Developing a Protein Scaffolding System for Rapid EnzymeImmobilization and Optimization of Enzyme Functions forBiocatalysisGuoqiang Zhang, Timothy Johnston, Maureen B. Quin,* and Claudia Schmidt-Dannert*

Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, St. Paul, Minnesota 55108, United States

*S Supporting Information

ABSTRACT: Immobilization of enzymes is required for mostbiocatalytic processes, but chemistries used in enzyme immobilizationare limited and can be challenging. Genetically encoded protein-basedbiomaterials could provide easy-to-use immobilization platforms forbiocatalysts. We recently developed a self-assembling protein scaffoldthat covalently immobilized SpyTagged enzymes by engineering thebacterial microcompartment protein EutM from Salmonella entericawith a SpyCatcher domain. We also identified a range of EutMhomologues as robust protein nanostructures with diverse architec-tures and electrostatic surface properties. In this work, we created amodular immobilization platform with tunable surface properties bydeveloping a toolbox of self-assembling, robust EutM-SpyCatcher scaffolds. Using an alcohol dehydrogenase as modelbiocatalyst, we show that the scaffolds improve enzyme activity and stability. This work provides a modular, easy-to-useimmobilization system that can be tailored for the optimal function of biocatalysts of interest.

KEYWORDS: protein scaffolds, immobilization, synthetic biology, biocatalysis, alcohol dehydrogenase, enzyme

During the past decade significant advances have beenmade in the discovery and engineering of enzymes as

biocatalysts for the synthesis of valuable chemicals ofinterest.1,2 Such enzymatic processes offer several advantagesover synthetic routes, including high substrate specificity andenantio- and stereoselectivity of reactions.3 Yet, relatively fewbiocatalytic reactions designed and tested in the laboratoryhave been developed into industrial scale processes, whichmandate that biocatalysts perform with high efficiency andconversion rates under harsh reaction conditions such as hightemperatures or solvents that can destabilize enzymes.4

Furthermore, in order to be economically viable, biocatalystsmust be recycled, and many biocatalytic processes require theuse of multiple enzymatic steps that for cost reasons are ideallyperformed in a combined process.5,6 A common approach toachieve biocatalyst stability and recycling is to immobilize theenzyme on a solid support, which can extend the lifetime andperformance of the reaction.7−9 Unfortunately, immobilizationof enzymes, and especially coimmobilization of multipledifferent enzymes, by chemical modifications can be time-consuming and technically challenging, requiring significantoptimization.10

To address these challenges, we have developed a rapid,straightforward method to immobilize (multi)enzyme systemson protein-based biomaterials. The concept for this approachderives from the fact that in nature, enzymes are oftencolocalized in close proximity by, e.g., sequestration,11

encapsulation,12 or scaffolding.13 Positioning enzymes togethercan increase reaction efficiency either by substrate channel-

ing,14 or by providing unique microenvironments that are idealfor enzyme function.15 Synthetic biology techniques nowenable the relatively easy adoption of nucleic acid and proteinbased supports to immobilize enzymes and improveyields.16−21

Previously, we took advantage of the self-assemblingproperties of the ethanolamine utilization (Eut) bacterialmicrocompartment (BMC) to colocalize proteins.22−24 Theouter shell of BMCs assembles from thousands of copies ofrepeating ∼10 kDa protein units known as BMC domainproteins. These proteins are known to self-assemble intocrystalline, tube, or filament-like nanoscale structures uponoverexpression and/or purification from a heterologoushost.18,25−31 One of these proteins, EutM from Salmonellaenterica (SE), will also self-assemble in vivo as large proteinfilaments or in vitro as crystalline arrays,22,23 making it an idealprotein for the creation of a modular scaffolding platform withdifferent nanoarchitectures. We have therefore engineeredEutM (SE) as an easy to use platform for immobilization ofmultienzyme biocatalytic cascades.23,32 By genetically fusing aSpyCatcher domain to EutM (SE), we created a protein-basedscaffolding system for the covalent immobilization of SpyTag-fused cargo proteins via spontaneous isopeptide bondformation.33,34 As proof-of-concept, we coimmobilized a self-sufficient dual enzyme cascade for amine synthesis,35 which

Received: April 26, 2019Published: July 15, 2019

Research Article

pubs.acs.org/synthbioCite This: ACS Synth. Biol. XXXX, XXX, XXX−XXX

© XXXX American Chemical Society A DOI: 10.1021/acssynbio.9b00187ACS Synth. Biol. XXXX, XXX, XXX−XXX

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consisted of an alcohol dehydrogenase (ADH) and an aminedehydrogenase (AmDH). We discovered that coimmobilizingthe cascade on EutM-SpyCatcher protein scaffolds significantlyreduced the time required to reach 90% conversion of alcoholto chiral amine, and that the enzymes were stabilized uponimmobilization.23 Parallel to this work, we built andcharacterized a toolbox of 12 additional EutM homologues,representing a diverse collection of sequences identified in thegenome sequences of extremophilic bacteria. We showed thatthe purified recombinant proteins self-assemble like EutM(SE) as robust protein-based scaffolds with differentnanostructures. We also found that EutM (SE) can self-assemble with other homologues to form hybrid scaffolds.36

