deficient of this amino acid. For these reasons, efforts are now being focused on the potential of L-lysine derived bacteria anti its application. In the present studies, the production of L-lysine was achieved through fermentation of an auxotrophic mutant developed from a locally isolated bacterial strain of Corynebacterium glutamicum.
For the isolation of Corynebacterium glutamicum sewage and soil samples (thirty each) were collected from different parts of Karachi city. In total, twenty three hundred (2300) bacterial strains, twelve hundred (1200) from sewage and eleven hundred (1100) from soil samples were isolated and screened through “Bio-autographic Technique”. Out of these, five (5) strains were identified by taxonomic comparison as Corynebacterium glutamicum The characteristic morphological, cultural, bio-chemical, physiological and chemical properties of the strains were taken into consideration for identification. For the comparative study, Corynebacterium glutamicum ATCC-I3032 was used as a reference strain. The strains were established according to the system as described in Bergey’s manual of Systematic Bateriology (1986).
The five (5) strains identified as Corynebacterium glutamicum were designated as Fermentation Research Laboratory (FRL) Nos. 44,54,263,625 and 781. Initially these strains produced significant quantities of L-glutamic acid but no L-lysine. However, auxotrophic mutants of these strains did accumulated high quantities of L-Iysine. One of the strain, FRL N0.44, yielded 21.50 g/l L-glutamic acid, was later utilized to develop auxotrophic mutants by "Penicillin Selection Technique" followed by UV irradiation so as to produce as well as to improve the yield of L-Iysine. The growth factor(s) requirements of the auxotrophic mutants was made “Auxanographically” using different amino acids and vitamins.
Sixteen hundred (1600) auxotrophic mutants isolated form Corynebacterium glutamicum FRL No.44 were tested for their L-Iysine producing ability. Each strain was cultivated in a medium containing 70 g/l glucose, 10 g/1 peptone, 5 g/l yeast extract supplemented with inorganic slats and growth factor (biotin). Fermentation was carried out in Erlenmeyer flasks with shaking at 200 rpm. for 69 hours at 30°C. Quantitative estimation of L-Iysine in the fermented broth was measured by “Turbidimetric Assay” using Pediococcus acidilactici ATCC - 8042 as test organism. Sixty six (66) auxotrophic mutants were found to produce L-Iysine. These include 34 homoserine, 10 methionine, 3 threonine, 12 methionine plus threonine, 2 leucine and 5 isoleucine auxotrophs, Eleven (II) homoserine auxtrophic mutants, out of 34 were identified as requiring vitamin B, as growth factor.
Based on highest L-Iysine producing ability, a homoserine auxotrophic mutant strain of Corynebacterium glutamicum FRL No. 2753, requiring vitamin B1 as growth factor was utilized for further studies. The strain FRL No.2753, yielding 26.40 g/l L-lysine was consequently selected for strain improvement by developing mutants resistant to S-(2 amino ethyl)-L-cysteine (AEC)-an L-Iysine analogue.
The AEC-resistant mutants were developed by treating the culture, FRL No.2753 with 2 mg/ml N methyl-N-nitrosoguanidine (MNNG) in 0.1 M phosphate buffer for 30 minutes at zero degree centigrade. The cells, after washing with sterile saline solution were inoculated into a medium supplemented with 2.0 mg/I AEC, 2.0 mg/l L-threonine and 50 μg/l DL-methionine, Colonies that appeared on the surface of agar plate during 2 to 7 days of incubation were picked as AEC-resistant strains. Out of eighty five(85) AEC-resistant strains isolated and tested for L-Iysine production, only five (5) strains gave a significant yield of L-Iysine higher than the auxotrophic mutant strain No. 2753. The highest yield of L-Iysine was achieved by the AEC-resistant strain FRL No. 3960, and thus was ultimately selected for the process optimization studies through modification in media composition to maximize the yield of L-Iysine.
During process optimization studies, effect of glucose plus ammonium sulphate (carbon and nitrogen source respectively), biotin, vitamin B, and aeration on L-Iysine production were investigated. A maximum yield of L-Iysine, 68.65 g/l was achieved with a medium containing glucose 110 g/l, ammonium sulphate 15 g/l, biotin 125 μg/l and Vitamin B1 10.mg/1 supplemented with inorganic salts. The process optimization studies were carried out in 250 ml Erlenmeyer flasks containing 30 ml of the above mentioned medium at 30°C for 96 hours. An increase in L-Iysine upto 74.75 g/1 was reached when fermentation was done in 500 ml Erlenmeyer flasks carrying 100 ml of the same medium for 96 hours at 30oC.
In order to further scale-up and to explore the possibility of using the strain for commercial production of L-Iysine, the strain FRL No.3960 of Corynebacterium glutamicum was cultivated in 2.0 I fermenters using glucose and cane molasses media. The composition of glucose medium was kept same as that used in process optimization studies. However, keeping in view of the high cost of organic nutrients, such as yeast extract peptone and meat extract, these were replaced in cane molasses medium by low cost and locally available raw materials such as corn steep liquor, soyabean protein acid hydrolysate and fish meal respectively.
Fermentation using both media was carried out under submerged condition with an agitation rate of 400 rpm.and air now of 1.0 III of medium I minute at 30°e. After 96 hours, the fermentation was terminated and the broths were subjected to separation and purification of L-lysine by ion exchange technique. The strain FRL No.3960 furnished 67.80 g/l and 65.20 (average of three experiments) crystals of L-Iysine monohydrochloride from glucose and cane molasses respectively. The percentage yield, Yp/s for glucose medium was recorder as 0.616 gg-1 (61.6% conversion efficiency), while for cane molasses medium it was 0.592 g g-1(59.2% conversion efficiency). The purity of L-Iysine monohydrochloride was calculated as 99.4% for glucose medium and 98.8% for cane molasses medium. The purity of the product was determined by the methods as given in the U.S. P XXII; the N.F.XVII (1990) and The Japan Pharmacopoeia (1986).
The quantities of L-Iysine produced by the strain FRL No. 3960 utilizing both media, as well as the purity of the products were quite comparable. Thus the locally available cane molasses justified its use as carbon source while corn steep liquor, soyabean protein acid hydrolysate and fish meal as organic nutrients for L-Iysine fermentation. In addition the locally isolated strain of Corynebacterium glutamicum FRL No. 3960 (a homoserine auxotrophic mutant and resistant to AEC), displayed the potential to he used for the commercial production of L-Iysine.