FTIR Microscopy for Direct Observation of Conformational Changes on Immobilized ω-Transaminase: Effect of Water Activity and Organic Solvent on Biocatalyst Performance

Enzyme immobilization is a key strategy to expand the scope of enzyme applications and to enable the recycling of biocatalysts, resulting in greener and more cost-efficient processes. The full exploitation of the technology advantages is strictly connected to the optimal selection of the carriers and the rational development of the immobilization protocol. The present study achieved such objectives by investigating the activity of a ω-transaminase in organic solvent (toluene) upon immobilization on commercially controlled porosity glass carriers (EziG™) with diverse porosity and surface functionalization. In addition to more conventional wet-chemistry approaches and confocal microscopy, infrared microspectroscopy and imaging were exploited to highlight the enzyme distribution in a label-free manner and provide details on the immobilized enzyme's conformation with respect to the native form. Contrary to what could be expected, the highest activity of the enzyme in organic solvent was achieved for the immobilization protocol on the most hydrophilic support that more severely affects the enzyme secondary structure, promoting a beta-sheet rich folding. Experimental data show that values of water activity above 0.90 in the reaction system had a positive effect on the efficiency of the transaminase reaction. The present study represents the first example of rational development of immobilization protocols relying on direct observation of the enzyme conformation upon immobilization, shedding light on the mutual interaction between the diverse process parameters and the carrier properties.酶固定化是拓展酶应用范围、实现生物催化剂回收利用的关键策略，可助力形成更环保且更具成本效益的工艺。充分发挥该技术的优势，与载体的优化选择及固定化方案的合理开发紧密相关。本研究通过探究ω-转氨酶（ω-transaminase）在有机溶剂（甲苯）中、固定于具备不同孔隙结构与表面官能化修饰的商业化可控多孔玻璃载体（EziG™）后的活性，达成了上述目标。除采用较为常规的湿化学方法与共聚焦显微镜技术外，本研究还借助红外显微光谱与成像技术，以无标记方式揭示酶的分布情况，并阐明固定化酶相较于天然酶的构象细节。与预期相悖的是，在最亲水的载体上采用该固定化方案时，酶在有机溶剂中的活性达到最高——尽管该载体更显著地影响酶的二级结构，促进形成富含β折叠的构象。实验数据表明，反应体系中水分活度高于0.90时，对转氨酶反应效率具有积极影响。本研究首次实现了基于固定化过程中酶构象直接观测的固定化方案合理开发，阐明了多样工艺参数与载体性质之间的相互作用机制。