Microbial production of vitamin B12 by methanol utilizing strain of

Pak J. Biochem. Mol. Biol., (2007); 40(1): 5-10. Microbial production of vitamin B12 by methanol utilizing strain of Pseudomonas specie Muhammad Riaz1, Zumir Ahmed Ansari1, Fouzia Iqbal2 and Muhammad Akram3 1 Department of Chemistry, University of Engineering and Technology, Lahore 2 PCSIR Laboratories Complex, Lahore 3 Department of Botany, University of the Punjab, Lahore, PAKISTAN Abstract. A gram-negative strain of Pseudomonas specie PCSIR-B-99 with profound ability of methanol utilization as carbon and energy source was taken from Pakistan Type of Culture Collection Laboratory. This strain was found to be an excellent producer of vitamin B12 on a modified basal medium. The effect of time course of fermentation, different concentrations of methanol and cobalt ions on the growth as well as vitamin B12 production of indigenously isolated bacteria was investigated. Maximum bacterial cell mass and vitamin B12 production was observed in medium containing 3.5% (v/v) methanol and 1.0 mg/L of Co++ ions concentration after 72 hours of fermentation. However, the yield of vitamin B12 was further enhanced up to 3500µg/L of medium by the addition of 200 mg/L of 5, 6-dimethybenzimidazole, the precursor of vitamin B12, as an optimum concentration. Key words: 5, 6-dimethybenzimidazole, Methanol utilizing bacterium, Vitamin B12, Fermentation. INTRODUCTION One of the most alluring and fascinating molecules in the world of science and medicine is vitamin B12 (cobalamin), which was isolated from liver extract in 1948 and reported to control pernicious anemia1. Its structure was elucidated in 1955. Chemically, vitamin B12 is a molecule with the formula of C63H90CoN14O14P. Cobalamin is the generic name of vitamin B12 because it contains the heavy metal cobalt, which gives this water-soluble vitamin its red color. Vitamin B12 is an essential growth factor and plays a role in the metabolism of cells, especially those of the gastrointestinal tract, bone marrow, and nervous tissue2. In addition to this vitamin B12 has been used in modern research of being an essential component of medium for cell line growth3. Several different cobalamin compounds exhibit vitamin B12 activity. The most stable form is cyanocobalamin, which contains a cyanide group that is well below toxic levels4. To become active in the body, cyanocobalamin must be converted to either methylcobalamin or adenosylcobalamin5. A protein in gastric secretions called intrinsic factor binds to vitamin B12 and facilitates its absorption. Without intrinsic factor, only a small percentage of vitamin B12 is absorbed. Once absorbed, relatively large amounts of vitamin B12 can be stored in the liver. In humans, the vitamin B12 is required in trace amounts (approximately 1µg/day) to assist the actions of only two enzymes, methionine synthase and -methylmalonylCoA mutase6; yet commercially more than 10 tons of B12 are produced each year from a number of bacterial species. Streptomyces griseus, a bacterium once thought to be yeast, was the commercial source of vitamin B12 for many years4. Since the chemical synthesis of vitamin B12 requires more than 70 steps7,8, the chemical synthesis of vitamin B12 on industrial scale is in principle very complicated and expensive. Therefore, vitamin B12 has been produced intracellularly or extracellularly on an industrial scale using the batch or fedbatch process of microbial fermentation9. Several microorganisms, including those of the genera Bacillus, Methanobacterium, Propionibacterium, and Pseudomonas, have been used to produce vitamin B12 on an industrial scale. Since some species of Propionibacterium have been granted GRAS (generally recognized as safe) 6 Riaz et al. status by the United States Food and Drug Administration and produce neither endotoxins nor exotoxins10, they are the preferred species for the production of vitamin B12. In current investigation, production of vitamin B12 was studied by a bacterial strain of Pseudomonas specie. The strain grows well on synthetic medium with methanol as a best source of carbon in aerobic condition and produce vitamin B12 with larger yield under optimized fermentation conditions. MATERIALS AND METHODS Microorganism The bacterial strain of pseudomonas specie was taken from Pakistan Type of Culture Collection (PTCC), Pakistan Council of Scientific and Industrial Research Laboratories Complex Lahore, Pakistan and used as good producer of vitamin B12. The strain was grown at 30oC and maintained on nutrient agar slants and was sub-cultured with four week intervals. Inoculum Preparation Inoculum was prepared by transferring 5 mL suspension prepared from 24 hours old slant culture, into Erlenmeyer flask containing 45 mL of sterile inoculum medium. The composition of medium was NH4PO4, 0.2%; KH2PO4, 0.2 %; CaCl2.2H2O, 0.001%; FeSO4.7H2O, 0.003%; MnSO4.nH2O, 0.002g; CoSO4.7H2Og, 0.005% and 0.5 mL methanol per 50 mL11. Fermentation Studies Fifty milliliters of 24 hours aged inoculum at the ratio of 10 % (v/v) was added to one liter of production medium in 2.0 liters glass jar fermentor having working