Meng Xiangqi, Liu Yan, Yang Lin, Li Rui, Wang Hualei, Shen Yaling, Wei Dongzhi
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, People's Republic of China.
Biotechnol J. 2023 May;18(5):e2200465. doi: 10.1002/biot.202200465. Epub 2023 Feb 20.
Enzymatic asymmetric synthesis of chiral amino acids has great industrial potential. However, the low catalytic efficiency of high-concentration substrates limits their industrial application. Herein, using a combination of substrate catalytic efficiency prediction based on "open to closed" conformational change and substrate specificity prediction, a novel leucine dehydrogenase (TsLeuDH), with high substrate catalytic efficiency toward benzoylformic acid (BFA) for producing l-phenylglycine (l-Phg), was directly identified from 4695 putative leucine dehydrogenases in a public database. The specific activity of TsLeuDH was determined to be as high as 4253.8 U mg . Through reaction process optimization, a high-concentration substrate (0.7 m) was efficiently and completely converted within 90 min in a single batch, without any external coenzyme addition. Moreover, a continuous flow-feeding approach was designed using gradient control of the feed rate to reduce substrate accumulation. Finally, the highest overall substrate concentration of up to 1.2 m BFA could be aminated to l-Phg with conversion of >99% in 3 h, demonstrating that this new combination of enzyme process development is promising for large-scale application of l-Phg.
手性氨基酸的酶促不对称合成具有巨大的工业潜力。然而，高浓度底物的低催化效率限制了它们的工业应用。在此，结合基于“开放到闭合”构象变化的底物催化效率预测和底物特异性预测，从公共数据库中的4695种假定的亮氨酸脱氢酶中直接鉴定出一种新型亮氨酸脱氢酶（TsLeuDH），其对苯甲酰甲酸（BFA）生产L-苯甘氨酸（L-Phg）具有高底物催化效率。TsLeuDH的比活性测定高达4253.8 U mg。通过反应过程优化，在单批次中90分钟内可将高浓度底物（0.7 m）高效且完全转化，无需添加任何外部辅酶。此外，设计了一种连续流加方法，通过进料速率的梯度控制来减少底物积累。最后，在3小时内，高达1.2 m BFA的最高总底物浓度可被胺化为L-Phg，转化率>99%，表明这种新的酶法工艺开发组合对于L-Phg的大规模应用具有前景。
