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。

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.