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The thermal stability of inulin fructotransferase (IFTase) subjected to high hydrostatic pressure (HHP) was studied. The value of inactivation rate of IFTase in the range of 70–80 °C decreased under the pressure of 100 or 200 MPa, indicating that the thermostability of IFTase under high temperature was enhanced by HHP. Far-UV CD and fluorescence spectra showed that HHP impeded the unfolding of the conformation of IFTase under high temperature, reflecting the antagonistic effect between temperature and pressure on IFTase. The new intramolecular disulfide bonds in IFTase were formed under a combination of HHP and high temperature. These bonds might be related to the stabilization of IFTase at high temperature. All the above results suggested that HHP had the protective effect on IFTase against high temperature.

BACKGROUND The demand for difructose anhydride III (DFA III), a novel functional sweetener, is growing continuously. It is produced from inulin by inulin fructotransferase (IFTase). In this study, high hydrostatic pressure (HHP), as a clean technology, was first applied to further improve the catalytic efficiency of IFTase in the process. RESULTS The maximum activity of IFTase was obtained under 200 MPa at 60 °C. Meanwhile, HHP lowered the energy barrier necessary for the enzymatic reaction and decreased the volume between the reactants and the transition state. Under this condition, the optimal pH for the enzymatic reaction shifted from 5.5 to 6.0. The activity was further enhanced by 65.2% in the presence of 1.5 mol L−1 NaCl. CONCLUSION The catalytic reaction of IFTase was performed under HHP for the first time. HHP, as a promising green technology for bioconversion, significantly accelerated the enzymatic reaction under the appropriate operational conditions. © 2015 Society of Chemical Industry

BACKGROUND Inulin fructotransferase (IFTase) catalyzes inulin conversion to difructose anhydride (DFA III), which is a natural low-calorie sweetener. Although heterologous expression of IFTase was achieved in Escherichia coli, the extracellular enzyme activity was very low, which limited the commercialization of IFTase. RESULTS Active IFTase of about 43 kDa molecular mass of subunit was extracellularly expressed by Pichia pastoris and was greatly regulated by the IFTase gene copy number integrated into the P. pastoris genome and by the methanol concentration in the induction phase. Under optimized culture conditions, multicopy P. pastoris exhibited a maximum extracellular IFTase activity of 105.4 U mL−1 in a 5 L fermenter, which was 8.9-fold the activity in shake flasks and 5.3-fold that obtained from wild-type strain. CONCLUSION IFTase was expressed in a eukaryotic P. pastoris system for the first time and achieved high-level extracellular expression using a high-cell-density fed-batch cultivation strategy. This demonstrated that P. pastoris was a good candidate for potential DFA III production as a novel IFTase expression system. © 2014 Society of Chemical Industry

This study investigated interactions of six type catechins monomers with human pancreatic α-amylase and the structural requirements for inhibitory activity. The in vitro and in silico results showed that inhibitory effects of catechins followed the order: (+)-gallocatechin-3-O-gallate > (−)-epicatechin-3-O-gallate > (−)-epigallocatechin-3-O-gallate > (−)-epicatechin > (−)-epigallocatechin > (+)-catechin. The A, B and C rings of catechins affected the activity by interact with the catalytic residues of the active site of α-amylase forming a phenols–protein complex, including hydroxyl on the 3-position or 5-position of A–C rings, number of hydroxyl substation on the B-ring or C ring, or 2,3-cis/trans isomerism. The galloylated catechins has higher binding affinity with α-amylase than non-galloylated catechins. The results showed that biological activity of catechins against α-amylase, which supported catechins monomer is a promising ingredient as a development strategy of health food for regulation of energy balance and reduction of related diseases risk.

The relationship between structure and activity of theaflavins against human pancreatic α-Amylase was investigated by in vitro and in silico methods. The IC50 and total energy value showed that inhibitory effects followed the order: theaflavin-3, 3’-di-O-gallate > theaflavin-3’-O-gallate > theaflavin-3-O-gallate > theaflavin. Inhibitory activity was depended on hydroxyl groups and galloyl moieties of theaflavins to interact with the catalytic residues of the active site of α-Amylase by hydrogen bonds and π–π (aromatic–aromatic) interactions. The galloylated theaflavin has higher binding affinity with α-Amylase than non-galloylated theaflavin. The study showed that theaflavins might act as natural enzyme inhibitors with potential health benefits, which provide a foundation for designing novel functional food for effective controlling of starch digestion and postprandial glucose levels.

