Stabilization of enzymes within single enzyme nanoparticles

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All wrapped up: Stabilization of enzymes within single enzyme nanoparticles Robert Chapman, and Martina H. Stenzel J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b10338 • Publication Date (Web): 09 Jan 2019 Downloaded from http://pubs.acs.org on January 9, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

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Journal of the American Chemical Society

All wrapped up: Stabilisation of enzymes within single enzyme nanoparticles Robert Chapman,* Martina H Stenzel* Centre for Advanced Macromolecular Design (CAMD), School of Chemistry, University of New South Wales, Sydney NSW 2052, Australia.

Abstract Enzymes are extremely usefulin many industrial and pharmaceutical areas due to their ability to catalyse reactions with high selectivity.In order to extend their life-time, significant effortshave been made to increase their stability using protein- or medium engineering as well as by chemical modification. Many researchers have explored the immobilization of enzymes onto carriers, or entrapment within a matrix, framework or nanoparticle with the hope of constricting the movement of the enzyme and shielding it from aggressive environments, thus delaying the denaturation. These strategies often balance three competing interests: i) maintaining high enzymatic activity, ii) ensuring good long term stability against temperature, dehydration, organic solvents, and or aggressive pH, and iii) enabling a tuning or reversible switching of enzyme activity. In almost all cases, multiple enzymes will be contained within a single nanoparticle or matrix, but in recent years researchers have begun to wrap up individualenzymes within single enzyme nanoparticles (SENs). In these nanoparticles the enzyme is stabilized by a thin shell, typically a polymer, prepared either by in-situ polymerization from the enzyme surface or by assembling a pre-formed polymer around it. Because of the increased control over the environment directly around the enzyme, and the possibility of more directly controlling substrate diffusion, many SENs show remarkable stabilitywhile retaining high initial activities even for quite fragile enzymes. Moreover, the activity of the enzyme can often be more easily fine-tuned by adjusting the layer properties. We believe this emerging field will offer exciting and elegant opportunities to both extend the catalytic lifetime of enzymes in aggressive solvents, temperatures and pH, and enable their activity to be switched on and off on demand by modulation of the outer material layer.

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Introduction Enzymes are crucial to life, taking on the important taskof catalyzing almost all metabolic processes. The ability of enzymes to do this with such high efficiency, specificity and selectivity has made them extremely valuable across a wide range of industries. They are commonly employed as tools for synthesis in the textile, biofuel, cleaning and food industries, as signal amplifiers in the medical devices and biosensing industries, and as therapeutics in the pharmaceutical industry.1The efficiency, specificity and high reaction rates at low temperatures also make enzymes ideal catalysts in green chemistry.2The main drawback in the use of enzymes is their often poor stability towards even mild temperature fluctuations, low or high pH, the presence of organic solvents, small molecule inhibitors, or even other enzymes.3 This poor stability reduces the number of cycles that the enzyme can catalyze in synthetic applications before it needs to be discarded. It limits the use of enzymes in point of care biosensing applications and in clinical settings outside the hospital in remote areas. It also restricts the effectiveness of therapeutic enzymes, which often have a half-life of only a few hours in the blood stream and so need to be administered in high and regular doses in order to show any effect. The fragility of enzymes stems from their extremely well-defined three-dimensional structure, determined by the amino acid sequence,which gives the enzyme its activity.3 External factors that lead to changes in the molecular structure of the underlying building blocks, or on the interactions between building blocks will have a detrimental and often irreversible effect on the activity.4To understand loss of enzymatic activity due to changes in the conformation, the interplay of enzymes with external factors need to be considered. Enzymes need some flexibility in order to function, but extensive conformal change need to be avoided and therefore enzyme stabilization is directed at reducing the ability to undergo extensive motions.The structure of enzymes is the result of a fragile equilibrium between the entropic loss and enthalpic contribution, which is mainly caused by the hydrogen bond formation. Small changes such as heat, pressure or the presence of solvent can compromise the thermodynamic balance,5although it needs to be noted that protein can display some kinetic stability and resist the unfolding process for some time.6Water is thought to play a pivotal role in the activity of enzymes,7 even when the enzymeis used in an organic solvent.810

