3.1 Purification of the recombinant levansucrase
As shown in Fig. 1, the purified enzyme had a single band clearly visible in SDS-PAGE (between 80kDa and 58kDa), indicating that no impurities were introduced in the purification process. GEM particle purification is a promising method, which can save purification time, simplify operation and reduce activity loss compared with traditional purification methods .
3.2 Effect of temperature, pH, metal ions on the levan formation activity of the recombinant levansucrase
The levan biosynthesis ability of the recombinant enzyme was evaluated at various temperatures, ranging from 20 ℃ to 60 ℃. As shown in Fig. 2A, the optimum activity temperature of the recombinase was 40 ℃, and the activity of the recombinase was severely inhibited when temperatures below 30°C and above 55°C. The levansucrase from Bacillus methylotrophicus SK 21.002 showed the highest levan production at around 37 ℃ . Lower optimum temperature for levan formation of 15 ℃ was reported for levansucrase from Halomonas smyrnensis AAD6T .
The effect of pH on the levan biosynthesis ability of the recombinant enzyme was studied at different pH values (4.6–5.6). The highest levan formation activity of enzyme was obtained at pH 5.2. As shown in the Fig. 2B, the activity was rather sensitive to pH which was only high at pH 5.2 but decreased sharply when pH below 5.0 or above pH 5.4. The optimum pH for levan synthesis was lower than that of levansucrase from Leuconostoc mesenteroides B-512 FMC  and levansucrase from Brenneria goodwinii , and their optimum pH values were 6.2 and 6.0, respectively. The optimal pH for most levansucrases is between 5.0 and 6.5 .
The effects of different concentrations and different kinds of metal ions on the levan formation activity of levansucrase were determined at pH 5.2 and 40 ℃ (Fig. 2C). 50 mM Ca2+ and K+ increased the activity to around 130% of the initial relative activity. However, metal ions such as Cu2+, Fe3+ and Zn2+ had an obvious inhibitory effect on the enzyme activity indicating that the recombinase was sensitive to the presence of Cu2+, Fe3+ and Zn2+. Levansucrase from Bacillus methylotrophicus SK 21.002 was tested for effects on metal ions on levan biosynthesis. 20 mM Mg2+ increased the enzyme activity to 115% of the initial relative activity, while Cu2+, Fe2+, and Zn2+ had a strong inhibitory effect on the enzyme activity . Hg2+ and Ag+ decreased the activity of levansucrase from Leuconostoc Mesenteroides B-512 FMC by 92% and 86%, respectively, while Zn2+, Fe2+ and Cu2+ slightly inhibited the activity of levansucrase .
Levan biosynthesis was carried out from 10% (w/v) sucrose at pH 5.2 and 40 ℃, using the recombinant levansucrase of 6.45 U/g sucrose. The highest production reached to 30.6 g/L after 2 h, which was higher than 15 g/L for Erwinia herbicolaand  and lower than 36 g/L for B. polymyxa (NRRL B-18475) .
3.3 Enzyme kinetics
Different concentrations of sucrose solution were prepared with sodium acetate-acetate buffer. The reaction rate of levansucrase with different concentrations of sucrose solution was determined according to the method described in 2.5. According to the regression equation (Fig. 3), the Michaelis constant of levansucrase to sucrose was 25.63 mM. The Km value of the enzyme in this study was similar to the Km value (24mM) of the levansucrase from Leuconostoc mesenteroides NTM048 . Moreover, the Km value was much lower than that of levansucrase from Halomonas smyrnensis AAD6T (104.79 ± 4.17 mM) . Therefore, the affinity of levansucrase from different microbial sources to sucrose is very different.
3.4 Monosaccharide Composition Analysis
Monosaccharide composition analysis usually requires hydrolysis of polysaccharides or oligosaccharides with appropriate acids before derivatization for gas chromatography (GC) and high-performance liquid chromatography (HPLC) analysis, or high-performance ion chromatography (HPIC) analysis without derivatization. The resulting chromatogram is shown in Fig. 4. By comparing the retention time of sample monosaccharides with that of standard monosaccharides, it was determined that the sample polysaccharide was composed of fructose, glucose and galactose, which accounted for 81.6%, 16.6% and 1.9%, respectively. The monosaccharide composition of polysaccharides is related to many factors, such as the hydrolysis temperature of the sample can affect the extraction of ketose. When the temperature was 30–70℃, the free fructose was relatively stable; when the temperature rose to 120℃, the free fructose would rapidly degrade to 80% .
3.5 Molecular weight and purity of levan
Gel permeation chromatography is the most commonly used method to detect the purity and molecular weight of polysaccharides. The molecular weight of levan was obtained by comparing the retention time of levan with the standard substance with different molecular weights. The retention time of levan was 16.667 min and it has a single elution peak in gel permeation chromatography (Fig. 5), indicating that the polymer is a homogeneous component. Based on the linear regression curve of PEG standards, the average molecular weight of levan was 1.56×106Da by calculation. In general, the molecular weight of polysaccharides is related to many factors, including strain type, fermentation conditions, medium composition, and extraction method. Malang et al  have shown levans synthesized by raffinose as carbon source in W. confusa E5/2 − 1 have higher molecular weight than levans synthesized from sucrose. Levan produced by Bacillus subtilis was reported to have two levan distributions: a high molecular weight levan (2.3×106 Da) and a low molecular weight levan (7.2×103 Da) . The molecular weight of levan in this study is between the two. Levan with different molecular weights has different applications in medicine, cosmetics and food. For example, levan with low Mw produced from Z. Mobilis had a stronger antibacterial inhibition in vitro, while levan with high Mw produced from Bacillus subtilis NRC1aza had the strongest DPPH free radical scavenging activity .
