Download bioengineering 938 pantothenic acid – applications, synthesis and

Rev. Med. Chir. Soc. Med. Nat., Iaşi – 2015 – vol. 119, no. 3
BIOENGINEERING
ORIGINAL PAPERS
PANTOTHENIC ACID – APPLICATIONS, SYNTHESIS
AND BIOSYNTHESIS
Mădălina Poştaru1*, D. Caşcaval 2, Anca-Irina Galaction 1
University of Medicine and Pharmacy “Grigore T. Popa” - Iasi
Faculty of Medical Bioengineering
1. Department of Biomedical Science
Technical University “Gheorghe Asachi” - Iasi
Faculty of Chemical Engineering and Environmental Protection
2. Department of Biochemical Engineering
*
Corresponding author. E-mail: [email protected]
PANTOTHENIC ACID – APPLICATIONS, SYNTHESIS AND BIOSYNTHESIS (Abstract): Pantothenic acid, also known as vitamin B5 or anti-stress vitamin, is a water-soluble
vitamin with various applications in pharmaceutical industry (ointments, solut ions for treating lesions and irritation of the skin or mucous membranes, multivitamin supplements - tablets, capsules, effervescent tablets, chewable tablets, syrups), food industry (dietetic foods,
baby products, cereals, juices), cosmetics (component of shampoos, hair masks, balms,
creams and sunscreen lotions, baby products), and widely used as feed additive. Currently,
pantothenic acid is produced by chemical synthesis using pantolactone and the calcium salt
of β - alanine, but it can also be obtained by fermentation processes which involve different
bacterial species, as well as a number of yeasts. Lately, the demand for pantothenic acid for
cosmetic and dermatocosmetic industry registered a significant increase. In this context, o btaining pantothenic acid by fermentation has become a promising method because it does not
have a negative environmental impact, it uses renewable resources and cheap raw materials,
and global consumer demand for organic products is increasingly higher. Keywords: PANTOTHENIC ACID, APPLICATIONS, PRODUCTION.
Pantothenic acid is an organic acid having the formula C9H17O5N (fig. 1). From the
chemical point of view, pantothenic acid is
the amide of pantoic acid with β-alanine.
Fig. 1. The chemical structure
of pantothenic acid
Pantothenic acid was discovered in 1931
by RJ Williams. In 1933, vitamin B5 was
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isolated from yeast, and a few years later, RJ
Williams successfully establish the chemical
structure and extract the compound from
liver (1). The main role of pantothenic acid
in cells is the synthesis of coenzyme A
(CoA). Coenzyme A acts as an acyl group
carrier to form acetyl-CoA and other related
compounds. CoA is very rich in energy and
enzymatically reacts on two distinct paths:
an anabolic pathway, where acetyl-CoA is
the starting point in many biosynthetic processes (in the biosynthesis of fatty acids,
cholesterol, and acetylcholine) and a cata-
Pantothenic acid – applications, synthesis and biosynthesis
bolic pathway that continues with the tricarboxylic acid cycle (Krebs cycle) and ends
with the respiratory chain. Pantothenic acid
in the form of CoA is also involved in signal
transduction and enzyme activation and
deactivation (2, 3) (fig. 1).
PANTOTHENIC ACID
APPLICATIONS
Regarding the human body, this compound is involved in the health of the digestive, nervous, circulatory, and skeletal systems, skin and hair, as well as in the synthesis
of hormones (insulin, adrenaline) (4). Combs
G.F. Jr. (5) consider that it also plays an important role in increasing the immunity of
human body. Panthenol, another form of
vitamin B5, is used in hair care products,
considering that it interferes in maintaining a
healthy aspect of it, acting as a humectant to
facilitate the increase of water content and
elasticity. At the same time, it protects the
skin from harmful effect of radiation, mois-
turizes the skin, mucous membranes and
cornea. In addition, the administration of high
doses of pantethine (500-1200 mg/day) to
subjects suffering from dyslipidemia (blood
lipid pathological change) and diabetes, it
was found that it is effective in reducing the
level of cholesterol and triglycerides in the
blood, treating fatty liver and related diseases. Some studies suggest that vitamin B5
supplements may speed the healing of superficial wounds, especially after surgery. This
quality is important in case of burns, when a
significant proportion of micronutrients are
lost, phenomenon that increases the risk of
infection and slows healing (4).
Pantothenic acid is found in all living
cells (pantos = everywhere) and is widely
found in foods, so deficiency of this substance is generally rare encountered. The
richest sources of vitamin B5 are: yeast,
royal jelly, cereals, vegetables, egg yolk,
liver and kidney, beef, soy, nuts/peanuts,
walnuts, mushrooms (4, 6) (tab. I).
