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252 Pharmaceutical Chemistry
Fermentative production of riboflavin from Lactococcus lactis
Sybesma et al. reported the overproduction of riboflavin and folate by metabolic engineering
and direct mutagenesis using various strains of L. lactis. Roseoflavin is the analogue when
L. lactis MG-1363 strain was exposed to riboflavin and thus the development of roseoflavin
resistant strain (L. lactis CB010) exhibits deregulated riboflavin biosynthesis that leads into
riboflavin production instead of consumption.
10.7 MICROBIAL PRODUCTION OF VITAMIN B12
Vitamins are one of the major essential and important nutritional constituents of living
organisms, viz., humans, animals and microbes. Even very small amount of the vitamin is
sufficient to the growth factors in the living organism. Vitamin B12 is one of the important
vitamins of B-complex group (B1, B2, B3, B6 and folic acid) which acts as coenzyme or
building blocks of several enzymes. Vitamin B12 is also known as cobamide or cobalamin
and consists of a very complex structure which helps in the formation of red blood cells
(RBCs). It contains co-porphyrin nucleus and is attached to ribose and phosphate. It also
contains purine, benzimidazole or other chemical groups. It is water soluble in nature thus
it needs to be supplied to the living organism.
Vitamin B12 is synthesized by commercial production of microbes. Various cobalamin
derivatives are synthesized in the form of coenzyme along with vitamin B12. Rickes et al.
in 1948, first time isolated vitamin B12 from Actinomycete and Streptomyces griseus. Vitamin
B12 is generally synthesized from bacteria, fungi and Actinomycetes while large-scale
production from intestinal and rumen bacteria were documented. Two types of vitamin
B12 are reproduced by the microorganisms; one responsible for microbial growth and
other normal growth in humans and animals. Previously, it was used for the treatment of
anaemic patients. Many modified fermentation processes have been employed for the
commercial production of vitamin B12 as follows:
1. Direct fermentation: Nutrient medium containing glucose is generally used with
Streptomyces olivaceus for carbon source where catalytic amount of cobalt chloride
(CoCl
At the end of incubation, growth is harvested before destruction of vitamin and
mycelial autolysis followed by the filtration of broth to collect vitamin. Filter the
mycelium while remaining mycelia are acidified (stabilize the vitamin during
sodium sulfite) or may be treated with ethanol to recover rest of the vitamin.
Sometimes mild heat is required for extraction due to the sensitiveness of vitamin
to the heat. Sometimes a mycelium-vitamin B12 complex is dried and used to feed
animals and birds. The mycelial growth is removed by filtering the acidified broth
and the filtrate is evaporated under vacuum at room temperature followed by
another purification using acetone and ion exchange resins. The commercial
production of vitamin B12 is also executed by Bacillus megaterium, Propionibacterium
freudenreichii, P. shermanii and Pseudomonas.
, <10 ppm) is added and then the process is carried out for five days at 27°C.
2
Fermentation 253
2. Indirect production: High concentration of vitamin B12 is found in sewage sludge.
It is also formed by various antibiotic fermentation processes as a by-product, viz.,
fermentation production of streptomycin, aureomycin, grisein, and acetonebutanol, etc.
3. Genetic engineering: Genetic engineering techniques or genome shuffling can play
a major role to modify the desired product. For example, rigorous strain mutations
and recombination techniques have been developed for Streptomyces griseus to get
better yield of vitamin B12.
10.8 PRODUCTION OF VITAMIN C (ASCORBIC ACID)
Vitamin C is also known by other names, viz., antisorbutic vitamin,
L-Ascorbic acid, 3-oxo-L-gulofuranolactone (enol form), L-xyloascorbic
acid and L-3-ketothreohexuronic acid lactone. It is a water-soluble
vitamin and useful in formation of protein called collagen (form
muscle, bones, blood vessels and cartilage, etc.) and also participates in
HO OH
O
O
Ascorbic acid
OH
OH
various biological regulation, maintenance, development and absorption of iron in the
body. It can donate H-atom frequently and stabilizes the ascorbyl free radical, thus it can
be used as a powerful antioxidant. It is also used as strong scavenger for the reactive
oxygen, singlet oxygen, nitrogen oxides, superoxide radical ion, hydroxyl radical and
hydrogen peroxide, etc. It also inhibits folic acid reductase enzyme, which is used to
convert folic acid to folinic acid. It is also used to facilitate and enhance the iron absorption
in the body. Severe deficiency of ascorbic acid may lead to scurvy and other common
symptoms such as loosening of teeth, dry itchy skin, follicular hyperkeratosis, swollen
gums, loss of hair and dryness of the eyes and mouth.
