Department of Chemistry, Visva-Bharati, Santiniketan, West Bengal 731235, India.
Tilka Manjhi Bhagalpur University, Bhagalpur, Bihar 812007, India.
Langmuir. 2022 Jun 7;38(22):6826-6840. doi: 10.1021/acs.langmuir.2c00022. Epub 2022 May 24.
At present, enzyme immobilization is a big issue. It improves enzyme stability, activity, specificity, or selectivity, particularly the enantioselectivity compared to the native enzymes, and by solving the separation problem, it helps in recovering the catalyst with good reusability as desired in vitro. Motivated by these facts, in this work, Jack bean urease (JBU) is immobilized on three-dimensional (3D)-network silica gel (SG) via multipoint covalent bonding employing dimethyldichlorosilane (DMDCS) and -nitrophenol, respectively, as the second-generation silane-coupling reagent and spacer. The homofunctional diazo group appearing at the functionalized SG unit cell makes a diazo linkage at the inert center, the ortho position of the phenolic-OH of the tyrosine moiety, where all of the amino, thiol, phenol, imidazole, carboxy, etc., groups of the enzyme residues, including those that belong to the active site, remain intact. The coupling process, analyzed using field emission scanning electron microscopy (FESEM), energy-dispersive X-ray analysis (EDX), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FT-IR) spectroscopy, ultraviolet-visible spectroscopy (UV-vis), and fluorescence spectroscopy, occurs without molecular aggregation in borate buffer at pH 8.8 ± 0.4, which is much higher than the iso-electric point (pH 5.1) of the macromolecule where it becomes soluble. Eventually, the immobilization is maximize and also the native-enzyme activities are restored remarkably. The immobilized catalyst converts urea (0.0625-0.15 mmol L) to ammonia appreciably (94.50 ± 1.5%) at 27 °C, and the efficiency is well comparable to that of the native enzyme (93.0 ± 0.4%). The efficiency gradually diminishes, coming down to 50% at the 40th cycle, and the enzyme returns to its native conformation within 72 h in tris-EDTA borate buffer at 27 °C for the next 40 cycles of reuse and so on. The efficiency becomes hindered by 8-10% in every 5th subsequent reuse to reach 50% on the 30th reuse, resulting in room-temperature catalytic sustainability of 90 days. The catalytic performances are well restored in rice extract and coconut water.
目前，酶固定化是一个大问题。它可以提高酶的稳定性、活性、特异性或选择性，特别是与天然酶相比的对映选择性，并通过解决分离问题，有助于体外回收具有良好可重复使用性的催化剂。受这些事实的启发，在这项工作中，通过使用二甲二氯硅烷 (DMDCS) 和 - 硝基苯酚分别作为第二代硅烷偶联试剂和间隔物，将豇豆脲酶 (JBU) 通过多点共价键固定在三维 (3D) 网络硅胶 (SG) 上。出现在功能化 SG 单元上的同官能团重氮基团在惰性中心处形成重氮键，即酪氨酸部分的酚-OH 的邻位，其中酶残基的所有氨基、巯基、酚、咪唑、羧基等基团，包括属于活性部位的基团，保持完整。使用场发射扫描电子显微镜 (FESEM)、能量色散 X 射线分析 (EDX)、X 射线光电子能谱 (XPS)、傅里叶变换红外 (FT-IR) 光谱、紫外-可见光谱 (UV-vis) 和荧光光谱分析耦合过程，在硼酸缓冲液中（pH 8.8 ± 0.4）在高于大分子的等电点（pH 5.1）的情况下，不会发生分子聚集，从而变得可溶。最终，实现了最大的固定化并显著恢复了天然酶的活性。固定化催化剂在 27°C 下将尿素（0.0625-0.15mmol L）显著转化为氨（94.50 ± 1.5%），效率与天然酶相当（93.0 ± 0.4%）。效率逐渐下降，在第 40 个循环时降至 50%，在 27°C 的 tris-EDTA 硼酸缓冲液中，酶在 72 小时内恢复到其天然构象，然后在接下来的 40 个循环中重复使用等等。在每次重复使用后的第 5 次，效率会降低 8-10%，在第 30 次重复使用时达到 50%，从而在 90 天的时间内实现了室温催化的可持续性。在大米提取物和椰子水中，催化性能得到了很好的恢复。
