The Study Of Recombinant Expression And Intracellular Activities Of Bacillus Subtilis CobA And NasF

S-adenosylmethionine-dependent uroporphyrinogen methyltransferase (SUMT) is abranchpoint enzyme which plays a key role in the biosynthesis of modified tetrapyrroles bycontrolling flux to compounds such as siroheam and vitamin B12. Recombinant expressioncells of SUMT in E.coli can emit red fluorescence under UV light illumination. The redfluorescence substance is the by-product from SUMT catalytic process, so fluorescenceintensity can reflect SUMT intracellular catalytic activity. Over-expression of SUMT cando help for VB12production. As a red fluorescence reporter protein, SUMT can be used todetect the intracellular activities downstream. The genes encoding SUMT representpolymorphism and they are cobA, cobA+hemD and cysG respectively. There are cobA andcobA+hemD, which are not reported yet until now in Bacillus subtilis. The genecobA+hemD of Bacillus subtilis was named nasF in this paper. The fluorescence intensityand intracellular activities of CobA and NasF were studied respectively in this paper. Theresults are as follows:1. The genes cobA and nasF were cloned, alignment of amino acid suggested that therewere both cobA and hemD domain in nasF.2. The expression vectors of CobA and NasF were constructed and red fluorescence wasobserved in recombinant cells.3. The UV scanning spectrum of the recombinant cell lysate supernatant with CobA andNasF suggested that there was a maximal peak in354nm and secondary peak in378nm.The fluorescence scanning spectrum suggested that the excitation peak was357nm, theemission peak was620nm.4. Quantitative analysis suggested that the fluorescence intensity of NasF recombinantcell is higher than CobA.5. The effect of IPTG concentration gradient to fluorescence was studied. The resultsdetected that0.5mM IPTG was the best concentration in this study; when lower than0.5mM, the fluorescence was low; while higher than0.5mM, the strains began degradation.6. Exogenous betaine was added to culture fluid when induced by IPTG and effect ofbetaine to fluorescence was analyzed. The results suggested that betaine had no help topromote fluorescence intensity.7. CobA and NasF were purified respectively with Ni-NTA, SDS-PAGE suggested theycan be purified and the protein subunit molecular weight was28kD and55kD respectively.In summary, both CobA and NasF have methylation enzyme properties andfluorescence intensity of NasF recombinant cell is higher than CobA. Exogenous betaineadded was no obviously help for promoting fluorescence intensity.