Pathway for Production Ascorbic Acid Synthesis

Metabolic pathway engineering for the production of L-ascorbic acid

1

Introduction

Ascorbic acid or L-Ascorbic acid, L-xylo-Ascorbic acid, L-threo-hex-2-enoic acid γ-

Lactone is IUPAC name for Vitamin C (ULLMANN’S Encyclopedia vol. 38).

All known disease called scurvy was known to human from ancient (Egyptians) age

occurred due to deficiency of Vitamin C. But this is not known up to 1753 when James Lind

described it to be cured by dietary mean (ULLMANN’S Encyclopedia vol. 38).

Later on many peoples were working on Ascorbic acid. In which Szent-Gyorgyi

(1928) first isolated Ascorbic acid form Adrenal gland and from plant tissue by Herbert et al.

(1933). Chemical synthesis of L-ascorbic acid from L-xylosone was achieved. This work also

done by Haworth and Hirst (1933). They also propose the chemical structure for Ascorbic

acid, Reichstein et al. (1933). Biochemical reaction for preparation of Ascorbic from D-

Glucose was introduced by Reichstein and Grössner (1934) and this sequence is adapted for

manufacturing of Ascorbic acid industrially (Hancock and R. Viola, 2001).

This play very important role in the human as conjunctive tissue formation, ion

transportation, and cell protection against free radicals. In plants, act against reactive oxygen

species that are formed from Photosynthesis and Respiratory processes. It also linked with

cell growth, cell cycle and cofactor for many enzymes (Anderson et al. 2004).

Wide range of reactive oxygen species, such as singlet oxygen, superoxide anion and

Hydroxyl radicals which have been implicated with many chronic disorders including cancer

and cardiovascular disease.

1Institute Of Chemical Engineering, Mumbai


2

Objective and scope

Ascorbic acid is used by human as dietary purpose to fulfill their nutrients

requirement and fight against disease called Scurvy and some fatal disease known to be

cancer and cardiovascular disease. It also act as antioxidant in human body which give free

electron to reactive oxygen formed from hydrogen peroxide.

For this purpose we synthesize ascorbic acid on natural way by means of plant which

gives direct feed to humans. This was also synthesizing by many methods using microbes

such as bacteria and algae by biosynthesis. These microbe used as it is but yield is low so for

this to overcome genetically modified strains (Acetobacter suboxidans, Bacterium xylinum,

Erwinia sp., Corynebacterium sp.) are used. Using algae we also synthesize Ascorbic acid.

The more well known and commercial adapted pathway is Reichstein process. This is

chemical oriented method have a single fermentation step. Due to its energy consumption,

and required high temperature, pressure for many steps in Ascorbic acid synthesis.

To overcome this intermediate compounds are biosynthetically synthesize to increase

the yield of the ascorbic acid with low energy consumption.



3

Pathways for production Ascorbic acid

3.1Microbiological fermentation

In microbiological fermentation different species are used to produce Ascorbic acid

these are Bacteria, and Algae. Different pathway has been proposed to describe how they

produce Ascorbic acid by initial starting materials.

3.1.1 Metabolic Pathway for BacteriaAt present there are six bacterial fermentation processes for the production of 2-keto-

L-gulonic acid, a direct precursor of L-ascorbic acid.

These are a) sorbitol pathway, b) L-idonic acid pathway, c) L-gulonic acid pathway, d) 2-

keto-D-gluconic acid pathway, e) 2, 5-diketo-D-gluconic acid pathway, and f) 2-keto-L-

gulonic acid pathway. The most commercially advanced methods are the oxidation of D-

sorbitol or L-sorbose to 2-keto-L-gulonic acid (2-KLG) (Shrikant et al. 2006) .

The pathway cycle for production of ascorbic acid is given below:

Figure 3.1 Bremus et al. gives different pathway cycle for production Ascorbic acid using Bacteria.



