Silica-binding Peptide Research Articles

A universal method for surface-based binding assays by preparing immobilized β2-adrenergic receptor stationary phase using solid binding peptide as a fusion tag

Protein functionalized surface has the potential to develop new assays for determining the drug-like properties of potential compounds and discovering specific partners of G protein-coupled receptors (GPCRs). However, a universal method for purifying and immobilizing functional GPCRs has remained elusive. To this end, we developed a general and rapid way to purify and immobilize β2-adrenergic receptor (β2AR) by silicon-specific peptide. We screened CotB1p as a tag from six silica-binding peptides (minTBP-1, CotB1p, SB7, Car9, and Si4–1) by examining their affinity to macroporous silica gel. We investigated the adsorption and desorption of CotB1p-tagged β2-adrenoceptor (β2AR-CotB1p) under diverse conditions to propose a protocol for receptor purification and immobilization. Under optimized conditions, β2AR immobilization were achieved by directly immersing cell lysates harboring the receptor with silica gel, and the elution of the receptor without demonstratable contaminants was realized by including l-arginine/L-lysine in the elutes. This allows purification of the receptor from Escherichia coli (E.coli) lysates with a purity of 95 %. The immobilized receptor was utilized as a stationary phase to reveal the tag impact on ligand-binding outputs by comparing the CotB1p-strategy with a typical covalent method. The KAs of salbutamol, chlorprenaline, tulobuterol, and terbutaline on β2AR-CotB1p column were 1.26 × 106, 6.59 × 106, 7.90 × 106, and 8.97 × 105 M−1 respectively, which were two orders of magnitude higher than those on the Halo-β2AR column. The whole immobilization was accomplished within 30 min without the requirement of any special treatment, resulting in enhanced accuracy for determining receptor-ligand binding parameters. Taken together, CotB1p-mediated strategy is simple, rapid, and universal for purification or immobilization of unstable biomolecules like GPCRs for analytical and biological applications.

High-Throughput Computational Screening of Solid-Binding Peptides.

Inspired by biomineralization, a naturally occurring, protein-facilitated process, solid-binding peptides (SBPs) have gained much attention for their potential to fabricate various shaped nanocrystals and hierarchical nanostructures. The advantage of SBPs over other traditionally used synthetic polymers or short ligands is their tunable interaction with the solid material surface via carefully programmed sequence and being solution-dependent simultaneously. However, designing a sequence with targeted binding affinity or selectivity often involves intensive processes such as phage display, and only a limited number of sequences can be identified. Other computational efforts have also been introduced, but the validation process remains prohibitively expensive once a suitable sequence has been identified. In this paper, we present a new model to rapidly estimate the binding free energy of any given sequence to a solid surface. We show how the overall binding of a polypeptide can be estimated from the free energy contribution of each residue based on the statistics of the thermodynamically stable structure ensemble. We validated our model using five silica-binding peptides of different binding affinities and lengths and showed that the model is accurate and robust across a wider range of chemistries and binding strengths. The computational cost of this method can be as low as 3% of the commonly used enhanced sampling scheme for similar studies and has a great potential to be used in high-throughput algorithms to obtain larger training data sets for machine learning SBP screening.

Modular Design for Proteins Assembling into Antifouling Coatings: Case of Gold Surfaces.

We analyze modularity for a B-M-E triblock protein designed to self-assemble into antifouling coatings. Previously, we have shown that the design performs well on silica surfaces when B is taken to be a silica-binding peptide, M is a thermostable trimer domain, and E is the uncharged elastin-like polypeptide (ELP), E = (GSGVP)40. Here, we demonstrate that we can modulate the nature of the substrate on which the coatings form by choosing different solid-binding peptides as binding domain B and that we can modulate antifouling properties by choosing a different hydrophilic block E. Specifically, to arrive at antifouling coatings for gold surfaces, as binding block B we use the gold-binding peptide GBP1 (with the sequence MHGKTQATSGTIQS), while we replace the antifouling blocks E by zwitterionic ELPs of different lengths, EZn = (GDGVP-GKGVP)n/2, with n = 20, 40, or 80. We find that even the B-M-E proteins with the shortest E blocks make coatings on gold surfaces with excellent antifouling against 1% human serum (HS) and reasonable antifouling against 10% HS. This suggests that the B-M-E triblock protein can be easily adapted to form antifouling coatings on any substrate for which solid-binding peptide sequences are available.

