State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, China.
State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, China; Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University, Nanjing 211816, China.
Colloids Surf B Biointerfaces. 2024 Jan;233:113658. doi: 10.1016/j.colsurfb.2023.113658. Epub 2023 Nov 22.
This study developed a surface functionalized lignin-based sub-microsphere as an innovative support for enzyme immobilization. Lignin was first modified with a silane reagent leading to lignin/SiO (LS) organic/inorganic hybrid particles, displayed as regular sub-micro spheres with a SiO shell as demonstrated in SEM images. The LS particles were further modified to introduce nickel ions, as evidenced in XPS spectra, facilitating affinity adsorption with a his-tagged enzyme. The immobilization of adenylate cyclase from Haloactinopolyspora alba (HaAC), expressed in Escherichia coli, was conducted on the surface functionalized LS (LS-G-NTA-Ni). The immobilization conditions were optimized to achieve the highest relative activity, which were determined to be using a Ni concentration of 62.5 mM, at pH=9.5 and 25 °C, with an enzyme-to-support ratio of 4.0 for a duration of 2 h. The immobilized HaAC shows maximum relative activity at pH=9.5 and 40 °C, and exhibits significantly improved thermal stability compared to the free enzyme. After undergoing five reusing cycles, the immobilized HaAC maintains a satisfactory activity (54.15%), which is due to the surface chemistry and the structural stability of the functionalized LS. This work provides a valuable exploration for high-value application of industrial lignin.
本研究开发了一种表面功能化的基于木质素的亚微米球，作为酶固定化的创新载体。木质素首先用硅烷试剂进行修饰，导致木质素/二氧化硅（LS）有机/无机杂化颗粒，如 SEM 图像所示，呈现出规则的亚微米球形状，具有二氧化硅壳。LS 颗粒进一步进行修饰以引入镍离子，如 XPS 光谱所示，有利于与带有 his 标签的酶进行亲和吸附。通过在表面功能化的 LS（LS-G-NTA-Ni）上进行固定化，实现了来自 Haloactinopolyspora alba 的腺苷酸环化酶（HaAC）的固定化，该酶在大肠杆菌中表达。优化了固定化条件以实现最高相对活性，结果表明，Ni 浓度为 62.5 mM、pH=9.5 和 25°C、酶与载体的比例为 4.0，持续 2 小时，可获得最佳相对活性。固定化 HaAC 在 pH=9.5 和 40°C 时表现出最大相对活性，与游离酶相比，其热稳定性显著提高。在经历了五次重复使用循环后，固定化 HaAC 仍保持令人满意的活性（54.15%），这归因于功能化 LS 的表面化学性质和结构稳定性。这项工作为工业木质素的高附加值应用提供了有价值的探索。
