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

作　　者：沈骥冬 徐宝萍 黄良刚 柳志强 郑裕国 SHEN Jidong;XU Baoping;HUANG Lianggang;LIU Zhiqiang;ZHENG Yuguo(College of Biotechnology and Bioengineering/The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals/Key Laboratory of Bioorganic Synthesis of Zhejiang Province,Zhejiang University of Technology,Hangzhou 310014,China)

机构地区：[1]浙江工业大学生物工程学院/手性生物制造国家地方联合工程研究中心/浙江省生物有机合成重点实验室,浙江杭州310014

出　　处：《食品科学技术学报》2025年第2期42-50,共9页Journal of Food Science and Technology

基　　金：国家重点研发计划项目(2022YFC2105400);浙江省“尖兵”“领雁”研发攻关计划项目(2023C01113)。

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(T I-DAE),制备了具有“核壳”结构的固定化酶(T I-DAE@Diatomite-PE I-GA),并对其结构和性能进行了表征。相关结果表明,固定化酶T I-DAE@Diatomite-PE I-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,T I-DAE@Diatomite-PE I-GA的半衰期大于24 h,相比游离酶提高了11倍。T I-DAE@Diatomite-PE I-GA可以重复催化500 g/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。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(T I-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(T I-DAE@Diatomite-PE I-GA),and its structure and performance were characterized.The result revealed that immobilized enzyme T I-DAE@Diatomite-PE I-GA exhibited better pH and thermal stability than the free enzyme T I-DAE.The half-life of T I-DAE@Diatomite-PE I-GA was greater than 24 h,11 times higher than free enzyme under 90℃.Furthermore,T I-DAE@Diatomite-PE I-GA retained over 70% relative activity after 20 batches of converting 500 g/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.

关 键 词：D-阿洛酮糖 D-阿洛酮糖3-差向异构酶 酶固定化 高温催化 稀有糖 

分 类 号：TS242.9[轻工技术与工程—制糖工程]