Catalytic activity and thermostability of enzymes immobilized on silanized surface: Influence of the crosslinking agent
Catalytic activity and thermostability of enzymes immobilized on silanized surface: Influence of the crosslinking agent
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DOI：
10.1016/j.enzmictec.2013.02.018
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发表时间：
2013-05-10
影响因子：
3.4
通讯作者：
Lambert, Jean-Francois
中科院分区：
文献类型：
--
作者：
Aissaoui, Nesrine;Landoulsi, Jessem;Lambert, Jean-Francois
在这项工作中，我们研究了交联剂对硅烷化硅表面固定化酶的操作和热稳定性的影响。为此，6-磷酸葡萄糖脱氢酶 (G6PDH)（一种模型多聚酶）通过双功能交联剂连接，该交联剂能够共价结合硅烷层中的 -NH2 和酶中的胺残基。使用了“X-间隔基-X”形式的五种双功能交联剂，其区别在于反应性官能团（X = 醛：-CHO、异硫氰酸酯：-NCS、异氰酸酯：-NCO）、间隔链的性质（芳香族或脂肪族）或几何形状（位于芳香环上的间位或对位的双官能团）。使用 1,4-亚苯基二异硫氰酸酯 (PDC) 和 1,4-亚苯基二异氰酸酯 (DIC) 固定化酶的热稳定性得到了增强。此外，使用后一种交联剂，在连续使用中大部分保留了活性，从而实现了最佳的操作稳定性。与 1,3-亚苯基二异硫氰酸酯（分别为 PDC 和 MDC）相比，改变交联剂的几何形状（即 1,4- 交联剂）对操作稳定性和热稳定性具有至关重要的影响。事实上，在所有使用过的交联剂中，MDC 的损失最为严重（使用 6 次后的残余活性约为 16%）。使用二醛交联剂：戊二醛 (GA) 和对苯二醛 (TE)，活性的保留效果明显不如 DIC 和 PDC（对于 GA 和 TE，在 30 摄氏度时损失约 50%，而 PDC 和 DIC 则没有损失）。这些效应可以通过多点附着模型来解释，其中更多数量的锚定点可以稳定三维结构，尤其是活性二聚体中两个亚基的结合，但代价是更大的刚性，这不利于绝对活动。交联剂观察到的差异主要是由于界面处的空间位阻，这似乎很大程度上受到连接体的结构和反应性的影响。 (C) 2013 Elsevier Inc. 保留所有权利。
In this work, we investigate the influence of crosslinkers on the operational and heat stability of immobilized enzymes on a silanized silicon surface. To this end, gluccise-6-phosphate dehydrogenase (G6PDH), a model multimeric enzyme, was attached through bifunctional crosslinkers able to bind covalently the -NH2 in the silane layer and of amine residues in the enzyme. Five bifunctional crosslinkers in the form of "X-spacer-X" were used, differing by the reactive functional groups (X = aldehyde: -CHO, isothiocyanate: -NCS, isocyanate: -NCO), by the nature of the spacer chain (aromatic or aliphatic) or by the geometry (bifunctional groups positioned in meta- or para- on an aromatic ring). A thermostability enhancement has been obtained for enzymes immobilized using 1,4-phenylene diisothiocyanate (PDC) and 1,4-phenylene diisocyanate (DIC). Moreover, using the latter crosslinker, activity was the mostly preserved upon successive uses, thus giving the best operational stability achieved. Changing the geometry of the cross-linker, i.e., 1,4- as compared to 1,3-phenylene diisothiocyanate (PDC and MDC, respectively), has a crucial effect on operational and thermal stabilities. Indeed, among all used crosslinkers, the most important loss was observed for MDC (residual activity after 6 times use is similar to 16%). Using dialdehyde crosslinkers: glutaraldehyde (GA) and terephtalaldehyde (TE), activity was significantly less well preserved than with DIC and PDC (for GA and TE, a loss of about 50% at 30 degrees C against no loss for PDC and DIC).These effects can be explained by a multipoint attachment model, in which a higher number of anchoring points stabilizes the three-dimensional structure and especially the binding of the two subunits in the active dimer, at the expense of a greater rigidity which is detrimental to the absolute activity. The differences observed with the crosslinkers are mainly due to steric hindrance at the interface which seems to be greatly influenced by the structure and the reactivity of the linkers. (C) 2013 Elsevier Inc. All rights reserved.