Non-covalent binding tags for batch and flow biocatalysis

Enzyme immobilization offers considerable advantage for biocatalysis in batch and continuous flow reactions. However, many currently available immobilization methods require that the surface of the carrier is chemically modified to allow site specific interactions with their cognate enzymes, which requires specific processing steps and incurs associated costs. Two carriers (cellulose and silica) were investigated here, initially using fluorescent proteins as models to study binding, followed by assessment of industrially relevant enzyme performance (transaminases and an imine reductase/glucose oxidoreductase fusion). Two previously described binding tags, the 17 amino acid long silica-binding peptide from the Bacillus cereus CotB protein and the cellulose binding domain from the Clostridium thermocellum, were fused to a range of proteins without impairing their heterologous expression. When fused to a fluorescent protein both tags conferred high avidity specific binding with their respective carriers (low nanomolar Kd values). The CotB peptide (CotB1p) induced protein aggregation in the transaminase and imine reductase/glucose oxidoreductase fusions when incubated with the silica carrier. The Clostridium thermocellum cellulose binding domain (CBDclos) allowed immobilization of all the proteins tested, but immobilization led to loss of enzymatic activity in the transaminases (< 2-fold) and imine reductase/glucose oxidoreductase fusion (> 80%). A transaminase-CBDclos fusion was then successfully used to demonstrate the application of the binding tag in repetitive batch and a continuous-flow reactor.

Biocatalytic synthesis and ordered self-assembly of silica nanoparticles via a silica-binding peptide.

Achieving scalable and economic methods for manufacturing ordered structures of nanoparticles is an ongoing challenge. Ordered structures of SiO2 nanoparticles have gained increased attention due to the great potential they offer in filtering, separation, drug delivery, optics, electronics, and catalysis. Biomolecules, such as peptides and proteins, have been demonstrated to be useful in the synthesis and self-assembly of inorganic nanostructures. Herein, we describe a simple Stöber-based method wherein both the synthesis and the self-assembly of SiO2 nanoparticles can be facilitated by a silica-binding peptide (SiBP). We demonstrate that the SiBP acts as a multirole agent when used alone or in combination with a strong base catalyst (NH3). When used alone, SiBP catalyzes the hydrolysis of precursor molecules in a dose-dependent manner and produces 17-20 nm SiO2 particles organized in colloidal gels. When used in combination with NH3, the SiBP produces smaller and more uniformly distributed submicrometer particles. The SiBP also improves the long-range self-assembly of the as-grown particles into an opal-like structure by changing the surface charge, without any need for further modification or processing of the particles. The results presented here provide a biomimetic route to the single-step synthesis and assembly of SiO2 nanoparticles into colloidal gels or opal-like structures.

Simple and efficient enzymatic procedure for p-coumaric acid synthesis: Complete bioconversion and biocatalyst recycling under alkaline condition

p-Coumaric acid (para-hydroxycinnamic acid, p-HCA), as a kind of natural active substance from plant cell walls, is widely used in medicine, food, and cosmetics with its antioxidant, anticancer, and other properties. Biological production of p-HCA can be achieved via one-step deamination of the L-tyrosine by tyrosine ammonia-lyase (TAL). Here, a new highly active TAL (Fc-TAL) from Flavobacterium columnare was identified via gene mining and heterologous expression. The purified Fc-TAL completely converts 22 mM L-tyrosine to p-HCA under alkaline condition (pH 9.5–10.0) at 55 °C. To increase enzymatic recyclability and reduce the bioproduction cost, Fc-TAL was immobilized on a silica nanospheres through silica-binding peptides (SiBP2) at pH 10. In comparison to free enzyme, the immobilized TALSiBP2 exhibited higher stability, over 50% activity was retained even after 10 consecutive catalytic cycles. Further experiments revealed that an aliquot of immobilized enzyme (about 200 mg TAL) can transform 20 g L-tyrosine into17.8 g p-HCA with 96% yields and 98% purity through 5 consecutive reaction cycles. Our studies not only provided a strategy for enzyme immobilization in alkaline condition, but also developed a green process for p-coumaric acid biosynthesis with the highest conversion rate and yield reported to date.

Directed Immobilization of PETase on Mesoporous Silica Enables Sustained Depolymerase Activity in Synthetic Wastewater Conditions.