3.6 SEM analysis
A scanning electron microscopy analysis was performed to observe the microstructure and surface morphology of the levan, which can help in understanding the physical properties of the levan. The surface morphology micrographs of levan at 400×, 1000×, 2000× were shown in Fig. 6. As observed by SEM images, levan in this study had a highly branched and porous structure. It was supposed that levan with a highly branched and porous structure was conducive to the formation of hydrated polymers and was most likely to be used in foods and cosmetics industries as texturing, thickening, stabilizing, and water-binding agent [36–38]. Besides, SEM images indicated that levan had a sheet-like smooth and glossy surface, which had the potential to prepare plasticized film . The part microstructure of the levan in this study was similar to the microstructure of glucan produced by Leuconostoc pseudomesenteroides XG5, which had a smooth and glittering surface and high branched structure , but there were a little differences between levan from Bacillus mojavensis and Brenneria sp. EniD312 exhibited uniform porous network [7, 40].
3.7 AFM analysis
AFM is a useful tool for characterizing polymer morphology with high resolution and simple operation, which is developed on the basis of SEM. The topographical AFM images of levan exhibited many ellipsoidal or spheroidal particles and spike-like lumps (Fig. 7), which indicated that polysaccharides had a strong affinity with water molecules [40–42]. The maximum peak height of rounded lumps was 55.7nm, the average roughness was 3.41 nm and the mean roughness was 1.48 nm. The maximum height of levan was much higher than the height of a single polysaccharide chain(0.1-1nm) suggesting that the tightly packed molecular structure formed in AFM images may be caused by the intermolecular and intramolecular aggregation of levan . A similar result was reported for the EPS polymer from Lactobacillus sakei L3  but different from Lactobacillus reuteri E81 glucan which had the tangled networks  and Mesona blumes gum EPS polymer which had an irregular shape like the worm .
3.8 FT-IR analysis
Fourier-transformed infrared spectroscopy is used to determine the glycoside bond configuration and the functional group on the sugar chain by using the relative vibrations within the molecule and molecular rotation information to analyze the structure of polysaccharides. Figure 8 showed the FT-IR spectrum of purified levan. The wide and strong peak at 3304 cm− 1 was caused by the stretching vibration of O-H [25, 46], indicating the existence of intermolecular hydrogen bonding. The weak peaks at 2931 cm− 1 and 2887 cm− 1 were the results of C-H stretching vibration and bending vibration respectively . The strong absorption peak at 1644 cm− 1 was caused by the O-H bending vibration, which might be caused by the presence of water in the sample . The absorption peaks at 1122 cm− 1 and 1009 cm− 1 were caused by C-O-C stretching vibration , which were the characteristic peaks of carbohydrates. The absorption peaks at 923 cm− 1 and 809 cm− 1 represented symmetric stretching vibration of furanose and D-type C-H bending vibration of furanose respectively, which were typical signal peaks of furanose [14, 50]. Thus it was proved that a furan ring was contained in the polysaccharide structure. Preliminary analysis showed that the polysaccharide was composed of D-furanose.
3.9 NMR analysis
Further analysis of the structure of purified polysaccharides was obtained by 1H and 13C NMR spectra. The 13C NMR spectra had several signals in the anomeric carbon signal region (95-110ppm). The peak at 96.31 ppm may be a signal for ɑ-glucose C1 . Two peaks at 104.19 and 103.65 ppm were derived from β-fructose C2 signal . Major signals in the ring carbon signal region (50-85ppm) at around 59.88, 76.28, 75.18, 80.27 and 63.36 ppm were attributed to the fructose groups C1, C3, C4, C5 and C6, respectively. Among them, signal at the 63.36 ppm (C6) confirmed the presence of the fructose β-(2,6)-linkage . These carbon chemical shift of levan produced by levansucrase reported in other literatures are shown in the table below, which are similar to the six signals in this paper.
According to the 1H NMR spectra, the signal at 4.70 ppm was due to D2O. The signals of 5.33 and 5.15 ppm in the anomeric proton region can be attributed to the characteristic signal of ɑ-glucose H1 . Seven major proton signals were observed at 4.10 ppm (H3), 4.01 ppm (H4), 3.87 ppm (H5), 3.82 ppm (H6a), 3.68 ppm (H1a), 3.60 ppm (H1b), and 3.48 ppm (H6b), respectively. All of them were in the ring proton region (3.4–4.2 ppm) indicating that the presence of fructose in the polysaccharide . The ratio of peak areas was approximately 1:1:1:1:1:1 which meant the same amount of every kind of H atom. No signal of galactose residue was found in the spectrum, which may be due to the low content of galactose residues, resulting in too little signal accumulation. All the information indicated that the polysaccharide synthesized by levansucrase was β-(2,6) levan.