Novel vitamin B12-producing Enterococcus spp. and preliminary in vitro evaluation of probiotic potentials.

Vitamin B12 is an essential nutrient required for crucial metabolic processes in humans. Vitamin B12-producing lactic acid bacteria (LAB) have been attracting increased attentions currently because of the generally recognized as safe (GRAS) status. Most of recent studies focused on Lactobacillus, and little is known about B12-producing Enterococcus. In the present study, five Enterococcus strains isolated from infant feces were identified as vitamin B12 producers. Among them, Enterococcus faecium LZ86 had the highest B12 production (499.8 ± 83.7 µg/L), and the B12 compound from LZ86 was identified as the biological active adenosylcobalamin, using reversed phase high-performance liquid (RP-HPLC) chromatogram. We examined basic probiotic and safety properties of E. faecium LZ86 and found that it was able to survive harsh environmental conditions (hot temperature, cold temperature, ethanol and osmotic stresses), tolerate gastric acid (pH 2.0, 3 h) and bile salts (0.3%), and adhere to Caco-2 cells. We also showed that E. faecium LZ86 is devoid of transferable antibiotic resistance and potential virulence factors. Together, here we report a B12-producing E. faecium strain LZ86 firstly, which has desirable probiotic properties and may serve as a good candidate for vitamin B12 fortification in food industry.

Occurrence of 5'-deoxyadenosylcobalamin and its physiological function as the coenzyme of methylmalonyl-CoA mutase in a marine eukaryotic microorganism, Schizochytrium limacinum SR21.

A marine eukaryotic microorganism, Schizochytrium limacinum SR21, had the ability to absorb and accumulate exogenous cobalamin, which was converted to the cobalamin coenzymes 5'-deoxyadenosylcobalamin (20.1%) and methylcobalamin (29.6%). A considerably high activity (about 38 mU/mg protein) of 5'-deoxyadenosylcobalamin-dependent methylmalonyl-CoA mutase (EC 5.4.99.2) involved in amino acid and odd-chain fatty acid metabolism was found in the cell homogenate of S. limacinum SR21. The enzyme was purified to homogeneity and characterized.

Isolation and identification of a cobamide coenzyme from the tapeworm Spirometra mansonoides.

A light-sensitive vitamin B12 derivative has been extracted from the adult cestode, Spirometra mansonoides. This corrinoid was identified as the cobamide coenzyme, adenosylcobalamin, by its chromatographic, chemical, and spectral properties.

Development of a method for the separation of corrinoids in ovine tissues by HPLC.

A method has been developed using a combination of high-performance liquid chromatography (HPLC) and a radioisotope dilution assay (RIDA) to routinely estimate the distribution of corrinoids (the cobalamins hydroxocobalamin, methylcobalamin and 5'-deoxyadenosylcobalamin, and cobalamin analogues) in liver, plasma, milk, intestinal fluid and faeces. Corrinoids were extracted with a sodium acetate buffer, separated by HPLC and quantified by RIDA. Recoveries of corrinoids were 29% for hydroxocobalamin, 50% for 5'-deoxyadenosylcobalamin and 64% for methylcobalamin. The method allows the routine analysis of many samples and maintains good standards of precision.

Isolation of 57Co-cobalamin coenzymes at high specific activity from Streptomyces griseus.

The distribution of radio-labelled cobalamins in Streptomyces griseus grown in medium containing 57Co-cobalt chloride has been estimated by two-dimensional thin-layer chromatography and bioautography. 57Co-Methylocobalamin (Me[57Co]Cbl) was the major form in the mycelium together with smaller amounts of 57Co-adenosylcobalamin (Ado[57Co]Cbl) and 57Co-hydroxocobalamin (OH[57Co]Cbl). The OH[57Co]Cbl was detected in three forms having, respectively, anionic, cationic and neutral properties. A simple technique has been developed to isolate and purify Me[57Co]Cbl and Ado[57Co]Cbl from the mycelium using column chromatography on ion-exchange celluloses. Small quantities of each cobalamin coenzyme have been obtained at 90--96% purity and specific activities of 190--230 muCi/microgram.

A specific radioimmunoassay for 5'-deoxyadenosyl cobalamin in serum.