Microplastic accumulation in terrestrial and aquatic environments is a growing environmental challenge. Biodegradation has shown promise as an intervention strategy for reducing the spread of microplastics. The wastewater treatment system is a key intervention point in microplastic biodegradation due to its pivotal role in the water cycle at the interface between human activity and the environmental. However, the best characterized microplastic degradation enzyme, PETase, lacks the stability to perform at scale in wastewater treatment. In this work, we show that genetic fusion of PETase to a silica binding peptide enables directed immobilization of the enzyme onto silica nanoparticles. PETase activity in simulated wastewater conditions is quantified by linear regression from time zero to the time of maximum fluorescence of a fluorescent oxidized product of PETase degradation of PET microfibers. Mesoporous silica is shown to be a superior support material to nonporous silica. The resulting biocatalytic nanomaterial has up to 2.5-fold enhanced stability and 6.2-fold increased activity compared to free enzyme in unbuffered, 40 °C simulated influent (ionic strength ∼15 mM). In unbuffered, 40 °C simulated effluent (ionic strength ∼700 μM), reaction velocity and overall catalytic activity were increased by the biocatalytic material 2.1-fold relative to free PETase. All reactions were performed in 0.2 mL volumes, and enzyme concentrations were normalized across both free and immobilized samples to 9 μg/mL. Site-directed mutagenesis is shown to be a complementary technique to directed immobilization, which may aid in optimization of the biomaterial for wastewater applications. PETase stabilization in application-relevant environments as shown here enables progress toward application of PETase for microplastic biodegradation in wastewater treatment.

Design of Polypeptides Self-Assembling into Antifouling Coatings: Exploiting Multivalency.

We propose to exploit multivalent binding of solid-binding peptides (SBPs) for the physical attachment of antifouling polypeptide brushes on solid surfaces. Using a silica-binding peptide as a model SBP, we find that both tandem-repeated SBPs and SBPs repeated in branched architectures implemented via a multimerization domain work very well to improve the binding strength of polypeptide brushes, as compared to earlier designs with a single SBP. At the same time, for many of the designed sequences, either the solubility or the yield of recombinant production is low. For a single design, with the domain structure B-M-E, both solubility and yield of recombinant production were high. In this design, B is a silica-binding peptide, M is a highly thermostable, de novo-designed trimerization domain, and E is a hydrophilic elastin-like polypeptide. We show that the B-M-E triblock polypeptide rapidly assembles into highly stable polypeptide brushes on silica surfaces, with excellent antifouling properties against high concentrations of serum albumin. Given that SBPs attaching to a wide range of materials have been identified, the B-M-E triblock design provides a template for the development of polypeptides for coating many other materials such as metals or plastics.

Superior Binding of Proteins on a Silica Surface: Physical Insight into the Synergetic Contribution of Polyhistidine and a Silica-Binding Peptide.

Controllable protein attachment onto solid interfaces is essential for the functionality of proteins with broad applications. Silica-binding peptides (SBPs) have emerged as an important tool enabling convenient binding of proteins onto a silica surface. Surprisingly, we found that removal of polyhistidines, a common tag for protein purification, dramatically decrease the binding affinity of a SBP-tagged nanobody onto a silica surface. We hypothesized that polyhistidines and SBPs can be combined to enhance affinity. Through a series of purposely designed SBPs, we identified that the relative orientation of amino acids is a key factor affecting the surface binding strength. One re-engineered SBP, SBP4, exhibits a 4000-fold improvement compared to the original sequence. Guided by physical insights, the work provides a simple strategy that can dramatically improve affinity between a SBP and a silica surface, promising a new way for controllable immobilization of proteins, as demonstrated using nanobodies.

Bare silica as an alternative matrix for affinity purification/immobilization of His-tagged proteins

“Green” protein purification/immobilization processes based on low-cost, earth-abundant, and eco-friendly affinity matrices are highly desirable. Unmodified silica matrices fit well these demands. Since histidine-rich silica-binding peptides are frequently isolated in biopanning experiments, this work aimed at assessing the viability of using bare silica as an alternative matrix for the purification/immobilization of His-tagged proteins. Adsorption and desorption studies with a purified His6-tagged EGFP shown that binding to bare silica particles of different size and porosity occurred under the conditions tested, and that elution could be accomplished with eco-friendly eluants containing L-arginine/L-lysine. Non-tagged EGFP did not bind to these matrices. Small-scale batch purification schemes using silica gel Davisil grade 643 or 646 as affinity matrices and a Tris-buffered saline eluant containing 0.5 M L-arginine (pH 8.5) allowed purifying His6-EGFP from Escherichia coli lysates with a purity of up to 96% and a recovery yield of ∼70% after just one elution step. EGFP tagged with the silica-binding peptide Car9 was recovered with comparable purity and yield. Other His-tagged proteins could also be purified to similar purity levels. The scale of this batch purification scheme was shown to be extendable. These results demonstrate that unmodified silica matrices can be used to effectively purify His-tagged proteins. Since the recovery of double tagged His6-EGFP-Car9 was only of 30–55%, the combination of tags revealed to be advantageous for immobilization purposes.

