在线微生物浊度法测定多维元素片中微量维生素B_(12)的含量

目的:建立在线微生物浊度法测定多维元素片中微量维生素B12的含量。方法:以莱士曼氏乳酸杆菌为实验菌,以缺乏维生素B12的培养基为检测培养基,采用浊度法在线检测。结果:维生素B12浓度在0.02～0.10 ng.mL-1范围内呈现良好线性关系(r=0.9985),平均回收率为101.8%。结论:该方法快速、简便、灵敏,在线检测结果准确可靠,适用于多维元素片中微量维生素B12的含量测定。     

VITAMIN B12 ANALOGUES AND INTESTINAL BACTERIA

Bacterial production of analogues of vitamin B₁₂, by small-bowel bacteria results in significant loss of vitamin B₁₂ to the host. Bacterial production of cobamides occurs de novo as well as from vitamin B₁₂ bound to intrinsic factor.     

Anaerobic biosynthesis of the lower ligand of vitamin B12

Vitamin B₁₂ (cobalamin) is required by humans and other organisms for diverse metabolic processes, although only a subset of prokaryotes is capable of synthesizing B₁₂ and other cobamide cofactors. The complete aerobic and anaerobic pathways for the de novo biosynthesis of B₁₂ are known, with the exception of the steps leading to the anaerobic biosynthesis of the lower ligand, 5,6-dimethylbenzimidazole (DMB). Here, we report the identification and characterization of the complete pathway for anaerobic DMB biosynthesis. This pathway, identified in the obligate anaerobic bacterium Eubacterium limosum, is composed of five previously uncharacterized genes, bzaABCDE, that together direct DMB production when expressed in anaerobically cultured Escherichia coli. Expression of different combinations of the bza genes revealed that 5-hydroxybenzimidazole, 5-methoxybenzimidazole, and 5-methoxy-6-methylbenzimidazole, all of which are lower ligands of cobamides produced by other organisms, are intermediates in the pathway. The bza gene content of several bacterial and archaeal genomes is consistent with experimentally determined structures of the benzimidazoles produced by these organisms, indicating that these genes can be used to predict cobamide structure. The identification of the bza genes thus represents the last remaining unknown component of the biosynthetic pathway for not only B₁₂ itself, but also for three other cobamide lower ligands whose biosynthesis was previously unknown. Given the importance of cobamides in environmental, industrial, and human-associated microbial metabolism, the ability to predict cobamide structure may lead to an improved ability to understand and manipulate microbial metabolism.Vitamin B₁₂ (cobalamin) is required by the majority of animals, protists, and prokaryotes, although B₁₂ and other cobamide cofactors are synthesized exclusively by a subset of prokaryotes (1). Cobalamin is a member of the cobamide family of cofactors that are composed of a central cobalt ion coordinated by a tetrapyrrolic corrin ring, an upper ligand, and a lower ligand covalently tethered to the corrin ring by a nucleotide loop (Fig. 1A) (1). The lower ligand of cobalamin is 5,6-dimethylbenzimidazole (DMB). Purines, phenolic compounds, and other substituted benzimidazoles have also been found as cobamide lower ligands (2).Open in a separate windowFig. 1.Vitamin B₁₂ structure and the labeling pattern of DMB. (A) Structure of cyanocobalamin (vitamin B₁₂). Cbi and the lower ligand DMB are indicated. (B) AIR is a branch point in the biosynthesis of thiamin (8), purines (27), and benzimidazoles (as shown in this study). The symbols represent the origins of atoms in each product: ●, formyltetrahydrofolate; #, glutamine; *, glycine; shaded circle, erythrose 4-phosphate or threose; ▪, methionine.Distinct oxygen-requiring (aerobic) and oxygen-sensitive (anaerobic) cobamide biosynthesis pathways have been characterized, and each contains ∼30 genes (1). The biosynthetic pathway for DMB remained elusive until the discovery of the bluB gene, the product of which fragments reduced flavin mononucleotide (FMNH₂) to produce DMB in an oxygen-dependent reaction (3–6). Currently, the only unidentified genes in the cobamide biosynthesis pathway are those required for the anaerobic biosynthesis of DMB and other benzimidazoles (7). Based on previous isotope-labeling studies, performed mainly in the anaerobic bacterium Eubacterium limosum, the biosynthesis of DMB was proposed to branch from the purine biosynthetic pathway, similar to the pyrimidine ring of the cofactor thiamin pyrophosphate (Fig. 1B) (2, 8–15). Here, we sought to identify the genes in Eubacterium limosum required for the biosynthesis of DMB.