Strictly G-Specific Alginate Lyase Aly7Sa for Efficient Preparation of Unsaturated Guluronate Oligosaccharides.

Alginate is a commercially valuable polysaccharide consisting of β-d-mannuronate (M) and its C5 epimer, α-l-guluronate (G). Alginate lyases are efficient tools for the degradation of alginate and the preparation of oligosaccharides. In this research, an endolytic alginate lyase Aly7Sa with strict G specificity was expressed and characterized with the optimum reaction conditions at 30 °C and pH 6.5. The main degradation products of Aly7Sa for alginate were trisaccharide to octasaccharide, and those of PolyG were disaccharide to heptasaccharide. By utilizing HPAEC-PAD/MS and NMR methods, we identified the structure of products obtained from alginate. Interestingly, the trisaccharide to hexasaccharide products of Aly7Sa contained only unsaturated guluronate oligosaccharides, which were different from all of the characterized G-specific alginate lyases. The absence of oligosaccharide products with M residues demonstrated the strict G specificity of Aly7Sa. The targeted preparation was carried out based on the regular oligosaccharide pattern of Aly7Sa. By single-step purification employing gel-permeation chromatography, 4.8 mg of ΔG, 6.8 mg of ΔGG, and 3.7 mg of ΔGGG were obtained with 100 mg of alginate as substrate. The strictly G-specific alginate lyase Aly7Sa provided an efficient tool for the preparation of unsaturated guluronate oligosaccharides, and the unique substrate specificity of the enzyme could also serve the research and development of alginate.