Biochemical characterization of isoprene synthase from Ipomoea batatas

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 (IspS_ib). In this work the biochemical properties of IspS_ib were analyzed and compared with those of isoprene synthase from Populus alba (IspS_pa) and other species. Firstly, IspS_ib and IspS_pa were expressed, purified, and identified by SDS-PAGE and western blot analysis. Secondly, pH and temperature dependence of IspS_ib 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 IspS_ib and IspS_pa were performed. The enzyme activity of IspS_ib and IspS_pa dropped very rapidly after incubation at 30 °C; almost 80% enzyme activity of IspS_ib was lost after 20 min of incubation. Moreover, the Michaelis-Menten constant was measured. IspS_ib showed a lower K_m, 0.2 mM, and a higher k_cat, 0.37 s^-1, as compared with IspS_pa. The high catalytic efficiency, which was reflected by the high k_cat/K_m ratio, indicates that IspS_ib 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.

Keywords: Characterization; Enzyme; Ipomoea batatas; Isoprene synthase; Thermostability.