QM/MM modeling of the hydrolysis and transfructosylation reactions of fructosyltransferase from Aspergillus japonicas,an enzyme that produces prebiotic fructooligosaccharide

Fructosyltransferases (FTs) act on sucrose by cleaving the β-(2 → 1) linkage, releasing glucose, and then transferring the fructosyl group to an acceptor molecule. These enzymes are capable of producing prebiotic fructooligosaccharides (FOSs) that are of industrial interest. While several FOS-synthesizing enzymes FTs have been investigated, their catalytic mechanism is not yet fully understood, especially the molecular details of how FOS are enzymatically synthesized from sucrose. Here, we present a comparative quantum mechanics/molecular mechanics (QM/MM) study on the hydrolysis and transfructosylation reactions catalyzed by A. japonicus FT using sucrose as donor and acceptor substrates. It is shown that the hydrolysis and transfructosylation reactions of the enzyme seem to be competitive with similar potential energy profiles. For all studied reaction steps, the fructosyl ring bound in the −1 position was observed to have a ⁴E conformation in the oxocarbonium ion-like transition state. Based on the SCC-DFTB/MM simulations of sucrose complexes of wildtype and D191A mutant FT, Asp191 is shown to be responsible for the productive sugar conformation (at subsite −1) required for catalysis. A key interaction, Asp119⋯nucleophile⋯1–OH (substrate), is proposed to facilitate the formation of fructosyl-enzyme intermediate. This is the first computational study for understanding the FOS synthesis process, and it can be applicable to related FOS-synthesizing enzymes.     

Kinetics and modeling of fructo‐oligosaccharide synthesis by immobilized fructosyltransferase from Rhodotorula sp.

BACKGROUND: Fructosyltransferase synthesizes fructo‐oligosaccharides from sucrose. Data used in this work were obtained by an enzyme produced by Rhodotorula sp., a microorganism isolated from fruit samples from the Brazilian Atlantic Forest, which was immobilized in an inorganic support, consisting of a niobium and graphite alloy. RESULT: All essays were conducted using enzymes at two purification grades, highly and partially purified enzymes, as comparison. The results were not significantly different between the two enzyme grades, mainly concerning the final fructo‐oligossacharides yield, which were around 46%. Concerning the kinetics, the enzyme follows the Michaelis–Menten equation with inhibition by sucrose (above 60%). Also, a competitive inhibition by glucose was observed on sucrose, kestose and nystose uptakes. The immobilization of the enzyme was by ion exchange on the surface of the particles, since the support is a charged and compact solid, with negligible porosity. The mathematic model includes mass balances, considering the resistance to external mass transfer. A parameter sensitivity analysis and parameter fitting were performed by simulations and the model was validated by comparison with experimental data. CONCLUSION: The model fitted experimental data well, with deviations lower than 5% concerning FOS concentrations, indicating that it can be used in the design and control of bioreactors, either using purified or partially purified enzyme. Copyright © 2010 Society of Chemical Industry     

日本曲霉菌体固定化及改性大豆低聚糖的研究

将日本曲霉经液体培养制备成1mm左右的菌球，以戊二醛为交联剂，牛血清蛋白为活性保护剂对其进行固定化．得到了果糖转移酶活力和稳定性均较高的固定化菌体细胞。利用固定化菌体细胞改性大豆低聚糖，蔗糖的转化率可达76％。