A novel glycosyl hydrolase family 32 exo-inulinase (InuAGN25) gene was cloned from Sphingobacterium sp. GN25 isolated from feces of Grus nigricollis. InuAGN25 showed the highest identity of 54.3% with a putative levanase recorded in GenBank. Molecular-Activity strategy was proposed to predict InuAGN25 to be a low-temperature-active exo-inulinase before experiments performance. Molecular analyses included progressive sequential, phylogenetic and structural analyses. InuAGN25 was effectively expressed in Escherichia coli. The purified recombinant InuAGN25 showed characteristics of low-temperature-active enzymes: (1) the enzyme retained 55.8% of the maximum activity at 20 degrees C, 35.8% at 10 degrees C, and even 8.2% at 0 degrees C; (2) the enzyme exhibited 75.8, 30.5 and 10.8% of the initial activity after preincubation for 60min at 45, 50 and 55 degrees C, respectively; (3) K-m values of the enzyme toward inulin were 2.8, 3.0, 3.2 and 5.8 mg ml(-1) at 0, 10,20 and 40 degrees C, respectively. Fructose was the main product of inulin and Jerusalem artichoke tubers hydrolyzed by the purified recombinant InuAGN25 at room temperature, 10 degrees C and 0 degrees C. These results suggested the Molecular-Activity strategy worked efficiently and made InuAGN25 promising for the production of fructose at low temperatures. (C) 2014 Elsevier Ltd. All rights reserved.