Kinetic model assisted synthesis of β-alanine from 3-aminopropionaldehyde by a novel single step enzymatic biotransformation

Cell-free enzymatic biotransformation has been the focus of industries for the synthesis of commercially valuable biological compounds. In this study, we explored a kinetic modeling approach to study the novel, single-step, cell-free enzymatic biotransformation of 3-aminopropionaldehyde (3-Apal) to a key drug intermediate, β-alanine. The substrate 3-Apal was converted to β-alanine by the enzyme 3-aminopropionaldehyde dehydrogenase (APALDH), while the enzyme NADH oxidase (NOX) was deployed for cofactor recycling. The enzyme candidates were expressed in recombinant <i>E. coli,</i> with the specific activity of crude lysate being 5 U/mg for APALDH and 1.7 U/mg for NOX. The optimum pH and temperature were identified as pH 7 and 37 °C, respectively. The kinetic parameters were also estimated, as the K_m values of APALDH for 3-apal and NAD⁺ were 0.0375 and 11.96 mM respectively, and the K_m value of NOX for NADH was 0.07 mM. A kinetic model was developed by incorporating the kinetic parameters obtained from the experiment and from the available literature. With insights from the enzyme kinetics and the simulated kinetic model, the biotransformation was designed in fed-batch mode, and the conversion of 3-Apal to β-alanine up to 15 mM was achieved within 80 min. The model was also validated with the experimental results obtained from the cell-free biotransformation. Further improvement in the yield of β-alanine can be obtained by optimizing the initial cofactor concentration and substrate feed flow rate. Conclusively, the kinetic modeling approach can eliminate the intimidating trial experiments to achieve the maximum product yield in cell-free enzymatic biotransformation.

无细胞酶促生物转化（cell-free enzymatic biotransformation）已成为工业界合成具有商业价值生物化合物的核心研究方向。
本研究采用动力学建模方法，探究了将3-氨基丙醛（3-aminopropionaldehyde, 3-Apal）一步转化为关键药物中间体β-丙氨酸的新型无细胞酶促生物转化体系。
底物3-Apal在3-氨基丙醛脱氢酶（3-aminopropionaldehyde dehydrogenase, APALDH）的催化下生成β-丙氨酸，同时借助烟酰胺腺嘌呤二核苷酸氧化酶（NADH oxidase, NOX）实现辅酶循环。
两种候选酶均在重组大肠杆菌（E. coli）中重组表达，其中APALDH的粗裂解液比酶活为5 U/mg，NOX的粗裂解液比酶活为1.7 U/mg。
本研究确定该反应的最优pH为7，最优温度为37 ℃。
本研究同时测定了相关动力学参数：APALDH对3-Apal与烟酰胺腺嘌呤二核苷酸（NAD+）的米氏常数（Km）分别为0.0375 mM与11.96 mM；NOX对烟酰胺腺嘌呤二核苷酸还原态（NADH）的米氏常数（Km）为0.07 mM。
本研究结合实验测得与公开文献中的动力学参数，构建了对应的动力学模型。
基于酶动力学分析与模拟得到的动力学模型，本研究将生物转化过程设计为补料分批（fed-batch）模式，最终在80 min内实现了最高15 mM的3-Apal向β-丙氨酸的转化。
本研究采用无细胞酶促生物转化的实验数据对该模型进行了验证。
通过优化初始辅酶浓度与底物补料流速，可进一步提升β-丙氨酸的产率。
综上，动力学建模方法可省去大量繁琐的试错实验，实现无细胞酶促生物转化体系中产物产率的最大化。