In this study we sought to extend our self-assemblingimmobilization platform beyond EutM (SE)-SpyCatcher toinclude other EutM homologues selected from our tool-box.23,36 We hypothesized that the different EutM homologuesmay provide scaffold surfaces with different charge distribu-tions that could modify the electrostatic and pH microenviron-ment for optimal function and stability of an immobilizedenzyme.15,37,38 To explore this idea, we selected eighthomologues representing the sequence diversity of ourpreviously characterized EutM homologues to create additionalEutM-SpyCatcher scaffolds for enzyme immobilization.Herein, we characterize the self-assembly of these new

scaffolds in vitro, and demonstrate loading of a SpyTag-labeledcargo enzyme onto these scaffolds. As a proof-of-concept cargoenzyme we chose to immobilize ADH, representing a class ofenzymes widely used in biocatalysis which we previouslycharacterized in detail and shown to be the least stable catalystin a dual enzyme cascade. Previously we found that ADH’sstability could be significantly increased by loading onto EutM(SE)-SpyCatcher scaffolds.23 We therefore characterize theeffect of immobilization of this biocatalyst on the differentEutM-SpyCatcher scaffolds in terms of enzyme activity andstability. Our results show that the new EutM-SpyCatcherproteins self-assemble as robust scaffolds that can sponta-neously and covalently immobilize SpyTag-ADH. Further-more, the individual protein scaffolds improve biocatalystactivity and stability to different degrees, presumably bycreating distinct, favorable microenvironments for biocatalystfunction. This work therefore further supports our self-assembling protein-based system as a versatile and easy-to-use platform for biocatalyst immobilization and optimization ofbiocatalytic systems.

■ MATERIALS AND METHODSChemicals and Reagents. All chemicals were purchased

from Millipore-Sigma (St. Louis, MO), unless stated otherwise.Q5 High-Fidelity DNA Polymerase for PCR amplifications andHiFi DNA assembly master mix for Gibson assembly of DNAfragments were purchased from New England Biolabs (NEB)(Ipswich, MA). Escherichia coli T7 Express (C2566) for proteinexpressions was also obtained from NEB. The Pierce BCAassay kit that was used to measure protein concentrations waspurchased from ThermoFisher Scientific (Waltham, MA).Cloning of Plasmids. All plasmids and strains used in this

study are described in Table S1. Sequences for EutM-SpyCatcher fusions cloned in this study are provided inTable S2. All plasmids were created using HiFi DNA assembly(NEB, Ipswich, MA) with fragments (EutM homologues) andplasmid backbone (pCT5BB with GS-SpyCatcher region)amplified from existing plasmids as templates. PCR and DNA

assembly reactions were carried out according to themanufacturer’s instructions. All gene sequences were con-firmed by Sanger sequencing (University of MinnesotaGenomics Center, MN).

Recombinant Protein Expression and Purification.E. coli T7 Express (C2566) cells were transformed withpCT5BB plasmids encoding different homologues of EutM-SpyCatcher and colonies were grown on Lysogeny Broth (LB)agar plates supplemented with ampicillin (100 μg mL−1). Foreach transformant, an individual colony was used to inoculate50 mL LB medium supplemented with ampicillin (100 μgmL−1) and the culture was incubated overnight at 30 °C withshaking at 220 rpm. The overnight cultures were diluted 100-fold into 500 mL LB medium supplemented with ampicillin(100 μg mL−1) and the cultures were incubated at 30 °C withshaking at 220 rpm to an optical density at 600 nm (A600) =0.4−0.6. Protein expression was induced by adding 4-isopropylbenzoic acid (cumate, 50 μM) and the cultures were grown at37 °C for a further 6 h. The cultures were centrifuged at 3500gfor 30 min to harvest the cells and pellets were stored at −20°C.For ADH (with or without SpyTag), pET28a plasmids

encoding the gene of interest were transformed into E. coli T7Express (C2566) and colonies were selected on LB agar platessupplemented with kanamycin (30 μg mL−1). A single colonywas used to inoculate 50 mL LB medium supplemented withkanamycin (30 μg mL−1), and the culture was incubatedovernight at 37 °C. The overnight culture was used to seed alarger volume of LB medium (700 mL) with a 1:100 inoculum,and cultures were grown at 37 °C. Protein expression wasinduced by addition of isopropyl β-D-1-thiogalactopyranoside(IPTG, 0.5 mM) once an OD of A600 = 0.6 was reached.Cultures were grown for a further 24 h at 170 rpm and 20 °C.Cells were harvested and were washed with 1× PBS (pH 7.4),and pellets were stored at −20 °C.

Protein Purification. For the purification of EutM-SpyCatcher scaffolds, cell pellets were defrosted and resus-pended in Scaffold Lysis Buffer (50 mM Tris-HCl, 250 mMNaCl, 20 mM imidazole, pH 8.0). Cells were lysed bysonication on ice (30 min, power 50%, pulse on 10 s, pulse off20 s with a Branson Sonifier). The lysed cells were centrifuged(12 000g, 40 min, 4 °C) in a Beckman J2-HS centrifuge andthe supernatant was passed through a 0.2 μm ultrafilter. Nickelaffinity chromatography was performed to purify all proteins,following standard HisTrap HP and AKTA FPLC techniques(GE Healthcare Life Sciences, Pittsburgh, PA), and asdescribed in our previous publications.23,32 Scaffold ElutionBuffer (50 mM Tris-HCl, 250 mM NaCl, 250 mM imidazole,pH 8.0) was used to release the 6xHis tagged scaffolds fromthe HisTrap column. The purity of the scaffold proteins wasanalyzed by 15% SDS-PAGE, and the proteins were bufferexchanged to EutM-SpyCatcher Storage Buffer (50 mM Tris-HCl pH 8.0) in Amicon-Ultra (10 000 MWCO) centrifugalfilters (Millipore-Sigma).For purification of (SpyTag-)ADH, cells were resuspended

in Cargo Lysis Buffer (50 mM Tris-HCl, 300 mM NaCl, 20mM imidazole, pH 8.0). Cells were treated with lysozyme (1mg mL−1) by shaking at 150 rpm, at 20 °C for 30 min andwere then disrupted by sonication (30 min, power 50%, pulseon 10 s, and pulse off 20 s). Protein purification from cell lysatefollowed the procedure described in our previous publica-tion.23