It can be synthesized by many methods using microbes, viz., biosynthesis of bacteria
and algae. These microbes may be used as such, but yield is the concern. To improve the
yield, genetically modified strains (Acetobacter suboxidans, Bacterium xylinum, Erwinia sp.,
Corynebacterium sp.) can be used. It requires high temperature and pressure to complete
the synthesis due to its high energy consumption. Usually, Reichstein process (single
fermentation step) is well known and adapted biosynthetic pathway for the production of
vitamin C. The biosynthetic pathways have been explored to overcome these problems
and need low energy.
10.8.1 Pathways for the Production of Ascorbic Acid
10.8.1.1 Microbiological Fermentation
Ascorbic acid can be produced by the microbiological fermentation (bacteria, and algae
etc.). It can be produced by using various metabolic pathways.
1. Metabolic Pathway for Bacteria
A key intermediate 2-keto-L-gulonic acid to produce L-ascorbic acid. The widely accepted
commercial bacterial fermentation processes to produce ascorbic acid are as follows:
254 Pharmaceutical Chemistry
(a) D-Sorbitol Pathway
Fermentation of 2-KLG via intermediate L-sorbosone is used in the production of sorbitol.
Bacterial strains, viz., Pseudomonas and Acetobacter, have been used to catalyze the oxidation
of D-sorbitol to 2-KLG which are usually catalyzed by either membrane bound or cystolic
sorbosone dehydrogenase enzymes. Glucano oxydans strain can also be used to produce
2-KLG (60 g/L) from L-sorbosone or D-sorbitol
used for these processes. Conversion of D-sorbitol to 2-KLG takes place in the presence of
dehydrogenase but often depends on strain to strain. The cystolic reductase is used to
determine the pathway of transferring D-sorbitol into cytoplasm of these strains, which
helps to open the path of these intermediates in the pentose cycle. For reducing such
problem, membrane cystolic enzyme may be replaced by bound dehydrogenase
recombinant with Glucanobacter oxydans.
(b) 2-Keto-D-Gluconic Acid Pathway
D-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). There are no effective bacterial strains reported for
catalyzing the complete conversion of D-glucose to 2-KLG. Basically, D-glucose to 2-KLG
step is carried out in three main steps using different microorganisms as follows:
(a) Conversion of D-glucose into 2-keto-D-gluconic acid: This conversion can be
done using Acetobacter melanogenus and Pseudomonas albosesamae bacteria.
(b) Oxidation of 2-keto-D-gluconic acid: Oxidation of 2,5-DKG is carried out using
Bacterium hoshigaki and B. gluconicum while A. albosesamae can be used to direct
conversion of D-glucose to 2,5-DKG.
(c) Oxidation of 2,5-DKG acid into 2-KLG: This conversion can be done using strains
of the genera Brevibacterium and Pseudomonas, and Corynebacterium.
Sometimes Erwinia strain (a mutant) may be used for conversion into 2,5-DKG. The
conversion of D-glucose into 2-KLG can also be done using a mutant strain of Corynebacterium
and genetically modified bacterial strains.
(c) Bioconversion of 2-KLG to L-Ascorbic Acid Pathway
Two methods have been used to convert 2-KLG into L-ascorbic acid. The first method
includes multiple steps as follows:
Under strong acidic condition, 2-KLG derivative esterifies to yield methyl-2-keto-L-
gulonate (MeKLG).
