Langmuir. 2018 Aug 7;34(31):9133-9140. doi: 10.1021/acs.langmuir.8b01657. Epub 2018 Jul 24.
Surface immobilized enzymes have been widely used in many applications such as biosensors, biochips, biofuel production, and biofuel cell construction. Many factors dictate how enzymes' structure, activity, and stability may change when immobilized, including surface functionalization, immobilization chemistry, nature of the solid support, and enzyme surface density. To better understand how immobilization affects enzyme structure and activity, we have developed a method to measure both surface-sensitive protein vibrational spectra and enzymatic activity simultaneously. To accomplish this, an optical/fluorescence microscope was incorporated into a sum frequency generation (SFG) spectrometer. Using β-glucosidase (β-Glu) as a model system, enzymes were covalently tethered to a self-assembled monolayer surface using cysteine-maleimide chemistry. Their orientations were determined by SFG spectroscopy, with a single native cysteine residue oriented toward the functionalized surface, and activity measured simultaneously using a fluorogenic substrate resorufin β-d-glucopyranoside, with a loss of activity of 53% as compared to comparable solution measurements. Measuring β-Glu activity and orientation simultaneously provides more accurate information for designing and further improving enzymatic activity of surface-bound enzymes.
表面固定化酶已广泛应用于生物传感器、生物芯片、生物燃料生产和生物燃料电池构建等多个领域。许多因素决定了酶在固定化时其结构、活性和稳定性可能发生变化，包括表面功能化、固定化化学、固体载体的性质和酶表面密度。为了更好地理解固定化如何影响酶的结构和活性，我们开发了一种同时测量表面敏感蛋白振动光谱和酶活性的方法。为了实现这一目标，我们将光学/荧光显微镜与和频产生（SFG）光谱仪结合在一起。使用β-葡萄糖苷酶（β-Glu）作为模型系统，通过半胱氨酸-马来酰亚胺化学将酶共价连接到自组装单层表面上。通过 SFG 光谱确定它们的取向，其中一个单一的天然半胱氨酸残基朝向功能化表面，同时使用荧光底物 Resorufin β-d-葡萄糖苷来测量活性，与可比的溶液测量相比，活性损失 53%。同时测量β-Glu 的活性和取向可提供更准确的信息，有助于设计和进一步提高表面结合酶的酶活性。