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Isopeptide Bond Formation Between SpyTag-ADHand EutM-SpyCatcher Tested by SDS-PAGE. Covalentisopeptide bond formation between purified EutM-SpyCatcherscaffolds and SpyTag-ADH was detected by denaturing SDS-PAGE analysis. Purified proteins were mixed at a 1:1, or 1:9molar ratio (10 μM each, or 10 μM and 90 μM respectively) in50 mM Tris-HCl (pH 8.0) and the suspensions wereincubated at 25 °C for 30 min. To stop reactions, sampleswere heated in denaturing SDS loading buffer at 95 °C for 10min. SDS-PAGE was performed using 15% denaturingpolyacrylamide gels to detect higher molecular weightspecies.23

Negative Stain Transmission Electron Microscopy.Negative staining of EutM-SpyCatcher scaffolds was con-ducted as previously described23,36 using protein at aconcentration of 1.2 mg mL−1. Grids were allowed to air-drybefore storage and imaging. Grids were visualized using aPhillips CM12 TEM (University Imaging Center, University ofMinnesota, MN) at magnifications of 19 500× and 53 000×.Activity and Stability of (SpyTag)-ADH Immobilized

on Scaffolds. Activity of purified (SpyTag-)ADH wasdetermined using a UV-microplate reader by monitoring thechange in NADH concentration at 340 nm (ε = 6.22 mM−1

cm−1) in Tris-HCl buffer (pH 8.0, 50 mM) with cofactorNAD+ (1 mM) as described.23 The reactions were started bythe addition of substrate (S)-2-hexanol (20 mM) to themixture and the absorbance was continuously recorded at 30°C for 3 min. One unit is defined as the amount of protein thatproduces 1 μmol of NADH per minute. Control reactions wereperformed under the same conditions without enzyme. Todetermine the stability of free and scaffold-immobilizedenzyme, (SpyTag-)ADH was mixed with EutM-SpyCatcherat 1:9 molar ratios (corresponding to 6 μM (SpyTag)-ADH at0.2 mg mL−1 and 54 μM EutM-SpyCatcher at 1.2 mg mL−1)and incubated at 30 °C and 150 rpm. Controls lacking anyEutM-SpyCatcher scaffolds were also included. Activities weremeasured at various time points for a total incubation time of48 h using the method described above. For each time pointmeasurement, a small aliquot was removed from an incubationmixture and diluted in the assay mixture at a 1:10 ratio.Activities were measured with two experimental and threetechnical replicates.

■ RESULTS AND DISCUSSION

Design and in Vitro Characterization of a Toolbox ofProtein Scaffolds. We previously demonstrated that differentEutM homologues self-assemble as robust protein-basedbiomaterials upon recombinant expression and purificationfrom E. coli.36 Furthermore, we developed EutM fromSalmonella enterica (SE) as a covalent enzyme immobilizationplatform23 by taking advantage of the well characterizedSpyCatcher-SpyTag protein fusion technology.33 Using thesame strategy, we decided to fuse the SpyCatcher domain (9.5kDa) to the C-terminus of EutM homologues to createdifferent EutM-SpyCatcher (20.5−21 kDa, Tables S2 and S3)scaffold building blocks that would form scaffolds withpotentially diverse properties for enzyme immobilization(Figure 1). The SpyCatcher domains on the different scaffoldsare expected to spontaneously and rapidly form (withinminutes) covalent isopeptide bonds with cargo proteins fusedwith a small SpyTag domain (1.5 kDa)34 as we had shownbefore with the EutM (SE)-SpyCatcher scaffolds.23

We selected a subset of eight proteins from our toolbox of12 EutM homologues to develop and characterize as enzymeimmobilization platforms based upon their previouslydescribed grouping into three phylogenetic clades, scaffoldstructures, and predicted surface electrostatic potentials36

(Figure 2). From clade I, we selected homologue EutM

(TL) as the most closely related protein to EutM (SE) interms of scaffold structure, and EutM (DP) as an outlier in thisclade because it did not appear to assemble into the same well-ordered scaffolds. Homologues EutM (AM), EutM (FG), andEutM (SA) were selected from clade II because of their diversesurface charges as well as scaffold structures (nanotubes to flatsheets). Finally, from clade II we chose all of its threepreviously characterized homologues EutM (CT), EutM(DT), and EutM (TS), again owing to their differences insurface charges and structures.36 For comparison and also asour third member of Clade I, we used EutM (SE)-SpyCatcher,which we had previously shown to self-assemble into scaffoldsthat can immobilize and stabilize23 the alcohol dehydrogenase

Figure 1. Overview of design of EutM-SpyCatcher constructs. EutMmonomer self-assembles into a hexamer (rainbow colored), whichforms the basic building block of the protein scaffolds. SpyCatcher(gray colored) can be genetically fused to the C-terminus of EutM,allowing isopeptide bond formation between scaffolds and SpyTag-labeled cargo. Sequences of EutM-SpyCatcher genetic constructs areprovided in Table S2.

Figure 2. EutM homologues used to create immobilization scaffoldsin this study. The nine EutM homologues were previously identifiedand characterized by us for their surface properties and self-assemblyinto protein structures. The EutM homologues are phylogeneticallyrelated within three distinct clades, as indicated by different coloredboxes (see ref 36 for details). Homology models of the proteinhexamers are shown as surface renderings displaying electrostaticpotentials (red = negatively charged, blue = positively charged).Negative stain TEM images of the purified EutM homologues showedthat they formed distinct nanoscale structures. The TEM images weretaken at a magnification of 53 000×, and the scale bar represents 100nm. Some figure elements have been modified from ref 36.

