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.

Synthesis of ß-D-fructofuranosyl-(2→1)-2-acetamido-2-deoxy-α-D-glucopyranoside (N-acetylsucrosamine) using ß-fructofuranosidase-containing Aspergillus oryzae mycelia as a whole-cell catalyst.

Using soft granules consisting of Celite 535 and dried Aspergillus oryzae NBRC100959 mycelia containing ß-fructofuranosidase as a whole-cell catalyst, N-acetylsucrosamine [ß-D-fructofuranosyl-(2→1)-2-acetamido-2-deoxy-α-D-glucopyranoside] was produced from sucrose and 2-acetamido-2-deoxy-D-glucose by enzymatic transfructosylation. The isolated yield of N-acetylsucrosamine from the reaction mixture was 22.1% (from sucrose). The result of N-terminal amino acid sequence analysis indicated that the enzyme involved in the synthesis of N-acetylsucrosamine is a product from gene (NCBI accession number; NW_001884675, locus tag; AOR_1_1114084) encoding putative ß-fructofuranosidase on chromosome 6 of strain NBRC100959. The N-acetylsucrosamine we produced is highly soluble in water and is more stable in acidic solution than sucrose. The disaccharide was also produced using dried mycelia prepared from another A. oryzae strains.