Optimized bioprocess for production of fructofuranosidase by recombinant Aspergillus niger.

A comprehensive approach of bioprocess design at various levels was used to optimize microbial production of extracellular fructofuranosidase, important as biocatalyst to derive fructooligosaccharides with broad application in food or pharmaceutical industry. For production, the recombinant strain Aspergillus niger SKAn1015 was used, which expresses the fructofuranosidase encoding gene suc1 under control of a strong constitutive promoter. In a first screening towards an optimized medium, glucose, nitrate, Fe(2+), and Mn(2+) were identified as beneficial for production. A minimal medium with optimized concentration of these key nutrients, obtained by central composite design experiments and quadratic modelling, provided a threefold increased fructofuranosidase activity in the culture supernatant (400 U/mL) as compared to the originally described medium. Utilizing the optimized medium, the process was then transferred from shake flask into a fed-batch-operated bioreactor. Hereby, the intended addition of talc microparticles allowed engineering the morphology of A. niger into a highly active mycelial form, which strongly boosted production. Fructofuranosidase production was highly specific as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The secreted enzyme activity of 2,800 U/mL, corresponding to about 3 g/L of fructofuranosidase, achieved by the microparticle-enhanced fed-batch process, is tenfold higher than that of any other process reported so far, so that the presented bioprocess strategy appears as a milestone towards future industrial fructofuranosidase production.