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ADH39,40 as part of a dual enzyme cascade to produce chiralamines.35

First, we verified that the newly constructed EutM-SpyCatchers self-assemble as scaffolds in vitro by purifyingthe recombinant proteins expressed in E. coli. All of the fusionproteins, which had an N-terminal 6xHis tag, were purified in asingle step by Ni2+ affinity chromatography. None of theEutM-SpyCatcher proteins required prior solubilization forpurification, unlike the unmodified EutMs which can only bepurified in the presence of 4 M urea.36 Therefore, addition ofthe SpyCatcher domain causes the EutM homologues tobehave similarly to our previously characterized EutM (SE)-SpyCatcher.23 A clear band was detected close to the expectedmolecular weight (20.5−21 kDa) when the purified proteinswere run on a denaturing SDS-PAGE gel (Figure 3a, Table

S3). However, EutM (TL)-SpyCatcher migrated slightlyhigher than its predicted size, and a very low level of a highermolecular weight band could also be detected for purifiedEutM (DP)-SpyCatcher, EutM (DT)-SpyCatcher, EutM(FG)-SpyCatcher and EutM (TS)-SpyCatcher, even afterdenaturation for 10 min at 95 °C for in SDS buffer. We hadpreviously observed a similar aberrant migration behavior onSDS-gels with purified EutMs.36 The purified proteins weredialysis into EutM-SpyCatcher Storage Buffer (50 mM Tris-HCl pH 8.0),32 which was used for all enzyme assays andcharacterization studies (see below).

We then confirmed scaffold assembly by negative stain TEMvisualization of the prepared protein samples (Figure 4a,

Figure S1). The concentration of the EutM-SpyCatcherproteins was normalized to 1.2 mg mL−1 to match theconcentration used for all of the enzyme immobilizationstudies performed below. Almost all of the EutM-SpyCatchersformed dense clusters of fibril-like structures that were thin andlong (approximately 40 nm in diameter, micrometers inlength) and appeared flexible, similar to the previouslyobserved fibrils of EutM (SE)-SpyCatcher.23 In some cases(e.g., EutM (AM)-SpyCatcher, EutM (DT)-SpyCatcher, EutM(FG)-SpyCatcher, EutM (TS)-SpyCatcher) it seemed that thefibrils may have assembled from rolled-up nanotubes, whichlooked like round donut-like structures (20−40 nm indiameter) alongside the fibrils. Compared to these homo-logues, EutM (SE) fibrils appeared to be the least defined.Notably, one of the homologues EutM (CT)-SpyCatcher didnot form fibrils, but instead formed masses of donut-likestructures (approximately 100 nm in diameter).

Rapid Covalent Enzyme Immobilization on EutM-SpyCatcher Scaffolds. Because our objective was to

Figure 3. Characterization of EutM-SpyCatcher proteins. (a)Purification of EutM-SpyCatcher proteins. The nine EutM-SpyCatch-er proteins were recombinantly overexpressed and were purified byNi2+ affinity purification. The 6xHis tagged proteins migrate as bandswith a size range of 20.5−21 kDa on an SDS-PAGE gel. DifferentEutM-SpyCatchers are labeled on the gel. Molecular weights andisoelectric points of the proteins are provided in Table S3. (b)Immobilization of SpyTag-ADH on EutM-SpyCatcher scaffolds.Purified SpyTag-ADH (31.5 kDa) and purified EutM-SpyCatchers(21 kDa) mixed at a 1:9 molar ratio form a covalent bond to generateSpyTag-ADH::EutM-SpyCatcher (size range of 52−53.5 kDa). Thedifferent EutM-SpyCatchers are labeled on the gel. Molar excessEutM-SpyCatcher that does not form a covalent bond remains asa 20.5−21 kDa band (see Table S3 for molecular weights of EutM-SpyCatcher homologues).

Figure 4. Visualization of EutM-SpyCatcher scaffold assemblies. (a)Characterization of EutM-SpyCatcher scaffold assembly in vitro.Negative stain transmission electron microscopy images of EutM-SpyCatchers were collected using purified proteins that were bufferexchanged into 50 mM Tris-HCl pH 8.0 and were normalized to aconcentration of 1.2 mg mL−1. Examples of protein fibrils and donut-like structures are indicated by black arrows.. Images were collected ata magnification of 53 000× and the scale bar represents 100 nm.Lower magnification images are also provided in Figure S1. (b) Effectof immobilizing SpyTag-ADH on EutM-SpyCatcher scaffolds.Negative stain transmission electron microscopy images of SpyTag-ADH alone, or SpyTag-ADH mixed with EutM-SpyCatchers at a 1:9molar ratio were collected from mixtures that had been incubated for30 min before staining. Examples of protein fibrils are indicated byblack arrows, examples of film-like material covering the surface offibrils are indicated by white arrows. Images were collected at amagnification of 53 000× and the scale bar represents 100 nm. Imagesof the corresponding control ADH + EutM-SpyCatcher are providedin Figure S3.