It is presumed that water acts to stabilizethe different equilibrium structures of the enzyme

as well asacting as plasticizer for the internal structure of the enzyme and hydrating the substrate.10However, the need for a hydration layer has recently been challenged by Mann 2 ACS Paragon Plus Environment

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and coworkers, who have shown the stabilization of a variety of enzymes within PEG surfactants in the total absence of water, suggesting that the right wrapping layer may negate the need for this hydration layer.11-13 Tremendous effort has been devoted to enhancing the stability of enzymes,byeither modifying

the

enzyme

structure,14-21tuning

the

microenvironment

around

the

enzyme,22immobilizingthem onto a surface,23-26 or encapsulating them within a matrix or nanoparticle (Figure 1).3, 26-36In the first of these approaches, the structure of the enzyme is altered either by modification of the amino acid sequence or by covalent attachment of small molecules to the existing sequence. Although we do not yet understand the rules governing how tertiary structure leads to function, it is clear from even a brief study of the natural world that not all enzymes are degraded at high temperatures and extreme environments. Organisms such as extremophiles use enzymes under very harsh conditions to catalyse reactions,37and some of these enzymes can be extracted and used in high pressure, high temperature industrial biocatalytic processes directly.38 Inspired by the ability of nature to adapt, protein engineers seek to design enzymes that are inherently stable. This can be done by manipulation of genes, often with the aid of computer-based models,30or by directed evolution as Arnold and others have famously shown.15-18Asthe interplay between protein sequence, structure and dynamics become clearer, it increasingly possible to design artificial enzymes that catalyze non-natural reactions.19-21, 39 Covalent modification of enzymes with small molecules can also enhance theirstability, as has been shown with α-chymotrypsin in organic solvent mixtures,40and increase catalytic efficiency,as has been shown on glycoside hydrolase after modification with heterocyclic aromatics.41 In the second approach, enzyme stability is enhanced by altering the microenvironment of the enzyme.22 Hydrophilic compounds such as sugars, salts or polyols are well known to augment hydrophobic interactions between hydrophobic amino acids resulting in strengthening of the tertiary protein structure.42-43These, and other compounds,44can act as cryoprotectantsaiding the recovery of activity after freeze-drying.43 Thirdly, enzymes can be stabilized byeithercovalent or non-covalent immobilization on a surface.23-26Unfortunately most of these modification reactions are not site-specific, which can lead to a mixture of products.45Immobilization onto surfaces or beads has been well established for the past century, although in the early days absorption,46 chemical conjugation or ionic interactions,26were used more to purify than to stabilize the enzyme.47One book on 3 ACS Paragon Plus Environment


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this topic suggests that more than 5000 publications have been published on enzyme immobilization techniques alone.48The process is widely used today in industrial settings as immobilized enzymes can have a range of superior properties such as enhanced stability, accelerated reaction rate and improved selectivity. The stability of the enzyme in these cases is dependent on:i) the properties of its interaction with the carrier, ii) the binding position and the number of the bonds, iii) the freedom of the conformation change in the matrix, iv) the microenvironment in which the enzyme molecule is located, v) the chemical and physical structure of the carrier, vi) the properties of the spacer (for example, charged or neutral, hydrophilic or hydrophobic, size, length) linking the enzyme molecules to the carrier, and vii) the conditions under which the enzyme molecules were immobilized.48 Enzyme stability can also be enhanced by immobilization on nanoparticles of graphene (oxide), fullerene, carbon nanotubes, iron oxide, gold, silver, silicon dioxide, quantum dots, platinum, cobalt oxide, ceria, copper compounds, magnetic titanium dioxide and zinc oxide,49onto polymeric nanoparticles such aspolystyrene or polyacrylonitrile,49 or onto the surface of metal organic frameworks (MOFs).50-51The size and surface curvature of the particle will regulate the packing process of the enzyme on the surface, as well as the most suitable method of conjugation. Both of these factors can affect the resulting enzymatic activity.48 Finally, enzymes may be stabilized by encapsulation within a matrix such as a cross-linked network, a film or bulk polymer,3,

