Framework for Optimizing Polymeric Supports for Immobilized Biocatalysts by Computational Analysis of Enzyme Surface Hydrophobicity

Abstract

Immobilization is a powerful strategy for improving enzyme usability and stability in various technologies that employ biocatalysis. However, the interactions leading to stabilization or destabilization remain poorly understood, and a support that may stabilize one enzyme may destabilize another. Employing chemically heterogeneous and complex random copolymer brushes as supports, we demonstrate a rational approach toward estimating the chemical composition of an optimally stabilizing enzyme immobilization support by computational analysis of enzyme surface hydrophobicity. This approach was tested by immobilizing a range of enzymes with diverse functions and hydrophobicity on tunable statistical random copolymer brush supports composed of poly(ethylene glycol) methacrylate (PEGMA) and sulfobetaine methacrylate (SBMA). Remarkably, we observed greatly improved enzyme performance as a function of brush composition with enhancements in the retention of catalytic activity at temperatures as high as 90 °C. Additionally, we observed an increase in activity at the optimal temperature by as much as 20-fold relative to the activity at the optimal temperature of the unimmobilized form of the enzyme. Most significantly, our results showed that the optimal composition of the brush support correlated with the overall hydrophobicity of the enzyme surface (ΔG solv,total/area), which was determined from computational analysis. This correlation provides a framework for the choice of polymer brush supports based on enzyme structure and stabilizing enzymes using complex synthetic materials.

Combinatorial High-Throughput Screening of Complex Polymeric Enzyme Immobilization Supports

“…3,4,29 These brushes also contained glycidyl methacrylate (GMA), an epoxide-containing monomer that allowed covalent attachment of enzymes. 7,37 While modeling enzyme surface hydrophobicity in conjunction with empirical experiments aided in the development of a predictive rule for selecting immobilization supports, 3,29 this approach in practice was limited to a widely spaced exploration of the unidimensional SBMA/PEGMA chemical space using a small number of two-component compositions. Consequently, it was limited in its ability to make precise predictions for more complex polymer supports involving additional components.…”

Characterization of a Model Bioreactive Membrane for the Simultaneous Separation and Hydrolysis of Lipopolysaccharides

“…A medium-sized enzyme (CALB) belonging to lipases (EC 3.1.1.3) was selected as part of the selective layer model. This lipase has also been implemented in various applications such as biodiesel production, polymer degradation, and triglyceride modifications. ,− The catalytic triad of CALB is composed of the three main amino acid residues Asp187, His224, and Ser105 (Asp–His–Ser) . Lipase B has a hydrophobic pocket near the catalytic site which enables it to undertake the cleavage actions at the interface, making it an interfacial enzyme …”

“…For example, a chemoenzymatic cascade reaction using glucose oxidase along with Fe 3 O 4 NPs has been proposed for foulant degradation in nanofiltration membranes . The immobilization of enzymes on surfaces has found fertile ground to catalyze reactions in a myriad of evolving biotechnologies such as biosynthetic production of compounds, biomedical applications, and bioremediation. , Using immobilized enzymes attached to solid supports facilitates its handling process after the intended reaction is accomplished. − Henceforth, enzyme immobilization is a preferred method over free enzyme catalysis to overcome the associated product recovery challenge. Other than solid support materials, enzymes can also be attached over porous polymeric thin films to design emerging materials for water treatment applications.…”

Simplification of Corticosteroids Biosynthetic Pathway by Engineering P450BM3

“…Modification of enzyme surface residues to other types may disrupt the surface charge and further affect the catalytic efficiency. 42 Overall, the B-factor analysis can reduce the number of candidate residue mutations in substrate channel engineering and further streamline the types of mutations at each residue by replacing the residues with others presenting similar properties. Natural or artificial evolution of the substrate access channel can, respectively, adjust the direction of substrate entry into the CYP5311B2 and FA11a-BM3 heme center, thereby achieving unprecedented hydroxylation sites.…”

“…In particular, engineering E435 into hydrophobic nonpolar residues did not result in a higher conversion rate during the engineering process of VD-BM3, whereas a smaller conformation, E435D, caused increased catalytic activity. Modification of enzyme surface residues to other types may disrupt the surface charge and further affect the catalytic efficiency . Overall, the B -factor analysis can reduce the number of candidate residue mutations in substrate channel engineering and further streamline the types of mutations at each residue by replacing the residues with others presenting similar properties.…”