MeKLG then reacts with base lead to metal ascorbate salt.
Metal ascorbate salt is treated with an acidulant leading to ascorbic acid.
The second method is a one-step process where acid-catalyzed cyclization leads to KLG.
Multiple steps process in the first method and huge amount of HCl gas consumption are
the main disadvantages of commercial production of ascorbic acid. To avoid these
disadvantages, hydrolase enzyme can be used to convert ester of 2-KLG to L-Ascorbic
acid. The conversion of 2-KLG to L-ascorbic acid is also performed using lactonases which
is extracted from Zymomonas mobilis, Escherichia coli and Fusarium oxysporium (Fig. 10.7)
. Genetically modified strains may also be
.
Fermentation 255
CHO
H OH
HO H
H OH
H OH
CH2OH
Glucose Gluconic acid 2-keto-D-
E1 = Glucose dehydrogenase
E2 = Gluconic acid dehydrogenase
E3 = 2-keto-D-Gluconic acid dehydrogenase
E4 = 2,5-diketo-D-Gluconic acid reductase
E1 E2
2-KLG recovery
via crystallization
COOH
H OH
H
HO
H OH
H OH
CH2OH
Fig. 10.7: Biosynthesis of ascorbic acid
HO H
H OH
H OH
gluconic acid
Esterification
Lactonization
recovery
COOH
O
CH2OH
E3
HO OH
O
COOH
O
HO H
H OH
O
CH2OH
2,5-diketo-Dgluconic acid
O
Ascorbic acid
E4
OH
OH
COOH
O
HO H
H OH
HO H
CH2OH
2-keto-L-Gulonic
acid (2KLG)
Production of L-Ascorbic Acid from Yeast
Yeast can be incubated with L-galactose, L-galactono-1,4-lactone or L-galactono-1,4lactone intermediates (from plants or animal pathway cells) produces L-ascorbic acid due
to the enzyme activity during D-erythroascorbic acid pathway. Overproduction of
L-ascorbic acid and D-erythroascorbic acid was obtained when supplied with D-galactono1,4-lactone and D-arabinono-1,4-lactone, respectively, by using D-arabinono-1,4-lactone
oxidase enzyme from Saccharomyces cerevisiae in E. coli. S. cerevisiae and Zygosaccharomyces
bailii on incubation with L-galactose may increase the intracellular L-ascorbic acid
accumulation.
Production of L-Ascorbic Acid from Algae
Many attempts are employed to use microalgae for the direct production of L-ascorbic acid
from inexpensive feedstock. Low quantity of L-ascorbic acid (~40 mg/L) was obtained
from heterotrophic green microalga (viz., Chlorella pyrenoidosa) in the fermentation process.
Continuous chemical mutagenesis and fermentation optimization techniques may improve
the amount by additional 2 g/L.
A colourless microalgae Prototheca moriformis is responsible to accumulate more
L-ascorbic acid in the fermentation medium. 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. Strain-improvement of Prototheca mutants may
increase and reduce the abilities to accumulate L-ascorbic acid. These mutants are used to
identify the pathway for L-ascorbic acid biosynthesis which involves mannose containing
intermediates. Similarly in the plants, l-galactono-1,4-lactone is produced from D-glucose
through GDP-D-mannose, GDP-l-galactose and l-galactose. Finally, l-galactono-1,4lactone is converted into L-ascorbic acid (Fig. 10.8).
256 Pharmaceutical Chemistry
Fig. 10.8: Production of ascorbic acid using algae
2. Metabolic Pathway for Plants
It was proposed that oxidation of L-galactose is used to synthesize ascorbate in plants. The
enzymatic steps of non-inverted conversion of glucose to ascorbic acid are summarized as
follows:
(a) Oxidation of the C1 of glucose,
(b) C2/C3 oxidation,
(c) C5 epimerization,
(d) Lactonization between C1 & C4.
