RESUMO
Chemo-enzymatic synthesis of lacto-N-biose I (LNB) catalyzed by ß-1,3-galactosidase from Bacillus circulans (BgaC) has been developed using 4,6-dimethoxy-1,3,5-triazin-2-yl ß-galactopyranoside (DMT-ß-Gal) and GlcNAc as the donor and acceptor substrates, respectively. BgaC transferred the Gal moiety to the acceptor, giving rise to LNB. The maximum yield of LNB was obtained at the acceptor : donor substrate ratio of 1:30.
Assuntos
Bacillus/enzimologia , beta-Galactosidase/metabolismo , Acetilglucosamina/análogos & derivados , Acetilglucosamina/metabolismo , Catálise , Glicosilação , Cinética , Estereoisomerismo , Especificidade por SubstratoRESUMO
A 38 kDa ß-1,3-glucanase allergen from Cryptomeria japonica pollen (CJP38) was recombinantly produced in E. coli and purified to homogeneity with the use of Ni-affinity resin. CJP38 hydrolyzed ß-1,3-glucans such as CM-curdlan and laminarioligosaccharides in an endo-splitting manner. The optimum pH and temperature for ß-1,3-glucanase activity were approximately 4.5 and 50 °C, respectively. The enzyme was stable at 30-60 °C and pH 4.0-10.5. Furthermore, CJP38 catalyzed a transglycosylation reaction to yield reaction products with a molecular weight higher than those of the starting laminarioligosaccharide substrates. The three-dimensional structure of CJP38 was determined using X-ray crystallography at 1.5 Å resolution. CJP38 exhibited the typical (ß/α)8 TIM-barrel motif, similar to allergenic ß-1,3-glucanases from banana (Mus a 5) and rubber tree latex (Hev b 2). Amino acid sequence alignment of these proteins indicated that the two-consensus IgE epitopes identified on the molecular surfaces of Mus a 5 and Hev b 2 were highly conserved in CJP38. Their conformations and surface locations were quite similar for these proteins. Sequence and structural conservation of these regions suggest that CJP38 is a candidate allergen responsible for the pollen-latex-fruit syndrome relating to Japanese cedar pollinosis.