Understanding Design Rules for Optimizing the Interface between Immobilized Enzymes and Random Copolymer Brushes

Abstract

A long-standing goal in the field of biotechnology is to develop and understand design rules for the stabilization of enzymes upon immobilization to materials. While immobilization has sometimes been successful as a strategy to stabilize enzymes, the design of synthetic materials that stabilize enzymes remains largely empirical. We sought to overcome this challenge by investigating the mechanistic basis for the stabilization of immobilized lipases on random copolymer brush surfaces comprised of poly­(ethylene glycol) methacrylate (PEGMA) and sulfobetaine methacrylate (SBMA), which represent novel heterogeneous supports for immobilized enzymes. Using several related but structurally diverse lipases, including Bacillus subtilis lipase A (LipA), Rhizomucor miehei lipase, Candida rugosa lipase, and Candida antarctica lipase B (CALB), we showed that the stability of each lipase at elevated temperatures was strongly dependent on the fraction of PEGMA in the brush layer. This dependence was explained by developing and applying a new algorithm to quantify protein surface hydrophobicity, which involved using unsupervised cluster analysis to identify clusters of hydrophobic atoms. Characterization of the lipases showed that the optimal brush composition correlated with the free energy of solvation per enzyme surface area, which ranged from −17.1 kJ/mol·nm2 for LipA to −11.8 kJ/mol·nm2 for CALB. Additionally, using this algorithm, we found that hydrophobic patches consisting of aliphatic residues had a higher free energy than patches consisting of aromatic residues. By providing the basis for rationally tuning the interface between enzymes and materials, this understanding will transform the use of materials to reliably ruggedize enzymes under extreme conditions.

Is enzyme immobilization a mature discipline? Some critical considerations to capitalize on the benefits of immobilization

“…302,326 Very recent advances in surface-sensitive spectroscopic techniques have provided evidence that push the determination of enzyme structure and orientation at the solid-liquid interface, in particular single-molecule studies showing that analyses sensitive to temporal and spatial heterogeneities in immobilized enzymes are useful to explain the effects of conformational stability and active-site accessibility on activity. 302,[504][505][506][507] In the interplay with the structural aspects of the immobilized enzyme the microenvironment is the other fundamental aspect determining enzyme performance. The microenvironment in which enzymes are acting when they are immobilized in solid materials is usually quite different from conditions in the bulk solution.…”

Activation and Stabilization of Lipase B from Candida antarctica by Immobilization on Polymer Brushes with Optimized Surface Structure

“…Different hydrophobic supports have been used to this purpose, like octyl-agarose beads [ 53 ], decaoctyl sepabeads [ 54 ], silica beads coated with acyl groups, polypropylene [ 55 ], or styrene/styrene divinylbenzene beads [ 17 , 56 ]. The activation of CalB on well-defined modified silica surfaces has been discussed previously in the context of silanized silica surfaces and [ 57 ] SBMA/PEGMA polymer brushes [ 36 ]. It has been suggested that hydrophobic surfaces lead to a more open conformation of CalB giving easier access to the substrate [ 57 ].…”

“…It has been suggested that hydrophobic surfaces lead to a more open conformation of CalB giving easier access to the substrate [ 57 ]. Yet, in a more generalized computational analysis, it was suggested that the optimum support for individual lipases can be predicted using the free surface energy of solvation as predictor and the mechanism driving hyperactivation exceeds the nature of the lid [ 36 ]. Interestingly, CalB showed the highest free energy of solvation of the lipases under investigation which represents a high hydrophobicity.…”

“…An increase in temperature stability with respect to the enzymes in solution was found for a variety of lipases, whereas CalB activity was slightly disturbed by the presence of the polymeric brushes. These differences were explained by the different surface energies of the lipases under investigation [ 35 , 36 ]. Yet, no silica-supported brushes of PMMA, which is ubiquitely used as carrier resins for lipase immobilization in biocatalysis, have been explored before.…”

Chemically Triggered Changes in Mechanical Properties of Responsive Liquid Crystal Polymer Networks with Immobilized Urease

“…Polymers whose physical and chemical properties respond to external cues are of widespread interest in many areas, including sensing, − drug delivery, − actuation, − and tissue engineering. , Such materials also have sizable implications in the design of smart materials that readily adapt to their environment, catalyze reactions on demand, and repair and/or regenerate upon damage. Over the past two decades, there has been extensive work on integrating enzymes into polymeric supports to create biocatalytic materials; however, the polymers in such cases have generally been chosen solely for their ability to stabilize enzymes. − As a result, such materials have been composed of simple matricese.g., poly­(ethylene glycol), poly­(styrene), polyurethanes, and poly­(methyl methacrylate)that lack functional (e.g., shape-reconfigurable and adaptable/switchable) properties. The scope of materials that could be envisioned would be significantly expanded if the polymer support enhanced the functionality of the enzyme-containing material.…”