Synthesis Of Chitooligosaccharides Using Recombinant Escherichia Coli

Chitooligosaccharides are alkaline amino sugars composed of2-10amino glucoses linked by β-1,4glycosidic bond. It can be widely used as preservatives, antioxidants and plant growth regulating factor in many yeilds because of its good water-solubility, easy absorption and other special characteristics. But high-cost and serious pollution restricted mass production greatly. So the purpose of this study was to explore a new way to get more chitooligosaccharides.Using recombinant Escherichia coli to synthesis chitooligosaccharides has been a new technology. Used S. Meliloti nodB gene as a template in this experiment, and then constructed two kinds recombinant vectors pET-nodB1and pET-nodB2(vector was pET-28a). NodBl protein carried T7-tag in N terminer and His-tag in C terminer, including two sites:BamHI and XhoI; NodB2protein only carried His-tag in C terminer, including two sites:NcoI and XhoI. And transformed two recombinant vectors into E. coli BL21. After cultivated under IPTG induce, separated and purified the recombinant proteins NodBl and NodB2, their molecular weights were30KD and26KD.In addition, synthesised1879bp of nodBC genes coming from A. Caulinodans. nodC gene could encode N-acetylglucosaminyltransferase (NodC protein), the function was that it could synthesis2-6N-acetyl glucosamine oligomers with UDP-GlcNAc as glycosyl donorl; NodB protein, encoding by nodB gene, could remove an acetyl group from the non-reducing of glucosamine oligomer. First connected the nodBC genes to the downstream of the Lac promoter of pUC18to construct recombinant plasmid pUC-nodBC, and then transformed it into E. coli DH5α, after cultivated DH5α, identified the nodBC gnes by digestion(EcoRI、BamHI) and sequenced. And then cultivated recombinant DH5α in medium MMY under IPTG induce, for identifing whether nodBC genes could encode corresponding proteins to produce chito-oligosaccharides. Use10L full automatic fermentor to culture by medium MMYNG, collected284.68g cell in total after centrifugalize. Then broke the cell wall through repeat freezing and thawing, afer centrifugalized the solution with hydrochloric acid, adsorped the chitooligosaccharide in the supernatant by activated carbon, after that, eluted the objective products by ethanol of different strengths, and then purified the target product through P4gel molecular chromatography further to detect by TLC assay, finally used mass spectrometry to determine the structure of products, in this study, three kinds of chitooligosaccharides were received: tetra-N-acetyl-chitotetraose,830; penta-N-acetyl-chitotetraose,1033; chitopentaose,991.