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characterize the effect of the different types of protein scaffoldson enzyme activity and stability, we chose as our modelenzyme an alcohol dehydrogenase (ADH) from A. aromati-cum23,35 that we knew from our previous studies suffers frominactivation over the course of a biocatalytic process, but couldbe stabilized by immobilization on EutM (SE)-SpyCatcherscaffolds.23 Further, ADH has been well-characterized kineti-cally by us and structurally by others, and it represents amember of an important class of NAD(P)H-dependentoxidoreductases that is widely used in biocatalysis for theproduction of chiral chemicals.41 The activity of this enzymehas been well characterized with a range of differentsubstrates.23,39,40,42 Using our previously characterized N-terminally SpyTagged ADH,23 we first confirmed whether theSpyCatcher domain on the different EutMs was available toform an isopeptide bond with the SpyTagged enzyme. Wemixed purified SpyTag-ADH (31.5 kDa) and EutM-SpyCatch-ers (20.5−21 kDa) at a 1:1 molar ratio and allowed thereactions to proceed at room temperature (25 °C) for 30 min.SDS-PAGE analysis confirmed that a covalent bond sponta-neously formed, as indicated by the presence of a highermolecular weight band (52−53.5 kDa) that was not observedin the negative controls (ADH without a SpyTag mixed withEutM-SpyCatchers at a 1:1 molar ratio) (Figure S2). Theefficiency of isopeptide bond formation varied betweendifferent EutM-SpyCatcher homologues, in some cases notall of the band corresponding to SpyTag-ADH or EutM-SpyCatcher disappeared, which is not uncommon for theSpyTag-SpyCatcher system.33

After confirming that the EutM-SpyCatcher scaffolds couldcovalently immobilize SpyTag-ADH, we next confirmedimmobilization of SpyTag-ADH on the different EutM-SpyCatcher scaffolds under the same conditions chosen tocharacterize their effect on ADH stability and activityperformed below. We tested immobilization of enzyme toscaffolds at a 1:9 molar ratio using protein concentrations (6.0μM ADH (0.2 mg mL−1) and 54 μM scaffold building block(1.2 mg mL−1)) based on our work of optimizing scaffolding ofADH together with an amine dehydrogenase for chiral aminesynthesis.23 In this previous work, we fixed the concentrationof SpyTag-ADH at 0.2 mg mL−1 (6.0 μM) and established thatimmobilization of SpyTag-ADH on EutM (SE)-SpyCatcherscaffolds at a molar ratio of greater than 1:6 increased enzymestability more than 40% after 48 h under amination reactionconditions in a 2 M ammonium chloride buffer (pH 8.7). Forthe purpose of this study, we used a 9-fold molar excess ofEutM-SpyCatcher corresponding to a concentration of 1.2 mgmL−1 sufficient for scaffold formation (Figure 4a, Figure S1).Instead of the previously used amination specific buffer system,we opted to use a 50 mM Tris-HCl pH 8.0 buffer as a moregeneric buffer system for the characterization of our enzymeimmobilization platform. Under these conditions, SpyTag-ADH self-immobilized onto all EutM-SpyCatchers as detectedby the formation of a higher molecular weight band (52−53.5kDa) after SDS-PAGE analysis (Figure 3b). This time, theSpyTag-ADH bands (31.5 kDa) completely disappeared, andas expected, an excess of EutM-SpyCatchers that did not forma covalent bond remained at 20.5−21 kDa.We then tested under these conditions whether the addition

of ADH, or SpyTag-ADH to EutM-SpyCatchers affected themorphology of the scaffolds by negative stain TEM (Figure 4b,Figure S3). There were no significant changes in the overallstructure of the fibril-like assemblies that we had observed with

the EutM-SpyCatcher scaffolds, these fibrils were still present.However, in some of the SpyTag-ADH + EutM-SpyCatchersamples (e.g., SpyTag-ADH + EutM (DT)-SpyCatcher, EutM(FG)-SpyCatcher, EutM (SA)-SpyCatcher and EutM (TS)-SpyCatcher) the fibrils were now covered in a film-like materialthat was not present in the EutM-SpyCatchers alone, or theSpyTag-ADH alone samples (Figure 4b). We also noted that itwas more difficult to focus on individual structures in thesesamples, making the structures appear slightly blurred.Interestingly, in the ADH (without SpyTag) + EutM (TL)-SpyCatcher sample protein aggregates were located eitherclose to or on the protein fibrils (Figure S3). Whether theseprotein aggregates arise from ADH is not clear; ADH itselfdoes not appear as aggregated protein by negative stain TEM.Finally, in the two EutM-SpyCatcher samples that did not formclearly defined fibrils (EutM (CT)-SpyCatcher and EutM(SE)-SpyCatcher), we did see a difference in the structures ofscaffolds (Figure 4). Specifically, SpyTag-ADH + EutM (CT)-SpyCatcher now appeared as protein aggregates as opposed tosharp edged donut-like structures, and SpyTag-ADH + EutM(SE)-SpyCatcher formed a gel-like material. Similarly, in ADH(without SpyTag) controls, these two scaffolds appeared as notvery well-ordered structures (Figure S3).An important aspect of our chosen model enzyme ADH is

that fact that it is a homotetramer.39 The finding that a SpyTagcan be added to oligomeric enzymes for covalent linkage to aSpyCatcher domain to form a functional biomaterial has beenshown by us and others.23,43−46 The SpyTag/SpyCatchertechnology therefore proves to be an elegant, easily adaptable,and versatile protein−protein ligation system whose utility hasbeen demonstrated with a diversity of proteins and enzymes47

and is therefore widely applicable for the production ofbiomaterials for biotechnological applications48·

EutM-SpyCatcher Scaffolds Have Different Effects onthe Activity of Free and Immobilized Enzyme. Thedifferent EutM-SpyCatcher scaffolds are predicted to havedifferent charge distributions on their surface (Figure 2), whichcan provide different pH microenvironments that may affectenzyme activity and/or stability.15,38,49 To test the effect ofimmobilization on SpyTag-ADH activity, we measured thespecific activities of free enzyme (6.0 μM, 0.2 mg mL−1), andenzyme immobilized on EutM-SpyCatcher scaffolds (at a 1:9ratio). As a control, we also tested the activity of ADH(without any SpyTag) in the absence and presence of EutM-SpyCatcher scaffolds. Reactions were carried out in 50 mMTris-HCl pH 8.0 with NAD+ as cofactor and (S)-2-hexanol assubstrate (Figure 5, see Table S4 for detailed data). Thespecific activity of free ADH at 0.2 mg mL−1 with no scaffoldspresent was 1100 mU mg−1, while the specific activity of freeSpyTag-ADH was slightly higher at 1500 mU mg−1. Thesevalues were 3 times lower than our previously reported specificactivities of ADH and SpyTag-ADH measured with the samesubstrate but in a buffer system optimized for a dual enzymecascade for chiral amine synthesis.23

