Abstract: Objective: Fructosyl hydrolase plays a significant role in the preparation of novel fructose derivatives.It is important to obtain an efficient fructosyl hydrolase with high activity.Methods: Five fructosyl hydrolase-encoding genes in the Aspergillus niger genome were selected and analyzed by MEGA 4.0, Clustal X2 and other softwares. Subsequently, these genes were successfully cloned and expressed in Pichia pastoris followed by comprehensive investigation of their biochemical properties.Results: At the shake-flask fermentation level, the activities of recombinant Fru1 and Fru5 were determined to be 1360 and1560 U/m L, respectively, which were much higher than those of the fructosyl hydrolases ever reported.The optimum temperature and p H of recombinant enzyme Fru1 were 45 ℃ and 5.5, respectively.Its enzymatic activity was slightly enhanced by EDTA and inhibited by Na+, Co²⁺, Cu²⁺and Ca²⁺. Interestingly, this enzyme showed both hydrolytic and transglycosylation activities toward sucrose, and hydrolyzed short-chain inulin with degree of polymerization of 2 ~ 5. The temperature and p H optima of recombinant Fru5 were 50 ℃ and 5.0, respectively.It was remarkably enhanced by Li⁺, Na⁺and EDTA, and strongly inhibited by Fe²⁺.Fru5 could also hydrolyze sucrose and almost all polysaccharides in inulin.Conclusions: Our results have paved the way for the industrial manufacturing of fructosyl hydrolases and their applications in the production of novel fructose derivatives.