volume of 1.0 liter. The fermentation medium used contained (g/L) NH4PO4, 2g; KH2PO4, 2g; CaCl2.2H2O, 0.01g; FeSO4.7H2O, 0.005g; MnSO4.nH2O, 0.005g; CoSO4.7H2O, 1.0 mg. The methanol was used as a carbon source with various concentration ranges from 0.5% to 4.5% (v/v). The pH of the cultures was maintained at 7.0 with a pH controller (metrohm AG, Herisau, Switzerland), using 3.0 M NaOH and 1.0 M HCl. Fermentation was carried out at 30oC for 5 days. The rate of aeration was kept at 1 liter/liter/minute. Growth of the bacterium and vitamin B12 production was measured at different methanol and cobalt ion concentration after 12 hours interval. Biomass Estimation An aliquot of 50 mL of culture sample was centrifuged (Backman; T2-HS centrifuge with rotor JA-20) at 7740 x g and 4oC for 15 minutes to collect the cells12. The cell free culture broth was stored for estimation of vitamin B12. The cells were washed with sterilized distilled water. The pellet was then desiccated in an electric oven (D 06060. Model 400; Memmert) at 105oC until constant weight was achieved. Extraction of Vitamin B12 The extraction of vitamin B12 was done by harvesting the cells from fermentation broth and centrifuging at 10000 rpm. The pellets were washed with 0.2 M potassium phosphate buffer (pH 5.5) and suspended in the same buffer containing 0.1 % KCN. The suspension was autoclaved for 15 minutes and 121oC. The supernatant containing extracted vitamin B12 was filtered through a cellular acetate membrane filter 0.2 µm. Estimation of Vitamin B12 The vitamin B12 was estimated microbiologically using E. coli 215 organism13. This assay was also used to differentiate between physiologically active form of vitamin B12 that can be used by human and analogous form of vitamin B12. All vitamin B12 assays were carried out in triplicate at three different dilutions, each was analyzed 5 times. The maximum standard deviation was found to be ± 6 %. Microbial production of vitamin B12 7 RESULTS AND DISCUSSION Effect of Fermentation Time course on Growth and Vitamin B12 Production Figure 1 shows the effect of fermentation time course on growth and vitamin B12 production. After 24 hours there was no marked increase in the cell mass of the bacterium, which shows lag phase of the bacteria. After this, bacteria started multiplying and growth reached maximum at 72 hours indicating the exponential phase of the growth. The cell mass remained constant up to 96 hours of the fermentation medium that seams to be stationary phase. The decrease in biomass indicates after 96 hours indicates the death phase of bacterium. From 12 to 24 hours of fermentation there was no any synthesis of vitamin B12. It was because of exclusively low growth of bacteria, however maximum growth and yield of vitamin B12 was observed at 72 hours (Figure 1). The concentration of vitamin B12 was high since there is high rate of multiplication of cells involving reduction of ribose nucleotides triphosphate to 2deoxyribose nucleotide triphosphate. This conversion was catalyzed by adynosylcobalamin14,15,16. Late exponential growth phase was occurring with higher intracellular yield of vitamin B12. This would be due to high requirement of co-enzyme dependent redox reactions taking place within the cell17. Effect of Methanol Concentration on Growth and Vitamin B12 Production Figure 2 indicates that methanol concentration had pronounced effect on the growth of bacterium and vitamin B12 production. Bacteria grew rapidly in the medium containing methanol at an initial concentration 2.75 % v/v. However, growth was markedly inhibited at the concentration of 4.5 %. The vitamin B12 synthesis was found to be high (815 µg/L of the medium) at elevated methanol concentration. This indicated that although high concentration of methanol had inhibitory effect on growth of bacterium but was quite good for vitamin B12 biosynthesis. Moreover, once corrinoids are formed, they quickly involve in methyl transfer from methanol to methyl coenzyme M18,19, the common precursor of methane from all substrates. However, the content of corrinoids in anaerobic bacteria varies greatly among species and substrate utilized but is always higher when cells are grown on methanol20. Effect of Co++ Concentration on Growth and Vitamin B12 Production Since vitamin B12 contains Co++ coordinated by corrin ring, the effect of various concentration of cobalt (II)sulfate-heptahydrate was studied on the growth and vitamin B12 production. Figure 3 indicates that vitamin B12 synthesis was dependent on cobalt concentration and maximum production of vitaminb12 was obtained at 1.0 mg/L of cobalt concentration whereas there was no discernible effect on the growth of the bacterium. However, concentration of the cobalt, higher then this limit had some detrimental effect on the growth of bacteria (Figure 3). 450 Vitamin B12 µg /g dry cell 400 Vitamin B12 µg /liter Growth g dry cell /liter 2 2.5 350 300 1.5 250 200 1 150 100 0.5 50 0 0 12 24 36 48 60 72 84 96 108 0 120 Fermentation growth time course (hours) FigureEffect of fermentation time course on growth and vitamin growth and Fig. 1. 