Crystal Structure Of Inulin Fructotransferase And The Effects Of High Hydrostatic Pressure On Its Conformation And Function

Inulin fructotransferase(IFTase) as an inulin specific hydrolase is used to produce the novel functional sweetener, difructose anhydride III(DFAIII). Current research focuses on the microorganism screening for IFTase. However, to date, little detailed information has been obtained regarding the structure of IFTase. It is well known that the structure of protein determines its function. Therefore, it is necessary to further study on the conformation of IFTase at the molecular level in order to improve the catalytic behaviour and provide theoretical basis for the following rational protein design research.The crystals of IFTase and IFTase- fructofuranosyl nystose(GF4) complex were prepared in this dissertation. The structure of IFTase was also decribed in detail. Furthermore, high hydrostatic pressure(HHP), as a thermodynamic parameter, could be used to obtain a more detailed description of the reactions catalysed by the enzyme and the regulation of enzyme behaviours. One objective of this study was to probe the relationships between enzyme conformation and the activities of IFTase at different pressure levels by HHP, usually on the order of 100-600 MPa. The main contents and conclusions were as follows:(1) The crystals were prepared with the sitting drop method. Crystal growth conditions for the final screening were obtained: 0.2 mol/L lithium sulfate monohydrate, Tris-HCl buffer(0.1 M, p H 8.5), polyethylene glycol 3350(25%, w/v). The crystal size of IFTase was 0.4 × 0.3 × 0.2 mm. The resolution of X-ray diffraction was 2.15 ?. IFTase, per asymmetric unit, consisted of three identical monomer molecules. This trimer was connected by intermolecular hydrogen bonds between the Ser 293 and Ser 269 at each subunit. The monomer was tapered, and mainly constituted by β-sheets. Compared with the reported IFTase structure, each IFTase molecule could combine seven GF4 molecules at three positions. Wherein, Asp 169 and Gln 216 might be the binding sites for GF4. π-σ interactions were formed between the benzene ring in Trp 103 and Pro 133 on the other subunit. This conjugated bond might stabilize IFTase molecules or its active pocket. The thermostability of the mutation from cysteine to alanine was detected at 80 oC. The results showed that Cys 144, Cys 205 and Cys 208 might be more vital for the thermostability.(2) The changes in the enzymatic properties and conformation of IFTase after the treatment of HHP were investigated. IFTase activity slightly increased by 13.6% after the treatment of 200 MPa and 60 oC for 15 min. With the increase in the pressure, the activity decreased gradually. When the pressure was 600 MPa, the residual activity was only 4.56%.When 3 mol/L sorbitol was added, the activity increased by 3.88 times after the treatment of 600 MPa. The experimental results showed that the high concentration of sorbitol could improve the high pressure-stability. After relief, the activity was reversed when IFTase suffered from low pressure. IFTase was irreversibly deactivated after high pressure treatment. In addition, the thermal stability was significantly improved at 200 MPa. The residual activity was only 50.5% with the incubation at 80 oC and atmospheric pressure for 25 min, while it remained approximately 70% at 200 MPa and 80 oC for 25 min. Thermal inactivation rate constant reduced by 55.9%, while the activation energy could increase by 1.41 times.The conformational changes were investigated using circular dichroism, intrinsic fluorescence spectra, fluorescence probe technique, size exclusion chromatography, electrophoresis, dynamic light scattering, and atomic force microscope. The results showed that the primary structure has not changed significantly, while the secondary structure gradually destroyed. Changes in the microenvironment of tryptophan were more complicated. When the pressure was less than 200 MPa, tryptophan might shift to hydrophobic microenvironment. The whole conformation was slightly purturbed, while the loops have been changed significantly. These loops were more ordered and impact, resulting in the slight increase in the activity of IFTase. When the pressure further increased, tryptophan contacted with more water moleculars, and the hydrophobic surface also increased. The trimer dissociated, the average particle size decreased, the π-σ interactions between Pro 133 and Trp 103 from different subunits, and the intermolecular hydrogen bonds were also disrupted, resulting in the destabilizing of the enzyme and the loss of the activity. The enzyme was more stable with the addition of sorbital which might provide amounts of hydrogen bonds. The mechanism of pressure treatment on the enzyme was different from that of heat treatment. The increasing thermal stability were partially attributed to the increase in the intramolecular disulfide bonds after the treatment of 200 MPa pressure at 80 oC for 15 min. Changes in non-covalent bond also played a crucial role in improving the thermal stability of the enzyme.(3) Based on the above research, thermodynamics of IFTase catalytic reaction under HHP were also studied. IFTase optimal catalytic conditions were 200 MPa and 60 oC. Michaelis constant, activation energy, activation enthalpy and activation volume were lowest, while turnover number and catalytic efficiency were highest. At 200 MPa, the optimum p H was 6.0. 1.5 mol/L Na Cl rather than 1.5 mol/L Na NO3 could further accelerate the catalytic rate under high pressure.