Methods for production of commercial products such as ascorbic acid and novel antibodies

1. SYNTHESIS OF COMMERCIAL PRODUCT:
ASCORBIC ACID AND NOVEL
ANTIBODIES

2. ASCORBIC ACID (VITAMIN C)
• Sources
• Functions
• Deficiency Causes
• Symptoms
• Medication

3. For example, some
bacteria (Acetobacter,
Gluconobacter, and
Erwinia) can convert
glucose to 2,5-diketo-d-
gluconic acid (2,5-
DKG), and others
(Corynebacterium,
Brevibacterium, and
Arthrobacter) have the
enzyme 2,5-DKG
reductase, which
converts 2,5-DKG to 2-
KLG.

4. CO-FERMENTATION OF SUITABLE ORGANISMS

5. Cloning of 2,5-Di Keto Gluconic reductase gene from
Conynebacterium sp.
1. Purification
2. Determination of sequence
3. Synthesis of DNA hybridisation probes
4. Screening
5. Expression of the construct
6. Transformation of microorganisms

6.  The transformed Erwinia cells
were able to convert D-glucose
directly to 2-Keto-L-Gulonic
acid.
 The endogenous Erwinia
enzymes, localized in the inner
membrane of the bacterium,
converted glucose to 2,5-DKG,
and the cloned 2,5-DKG
reductase, localized in the
cytoplasm, catalysed the
conversion of 2,5-DKG to 2-
KLG.

7.  From the primary amino acid sequence, computer modelling predicted an
enzyme structure with an eight-stranded α/β barrel.
Predicted structure of 2,5-DKG Reductase enzyme.

8.  Comparison with the other known enzyme structure: Observed in 17 known
crystal enzymes structures.
Development in the clone of 2,5-Di Keto Gluconic reductase
gene from Conynebacterium sp.

9.  Oligonucleotide-directed mutagenesis:12 Mutants
• 11 mutants showed lower specific
activity for the production of the
enzyme
• 12th mutant showed twice the
specific activity.
• Kinetics of the reaction:1.8 fold
increase in Vmax and 25%
decrease in the Michaelis
constant in the enzyme catalysed
reaction.

10.  Modifications in the Cofactors used for the reaction
• NADH is financially
beneficial then
NADPH for the
production of
ascorbic acid.
• The difference is the
presence or absence
of phosphate at the
2’site.
• From the 3D structure
it is observed that 5
amino acids are in
contact with the
2’phosphate site.

11.  Cassette Mutagenesis: 40
different mutants were
synthesized. Final product, a
double mutant gave a 72 times
higher product then the wild
type.

12. REFERENCE
1. Molecular Biotechnology, Principles and applications of Recombinant DNA, Glick
[4th Edition] (507-513)
2. Principles of Gene Manipulation, Primrose [6th Edition] (507-509)
3. M.J. McPherson, Directed Mutagenesis.