*3.6. Kinetic Parameters Determination*

Differently from previous studies, the kinetics of fructose and glucose "release" in the reaction have been calculated. The rate of glucose release is the hydrolysis rate plus the transferase reaction rate. The *K*<sup>m</sup> of hydrolysis and transferase reaction is expressed as "*K*mH" and "*K*mT", respectively [36]. The kinetic parameters of Cedi-LS and Psor-LS are shown in Table 2. The determined *<sup>K</sup>*m<sup>H</sup> and *<sup>K</sup>*m<sup>T</sup> values of Cedi-LS were 57 ± 2 and 202 ± 7 mM, respectively. Cedi-LS showed higher *k*cat values for transfructosylation than for hydrolysis, and its *k*cat H/*k*cat <sup>T</sup> was 1.35, significantly higher than that of *B. subtilis* LS (0.37) [36]. The *<sup>K</sup>*m<sup>H</sup> (117 ± 8 mM) and *<sup>k</sup>*cat <sup>H</sup> (620 ± 12 s−1) of Psor-LS were higher than Cedi-LS, indicating that Psor-LS is more favorable to hydrolyze sucrose than Cedi-LS. Nevertheless, the kinetic parameter of the transferase reaction of Psor-LS did not conform to the nonlinear least square regression method.

**Table 2.** Kinetic parameters of Cedi-LS and Psor-LS.


#### *3.7. The Effect of Sucrose Concentration on the Activity and T/H of Psor-LS*

Sucrose concentration is an essential factor in the activity and T/H of LSs. As a result, Psor-LS exhibited the highest activity at 30% sucrose (Figure 6A). The Psor-LS could retain over 80% of its activity at substrate concentrations ranging from 10 to 60%, suggesting that Psor-LS showed a broad sucrose concentration spectrum for its activity. In this study, the total activity of Psor-LS could be saturated at 30% sucrose. A similar result was reported in the LSs from *B*. *methylotrophicus* SK 21.002 [7] and *Z. mobilis* [37]. The "E-F·F-G" complex accumulation and the nonproductive binding are possibilities for this inhibitory phenomenon [37]. Although the activity was decreased, the T/H of the Psor-LS showed an upward trend with the increase in sucrose concentration (Figure 6B). The Psor-LS showed relatively high T/H (>1) at 40% sucrose and exhibited the highest T/H at 60% sucrose (1.3). By comparison, the *E*. *amylovora* LS had a T/H of about 4 at 100 mM sucrose, and its transfructosylation reaction could reach a maximum of 97% at 1.5 M sucrose [5].

**Figure 6.** Effect of sucrose concentration on the activity and T/H of the recombinant LSs. (**A**) Effect of sucrose concentration on the total activity of Psor-LS. (**B**) Effect of sucrose concentration on the T/H of Psor-LS. All of the values were the mean of triplicate experiments.

#### *3.8. Effect of Enzyme Concentration on the Levan Production and T/H of Psor-LS*

Different enzyme dosages ranging from 10 to 100 μg/mL at 30% sucrose were employed to optimize the levan production. Since enzyme concentration was related to levan production, the ratio of levan production and enzyme concentration (P/E) was evaluated as "input–output" in this study. As a result, the highest P/E value of Psor-LS was exhibited at 25 μg/mL enzyme, suggesting that both enzyme and substrate were the maximum output in this enzyme dosage (input) (Figure 7A). A comparable P/E ratio was observed at 30 μg/mL enzyme concentration compared to 25 μg/mL. However, it dropped remarkably when enzyme concentration was below 25 or above 30 μg/mL. The Psor-LS showed a growing T/H with increased enzyme concentration (Figure 7B), and it exhibited an equivalent transfructosylation and hydrolysis reaction at 100 μg/mL enzyme. A similar result for IS from *Lactobacillus jensenii* was reported when the T/H was increased with increased enzyme dosage [38]. By contrast, the *B*. *subtilis* LS showed high T/H (2.7) at 0.1 U/mL enzyme concentration but low T/H (0.7) when enzyme concentration is increased to 10 U/mL.

**Figure 7.** Effect of enzyme concentration on the P/E and T/H of the recombinant LS; P/E is the ratio of levan production and enzyme concentration. (**A**) Effect of enzyme concentration on P/E of Psor-LS. (**B**) Effect of enzyme concentration on T/H of Psor-LS. All of the values are the mean of triplicate experiments.

