Fan Yucheng, Wei Zijia, Zhang Yuhua, Duan Xuguo
College of Light Industry and Food Engineering, Nanjing Forestry University, Nanjing, 210037, Jiangsu, China.
Appl Biochem Biotechnol. 2024 Dec;196(12):8685-8699. doi: 10.1007/s12010-024-04982-8. Epub 2024 Jun 20.
L-asparaginase synthetase, an ATP-dependent enzyme, necessitates ATP for its catalytic activity. However, the integration of L-asparaginase synthetase into industrial processes is curtailed by the prohibitive cost of ATP. To address this limitation, this study explores the construction of an efficient ATP regeneration system using the glucose metabolism of Escherichia coli, synergistically coupled with L-asparaginase synthetase catalysis. The optimal conditions for L-asparagine yield were determined in shake flasks. A total of 2.7 g/L was the highest yield achieved under specific parameters, including 0.1 mol/L of substrate, 0.2 mol/L glucose, 0.01 mol/L MgCl at pH 7.5, a temperature of 37 °C, and agitation at 300 r/min over 12 h. The process was then scaled to a 3-L fermenter, optimizing the addition rates of the substrate and magnesium chloride, and employing a constant glucose feed of 10 g/L/h. The scale-up process led to a significant enhancement in the production of L-asparagine. The yield of L-asparagine was increased to 38.49 g/L after 20 h of conversion, and the molar conversion rate reached 29.16%. This strategy has proven to be effective in improving the efficiency of L-asparagine production. When compared to in vitro ATP regeneration methods, this in vivo approach showcased superior efficiency and reduced costs. These findings furnish pivotal insights that may propel the enzymatic synthesis of L-asparagine toward viable industrial application.
L-天冬酰胺酶合成酶是一种依赖ATP的酶，其催化活性需要ATP。然而，ATP的高昂成本限制了L-天冬酰胺酶合成酶在工业生产中的应用。为了解决这一限制，本研究探索了利用大肠杆菌的葡萄糖代谢构建高效ATP再生系统，并与L-天冬酰胺酶合成酶催化协同耦合。在摇瓶中确定了L-天冬酰胺产量的最佳条件。在特定参数下，包括0.1 mol/L底物、0.2 mol/L葡萄糖、0.01 mol/L MgCl，pH 7.5，温度37°C，300 r/min搅拌12 h，最高产量达到2.7 g/L。然后将该过程扩大到3-L发酵罐，优化底物和氯化镁的添加速率，并采用10 g/L/h的恒定葡萄糖进料。放大过程显著提高了L-天冬酰胺的产量。转化20 h后，L-天冬酰胺产量提高到38.49 g/L，摩尔转化率达到29.16%。该策略已被证明在提高L-天冬酰胺生产效率方面是有效的。与体外ATP再生方法相比，这种体内方法显示出更高的效率和更低的成本。这些发现提供了关键的见解，可能推动L-天冬酰胺的酶促合成走向可行的工业应用。
