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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 938 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 5.3-11 939 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; 940 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- 941 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 942 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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