Fructosyltransferases catalyze the transfer of a fructose unit from one sucrose/fructan to another and are engaged in the production of fructooligosaccharide/fructan. The enzymes belong to the glycoside hydrolase family 32 (GH32) with a retaining catalytic mechanism. Here we describe the crystal structures of recombinant fructosyltransferase (AjFT) from Aspergillus japonicus CB05 and its mutant D191A complexes with various donor/acceptor substrates, including sucrose, 1-kestose, nystose, and raffinose. This is the first structure of fructosyltransferase of the GH32 with a high transfructosylation activity. The structure of AjFT comprises two domains with an N-terminal catalytic domain containing a five-blade ␤-propeller fold linked to a C-terminal ␤-sandwich domain. Structures of various mutant AjFT-substrate complexes reveal complete four substrate-binding subsites (؊1 to ؉3) in the catalytic pocket with shapes and characters distinct from those of clan GH-J enzymes. Residues Asp-60, Asp-191, and Glu-292 that are proposed for nucleophile, transition-state stabilizer, and general acid/base catalyst, respectively, govern the binding of the terminal fructose at the ؊1 subsite and the catalytic reaction. Mutants D60A, D191A, and E292A completely lost their activities. Residues Ile-143, Arg-190, Glu-292, Glu-318, and His-332 combine the hydrophobic Phe-118 and Tyr-369 to define the ؉1 subsite for its preference of fructosyl and glucosyl moieties. Ile-143 and Gln-327 define the ؉2 subsite for raffinose, whereas Tyr-404 and Glu-405 define the ؉2 and ؉3 subsites for inulin-type substrates with higher structural flexibilities. Structural geometries of 1-kestose, nystose and raffinose are different from previous data. All results shed light on the catalytic mechanism and substrate recognition of AjFT and other clan GH-J fructosyltransferases.

Oxidation of benzoic acid (BA) by H2O2 was performed with a novel supported gamma-FeOOH catalyst in a circulating fluidized-bed reactor (CFBR). This study focused mainly on determining the proportions of homogeneous catalysis and heterogeneous catalysis in this CFBR. Also studied herein was how pH, H2O2 concentration, and BA concentration affect the oxidation of BA. Experimental results indicate that the decomposition rate of H2O2 was proportional to its concentration and that the oxidation rate of BA depended on both H2O2 and BA concentrations. The change in the rate constant of heterogeneous catalysis by pH was described in terms of ionization fractions of surface hydroxyl group. From the mathematical deduction, we can infer thatthe reaction rate associated with ...Fe(III)OH2+ is markedly higher than that with ...Fe(III)OH. Conclusively, although heterogeneous catalysis contributes primarily to the oxidation of BA at pH 4.4-7.0, the homogeneous catalysis is of increasing importance below pH 4.4 because of the reductive dissolution of gamma-FeOOH.

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