Vitamin B12 ELISA Kit

Background: As a very important coenzyme in the cell metabolism, Vitamin B-12 (cobalamin, VB12) has been widely used in food and medicine fields. The complete biosynthesis of VB12 requires approximately 30 genes, but overexpression of these genes did not result in expected increase of VB12 production. High-yield VB12-producing strains are usually obtained by mutagenesis treatments, thus developing an efficient screening approach is urgently needed. Result: By the help of engineered strains with varied capacities of VB12 production, a riboswitch library was constructed and screened, and the btuB element from Salmonella typhimurium was identified as the best regulatory device. A flow cytometry high-throughput screening system was developed based on the btuB riboswitch with high efficiency to identify positive mutants. Mutation of Sinorhizobium meliloti (S. meliloti) was optimized using the novel mutation technique of atmospheric and room temperature plasma (ARTP). Finally, the mutant S. meliloti MC5-2 was obtained and considered as a candidate for industrial applications. After 7 d's cultivation on a rotary shaker at 30 degrees C, the VB12 titer of S. meliloti MC5-2 reached 156 +/- 4.2 mg/L, which was 21.9% higher than that of the wild type strain S. meliloti 320 (128 +/- 3.2 mg/L). The genome of S. meliloti MC5-2 was sequenced, and gene mutations were identified and analyzed. Conclusion: To our knowledge, it is the first time that a riboswitch element was used in S. meliloti. The flow cytometry high-throughput screening system was successfully developed and a high-yield VB12 producing strain was obtained. The identified and analyzed gene mutations gave useful information for developing high yield strains by metabolic engineering. Overall, this work provides a useful high-throughput screening method for developing high VB12-yield strains.

In this paper, the electrochemical behavior of vitamin B-12, ie cyanocobalamin (abbr. VB12) in a weak acidic aqueous solution and adsorbed on glassy carbon (GC) surface (abbr. VB12(ad)/GC) in different pH buffer solutions have been described by using cyclic voltammetry (cv). It is found that VB12 and VB12(ad)/GC exhibit catalytic activity for the electroreduction of O2 according to two reduction peaks at -0.50 and -1.00 V vs. sce; but their electrocatalytic activity is very unstable. Based on the method of hydrodynamic amperometry [B. Miller and S. Bruckenstein, J. electrochem. Soc. 117, 1033 (1970)], some kinetic parameters for the electrocatalytic reduction of O2 by VB12(ad)/GC have been determined rapidly by using a linear rotation-scan method [Rongzhong Jiang and Shaojun Dong, Electrochim. Acta 35, 1451 (1990)]. These kinetic parameters indicate that the reduction of O2 on VB12(ad)/GC gives water predominantly in both potential ranges which correspond to those two reduction peaks. Possible reaction mechanisms have been suggested.