TABLE I
Sources of vitamin B5 and content
Sources
Content, mg/100g
Meat
beef
0.3-2
pork
0.4-3.1
young cattle kidney
3.9
chicken liver
9.7
young cattle liver
8
pork liver
7
Cereals
wheat flour
0.21
wheat bran
2.1
whole wheat
1.2
soy flour
1.4
oatmeal
1.1
whole rice
1.1
Fruits
apples
0.1
strawberry
0.3
oranges
0.25
blackcurrants
0.4
Sources
Content, mg/100g
Vegetables
avocado
1.1
broccoli
1.2
cabbage
0.1-1.4
carrots
0.27
cauliflower
1
lentil
1.4
potatoes
0.3
soybeans
1.7
tomatoes
0.3
Nuts
Cashew
1.3
Peanuts
2.8
walnuts
0.8
Others
eggs
2.9
milk
0.2
mushrooms
2.1
yeast
5.3-11
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Mădălina Poştaru et al.
Recommended daily doses of vitamin B5
(tab. II) depend on, besides age and sex, on
daily caloric consumption and physiological
state of the body (7). Vitamin B5 deficiency
symptoms include: fatigue, insomnia, depression, irritability, listlessness, nausea and
vomiting, abdominal cramps, muscle weak-
ness, burning sensation in the extremities. In
hypovitaminosis, at molecular level, changes occur in the biosynthesis of corticosteroids and cholesterol, inhibition of the oxidative decarboxylation pathway of pyruvate
and metabolism of fatty acids, as well as in
the antibody biosynthesis process.
TABLE II
Recommended daily doses of pantothenic acid
Pediatrics
0 - 6 months
6 months - 1 year
1 - 3 years
4 - 8 years
9 - 13 years
14 - 18 years
Recommended
daily intake, mg
1.7
1.8
2
3
4
5
THE CHEMICAL SYNTHESIS
OF PANTOTHENIC ACID
The production of calcium pantothenate, pantothenic acid commercial form,
is performed chemically, by heating pantolactone with β-alanine (calcium or sodium
salt) in methanol or ethanol (fig. 2).
Racemic pantolactone was obtained in a
Adults
19 years and older
pregnant women
breastfeeding women
Recommended
daily intake, mg
5
6
7
yield of about 90% from isobutyraldehyde,
formaldehyde and hydrogen cyanide, without the need for separation of the intermediate products. Saponification of the second intermediate with a strong acid causes
the formation of pantolactone in the reaction media, which can be separated by
extraction and/or distillation (6).
Fig. 2. Calcium pantothenate production by chemical synthesis
For obtaining biologically active enantiomer, R (D)-pantolactone, several methods were developed (6):
1. fractional crystallization of diastereoisomeric salts or amides of pantothenic
acid with alkaloids or synthetic chiral amines;
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2. the cleavage of the racemic mixture
and recycling of S-enantiomer - can be made
either by heating with an aqueous solution
of NaOH at 150°C for a few hours, until the
obtaining of sodium pantoat, or by treatment
with very alkaline amines or alkoxides;
Pantothenic acid – applications, synthesis and biosynthesis
3. stereo selective hydrogenation of 2oxolactone (obtained either by oxidation of 2aminolactone or pantolactone, or by acid
treatment with dimethylpiruvic formaldehyde).
The industrial synthesis of β-alanine
can be performed by two methods (6):
a) the addition of ammonia to acrylonitrile, followed by basic hydrolysis of the
nitrile with formation of the sodium salt of βalanine; after sulfuric acid treatment, evaporation and extraction of the residue with
methanol, crystalline β-alanine is obtained;
b) ammonia addition to acrylic acid, in
which case the sodium salt formation is
avoids.
An important problem associated with
industrial obtain of calcium pantothenate,
apart from the use of toxic chemical reagents - hydrocyanic acid, is the laborious
and expensive step of optical resolution of
racemic pantolactone. Thus, numerous
studies have concentrated on the development of more efficient methods for the
preparation of D-pantolactone, the biologically active isomer (8, 9).
Therefore, a more economical alternative to conventional resolution, that uses a
quite expensive synthetic alkaloid series, is
the biotransformation of racemic pantolactone using a fungal enzyme, lactonohydrolaze, as biocatalyst. The stereospecific hydrolysis of D-lactone is catalysed by
this enzyme. Because D-pantolactone is
one of the preferred substrates, lactonohydrolaze rapidly converts it in D-pantoic
acid, while the L-enantiomer of the racemic
mixture remains unchanged and will be
racemized and recycled for a new cycle of
optical resolution (8, 10).
Some strains of fungi belonging to the
genera Fusarium, Gibberella and Cylindrocarpon have a high activity of this enzyme
(8, 9, 11). For example, by incubating the
Fusarium oxysporum cells for 24 hours at
30°C and a pH value of 7, D-pantolactone
from the racemic mixture (700 g/l) is 90%
hydrolyzed to D-pantoic acid, while the Lisomer remains unchanged (8, 9).