26-32

This gives very good control over the physical

environment surrounding the enzyme, and can yield high stabilities, but can also reduce the ability of substrate to diffuse to the enzyme thus lowering its activity. Using inherently porous materials such MOFs,50-51or reducing the size of the matrix to the nanoscale can offset this problem, and much research has gone into the stabilization of enzymes within inverse micelles and emulsions,32 as well as in compartmentalized spaces such as a liposomes,3334

layer by layer capsules, and polymersomes.35-36It should be noted,however, that even

nanoparticles offer relatively little control over the number of enzymes encapsulated per particle.More recently, a new class of physically encapsulated enzymes known as single enzyme nanoparticles (SENs) has emerged. In these systems each enzyme is individually wrapped in a protective coating, and this offers several advantages in terms of controlling both the activity and stability of the enzyme. In this review we will discuss single enzyme nanoparticles within the context of the enzyme encapsulation literature, and offer our perspectives on the exciting new opportunities these materials can offer to not only stabilize


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enzymes, but to do so without disrupting the initial activity of the enzyme, and in a way that gives access to tunable and stimuli-responsive activity. a)

b) Ionic

Covalent modification

Surface / bead

Crosslinking / crystalisation

MOF

Nanoparticle

IMMOBILISATION

SINGLE ENZYME NANOPARTILCE (SEN)

R-CO2H R-CO2Cl

O HN

H-bonding NH

O O

O

CO2H

O

R-NH2

NH R

HN HN

Glu, Asp

OH R1

O

R

Hydrophobic pockets/patches Liposome / polymersome / capsuls

R1

O HN

MOF

R

Lys

OH

Polymer matrix

HN

R

R

ENCAPUSULATION

O

NH2

R

H N Cys

Tyr

H

H2 N

HN

R2

R2

O

SH

S S R R-SH

Inverse micelle O N

O

R O

S

N

R O

cm

µm

nm

low nm

Figure 1.a) Immobilisation and encapsulation strategies to enhance enzyme stability. b) Methods for attaching functionality to enzymes including common covalent couplings to natural amino acids

Factors to consider when encapsulating enzymes There are three factors which may be desirable when encapsulating enzymes within a matrix: improving the stability of the enzyme against time or environmental stressors, maintaining high levels of initial activity relative to the native enzyme, and enabling tunable or switchable control over the enzyme activity (Figure 2). These three criteria are often in tension with each other, and while traditional methods of immobilisation or encapsulation may be capable of maintaining one or even two of them; it is rare for a system to produce all three effects at once. Initial activity Native enzyme

Substrate turnover

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


High ACTIVITY Good STABILITY

TUNABLE activity Time / Environmental stress

Figure 2. Representative trends showingsubstrate turnover against time or environmental stressors for encapsulated enzymes with good stability (but poor initial activity and tunability), high initial activity (but poor stability and tunability), and high tunability (but poor initial activity and stability) relative to the native enzyme. An ideal encapsulation strategy should enable all three properties.



