Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300354, China.
Department of Engineering, Aarhus University, DK-8000 Aarhus, Denmark.
J Colloid Interface Sci. 2018 Jun 1;519:145-153. doi: 10.1016/j.jcis.2018.02.039. Epub 2018 Feb 14.
Enzymes are promising biocatalysts for the production or degradation of chemical compounds, but low stabilities of free enzymes restrict their industrial applications. Therefore, development of effective immobilization methods to maintain or increase enzyme activity and stability remains a challenge. In this work, a novel support made of zwitterionic polymer-grafted silica nanoparticles (p-SNPs) was fabricated and Candida rugosa lipase (CRL) was covalently attached onto the p-SNPs. The zwitterionic polymer was a product of the reaction between poly(maleic anhydride-alt-1-octadecene) and N,N-dimethylenediamine and contained a cetane side chain. The hydrolytic activity, reaction kinetics, thermal stability, pH tolerance, storage stability and reusability of the immobilized CRL (SNPs-CRL) were investigated. It revealed that the specific activity of SNPs-CRL was two to four times higher than the free CRL in the temperature range of 25-60 °C. It is considered mainly due to the interfacial activation effect regulated by the cetane side chains of the zwitterionic polymer. Kinetic studies revealed remarkable improvement of the enzymatic reaction efficiency by the immobilized enzyme as demonstrated by the significant increases of the reaction rate constant and the decreases of Michaelis constant (i.e., increase of enzyme-substrate affinity) determined with two different substrates (p-nitrophenyl acetate and p-nitrophenyl palmitate). Moreover, the immobilization improved the enzyme stabilities and SNPs-CRL displayed good reusability. Finally, the SNPs-CRL was proven to catalyze the hydrolysis of methyl mandelate to produce mandelic acid at an activity three times higher than the free enzyme. The results indicate that zwitterionic polymers deserved further development for enzyme immobilization.
酶是生产或降解化合物的有前途的生物催化剂，但游离酶的稳定性低限制了它们的工业应用。因此，开发有效的固定化方法以维持或提高酶的活性和稳定性仍然是一个挑战。在这项工作中，制备了一种由两性离子聚合物接枝的二氧化硅纳米粒子（p-SNPs）制成的新型载体，并将 Candida rugosa 脂肪酶（CRL）共价连接到 p-SNPs 上。两性离子聚合物是聚（马来酸酐-alt-1-十八烯）和 N,N-二甲基乙二胺反应的产物，含有十六烷侧链。研究了固定化 CRL（SNPs-CRL）的水解活性、反应动力学、热稳定性、pH 值耐受性、储存稳定性和可重复使用性。结果表明，在 25-60°C 的温度范围内，SNPs-CRL 的比活性是游离 CRL 的两到四倍。这主要归因于两性离子聚合物的十六烷侧链调节的界面活化效应。动力学研究表明，固定化酶显著提高了酶促反应效率，这表现为两个不同底物（对硝基乙酸酯和对硝基棕榈酸酯）的反应速率常数显著增加，米氏常数（即酶-底物亲和力增加）显著降低。此外，固定化提高了酶的稳定性，SNPs-CRL 具有良好的可重复使用性。最后，证明 SNPs-CRL 能够催化甲酯的水解生成扁桃酸，其活性是游离酶的三倍。结果表明，两性离子聚合物值得进一步开发用于酶固定化。
