Research School of Chemistry, Australian National University, Canberra, ACT, Australia.
CSIRO Synthetic Biology Future Science Platform, Australian National University, Canberra, ACT, Australia.
Methods Mol Biol. 2022;2487:297-315. doi: 10.1007/978-1-0716-2269-8_18.
The utility of ATP-dependent multi-enzymatic reactions is limited by their requirement for stoichiometric amounts of this expensive cofactor or additional purified enzymes for its recycling. Here we describe a simple method for the production of recombinant cell-free extracts (or lysates) of E. coli that support ATP-dependent biotransformations. The inexpensive preparation described is obtained with modest processing from a single recombinant bacterial culture of E. coli. In addition to recombinantly overexpressed enzymes that catalyze the primary ATP-dependent reactions of interest, endogenous kinases that are naturally present in the extract catalyze recycling of the requisite ATP. This means that only catalytic amounts of cofactor are necessary to drive the biotransformation, and without the requirement for additional purified enzymes. This approach has been applied successfully to an array of in vitro enzymatic cascades with multiple ATP-dependent steps.
三磷酸腺苷（ATP）依赖性多酶反应的实用性受到其对这种昂贵辅助因子的化学计量需求或用于其循环回收的额外纯化酶的限制。在这里，我们描述了一种简单的方法，用于生产支持三磷酸腺苷依赖性生物转化的重组大肠杆菌无细胞提取物（或裂解物）。所描述的廉价制剂是通过对单个重组大肠杆菌培养物进行适度处理获得的。除了催化感兴趣的主要三磷酸腺苷依赖性反应的重组过表达酶外，提取物中天然存在的内源性激酶还催化必需的三磷酸腺苷的循环利用。这意味着仅需要催化量的辅助因子即可驱动生物转化，而无需额外的纯化酶。该方法已成功应用于一系列具有多个三磷酸腺苷依赖性步骤的体外酶级联反应。