#### *3.9. Biological Production of Psor-LS*

The biotransformation process of Psor-LS is shown in Figure 8A. Rapid sucrose consumption was shown in the first 1.5 h. The consumption rate slowed down in the next 1.5 h. After 3 h, the sucrose concentration was almost unchanged, consuming at a very low rate. When the reaction reaches equilibrium, the maximum conversion ratio of the transfructosylated product to sucrose was 29.2% at 3 h (Figure 8B). Like Cedi-LS, the product produced by Psor-LS decreased slowly after 3 h. By comparison, the LSs from *B*. *methylotrophicus* SK 21.002 and *B*. *licheniformis* NS032 could also produce levan effectively, and their conversion ratios were 33 and 25%, respectively [7,39].

**Figure 8.** Biotransformation process and conversion ratio of recombinant LS. (**A**) The variation in sucrose, glucose, and fructose concentration in the biotransformation process of Psor-LS. (**B**) The variation in conversion ratio in the biotransformation process of Psor-LS. All of the values are the mean of triplicate experiments.

As reported, the T/H of Cedi-LS was 1.3 at 65 ◦C, higher than that of Psor-LS (0.8). T/H is considered to reflect the transfructosylation ability of LS and continue to the product distribution of LS. For instance, the *B*. *subtilis* LS showed higher T/H (2.7) at 0.1 U/mL enzyme concentration and produced HMW levan. When the enzyme concentration was 1 U/mL, the enzyme produced LMW levan with lower T/H (1.0) [15]. As shown in Figure 8A, the residual fructose in the Psor-LS system is significantly higher than that in Cedi-LS, consistent with its relatively lower T/H value. Meanwhile, the residual sucrose and glucose in Psor-LS system are lower than that in Cedi-LS, which indicates that Psor-LS has higher glucose utilization than Cedi-LS.

## *3.10. Effect of Temperature on the Product Distribution of Cedi-LS and Psor-LS*

Many factors were considered to be potential reasons for the product specificity of LSs, such as sucrose concentration [5] and enzyme concentration [15]. The temperature could also affect the product specificity [17]. However, how temperature could affect the product specificity of LS remains unclear. To investigate the effect of temperature on the product distribution of LS, we reduced the reaction temperature from 65 to 35 ◦C. At 35 ◦C, the product conversion ratios of Cedi-LS and Psor-LS were 41.4 and 37.1%, respectively. The T/H values of the two enzymes were 2.3 and 1.0. Moreover, the viscosity of the reaction solution of Cedi-LS was increased when the temperature was decreased to 45 and 35 ◦C. At 2 h reaction time, the solution showed a "gel-similar" phenomenon (Figure 9C), which is much different from that of the Psor-LS solution (Figure 9D).

**Figure 9.** Effect of temperature on the product distribution and solution status of the recombinant LSs. (**A**) Effect of temperature on the product distribution of Cedi-LS. (**B**) Effect of temperature on the product distribution of Psor-LS. (**C**) Effect of temperature on the reaction mixture of Cedi-LS. (**D**) Effect of temperature on the reaction mixture of Psor-LS.

To further determine the possible change in product distribution at different temperatures, the reaction mixture components were analyzed by HPGFC in detail (Figure 9A,B). At its optimal temperature of 65 ◦C, the Cedi-LS could simultaneously produce FOS, LMW (4.1 × 104 Da), and HMW (1.8 × 106 Da) levan in the reaction mixture. On the contrary, the Psor-LS specifically produced FOS and HMW (1.4 × <sup>10</sup><sup>6</sup> Da) levan without LMW levan. When the temperature was decreased, the levan produced by Cedi-LS showed a

higher *<sup>M</sup>*<sup>W</sup> that reached 8.4 × <sup>10</sup><sup>6</sup> Da at 35 ◦C. Simultaneously, a low temperature results in higher production of HMW levan. Therefore, the increase in *M*<sup>W</sup> and production of HMW levan were supposed to result in a higher viscosity of the reaction solution, as shown in Figure 9C. The low temperature increased the HMW levan in many LSs [40]. For instance, the LS from *R*. *aquatilis* ATCC 33071 mainly produced FOS at 55–60 ◦C, while it synthesized HMW levan (1 × <sup>10</sup><sup>6</sup> Da) at low temperature (37 ◦C) [41]. The production of HMW levan increased obviously at low temperatures (4 ◦C) in *Z. mobilis* LS [17]. On the contrary, the production of HMW levan in Psor-LS decreased as the temperature decreased. This means that lower temperatures promoted the synthesis of FOS in Psor-LS. As far as the authors are concerned, this is the first LS that prefers to produce FOSs rather than HMW levan when the temperature is decreased. Moreover, the *M*<sup>W</sup> of the levan from Psor-LS was not changed, indicating that the temperature has different effects on the product distribution of Cedi-LS and Psor-LS.

