6/13/2008 Vitamin Production (NRM) 1

Transcription

1 6/13/2008 Vitamin Production (NRM) 1

2 A vitamin is an organic compound required as a nutrient in tiny amounts by an organism. Vitamins are classified by their biological activity, not their structure. Vitamins have diverse biochemical functions, including function as: 1. a precursors for enzyme cofactor biomolecules (coenzymes) (e.g. B complex vitamins), 2. hormones (e.g. vitamin D), 3. antioxidants (e.g. vitamin E), 4. mediators of cell signaling and regulators of cell and tissue growth and differentiation (e.g. vitamin A). 6/13/2008 Vitamin Production (NRM) 2

3 Vitamins may be grouped as follows: The fat-soluble vitamins The water-soluble vitamins Vitamin A Vitamin D Vitamin E Vitamin K Choline Folacin (folic acid) Niacin (nicotinic acid) Panthotenic acid Riboflavin Thiamin Pyridoxine Cobalamin Ascorbic acid 6/13/2008 Vitamin Production (NRM) 3

4 The discovery of vitamins and their sources Year of discovery Vitamin Source Vitamin A (Retinol) Vitamin B 1 (Thiamin)* Vitamin C (Ascorbic acid) Cod liver oil Rice bran Lemons * also produced by microbes 1918 Vitamin D (Calciferol) Cod liver oil 1920 Vitamin B 2 (Riboflavin)* Eggs 1922 Vitamin E (Tocopherol) Wheat germ oil 1926 Vitamin B 12 (Cobalamine)* Liver 1929 Vitamin K (Phyllochinone) Luzerne 1931 Vitamin B 5 (Pantothenic acid)* Liver 1931 Vitamin B 7 (Biotin) Liver 1934 Vitamin B 6 (Pyridoxine)* Rice bran 1936 Vitamin B 3 (Niacin) Liver 1941 Vitamin B 9 (Folic acid)* Liver 6/13/2008 Vitamin Production (NRM) 4

5 Vitamin B 12 (Cyanocobalamin) The term cobalamin is all of them contain cobalt. Corrin is the base (central) structure of cobalamin,, composed of a tetrapyrrole ring (four pyrrole units). Cobalamin can be considered in 3 parts: 1. a central corrin ring 2. a lower ligand (benzimodazole) 3. an upper ligand 6/13/2008 Vitamin Production (NRM) 5

6 Natural forms of cobalamin depending on the upper ligand are: 1. Adenosylcobalamin (coenzyme B 12, AdoCbl) 2. Methylcobalamin (MeCbl) 3. Hydroxycobalamin (OHCbl) Cyanocobalamin (Vitamin B 12 ) is the industrially produced stable cobalamin form, which is a synthetic compound not found in nature. 6/13/2008 Vitamin Production (NRM) 6

7 The biosynthesis of cyanocobalamin is intricate and confirmed to certain members of the prokaryotic worldmembers of the Archaea and certain eubacteria. Animals, humans, and protists require cobalamin but apparently do not synthesize it, whereas plant and fungi are thought to neither synthesize nor use it. Humans require cobalamin between 1-2 g per day. Cobalamin is anti-pernicious anaemia factor. Cobalamin is mainly found in animal products, such as meat, poultry, fish, egg, and milk. The cobalaminproducing bacteria often live in bodies of water and soil, and animals get cobalamin by eating food contaminated with these microorganisms. 6/13/2008 Vitamin Production (NRM) 7

8 The biosynthesis of cobalamin requires somewhere around 70 enzyme-mediated steps involving more than 30 genes for its complete de novo synthesis. In 1993 the Everest Cobalamin was conquered, meaning that all the intermediates on the biosynthetic pathway in Pseudomonas denitrificans were isolated and their structures determined. A genetically engineered highly effective cobalaminproducing strain of P. denitrificans has a productivity that reaches 300 mg/l and accounts for 80% of the cobalamin production in the world. 6/13/2008 Vitamin Production (NRM) 8

9 Flow chart for production of Vitamin B 12 from P. denitrificans P. denitrificans Inoculum cultivation Inoculum cultivation on agar slant with medium contain sugar beet molasses, yeast extract, etc. Preculture in erlenmeyer flask with medium the same as for inoculum cultivation, without agar Preculture Production culture Production in erlenmeyer with medium contain sugar beet molasses, yeast extract, etc. Cobalt and 5,6-dimethyl benzimidazole must be added as supplemen. Betaine is assumed to cause an activation of biosynthesis or an increase in membrane permeability. Sugar beet molasses is used as a low-cost betaine source. 6/13/2008 Vitamin Production (NRM) 9

10 Vitamin B 12 from Propionibacterium shermanii or P. freudenreichii These strains are used in a two stage process with added cobalt. In a preliminary anaerobic phase (2-4 days), 5 -deoxyadenosylcobinamide is mainly produced. In a second, aerobic phase (3-4 days) the biosynthesis of 5,6- dimethylbenzimidazole to produce 5 -deoxyadnosylcobalamine (coenzyme B 12 ) Isolation and Purification Cells are lysed by heat treatment at C for minutes at ph The cells on lysis release various cobalamin. The obtained of cobalamin is converted into cyanocobalamin. The purification of the product is done using adsorption method for substances like amberlite, alumina, silanized silica gel follwed by elution with water-alcohol or water-phenol mixtures. 6/13/2008 Vitamin Production (NRM) 10

