Lipid composition and amino acid sequence in microbial taxonomy

2. LIPID
COMPOSITION
NTRODUCTION
Insoluble in water and soluble in alcohol, ether, chloroform
and easily stored in body
Fatty acids, neutral fats, waxes, steroids
Compound lipids are lipoproteins, glycolipids, phospholipids
Serve as source of fuel
 Important constituent of structure of cells
Commonly found in bacteria are amphiphatic molecules
Chemical composition of bacterial components affected by
external factors
KKR1116 2

3. EXAMPLES :
KKR1116 3

4. FAME ANALYSIS
Fatty acid methyl ester
Characterizing fats and oils and for determining the total
fat content in food
FAME PRODUCTION
Produced by transesterification
They form mixture of fatty acids
KKR1116 4

5. OBJECTIVE
Obtained from vegetable oils, animal fats, waste cooking
oils
Characterizing new species of bacteria and identifying
pathogenic strains
Quality and quantity of fatty acid
KKR1116 5

6. PROCEDURE
 Fatty acids are extracted
 Saponification and methylation
 Extraction from aqueous phase
 Injected on a gas chromatography
 Identification of bacteria by sherlock software
KKR1116 6

7. SHERLOCK FAME ANALYSIS
A) SHERLOCK INSTANT FAME (I-FAME)
 Useful for gram positive aerobes and non microbes
 Turn around time (TAT) is less than 15 min
B) SHERLOCK QUALITY FAME(Q-FAME)
 Useful for aerobic bacteria
 Turn around time is less than 25 min
KKR1116 7

8. SHERLOCK SYSTEM LIBRARY
 Capable of identifying a wide range of microorganisms
 Collecting cultures from many areas like clinical, environmental, industrial,
drinking water, waste water, and food
 1,500 bacterial species along with 200 species of yeast
KKR1116 8

9. AMINO ACID SEQUENCING
 A group of organic molecules consist of a amino group , an acidic carboxyl
group and an organic R group
 Building blocks of Polypeptides and Proteins
 Functions on their own , but commonly act as monomer
 22 amino acid invovled in Protein production
 Ex : Lysine, Glycine, Tryptophan
KKR1116 9

10. N-TERMINAL ANALYSIS
 Each Polypeptide chain has an N- Terminal residue
 Establish the number of chemically distinct Polypeptides in a protein
 Insulin has an equal amount of N- Terminal residues Gly and Phe it
indicates that has equal amount of two Non- Identical Polypeptide chains
METHODS
1) SANGERS METHOD
2) EDMAN DEGRADATION METHOD
KKR1116 10

11. SANGERS METHOD
 He used a reagent 2,4-dinitroflurobenzene (DNFB)
 The DNFB reacts with free NH2 group of N- Terminus
 Upon hydrolysis a yellow coloured dinitrophenol (DNP) derivative of
N- Terminal amino acid is produced
 The DNP amino acid is identified by gel chromatography
KKR1116 11

12. EDMAN DEGRADATION
 In 1950, Pehr Edman developed a method of protein sequencing
 Phenyl isothyocyanate (PTC) reagent is used
 PTC reacts with uncharged N-Terminal group of the peptide to form a PTC
derivative
 In presence of anhydrous trifluroacetic acid PTC derivative form to Thiozolinone
derivative
 Under mild acidic in nature the cyclic Phenyl thiohydranation of the terminal amino
acid is liberated
 The released PTH amino acid is identified by HPLC
 50 residues from the N- Termines of a protein can be sequenced
KKR1116 12

13. MECHANISM :
KKR1116 13

14. REFERENCES :
 WWW.Sciencedirect.com
 WWW.Researchgate.net
 WWW.aklectures.com
 WWW.m.restek.com
 WWW.Slideshare.net
KKR1116 14