d-Amino acids are important intermediates for the synthesis of β-lactam antibiotics and other vital pharmaceuticals, which can be synthesized by various methods including biocatalysis. In this work, we have demonstrated using immobilized multienzyme cofactor-driven cascade reaction for the synthesis of a model d-amino acid, (R)-2-amino-3-(2-bromophenyl)propanoic acid. In the present study, three enzymes, namely, d-amino acid amino transaminase from Bacillus cereus (bcDAAT), a d-lactate dehydrogenase (lhD-LDH) from Lactobacillus helveticus, and a formate dehydrogenase (cbFDH) from Candida boidinii, were successfully demonstrated in a practical and scalable immobilization protocol on glutaraldehyde-activated amino polymer beads LX1000HA. From the results, it was evident that the sequentially co-immobilized cbFDH along with the other two enzymes exhibited excellent stability at >90% for 10 cycles (150 h). Pilot-scale batches conducted at 50 g scale using the above immobilized multienzyme resulted in an overall isolated yield of 65% of (R)-2-amino-3-(2-bromophenyl)propanoic acid (∼33 g white powder; HPLC purity, >99%; ee, 99.0%). The application of the immobilized enzyme was also evaluated in PBR continuous-flow reaction, in which a stable conversion of >95% for 36 h was achieved (space–time yield, 323.3 g L^–1 day^–1]. d-Amino acids are vital building blocks used in pharmaceuticals and fine chemicals. Hence, we believe that immobilized multienzyme cofactor-driven cascade reaction could be a potential manufacturing platform for d-amino acids.

中文翻译：

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用于辅助因子驱动级联合成 (R)-2-Amino-3-(2-bromophenyl)propanoic 酸的共固定化多酶系统：模型反应

d-氨基酸是合成β-内酰胺类抗生素和其他重要药物的重要中间体，可以通过包括生物催化在内的多种方法合成。在这项工作中，我们展示了使用固定化多酶辅助因子驱动的级联反应来合成模型d-氨基酸 ( R )-2-amino-3-(2-bromophenyl)propanoic acid。在本研究中，三种酶，即来自蜡样芽孢杆菌的d-氨基酸氨基转氨酶( bc DAAT)、来自瑞士乳杆菌的d-乳酸脱氢酶 ( lh D-LDH) 和来自瑞士乳杆菌的甲酸脱氢酶 ( cb FDH)Candida boidinii，在戊二醛激活的氨基聚合物珠 LX1000HA 上的实用且可扩展的固定化方案中得到成功证明。从结果中可以明显看出，顺序共固定的cb FDH 与其他两种酶在 10 个循环（150 小时）内表现出 >90% 的出色稳定性。使用上述固定化多酶以 50 g 规模进行的中试规模批次导致 ( R ) -2 -amino-3-(2-bromophenyl)propanoic acid 的总分离产率为 65%（~33 g 白色粉末；HPLC 纯度, >99%; ee, 99.0%)。还在 PBR 连续流动反应中评估了固定化酶的应用，其中实现了 36 小时 >95% 的稳定转化（时空产率，323.3 g L ^–1第^–1天]。d-氨基酸是用于制药和精细化学品的重要组成部分。因此，我们认为固定化多酶辅助因子驱动的级联反应可能是d-氨基酸的潜在制造平台。