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1. Stability:In many cases the aim of encapsulation is to stabilize the enzyme against loss of activity over time, and or after exposure to high temperature, dehydration, pH or aggressive solvents. This is typically done by restricting the conformational flexibility of the enzyme, preventing it from denaturing and or by trapping the enzyme within a matrix where it is stable and allowing substrate to diffuse to the enzyme from the less friendly environmental surroundings. The simplest way of trapping enzymes is by cross-linking the crystalline forms of them with a bifunctional molecule such a dialdehyde, dithiobiimidate and diamineorwith a heterobifunctional crosslinker, which can react with two different functional groups. Depending on the type of enzyme, more unusual strategies can be applied such as the oxidation of sugar residues to aldehydes, which can then be quickly reacted with diamines, or the stabilization by metal complex formation.31The resulting cross-linked enzyme crystals (CLEC) can display good stability against elevated temperatures, organic solvents, pH changes and mechanical impact thanks to the enzyme-enzyme interactions in the crystal that limit the enzyme‘s mobility. The well defined channels in the crystal lattice enable diffusion of substrate to the active site.31Enzymes which do not crystallise can also be cross-linked to form enzyme aggregate (CLEA).28 Enzymes can also be encapsulated within polymer matrices by physical entrapment,52-53 ionic interactions as a polyelectrolyte-complex54-55and or layer-by-layer assembly,56-60or by chemical conjugation with polymers.61Polymers can moreover be used to form layer by layer assemblies which sandwich the enzyme between layers,62or enclosed them within a capsule,35-36 or to trap enzymes within hydrogels,63 as Maynard and coworkers have recently shown with the use of trehalose functionalized hydrogels to protect phytase.64 Enzymes can also be encapsulated within sol-gel networks by hydrolytic polymerization of tetraethoxysilane in the presence of enzymes.29, 65This approach to enzyme stabilization is one of the oldest techniques, dating back to the 1950s, and it is now widely used to stabilize not only enzymes but also other fragile biomaterials such as DNA and cells.66-67 Emulsion techniques have been particularly effective at stabilizing enzymes in organic solvents. Several studies have shown the stabilization of enzymes within water-in-oil emulsions by extraction of enzymes from a water phaseto an organic solvent containing surfactants such as dioctyl sodium sulfosuccinate; by addition of an aqueous enzyme solution to an organic solution containing inverse micelles; or by the dissolution of the enzyme directly into a microemulsion.32 In all cases the enzyme is situated in an aqueous 6 ACS Paragon Plus Environment

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pool(typically 5-30 nm) surrounded by the surfactant, which forms the membrane barrier against the organic solvent, and prevents denaturation of the enzyme. It has been noted that it is possible to have only one enzyme per inverse micelle, although this aspect is often not discussed.32 Thanks to the presence of hydrophilic and hydrophobic media, enzyme microemulsions can enable enzymatic reactions on substrates of different polarities such as sugars and fatty acids.68 As the self-assembled membrane is dynamic, the substrate can easily diffuse between the two phases, although at times this membrane can impede isolation of the product. Unfortunately the micelle itself is not stable over long periods of time when stabilized by surfactants and this can be addressed by using amphiphilic block copolymers as the stabilizing agent. Such emulsions can be further stabilized by crosslinking of the polymer shellas is the case in inverse miniemulsion periphery RAFT polymerization (IMEPP).69-70 Due to the large size of the block copolymer and the nature of the preparation the sizes of the resulting capsules are typically between 100-200 nm. These emulsions can be prepared by sonication,68,

70

or using aporous glass membrane.71While the latter approach gives tunable

particle sizes, these tend to be >500nm in diameter. Surfactant micelles can also be stabilized by the use of polymers such as gelatin, cellulose or agar whichare able to form a gel at the water-oil interface.72If the enzyme is pre-stabilized with a grafted polymer it is also possible to invert the system and prepare enzyme loaded particles via oil-in-water emulsion as the Wich group has recently shown (Figure 3).73-74In this case PEGylation allows the enzyme (lysozyme) to be dissolved an organic solvent such as dichloromethane with only a small loss in activity. Subsequent evaporation of dichloromethane yields ~100 nm enzyme loaded particles without the need for any additional cross-linking.

Figure 3.Encapsulation of PEGylated lysozyme within nanoparticles prepared by emulsification.Reprinted with permission from ref73. Copyright 2016 American Chemical Society