3.1.1.1 D-Sorbitol pathwaySorbitol is transformed by fermentation of 2-KLG (2-Keto-L-Gluconic acid) via

intermediate L-Sorbosone. This is done by many strains of bacteria namely Pseudomonas and

Acetobacter, which are catalyze the oxidation of D-Sorbitol to 2-KLG via the series of

membrane bound hydrogenase to produce L-Sorbosone. The final oxidation to 2-KLG is

catalyzed by either membrane bound or cystolic Sorbosone dehydorgenase. Using the strain

Glucano Oxydans that produce up to 60g/l. of 2-KLG from L-Sorbosone or D-Sorbitol with

60% conversion (Hancock and R. Viola, 2001).

Genetically modified strains are also used to carry out above steps. The location of

dehydrogenase required for conversion of D-Sorbitol to 2-KLG varies from strain to strain.

The transfer of D-Sorbitol pathway intermediate into cytoplasm of these strain determined by

the presence of cystolic reductase which give path to the intermediate in the pentose cycle. To

overcome this problem membrane bound dehydrogenase recombinant with Glucanobacter

Oxydans is replace this or substitute cystolic enzymes. For example Acetobacter liquifaciens

a membrane bound Sorbosone dehydrogenase is expressed in Glucanobacter Oxydans which

is membrane bound sorbitol dehydrogenase and sorbose dehydrogenase but not cystolic

dehydrogenase. A significant yield is observed from L-Sorbose (68-81%) and L-Sorbosone

(23-83%). But there is no yield improvement under fermentation condition (Shrikant et al.,

2006).

3.1.1.2 2-Keto-D-Gluconic Acid PathwayD-Glucose is converted to 2-KLG via D-Gluconic acid, 2-Keto-D-Gluconic acid and

2,5-Diketo-D-Gluconic acid (2,5-DKG). Until now there is no bacterial strains capable of

efficiently catalyzing the complete conversion of D-Glcose to 2-KLG have been isolated.

This is carried out in three main steps; each step is carried out by using different

microorganisms.

(i) Transformation of D-glucose to 2-keto-D-gluconic acid: the transformation of glucose to

2-keto-D-gluconic acid is carried out by Acetobacter melanogenus and Pseudomonas

albosesamae. Some Acetobacter strains also synthesize 2-keto-D-gluconic acid;

(ii) Oxidation of 2-keto-D-gluconic acid: this oxidation is carried out by Bacterium hoshigaki

and Bacterium gluconicum with 2, 5-DKG as a product. In addition, Acetomonas

albosesamae can directly transform D-glucose to 2, 5-DKG;

(iii) Oxidation of 2, 5-DKG acid: conversion of 2, 5-DKG into 2-KLG. The mentioned strain

is of the genera Brevibacterium and Pseudomonas, and maximum yield was obtained from

Brevibacterium ketosoreductum. The use of Corynebacterium has also been suggested.4

Institute Of Chemical Engineering, Mumbai


A mutant Erwinia strain to convert D-glucose into 2, 5-DKG. The whole culture broth

is then treated with sodium dodecyl sulphate to reduce cell viability, mixed with glucose and

then supplied to a mutant strain of Corynebacterium for conversion to 2-KLG. The whole

process achieved yields of 2-KLG of up to 85 % from D-glucose with final 2-KLG

concentrations of 10.5 g/L. bacterial strain via genetic engineering. Anderson et al. cloned a

cytosolic 2, 5-DKG reductase gene from Corynebacterium species. And expressed it in

Erwinia herbicola. The recombinant organism was capable of synthesizing 2-KLG from D-

glucose but the yields attained were very poor (1 g/L of 2-KLG from 20 g/L of D-glucose).

Recombinant E. citreus expressing cytoplasmic 2, 5-DKG reductase from Corynebacterium

species. could accumulate

19.8 g/L of 2-KLG with a 49 % conversion efficiency from glucose (Hancock and R. Viola 2001). Such improvements in yield were attained by optimization of the fermentation conditions, careful selection of the promoter controlling the expression of the 2, 5-DKG reductase gene and by the use of mutant strains of E. citreus which were unable to use 2, 5-DKG or 2-KLG for growth.