A specific radioimmunoassay for 5'-deoxyadenosyl cobalamin (ado-Cbl) has been developed using an antiserum raised to this cobalamin (Cbl). At a 1:100 or greater dilution the antiserum did not bind radiolabelled methyl-, hydroxo-, sulphito- or cyano-Cbl and these Cbl analogues did not compete in the radioimmunoassay even at 100-fold higher concentration. The serum concentration (range and mean +/- SD) of ado-Cbl in 30 normal subjects was 47-134 and 81 +/- 16 pg/ml. The corresponding values for total Cbl in these sera were 189-610 and 355 +/- 144 pg/ml, and the computed values for methyl-Cbl were 142-476 and 274 +/- 127 pg/ml. The coefficient of variation was substantially greater for methyl-Cbl than for ado-Cbl (46% v. 21%, respectively). The ado-Cbl concentration was in the normal range (75-95 pg/ml) in five healthy subjects with a low normal concentration (189-217 pg/ml) of total Cbl. In two subjects with low total Cbl (118 and 170 pg/ml) and without any clinical evidence of Cbl deficiency, ado-Cbl was normal (63 and 95 pg/ml, respectively). Thus, in this group, low methyl-Cbl accounted for the lower total Cbl. The concentration (mean +/- SD) of serum ado-Cbl and methyl-Cbl in six patients with low total Cbl (57 +/- 25 pg/ml) and clinical evidence for Cbl deficiency was 35 +/- 12 pg/ml and 22 +/- 22 pg/ml, respectively. This difference from the normal mean for each cofactor was highly significant (P less than 0.001). The decrease in the concentration of methyl-Cbl in Cbl deficiency was relatively greater than the decrease in ado-Cbl (92% v. 57%, respectively) which raised the relative concentration of ado-Cbl in Cbl deficiency to 61% of the total Cbl. Although a low serum methyl-Cbl is a sensitive indicator of Cbl deficiency, it may not be as specific as a decrease in serum ado-Cbl. Factor(s) other than tissue stores of Cbl may lower serum methyl-Cbl below the 95% confidence limit in the absence of clinical Cbl deficiency.

Isolation and analysis of bacterial cobamides by high-performance liquid chromatography.

Cyanocobamides were extracted from diverse bacterial species, purified by XAD-4 and neutral aluminum oxide column chromatography, and separated by isocratic reversed-phase high-performance liquid chromatography (HPLC). Retention times are given for seven cobamide types: dicyanocobinamide (factor B), Co alpha-(alpha-benzimidazolyl)-Co beta-cyanocobamide, Co alpha-(5-hydroxybenzimidazolyl)-Co beta-cyanocobamide (factor III), Co alpha-(5-methoxybenzimidazolyl)-Co beta-cyanocobamide (factor IIIm), Co alpha-(5-methylbenzimidazolyl)-Co beta-cyanocobamide, cyanocob(III)alamin (vitamin B-12) and Co alpha-(naphthimidazolyl)-Co beta-cyanocobamide. Other Co beta-ligandyl-cobamides such as hydroxycobamide and the light-sensitive methyl-, acetyl-, propyl-, and adenosylcobamides were separated by HPLC in a gradient mode. The recovery of total cell cobamide after extraction, purification, and separation was 75-80%. The method was useful in preparative and analytical work. Less than 10 ng cyanocobamide was detectable.

Purification and analysis of cobamides of Methanobacterium bryantii by high-performance liquid chromatography.

Cobamides from Methanobacterium bryantii were purified by isocratic reverse-phase high-performance liquid chromatography at neutral pH in readily extractable buffers. A gradient adaptation of this chromatography system on C-18 muBondapak columns in buffers containing aqueous methanol and 100 mM LiCl readily separated Co alpha-(5-hydroxybenzimidazoyl)-Co beta-cyanocobamide, Co alpha-(5-hydroxybenzimidazoyl)-Co beta-adenosylcobamide, cyanocobalamin, adenosylcobalamin, and methylcobalamin. Aquacobalamin was not resolved from cyanocobalamin by this procedure. The gradient procedure was also useful in following the fate of tritiated cyanocobalamin in cell-free extracts of M. bryantii.

Two new vitamin B-12 factors from sewage sludge containing 2-methylsulfinyladenine or 2-methylsulfonyladenine as base component.

Two hitherto unknown vitamin B-12 factors were isolated from sewage sludge. They were degraded with cerous hydroxide to cobinamide and the corresponding nucleoside. The nucleosides were further split with dilute hydrochloric acid to the bases and D-ribose. The structure of the two bases was found to be 2-methylsulfinyladenine and 2-methylsulfonyladenine. This was revealed by mass, infrared and nuclear magnetic resonance spectroscopy, and by comparison with the synthetic compounds. On addition of the synthetic bases to fermentations with Propionibacterium acidi-propionici the vitamin B-12 factors containing the corresponding base were formed. They were identical with the 2-methylsulfonyladenylcobamide and 2-methylsulfonyladenylcobamide originally isolated from sewage sludge.

Diversity of corrinoids in acetogenic bacteria. P-cresolylcobamide from Sporomusa ovata, 5-methoxy-6-methylbenzimidazolylcobamide from Clostridium formicoaceticum and vitamin B12 from Acetobacterium woodii.