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2. Activity: Enzyme activity, and the loss thereof, is typically described using MichaelisMenten kinetics, which incorporates both substrate binding and catalytic rate terms KM and kcat. A lower kcat is indicative of a reduced catalytic activity, expressed in a slower turnover of bound substrate to product in the active site. A lower KM (at the same kcat) is indicative of a reduced substrate binding affinity in the active site. These terms can be combined to produce a single number kcat / KM, which is used to describe an enzymes ‗catalytic efficiency‘. Despite the high stability of the enzymes in the above examples towards aggressive pH, temperatures, dehydration and organic solvents, the overall catalytic performance of these systems can be quite poor. This is because, in the same way as in a native enzyme, encapsulation often causes eitheri) denaturation of the enzyme structure and or restriction of the movement of the enzyme around the active site (a reduction in catalytic activity); or ii) restriction of substrate and product diffusion through the matrix (a reduction in substrate concentration / binding). By reducing the size of the encapsulating matrix to the nanoscale, the diffusion problem can be reduced, but even the 100 nm PEGylated lysozyme particles described at the end of the previous section show an initial enzyme activity of only 19% relative to the free enzyme due to restricted diffusion.73Diffusion can also be improved by loosening the density of the crosslinking gel, as in the copolymerization of sol-gel materials with alkyl siloxanes to produce ambigels.29, 75This typically results in higher enzymatic activity, although can come at the cost of stability. Activity and stability are often in tension with each other. Immobilisationof an enzyme onto a surface will give very good initial activities, provided the crosslinking chemistry doesn‘t impact the enzyme structure, but will typically result in poor stabilities against organic solvents or aggressive pH.30Applying athick matrix layer around the enzyme may preserve enzyme structure, but reduce substrate diffusion and the catalytic mobility of the active site. Conjugation of polymers or other groups to the surface of the enzyme may protect it against dehydration,76 but also result in some off-target modifications and destruction of the structure around the active site. An ideal enzyme encapsulation would maintain the activity at levels close to that of the native enzyme. Encapsulation of enzymes within coordination networks such as metal organic frameworks (MOFs) offers an interesting approach to maintaining both stability and high substrate diffusion because of the ease with which the dimensions of the pores can be tuned and independently filled (Figure 4).50-51, 77The cavity size of MOFs are generally small enough to 8 ACS Paragon Plus Environment

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ensure that each enzyme is contained within its own ‗cage‘, while leaving still smaller cavities empty for substrate diffusion.In one of the earliest examples of this the Ma group encapsulated cytochrome c inside a Tb-mesoMOF by simply mixing the enzyme with the MOF.78 While the cavities in this MOF (3.9 and 4.7 nm) are large enough to accommodate the enzyme (2.6 × 3.2 × 3.3 nm), the connecting windows are much smaller (1.3 - 1.7 nm in diameter). Despite this, the enzyme was able to translocate into the pores when simply mixed with the MOF in solution, and this was attributed to a partial unfolding and refolding of the enzyme. Similar results were observed with cytochrome c and horseradish peroxidase in an aluminium based PCN-333 MOF (1.1, 3.4, 5.5 nm cavities, 2.6-3.0 nm windows),79 with glucose oxidase and horseradish peroxidase in a PCN-888 MOF (2.0, 5.0, 6.2 nm cavities, 2.5-3.6 nm windows),80 and with organophosphorus acid anhydrolasein a zirconium PCN128y MOF (1.2, 4.4nm channels).81 In these cases close to the maximum theoretical loading of enzyme in the framework could be achieved. Compared to the free enzymes, MOF encapsulated enzymes typically demonstrate lower kcat values (suggesting slower diffusion of substrate), but also a lower km (indicative of stronger substrate binding), and so overall show similar catalytic efficiencies.50,

79

The thermal stability of the enzyme and the recovery of

activity after lyophilisation is often improved by encapsulation. In the case of encapsulated organophosphorus acid anhydrolase,81 the enzyme was able to maintain ~80% of its initial activity after 3 days of storage as a dry powder at room temperature or 30 min at 70°C. Under the same conditions the free enzyme activity dropped to between 20 and 50%. To date, however, this kind of encapsulation has failed to impart the same degree of thermal and solvent stabilization enzyme stabilization as has been shown by encapsulation within a polymer. This may be because the cage is rigid and does not conform to the shape of the enzyme, although the potential to functionalize the cavities and tune diffusion makes this approach attractive and worthy of further attention.

Figure 4.Various methods for encapsulation of enzymes within metal organic frameworks (MOFs). Adapted with permission from ref 51. Copyright 2017 Royal Society of Chemistry.