3.1.1.3 Bioconversion Of 2-KLG to L-Ascorbic Acid (Hancock and R. Viola 2001), (Shrikant et al. 2006)

2-KLG is converted to L-ascorbic acid chemically via two methods.

The first involves multiple steps including:

• Esterification of a 2-KLG derivative under strongly acidic conditions to produce methyl 2-

keto-L-gulonate (MeKLG);

• Reaction of MeKLG with a base to produce a metal ascorbate salt;

• Treatment of metal ascorbate salt with an acidulant to obtain ascorbic acid.

The second way is a one-step method comprising acid-catalyzed cyclization of KLG.

Both methods are commercially undesirable due to the requirement for multiple chemical

steps for first method, or the use of large amounts of gaseous hydrogen chloride or

requirement for very expensive process equipment for second method.

To overcome this situation hydrolase enzyme is used to convert ester of 2-KLG to L-

Ascorbic acid. In similar way lactonases is extracted from Zymomonas mobilis, Escherichia

coli and Fusarium oxysporium are capable to convert 2-KLG to L-Ascorbic acid.



3.1.2 Metabolic Pathway for Algae

The pathway for the production of ascorbic acid from D-Glucose using Algae is shown

below.

Figure 3.2 Bremus et al. gives (2006) Pathway cycle for production of Ascorbic acid using Algae.

Many attempts are employed to use microalgae for the direct production of L-

Ascorbic acid from inexpensive feedstock. One-step fermentation process for the production

of L-Ascorbic acid using the heterotrophic green microalga Chlorella pyrenoidosa produce

low quantity up to 40 mg/l L-Ascorbic acid.. After continuous chemical mutagenesis and

fermentation optimization, an improved amount of up to 2 g/l L-Ascorbic acid is obtained.

Accumulation of L-Ascorbic acid in the fermentation medium was obtained by the use of the

colorless microalgae Prototheca moriformis. Using this alga it is shown that a pH reduction

could stabilize L-Ascorbic acid in the fermentation reactor, so most of the L-Ascorbic acid

became harvestable from the medium.

For strain-improvement Prototheca used for many mutant strains with increased and

reduced abilities to accumulate L-Ascorbic acid. These mutants are used to identify the

pathway for L-Ascorbic acid biosynthesis that involves mannose-containing intermediates.

Similarly in plants, l-galactono-1, 4,-lactone is produced from d-glucose through GDP-d-

mannose, GDP-l-galactose and l-galactose. Finally l-galactono-1,4,-lactone is converted into

l-AA by l-GL-DH. This is shown by Wheeler et al. (1998) in higher plants.



3.2 Metabolic Pathway for Plants (Hancock and R. Viola 2002), (Ishikawa1 et al.,

2006)]

The pathway cycle for production of Ascorbic acid from D-Glucose is given below:

Figure 2.3 Hancock and R Viola (2002) gives Pathway cycle for production of Ascorbic acid in Plants.

It was proposed that ascorbate is synthesized in plants by oxidation of L-galactose

(Wheeler et al. 1998).

The enzymatic steps are summarized below.

In this pathway, four chemical events are minimally required for the non inverted conversion

of glucose to Ascorbic acid are present:

(1) Oxidation of the C1 of glucose;

(2) Oxidation at C2 or C3;

(3) Epimerization at C5; and

(4) Lactonization between C1 and C4.

