In vitro synthesis of a crystalline (1 3,1 4)-beta-d-glucan by a mutated (1 3,1 4)-beta-d-glucanase from Bacillus

Abstract

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.

Glycosynthase Activity of Geobacillus stearothermophilus GH52 β‐Xylosidase: Efficient Synthesis of Xylooligosaccharides from α‐D‐Xylopyranosyl Fluoride through a Conjugated Reaction

“…3, 1!4)-b-d-glucanase from Bacillus licheniformis and a-laminaribiosyl fluoride to produce oligosaccharides (degree of polymerization 6-12). [33] Recently, it was demonstrated that GH10 xylanases can also serve as glycosynthases. Kim et al [18] converted Cellulomonas fimi xylanase into a glycosynthase by replacing its nucleophile Glu235 by Gly.…”

Preparation of a glycosynthase from the β-glycosidase of the ArchaeonPyrococcus horikoshii

“…In both cases these novel catalysts, named glycosynthases , synthesize oligosaccharides with almost quantitative yields. Interestingly, a novel b-glucan containing mixed b-1,3 and b-1,4 bonds was recently obtained by using glucansynthases , indicating that the synthesis of biomaterials can be another useful application of this approach (Faijes et al 2004).…”

Determination of Biomass Composition of Catharanthusroseus Hairy Roots for Metabolic Flux Analysis

“…A 2-D [ 13 C, 1 H] heteronuclear single quantum correlation (HSQC) spectrum (not shown) revealed that the carbon atoms of the biomass components were covalently bonded to protons with 1 H chemical shifts in the range 3.26-5.43 ppm. These chemical shifts and spectra compared well with published 13 C and 1 H chemical shifts and spectra of -glucans (24)(25)(26)(27)(28), which are polymers of glucose. The presence of many 13 C signals near 80 ppm indicated that the -glucans were a mixture of -(1,3) and -(1,4) glucans (24,26,28).…”