Optimized Silica-Binding Peptide-Mediated Delivery of Bactericidal Lysin Efficiently Prevents Staphylococcus aureus from Adhering to Device Surfaces.

Staphylococcal-associated device-related infections (DRIs) represent a significant clinical challenge causing major medical and economic sequelae. Bacterial colonization, proliferation, and biofilm formation after adherence to surfaces of the indwelling device are probably the primary cause of DRIs. To address this issue, we incorporated constructs of silica-binding peptide (SiBP) with ClyF, an anti-staphylococcal lysin, into functionalized coatings to impart bactericidal activity against planktonic and sessile Staphylococcus aureus. An optimized construct, SiBP1-ClyF, exhibited improved thermostability and staphylolytic activity compared to its parental lysin ClyF. SiBP1-ClyF-functionalized coatings were efficient in killing MRSA strain N315 (>99.999% within 1 h) and preventing the growth of static and dynamic S. aureus biofilms on various surfaces, including siliconized glass, silicone-coated latex catheter, and silicone catheter. Additionally, SiBP1-ClyF-immobilized surfaces supported normal attachment and growth of mammalian cells. Although the recycling potential and long-term stability of lysin-immobilized surfaces are still affected by the fragility of biological protein molecules, the present study provides a generic strategy for efficient delivery of bactericidal lysin to solid surfaces, which serves as a new approach to prevent the growth of antibiotic-resistant microorganisms on surfaces in hospital settings and could be adapted for other target pathogens as well.

Uranyl-Binding Proteins on Silica Nanoparticles for Repeatable Capture of Uranyl Ions

Successful protein-based enrichment of uranyl, the predominant form of uranium from seawater, relies not only on selective binding of uranyl with a high affinity but also on the reusability of protein binder and a supporting matrix that is abundantly available at low cost. In this work, we propose a silica-binding peptide-enabled approach that allows the non-covalent immobilization of super uranyl-binding protein (SUP) onto silica nanoparticles for their repeated use. We first thoroughly examined solution conditions that affect the stability of uranyl-binding proteins and identified suitable physical conditions that are beneficial for the non-covalent SUP immobilization and subsequent capture of uranyl. We found that the molecular linker between SUP and silica binding peptide plays an important role in improving interaction strength between the silica nanoparticles and the engineered proteins. Consequently, we have demonstrated repeatable recovery and enrichment of uranyl ions from synthetic seawater using the engineered protein on silica nanoparticles. Our approach does not require chemical modification of silica nanoparticles, yet offers strong attachment of SUP to the silica interface and thus attractive reusability of protein-silica nanomaterials.

Self-Assembly of Elastin-like Polypeptide Brushes on Silica Surfaces and Nanoparticles

Control over the placement and activity of biomolecules on solid surfaces is a key challenge in bionanotechnology. While covalent approaches excel in performance, physical attachment approaches excel in ease of processing, which is equally important in many applications. We show how the precision of recombinant protein engineering can be harnessed to design and produce protein-based diblock polymers with a silica-binding and highly hydrophilic elastin-like domain that self-assembles on silica surfaces and nanoparticles to form stable polypeptide brushes that can be used as a scaffold for later biofunctionalization. From atomic force microscopy-based single-molecule force spectroscopy, we find that individual silica-binding peptides have high unbinding rates. Nevertheless, from quartz crystal microbalance measurements, we find that the self-assembled polypeptide brushes cannot easily be rinsed off. From atomic force microscopy imaging and bulk dynamic light scattering, we find that the binding to silica induces fibrillar self-assembly of the peptides. Hence, we conclude that the unexpected stability of these self-assembled polypeptide brushes is at least in part due to peptide–peptide interactions of the silica-binding blocks at the silica surface.

Ultrahigh Adhesion Force Between Silica-Binding Peptide SB7 and Glass Substrate Studied by Single-Molecule Force Spectroscopy and Molecular Dynamic Simulation.

