Purification and biochemical characterization of a novel β-fructofuranosidase from Penicillium oxalicum with transfructosylating activity producing neokestose

•A novel β-fructofuranosidase from P. oxalicum was purified and characterized.•The enzyme could efficiently convert sucrose into the prebiotic neokestose.•The enzyme exhibited remarkable stability over a broad pH range. Neokestose is a novel fructooligosaccharide (FOS) exhibiting greater prebiotic effects and chemical stability than commercial FOSs. In this study, a neokestose-producing β-fructofuranosidase was purified from Penicillium oxalicum GXU20. The enzyme is a glycoprotein with an approximate 111kDa molecular weight, and it has an N-linked carbohydrate composition that accounts for approximately 38% of its total mass. Optimal enzymatic activity occurred at pH 5.5 and 60°C. The enzyme remained stable over a wide pH range (2–9.5). Metal ions and chemical reagents had no significant effect on its enzymatic activity, with the exception of silver (Ag+). The enzyme could hydrolyze fructosyl-(2-1)-linked carbohydrates. Using sucrose as a substrate, the Km and Vmax values for a transfer reaction were 163.9±10.3mmol/L and 800.1±19.8μmol/(minmg), respectively, whereas the Km, Vmax, and Ki values for a hydrolysis reaction with substrate inhibition were 48.3±1.7mmol/L, 1631.3±28.2μmol/(minmg), and 162.6±4.9mmol/L, respectively. In the catalysis of 500g/L sucrose, the maximum concentration of neokestose and total FOS were 94.2g/L and 224.7g/L, respectively. Finally, the gene encoding the β-fructofuranosidase was cloned, analyzed, and functionally expressed in Pichia pastoris.