## 3 Results and Discussion
### Fusion of the Prokaryotic SuSy with UGT76G1  
As shown in Table 3, only a small amount of RebA was detected in the reaction catalyzed by Ac-76G1, but _Ac_SuSy showed the best specific activity among four SuSys under the investigated conditions. In the assays of SuSy activity, 500 mM sucrose and 10 mM UDP were added [15], which would not available in the one-pot two-enzyme reactions that used 175.3 or 262.9 mM (60 or 90 g/L) sucrose, and the UDP and UDPG from the cell lysate. Afterward, a double mutant of _Ac_SuSy (L637M-T640V), which has the reduced K\({}_{\text{m}}\) value to UDP [28] and named _Ac_SuSym in this study, was coupled with UGT76G1 instead of _Ac_SuSy. It was shown in HPLC analysis, that only a small amount of RebA was synthesized in the reaction catalyzed by Ac-76G1 (Figure S4a), while stevioside had been mostly converted into RebA in the reaction catalyzed by Acm-76G1 in 12 h (Figure S4b). The RebA concentration of Acm-76G1 was about seven times that of Ac-76G1, indicating that the mutant (_Ac_SuSym) can effectively improve RebA productivity in UGT-SuSy reactions. Then, another fusion construct named Acm-R3-76G1 was created by fusing the \(C\)-terminal of _Ac_SuSym and the \(N\)-terminal of UGT76G1 with the rigid linker (EAAAK)3. To enhance the catalytic ability to produce RebA, the influence factors in terms of reaction temperature, pH, substrate ratio, and enzyme concentrations on the reaction catalyzed by Acm-R3-76G1 were investigated. Temperature is critical for enzymatic reactions; the speed of the glycosylation reaction is positively related to temperature in a certain range. Increasing reaction temperature will accelerate the glycosylation of stevioside, but it will also shorten the half-life of the enzyme, making the enzyme unstable and prone to inactivation. Considering the temperature preferences of bacterial _Ac_SuSym and plant origin UGT76G1, the temperature for Acm-R3-76G1 was set in the range of 20 \({}^{\circ}\)C to 60 \({}^{\circ}\)C. As was shown in Figure 3a, the yield of RebA continued to rise at 20-50 \({}^{\circ}\)C under the same conditions, and reached the peak at 50 \({}^{\circ}\)C, which was 9 times that at 20 \({}^{\circ}\)C. However, the yield dropped rapidly at 60 \({}^{\circ}\)C, possibly because the high temperature led to the inactivation of the fusion protein Acm-R3-76G1. Figure 3: Effects of temperature (**a**), pH (**b**), substrate ratios (**c**), and crude enzyme concentrations (**d**) on RebA synthesis catalyzed by Acm-R3-76G1. SuSys involve in a reversible reaction, whose direction was affected by pH [12]. The reactions catalyzed by Acm-R3-76G1 were conducted at a pH range of 6-9. As was shown in Figure 3b, the highest yield of RebA was obtained at pH 7.2, which was 1.85-fold that at pH 9. Under the experimental conditions in this study, pH preference of Acm-R3-76G1 was pH 7.2 > pH 6 > pH 8 > pH 9 for RebA formation. As for the substrate mass ratios of stevioside to sucrose, it looked like the higher amount of sucrose used, the more RebA was generated below the ratio of 1:20 (stevioside: sucrose) (Figure 3c). In the reactions catalyzed by Acm-R3-76G1, the yield was relatively low when the initial substrate ratio was 1:3 or 1:5; it reached about 54.4% at the ratio set to 1:30. While the substrate ratio was 1:20, and sucrose was up to 400 g/L, the yield reached the highest at the reaction of 16 h, which was 4.2 times that at the ratio of 1:3. The product yield is positively relevant to the enzyme concentration in the glycosylation reactions (Figure 3d). The increase in crude enzyme concentration accelerated the accumulation of RebA.