Study On The Purification Of Fructosyltransferase From Aspergillus Oryzae And Analysis Of Its Catalytic Activity

Fructosyltransferases are a group of enzymes(EC2.4.1.9)that catalyze the transfer of fructosyl units from one sucrose to another one. They belong to the glycoside hydrolase 32(GH32) and 68(GH68) families. The fructosyltransferases obtaining from microorganisms have a high catalytic activity, bear a high temperature and are not sensitive to seasonal changes, so they are significant to industry. Sucralose is a new non-nutritive oligofructose, and it has huge marketing potential as its high sweetness, safety good taste and so on. Otherwise it has good physical and chemical properties, such as bearing heat and acid, so it is suitable for baking foods, drinking, candy and medicines, and it is one of the most perfect and competitive sweetening agent by far. The sucrose-6-acetate is a key intermediate in the conventional synthesis pathway. Biological enzyme method to product sucrose-6-acetate is a hot topic recently, so fructosyltransferases is capable of synthesis sucrose-6-acetate from glucose-6-acetate and sucrose. The method has many advantages, such as specificity, efficient, less by-product, high percent conversion, so the method has a strong application potential.In our previous experiments, we screened a new fructosyltransferase(Ao FT) from the fermentation broth of a strain of Aspergillus oryzae, and researches show that crude cell extract has a high activity in synthesis of sucrose-6-acetate. In order to obtain the purified fructosyltransferase Ao FT, the purification of the fructosyltransferase Ao FT is summarized in next procedures,(NH4)2SO4 precipitation, Q-Sepharose Fast Flow column and Sephacryl S-200 HR. Single protein band was excised from SDS-PAGE, and the molecular mass is about 50 k Da. The peptide sequences determined by MALDI-TOF MS from 50 k Da Ao FT, the enzyme contained two well-conserved FRDPXVFW and GXXXECPX motifs found in the activity sites of enzymes belonging to the GH32 families. The activity of purified fructosyltransferase Ao FT is measured by HPLC and LC-MS, the results show that it has a high activity.The properties of purified fructosyltransferase Ao FT are characterized. The optimum temperature and p H of the enzyme are 45 and 6.0, and more than 80% of its activity was ℃recovered at 30-50 ℃after treated for 1h. Fructosyltransferase Ao FT also has high p H stability, and more than 80% of its activity was still kept after treated at p H 4.5-7.5 for 1 h. In addition, the enzyme has a good resistance to the majority of the metal ions, surfactant(Tween20, Triton- X100) and organic solvent(methanol, ethanol, acetone, n-butyl alcohol, formaldehyde, DMF, DMSO etc.). The enzyme activity was strongly inhibited by SDS and urea, but the enzyme activity is not affected by EDTA, which shows that the enzyme is not a metal enzyme.Through the analysis of its catalytic activity in the ionic liquids, we found the enzyme has different catalytic activity in different ionic liquids. Its activity is obviously activating in hexafluorophosphate ionic liquid, especially in the [Dmim][PF6] ionic liquid, and the yield of sucrose-6-acetate increased to 30.5%, while in the tetrafluoroborate salt ionic liquids its activity is obviously inhibited, and the yield of sucrose 6-acetate dropped to 3.73% in [Om Py][BF4] ionic liquid, in other ionic liquids the activity of the enzyme has little changes. Using Fourier transform infrared FT-IR spectroscopy and circular dichroism CD to analyse molecular structure of the enzyme Ao FT, we found the changes of its secondary structure is closely related to its catalytic activity in ionic liquids. In the hydrophobic hexafluorophosphate ionic liquid, the α-helix content of enzyme decrease, the β-folding content of the enzyme is more, and the catalytic activity of the enzyme is higher, as in the [Dmim][PF6] ionic liquid, the α-helix content of enzyme decreases to 9.4% and the β-folding content of the enzyme increases to 46.2%, the activity of the enzyme has increased 54.8%. While in the [Om Py][BF4] ionic liquid, the α-helix content of enzyme is 12.3% and the β-folding content of the enzyme is 42.5%, so the activity of the enzyme has decreased 82.2%. The secondary structure content has an impact on the catalytic activity of the enzyme in the ionic liquids.