Gasser Christoph A, Ammann Erik M, Schäffer Andreas, Shahgaldian Patrick, Corvini Philippe F-X
Institute for Ecopreneurship, School of Life Sciences, University of Applied Sciences and Arts Northwestern Switzerland, Gründenstrasse 40, 4132, Muttenz, Switzerland.
Institute for Environmental Research, RWTH Aachen, Worringerweg 1, 52074, Aachen, Germany.
Appl Microbiol Biotechnol. 2016 Aug;100(16):7281-96. doi: 10.1007/s00253-016-7479-7. Epub 2016 Apr 23.
Immobilization of enzymes on solid supports is a convenient method for increasing enzymatic stability and enabling enzyme reuse. In the present work, a sorption-assisted surface conjugation method was developed and optimized to immobilize enzymes on the surface of superparamagnetic nanoparticles. An oxidative enzyme, i.e., laccase from Trametes versicolor was used as model enzyme. The immobilization method consists of the production of superparamagnetic nanoparticles by co-precipitation of FeCl2 and FeCl3. Subsequently, the particle surface is modified with an organosilane containing an amino group. Next, the enzymes are adsorbed on the particle surface before a cross-linking agent, i.e., glutaraldehyde is added which links the amino groups on the particle surface with the amino groups of the enzymes and leads to internal cross-linking of the enzymes as well. The method was optimized using response surface methodology regarding optimal enzyme and glutaraldehyde amounts, pH, and reaction times. Results allowed formulation of biocatalysts having high specific enzymatic activity and improved stability. The biocatalysts showed considerably higher stability compared with the dissolved enzymes over a pH range from 3 to 9 and in the presence of several chemical denaturants. To demonstrate the reusability of the immobilized enzymes, they were applied as catalysts for the production of a phenoxazinone dye. Virtually, 100 % of the precursor was transformed to the dye in each of the ten conducted reaction cycles while on average 84.5 % of the enzymatic activity present at the beginning of a reaction cycle was retained after each cycle highlighting the considerable potential of superparamagnetic biocatalysts for application in industrial processes.
将酶固定在固体载体上是提高酶稳定性和实现酶重复使用的便捷方法。在本研究中，开发并优化了一种吸附辅助表面偶联方法，用于将酶固定在超顺磁性纳米颗粒表面。使用一种氧化酶，即来自云芝的漆酶作为模型酶。固定化方法包括通过共沉淀FeCl2和FeCl3制备超顺磁性纳米颗粒。随后，用含氨基的有机硅烷修饰颗粒表面。接下来，在添加交联剂（即戊二醛）之前，将酶吸附在颗粒表面，戊二醛将颗粒表面的氨基与酶的氨基连接起来，同时也导致酶的内部交联。使用响应面法对酶和戊二醛的最佳用量、pH值和反应时间进行了优化。结果得到了具有高比酶活性和更高稳定性的生物催化剂。在pH值为3至9的范围内以及在几种化学变性剂存在的情况下，与溶解的酶相比，这些生物催化剂表现出显著更高的稳定性。为了证明固定化酶的可重复使用性，将它们用作生产吩恶嗪酮染料的催化剂。实际上，在进行的十个反应循环中的每一个循环中，几乎100%的前体都转化为了染料，而在每个循环后，平均保留了反应循环开始时存在的84.5%的酶活性，这突出了超顺磁性生物催化剂在工业过程中应用的巨大潜力。
