Enzyme Immobilization on Graphite Oxide (GO) Surface via One-Pot Synthesis of GO/Metal–Organic Framework Composites for Large-Substrate Biocatalysis

Abstract

Although enzyme immobilization has improved many areas, biocatalysis involving large-size substrates is still challenging for immobilization platform design because of the protein damage under the often “harsh” reaction conditions required for these reactions. Our recent efforts indicate the potential of using Metal–Organic Frameworks (MOFs) to partially confine enzymes on the surface of MOF-based composites while offering sufficient substrate contact. Still, improvements are required to expand the feasible pH range and the efficiency of contacting substrates. In this contribution, we discovered that Zeolitic Imidazolate Framework (ZIF) and a new calcium-carboxylate based MOF (CaBDC) can both be coprecipitated with a model large-substrate enzyme, lysozyme (lys), to anchor the enzyme on the surface of graphite oxide (GO). We observed lys activity against its native substrate, bacterial cell walls, indicating lys was confined on composite surface. Remarkably, lys@GO/CaBDC displayed a stronger catalytic efficiency at pH 6.2 as compared to pH 7.4, indicating CaBDC is a good candidate for biocatalysis under acidic conditions as compared to ZIFs which disassemble under pH < 7. Furthermore, to understand the regions of lys being exposed to the reaction medium, we carried out a site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy study. Our data showed a preferential orientation of lys in GO/ZIF composite, whereas a random orientation in GO/CaBDC. This is the first report on immobilizing solution-state large-substrate enzymes on GO surface using two different MOFs via one-pot synthesis. These platforms can be generalized to other large-substrate enzymes to carry out catalysis under the optimal buffer/pH conditions. The orientation of enzyme at the molecular level on composite surfaces is critical for guiding the rational design of new composites.

Protocol for resolving enzyme orientation and dynamics in advanced porous materials via SDSL-EPR

“…Lysozyme is a good model because both large (∼μm) and small (∼nm) size substrates can be catalyzed by this enzyme ( Vocadlo et al., 2001 , Kao et al., 2014 ). Meanwhile, MOFs/COFs are advanced porous platforms for enzyme encapsulation ( Howarth et al., 2016 , Majewski et al., 2017 , Drout et al., 2019 , Wang et al., 2020 , Gkaniatsou et al., 2017 , Lyu et al., 2014 , Lian et al., 2017 , Li et al., 2020 , Farmakes et al., 2020 , Neupane et al., 2019 ). Note that, a unique feature of SDSL-EPR is that it is immune of the background matrices (under low water volume; see below) ( Pan et al., 2021a ).…”

Current Status and Future Perspectives of Supports and Protocols for Enzyme Immobilization

“…Generally, the preparation of enzymes immobilized in MOFs is based on three strategies: encapsulation, surface immobilization, and pore trapping with presynthesized MOFs [180]. Enzymes encapsulated in porous materials such as porous nanoparticles or reversible micelles have better stability under adverse conditions such as high temperature, organic solvents, or extreme pH [181][182][183]. Figure 3 presents enzyme encapsulation in MOFs.…”

De Novo Approach to Encapsulating Biocatalysts into Synthetic Matrixes: From Enzymes to Microbial Electrocatalysts

“…This phenomenon is attributed to the π–π interaction or other hydrophobic interactions between the enzyme active sites and the CNT. Later, the same group deployed similar strategy and coprecipitated lysozyme and MOFs in the presence of graphite oxide (GO) . Two kinds of MOFs (ZIF-8 and CaBDC) were tested for comparison, and CaBDC showed superior performance under acidic condition.…”

“…Later, the same group deployed similar strategy and coprecipitated lysozyme and MOFs in the presence of graphite oxide (GO). 85 Two kinds of MOFs (ZIF-8 and CaBDC) were tested for comparison, and CaBDC showed superior performance under acidic condition. Similar to CNT, the GO allows lysozyme to stay and function at the composite's surface.…”