SYNTHESIS OF COMMERCIAL PRODUCT: ASCORBIC ACID AND NOVEL ANTIBODIES

ASCORBIC ACID (VITAMIN C)

• Sources

• Functions

• Deficiency Causes

• Symptoms

• Medication

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.

CO-FERMENTATION OF SUITABLE ORGANISMS

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

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.

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.

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.

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.

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.

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

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.