Many proteins and peptides have been identified to effectively and specifically bind on certain surfaces such as silica, polystyrene and titanium dioxide. It is of great interest, in many areas such as enzyme immobilization, surface functionalization and nanotechnology, to understand how these proteins/peptides bind to solid surfaces. Here we use single-molecule force spectroscopy (SMFS) based on atomic force microscopy to directly measure the adhesion force between a silica-binding peptide SB7 and glass surface at single molecule level. SMFS results show that the adhesion force of a single SB7 detaching from the glass surface distributes in two populations at ~220 pN and 610 pN, which is higher than the unfolding forces of most mechanically stable proteins and the unbinding forces of most stable protein-protein interactions. Molecular dynamics simulation reveals that the electrostatic interactions between positively charged arginine residues and the silica surface dominates the binding of SB7 on silica. Our study provides experimental evidence and molecular mechanism at the single-molecule level for the SB7-based immobilization of proteins on silica-based surface, which is able to withstand high mechanical forces, making it an ideal fusion tag for silica surface immobilization or peptide-base adhesive materials.

Affinity purification of Car9-tagged proteins on silica-derivatized spin columns and 96-well plates

The Car9 affinity tag is a dodecameric silica-binding peptide that can be fused to the N- and C-termini of proteins of interest to enable their rapid and inexpensive purification on underivatized silica in a process that typically relies on l-lysine as an eluent. Here, we show that silica paper spin columns and borosilicate multi-well plates used for plasmid DNA purification are suitable for recovering Car9-tagged proteins with high purity in a workflow compatible with high-throughput experiments. Spin columns typically yield 100 μg of biologically active material that can be recovered in minutes with low concentrations of lysine. Because of their short bed length, spin columns also offer unique advantages, as evidenced by the selective recovery of functional Car9-tagged tobacco etch virus (TEV) protease from a fused and auto-cleaved maltose binding protein (MBP) folding partner that nonspecifically binds to silica in the presence of NaCl. These additional purification modalities should increase the versatility and appeal of the Car9 tag for affinity protein purification.

Sequence–Structure–Binding Relationships Reveal Adhesion Behavior of the Car9 Solid-Binding Peptide: An Integrated Experimental and Simulation Study

Solid-binding peptides (SBPs) recognizing inorganic and synthetic interfaces have enabled a broad range of materials science applications and hold promise as adhesive or morphogenetic control units that can be genetically encoded within desirable or designed protein frameworks. To date, the underlying relationships governing both SBP-surface and SBP-SBP interactions and how they give rise to different adsorption mechanisms remain unclear. Here, we combine protein engineering, SPR characterization, and molecular dynamics (MD) simulations initiated from Rosetta predictions to gain insights on the interplay of amino acid composition, structure, self-association and adhesion modality in a panel of variants of the Car9 silica-binding peptide (DSARGFKKPGKR) fused to the C-terminus of superfolder green fluorescent protein (sfGFP). Analysis of kinetics, energetics and MD-predicted structures shows that the high affinity binding of Car9 to the silanol-rich surface of silica is dominated by electrostatic contributions and a spectrum of several persistent interactions that, along with a high surface population of bound molecules, promote cooperative interactions between neighboring SBPs and higher order structure formation. Transition from cooperative to Langmuir adhesion in sfGFP-Car9 variants occurs in concert with a reduction of stable surface interactions and self-association, as confirmed by AFM imaging of proteins exhibiting the two different binding behaviors. We discuss the implications of these results for the de novo design of SBP-surface binding systems.

Elucidating the Binding Mechanism of a Novel Silica-Binding Peptide.

Linker-protein G (LPG) is a bifunctional fusion protein composed of a solid-binding peptide (SBP, referred as the “linker”) with high affinity to silica-based compounds and a Streptococcus protein G (PG), which binds antibodies. The binding mechanisms of LPG to silica-based materials was studied using different biophysical techniques and compared to that of PG without the linker. LPG displayed high binding affinity to a silica surface (KD = 34.77 ± 11.8 nM), with a vertical orientation, in comparison to parent PG, which exhibited no measurable binding affinity. Incorporation of the linker in the fusion protein, LPG, had no effect on the antibody-binding function of PG, which retained its secondary structure and displayed no alteration of its chemical stability. The LPG system provided a milder, easier, and faster affinity-driven immobilization of antibodies to inorganic surfaces when compared to traditional chemical coupling techniques.

Interfacial Interaction between Suolunite Crystal and Silica Binding Peptide for Novel Bioinspired Cement.