The Co beta-cyanocobamides obtained by cyanide extractions from several acetogenic bacteria were structurally characterized by ultraviolet/visible spectra, proton-nuclear-magnetic-resonance spectra and fast-atom-bombardment mass spectra. p-Cresolycobamide was detected as a major corrinoid from Sporomusa ovata. This 'complete' corrinoid was isolated from an organism for the first time. Instead of the common Co alpha bases of the known and biologically active cobamides, p-cresolylcobamide contained a glycosidically bound cresolyl function that was unable to coordinate to the cobalt of the corrin ring. An additional, previously unknown corrinoid from natural sources, Co alpha-[alpha-(5-methoxy-6-methylbenzimidazolyl)]-Co beta-cyanocobamide, was isolated along with vitamin B12 from Clostridium formicoaceticum. Both homoacetogenic eubacteria were grown on methanol and contained high amounts of corrinoids (greater than 950 nmol/g cell dry mass). Less corrinoid was isolated from Acetobacterium woodii and characterized as vitamin B12.

Identification of phenolyl cobamide from the homoacetogenic bacterium Sporomusa ovata.

Phenolyl cobamide was isolated from cyanide extractions of the anaerobic eubacterium Sporomusa ovata. The proposed corrinoid structure [Co alpha,Co beta-(monocyano,monoaquo)-phenolyl cobamide] has been deduced from 1H NMR, fast-atom-bombardment mass spectroscopy and ultraviolet/visible spectroscopy data. The complete corrinoid resembled p-cresolyl cobamide [Co alpha,Co beta-(monocyano,monoaquo)-p-cresolyl cobamide], which recently has been obtained from cyanide extractions of the same bacterium. The structures and chemical properties of both cobamides with uncoordinated nucleotides differed significantly from those of vitamin B12 [Co alpha-[alpha-(5,6-dimethylbenzimidazolyl)]-Co beta-cyanocobamide]. Sporomusa synthesized coenzymes of phenolyl cobamide and p-cresolyl cobamide in considerable amounts of 400 nmol/g and 1700 nmol/g dry cells, respectively. More than 90% of the complete corrinoid pool of the homoacetogenic bacterium consisted of these two corrinoids, indicating that they are physiologically important coenzymes of the bacterial metabolism.

Purification of the coenzyme B12-containing 2-methyleneglutarate mutase from Clostridium barkeri by high-performance liquid chromatography.

Two methods are described by which the enzymes 2-methyleneglutarate mutase and 3-methylitaconate delta-isomerase from Clostridium barkeri have been separated by high-performance liquid chromatography on a much larger scale than reported previously. First, the mutase eluted before the delta-isomerase after incubation with the mild detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS) followed by high-performance anion-exchange chromatography on Mono Q in the presence of the same detergent. Second, an even better separation, although with a lower yield of mutase, was obtained by hydrophobic interaction chromatography on phenyl-Sepharose HiLoad, whereby the enzymes were eluted in the reverse order. Final high-performance anion-exchange chromatography of the latter preparation on Mono Q at pH 8 gave highly purified 2-methyleneglutarate mutase (greater than 95% purity) which had a pink-orange colour (lambda max 280, 375, 470 and 532 nm). The enzyme was active in the absence of coenzyme B12 (adenosylcobalamin) and contained 2.1 mol of this coenzyme per homotetramer (molecular mass, m = 300 kilodalton).

Purification and characterization of dimethylamine:5-hydroxybenzimidazolyl-cobamide methyltransferase from Methanosarcina barkeri Fusaro.

Dimethylamine:5-hydroxybenzimidazolylcobamide methyltransferase (DMA-MT) was purified from cells of Methanosarcina barkeri Fusaro grown on trimethylamine. In the presence of methylcobalamine:coenzyme M methyltransferase isoenzyme II [MT2(II)] the enzyme quite specifically catalyzed the stoichiometric conversion of dimethylamine (apparent Km = 0.45 mM) and 2-mercaptoethane-sulfonate (coenzyme M) to monomethylamine and methyl-coenzyme M. Monomethylamine was a competitive inhibitor of the reaction (Ki = 4.5 mM). The apparent molecular mass of DMA-MT was 100 kDa and the enzyme was found to be a dimer, composed of identical 50-kDa subunits. A corrinoid content of 0.9 +/- 0.1 mol B12/mol holoenzyme was calculated from HPLC analysis. The as-isolated methyltransferase was inactive, but it could be reductively reactivated. Activation required the presence of methyltransferase-activating protein, ATP and dimethylamine. Incubation with these compounds resulted in the methylation of the corrinoid prosthetic group.