Inulin Fructotransferase

Difructose anhydride III （DFA III） is a novel type of attractive sweetener. It has been shown to have numerous health and medical benefits, including improving the absorption of calcium, magnesium, zinc, copper and other minerals, accelerating bone formation, stimulating diuretic action, improving bowel movement and inhibiting dental caries. It has half the sweetness but only 1/15 the calories of sucrose. Based on these properties, DFA III has attracted a great deal of attention in recent years as a low calorie sugar-substituting sweetener, and an additive in baked foods, beverage, candy, as well as in pharmaceutical formulations. DFA III can be produced from inulin by inulin fructotransferase （IFTase, EC 4.2.2.18）. Much emphasis has been put on the biotransformation of inulin in recent years, and inulin fructotransferase is considered to have the most potential use for DFA III production, since it can effectively hydrolyze inulin.In this work, we isolated a new strain SK 8.001 from soil for IFTase production. According to its morphological and biochemical characteristics, as well as 16S rDNA, it was identified as Arthrobacter aurescens and deposited at China Center for Type Culture Collection （CCTCC） under the accession number M 207185. The structural identification of the reaction products was assigned using the method of LC-MS-MS and （13）^C-NMR. The main reaction product was identified as DFA III, with minor products being 1-kestose （GF₂）, nystose （GF₃）, and fructofuranosyl nystose （GF₄）.The purification of difructose anhydride III using cation exchange resin DTF-02 was studied. The effects of column temperature, sample concentration, sample volume and elution flow rate were discussed. It showed that, when 5 ml difructose anhydride III containing a soluble solid content of 20%, difructose anhydride III could completely separate from fructo-oligosaccharides with elution flow rate of 1 ml/min at 60 oC. Under such condition, the fractions containing DFA III were pooled and determined with HPLC. The collected DFA III purity was over 98% and its recovery rate was above 95%.Effects of various carbon sources on the formation of IFTase were investigated and it was shown that inulin and DFA III markedly stimulated the synthesis of IFTase while fructo-oligosaccharides （FOS） induced at a low level. The time courses of IFTase induced with inulin or DFA III at different concentrations were studied. Even with 30-50 g/l inulin as carbon source, bacteria grew well and IFTase was normally synthesized. When the concentration of DFA III was above 30 g/l, bacteria growth and IFTase synthesis was inhibited.The enzyme was purified to electrophoresis homogeneity by a combination of ethanol precipitation, DEAE Sepharose Fast Flow column and Superdex 200 column. The molecular mass estimated to be 35 kDa by size exclusion chromatography and 40 kDa by SDS-PAGE. From these results, IFTase from A. aurescens SK8.001 was considered to be a monomer.The properties of purified IFTase were characterized. It exhibited maximal activity at 60-70°C and pH 5.5. More than 50% activity was retained at pH 4.5-8.0 after incubation at 4°C for 24 h and 86.5% of its initial activity was still kept after incubation at 60°C for 4 h. Chemical modification indicated that tryptophan residues of IFTase were essential for the activity. The purified IFTase was neither inhibited by EDTA nor activated by any metal ion, suggesting it was not a metalloenzyme. K_m and V_max were estimated to be 0.52 mM and 0.3μmoles DFA III/ ml·min. Its smallest substrate was GF₃. The N-terminal amino acid sequence of the purified enzyme was AEGAKASPLNSPNVYDVT, which is different from that of other known IFTases. According to the homology analysis of N-terminal amino acid sequence and enzyme properties, IFTase from A. aurescens SK8.001 was identified as a new member of DFA III-forming IFTase family.The IFTase gene was obtained through PCR amplification. DNA sequence analysis revealed an open reading frame of 1,353 bp, encoding a polypeptide of 450 amino acid residues. This sequence data was submitted to the GenBank databases under accession no. HM138085. Three plasmids haboring, respectively, the native IFTase signal peptide, pelB signal peptide and no peptide were constructed in order to characterize the effect of signal peptides on the active IFTase expression in E. coli. These three transformants all expressed active IFTase. Transformant with the native IFTase signal peptide, BL21（pET22b-ift-W）, was a useful system for extracelluar over-expression of IFTase, and its maximum extracellular IFTase activity reached 81.0 U/ml. This value was 4.1-fold of that obtained with A. aurescens SK 8.001 for IFTase production. Its maximum intracellular activity, 119 U/ml, occurred at 12 h. The recombinant IFTase was purified to electrophoretical homogeneity and characterized. The enzyme showed maximum activity at pH 6.0 and 55°C, and retained 81% of its initial activity after incubation at 60°C for 4 h. And more than 70% activity was still kept at pH 4.5-8.0 after incubation at 4°C for 24 h...