Immobilization of SpyTag-ADH on our control scaffoldsEutM (SE)-SpyCatcher had minimal effect on the enzymespecific activity. A similar trend was observed for ADH mixedwith EutM (SE)-SpyCatcher scaffolds (Figure 5, Table S4).Likewise, immobilization of SpyTag-ADH on EutM (CT)-SpyCatcher scaffolds did not result in a significant change inspecific activity. On the other hand, immobilization of SpyTag-ADH on the other seven EutM-SpyCatchers did increase thespecific activity of the enzyme up to 1.6-fold. The general trend

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observed was that mixing the enzyme with the scaffoldsimproved the specific activity to different extents, ranging fromno change to more than a 50% increase in relative specificactivity compared to the enzyme only sample.Immobilization on EutM (DP)-SpyCatcher had the least

effect on SpyTag-ADH specific activity. In contrast, immobi-lization of SpyTag-ADH on EutM (DT)-SpyCatcher scaffoldsled to a 1.6-fold increase in specific activity of SpyTag-ADHcompared to the free SpyTag-ADH. Yet, in both of these cases,the increase in enzyme specific activity cannot be completelyattributed to immobilization. For example, the specific activityof ADH without any SpyTag also increased when mixed withEutM (DP)-SpyCatcher, and it also increased 1.6-fold whenmixed with EutM (DT)-SpyCatcher. It could be that theseparticular scaffolds generate a favorable pH microenviron-ment15,37 that supports the catalytic function of the enzymeregardless of whether it is attached to the scaffolds.The other five EutM-SpyCatchers also increased the specific

activity of SpyTag-ADH/ADH compared to the SpyTag-ADH/ADH alone controls. But unlike the EutM (DP)- andEutM (DT)-SpyCatcher scaffolds, immobilization of SpyTag-ADH to these scaffolds led to a larger increase in specificactivity compared to the free ADH mixed with scaffolds. Themost prominent effect on SpyTag-ADH in comparison toADH was with EutM (TS)-SpyCatcher. With this scaffold, theimprovement in specific activity of SpyTag-ADH was 1.3-foldand with ADH it was less than 1.1-fold, representing a morethan 1.2-fold improvement of SpyTag-ADH activity over ADHactivity upon mixing with scaffolds. Similarly, EutM (FG)-SpyCatcher improved activity of SpyTag-ADH by 1.5-fold,which was a greater effect than the 1.3-fold increase in activitymeasured for ADH. It appears that immobilization of SpyTag-ADH on these scaffolds pronounces the effect of improvingenzyme specific activity afforded by the scaffold microenviron-ment alone without enzyme attachment. Overall, immobiliza-tion of the enzyme on scaffolds formed by the three EutMhomologues (AM, FG, and SA) from Clade II results in thelargest increase in specific activity, likely due to similar surfaceelectrostatic properties (Figure 2). EutM (DT)-SpyCatcher

scaffolds from Clade III significantly increased the specificactivity of both ADH and SpyTag-ADH.It is important to note that ADH activity is dependent on

both substrate and buffer composition and that in this work weopted to use a buffer system commonly employed for enzymepurification and characterization, which we had also previouslyused for the characterization of our scaffold toolbox. For eachimmobilized biocatalytic system, testing different buffersystems to find the most suitable conditions for catalystfunction would therefore be preferable. We have already shownthat EutM (SE)-SpyCatcher scaffolds can form at a variety ofpHs,23 and while beyond the scope of this study, future workwill explore the effect of a wide range of conditions includingbuffer type, pH, salt concentrations on the self-assembly of ourEutM-SpyCatcher toolbox.

EutM-SpyCatcher Scaffolds Increase Enzyme Stabil-ity. Previously we found that immobilizing SpyTag-ADH onEutM (SE)-SpyCatcher scaffolds improved the stability of theenzyme.23 To test to which extend the new EutM-SpyCatchersaffected enzyme stability, we measured the specific activity ofSpyTag-ADH alone (control) in solution, or immobilized onEutM-SpyCatcher scaffolds at a 1:9 ratio, every 12 h for a totalincubation time of 48 h at 30 °C with mixing at 150 rpm tosimulate biocatalysis relevant conditions (Figure 6a, see TableS5 and Figure S4 for detailed data). To discern the effect ofscaffold immobilization on enzyme stability, we also measuredthe specific activity of ADH without a SpyTag in the absence(control) or presence of EutM-SpyCatcher scaffolds at thesame time points under the same conditions (Figure 6b, TableS5).

Figure 5. Effect of EutM-SpyCatcher scaffolds on specific activity ofenzyme. Specific activities of purified ADH (blue bars) and SpyTag-ADH (orange bars) were measured with (S)-2-hexanol (20 mM) as asubstrate and NAD+ (1 mM) as a cofactor in 50 mM Tris-HCl (pH8.0) buffer. Specific activities were measured in the absence (control)and presence of the EutM-SpyCatcher scaffolds (at a 1:9 molar ratio).Dotted lines visualize specific activities of enzyme only samples forcomparison. The measured specific activities and calculated relativespecific activities, including standard deviations, are also provided inTable S4 along with statistical data.

