Exploiting Silica-Binding and Silica-Forming Proteins as Versatile Tools for One-Step Enzyme Immobilization on Siliceous Materials

Abstract

Enzyme immobilization has emerged as an essential technique in industrial applications of enzymes. Silica (SiO₂) serves as a prominent support material for enzyme immobilization. Recent advancements have led to the development of various silica-binding proteins (SBPs) and silica-forming proteins (SFPs) that are invaluable tools in immobilizing enzymes on siliceous materials in a fast and simple manner. SBPs facilitate the immobilization of enzymes with controlled orientation on silica surfaces, while SFPs enable the biomimetic synthesis and encapsulation of enzymes within silica particles. In this review, we explore recent advances in the use of SBPs and SFPs in enzyme applications. We provide a comprehensive overview of their mechanisms and sequence characteristics relevant to enzyme immobilization. Additionally, we summarize the recombinant production and immobilization procedures for enzymes with SBPs or SFPs. We then categorize the available SBPs and SFPs into naturally occurring and artificially engineered types, presenting recent examples that demonstrate their utilization in enzyme immobilization. Our review highlights the strengths and limitations of various SBPs and SFPs and sheds light on future directions for the development of tailor-made biocatalytic silica.

Keywords: biomimetic silica; enzyme immobilization; fusion protein; silica-binding peptide; silica-forming peptide.

Figure 1

(a) Oriented enzyme immobilization on silica surface by a silica-binding peptide/protein (SBP) fusion. (b) Enzyme encapsulation within silica particles using a silica-forming peptide/protein (SFP) either in a genetically fused form (left) or in a physically mixed form (right) with the SFP.

Figure 2

Schematic representation of silica-binding and silica-forming peptides/proteins, each categorized into naturally occurring and artificially engineered types.

Figure 3

Adsorption of positively charged silica-binding peptides/proteins onto silica surface (a) primarily via ionic interactions at near-neutral pH or (b) via van der Waals interactions and hydrogen bonds at the near point of zero charge (redrawn from reference [14]).

Figure 4

Recombinant production and immobilization of SBP-fused enzyme. One-step enzyme immobilization is achieved by mixing either (a) purified enzyme solution or (b) cell lysate with a silica matrix.

Figure 5

Silica polycondensation by silica-forming peptides/proteins. Red arrow indicates nucleophilic attack. (a) The formation of reactive silanolate by deprotonated amino group and the polycondensation through nucleophilic substitution reaction between silicic acid molecules (redrawn from reference [43]). (b) The charge relay effect between the amino and the carboxyl groups on adjacent amino acids promotes the activation of the lone-pair electrons on the amine nitrogen (redrawn from reference [44]). (c) The hydrolysis of silicon alkoxide (upper, redrawn from reference [45]) and the polycondensation of orthosilicic acids (lower, redrawn from reference [46]) catalyzed by silicatein-α.

Figure 6

Production and immobilization of (a) SFP-fused or (b) untagged enzyme. To immobilize untagged enzymes, separate preparation and blending of sole SFP are required to induce silica coprecipitation. Sole SFP can be produced via (c) chemical synthesis or (d) recombinant expression. Abbreviations: MBP, maltose-binding protein; SFP, silica-forming peptide; TEVP, tobacco etch virus protease; TEVcs, TEVP cleavage site.