Hexose-phosphates can be converted into D-mannose in the presence of catalyst
phosphomannose isomerase (PMI, isolated from E. coli) enzyme. Conversion of D-mannose6-phosphate to D-mannose-1-phosphate is generally catalyzed by phosphomannose
mutase (PMM) enzyme. D-mannose-1-phosphate resulted into GDP-D-mannose where
GTP is catalysed by GDPD-man-pyrophosphorylase (GMP) enzyme. GDP-D-mannose3,5-epimerase (GME) (isolated from Chlorella pea, algae Prototheca and also cloned from A.
thaliana) is used to convert GDP-D-mannose to GDP-L-galactose by a reversible double
epimerization. GDP-D-mannose and GDP-L-galactose are also involved in the synthesis of
polysaccharide and protein glycosylation. GDP-L-galactose is converted to L-galactose-1phosphate and GDP by a specific phosphate-dependent GDP-L-galactose phosphorylase
enzyme. Finally, oxidation of L-galactose takes place in two steps, first cytosolic
NAD-dependent L-galactose dehydrogenase (L-GalDH) enzyme at C-1 is used to form
L-galactono-1,4-lactone (L-GalL) and then L-GalL dehydrogenase (L-GalLDH) at C-2/C-3
used for the ascorbate production while antisense suppression of LGalDH and L-GalLDH
leads to reduction in ascorbate concentration. Thus, the predominant pathways for the
production of ascorbate are D-mannose and L-galactose while uronic acid intermediates
may lead to the ascorbate content of plant tissues.
3. Metabolic Pathway for Animals
The production of ascorbic acid from D-glucose in animals is given as
Fermentation 257
:
UDP-Glucose
UDP-
Glucuronate
D-Glucuronate L-Gulonate
L-Gulono-
1,4-lactone
L-Ascorbic acid
Another source of ascorbic acid is sea lamprey (vertebrates), but most of the species,
viz., teleost fishes, bats, Passeriformes birds, guinea pigs, humans, etc., have lost their
biosynthetic capacity to produce ascorbate while amphibians, fishes and reptiles are able
to synthesize ascorbate in the kidney, and some mammals are able to produce it in the
liver. In animals, D-glucuronate was synthesized biosynthetically from UDP-glucuronate
which later further reduced to ascorbate precursor called L-gulonate. In plants, L-galactono
lactone was synthesized biosynthetically to ascorbate precursor called GDP-D-mannose
using L-galactono lactone dehydrogenase enzyme.
Commercial Production of Ascorbic Acid
Earlier, ascorbic acid was extracted from plant in commercial production (Fig. 10.9) while
major commercial production of ascorbic acid is, by chemical synthesis. Presently, it is
produced by two processes:
(a) Reichstein process: It is a traditional process generally used to convert glucose to
sorbitol by heat treatment. Then sorbitol is fermented and oxidised in the presence
of a microorganism to yield sorbose thereby, reaction between sorbose and acetone
yielded in di-acetone sorbose which further gets oxidised to di-acetone keto gulonic
acid (DAKS). Then DAKS is dissolved in a mixture of organic solvents using catalyst
to yield vitamin C followed by recrystallization.
Glucose Sorbitol
Δ
Fermentation
Sorbose Diacetone sorbose
DAKS Crude Vitamin C
Reichstein-Grussner synthesis Sorbitol fermentation Glucose fermentation
D-Glucose
Hydogenation
D-Sorbitol
Fermentation
L-Sorbose
Acetonization
Diacetone-L-sorbose
Oxidation Hydrolysis
2-Keto-L-gulonic acid
Esterification
Methyl-2-keto-L-gulonic acid
Lactonization
Fig. 10.9: Process routes to ascorbic acid
Fermentation
Fermentation
Recrystallisation
L-Sorbose Fermentation
Ascorbic acid
Pure Vitamin C
one-step process
Chemical processing
technology
258 Pharmaceutical Chemistry
(b) Two-step fermentation process: This process was developed in China. In this
process, fermentation of sorbose yielded in KGA which later converted into crude
vitamin C followed by recrystallization. This process is more appealing and cost
effective for the production of ascorbic acid.