1: Effect of fermentation time course on B12 production. vitamin B12 was cultivated for 120 hours at pH 7.0 and temperatue 30 degree production. The bacterium was cultivated for The bacterium 120 hrsaeration rate(pH 7.0) with aeration rate 1L/L/min. C with at 30? C 1 liter/ liter/ minute This might be due to some toxic effect of Co++ on protein synthesis of bacterial 21. The requirement of cobalt ion for vitamin B12 synthesis was evident that Growth (g dry cells/ liter) Vitamin B12 production 8 Riaz et al. cobalt is the central atom in corrinoids present in all methanogens and acetogens22 and a number of corrinoiddependent reactions are known to take place in the intermediary metabolism of substrates by methanogens and acetogens23. Thus, the high requirement for cobalt found in current investigation is likely to be due to the production of unique corrinoid containing enzymes or coenzymes that are only present in methylotrophs. In methanogens, corrinoids are involved in methyl transfer from methanol to methyl coenzyme M18,19, the common precursor of methane from all substrates. In acetogens, corrinoids participate in the formation of acetyl coenzyme A, the precursor intermediate of acetate and cell synthesis23. However, the content of corrinoids in anaerobic bacteria varies greatly among species and substrate utilized but is always higher when cells are grown on methanol20. The initial step of methanol conversion in methanolconsuming bacteria, such as the methanogen Methanosarcina barken and the acetogen eubacterium limosum, proceeds in a similar way and is catalyzed by an additional corrinoid-containing enzyme known as methyltransferase24. Recently, an induced corrinoid-containing protein was reported to occur only in methanol-grown cells of an acetogenic bacterium Sporomusaovata25. 4000 Vitamin B12 Production µg /g dry cell Vitamin B12 Production µg /liter Growth g dry cell /liter 2 2.5 900 Vitamin B12 µg /g dry cell 800 Vitamin B12 µg /liter Growth g dry cell /liter 2.5 700 2 600 Vitamin B12 production 1.5 500 400 1 300 200 0.5 100 0 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 0 Initial methanol concentration (% v/v) Figure 2: Effect of initial methanol concentration on growth Fig.and vitamin B12 production. growth and vitamin B12was cultivated 2. Effect of initial methanol concentration on The bacterium production. The bacteruim120cultivated for 120 hours at7.0) with aeration rateC with aeration rate 1 for was hrs at 30? C (pH pH 7and temperature 30 degree 1L/L/min. liter/ liter/ minute. 1200 V itamin B12 µg /g dry cell V itamin B12 µg /liter 1000 Growth g dry cell /liter 2 2.5 Growth (g dry cells/ liter) 3500 800 Growth (g dry cells/ liter) 3000 Vitamin B12 production 1.5 Vitamin B12 production 2500 1.5 600 2000 1 1 400 1500 1000 0.5 500 0.5 200 0 0 50 100 150 200 250 300 350 400 450 0 500 5,6-dimethyl bemzimidazole (mg/liter) 0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 0 1.8 Co(II)-sulfate-heptahydrate concentration (mg/liter) Fig. Figure 5,6-dimethyl benzimidzol on growth and vitamin B12 production.growth 4. Effect of 4: Effect of 5,6-dimethybenzimidazole on The and cultivated B12 production. The bacterium was bacterum wasvitaminfor 120 hours at pH 7 and temperature 37 degree C with aeration rate 1liter/ liter/ minute 120 hrs at 30? C (pH 7.0) with aeration rate cultivated for 1L/L/min. Figure of Cobalt(II)- sulfate-heptahydrate concentration on growth and vitamin B12 Fig. 3. Effect 3: Effect of Cobalt (II)-sulfate-heptahydrate concentration on growthforandhours at pH 7and temperature 37 degree C with production. The bacterium was cultivated 120 vitamin B12 production. The aeration rate 1 was minute. bacteriumliter/ liter/cultivated for 120 hrs at 30? C (pH 7.0) with aeration rate 1L/L/min. Growth (g dry cells/ liter) Microbial production of vitamin B12 9 Effect of 5, 6-dimethybenzimidazole on Growth and Vitamin B12 Production Independent of the employed production strains and culture conditions, it seems to be necessary to add some essential compounds to the medium for efficient biosynthesis of vitamin B12. The 5, 6- dimethylebezimidazole is believed to be a precursor of vitamin B12 utilized in the vitamin B12 biosynthesis in all living organisms. Since the addition of this precursor bypasses several of the biosynthetic reactions required for its formation as a precursor molecule for proficient biosynthesis of vitamin B12, so its addition to the medium offer a theoretical advantage. Figure 4 indicates that the yield of incorporation of 5, 6dimethybenzimidazole into vitamin B12 was very high and 3500 µg /L of vitamin B12 was obtained at its concentration of 200 mg/L of growth medium without having significant affect on the number cells. However increase in its concentration had limited the growth of bacterium and hence lower the over all production of vitamin B12. The 5, 6dimethybenzimidazole stimulates the production of vitamin B12 by Propionibacterium freudenreichii at concentrations which is subinhibitory for growth. The stimulatory effect of the compound depended not only on their concentration, but also on the time of addition. 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