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In some cases encapsulation can even lead to higher initial activities than observed for the unmodified enzyme, by promoting enhanced substrate binding or locally elevated substrate concentrations. This is particularly evident in cases where multiple involved in sequential, coupled, divergent, or convergent reactions are encapsulated together.82In this case trapping the enzymes in precise spatial arrangement on a scaffold or within a matrix or nanoparticlecan lead to much higher activities than observed for simple mixtures of the native enzymes. The same is true for cascade reactions conducted in MOFs. Where cavities of different sizes exist in the framework it is possible to first fill the largest cavities with a large enzyme, and then the smaller cavities with a smaller enzyme so as to precisely position multiple different enzymes, while still leaving the smallest cavities empty for the diffusion of substrate. In the case of both a cytochrome c / horseradish peroxidase mixed MOF,79 and a glucose oxidase / horseradish peroxidase mixed MOF.80 co-localization of the enzymes dramatically increased their activity in cascade reactions.

3. Tunability:The rapid growth in stimuli-responsive polymers, which can change their confirmation in response to pH, light, temperature and other environmental conditions, has opened up the possibility of controlling the enzyme activity via the encapsulating layer. This may be desirable for the delivery of therapeutic enzymes, such that they are only active at the desired site, or for maintaining long term stability of enzymes by ‗switching them off‘ when they are not needed. Enacapsulation of enzymes within polymersomeshas been a particularly attractive technique to use where this feature is required.35-36 Polymersomes not only produce vesicles of higher stability than liposomes but also make it possible to tune in any number of membrane properties including stimuli-responsiveness,83-85and permeability.84-96To some extent the permeability of the membrane can be tuned through the polymer structure, and some polymersomes are inherently permeable to small molecule enzyme substrates.97A variety of membrane proteins have been incorporated into membrane of polymersomes to facilitate substrate diffusion to an enzyme (Figure 5),35 since the concept was first introduced by Meier and coworkers.89, 98One of the earliest and most widely used of these is the outer membrane porinOmpFwhich acts as a size selective filter for molecules below 400g.mol-1and has been used to tune membrane permeability in polymersomes containing -lactamase,85acid phosphatase,93,

96

and nuclease hydrolase.94 The permeability of this channel can be tuned 10 ACS Paragon Plus Environment

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electrically allowing switchable control over the enzyme activity.85Other membrane proteinssuch as Tsx,94 and an engineered variant of FhuA,86, 91have also been used to tune membrane permeability in polymersomescontaining nuclease hydrolase,and

horseradish

peroxidase, respectively.

Figure 5.Example membrane porins that have been incorporated into polymersomes to tune the membrane porosity. A) LamB (PDB :1AF6), B) Tsx (PDB : 1TLY), C) OmpF (PDB : 2OMF), D) FluA variant with reducing trigger, E) ompG with pH trigger, and G) MscL which opens in response to mechanical stimuli (PDB : 1MSL). Reproduced with permission from ref. 35.

Membrane permeability in polymersomes can also be controlled using a variety of chemical switches. In one early example, stimuli responsive blocks containing boronic acid side chains were incorporated PEG-poly(styrene) polymersomes containing candida antarctica lipase B (CALB).83In the presence of sugars such as fructose, the boronic ester was ionized, causing it to become hydrophilic. The resulting pores formed in the membrane enabled substrate diffusion and the switch on of CALB activity. In a similar way, the permeability of PDMS membranes in horseradish peroxidase containing polymersomes have been tuned by a UV triggered reaction with a propiophenone.90Self –immolative polymers can be used to create polymersomes that will entirely disassemble upon exposure to triggers such as light or redox active species,92 and this has been used to control the release of lipase and alkaline phosphatase.99 While all of the above examples are irreversible, it is also possible to design polymersomes where the permeability of the membrane is reversibly tunable. In a prominent recent example, Bruns, Boesel and coworkers incorporated donor-acceptor Stenhouse adducts (DASAs) into polymersomes containing glucose oxidase and horseradish peroxidase.84 The two different DASAs used could be cyclized independently using two different wavelengths of light. In the cyclized form, membrane permeability increased, switching substrate diffusion 11 ACS Paragon Plus Environment


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and thus enzyme activity on, and in the absence of light, both returned to their linear form, restoring the membrane to its impermeable state (Figure 6).

