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过程工程学报 ›› 2025, Vol. 25 ›› Issue (8): 862-871.DOI: 10.12034/j.issn.1009-606X.224362

• 研究论文 • 上一篇    下一篇

多酶级联催化制备D-丙氨酸

宋显炳1,2,3， 杨宇2,3,4， 汪曼曼2,3,5， 何然峰2,3， 张玉明1*， 王自强2,3*， 李小连2,3， 王云山2,3   

  1. 1. 河北大学生命科学学院，河北 保定 071000 2. 中国科学院过程工程研究所，生化工程国家重点实验室，北京 100190 3. 中国科学院过程工程研究所，生物药制备与递送重点实验室(中国科学院)，北京 100190 4. 河北科技大学化学与制药工程学院，河北 石家庄 050018 5. 山西大学生命科学学院，山西 太原 030006
  • 收稿日期:2024-11-22 修回日期:2025-03-02 出版日期:2025-08-28 发布日期:2025-08-26
  • 通讯作者: 王自强 zqwang@ipe.ac.cn
  • 基金资助:
    河北省自然科学基金生物农业联合基金重点项目

Multi-enzyme catalytic preparation of D-alanine

Xianbing SONG1,2,3,  Yu YANG2,3,4,  Manman WANG2,3,5,  Ranfeng HE2,3,  Yuming ZHANG1*,  #br# Ziqiang WANG2,3*,  Xiaolian LI2,3,  Yunshan WANG2,3   

  1. 1. School of Life Sciences, Hebei University, Baoding, Hebei 071000, China 2. State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China 3. Key Laboratory of Biopharmaceutical Preparation and Delivery (Chinese Academy of Sciences), Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China 4. School of Chemical and Pharmaceutical Engineering, Hebei University of Science and Technology, Shijiazhuang, Hebei 050018, China 5. School of Life Sciences, Shanxi University, Taiyuan, Shanxi 030006, China
  • Received:2024-11-22 Revised:2025-03-02 Online:2025-08-28 Published:2025-08-26
  • Contact: Zi QiangWANG zqwang@ipe.ac.cn

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摘要： D-丙氨酸(D-Ala)作为一种重要的手性氨基酸，在医药、食品、化工等领域具有广泛应用。本研究设计了一种多酶级联催化制备D-Ala的工艺路径，建立了pRMA与pEAD2菌株的分批补料发酵工艺，实现了马来酸顺反异构酶(MaiA)/天冬氨酸酶(AspA)和天冬氨酸消旋酶(AspR)/D-氨基酸转氨酶(DaaT)的共表达，并优化了以马来酸酐(MA)为底物的多酶级联制备D-Ala的工艺参数，开发出高效的D-Ala酶促转化工艺。实验结果表明，pRMA发酵培养23 h时，菌体浓度和表观活性均达到峰值，分别为72.56 g/L和554.49±30.96 U；pEAD2发酵培养9 h时，表观活性可达到513.74±38.25 U，此时菌体浓度为30.75 g/L。多酶级联催化制备D-Ala时，最适工艺参数为：底物MA浓度为1.5 mol/L，加入5.0 mmol/L磷酸吡哆醛(PLP)，3.52 g/L丙酮酸(PA)和6.60 g/L pRMA菌体细胞，于pH为8.0、温度为50℃、转速为200 r/min条件下反应3 h，再加入9.80 g/L pEAD2菌体细胞继续反应至24 h。此时，底物转化率高达99.00%以上，D-Ala产率达到93.97%。将该催化体系放大40倍至4 L规模后，底物转化率仍保持在99.00%以上，D-Ala产率达93.19%，与小试水平无明显差异，可为D-Ala的工业生产提供参考。

关键词: D-丙氨酸, 马来酸酐, 多酶级联反应, 催化工艺优化

Abstract: D-Alanine (D-Ala) is an important chiral amino acid with a wide range of applications in the fields of medicine, food, and chemical industry. In this project, a "two-bacteria, four-enzyme" catalytic process for the preparation of D-Ala, a fed-batch fermentation process with pRMA [E.coli BL21(DE3)Alr-Dadx--pRSFDuet-1-MaiA-AspA] and pEAD2 [E.coli BL21(DE3)Alr-Dadx--pETDuet-1-AspR-DaaT] trains was established to achieve co-expression of maleic acid cis?trans isomerase (MaiA)/asparaginase (AspA) and aspartate racemase (AspR)/D-amino acid transcarbamylase (DaaT). Additionally, process parameters for multiple enzyme preparation of D-Ala with maleic anhydride (MA) as the substrate were optimized, including temperature, pH, and concentrations of pyruvate, pyridoxal phosphate, maleic anhydride, and pRMA/pEAD2 cells, leading to the development of an efficient enzymatic conversion process for D-Ala. The results showed that the cell concentration and apparent activity of pRMA reached the maximum value of 72.56 g/L and 554.49±30.96 U, respectively, at 23 h. In contrast, the apparent activity of pEAD2 could reach 513.74±38.25 U at 9 h, and the cell concentration was 30.75 g/L. For the multi-enzyme preparation of D-Ala, the optimized catalytic system was composed of 1.5 mol/L MA, 5.0 mmol/L pyridoxal phosphate and 3.52 g/L pyruvic acid. The dosages of pRMA and pEAD2 cells were 6.60 g/L and 9.80 g/L, added at 3 h intervals. The optimum reaction conditions were pH=8.0, temperature of 50℃, and rotation speed of 200 r/min for 24 h. The substrate conversion rate was up to 99.00%, and the yield of D-Ala reached 93.97%. Under the above optimal conditions, when the reaction volume was scaled up 40-fold, from 100 mL to 4 L, there was no significant difference in substrate conversion and product yield, which laid the foundation for its industrial application.

Key words: D-alanine, maleic anhydride, multi-enzyme cascade reaction, catalytic process optimization