*3.2. Expression and Purification of Psor-LS*

The sequence of the gene encoding the Psor-LS has been deposited in the GenBank database. SDS-PAGE analysis of the recombinant protein from *P*. *orientalis* indicated a band around 44 kDa (Figure 2A), consistent with the calculated molecular weights of the LS (ExPASy Computer *M*w tool, https://web.expasy.org/compute\_pi/), suggesting a triumphant expression of the target enzyme in *E*. *coli*.

**Figure 2.** SDS-PAGE and phylogenetic tree. (**A**) Lane P represents the purified LS from *P*. *orientalis*, and lane M represents the protein marker with standard enzymes with the following molecular weights: 250, 150, 100, 70, 50, 40, 35, 25, 20, 15, and 10 kDa. (**B**) Phylogenetic tree of LSs from different sources. The phylogenetic tree was constructed by MEGA 5.1. The content in the bracket is the accession numbers in the GenBank database.

Amino acid sequence identity analysis was performed by EMBI 225 (https://www. ebi.ac.uk/Tools/services/web/tool). The Psor-LS showed the highest identity of 80% with the *R. aquatilis* LS and had more than 70% identity to LSs from *C. diazotrophica*, *Brenneria* sp. EniD312 and *E. amylovora*. By contrast, the Psor-LS showed the lowest identity of 24% with the *Leuconostoc mesenteroides* LS. They only had less than 30% identity to the LSs from *B*. *subtilis*, *B. amyloliquefaciens*, and *Clostridium arbusti*. The evolutional relationship to LSs from different sources is shown in Figure 2B.

### *3.3. Effect of pH on the Activity and T/H Ratio of Psor-LS*

As shown in Figure 3A, Psor-LS showed relatively high activity (>80%) at pH values ranging from 5.0 to 7.5 but dropped when pH was below 5.0 or above 7.5. Figure 3B shows the effect of pH on the transfructosylation activity of Psor-LS. Unlike Cedi-LS, the Psor-LS was sensitive to pH since it exhibited the maximum transfructosylation activity at pH 6.0. However, less than 50% of the activity remained when pH was shifted from 6.0 to 4.5 or 8.0. Unlike Cedi-LS, the T/H of Psor-LS was lower than 1.0 in the whole pH range of 4.0 to 9.0, which means that the hydrolysis reaction was dominant for Psor-LS (Figure 3C). Most LSs exhibited optimal activity at slightly acidic (5.5) or neutral pH (7.0). For instance, the LS from *Brenneria goodwinii* [24] showed optimal activity at pH 5.5 and 6.0. The *L*. *mesenteroides* MTCC10508 LS had the highest activity at pH 5.5 [25].

### *3.4. Effect of Temperature on the Activity and T/H of Psor-LS*

The effect of temperature on Psor-LS activity was measured at an optimal pH of 6.5. As a result, the optimal temperature of Psor-LS was 65 ◦C (Figure 4A), the same as Cedi-LS. Psor-LS could continue relatively high activity (>70%) at temperatures ranging from 45 to 70 ◦C, but this dropped when the temperature was above 75 ◦C. A slight decrease was observed below 45 ◦C, but Psor-LS could retain more than 50% of its relative activity at 35 ◦C. The variation in transfructosylation activity is shown in Figure 4B. Psor-LS could retain 80% of its transfructosylation activity at 60–70 ◦C, suggesting that Psor-LS exhibits excellent transfructosylation ability at high temperatures. Unlike Cedi-LS, a relatively high T/H value of Psor-LS (>1.0) was obtained below 45 ◦C (Figure 4C).

**Figure 3.** Effect of pH on the activity and T/H of the recombinant LS. (**A**) Effect of pH on the total activity of Psor-LS. (**B**) Effect of pH on the transfructosylation activity of Psor-LS. (**C**) Effect of pH on the T/H of Psor-LS. All of the values were the mean of triplicate experiments.

**Figure 4.** Effect of temperature on the activity and T/H of the recombinant LS. (**A**) Effect of temperature on the total activity of Psor-LS. (**B**) Effect of temperature on the transfructosylation activity of Psor-LS. (**C**) Effect of temperature on the T/H of Psor-LS. All of the values were the mean of triplicate experiments.

Many previous studies have shown that LS showed higher transfructosylation activities at lower temperatures, while hydrolysis activity shows the opposite. For example, the LS from *Z. mobilis* exhibited the highest transfructosylation activity at 30 ◦C, while its hydrolase activity optimal temperature was 50 ◦C [26]. The *L*. *reuteri* LTH5448 LS showed the highest transfructosylation and hydrolase activity at 35 ◦C and 45 ◦C, respectively [9]. This characteristic is also related to the T/H of LS. At higher temperatures (>45 ◦C), hydrolysis is the dominant reaction for *B. goodwinii* LS [24]. The LS from *L. reuteri* LTH5448 exhibited a higher hydrolysis ability above 50 ◦C, with its T/H below 1.0 [9]. By contrast, Psor-LS exhibited high transfructosylation activity at high temperatures (>55 ◦C), which was better than most LSs.

#### *3.5. Thermostability Determination of Psor-LS*

Melting temperature (*T*m) is related to the structural stability of enzymes, and high *T*m generally represents high structural stability and thermostability [27]. The *T*<sup>m</sup> of Psor-LS was determined to be 65.1 ◦C (Figure 5A), which was significantly higher than that of FSs from *L*. *reuteri* 121 (50 ◦C) and IS from *L*. *gasseri* (55 ◦C) [27,28]. To further test the stability of Psor-LS at different temperatures, the enzyme was incubated at 45, 55, and 65 ◦C, respectively. Psor-LS retained 46% of initial activity at 55 ◦C for 9 h and half of the initial activity at 45 ◦C for 60 h (Figure 5B). The half-life was 69 h at 45 ◦C and 7.5 h at 55 ◦C (Figure 5C). However, Psor-LS was almost inactive when solely incubated at 65 ◦C for 10 min.

**Figure 5.** *T*m and thermostability of recombinant LS. (**A**) *T*m of Psor-LS. (**B**) Thermostability of Psor-LS at 45 and 55 ◦C. (**C**) The *t*1/2 value of Psor-LS at 45 and 55 ◦C.

Except for the LS from *L. reuteri* LTH5448, most LSs showed low thermostability at temperatures above 50 ◦C (Table 1). For instance, the *B*. *licheniformis* RN-01 LS retained less than 50% of its initial activity after 1 h of incubation at 50 ◦C [29]. The LS from *Bacillus* sp. TH4-2 lost 50% of its initial activity at 60 ◦C for 30 min [30]. The LS from *B*. *subtilis* NRC could retain 60% of its activity after incubating at 50 ◦C for 2 h [31]. Before this study, the LS from *Geobacillus stearothermophilus* was reported as the most thermostable LS since it could retain more than 95% of initial activity at 4-47 ◦C for 6 h [32]. However, the enzyme would rapidly lose activity at higher temperatures (57 ◦C).


