Federal University of Espírito Santo, Av Marechal Campos 1468, Vitória, ES, 29040-090, Brazil.
Federal University of Espírito Santo, Av Fernando Ferrari 514, Vitória, ES, 29075-910, Brazil.
Sci Rep. 2022 Apr 26;12(1):6815. doi: 10.1038/s41598-022-10721-y.
The use of enzymes immobilized on nanomagnetic supports has produced surprising results in catalysis, mainly due to the increase in surface area and the potential for recovery and reuse. However, the meticulous control of the process and difficulties in reproducibility have made industrial-scale applications unfeasible. Furthermore, the role of conjugation strategies in the catalytic activity and recycling of catalysts is unclear. Therefore, the objective of this study was to compare the conjugation of enzymes on nanomagnetic supports through physical adsorption (naked) or covalent bonding with mercaptopropyltrimethoxysilane (MPTS) and aminopropyltriethoxysilane (APTS) ligands. The free lipase obtained from Rhizomucor miehei was used as a model enzyme. Total protein and enzyme activity were determined using spectrophotometry (UV-Vis) and the p-nitrophenyl palmitate (p-NPP) hydrolysis method. The results indicated that a more significant enzyme surface loading does not always mean better immobilization success. The physical adsorption binding strategy had higher surface loading and low catalytic activity. On the other hand, covalent coupling with free NH2 had an excellent catalytic activity with very low surface loading. Finally, we show that recyclability can be improved with conjugation mediated by disulfide bonds. The findings presented here are essential for developing nanoconjugates with high enzymatic activity, which can guarantee the success of several industrial applications.
固定在纳米磁载体上的酶的使用在催化方面产生了惊人的结果，主要是由于表面积的增加以及回收和再利用的潜力。然而，对该过程的细致控制和重现性的困难使得工业规模的应用变得不可行。此外，缀合策略在催化剂的催化活性和循环利用中的作用尚不清楚。因此，本研究的目的是比较通过物理吸附（裸）或通过巯丙基三甲氧基硅烷（MPTS）和氨丙基三乙氧基硅烷（APTS）配体与纳米磁载体的共价键合固定化酶。使用从米根霉中获得的游离脂肪酶作为模型酶。使用分光光度法（UV-Vis）和对硝基苯棕榈酸酯（p-NPP）水解法测定总蛋白和酶活性。结果表明，较高的酶表面负载量并不一定意味着更好的固定化效果。物理吸附结合策略具有较高的表面负载量和较低的催化活性。另一方面，与游离 NH2 的共价偶联具有非常低的表面负载量和极好的催化活性。最后，我们表明通过二硫键介导的缀合可以提高可回收性。这里提出的发现对于开发具有高酶活性的纳米缀合物至关重要，这可以保证许多工业应用的成功。