Glucose Sorbitol
Δ
Crude Vitamin C
Fermentation
Sorbose KGA
Recrystallisation
Pure Vitamin C
QUESTIONS
1. What is submerged fermentation? Explain in detail.
2. What are the advantages of solid state fermentation over submerged fermentation?
3. How you will explain anaerobic fermentation?
4. What are the differences between aerobic fermentation and anaerobic fermentation?
5. What are immobilization cell bioreactors? Explain its advantages.
6. What are the advantages of immobilized enzyme bioreactors?
7. Write a short note on ethanol fermentation.
8. How is citric acid production carried out by using SmF and SSF?
9. What are antibiotics and how are they isolated by fermentation process?
10. Write a short note on liquid fermentation.
11. Explain the fermentation process of the following:
(a) Cephalosporin
(b) Penicillins
(c) Chloramphenicol
(d) Streptomycin
(e) Lysine
(f) Glutamic acid
(g) Vitamin B2 (Riboflavin)
(h) Vitamin B12
(i) Vitamin C
12. How does biosynthesis of streptomycin work?
13. How can raw protein material be hydrolysed?
14. How is glutamic acid produced using alkaline hydrolysis of Steffen’s molasses?
15. Describe the metabolic pathways for bacteria for the synthesis of ascorbic acid.
16. Describe the metabolic pathways for animals for the synthesis of ascorbic acid.
17. How is Reichstein process useful for the commercial production of ascorbic acid?
11
Medicinal Importance of Curcumin,
Neem, Vitamin C, Ranitidine, Ginger, Tulsi,
Garlic and Ajwain
11.1 MEDICINAL IMPORTANCE OF HALDI OR CURCUMIN (Curcuma longa)
Curcumin, generally obtained from turmeric, has characteristic
crystalline yellow colour (sometimes orange yellow colour) due to
the curcuminoids. Curcumin (C
component from turmeric. The turmeric plant Curcuma longa belongs
to the ginger family called Zingbereceae. This perennial herb is
extensively cultivated specially in South Asia. The rhizome (root) is
the most active and medicinally important part of the plant.
Curcumin (Curcumin-I) was first isolated in 1815, and identified as 1,6-heptadiene-3,5dione-1,7-bis(4-hydroxy-3-methoxyphenyl)-(1E,6E) or diferuloylmethane (Fig. 11.1) in
1870 while structurally described in 1913 by Lampe & Milobedeska. Demethoxycurcumin
(Curcumin-II), bisdemethoxycurcumin (Curcumin-III) and cyclocurcumin are the major
curcuminoids isolated from turmeric with a ratio of 77:17:3% respectively. It possesses
several functional groups, viz., phenols, D,E-unsaturated carbonyl groups, etc. The diketone
forms stable enols, which can be easily deprotonated and form enolates, whereas
D,E-unsaturated carbonyl groups can undergo nucleophilic addition easily.
The major use of turmeric is in the treatment for wide range of ailments, colouring
agents and dietary spices (Fig. 11.2). It can be used as pain reliever in case of osteoarthritis
when 2 gm dose of Curcuma domestica extract is used daily. This medication relieves pain
at the same rate as ibuprofen. It also helps to inhibit blood clotting. Administration of
crude turmeric with milk is the best home remedy for the treatment of variety of wounds
and inflammations. It can also be used as an antioxidant, free-radical scavenger, oxidative
DNA damage and lipid peroxidation inhibition. Since ancient times, it has been widely
used in traditional Indian medicine in the treatment of biliary disorders, hepatic disorders,
anorexia, cough, rheumatism, sinusitis, diabetic wounds, etc. It also helps to reduce blood
cholesterol to prevent LDL oxidation, Alzheimer’s disease, myocardial infarction, inhibits
platelet aggregation, rheumatoid arthritis, suppresses thrombosis and type-II diabetes
suppression, multiple sclerosis, increases bile secretion, inhibition of HIV-replication,
21H20O6
) is the most active bioactive
260 Pharmaceutical Chemistry
Arthritis
Cardiovascular diseas
protects from liver injury, and protects from pulmonary toxicity and fibrosis, protects from
cataract formation. It also acts as an antileishmaniasis and an antiatherosclerotic.
