Investigating the Effect of Two-Point Surface Attachment on Enzyme Stability and Activity

Abstract

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.

Implicit Modeling of the Impact of Adsorption on Solid Surfaces for Protein Mechanics and Activity with a Coarse-Grained Representation

“…disruption of the initial pattern observed inFigure 5bmay be related to the decrease in activity observed experimentally in ref (69)…”

“…Nitroreductase (NfsB) is a FMN-dependent homodimeric enzyme which catalyzes the reduction of a wide range of substrates containing nitro-groups. It is well characterized both structurally and functionally, and often serves as a model system to study how surface interactions will affect enzyme stability and activity [66][67][68][69] . Recently, Zou et al…”

“…investigated the impact of two-point surface attachment on protein function by producing NfsB mutants with neighboring tethering sites and using spectroscopic approaches to deduce their orientation and stability 69 . Interestingly, two mutants, with the anchor residue positions (111, 423) and (247, 423), display better structural stability than any single-site immobilized enzyme.…”

Surface Immobilization Chemistry of a Laminin-Derived Peptide Affects Keratinocyte Activity

“…Similar orientation effects are well known for enzyme and the vastly explored RGD peptide immobilization [ 65 , 66 ]. Other factors such as the specific residue anchoring point (and number of them) affect activity as well, including peptide density [ 67 , 68 ]. Previous work has shown that peptide density, which we did not control here, can affect cell adhesion, differentiation, and focal adhesion formation [ 48 , 69 , 70 ].…”

Between two walls: Modeling the adsorption behavior of β-glucosidase A on bare and SAM-functionalised gold surfaces

“…Altogether, the molecular mechanisms taking place upon enzyme immobilization are not well understood. While most experiments on immobilized proteins, either via a covalent [28][29] or non-covalent [30][31][32] strategy, report a decrease in their catalytic activity, sometimes enzyme immobilization can also result in an important increase of the catalytic performance. 33 Numerous enzyme immobilization strategies have been developed, that involve simple adsorption via electrostatic or van der Waals interactions, the use of porous materials, crosslinking, or multipoint-covalent attachment, [33][34][35] and many experimental approaches are now available to investigate protein immobilization on solid surfaces, including atomic force microscopy, mass spectrometry and spectroscopic methods.…”