An inulinase-producing strain, Paenibacillus polymyxa ZJ-9, was isolated from natural sources to produce R,R-2,3-butanediol via one-step fermentation of raw inulin extracted from Jerusalem artichoke tubers. The inulinase gene from P. polymyxa ZJ-9 was cloned and overexpressed in Escherichia coli BL21 (DE3), and the purified recombinant inulinase was estimated to be approximately 56 kDa by both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. This result suggests that the active form of the inulinase is probably a monomer. Terminal hydrolysis fructose units from the inulin indicate that enzymes are exo-inulinase. The purified recombinant enzyme showed maximum activity at 25 A degrees C and pH 6.0, which indicate its extreme suitability for industrial applications. Zn2+, Fe2+, and Mg2+ stimulated the activity of the purified enzyme, whereas Co2+, Cu2+, and Ni2+ inhibited enzyme activity. The K (m) and V (max) values for inulin hydrolysis were 1.72 mM and 21.69 mu mol min(-1) mg(-1) protein, respectively. The same parameters toward sucrose were 41.09 mM and 78.7 mu mol min(-1) mg(-1) protein, respectively. Considering its substrate specificity and other enzymatic characteristics, we believe that this inulinase gene from P. polymyxa ZJ-9 could be transformed into other special bacterial strains to allow inulin conversion to other biochemicals and bioenergy through one-step fermentation.