Figure 6. Effect of EutM-SpyCatcher scaffolds on stability of enzyme.Specific activities of purified (a) SpyTag-ADH and (b) ADH weremeasured with (S)-2-hexanol (20 mM) as a substrate and NAD+ (1mM) as a cofactor in 50 mM Tris-HCl (pH 8.0) buffer every 12 h fora total of 48 h. Specific activities were measured in the absence andpresence of the EutM-SpyCatcher scaffolds (at a 1:9 molar ratio).Relative activity (expressed as a percentage) assumes 100% activity ofthe enzyme at the beginning of the experiment (0 h). The measuredspecific activity values, including standard deviations, are provided inTable S5 (including statistical analysis) and Figure S4.

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Both SpyTag-ADH and ADH alone controls (i.e., noscaffolds present) displayed significant decreases in activityafter only 24 h of incubation and had completely lost activityby 36 h. On the other hand, immobilization of SpyTag-ADHon EutM-SpyCatcher scaffolds stabilized the enzyme (Figure6a). Relative activities measured after 48 h of incubationranged from 80% remaining relative activity when SpyTag-ADH was immobilized on EutM (SE)-SpyCatcher scaffolds, toalmost no remaining activity when SpyTag-ADH wasimmobilized on EutM (DT)-SpyCatcher scaffolds (Figure6a). Interestingly, ADH (without SpyTag) was also stabilizedin the presence of EutM-SpyCatchers (Figure 6b), corroborat-ing our suggestion that the microenvironments afforded by thescaffolds play a role in supporting enzyme function (Figure 5).Notably, immobilization of SpyTag-ADH on almost allscaffolds further increased enzyme stability (Figures 6a and6b, compare final relative remaining activities) except for EutM(FG)-SpyCatcher where both SpyTag-ADH and ADH had∼60% remaining activity after 48 h, and EutM (DT)-SpyCatcher, which stabilized the free ADH to a greater extentthan the immobilized SpyTag-ADH.Importantly, the highest remaining specific activity after 48 h

was measured with SpyTag-ADH immobilized on EutM (SA)-SpyCatcher, which was 1.1-fold higher than the startingspecific activity of SpyTag-ADH in the absence of any scaffolds(Table S5). Consequently, EutM (SA)-SpyCatcher is the bestperformer of our scaffold toolbox in terms of improvement ofSpyTag-ADH activity over time. Contrastingly, the lowest finalspecific activity (other than the controls that lost all activity)was measured with SpyTag-ADH immobilized on EutM (DT)-SpyCatcher (Table S5). Therefore, although immobilization ofSpyTag-ADH on this scaffold significantly improves initialenzyme activity (Figure 5), it does not stabilize the enzymeover time and in fact, it appears to destabilize the immobilizedSpyTag-ADH compared to the free enzyme mixed withscaffolds. Taken together, a similar pattern observed withClade II (AM, FG, and SA) and Clade III DT scaffolds forpromoting ADH specific activity is not apparent for enzymestabilization. Only EutM (SA)-SpyCatcher scaffolds increaseboth activity and stability, while EutM (DT)-SpyCatcherscaffolds have the opposite effect on ADH activity and stability.Finally, we confirmed whether the scaffolds themselves

remained stable after 48 h reaction time by visualizing themixtures by negative stain by TEM and SDS-PAGE analysis(Figure S5 and Figure S6). We found that the TEM gridscontaining the enzyme only (SpyTag-ADH, or ADH alone)controls did not have any discernible features any longer(compare to Figure 4b, Figure S3), suggesting that the enzymemay have unfolded or degraded over time. This may explainwhy no activity was detected by the end of the experiment(Figure 6, Table S5). Contrastingly, almost all other ADH/SpyTag-ADH + EutM-SpyCatcher samples still had the sameclear nanoscale structures (fibrils, donuts) that we previouslyobserved (Figure 4), confirming that these scaffolds are robustover time. The only differences in structures were observed insamples containing EutM (CT)-SpyCatcher and EutM (SE)-SpyCatcher, which had previously changed structure. EutM(CT)-SpyCatcher + ADH now appeared as an amorphous gel-like material after 48 h (Figure S5) as opposed to the proteinaggregates observed earlier (Figure S3), and EutM (CT)-SpyCatcher + SpyTag-ADH appeared as round donut-likestructures after 48 h (Figure S5) as opposed to the proteinaggregates observed earlier (Figure 4b). Meanwhile, while

EutM (SE)-SpyCatcher + ADH remained as not well-orderedscaffolds after 48 h (Figure S5) as observed earlier (Figure S3),EutM (SE)-SpyCatcher + SpyTag-ADH structures changedfrom being disordered (Figure 4b) to well-ordered fibrils after48 h (Figure S5). It is not clear to us why incubation withSpyTag-ADH with these scaffolds would improve the scaffoldassembly over time, perhaps the isopeptide bond formationbetween SpyTag and SpyCatcher helps to stabilize the self-assembling nanostructures.50,51 EutM (DT)-SpyCatcher scaf-folds with immobilized SpyTag-ADH, however, seem to havedeteriorated after 48 h (Figure S5) compared to the structuresseen earlier (Figure 4b). SDS-PAGE analysis (Figure S6)confirms the observations made by TEM. Enzyme in controlsamples without scaffolds was completely degraded, whileproteins in samples with scaffolds remained stable after 48 hwith the exception of the sample containing SpyTag-ADHimmobilized on EutM (DT)-SpyCatcher. It remains unclearwhy immobilization onto this scaffold leads to proteindegradation and, consequently, loss of activity observed duringthe enzyme stability measurements (Figure 6a).

