Preparation method for various novel fucosyl oligosaccharides and use thereof
A technology of fucosyl and oligosaccharides, applied in biochemical equipment and methods, glycosyltransferases, skin care preparations, etc., can solve the problems that drugs cannot meet all needs, and there is no basic treatment
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example 1
[0098] Synthesis of fucosyl oligosaccharides
[0099] To produce various fucosyl oligosaccharides, an enzymatic reaction of α-1,2-fucosyltransferase ( figure 1 ).
[0100] As the α-1,2-fucosyltransferase, Escherichia coli (E. coli)-derived WbgL (GenBank: ABE98421.1), Helicobacter pylori-derived FucT2 (GenBank: AAC99764. 1) and WcfB derived from Bacteroides fragilis (GenBank: AAD40713.1). The pET21a plasmid vector containing the genes encoding each enzyme was transformed into E. coli BL21(DE3) star. Each recombinant E. coli was cultured at 37° C. and 250 rpm until OD 0.5, and then the enzyme expression was induced using 0.1 millimolar IPTG at 16° C. and 250 rpm for 18 hours.
[0101] figure 1 A view showing the production of different types of fucosyl oligosaccharides using a GDP fucose donor and various sugar acceptors by an enzymatic reaction of α-1,2-fucosyltransferase, using the sugar Receptors were tested for their reactivity, sugar receptors such as glucose, galacto...
example 2
[0105] Enzyme reaction of α-1,2-fucosyltransferase of various sugar acceptors
[0106] Fucosyl oligosaccharides are produced by the enzymatic reaction of WbgL, which is α-1,2-fucosyltransferase, using a GDP-fucose donor and galactose or glucose as an acceptor. The sugar acceptors used in the enzyme reaction are glucose, galactose, cellobiose, lactose, fructose, sucrose, maltose, mannose, xylose, and agarobiose. Enzyme reaction conditions were as follows: use 1 mg / ml of each recombinant E. coli intracellular crude enzyme solution, 0.2 mmol of GDP-fucose donor, 0.5 mmol of sugar acceptor, and 20 mmol of sodium phosphate buffer reagent (pH 6.0) to allow the enzyme reaction to remain at 30°C for 12 hours. LC / MS analysis was performed on the reaction product to confirm accurate mass values. In LC / MS analysis, a Thermo Fisher porous graphite carbon LC column (Thermo Fisher Scientific) was used and positive ion mode was used. Two mobile phases, eg 25 micromolar lithium chloride s...
example 3
[0108] using metabolic engineering technology to produce fucosyl galactose
[0109] FGal, which is a biologically active substance, was produced from recombinant E. coli using metabolic engineering and then subjected to fermentation. Figure 6 A is a view of the production of FGal using recombinant E. coli, in which to produce the GDP-fucose donor in E. coli cells, the enzymes ManB, ManC, Gmd and WcaG involved in the de novo pathway were introduced and half Lactose is supplied as the acceptor. To use galactose as a receptor, an E. coli (E. coli) BL21(DE3) strain incapable of metabolizing galactose was used as a host. Helicobacter pylori-derived FucT2 acts as an α-1,2-fucosyltransferase and supplies glycerol as a carbon source. Recombinant Escherichia coli was cultured at 37°C for 6 hours in an LB broth medium containing 2 g / L of galactose and 5 g / L of glycerol, and IPTG was added thereto, and then after the temperature was lowered to 25°C To cultivate.
[0110] Such as ...
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