基于金属有机框架的固定化酰胺酶制备及催化合成(S)-4-氟苯甘氨酸

Synthesis of (S)-4-fluorophenylglycine by using immobilized amidase based on metal-organic framework

摘要:

以微水溶剂热法快速制备的稳定锆基金属有机框架为载体，戊二醛为交联剂，采用交联法对酰胺酶进行固定化，考察了不同条件对酰胺酶固定化效率的影响。结果表明，戊二醛浓度为1.0%、交联时间为180 min、载体与酶的质量比为8︰1，固定化效率最佳，固定化酶活力回收率达86.4%，蛋白负载量达115.3 mg/g。固定化酶最适温度为40 ℃，最适pH值为9.0，在40 ℃下半衰期为72.2 d，该固定化酶的Km为58.32 mmol/L，Vmax为16.23 μmol/(min·mg)，kcat为1 670 s–1。此外，考察了固定化酶催化合成 (S)-4-氟苯甘氨酸的工艺：最适底物浓度300 mmol/L，固定化酶用量10 g/L，反应时间180 min，在最佳反应条件下转化率达49.9%，对映体过量 (Enantiomeric excess，e.e.) 为99.9%。进一步考察了该固定化酶分批催化反应性能，重复使用20批次后，固定化酶活力仍保留95.8%。

Abstract:

A stable Zr-based metal-organic framework (MOF, UiO-66-NH2) synthesized via micro-water solvothermal method was used to immobilize amidase by using the glutaraldehyde crosslinking method. The effect of immoblization conditions on enzyme immoblization efficiency was studied. An activity recovery rate of 86.4% and an enzyme loading of 115.3 mg/g were achieved under the optimal conditions: glutaraldehyde concentration of 1.0%, cross-linking time of 180 min, and the weight ratio of MOF to enzyme of 8:1. The optimal temperature and optimal pH of the immobilized amidase were determined to be 40 °C and 9.0, respectively, and the Km, Vmax and kcat of the immoblized amidase were 58.32 mmol/L, 16.23 μmol/(min·mg), and 1 670 s–1, respectively. The immobilized enzyme was used for (S)-4-fluorophenylglycine synthesis and the optimal reaction conditions were 300 mmol/L of N-phenylacetyl-4-fluorophenylglycine, 10 g/L of immobilized enzyme loading, and reacting for 180 min at pH 9.0 and 40 °C. A conversion rate of 49.9% was achieved under the optimal conditions, and the conversion rate can be increased to 99.9% under the conditions of enantiomeric excess. The immobilized enzyme can be repeatedly used, 95.8% of its original activity can be retained after 20 cycles.