| 摘要： |
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 degrees 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 Isp(Sp)., were performed. The enzyme activity of IspS(ib) and IspS(pa) dropped very rapidly after incubation at 30 degrees 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. (C) 2018, The Society for Biotechnology, Japan. All rights reserved. |
