3.2 Purification and enzymatic properties of McSuSy
The Mc SuSy fused with a C -terminal histidine-tag that was overexpressed in E. coli BL21(pRSF-Mc SuSy), was purified by Ni-NTA affinity chromatography. The specific activity of rMc SuSy is 8.65 U/mg at 37 °C and pH 7.0. In SDS-PAGE,Mc SuSy with a C -terminal 6-histidine shows a band of roughly 100 kDa (Fig. S2).
According to the pH profile (Fig. 1A), Mc SuSy reached its maximum activity at pH 7.0 and showed high enzymatic activity (>70% of maximum value) between pH 7 and pH 7.5. From pH 6.0–7.5, its activity was still higher than 40% of the maximum value, while the activity was undetectable at pH 8.5. The temperature optima ofMc SuSy was 60°C ranging from 20 °C to 70 °C (Fig. 1B). After incubating the enzymes for 15 min between 30 and 60 °C with or without sucrose, the thermostability of Mc SuSy was determined by measuring the residual activity. The enzyme remained stable up to 42 °C after 15 min of incubation without substrates, but its activity sharply decayed beyond 50 °C (Fig. 1C). It is worth mentioning that sucrose is known to act as a stabilizing agent, and the result found that sucrose plays a positive role in maintaining enzyme activity. Addition of 200 mM sucrose enhanced enzyme activity by about 2 U/mg at the lower incubation temperature (30, 37, 42 °C) compared to the case without sucrose.
In terms of the effect of divalent metal ions, adding 2 mM of EDTA, Mg2+ and Ca2+, Ni2+, Cu2+, and Zn2+, the activities of Mc SuSy were observed to decrease (Fig. 1D). The activity was especially strongly inhibited by Ni2+, Cu2+, and Zn2+, resulting in undetectable activity, since these ions may influence the interaction with clusters of histidine on the protein surface.[15] EDTA had the least effect on the enzyme activity, followed by Ca2+ and Mg2+.The enzyme activity displayed 35% of the maximum value in the presence of 2 mM Mg2+ and Ca2+, while 74% was for EDTA.