# A Novel Sucrose Isomerase Producing Isomaltulose from _Raoultella terrigena_
## 3 Results and Discussion
### Characterization of Pal-2  
#### 3.3.1 Optimal Temperature and Thermostability  
Temperature is one of the crucial factors affecting the catalytic efficiency. Higher temperature usually enhances the substrate solubility to promote the reaction rate. However, higher temperatures can also accelerate enzyme denaturation, thus reducing the catalytic efficiency [33]. The optimum temperature of Pal-2 was assayed at temperatures ranging from 20 \({}^{\circ}\)C to 60 \({}^{\circ}\)C. As shown in Figure 3a, recombinant Pal-2 had maximal activity at approximately 40 \({}^{\circ}\)C, which is similar to that of _Erwinia_ sp. D12 [34]. At 35-50 \({}^{\circ}\)C, enzyme activity of recombinant Pal-2 was maintained above 80% and enzyme activity decreased rapidly when the temperature was higher than 50 \({}^{\circ}\)C. As shown in Table 3, it can exacerbate  
\begin{table}
\begin{tabular}{c c c c c c c c c c c} \hline \hline  & & \multicolumn{8}{c}{**Percentage Identity**} \\ \hline  & Gluha & Pecto & Rhizo & Pandi & Ente & Rate & KleLX3 & Klepn & Erwi & Serra \\ Gluha & 100 & 49.65 & 51.09 & 51.39 & 51.13 & 51.31 & 50.71 & 50.17 & 51.3 & 50.88 \\ Pecto & 49.65 & 100 & 68.04 & 64.72 & 65.33 & 64.66 & 67.61 & 65.27 & 65.11 & 67.52 \\ Rhizo & 51.09 & 68.04 & 100 & 67.15 & 68.47 & 70.09 & 69.49 & 69.49 & 67.5 & 70.05 \\ Pandi & 51.39 & 64.72 & 67.15 & 100 & 67.28 & 70.47 & 71.25 & 68.74 & 68.73 & 71.38 \\ Ente & 51.13 & 65.33 & 68.47 & 67.28 & 100 & 79.63 & 82.63 & 81.27 & 69.62 & 71.86 \\ Rate & 51.31 & 64.66 & 70.09 & 70.47 & 79.63 & 100 & 88.25 & 87.12 & 70.95 & 73.22 \\ KleLX3 & 50.71 & 67.61 & 69.49 & 71.25 & 82.63 & 88.25 & 100 & 99.3 & 71.93 & 74.2 \\ Klepp & 50.17 & 65.27 & 69.49 & 68.74 & 81.27 & 87.12 & 99.3 & 100 & 69.9 & 71.82 \\ Erwi & 51.3 & 65.11 & 67.5 & 68.73 & 69.62 & 70.95 & 71.93 & 69.9 & 100 & 78.66 \\ Serra & 50.88 & 67.52 & 70.05 & 71.38 & 71.86 & 73.22 & 74.2 & 71.82 & 78.66 & 100 \\ \hline \hline \end{tabular} Note: The symbols Rate, Ente, Erwi, Gluha, KleLX3, Klepn, Pandi, Pecto, Rhizo, and Serra represent microorganisms of _Raudellella tertergena, Entrobuc_ sp. FMB-1, _Erwinia phaponica, Glucobacter hinalinalicus_ NBRC 3257, _Klebsiella_ sp. LX3, _Klebsiella pneumonia_ sp., _Pantoa diispersa, Pectobacterium atrospepticum, Rhizobium_ sp. MX-45, and _Serraia plumuthica_ A30, respectively.  
\end{table}
Table 2: Comparison of the amino acid sequences of identified Slases from different microorganisms.  
Figure 2: Construction and expression of the recombinant Pal-2 (**a**) Construction process of plasmid pET-28a(+)-_pal_ 2. (**b**) Enzymatic digestion analysis of pET-28a(+)-_pal_ 2. (**c**) SDS-PAGE verification of the recombinant Pal-2. Lane M: protein size marker, Lane 1: Pal-2.  
As shown in Figure 3b, only 39.2% of the residual activity of Pal-2 was retained after incubation for 2 h at 45 \({}^{\circ}\)C. The thermostability remarkably decreased when the incubation temperature was 50 \({}^{\circ}\)C. Pal-2 displayed a thermostability with a half-life duration of 103 min at 45 \({}^{\circ}\)C and 48 min at 50 \({}^{\circ}\)C. Previous reports have shown that Slase is thermolable. Slase from _Klebsiella pneumoniae_ sp. was completely deactivated after incubation at 50 \({}^{\circ}\)C for 40 min [35] and the half-life of the Slase from _Ervinia_ sp. D12 was 30 min at 45 \({}^{\circ}\)C. However, in industrial production, Slase is required to have good thermostability to maintain catalytic efficiency and to reduce consumption of enzyme. Therefore, the thermostability of Pal-2 should be improved.