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Title of Thesis

Abdul Haleem Shah
Institute/University/Department Details
Department of Biological Sciences/ Quaid-i-Azam University, Islamabad
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
lysine, corynebacterium glutamicum, thialysine, arginine, l-iysine, mrlh, serine-alanine, methionine, threonine, alanine, methionine-threonine-alanine, methionine-threonine, threonine auxotrophs, homoserine-alanine, leucine auxotrophs

Lysine is an essential, economically important amino acid used as food and feed supplement. It is produced by chemical, enzymatic and fermentation processes. In this study, a glutamate producing strain of Corynebacterium glutamicum MRLH was isolated and investigated for the production of lysine. The wild strain of Corynebacterium glutamicum cannot accumulate lysine due to the concerted feed back inhibition by lysine and threonine. Thialysine and arginine (lysine analogues) act as false feed back inhibitors. The effect of different concentration of thialysine and arginine, alone and in combination with threonine, on growth was studied. Thialysine and threonine in 1: 1 ratio (2 mg / ml) completely inhibited the growth of the isolated strain. The wild strains were then treated for 30 sec. with DV radiation. As a result, a total of 50 thialysine resistant colonies were isolated. The most potent mutant of thialysine resistance (MRLH-TR1) produced 6.8 g/L L-Iysine in the fermentation broth. Exponentially growing cells of MRLH-TRl were further exposed to DV radiation followed by the addition of penicillin, penicillinase and amino acids of the desired auxotrophs. When glutamate, homo serine and alanine were added for enrichment then 14 triple auxotrophs of glutamate-homo serine-alanine (MRLH-GHA), 18 double auxotrophs of glutamate homo serine (MRLH-GH), 70 double auxotrophs of homo serine-alanine (MRLH-HA), 30 auxotroph of homo serine and 7 auxotroph of alanine were isolated and investigated for maximum lysine production.

On addition of methionine, threonine and alanine for enrichment, 29 triple auxotrophs of methionine-threonine-alanine (MRLH-MT A), 38 auxotrophs of methionine-threonine (MRLH-MT), 42 threonine auxotrophs (MRLH- T) and 13 double auxotrophs of homoserine-alanine were isolated and studied for maximum lysine production. Finally, leucine was added for the enrichment of leucine auxotroph. As a result, 31 leucine auxotrophs (MRLH-L) were isolated and screened for lysine. The most potent mutants namely, MRLH-GHAIO, MRLH-GH13, MRLH-HA5, MRLH-HI6, MRLH-MTA2, MRLH-MT8, MRLH-T3, MRLH-L and MRLH-A5 produced 12.9, 11.8, 11.32, 10.5, 10.38, 10, 8.85, 8.9 and 7.8 g per liter L-Lysine, respectively in the fermentation broth.

Triple auxotrophic mutant of glutamate-homo serine-alanine (MRLH-GHA10) were found to be the most potent for lysine production. Optimization of the culture condition for maximum production of lysine was carried for MRLH-GHA10. The time course of all the mutants were determined in shake flask and stirred tank fermentation using glucose (FMl) medium, molasses as total sugar (FM2) medium and starch hydrolyzate medium (FM3). Maximum production of lysine was observed in FMl medium for all the mutants. High yield was observed on the 6th day in shake flask and on the 4th day in stirred tank fermentation.

Profile of total yield, yield based on total sugar, percentage conversion of consumed sugar to lysine and yield based on per gram dry cell weight of each mutant was recorded. In shake flask, MRLH-GHA10 produced 28.64 g/L L-lyslne with 33.3% conversion, 29.2% yield based on total sugar and 3.36 g lysine per gram dry cell weight in FM 1 medium and 26.35 g/L L-Iysine with 30.42% conversion, 27% yield based on total sugar and 2.77 g L lysine per gram dry cell weight in FM2 medium and even lower yield was detected in FM3 medium. In stirred tank, 38.4 g/L L-Lysine with 43.8% conversion, 39.3% yield based on total sugar and 2.4 g L-Iysine per gram dry cell weight in FM 1 medium and 32.8 g/L L-lysine with 39.1% conversion, 33.6% yield based on total sugar and 2.1 g L-Iysine per gram dry cell weight and even lower yield was detected in FM3 medium.

