Antibacterial Mechanism Of Vitamin B₁₂ Analogues In Food Microbes

As a coenzyme,vitamin B12 is involved in many important metabolic activities of microorganisms in the form of adenosylcobalamin and methylcobalamin,so vitamin B12 is considered as an important resource that as cross-feeding metabolites among microorganisms to achieve the regulation of microbial growth and metabolic activities.Studies have found that vitamin B12 analogue C-lactone has significant inhibition to E.coli,but the regulation and antibacterial mechanism of vitamin B12 analogues in food microorganisms has not been studied systematically.Therefore,the inhibitory effect and mechanism of vitamin B12 and its analogues on common food microorganisms are crucial research and application for the development of novel antibacterial agents.Vitamin B12 and its analogues were used as research objects to investigate the influence on the growth of food microorganisms and its mechanism.We successfully obtained vitamin B12 analogues by chemical modification.In experiments,MeCbl-C-10-Br was found to have a significant inhibitory effect on Listeria monocytogenes.In order to investigate the antibacterial mechanism of MeCbl-C-10-Br,we studied the vitamin B12-dependent enzyme and riboswitch in turn.The main research contents and results are as follows:1)Reaction of cyano/methylcobalamin with a halogenating agent under different reaction conditions to obtain structurally similar vitamin B12 analogues CN-y-lactone and MeCbl-C-10-Br.The preferred reaction conditions were as follows:the ratio of the reaction material cyanocobalamin/methylcobalamin and halogenating agent is from 1:1 to l:1.2(mole based),the reaction temperature was 30 ?,and the reaction was stirred under weak acid conditions for 2.5 h.The reaction product and the four vitamin B12 were purified by SPE cartridge then detected by HPLC,followed by obtaining the optimised HPLC analysis conditions:column;C18 4.6*250 mm,mobile phase:0.1%formic acid aqueous solution-acetonitrile(A-B),column temperature:25 ?,flow rate:1.0 mL/min,injection volume:20 ?L,detection wavelength:361 nm,elution gradient:0-5 min,90%A,5-25 min,70%A,25-30 min,30%A,30-40 min,90%A.The products and vitamin B12 were structurally verified by LC-MS.2)96-well plate was used to measure the growth curve of Escherichia coli,Listeria monocytogenes,Staphylococcus aureus,Salmonella enterica,Bifidobacterium infantis and Lactobacillus reuteri.Compared the effect of CN-?-lactone,MeCbl-C-10-Br,cyanocobalamin,methylcobalamin on the above bacteria.The experimental results showed that MeCbl-C-10-Br inhibited the growth of gram-positive bacteria Listeria monocytogenes,Staphylococcus aureus,and Lactobacillus reuteri,and promoted the growth of Escherichia coli,but has no effect on Salmonella enterica and Bifidobacterium infantis.The CN-y-lactone slightly inhibited the growth of Staphylococcus aureus,Listeria monocytogenes and Lactobacillus reuteri,and had no significant effect on Escherichia coli,Salmonella enterica,and Bifidobacterium infantis.Compared with CN-y-lactone,the inhibitory effect of MeCbl-C-10-Br is more obvious,especially for Listeria monocytogenes.The inhibitory effect of CN-y-lactone and MeCbl-C-10-Br on Staphylococcus aureus,Listeria monocytogenes and Lactobacillus reuteri was best in the neutral environment by changing the pH value of the medium,otherwise the bacteria cannot grow normally or results show no difference.Changed the concentration of MeCbl-C-10-Br to detect the MIC for harmful bacteria Staphylococcus aureus and Listeria monocytogenes.The results showed that the MICs were 1 ng/mL and 0.125 ng/mL,respectively,indicating that the vitamin B12 analogue MeCbl-C-10-Br was more effective against Listeria monocytogenes.3)To investigate the effect of vitamin B12 and its analogues on the enzymatic activity of the vitamin B12 dependent enzyme by using the crude enzyme solution system of the adenosylocobalamin-dependent enzyme glycerol dehydratase and the methylcobalamin-dependent enzyme methionine synthase.The results showed that the catalytic activity of vitamin B12 and its analogues from high to low was MeCbl-C-10-Br,CN-?