#### *3.11. Product Purification and Analysis*

The residual enzyme in the mixture was removed by Sevage reagent, and the polysaccharide was separated by multiple ethanol precipitation. When the final ethanol concentration was 60%, the polysaccharide produced from Psor-LS was obtained. The obtained precipitate was vacuum freeze-dried for 48 h to remove moisture altogether (Figure 10A). The products of Psor-LS were identified as *β*-(2, 6) levan and levan-type FOSs by the NMR analysis (Figure S1). The 1H spectrograms were compared with those of *L. reuteri* LTH5448 LS and *L. jensenii* IS [9,38]. Meanwhile, the 13C chemical shifts reported for biosynthesized levan are compared in Table S2. The result revealed that the polysaccharide synthesized by Psor-LS was *β*-(2, 6) levan.

**Figure 10.** Products from the recombinant LSs. (**A**) The purified HMW levan from Psor-LS. (**B**) The purified FOS from Psor-LS. (**C**) The component analysis of products from Cedi-LS and Psor-LS. (**D**) The HPIC spectrogram of purified FOS from Psor-LS.

The *<sup>M</sup>*<sup>W</sup> of levan synthesized by Psor-LS was 1.4 × 106 Da (65 ◦C). Generally, the *<sup>M</sup>*<sup>W</sup> of HMW levan produced by LS from different microorganisms were different, such as the

LSs from *<sup>T</sup>*. *sakaeratensis* (1.0−6.8 × 105 Da) [42], *<sup>A</sup>*. *diazotrophicus* SRT4 (2.0 × <sup>10</sup><sup>6</sup> Da) [43] and *Bacillus aryabhattai* (5.3 × <sup>10</sup><sup>7</sup> Da) [44]. FOS was purified by activated carbon chromatography to remove the sucrose and monosaccharides and dried in a freeze dryer for 48 h. The purified FOS is shown in Figure 10B. The purity of FOS produced by Psor-LS with DP ≤ 16 could reach more than 90%, but quantitative comparison cannot be carried out due to the significant loss in the purification process (Figure 10D).

#### **4. Conclusions**

In this study, a novel thermostable LS from *P*. *orientalis* was identified. The Psor-LS retained 46% of its initial activity at 55 ◦C for 9 h and 50% at 45 ◦C for 60 h. Meanwhile, there are noticeable differences in the product distribution between the Cedi-LS and Psor-LS. The *<sup>M</sup>*<sup>W</sup> of levan synthesized by Cedi-LS was increased from 1.8 × 106 Da (65 ◦C) to 8.4 × 106 Da (35 ◦C). On the contrary, the decrease in temperature did not significantly affect the product distribution of Psor-LS. At 65 °C, the Psor-LS would specifically produce FOSs and HMW levan without LMW levan. Notably, at 35 ◦C, the reaction equilibrium of Psor-LS from sucrose (30%) was 37%, and a certain amount of FOS (DP ≤ 16) was obtained among them.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/polym15061435/s1, Figure S1: Nuclear magnetic resonance (NMR) analysis spectrogram of the products from Psor-LS; Table S1: The genbank number and sequnce length of different microbial FSs; Table S2: 13C chemical shifts reported for biosynthesized levan [7,45].

**Author Contributions:** C.G.: Supervision. X.Z.: Data analysis, writing—original draft. D.N.: Software. W.Z.: Methodology and software. W.X.: Writing—review and editing. W.M.: Co-Supervision, project administration. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Natural Science Foundation of China (22278183), the National Key R&D Program of China (2022YFD2101400), the Natural Science Foundation of Jiangsu Province (BK20210463), the Special Fund from Post-doctor Innovation Research Program of Shandong Province (SDCX-ZG-202203049), and the Independent Projects for Young Scholars at Jiangnan University (JUSRP122011).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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