A simplified procedure ensures biocatalytic production of D-pantolactone, with an
enantiomeric excess, from 3-hydroxy-2,2dimethyl-propionaldehyde and hydrogen
cyanide, followed by acid hydrolysis. As
biocatalyst, the hydroxyl-nitrilase isozyme
from Prunus amygdalus is used, the reaction being performed at a low pH value to
avoid spontaneous formation of the racemic
nitrile. A suitable biocatalyst for this process, which is stable and has a high activity
to acidic pH, may be obtained from hydroxyl-nitrilase isoenzyme V (10, 12).
BIOSYNTHESIS OF
PANTOTHENIC ACID
The development of genetic engineering
techniques and the progress of DNA manipulation help researchers, who by modifying chromosomal genes or introducing
additional extra chromosomal genetic material, managed the development new mutant strains with the ability to synthesize
much larger amount of pantothenic acid as
compared with the wild strains.
Gram positive bacteria Coryne bacterium glutanicum, used for large-scale production of amino acids (L-glutamic acid, Llysine) was studied for the biosynthesis of
vitamin B5. Thus, if the wild strain produces about 9 mg/l pantothenic acid, by chromosomal deleting the ilvA gene coding
threonine-dehydratase and over expression
combined ilvBNCD and panBC genes, scientists were able to accumulate more than 1
g/l vitamin in the supernatant (13, 14).
Saccharomyces cerevisiae has the capacity to produce endogenous β-alanine
and, therefore, to biosynthesize de novo
pantothenic acid, as long as FMS1 gene,
encoding amine oxidase that ensures the
transformation of spermine in the β-
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Mădălina Poştaru et al.
alanine, is over expressed (15). Over expression of FMS1 caused excess pantothenic acid to be excreted into the medium,
whereas deletion mutants required βalanine or pantothenic acid for growth.
White et al. concluded that yeast is naturally capable of pantothenic acid biosynthesis,
and that β-alanine is derived from methionine via a pathway involving spermine.
For Escherichia coli, the responsible
genes for encoding enzymes needed for the
biosynthesis of vitamin B5 are: panB, panC,
panD and panE. By overexpressing these
genes, a new strain with superior pantothenic acid productivity has been developed (10,
11). Laudert and Hohmann used glucose as a
substrate and β-alanine as a precursor and
after 72 hours of fermentation was obtained
a concentration of 66 g/l pantothenic acid.
The main disadvantage of this process, beside the fact that β-alanine is added as a cosubstrate, is that over 40 % of the amount of
useful product does not come from carbohydrate substrate, but from the synthetized βalanine.
A promising method for obtaining vitamin B5 is by fermentation processes using
the Bacillus subtilis gram-positive bacteria.
Normally, Bacillus subtilis synthesizes only
small amounts of pantothenic acid required
to refill their needs. By genetic engineering
the over expression of the genes panBCDE,
ilvBNC and ilvD of bacteria can be
achieved, thereby obtaining a higher productivity - 86 g/l in only 48 hours, compared to
the fermentation of mutant strain of E. coli.
In addition, in the case of Bacillus subtilis, it
is not necessary to add exogenous β-alanine.
Therefore, the biotechnologies methods are
a feasible alternative and economically solution in the production of vitamin B5 vs.
chemical synthesis (10, 16).
The pantothenic acid obtained by these
methods has to be further extracted from
natural extracts or biosynthesis media. The
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separation of pantothenic acid can be carried
out by crystallization, ion exchange, chromatography, and physical extraction (17-20).
Further studies focused on the separation of
pantothenic acid by reactive extraction,
method that has been used for the recovery of
biosynthesis products (organic acids, antibiotics, vitamins, amino acids etc.) from aqueous
solution using an organic phase in which
different extractants are added (21).
Although biosynthesis is an environmentally friendly and fairly cheap alternative to
obtain vitamin B5, it is not currently applied
to industrial level due to much lower productivity compared with chemical methods.
Further efforts are needed to develop new
strains whose metabolic flux to be directed
mainly towards the synthesis of pantothenic
acid, so as to ensure a higher yield (10).
CONCLUSIONS
Industrial production of pantothenic acid is based on chemical synthesis, but an
attractive alternative for obtaining this
compound, in terms of required steps and
consumption of raw materials and energy,
is biosynthesis by microorganisms.
With the use of chemical and genetic
engineering, scientists were able to improve the properties of the biosynthetic
product and also to achieve much higher
production yields for pantothenic acid. By
modifying chromosomal genes, Coryne
bacterium glutanicum can produce more
than 1 g/l pantothenic acid, compared with
about 9 mg/l produced by the wild strain.
Introducing additional extra chromosomal
genetic material and over expressing some
genes led to the development of new strains
with superior pantothenic acid production.
Thus, Escherichia coli can produce in 72
hours of fermentation 66 g/l pantothenic
acid, using glucose as a substrate and βalanine as a precursor, and Bacillus subtilis
was able to produce 86 g/l vitamin B5 in
Pantothenic acid – applications, synthesis and biosynthesis
only 48 hours.
ACKNOWLEDGEMENTS
This paper was published under the
frame of European Social Found, Human
Resources Development Operational Programme
2007-2013,
project
no.
POSDRU/159/1.5/S/136893.
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