Takahashi Natsuko, Fukushi Eri, Onodera Shuichi, Benkeblia Noureddine, Nishimoto Tomoyuki, Kawabata Jun, Shiomi Norio
Department of Food and Nutrition Sciences, Graduate School of Dairy Science Research, Rakuno Gakuen University, Ebetsu, Japan.
Chem Cent J. 2007 Jun 28;1:18. doi: 10.1186/1752-153X-1-18.
Recently synthesized novel oligosaccharides have been produced primarily by hydrolases and glycosyltransferases, while phosphorylases have also been subject of few studies. Indeed, phosphorylases are expected to give good results via their reversible reaction. The purpose of this study was to synthesis other novel oligosaccharides using kojibiose phosphorylase.
Three novel oligosaccharides were synthesized by glucosyltransfer from beta-D-glucose 1-phosphate (beta-D-G1P) to xylosylfructoside [O-alpha-D-xylopyranosyl-(1-->2)-beta-D-fructofuranoside] using Thermoanaerobacter brockii kojibiose phosphorylase. These oligosaccharides were isolated using carbon-Celite column chromatography and preparative high performance liquid chromatography. Gas liquid chromatography analysis of methyl derivatives, MALDI-TOF MS and NMR measurements were used for structural characterisation. The 1H and 13C NMR signals of each saccharide were assigned using 2D-NMR including COSY (correlated spectroscopy), HSQC (herteronuclear single quantum coherence), CH2-selected E-HSQC (CH2-selected Editing-HSQC), HSQC-TOCSY (HSQC-total correlation spectroscopy) and HMBC (heteronuclear multiple bond correlation).
The structure of three synthesized saccharides were determined, and these oligosaccharides have been identified as O-alpha-D-glucopyranosyl-(1-->2)-O-alpha-D-xylopyranosyl-(1-->2)-beta-D-fructofuranoside (saccharide 1), O-alpha-D-glucopyranosyl-(1-->2)-O-alpha-D-glucopyranosyl-(1-->2)-O-alpha-D-xylopyranosyl-(1-->2)-beta-D-fructofuranoside (saccharide 2) and O-alpha-D-glucopyranosyl-(1-->[2-O-alpha-D-glucopyranosyl-1]2-->2)-O-alpha-D-xylopyranosyl-(1-->2)-beta-D-fructofuranoside (saccharide 3).
最近合成的新型低聚糖主要是通过水解酶和糖基转移酶产生的，而磷酸化酶的相关研究较少。实际上，磷酸化酶有望通过其可逆反应产生良好的效果。本研究的目的是利用 kojibiose 磷酸化酶合成其他新型低聚糖。
使用嗜热栖热放线菌 kojibiose 磷酸化酶，通过将β-D-葡萄糖 1-磷酸（β-D-G1P）上的葡萄糖基转移到木糖基果糖苷[O-α-D-吡喃木糖基-(1→2)-β-D-呋喃果糖苷]上，合成了三种新型低聚糖。这些低聚糖通过碳-硅藻土柱色谱和制备型高效液相色谱进行分离。使用甲基衍生物的气相色谱分析、基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）和核磁共振测量对其结构进行表征。通过包括 COSY（相关光谱）、HSQC（异核单量子相干）、CH2 选择的 E-HSQC（CH2 选择的编辑-HSQC）、HSQC-TOCSY（HSQC-全相关光谱）和 HMBC（异核多键相关）在内的二维核磁共振对每种糖类的 1H 和 13C 核磁共振信号进行归属。
确定了三种合成糖类的结构，这些低聚糖已被鉴定为 O-α-D-吡喃葡萄糖基-(1→2)-O-α-D-吡喃木糖基-(1→2)-β-D-呋喃果糖苷（糖类 1）、O-α-D-吡喃葡萄糖基-(1→2)-O-α-D-吡喃葡萄糖基-(1→2)-O-α-D-吡喃木糖基-(1→2)-β-D-呋喃果糖苷（糖类 2）和 O-α-D-吡喃葡萄糖基-(1→[2-O-α-D-吡喃葡萄糖基-1]2→2)-O-α-D-吡喃木糖基-(1→2)-β-D-呋喃果糖苷（糖类 3）。
