修饰载体的功能基团对酶促甘油解合成甘油二酯的影响规律与调控机制

During the enzymatic production of diacylglycerols (DAG) through glycerolysis, it is crucial to improve the glycerolysis activity as well as increase the glycerolysis selectivity towards DAG formation, which speeds up the industrialization of DAG production. Our previous study indicated that, compared with the parent SBA-15 immobilized RML (Rhizomucor miehei lipase), the activity and thermal stability of the dodecyl–functionalized SBA-15 immobilized RML had dramatically improved. In this proposal, serious of organic functional groups will be grafted to the SBA-15 through silane coupling agents, the modified SBA-15 will then be used to immobilize lipases. The enzymatic properties of the obtained immobilized lipases, including activity, thermal stability, reusability, et al., will be studied. The obtained immobilized lipases will then be used to catalyze glycerolysis for DAG production, and the effects of the grafted organic functional group on the glycerolysis activity as well as DAG selectivity, will be studied systematically. In addition, in order to look into the mechanism behind the functional group affecting the DAG production through enzymatic glycerolysis, the effects of the grafted organic functional group on the structure of lipases as well as the bound state between lipases and SBA-15, will be also investigated. The implementation of the present proposal is with the aid of multi-disciplinary crossing, it will develop the related scientific theories, and provide theoretical foundation for the bioprocess of oils and fats, as well as promote the industrial development of nutritious & healthy oil.

提高酶促甘油解反应活性及反应对甘油二酯（Diacylglycerols, DAG）的选择性将推进生物制备DAG的工业化。申请者前期研究表明，正十二烷基修饰载体SBA-15极大提高了SBA-15负载RML酶的活性和稳定性。本项目拟通过硅烷偶联办法将系列有机功能基团修饰介孔材料SBA-15，然后再负载脂肪酶。研究功能基团对所得固定化酶的活性、稳定性、重复利用性等酶学性质的影响，研究固定化酶催化甘油解反应的活性及选择性与功能基团的变化关系，揭示功能基团对酶促甘油解合成DAG的影响规律；研究功能基团对脂肪酶蛋白结构及脂肪酶与载体结合状态的影响，阐明功能基团调控酶促合成DAG的内在机制。项目研究借助学科交叉，将促进学科发展，为食品油脂的生物加工提供理论基础，推动我国营养健康油脂产业的进步。

我国食用植物油的年人均消费量达22.5kg，超过世界平均水平。油脂高摄入量容易导致肥胖，危害人类健康。甘油二酯（DAG）是一种功能性油脂，能降低体内脂肪堆积，预防糖尿病等相关疾病。酶促甘油解反应是制备DAG最具前景的途径之一。提高酶促甘油解反应活性及反应对DAG的选择性是推进酶促制备DAG工业化的关键。. 本项目采用有机功能基团修饰SBA-15，然后负载脂肪酶。深入研究了修饰载体的功能基团对固载酶催化甘油解制备DAG的影响规律与机制。研究得出“适度极性”的功能基团有助于提高甘油解活性和DAG的选择性；但是，对于不同的脂肪酶，所需的“适度极性”基团并不相同。研究获得了系列能高效催化甘油解制备DAG的固定化脂肪酶促体系。例如，异氰酸丙酯基团修饰的SBA-15负载CALB和RML催化甘油解，可获得DAG含量分别为61.9和59.0%。此外，所得系列固载酶的稳定性和重复利用性好。例如，SBA-15-CH2CH2(CF2)5CF3修饰SBA-15后负载的CALB固定化酶，催化甘油解反应重复使用10次后，酶活没有损失。. 详细研究了核苷酸-金属配位聚合物固载脂肪酶催化甘油解反应和酯化反应的性能。表明，配位聚合物固载CALB能选择性催化酯化反应而几乎不能催化甘油解反应。该研究发现为选择性催化的固定化酶促体系的设计提供了理论指导，也为酶促定向酯化合成系列甘油酯产品提供了技术支撑和技术储备。此外，配位聚合物固载的TLL, AOL, RML, BCL, CALA和LU，特别是固载的CALA，则能高效催化甘油解反应，可获得58.62-64.88%的DAG。. 研究了从高酸价油脂中酶促脱酸同时制备DAG。表明，Novozym 435催化能将油酸含量从25%降低至0.36-0.66%，同时可获得60-62%的DAG；大孔树脂NKA-9负载RML能将25%的油酸降低至1.5%，同时DAG含量达58.95%。此外，酶重复利用性好，Novozym 435重复使用10次后酶活无损失。. 本项目的研究成果为选择性催化的固定化酶促体系的设计提供了理论基础，也为酶促制备高含量DAG产品提供了技术支撑和技术储备。研究获得了系列能高效催化甘油解制备DAG的固定化脂肪酶促体系，其反应条件温和，固载酶重复利用性好，具有良好的应用前景。