Phosphomannose isomerase (PMI) catalyses the first step in directing hexose phosphates into

D-Mannose metabolism. PMI from Escherichia coli has been used as a selectable marker for

plant transformation, because it renders plants mannose resistant. However, A. thaliana plants

expressing E. coli PMI do not have increased ascorbate. Conversion of D-Man 6-P to D-Man

1-P is catalysed by phosphomannose mutase (PMM). GDP-D-Man synthesis from D-Man 1-

P and GTP is catalysed by GDPD- Man pyrophosphorylase (GMP). GDP-D Man is converted 7



to GDP-L-Gal by a reversible double epimerization, catalysed by GDP-D-Man-3,5-epimerase

(GME) that was first identified in Chlorella pea This enzyme has recently been purified and

cloned from A. thaliana and purified from the alga Prototheca (Running et al. 2004). As well

as being involved in ascorbate synthesis, GDP-D-Man and GDP-L-Gal are substrates for

polysaccharide synthesis and protein glycoslyation. The steps subsequent to GDP-L-Gal are

likely to be dedicated to ascorbate synthesis. GDP-L-Gal is initially broken down to L-Gal 1-

P, which is subsequently hydrolysed to L-Gal. Enzymes catalysing these steps have been

recently purified and characterized. GDP-L-Gal is converted to L-Gal 1-P and GDP by a

novel and highly specific phosphate-dependent GDP-L-Gal phosphorylase. The released L-

Gal is then oxidized in two steps, first by a cytosolic NAD-dependent L-Gal dehydrogenase

(L-GalDH) at C1 to form L-galactono-1,4-lactone (L-GalL) (Wheeler et al. 1998) and then by

L-GalL dehydrogenase (L-GalLDH) at C2/C3 resulting in the production of ascorbate.

The antisense suppression of LGalDH and L-GalLDH decreases ascorbate concentration

Although the D-Man/L-Gal pathway appears to be the predominant pathway, there is some

suggestion that other biosynthetic pathways via uronic acid intermediates contribute to the

ascorbate content of plant tissues and that these may be developmentally regulated.

The authors proposed that the hydrolysis of GDP-L-gulose would result in the production of

L-gulose which could be converted to L-gulono-1,4-lactone by L-GalDH and subsequently

into ascorbate by the L-gulono-1,4-lactone dehydrogenase activity known to exist in plants.



3.3 Metabolic Pathway for Animals

The pathway cycle for production of ascorbic acid from D-glucose is given below:

Figure 2.4 Linster and E V Schaftingen (2006) gives Pathway cycle for production of Ascorbic acid in Animals.

Ascorbic acid is synthesized by many vertebrates. The occurrence of ascorbic acid

biosynthesis in sea lamprey suggests that this feature appeared early in the evolutionary

history of fishes (590–500 million years ago), i.e. prior to terrestrial vertebrate emergence [2].

The biosynthetic capacity has, however, subsequently been lost in a number of species, such

as teleost fishes, passeriform birds, bats, guinea pigs, and primates including humans, for

whom ascorbate has thus become a vitamin. Fish, amphibians and reptiles synthesize

ascorbate in the kidney, whereas mammals produce it in the liver.

In animals, d-glucuronate, derived from UDP-glucuronate, is reduced to l-gulonate, which

leads to inversion of the numbering of the carbon chain (‘inversion of configuration’) since

the aldehyde function of d-glucuronate (C1) becomes a hydroxymethyl group in the resulting



l-gulonate. In plants, the pathway starts with GDP-d-mannose, which is converted (without

change in carbon numbering) to l-galactonolactone, the substrate for the plant homologue of

GLO, l-galactonolactone dehydrogenase.

Formation of glucuronate from UDP-glucuronate (Linster and E. V. Schaftingen

2006)

The formation of glucuronate from UDP-glucuronate could hypothetically involve

following three mechanisms to convert glucuronate from UDP-glucuronate.

(a) The cleavage of UDPglucuronate to glucuronate 1-phosphate, followed by

dephosphorylation of the latter by a glucuronate-1- phosphatase;

(b) The formation of a glucuronidated intermediate followed by its hydrolysis by b-

glucuronidase or esterases (which could hydrolyze acyl-glucuronides); or

(c) Direct hydrolysis of UDP-glucuronate to UDP and glucuronate.

