关键词: 吡咯啉-5-羧酸, 哌啶-6-羧酸, 薄层层析, 产物分析, 工艺优化

Abstract: 1-Pyrroline-5-carboxylate (P5C) and 1-piperidine-6-carboxylate (P6C) are cyclic imine compounds closely related to metabolic reactions and disease diagnosis and treatment. They are respectively involved in the metabolism of L-proline and L-lysine and play an essential role in maintaining the balance of cell oxidation, regulating the concentration of NADP(H)/NADP, and inducing cell apoptosis. Therefore, it is necessary to develop a green, efficient and sustainable preparation method. Biocatalysis is widely used in various industries such as medicine, chemicals, energy, and materials for its advantages of environment friendliness, economy, mildness, and specificity. In this work, an effective enzymatic method for the preparation of P5C and P6C is established and optimized based on the catalytic ability of the sarcosine oxidase (SOX) against the -C-N- bond. TrSOX is derived from Thermomicrobium roseum, which possesses excellent stability and tolerance. The target compounds are separated and identified by thin-layer chromatography (TLC), liquid chromatography-mass spectrometry (LC-MS), and 1H-NMR, and the concentration determination method was created. The single-factor experiment is conducted to analyze the influence of reaction temperature, pH, and substrate concentration on the product yield, and it is discovered that the optimum reaction temperature, pH and substrate concentration was respective 80℃, 9.5, and 100 mmol/L. Furthermore, reliable models were obtained through response surface design by taking the product yield as the response value. It is found that the best reaction conditions of P5C are 79.33℃, pH=8.54, substrate concentration 112.82 mmol/L, and that of P6C are 80.19℃, pH=8.31, and substrate concentration 83.58 mmol/L. After a 30 min reaction under the above conditions, the yield of P5C and P6C is 340.15±9.12 and 450.87±8.54 mg/L, respectively, which is 1.35% and 1.47% different from the theoretical value. Finally, after 48 h of reaction, the conversion rate of L-proline and L-homoproline reached 81.94% and 99.99% respectively. This study provides a new preparation method for P5C and P6C with a high conversion rate and mild reaction conditions. Taken together, the research results laid a theoretical foundation for the enzymatic preparation of P5C and P6C, provided a reference model for industrial applications, and offered a new method for synthesizing pyrroline and piperidine compounds.

Key words: Pyrroline-5-carboxylate, Piperideine-6-carboxylate, Thin layer chromatography, Product analysis, Process optimization