11 Vitamin B 2 (Riboflavin, Lactoflavin) Riboflavin (6,7-dimethyl-9-(D- 1 -ribityl)-isoalloxazine is an alloxazine ring linked to alcohol derived from the pentose sugar ribose. The isoalloxazine ring acts as a reversible redox system. Riboflavin has an essential role in the oxidative mehanism in the cell. 6/13/2008 Vitamin Production (NRM) 11

12 Riboflavin is a water-soluble yellow-orange fluorescent pigment, heat-stable in neutral or acid solution, but heating in alkaline solutions may destroy it. It is easily destroyed by light, especially ultraviolet. Humans require cobalamin between 1 mg per day. Deficiency causes ariboflavinosis, characterized by cracked skin and eye problems including blurred vision. Riboflavin is present in milk as free riboflavin, but is present in other foods (liver, heart, kidney, eggs, or leafy vegetables) as part of flavoproteins which contain the protestic groups FMN (flavin mononucleotide) or FAD (flavin adenin dinucleotide). 6/13/2008 Vitamin Production (NRM) 12

13 Riboflavin is produced industrially by several processes: 1. chemical synthesis for pharmaceutical use (20% of world wide production) 2. biotransformation of glucose to D-ribose and subsequent chemical conversion to riboflavin (about 50% of world wide production) 3. direct fermentation (30% of world wide production) Riboflavin is synthesized by many microorganisms, including bacteria, yeasts, and fungi, such as: - Clostridium acetobutylicum (97 mg/l) - Candida flareri (567 mg/l). - Ascomycetes: Eremothecium ashbyii (2480 mg/l) constitutive ribo- Ashbya gossypii (6420 mg/l) flavin-synthesizing enzymes 6/13/2008 Vitamin Production (NRM) 13

14 Production by fermentation of Ashbya gossypii About 30% of the world industrial riboflavin output is produced by direct fermentation with A. gossypii and up to can produce riboflavin up to 15 g/l after 10 days to be the maximum yield. The hypae can accumulate large amounts of riboflavin released from the cells by heat treatment (1 h, C, ph 4.5) the mycelium is separted and discarded riboflavin is then further purified. The fermentation is conducted in four phases: 1. Phase one (the initial rapid growth of A. gossypii) glucose is utilized and pyruvic acid accumulates. 2. Phase two (the production phase) the level of the pyruvate reduces, ammonia in the medium accumulates. 3. Phase three the synthesis of cell bound riboflavin in the form FAD and FMN. 4. Phase four autolysis of the cells release of free riboflavin into medium. 6/13/2008 Vitamin Production (NRM) 14

15 -Carotene (Provitamin A) Carotenoids are not just another group of natural pigments but substances with very special and remarkable properties that no other groups of substances possess. They perform important functions in nature, including light-harvesting, photoprotection, protective and sexrelated coloration patterns in many animal species and as precursors of vitamin A in vertebrates. They may serve protective roles as well against agerelated diseases in humans, being implicated in the prevention or protection against serious human health disorders such as cancer and heart disease. 6/13/2008 Vitamin Production (NRM) 15

16 Carotenoids are found in many animal and plant tissues, but originate exclusively from plants or microbes. -carotene is converted into vitamin A in the intestinal mucous membrane and is stored in the liver as the palmitate ester. Humans require cobalamin between mg per day. 6/13/2008 Vitamin Production (NRM) 16

17 Structures of several carotenoids that can be produced by fermentation Carotenoids are highly unsaturated isoprene derivatives. The conjugated double bond system determines the photochemical properties and chemical reactivity that are the basis of carotenoid biological functions. Only compounds with the -ionone structure (the ring structure found at each end of the -carotene molecule) are effective as provitamin A. Two molecules of vitamin A can be formed from -carotene. Only one molecule of vitamin A can be formed from - and -carotene. 6/13/2008 Vitamin Production (NRM) 17

18 Production processes for -carotene using Blakeslea trispora B. t. (+) B. t.(-) Culture on agar slant Preculture Mixed preculture Production culture Culture on agar slant Preculture Production is induced by trisporic acids (act as (+) gamones/sexual hormones). Activator of -carotene synthesis is isoniazid, in combination with -ionon. The addition of purified kerosene to the medium doubles the yield. The addition of antioxidant to increase the stability of -carotene within the cells. 6/13/2008 Vitamin Production (NRM) 18

19 Creation of novel carotenoid biosynthetic pathways in E. coli. Novel carotenoid structures are in red; red arrows indicate in vitro evolved gene functions 6/13/2008 Vitamin Production (NRM) 19

20 Identification of a novel carotenoid oxygenase leads to the synthesis of novel oxygenated carotenoid structures by recombinant E. coli. Directed evolution of this enzymes creates novel E. coli color phenotypes. ulty/csd/html/research_isoprenoid.htm ( ) 6/13/2008 Vitamin Production (NRM) 20

21 Cyanobacterial carotenoids are tetraterpenoid (C-40) compounds with poly-ene chromophores. There is still no cyanobacterium for which the entire carotenoid biosynthetic pathway has been fully described. Synechococcus sp. PCC 7002 produces seven carotenoids that accumulate to significant amounts during standard exponential growth: -carotene, zeaxanthin, cryptoxanthin, echinenone, hydroxy-echinenone, myxoxanthophyll, and a newly discovered aromatic carotenoid, synechoxanthin. Synechoxanthin, c,c-caroten- 18,18 -dioic acid, is the first aromatic carotenoid to be documented in cyanobacteria. 6/13/2008 Vitamin Production (NRM) 21