Yim Sung-Kun, Jung Heung-Chae, Pan Jae-Gu, Kang Hyung-Sik, Ahn Taeho, Yun Chul-Ho
Hormone Research Center and School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea.
Protein Expr Purif. 2006 Oct;49(2):292-8. doi: 10.1016/j.pep.2006.05.013. Epub 2006 Jun 6.
To develop a whole-cell oxidoreductase system without the practical limitation of substrate/product transport, easy preparation, stability of enzymes, and low expression levels, we here report the development of a whole cell biocatalyst displaying rat NADPH-cytochrome P450 oxidoreductase (CPR, 77-kDa) on the surface of Escherichia coli by using ice-nucleation protein from Pseudomonas syringae. Surface localization and functionality of the CPR were verified by flow cytometry, electron microscopy, and measurements of enzyme activities. The results of this study comprise the first report of microbial cell-surface display of diflavin-containing mammalian enzymes. This system will allow us to select and develop oxidoreductases, containing bulky and complex prosthetic groups of FAD and FMN, into practically useful whole-cell biocatalysts for broad biological and biotechnological applications including the selective synthesis of new chemicals and pharmaceuticals, bioconversion, bioremediation, and bio-chip development.
为了开发一种不受底物/产物运输实际限制、易于制备、酶稳定性好且表达水平低的全细胞氧化还原酶系统，我们在此报告通过使用丁香假单胞菌的冰核蛋白在大肠杆菌表面展示大鼠NADPH-细胞色素P450氧化还原酶（CPR，77 kDa）的全细胞生物催化剂的开发。通过流式细胞术、电子显微镜和酶活性测量验证了CPR的表面定位和功能。本研究结果首次报道了含双黄素的哺乳动物酶在微生物细胞表面的展示。该系统将使我们能够选择和开发含有庞大而复杂的FAD和FMN辅基的氧化还原酶，将其转化为实际有用的全细胞生物催化剂，用于广泛的生物学和生物技术应用，包括新化学品和药物的选择性合成、生物转化、生物修复和生物芯片开发。
