Investigating the Effect of Two-Point Surface Attachment on Enzyme Stability and Activity
| Autor: | Joshua Jasensky, Charles L. Brooks, E. Neil G. Marsh, McKenna Schroeder, Zhan Chen, Shuai Wei, Xingquan Zou, Somayesadat Badieyan |
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| Rok vydání: | 2018 |
| Předmět: |
02 engineering and technology
Molecular Dynamics Simulation Protein Engineering 010402 general chemistry 01 natural sciences Biochemistry Catalysis Polyethylene Glycols Maleimides Colloid and Surface Chemistry Catalytic Domain Enzyme Stability Thermal stability Cysteine Downstream processing biology Chemistry Substrate (chemistry) General Chemistry Protein engineering Nitroreductases Enzymes Immobilized 021001 nanoscience & nanotechnology Combinatorial chemistry Enzyme structure Enzyme assay 0104 chemical sciences Covalent bond Attenuated total reflection Mutation biology.protein 0210 nano-technology |
| Zdroj: | Journal of the American Chemical Society. 140:16560-16569 |
| ISSN: | 1520-5126 0002-7863 |
| Popis: | Immobilization on solid supports provides an effective way to improve enzyme stability and simplify downstream processing for biotechnological applications, which has been widely used in research and in applications. However, surface immobilization may disrupt enzyme structure due to interactions between the enzyme and the supporting substrate, leading to a loss of the enzyme catalytic efficiency and stability. Here, we use a model enzyme, nitroreductase (NfsB), to demonstrate that engineered variants with two strategically positioned surface-tethering sites exhibit improved enzyme stability when covalently immobilized onto a surface. Tethering sites were designed based on molecular dynamics (MD) simulations, and enzyme variants containing cysteinyl residues at these positions were expressed, purified, and immobilized on maleimide-terminated self-assembled monolayer (SAM) surfaces. Sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy were used to deduce the NfsB enzyme orientations, which were found to be consistent with those predicted from the MD simulations. Thermal stability analyses demonstrated that NfsB variants immobilized through two tethering sites exhibited generally improved thermal stability compared with enzymes tethered at only one position. For example, NfsB enzyme chemically immobilized via positions 423 and 111 exhibits at least 60% stability increase compared to chemically immobilized NfsB mutant via a single site. This research develops a generally applicable and systematic approach using a combination of simulation and experimental methods to rationally select protein immobilization sites for the optimization of surface-immobilized enzyme activity and stability. |
| Databáze: | OpenAIRE |
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