Tseng Chih-Wen, Liao Chien-Yi, Sun Yuanxia, Peng Chi-Chung, Tzen Jason T C, Guo Rey-Ting, Liu Je-Ruei
Institute of Biotechnology, National Taiwan University , Taipei, Taiwan.
J Agric Food Chem. 2014 Jul 16;62(28):6771-6. doi: 10.1021/jf502022w. Epub 2014 Jul 8.
The rare sugar D-psicose possesses several fundamental biological functions. D-Psicose 3-epimerase from Clostridium cellulolyticum (CC-DPEase) has considerable potential for use in D-psicose production. In this study, CC-DPEase was fused to the N terminus of oleosin, a unique structural protein of seed oil bodies and was overexpressed in Escherichia coli as a CC-DPEase-oleosin fusion protein. After reconstitution into artificial oil bodies (AOBs), refolding, purification, and immobilization of the active CC-DPEase were simultaneously accomplished. Immobilization of CC-DPEase on AOB increased the optimal temperature but decreased the optimal pH of the enzyme activity. Furthermore, the AOB-immobilized CC-DPEase had a thermal stability and a bioconversion rate similar to those of the free-form enzyme and retained >50% of its initial activity after five cycles of enzyme use. Thus, AOB-immobilized CC-DPEase has potential application in the production of d-psicose at a lower cost than the free-form enzyme.
稀有糖D-阿洛酮糖具有多种基本生物学功能。来自解纤维素梭菌的D-阿洛酮糖3-表异构酶（CC-DPEase）在D-阿洛酮糖生产中具有相当大的应用潜力。在本研究中，CC-DPEase与油质蛋白（一种种子油体的独特结构蛋白）的N端融合，并作为CC-DPEase-油质蛋白融合蛋白在大肠杆菌中过表达。在重构为人工油体（AOB）后，活性CC-DPEase的重折叠、纯化和固定化同时完成。将CC-DPEase固定在AOB上提高了酶活性的最适温度，但降低了最适pH。此外，AOB固定化的CC-DPEase具有与游离形式酶相似的热稳定性和生物转化率，并且在酶使用五个循环后仍保留其初始活性的50%以上。因此，AOB固定化的CC-DPEase在以低于游离形式酶的成本生产D-阿洛酮糖方面具有潜在应用。
