Enzymatic synthesis of novel oligosaccharides from N-acetylsucrosamine using ß-fructofuranosidase from Aspergillus oryzae.

Mycelia of Aspergillus oryzae NBRC100959 contain 2 types of ß-fructofuranosidases, transfructosylation-catalyzing enzyme (ßFFaseI), and hydrolysis-catalyzing enzyme (ßFFaseII). Using ßFFaseI extracted from the mycelia of strain NBRC100959, two novel oligosaccharides consisting of GlcNAc and fructose, ß-d-fructofuranosyl-(2→1)-ß-d-fructofuranosyl-(2↔1)-2-acetamido-2-deoxy-α-d-glucopyranoside (N-acetyl-1-kestosamine, 1-KesNAc) and ß-d-fructofuranosyl-(2→1)-ß-d-fructofuranosyl-(2→1)-ß-d-fructofuranosyl-(2↔1)-2-acetamido-2-deoxy-α-d-glucopyranoside (N-acetylnystosamine, NysNAc), were synthesized from ß-d-fructofuranosyl-(2↔1)-2-acetamido-2-deoxy-α-d-glucopyranoside (N-acetylsucrosamine, SucNAc). We next planned to synthesize 1-KesNAc and NysNAc using A. oryzae mycelia. However, it was thought that the presence of ßFFaseII is disadvantageous for the production of these oligosaccharides by ßFFaseI, because ßFFaseII hydrolyzed 1-KesNAc and NysNAc. We succeeded to produce A. oryzae mycelia containing ßFFaseI as the major ß-fructofuranosidase, by increasing sucrose concentration in the culture medium. Then, using a dried sample of these A. oryzae mycelia, reaction for the oligosaccharide production was performed. As the results, 190mg of 1-KesNAc and 60mg of NysNAc were obtained from 0.6g of SucNAc. This whole-cell catalysis method facilitates the synthesis of 1-KesNAc and NysNAc because extraction and purification of ßFFaseI from mycelia are unnecessary.