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acid (vitamin C)
The biotransformation of sorbitol to sorbose in the manufacture of ascorbic acid (vitamin C).
H2 H H2
C C C
OH OH OH Polyol dehydrogenase
OH O OH
The biotransformation of glycerol to dihydroxyacetone.
Most vitamins were previously prepared from animal and plant tissues, although dried baker’s and brewer’s yeast preparations (both are strains of Saccharomyces cerevisiae) have long been employed as a rich source of B vitamins. Microorganisms are now used as sources of a wide range of these important compounds, including thiamin (vitamin B1), riboﬂavin (vitamin B2), pyridoxine (vitamin B6), cobalamin (vitamin B12), biotin, folic acid, l-ascorbic acid (vitamin C), b-carotene (provitamin A), ergosterol (provitamin D2) and pantothenic acid. For the production of some vitamins, direct fermentation processes are operated, whereas for others, biotransformations or combined chemical and microbiological processes are employed.
Ascorbic acid (vitamin C)
Annual production of ascorbic acid is now over 40 000 tonnes. The established process involves chemical stages and a microbial biotransformation. It starts with the chemical catalytic reduction of d-glucose to d-sorbitol,
a polyhydric alcohol. d-Sorbitol is then oxidized to lsorbose using Acetobacter suboxydans or Acetobacter xylinum (Fig. 13.3). Media for this biotransformation step consist of glucose, yeast extract or corn steep liquor, a slight excess of calcium carbonate and 15–30% (w/v) d-sorbitol. The biotransformation is performed at 30°C under vigorous aeration and within 1–2 days a 90–95% conversion is achieved (note: a similar biotransformation is used for producing dihydroxyacetone, a component of suntanning cosmetics, from glycerol using A. suboxydans or Gluconobacter melanogenus, Fig. 13.4). l-Sorbose is recovered from the medium following ﬁltration and concentration of the ﬁltrate to a syrup. This sugar crystallizes on cooling and about 65% is recovered, based on starting material. The l-sorbose is then chemically converted to ascorbic acid via 2-keto-lgluconic acid. A much more direct route from glucose to ascorbic acid has now been made possible by the introduction of a gene encoding 2,5-diketo-d-gluconic acid reductase from Corynebacterium into Erwinia. This simpliﬁes the process to a single biotransformation and a single chemical step. The genetically modiﬁed Erwinia strain transforms glucose to 2-keto-l-gulonic acid (Fig. 13.5), which can then be simply converted to ascorbic acid in one chemical step.
The reaction steps are:
• • • • • •
hydrogenation of D-glucose to D-sorbitol, an organic reaction with nickel as a catalyst under high temperature and high pressure. Microbial oxidation or fermentation of sorbitol to L-sorbose with acetobacter  at pH 4-6 and 30 °C. protection of the 4 hydroxyl groups in sorbose by formation of the acetal with acetone and an acid to Diacetone-L-sorbose (2,3:4,6−Diisopropyliden−α−L−sorbose) Organic oxidation with potassium permanganate followed by heating with water gives the 2-Keto-L-gulonic acid The final step is a ring-closing step or gamma lactonization with removal of water . Intermediate 5 can also be prepared directly from 3 with oxygen and platinum
The microbial oxidation of sorbitol to sorbose is important because it provides the correct stereochemistry.
'Corynebacterium' 2,5-diketo-D-gluconic acid reductase 2-keto-L-gulonic acid
Production of 2-keto-l-gulonic acid by genetically modiﬁed strain of Erwinia that expresses 2,5-diketo-d-gluconic acid reductase from Corynebacterium.