TY - JOUR
T1 - In vitro synthesis of a crystalline (1 → 3, 1 → 4)-β-D-glucan by a mutated (1 → 3, 1 → 4)-β-D-glucanase from Bacillus
AU - Faijes, Magda
AU - Imai, Tomoya
AU - Bulone, Vincent
AU - Planas, Antoni
PY - 2004/6/15
Y1 - 2004/6/15
N2 - 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)-β-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 α-laminaribiosyl fluoride via the formation of (1 → 4)-β-linkages to yield a new linear crystalline (1 → 3,1 → 4)-β-D-glucan with a repeating 4βG3βG 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)-β-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 γ = 90.5°. The dimensions of the ab plane are similar to those of cellulose I β, 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.
AB - 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)-β-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 α-laminaribiosyl fluoride via the formation of (1 → 4)-β-linkages to yield a new linear crystalline (1 → 3,1 → 4)-β-D-glucan with a repeating 4βG3βG 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)-β-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 γ = 90.5°. The dimensions of the ab plane are similar to those of cellulose I β, 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.
KW - (1 → 3,1 → 4)-β-D-glucan
KW - Carbohydrate
KW - Glycosynthase
KW - Polysaccharide structure
KW - Polysaccharide synthesis
UR - http://www.scopus.com/inward/record.url?scp=3042823386&partnerID=8YFLogxK
UR - https://www.webofscience.com/api/gateway?GWVersion=2&SrcApp=pure_univeritat_ramon_llull&SrcAuth=WosAPI&KeyUT=WOS:000222338100006&DestLinkType=FullRecord&DestApp=WOS
U2 - 10.1042/BJ20040145
DO - 10.1042/BJ20040145
M3 - Article
C2 - 15038792
AN - SCOPUS:3042823386
SN - 0264-6021
VL - 380
SP - 635
EP - 641
JO - Biochemical Journal
JF - Biochemical Journal
IS - 3
ER -