Difructosan anhydrides III (DFA III) preparation was usually obtained by inulin hydrolysis with inulin fructotransferase (IFTase). The fructofuranosidic linkages of inulin were the same as fructooligosaccharides (FOS), which was synthesized by sucrose with fructosyltransferase (FTase). FOS was mainly composed of 1-kestose (GF(2)), nystose (GF(3)) and fructofuranosylnystose (GF(4)), and nystose was observed to be the smallest substrate for IFTase to synthesize DFA III. So sucrose, much cheaper than inulin, was considered to produce DFA III by coupled FTase and IFTase reaction. DFA III yield was obtained about 100mg/g (DFA III weight/sucrose weight) through this method. The results demonstrated the high potential of the coupled enzyme reaction as a novel DFA III producing method.

A new heteropolymolybdate, (Him)8Na7H7[Cu(im)4(CuGeMo9O33)2][(CuGeMo9O33)2]·41H2O (1) (im = imidazole), was synthesized and structurally characterized by elemental analysis, IR spectra, UV electronic spectroscopy and X-ray diffraction. Compound 1 exhibits a one-dimensional chain-like framework [Cu(im)4(CuGeMo9O33)2]10− with isolated [(CuGeMo9O33)2]12− units residing among the chains, which is first observed in sandwich-type germanomolybdate system. Electrocatalytic experiments show that compound 1 exhibits good electrocatalytic activity for nitrite reduction. The magnetic investigation indicates the existence of antiferromagnetic interactions for 1.

Two new polyoxotungstates Na3H(C3H5N2)4[{Cu(C3N2H4)2}2Cu4(H2O)2(GeW9 O34)2]·27H2O (1) and Na10H8[Mn6Ge3W24O94(H2O)2]·37H2O (2) have been synthesized in aqueous solution. The polyoxoanion frameworks of the two compounds are built on the lacunary Keggin-type polyanion [GeW9O34]10−. The structure of compound 1 is a Weakley-type species capped by two [Cu(C3N2H4)2]2+ units through four bridging oxygen atoms on two opposite W4O4 faces. Compound 2 is a banana-shaped polyoxometalate consisting of two tri-Mn substituted [Mn3GeW9O34]4− Keggin units and a hexavacant bridging cluster [GeW6O26]12−. The magnetic investigations demonstrate the presence of both antiferromagnetic and ferromagnetic exchange interactions for 1 and weak antiferromagnetic interactions for 2.

Difructosan anhydrides III (DFA III) are usually obtained by inulin conversion with inulin fructotransferase (IFTase). IFTase liquor is difficult to store for a long time, which could greatly restrict its application and DFA III production. To meet DFA III scale-up preparation, this work was explored to research dry powder preparation of IFTase from Arthrobacter aurescens SK 8.001 fermented liquor by ultrafiltration concentration, ammonium sulfate precipitation and freeze drying. IFTase powder (10.2 g) was obtained from IFTase precipitation (126.4 g) and its specific activity determined was 16.4 U/mg. Dry powder of IFTase could maintain over 120 days at different temperatures. These results showed that it is easy to scale up DFA III preparation for industrial capacity.

Inulin fructotransferase (IFTase), a novel hydrolase for inulin, was exposed to high hydrostatic pressure (HHP) at 400 and 600 MPa for 15 min in the presence or absence of sorbitol. Sorbitol protected the enzyme against HHP-induced activity loss. The relative residual activity increased nearly 3.1- and 3.8-fold in the presence of 3 mol/L sorbitol under 400 MPa and 600 MPa for 15 min, respectively. Circular dichroism results indicated that the original chaotic unfolding conformation of the enzyme under HHP shifted toward more ordered and impact with 3 mol/L sorbitol. Fluorescence and UV spectra results suggested that sorbitol prevented partially the unfolding of the enzyme and stabilized the conformation under high pressure. These results might be attributed to the binding of sorbitol on the surface of IFTase to rearrange and strengthen intra- and intermolecular hydrogen bonds.

New-type two lanthanide complexes based on a germanotungstate ligand, Na12[Ln4(α(1,4)-GeW10O38)2 (H2O)6]·nH2O (Ln = Dy (1); Er (2)) which contain four trivalent lanthanide ions and two [α(1,4)-GeW10O38]12− units, have been synthesized and structurally characterised, and their magnetic properties have also been investigated. Each lanthanide ion resides in the vacant site of the [α(1,4)-GeW10O38]12− fragment through bonding to five oxygen atoms, four of which are from the WO6 octahedra, while the other one is from the central GeO4 tetrahedron group.