acib - Austrian Center of Industrial Biotechnology, Krenngasse 37, A-8010 Graz, Austria.
acib - Austrian Center of Industrial Biotechnology, Krenngasse 37, A-8010 Graz, Austria; Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Petersgasse 12, A-8010 Graz, Austria.
J Biotechnol. 2024 Feb 20;382:88-96. doi: 10.1016/j.jbiotec.2024.01.011. Epub 2024 Jan 26.
l-Lactate oxidase has important applications in biosensing and finds increased use in biocatalysis. The enzyme has been characterized well, yet its immobilization has not been explored in depth. Here, we studied immobilization of Aerococcus viridansl-lactate oxidase on porous carriers of variable matrix material (polymethacrylate, polyurethane, agarose) and surface functional group (amine, Ni-loaded nitrilotriacetic acid (NiNTA), epoxide). Carrier activity (A) and immobilized enzyme effectiveness (ɳ) were evaluated in dependence of protein loading. Results show that efficient immobilization (A: up to 1450 U/g carrier; ɳ: up to 65%) requires a hydrophilic carrier (agarose) equipped with amine groups. The value of ɳ declines sharply as A increases, probably due to transition into diffusional regime. Untagged l-lactate oxidase binds to NiNTA carrier similarly as N-terminally His-tagged enzyme. Lixiviation studies reveal quasi-irreversible enzyme adsorption on NiNTA carrier while partial release of activity (≤ 25%) is shown from amine carrier. The desorbed enzyme exhibits the same specific activity as the original l-lactate oxidase. Collectively, our study identifies basic requirements of l-lactate oxidase immobilization on solid carrier and highlights the role of ionic interactions in enzyme-surface adsorption.
L-乳酸氧化酶在生物传感中有重要的应用，并在生物催化中得到了越来越多的应用。该酶已得到很好的表征，但尚未深入研究其固定化。在这里，我们研究了 Aerococcus viridansl-乳酸氧化酶在不同基质材料（聚丙烯酸酯、聚氨酯、琼脂糖）和表面功能基团（胺、负载镍的次氮基三乙酸（NiNTA）、环氧化物）的多孔载体上的固定化。根据蛋白质负载，评估了载体活性（A）和固定化酶效率（ɳ）。结果表明，高效固定化（A：高达 1450 U/g 载体；ɳ：高达 65%）需要配备胺基的亲水性载体（琼脂糖）。ɳ的值随着 A 的增加而急剧下降，可能是由于过渡到扩散状态。未标记的 l-乳酸氧化酶与 N 端组氨酸标记的酶类似地结合到 NiNTA 载体上。溶出研究表明，酶几乎不可逆地吸附在 NiNTA 载体上，而从胺载体上部分释放出活性（≤25%）。解吸的酶表现出与原始 l-乳酸氧化酶相同的比活性。总的来说，我们的研究确定了 l-乳酸氧化酶在固体载体上固定化的基本要求，并强调了离子相互作用在酶-表面吸附中的作用。
