Faijes Magda, Imai Tomoya, Bulone Vincent, Planas Antoni
Laboratory of Biochemistry, Institut Químic de Sarrià, Universitat Ramon Llull, 08017 Barcelona, Spain.
Biochem J. 2004 Jun 15;380(Pt 3):635-41. doi: 10.1042/BJ20040145.
Oligo- and poly-saccharides have a large number of important biological functions, and they occur in natural composite materials, such as plant cell walls, where they self-assemble during biosynthesis in a poorly understood manner. They can also be used for the formation of artificial composite materials with industrial applications. Fundamental and applied research in biology and nanobiotechnology would benefit from the possibility of synthesizing tailor-made oligo-/poly-saccharides. In the present paper, we demonstrate that such syntheses are possible using genetically modified glycoside hydrolases, i.e. glycosynthases. The ability of the endoglycosynthase derived from Bacillus (1-->3,1-->4)-beta-D-glucanase to catalyse self-condensation of sugar donors was exploited for the in vitro synthesis of a regular polysaccharide. The specificity of the enzyme allowed the polymerization of alpha-laminaribiosyl fluoride via the formation of (1-->4)-beta-linkages to yield a new linear crystalline (1-->3,1-->4)-beta-D-glucan with a repeating 4betaG3betaG unit. MS and methylation analyses indicated that the in vitro product consisted of a mixture of oligosaccharides, the one having a degree of polymerization of 12 being the most abundant. Morphological characterization revealed that the (1-->3,1-->4)-beta-D-glucan forms spherulites which are composed of platelet crystals. X-ray and electron diffraction analyses allowed the proposition of a putative crystallographic structure which corresponds to a monoclinic unit cell with a =0.834 nm, b =0.825 nm, c =2.04 nm and gamma=90.5 degrees. The dimensions of the ab plane are similar to those of cellulose I(beta), but the length of the c -axis is nearly twice that of cellulose I. It is proposed that four glucose residues are present in an extended conformation along the c -axis of the unit cell. The data presented show that glycosynthases represent promising enzymic systems for the synthesis of novel polysaccharides with specific and controlled structures, and for the analysis in vitro of the mechanisms of polymerization and crystallization of polysaccharides.
寡糖和多糖具有大量重要的生物学功能，它们存在于天然复合材料中，如植物细胞壁，在生物合成过程中它们以一种尚不清楚的方式进行自组装。它们还可用于形成具有工业应用价值的人工复合材料。生物学和纳米生物技术领域的基础研究与应用研究将受益于合成定制寡糖/多糖的可能性。在本文中，我们证明了使用基因工程改造的糖苷水解酶，即糖基合成酶，进行此类合成是可行的。利用源自芽孢杆菌的内切糖基合成酶（1→3,1→4）-β-D-葡聚糖酶催化糖供体的自缩合能力，在体外合成了一种规则的多糖。该酶的特异性使得α-层叠二糖氟化物通过形成（1→4）-β-连接进行聚合，生成一种新的线性结晶（1→3,1→4）-β-D-葡聚糖，其重复单元为4βG3βG。质谱和甲基化分析表明，体外产物由寡糖混合物组成，其中聚合度为12的寡糖最为丰富。形态学表征显示，（1→3,1→4）-β-D-葡聚糖形成由片状晶体组成的球晶。X射线和电子衍射分析提出了一种假定的晶体结构，该结构对应于一个单斜晶胞，其a = 0.834 nm，b = 0.825 nm，c = 2.04 nm，γ = 90.5°。ab平面的尺寸与纤维素I（β）的相似，但c轴的长度几乎是纤维素I的两倍。有人提出沿着晶胞的c轴存在四个呈伸展构象的葡萄糖残基。所呈现的数据表明，糖基合成酶是用于合成具有特定和可控结构的新型多糖，以及在体外分析多糖聚合和结晶机制的有前景的酶系统。