-lactone,methylcobalamin,cyanocobalamin and coenzyme adenosylcobalamin in the glycerol reaction system,indicating that all vitamin B12 and its analogues can catalyse the dehydration reaction of glycerol to Acrylic aldehyde.In the methionine reaction system,each reaction produced methionine,indicating that in the crude enzyme solution in vitro,all five analogues can act as co-enzyme to catalyse the reaction,and the catalytic activity of MeCbl-C-10-Br was the strongest.The results showed that MeCbl-C-10-Br has a higher catalytic activity for glycerol dehydratase and methionine synthase than methylcobalamin,which is contrary to the inhibition of MeCbl-C-10-Br on the growth of Listeria monocytogenes.It suggested that the antibacterial effect of MeCbl-C-10-Br is not through the regulation of vitamin B12 dependent enzyme activity.4)Used bioinformatics methods to search for the target gene of vitamin B12 riboswitch related to transporter protein,synthetic protein as well as functional protein,then constructed the recombinant B12 riboswitch-p519ngfp vector plasmid.The synthetic target gene was constructed on the pUC57 vector plasmid.After the PCR,the vitamin B12 riboswitch gene sequence with restriction enzyme sites was obtained,and a large number of target gene fragments with sticky ends were obtained by double enzyme digestion.The p519ngfp vector plasmid was extracted from the expanded E.coli DH5a using a kit,and double-enzyme digested to obtain a linear plasmid.The target gene fragment having the same sticky end and the linear vector plasmid were recombined to obtain a recombinant vector plasmid.5)The riboswitch is a non-expression fragment located at the 5'end of the mRNA,with its structural changes having the ability to regulate protein expression.This study evaluated the effects of different concentrations of cyanocobalamin,methylcobalamin,CN-?-lactone and MeCbl-C-10-Br on the vitamin B12 riboswitch of Listeria monocytogenes.For the riboswitch associated with the vitamin B12 transporter,CN-y-lactone cobalamin had a promoting effect.but cyanocobalamin,methylcobalamin,and MeCbl-C-10-Br showed inhibition,and high concentration ofMeCbl-C-10-Br had a stronger inhibitory effect on transporter-type riboswitch than cyanocobalamin and methylcobalamin.For the riboswitch associated with the vitamin B12 synthesis protein,cyanocobalamin and MeCbl-C-10-Br had inhibitory effect,and methylcobalamin had a promoting effect,but CN-?-lactone cobalamin had no obvious regulation.The riboswitch related to the vitamin B12 functional protein,methylcobalamin and MeCbl-C-10-Br showed significant inhibitory effect,and the latter had stronger inhibition.Cyanocobalamin showed a more pronounced promoting effect,while the CN-y-lactone did not have a significant promoting effect.The results indicated that MeCbl-C-10-Br has a significant inhibitory effect on the riboswitch associated with the vitamin B12 transporter,the synthetic protein and the functional protein along with methylcobalamin has a catalytic effect on the synthesis of the related vitamin B12 riboswitch but showed an inhibitory effect on the function-related riboswitch,and the intensity was weaker than that of MeCbl-C-10-Br,It indicated that antibacterial site of vitamin B12 analogue MeCbl-C-10-Br on Listeria monocytogenes is in vitamin B12 riboswitch.In summary,the vitamin B12 analogue MeCbl-C-10-Br had a significant inhibitory effect on Listeria monocytogenes,and the mechanism of inhibition was not by regulating the enzyme activity of vitamin B12 dependent enzyme glycerol dehydrate and methionine synthase,but by inhibiting the riboswitch of vitamin B12,thus inhibiting the expression of related protein,and further affecting the normal growth of Listeria monocytogenes.