Cement and concrete have been important construction materials throughout human history. There is an urgent need to explore novel and untraditional cementitious materials to enhance the durability of building materials and structures in response to increased infrastructure demand worldwide. We report an exploratory study on a biocomposite cement based on a large-scale computational study using density functional theory. An explicitly solvated mixture of a mineral calcium silicate hydrate (C-S-H) crystal suolunite (Ca2Si2O5(OH)2·H2O) and a silicon binding peptide with amino acid sequence PRO-PRO-PRO-TRP-LEU-PRO-TYR-MET-PRO-PRO-TRP-SER is constructed using ab initio molecular dynamics (AIMD). Detailed analysis on the interface structure, interatomic bonding, mechanical properties, and solvent effect of this model reveals a complex interplay of different types of covalent and ionic bonding, including ubiquitous hydrogen bonding which plays a crucial role in their properties. The use of the total bond order density (TBOD), a single quantum mechanical metric, for assessing the interfacial cohesion for this composite biocement is proposed. We find that the solvated model has a slightly larger TBOD than the dried one. These results could lead to a systematic search and rational design for different types of bioinspired and hybrid functional materials with other inorganic minerals and organic peptides.

PAS1-modified optical SIS sensor for highly sensitive and specific detection of toluene

We report on a novel solution immersed silicon (SIS) sensor modified with bio-receptor to detect toluene. To perform this approach, bio-receptor PAS1 which specifically interacts with toluene was chosen as a capture agent for SIS ellipsometric sensing. We constructed wild PAS1 and mutant PAS1 (F46A and F79Y) which are toluene binding-defective. Especially, we utilized an easily accessible capturing approach based on silica binding peptide (SBP) for direct immobilization of PAS1 on the SiO2 surfaces. After the immobilization of SBP-tagged PAS1 to the sensing layers, PAS1-based SIS sensor was evaluated for its ability to recognize toluene. As a result, a significant up-shift in Psi (Ψ) was clearly observed with a low limit of detection (LOD) of 0.1 μM, when treated with toluene on wild PAS1-surface, but not on mutant PAS1-sensing layers, indicating the selective interactions between PAS1 and toluene molecule. The PAS1-SIS sensor showed no changes in Psi (Ψ), if any, negligible, when exposed to benzene, phenol, xylene and 4-nitrophenol as negative controls, thereby demonstrating the specificity of interaction between PAS1 and toluene. Taken together, our results strongly indicate that PAS1-modified ellipsometry sensor can provide a high fidelity system for the accurate and selective detection of toluene.

Flexible Peptide Linkers Enhance the Antimicrobial Activity of Surface-Immobilized Bacteriolytic Enzymes.

Chemical linkers are frequently used in enzyme immobilization to improve enzyme flexibility and activity, whereas peptide linkers, although ubiquitous in protein engineering, are much less explored in enzyme immobilization. Here, we report peptide-linker-assisted noncovalent immobilization of the bacteriolytic enzyme lysostaphin (Lst) to generate anti- Staphylococcus aureus surfaces. Lst was immobilized through affinity tags onto a silica surface (glass slides) and nickel nitrilotriacetic acid (NiNTA) agarose beads via silica-binding peptides (SiBPs) or a hexahistidine tag (His-tag) fused at the C-terminus of Lst, respectively. By inserting specific peptide linkers upstream of the SiBP or His-tag, the immobilized enzymes killed >99.5% of S. aureus ATCC 6538 cells (108 CFU/mL) within 3 h in buffer and could be reused multiple times without significant loss of activity. In contrast, immobilized Lst without a peptide linker was less active/stable. Molecular modeling of Lst-linker-affinity tag constructs illustrated that the presence of the peptide linkers enhanced the molecular flexibility of the proximal Lst binding domain, which interacts with the bacterial substrate, and such increased flexibility correlated with increased antimicrobial activity. We further show that Lst immobilized onto NiNTA beads retained the ability to kill ∼99% of a 108 CFU/mL microbial challenge even in the presence of 1% of a commercial anionic surfactant, C12-14 alcohol EO 3:1 sodium sulfate, when the Lst construct contained a decapeptide linker containing glycine, serine, and alanine residues. This linker-assisted immobilization strategy could be extended to an unrelated lytic enzyme, the endolysin PlyPH, to target Bacillus anthracis Sterne cells either in buffer or in the presence of anionic surfactants. Our approach, therefore, provides a facile route to the use of antimicrobial enzymes on surfaces.