Research Of Bifidobacterium Longum NCC2705 Fructose Metabolism

The strain NCC2705 of Bifidobacterium longum can utilize undigested fructose and oligosaccharide palys the role of scarvagte. The study aimed to answer the question of how fructose has been transported, utilized and regulated within B.longum NCC2705. At the same time, its adaptation within gut will be defined at the proteomic level. Proteomics and genomics were exploited for the research of B. longum NCC2705 fructose metabolism.Firstly, Immobilized pH gradient (IPG) strips were used at the first step of the 2-dimensional electrophoresis, with the proteins from the cells of B.longum NCC2705 grown on fructose as samples. All the enzymes presented during the process of glucose degradation (bifido shut) were identified by matrix-assisted desorption ionization - time of flight (MALDI-TOF MS). The seven enzymes existed during the degradation of fructose indicate that fructose was metabolized with the same pathway as glucose at the level of proteomics. Secondly, differently expressed proteins were analyzed by the ImageMaster 2D Platinum 5.0 on the sodium codicil sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with reference of proteins from the cells grown on glucose. And only which expression amount on fructose higher than on glucose as higher as 3 times were termed as different one. 18 proteins were identifdied by electrospray ionization tandem mass spectrometer (ESI–MS/MS). Meanwhile, reverse transcript polymerase chain reaction (RT-PCR) determined 6 genes which encoded different proteins. They were BL0033,BL0034,BL0715,BL1339,BL1340 and BL1341 correspondingly, and all of them were located on the chromosome. Especially, the sugar-binding protein specific to fructose (BL0033) and Frk (BL1339) showed higher levels of expression in cells grown on fructose than on glucose, and BL0033 with more than 5 isoforms.Furtherly, comparative proteomics research and semi-quantitative RT-PCR showed that induction time and higher fructose concentration correlate to increasing BL0033 expression. At the same time, an ABC transporter ATP binding protein (BL0034) was slightly up-regulated in cells grown on fructose compared to glucose. All of the above results suggest that the uptake of fructose into the cell may be conducted by a specific transport system (fructose ABC transport system), in which BL0033 and BL0034 might play an important role. Considering its novelty, we cloned BL0033 into expressional vector pET-32a which with a His tag. Recombinant plasmid was induced for expression within the E.coli BL21. Hetero-expressed His fused BL0033 protein were purified and injected into rabbit for its polyclonal antibody. At last, co-immunoprecipitation experiment supported some proteins may interact with BL0033 in vivo.All the findings above were useful for investigating the mechanism of fructose translocation and uptake/utilization. Some proteins and regulators with unknown function were interpreted which has much significance for Bifidobacteria.spp living in the gastrointestinal tracts (GIT).