超嗜热D-阿洛酮糖3-差向异构酶的固定化及固定化酶性能研究

Study on Immobilization of Hyperthermophilic D-Allulose 3-Epimerase and Properties of Immobilized Enzyme

摘要:

D-阿洛酮糖是一种应用前景广阔的低热量天然甜味剂,其作为稀有糖家族的重要成员具有抑制多种疾病的生理益处。D-阿洛酮糖是D-果糖的C-3位差向异构体,由D-阿洛酮糖3-差向异构酶(D-allulose 3-epimerase,DAE)催化合成。工业生产中的糖差向异构化反应需要高温条件(大于65℃),现有的DAE难以满足工业化生产需求。为了突破这一瓶颈,以硅藻土(diatomite)为固体核心、聚乙烯亚胺(polyethyleneimine,PEI)为保护壳、戊二醛(glutaraldehyde,GA)为交联剂,负载超嗜热DAE(TI-DAE),制备了具有“核壳”结构的固定化酶(TI-DAE@Diatomite-PEI-GA),并对其结构和性能进行了表征。相关结果表明,固定化酶TI-DAE@Diatomite-PEI-GA相比游离酶表现出较好的pH稳定性和热稳定性。在90℃条件下,TI-DAE@Diatomite-PEI-GA的半衰期大于24h,相比游离酶提高了11倍。TI-DAE@Diatomite-PEI-GA可以重复催化500g/L D-果糖生成D-阿洛酮糖20批次,且仍保持70%以上的相对酶活。研究结果证明基于硅藻土共价交联的酶固定化技术能大幅提高DAE的热稳定性,降低D-阿洛酮糖的工业生产成本,可突破目前工业化生产的技术瓶颈,为推进酶法合成D-阿洛酮糖的产业化奠定基础。

Abstract:

D-allulose, a promising low-calorie natural sweetener and a key member of the rare sugar family, exhibits physiological benefits in mitigating multiple diseases. As the C-3 epimer of D-fructose, D-allulose was synthesized through the catalytic action of D-allulose 3-epimerase (DAE). However, industrial-scale sugar epimerization required high-temperature conditions (greater than 65℃), and existed DAEs could not meet this requirement. To address this bottleneck, a hyperthermophilic DAE (TI-DAE) was immobilized using diatomite as a solid core, polyethyleneimine (PEI) as a protective shell, and glutaraldehyde (GA) as a cross-linking agent, resulting in the formation of a core-shell-structured immobilized enzyme (TI-DAE@Diatomite-PEI-GA), and its structure and performance were characterized. The result revealed that immobilized enzyme TI-DAE@Diatomite-PEI-GA exhibited better pH and thermal stability than the free enzyme TI-DAE. The half-life of TI-DAE@Diatomite-PEI-GA was greater than 24h, 11 times higher than free enzyme under 90℃. Furthermore, TI-DAE@Diatomite-PEI-GA retained over 70% relative activity after 20 batches of converting 500g/L D-fructose into D-allulose. The results proved that the enzyme immobilization technology based on diatomite covalent cross-linking could greatly improve the thermostability of DAE, reduce the cost of industrial production of D-allulose, break through a technical bottleneck of current industrial production, and lay a foundation for promoting the industrialization of enzymatic synthesis of D-allulose.