Walton Adam Z, Stewart Jon D
Department of Chemistry, University of Florida, Gainesville 32611-7200, USA.
Biotechnol Prog. 2002 Mar-Apr;18(2):262-8. doi: 10.1021/bp010177c.
Economical methods of supplying NADPH must be developed before biotransformations involving this cofactor can be considered for large-scale applications. We have studied the enzymatic Baeyer-Villiger oxidation of cyclohexanone as a model for this class of reactions and developed a simple approach that uses whole, non-growing Escherichia coli cells to provide high productivity (0.79 g epsilon-caprolactone/L/h = 18 micromol epsilon-caprolactone/min/g dcw) and an 88% yield. Glucose supplied the reducing equivalents for this process, and no exogenous cofactor was required. The volumetric productivity of non-growing cells was an order of magnitude greater than that achieved with growing cells of the same strain. Cells of an engineered E. coli strain that overexpresses Acinetobacter sp. cyclohexanone monooxygenase were grown under inducing conditions in rich medium until the entry to stationary phase; the subsequent cyclohexanone oxidation was carried out in minimal salts medium lacking a nitrogen source. After the biotransformation was complete, the lactone product was adsorbed to a solid support and recovered by washing with an organic solvent.
在考虑将涉及该辅因子的生物转化用于大规模应用之前，必须开发出经济的供应NADPH的方法。我们研究了环己酮的酶促拜耳-维利格氧化反应，以此作为这类反应的模型，并开发了一种简单的方法，即使用完整的、不生长的大肠杆菌细胞来实现高生产率（0.79克ε-己内酯/升/小时 = 18微摩尔ε-己内酯/分钟/克干细胞重）和88%的产率。葡萄糖为此过程提供还原当量，无需外源辅因子。不生长细胞的体积生产率比同一菌株的生长细胞高一个数量级。一株过表达不动杆菌属环己酮单加氧酶的工程化大肠杆菌菌株的细胞在富含培养基的诱导条件下生长至进入稳定期；随后的环己酮氧化反应在缺乏氮源的基本盐培养基中进行。生物转化完成后，内酯产物吸附到固体支持物上，并用有机溶剂洗涤回收。