Mechanism (a), It is show that a series of nonglucuronidable xenobiotics rapidly

stimulate the formation of glucuronate in isolated hepatocytes, the same xenobiotics also

stimulated the formation of glucuronate from UDP-glucuronate in liver cell-free extracts

enriched with ATP or in liver microsomes supplemented with ATP and a heat-stable cofactor

identified as coenzyme A. Quantitatively, the synthesis of glucuronate observed under these

conditions accounted for the formation of glucuronate observed in intact cells, indicating that

glucuronate is formed from UDP-glucuronate by a microsomal enzyme. These enzymes are

present in the endoplasmic reticulum as, similarly to UGTs, it is stimulated by UDP-N-

acetylglucosamine, which enhances the transport of UDP-glucuronate into vesicles derived

from the endoplasmic reticulum

Mechanism (b), it is ruled out by the fact that glucuronate formation from UDP-

glucuronate occurs in rat liver microsomes in the absence of UGT substrates and is actually

inhibited by such substrates. Furthermore, inhibitors of β-glucuronidase and esterases do not

affect the formation of glucuronate from UDP-glucuronate by microsomal preparations.

Mechanism (c). Taken together, these observations lead to the conclusion that

glucuronate is formed by direct hydrolysis of UDP-glucuronate by a UDP-glucuronidase. It is

likely that the ability of UGTs to hydrolyze UDP-glucuronate varies among isoforms and

depends on the phospholipidic environment. This last point may explain the observation that

UDPglucuronidase is inhibited in rat liver microsomes by the addition of ATP and coenzyme

A, the latter combination of cofactors could allow the reesterification of lipids in a subcellular

fraction known to contain free fatty acids, acyl-CoA synthetase and acyltransferases.



Glucuronate reductase (aldehyde reductase) (Linster and E. V. Schaftingen 2006)

The reduction of d-glucuronate to L-gulonate is catalyzed by an NADPH-dependent

reductase, with broad specificity, known as aldehyde reductase or TPN L-hexonate

dehydrogenase and now referred to as aldo-keto reductase for the human enzyme.

The compound D-glucuronate and D-glucuronolactoneare converted by aldehyde reductase to

L-gulonate and L-gulonolactone, respectively. Aldehyde reductase belongs to the large group

of monomeric NADPH-dependent oxidoreductases, known as aldo-keto reductase, which

comprise many members in the human genome, including aldose reductase and

hydroxysteroid dehydrogenases. These enzymes show broad substrate specificities and it

would therefore not be surprising that, besides aldehyde reductase, other members of the

aldo-keto reductase super family participate in the reduction of d-glucuronate. Aldose

reductase appears to be much less efficient than aldehyde reductase in this respect.

Urono- and gulonolactonase:The conversion of D-glucuronate to L-gulonolactone requires the action of two

enzymes, a reductase and a lactonase, proceeding either via D-glucuronolactone if the

lactonization is the first step, or via l-gulonate if the first reaction is the reduction. Three

different types of lactonases acting on sugar derivatives have been characterized in

mammalian tissues: 6-phosphogluconolactonase, uronolactonase and aldonolactonase. The

first one is an enzyme of the pentose phosphate pathway, which belongs, in mammals, to the

same family of proteins as glucosamine 6-phosphate isomerase and has no direct role to play

in the formation of vitamin C. Uronolactonase, a microsomal enzyme, hydrolyzes D-

glucurono-3,6-lactone, but is inactive against aldonolactones. Aldonolactonase

(gulonolactonase) is also a metal dependent enzyme, acting best with Mn2+, which is present

in the cytosol and hydrolyzes a number of C- and D-lactones of a variety of 5-, 6-, and 7-

carbon aldonates, including L-gulono-1, 4-lactone and D-glucono-1, 5- lactone. It also

catalyzes the lactonization of aldonates, e.g. of L-gulonate, and can therefore participate in

the formation of vitamin C.

