Abstract
The bio-production process of isoprene, an essential chemical used in industry, is strongly limited by isoprene synthase. In our previous work, relatively high isoprene production was observed with isoprene synthase from Ipomoea batatas (IspSib). In this work the biochemical properties of IspSib were analyzed and compared with those of isoprene synthase from Populus alba (IspSpa) and other species. Firstly, IspSib and IspSpa were expressed, purified, and identified by SDS-PAGE and western blot analysis. Secondly, pH and temperature dependence of IspSib were performed and an optimum pH of 8.6 and an optimum temperature of 42 °C were resulted. Mg2+ with optimum concentration of 56 mM was proved to be needed for enzyme activation. In addition, in vivo and in vitro study of the thermostabilities of IspSib and IspSpa were performed. The enzyme activity of IspSib and IspSpa dropped very rapidly after incubation at 30 °C; almost 80% enzyme activity of IspSib was lost after 20 min of incubation. Moreover, the Michaelis–Menten constant was measured. IspSib showed a lower Km, 0.2 mM, and a higher kcat, 0.37 s−1, as compared with IspSpa. The high catalytic efficiency, which was reflected by the high kcat/Km ratio, indicates that IspSib is a good candidate for the bio-isoprene production, while its thermal instability remains as a challenge. Enzyme engineering efforts, such as direction evolution or semi-rational evolution, are planned for further research.