■ CONCLUSIONSOur objective is to create a modular, self-assembling and easy-to-use protein scaffold platform for biocatalyst immobilizationthat improves biocatalytic reaction efficiency and whichremains stable under reaction conditions. Toward this goal,we expanded our immobilization platform of self-assemblingEutM (SE)-SpyCatcher protein building blocks with additionalblocks with different assembly properties and surface chargedistributions provided by the EutM protein hexamer.52,53 ThepH microenvironment is known to play a key role inpromoting the catalytic function and stability of en-zymes.15,38,54,55 Therefore, having a range of scaffold buildingblocks at our disposal will allow the design of tailored scaffoldsto support optimal biocatalyst function. The results from thiswork confirm that our EutM-SpyCatcher design is modular,and that the original EutM (SE) can be replaced by otherhomologues that also self-assemble as nanoscale scaffolds thatare robust over time. Furthermore, a SpyTag labeled enzymecovalently and spontaneously links to the SpyCatcher domainon the new scaffolds, making this a very straightforward systemfor quick and easy immobilization of biocatalysts. Importantly,we found that immobilization on the EutM-SpyCatcherscaffolds can promote activity and stabilize a biocatalyst toextend reaction lifetime, to varying degrees depending onscaffold building blocks.We found that it is necessary to test the effect of the entire

toolbox of EutM-SpyCatcher scaffolds on both the activity andstability of an enzyme in order to select a scaffold for abiocatalyst. The scaffolds affected enzyme activity and stabilitydifferently, and in some cases in opposite ways. The scaffoldshad a statistically significant effect on enzyme specific activity,with the exception of EutM (CT)-, EutM (SE)-, and EutM(DT)-SpyCatchers (see calculated p-values in Table S4). Thestatistical significance of their effect on the relative enzymeactivity over time varied because of the large variance inactivities as a result of the heterogeneous nature of the system(see calculated p-values in Table S5 for 24 and 48 h data). Wecan conclude with statistical significance though that thestabilizing effect of EutM (AM)-, EutM (CT)-, EutM (DP)-,and EutM (DT)-SpyCatcher scaffolds of SpyTag-ADH is lessthan that of our original EutM (SE)-SpyCatcher scaffolds. Thestabilizing effect observed after 48 h for samples where the

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enzyme (SpyTag-ADH as opposed to the untagged ADH) wascovalently immobilized on EutM (AM)-, EutM (SA)-, EutM(SE)-, and EutM (TL)-SpyCatcher scaffolds was statisticallysignificant. Interestingly, covalent attachment to EutM (DT)-scaffolds destabilized the enzyme even further. From thesedata, we expect that for each enzyme in a biocatalytic system asimilar initial screening approach will be required to select themost appropriate scaffold building block(s) for immobilizationthat provides optimal overall biocatalytic performance basedon its combined effect on activity and stability. Because EutMproteins can be readily produced and isolated36 fromrecombinant cells, a standardized screening platform may bedeveloped to quickly identify optimal scaffolds and conditionsfor biocatalytic reactions. In future work we will expand therepertoire of (multienzyme) biocatalytic cascades that we willimmobilize using our scaffold platform.6 Previously we havealready shown that hybrid scaffolds can be formed36 by mixingdifferent EutM homologues with EutM (SE). Going forward,we will therefore explore the formation of customizedSpyCatcher-functionalized scaffolds composed of differentEutM hexamer building blocks chosen to provide optimalsupport for each individual, immobilized biocatalyst in amultienzyme system. With EutM (SA)-SpyCatcher identifiedas the best building block for increasing ADH specific activity,our next efforts will focus on developing a scaled-up, two-enzyme process for chiral hydrogen-borrowing amination andcompare its performance to results from our proof-of-conceptwork with EutM (SE)-SpyCatcher scaffolds23 and to recentresults obtained for the same system immobilized on porousglass beads.56 We envision that the modularity of ourgenetically encoded protein scaffolding system will make it apowerful plug-and-play system for rapid prototyping andoptimization of immobilized enzyme systems suitable forscaled-up process development.In conclusion, synthetic biology is entering a new realm with

the tailored design and production of “engineered livingmaterials”, i.e., hybrid functionalized biomaterials that can beproduced by a living organism and will self-assemble ondemand.57,58 Our future efforts will be to create abiotechnologically relevant system that will enable scalableproduction, secretion, and immobilization of biocatalysts onprotein scaffolds.59,60 Furthermore, it will also becomenecessary to design hybrid biomaterials that have mechanicalproperties suitable for industrial processes, and can enablelong-term reusability of the system.61 We envision that ourprotein immobilization platform will lend itself to suchapplications.

■ ASSOCIATED CONTENT

*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acssyn-bio.9b00187.

Supplementary Tables S1−S4 provide information onstrains, plasmids and sequences and biochemical data ofEutM proteins used in this study; Supplementary TablesS4 and S5 provide detailed data and analysis of enzymeactivities measured for the scaffolded and unscaffoldedsystems characterized; Supplementary Figures S1−S6provide additional data graphics, TEM and SDS-PAGEgel images (PDF)

■ AUTHOR INFORMATIONCorresponding Authors*E-mail: mquin81@gmail.com.*E-mail: schmi232@umn.edu. Tel: 612-625-5782. Fax: 612-625-5780.ORCIDClaudia Schmidt-Dannert: 0000-0002-0559-3656NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSTEM imaging of scaffolds was conducted using equipmentprovided by University Imaging Center, University ofMinnesota. This work was supported by funds provided byDefense Threat Reduction Agency Grant HDTRA1-15-0004and Defense Advanced Research Projects Agency ContractHR0011-17-2-0038. T.J. was supported by funding from aGrand Challenge research award from the University ofMinnesota.

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ACS Synthetic Biology Research Article

DOI: 10.1021/acssynbio.9b00187ACS Synth. Biol. XXXX, XXX, XXX−XXX

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