L-Gulonolactone oxidase (Linster and E. V. Schaftingen 2006)



GLO, a microsomal enzyme, catalyzes aerobically the conversion of L-gulonolactone

to L-ascorbate with production of H2O2. The immediate oxidation product of GLO is 2-keto-l-

gulonolactone, an intermediate that spontaneously isomerizes to L-ascorbate. The preferred

substrate of the enzyme is L-gulono- 1, 4-lactone, but it also acts on L-galactono-, D-

mannono- and d-altrono-1, 4-lactone. Other C-lactones, including L-idono- and D-

gluconolactone, were not oxidized by the enzyme, indicating its configurationally specificity

for the hydroxyl group at C2. The production of H2O2 is unusual for an enzyme of the

endoplasmic reticulum, and one may wonder if this membrane-bound oxidoreductase does

not transfer its electrons to another acceptor in intact cells, most particularly because its plant

homologues do so. The latter share 30% sequence identity with mammalian GLO and differ

from this enzyme in three main aspects:

(1) They act specifically on l-galactono-1, 4-lactone, which is their physiological substrate;

(2) They are bound to the inner mitochondrial membrane; and

(3) They do not transfer electrons directly to O2, but to cytochrome c.

3.4 Commercial method for Ascorbic acid synthesis

Reichstein Process

The use of Reichstein’s procedure into an industrial process is marked by great efforts

to improve each reaction step. As a result of many technical and chemical modifications each

step gives over 90% yields. The overall yield of ascorbic acid from d-glucose is now 60%

(ULLMANN’S Encyclopedia vol. 38).

D-Sorbitol.

The catalytic hydrogenation of D-glucose to D-sorbitol is accomplished at high

pressures and elevated temperatures in the presence of a Raney nickel catalyst. The

hydrogenation is carried out in batch or continuous operations and affords almost quantitative

yields with minimal formation of D-mannitol and L-iditol. After removal of the catalyst, the

sorbitol solution is employed in fermentation without any further purification. Therefore,

manufacturers of ascorbic acid make high demands on the quality of D-glucose; crystalline

dextrose or purified starch hydrolysates are used 8, (ULLMANN’S Encyclopedia vol. 38).

L-Sorbose



L-sorbose is obtained in the pyranose form by microbiological oxidation of sterile

aqueous solutions of D-sorbitol in batch or continuous operations in the presence of air. The

most frequently used strains belong to the Gluconobacter oxydans family, which converts

dsorbitol to L-sorbose with more than 90% efficiency. Large-scale fermentations have to be

carried out at pH 4–6 and at 30 – 35oC under sterile conditions to avoid loss of product during

oxidation and work-up by filtration and crystallization (ULLMANN’S Encyclopedia vol. 38).

Figure 2.5 Hancock and R Viola (2002) gives Pathway cycle for production of Ascorbic acid by Reichstein

Process.

2,3:4,6-Di-O-isopropylidene-α-l-sorbofuranose.

The protection of the 2,3-and 4,6-hydroxyl groups by forming cyclic ketals is

achieved in acetone with excess sulfuric acid as catalyst and dehydrating agent, generally at

low temperatures (e.g., 4oC). In the final reaction mixture 2,3:4,6-di-O-isopropylidene-α-

lsorbofuranose is obtained as main product. Byproducts are 2,3-O-isopropylidene-α-l-

sorbofuranose and 1,2-O-isopropylidene-α- l-sorbopyranose. After neutralization, excess

acetone is recovered by distillation. 2, 3:4, 6-di-O-isopropylidene-α-l-sorbofuranose is

extracted from the aqueous solution with aromatic solvents (e.g., toluene). The remaining

monoisopropylidenesorboses are also recovered from the aqueous solution and returned to

the Process.

2, 3:4, 6-Di-O-isopropylidene-2-ketol-gulonic acid.



Originally, the oxidation of 2,3 : 4,6-di-O-isopropylidene-α-lsorbofuranose to 2,3 :

4,6-di-O-isopropylidene- 2-keto-l-gulonic acid was performed at elevated temperatures in

dilute sodium hydroxide withKMnO4, yielding ca. 90% of product. Much less expensive

oxidation methods are applied in modern continuous processes: sodium hypochlorite,

electrochemical oxidation, or catalytic air oxidation. Oxidation with hypochlorite in the

presence of catalytic amounts of nickel chloride or sulfate at 60oC yields >93% of 2,3:4,6-di-

O-isopropylidene-2-keto-l-gulonic acid. The active oxidant is presumably nickel peroxide.