Fig. 11.1: Structure of curcumin
Antiagiogenic
Antiinammatory
Inhibits scarring
Cataract formation
Gall stones
formation
Inammatory
bowel disease
(Cholesterol, platelet
aggregation)
HIV replication
Multidrug resistance
Diabetes
Septic shock
CURCUMIN
e
Lung brosis
Cardiotoxicity
Fig. 11.2: Medicinal properties of curcumin
Antioxidant
Liver injury
Nephrotoxicity
Chemotherapeutic
Chemopreventive
(Skin, liver, colon,
smooth muscle cell
Stimulates muscle
Multiple sclerosis
Immunosuppressive
Wound healing
stomach)
Inhibits vascular
proliferation
Alzheimer’s disease
regeneration
Medicinal importance of Curcumin, Neem, Vitamin C, Ranitidine, Ginger, Tulsi, Garlic and Ajwain 261
11.2 MEDICINAL IMPORTANCE OF NEEM (Azadirachta indica)
The evergreen and fast growing tree neem is commonly found in India, Africa and America.
This plant plays a key role in the prevention and control of various diseases. Since last 400
decades, it has been used continuously in Indian classical system of healing, i.e., Ayurveda,
due to its diverse medicinal characteristics. In Sanskrit neem is named as ‘arista’ that means
‘perfect’, ‘reliever of sickness’ and ‘Sarbarogaribarini’. Neem tree is also regarded as ‘Village
dispensary’, ‘Village pharmacy’, and ‘Panacea for all diseases’ in India. In 1992, US National
Academy of Sciences mentioned a slogan, “Neem: a tree for solving global problems” due to
its diverse medicinal importance. This plant is used in several ceremonies, worship and
rituals of Hindu religion. Each part of the plant is used traditionally as medicine.
The botanical name of neem is Azadirachta indica, given by De Jussieu in 1830. The
taxonomic description of neem is as follows: Order: Rutales, Suborder: Rutinae, Family:
Meliaceae (Mahogany family), Subfamily: Melioideae, Tribe: Melieae, Genus: Azadirachta,
Species: indica.
Two species, Azadirachta indica and A. Juss belong to the Indian subcontinent whereas
A. excels belong to Philippines and Indonesia. Numerous medicinally important components
are isolated from the neem plant and can be classified into three classes on the basis of their
structure: isoprenoids, nonisoprenoids and others. Isoprenoid class of bioactive
components includes diterpenoids and triterpenoids (viz., azadirachtin, azadirone
derivatives, protomeliacins, salanin, limonoids, gedunin derivatives and vilasinin and
csecomeliacins like nimbin) whereas nonisoprenoid class of bioactive components
includes carbohydrates (polysaccharides), proteins (amino acids), sulphur containing
compounds, polyphenolics (coumarins and tannins, flavonoids and their glycosides,
dihydrochalcone and aliphatic compounds, etc.). Other bioactive components extracted
from neem tree are azadirachtin, nimbin, salanin, meliacin, valassin, gedunin and tignic
acid (5-methyl-2-butanic acid from neem seed oil), etc. Although a huge number of
bioactive components have been isolated from neem as a whole, few are studied for their
biological activity as shown in Table 11.1 and Fig. 11.3 and also discussed as follows:
1. A major and bitter pharmacophore Nimbidin is extracted from the neem oil. Other
bioactive components such as nimbidic acid, nimbolide, nimbin, nimbidinin,
nimbinin and tetranortriterpenes are also isolated from the neem extract.
2. Neem gum is a rich source of proteins.
3. Nimbidin and sodium nimbidate are active as anti-inflammatory against formalin-
induced arthritis and carrageenan-induced acute paw-oedema in rats as a dose
dependent manner.
4. Nimbidin is very useful to prevent stress, serotonin induced gastric lesions,
acetylsalicylic acid, histamine/cysteamine-induced duodenal ulcers, indomethacin
and carbachol-stimulated gastric acid output.