甲醛多酶级联合成L-苏糖醇的反应体系优化

Optimization of the multi-enzyme cascade for synthesis of L-threitol from formaldehyde

摘要:

L-苏糖醇(L-threitol)是一种重要的药物合成中间体。本课题组前期开发了以甲醛为原料的一锅两步多酶级联合成路线，该路线由甲醛裂合酶(benzoylformate decarboxylase, BFD)、D-果糖-6-磷酸醛缩酶(fructose-6-phosphate aldolase, FSA)催化甲醛生成L-赤藓酮糖(L-erythrulose)，在L-苏糖醇脱氢酶(L-threitol dehydrogenase, TDH)和甲醇脱氢酶(methanol dehydrogenase, MDH)/异丙醇辅酶循环体系的驱动下，将L-赤藓酮糖还原为L-苏糖醇。该路线具有原子经济性高、副产物少的优势，但亟需优化各酶的适配性及最佳浓度。目的 系统优化甲醛转化为L-苏糖醇的多酶级联反应体系中各酶的用量及适配性，提升反应速率与转化率。方法 通过对反应途径中的BFD、FSA、TDH和MDH 4个关键酶进行酶活评估，进而对酶用量、辅酶循环体系、反应时间及反应温度等进行逐步优化，有效提升了反应体系中各因素的适配性。结果 最佳反应条件：BFD、FSA、TDH及MDH的酶用量分别为10、1、1、8 mg/mL，其中FSA、TDH用量较优化前分别降低87%和67%；辅酶NAD⁺用量为2 mmol/L，反应温度30 ℃，反应时长仅需8 h，较原体系大幅缩短了60%。在此最优条件下，L-苏糖醇产量最高达166.76 mmol/L，产率为89%。放大实验中L-苏糖醇产率仍保持在80%，较优化前提升了43%。结论 本研究通过对多酶体系中酶的适配性及反应条件的系统优化，显著降低了甲醛转化为L-苏糖醇的关键酶用量并缩短了反应时间，同时大幅提高了L-苏糖醇的生成效率与产率，为酶法催化甲醛合成L-苏糖醇的工业化应用奠定了重要基础。

Abstract:

L-threitol is a significant intermediate in pharmaceutical synthesis. Previously, we developed a one-pot, two-step multi-enzyme cascade for synthesizing L-threitol from formaldehyde. In this pathway, benzoylformate decarboxylase (BFD) and fructose-6-phosphate aldolase (FSA) catalyze the conversion of formaldehyde into L-erythrulose. This is followed by the reduction of L-erythrulose to L-threitol, driven by L-threitol dehydrogenase (TDH) and a methanol dehydrogenase (MDH)/isopropanol cofactor regeneration system. While this route boasts high atom economy and minimal by-products, the compatibility and optimal concentrations of the enzymes required optimization.Objective To systematically optimize the dosages and compatibility of enzymes in the multi-enzyme cascade to enhance both the reaction rate and conversion efficiency.Methods The activities of four key enzymes—BFD, FSA, TDH, and MDH—were assessed. Subsequently, factors including enzyme dosage, the cofactor regeneration system, reaction duration, and temperature were optimized step-by-step to improve the system compatibility.Results The optimal reaction conditions were determined as follows: enzyme dosages of BFD, FSA, TDH, and MDH being 10, 1, 1, and 8 mg/mL, respectively. Notably, the dosages of FSA and TDH were reduced by 87% and 67%, respectively, compared with pre-optimization levels. Other optimal parameters included a NAD⁺ concentration of 2 mmol/L, a reaction temperature of 30 ℃, and reaction duration of 8 h (representing a 60% decrease from that of the original system). Under these conditions, the maximum L-threitol concentration reached 166.76 mmol/L, with a yield of 89%. In a scale-up experiment, the L-threitol yield remained at 80%, representing a 43% increase compared with the pre-optimization level.Conclusion By systematically optimizing enzyme compatibility and reaction conditions, this study significantly reduces the enzyme dosages and reaction duration for converting formaldehyde to L-threitol. Simultaneously, it substantially improves the production efficiency and yield, establishing a robust foundation for the enzymatic synthesis of L-threitol from formaldehyde.