新型层状交联酶聚集体的制备条件优化与性质研究

以假丝酵母脂肪酶（Candida sp.lipase）为研究对象，开发了一种新型层状交联酶聚集体。用蛋白或氨基酸对纳米氧化锌粒子进行修饰，继以交联剂交联后作为核芯，酶分子再交联在纳米核表面形成层状结构。实验结果表明牛血清白蛋白（BSA）是纳米氧化锌适宜的修饰剂。并且对纳米芯层状交联酶聚集体（BSA-N-LCLEAs）其他制备条件进行了优化，优化后BSA-N-LCLEAs制备条件为：沉淀剂硫酸铵饱和度为58%，交联剂戊二醛浓度为3.5%，交联温度和时间分别为0℃和2 h。BSA-N-LCLEAs酶活收率较传统CLEAs提高了196.5%。扫描电镜表征表明BSA-N-LCLEAs较传统CLEAs孔道大幅增加。纳米芯层状CLEAs的pH稳定性和热稳定性也都比传统CLEAs有所提高，并将该固定化酶用于催化维生素E琥珀酸酯的合成，反应五批次后反应产率还能达90%左右，说明该新型交联酶聚集体具有良好的催化活性和操作稳定性。

Optimized preparation and characterization of novel layered cross-linked enzyme aggregates of Candida sp. lipase

A novel layered cross-linked enzyme aggregates (CLEAs) of Candida sp. lipase was developed. In the preparation of the novel enzyme aggregates, nano-ZnO particles were modified and cross-linked, which were used as the core of the immobilized lipase, then enzyme molecules were cross-linked on the surface of the nanoparticles, which formed a layered structure. The obtained results showed the bovine serum albumin (BSA) was the suitable agent to modify nano-ZnO particles among the test agents. The other factors affecting the activity of nano layered CLEAs (BSA-N-LCLEAs), such as ammonium sulfate saturation, concentration of glutaraldehyde, temperature and time for cross-linking reaction, were investigated. The optimum conditions were as follows:ammonium sulfate saturation 58%, glutaraldehyde concentration 3.5%, cross-linking temperature 0℃ and time 2 h. Compared with the traditional CLEAs, the recovered activity of BSA-N-LCLEAs increased about 196.5%. Scanning electron microscopy (SEM) characterization indicated that BSA-N-LCLEAs increased significantly compared to conventional CLEAs. Both pH stability and thermo stability of BSA-N-LCLEAs were improved. Furthermore, the layered CLEAs was used to synthesize astaxanthin succinate. The yields of astaxanthin were about 90%. The immobilized lipase could be reused for five batches, which indicated it had good operational stability.