Electrochemical oxidation is performed with nickel or nickel oxide electrodes in alkaline

solution. Hydrogen, evolved at the cathodes, can be used for hydrogenation of D-glucose. It

can be advantageous to stop electrochemical conversion to 2,3:4,6-di-O-isopropylidene-2-

keto-l-gulonic acid at 90% to avoid a drop in selectivity at the end of electrolysis. Oxidation

can be completed with sodium hypochlorite solution. At the end of the electro oxidation,

selectivity can be improved by electrolytic cells with high electrode surfaces, such as Swiss

roll cells or cells with three-dimensional electrodes. Alternatively, the oxidation has been

performed by using air or oxygen and a metal catalyst in alkaline solution. Good yields are

achieved with palladium or platinum on carbon. The reaction product 2,3:4,6-di-O-

isopropylidene-2-keto-l-gulonic acid is isolated by acidification and precipitation as the

monohydrate.

L-Ascorbic acid.

The conversion of 2,3:4,6-di-O-isopropylidene-2-keto-l-gulonic acid to l-ascorbic

acid is achieved by two different procedures:

1) Deprotection to give 2-keto-l-gulonic acid, followed by esterification with methanol and

base-catalyzed cyclization.

2) Acid-catalyzed cyclization to ascorbic acid directly from the protected or released 2-keto-l-

gulonic acid.

The starting material for base-catalyzed reactions is methyl 2-keto-l-gulonate,

prepared by treatment of the 2,3:4,6-di-O-isopropylidene-2-keto-l-gulonic acid with acidic

methanol. Finally, reaction of the methyl ester with sodium hydrogencarbonate or sodium

acetate affords sodium ascorbate in high yield. The first acid-catalyzed route to ascorbic acid

was published only few years after discovery of the Reichstein process. Today, the industrial

process is performed with an inert solvent (e.g., trichloromethane or toluene) in the presence

of hydrochloric acid. The advantage of this method is that ascorbic acid precipitates from the



mixture as it is formed, minimizing decomposition during reaction. The crude product is

obtained by filtration in high yield and high purity. After dissolution of crude ascorbic acid in

water, impurities are removed by refining with activated carbon, decolorizing resins, or ion

exchange resins, followed by crystallization.

415



Conclusion

Ascorbic acid is synthesized by different pathways using Bacteria, Algae and by

means of chemical reactions called Reichstein process. They all have their merits as well

demerits. In Bacterial biosynthesis the overall yield is 60 g/l. of 2-KLG from L-Sorbose or D-

sorbitol with 60% conversion in D-Sorbitol pathway (Shrikant et al., 2006).while that of 2-

Keto-D-Gluconic Acid Pathway have 85% yield using genetically modified strain

(Glucanobacter Oxydans)(Shrikant et al., 2006) and 10.5 g/l concentration of 2-KLG from D-

Glucose. Using genetically modified strain erwina citerus gives 19.8 g/l of 2-KLG with 49%

conversion (Hancock and R. Viola, 2001). So the D-Sorbitol pathway is more economical

pathway in bacterial biosynthesis of Ascorbic acid.

The other pathway is Reichstein pathway in these process seven steps is included of

which one is fermentation and others are chemical oriented. The yield of each step is 90%

(ULLMANN’S Encyclopedia vol. 38). Overall yield of the process is 50% (Hancock and R.

Viola, 2001). So that Reichstein process is more favorable for production of Ascorbic acid

industrially.

The intermediate steps in Reichstein process are energy consuming and required high

temperature , pressure condition make it non feasible for synthesis of ascorbic acid. For this

reason the intermediate are synthesize biotechnologically for higher yield and good

conversion.



5

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Pathway for Production Ascorbic Acid Synthesis