The other auxotrophic mutants MRLH-Ll, T3, MT8, MTA2, H16, HA5 and GH13 produced 18.8-27.53 g/L L-Iysine with 24 - 31.7% conversion, 15.5 - 24.17 g/L L-lysine with 17.8 - 28.3% conversion in FMl and FM2 medium, respectively in shake flask. In stirred tank these mutants produced, 27.5 - 33 g/L L-Iysine with 32.22 - 36.8% conversion in FMl medium and 25 - 30.8 g/L L-Iysine with 30.4 - 37% conversion in FM2 medium. The yield based on per gram dry cell weight was 2-3.2 gig in FMl, 1.74-2.6 gig in FM2 medium in shake flask and 1.5-1.98 gig in FMl and FM2 medium, in stirred tank fermentor. The yield based on total sugar increased as the amount of lysine increased. The parent strain of thialysine resistant mutant (regulatory mutant) MRLH-TRl produced 14 g/L L-Iysine with 18% conversion, 12.8 g/L L-Iysine with 16.3% conversion in FMl and FM2 medium, respectively in shake flask. In stirred tank fermentor this mutant produced 22 g/L L-Iysine with 26% conversion in FMl while 20.2 g/L lysine with 23.8% conversion was found in FM2. The yield per gram dry cell weight was 1.26 - 1.55 g/g.

All these observations showed that mutants which were regulatory, as well as auxotrophic, especially those that were glutamate negative, gave a higher yield of L-lysine than the parent strain, which is only a regulatory mutant. The highest yield of L-lysine were detected in the stirred tank fermentor. In all cases the lowest L-lysine yield was obtained in FM3 medium, both in shake flask and stirred tank fermentor. Lysine was extracted by cation exchange chromatography, after purification 78.2% recovery was obtained.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents 0
205.41 KB
2 1 Introduction 4
118.67 KB
3 2 Review Of Literature 12
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  2.1 Discovery And Isolation Of Lysine From Natural Source 13
  2.2 Methods Of Synthesis 14
  2.3 Chemical Synthesis 14
  2.4 Enzymatic Method Of Synthesis 17
  2.5 Microbial Synthesis 18
  2.6 Protoplast Fusion Technique 50
  2.7 Recombinant Dna Technology 51
4 3 Mateirau And Methods 52
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  3.1 Isolation Of Bacterial Strain 52
  3.2 Taxonomic Identification Of The Selected Strains 52
  3.3 Media Composition 54
  3.4 Culture Method 58
  3.5 Mutation Treatment 59
  3.6 Clarification Of Molasses 68
  3.7 Analytical Methods 68
  3.8 Determination Of Dry Cellweight 69
  3.9 Extraction And Purification Of Lysine 70
  3.10 Calculation 70
5 4 Results 71
217.76 KB
  4.1 Isolation And Characterization Of Bacterial Strain 71
  4.2 Effect Of UV Dosage On Percentage Inactivation 71
  4.3 Growth Inhibition By Thialysine And Arginine 74
  4.4 Development Isolation And Screening Of Thialysine Resistant Mutant. 81
  4.5 Development Of Different Auxotropic Mutants. 81
  4.6 Screening Of Different Auxotrophic Mutants For Lysine Production 88
  4.7 Optimization Of Culture Condition For L-Lysine Production 92
  4.8 Profiles Of Shake Flask Fermention 128
  4.9 Profiles Of Stirred Tank Fermention 142
  4.10 Profiles Of Mutants For L-Lysine Production, Yield Base On Total Sugar ,Coversion Of Consumed Sugar To Lysine And Yeild Based On Per Gram Dry Cell Weight 143
  4.11 Extraction And Purification Of L-Lysine 146
6 5 Discussion 164
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7 6 References 183
27.06 KB
8 7 Appendix 201
128.02 KB
  7.1 Publications 214