Figure 6.Schematic showing two enzyme containing polymersomes in which the membrane permeability can be controlled by light of narrow wavelengths.Reprintedwith permission from ref 84. Copyright 2018 American Chemical Society.

Single enzyme nanoparticles Over the last 10-15 years, increasing attention has been focused on building well-defined shells around each individual enzyme rather than trapping them statistically within a larger particle. By careful control of the chemical environment around each enzyme greatly enhanced stability with respect to temperature, solvents and pH has been shown (Tables 1-2). The shell may be built from a range of soft polymers or from more rigid materials such as metal organic frameworks (MOFs). Some methods require prior modification of the enzyme, but quite often the shell can be formed around the native unmodified enzyme. We have grouped the strategies into i) polymer conjugates, ii) in-situ polymerization and crosslinking, where the shell is polymerised around the enzyme from small monomeric components and;iii) assembly methods, where the shell is constructed by the assembly of pre-made polymers around the enzyme (Figure 7). In many cases, single enzyme nanoparticles (SENs) are able to enhance the stability or initial activity or the tunability of an encapsulated enzyme relative to the systems described above. More than this, however, we expect that the control present in these systems may provide an opportunity to access structures capable of producing two or even three of these features at once. 12 ACS Paragon Plus Environment

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Post polymerization assembly +

+ -

- -

In situ polymerization and crosslinking

+

R

+ + + + +

FRP

NH2

H2 N

O

R

R = Cl or NHS

+

R

O

+

R = Cl or NHS -

- - -

NH2

FRP, sugar

Polyion complex micelles (PIC)

O

O

O

R O

O

Single enzyme nanogels (SEN)

ZIF

Excipients and solvophobic assembly

Inclusion frameworks (ZIFs)

Polymer chain grafting

Polymer conjugates

Figure 7. Summary of approaches to the preparation of single enzyme nanoparticles, either by grafting of a polymer chain,in situpolymerization of a crosslinked shell, or postpolymerisationassembly.

Covalent attachment of polymers Owing to the large number of functional groups present on the enzyme surface, conjugation of carbohydratesor polymers such as PEG, pNIPAAm, pMPC and pHPMAto proteins is now standard practice.61,

100-102

Several enzymes have been modified in this way through their

lysine and or cysteine residues includinglysozyme,73, 108

103-104

papain,105-106α-chemotrypsin,107-

trypsin,109-111 and L-asparaginase.112-117 Gauthier and Klok‘s detailed review of this field

demonstrates that in many cases polymer conjugation results in 60% activity after 1h of incubation at pH 1. Polymers can also be grafted to the enzymes via their side chains, although this inevitablycauses some level of aggregation into ―multi-enzyme‖ nanoparticles. For example, Kumar, Kasi and coworkers grafted simple poly(acrylic acid) polymers to the lysine residues of catalase, to produce a trimodal distribution of particles at 80, 12, and 2.4 nm.120 Despite the distribution of particle sizes, in some cases close to 100% activity could be retained after grafting, along with increased shelf life and and good resistance against heat and trypsin degradation. Similar results have been observed with the use of this approach on a range of other enzymes including glucose oxidase, HRP,lactate dehydrogenase, acid phosphatase and lipase.121

In situ polymerization and crosslinking An alternative to grafting polymers to or from an enzyme is to polymerize a cross-linked network or gel around the enzyme. This has the advantage of more fully enclosing the enzyme and can lead to higher levels of stabilization while still retaining enough flexibility for the enzyme to perform its function. In the simplest of these approaches, an enzyme iscovalently modified with a polymerizable group,and then polymerized in the presence of a free monomer and cross-linker.122-123Because each enzyme is individually wrapped, rapid diffusion of the substrate to the enzyme can be maintained, provided sufficient porosity of the polymer shell. This approach was first shown by Kim and Grate who conjugated acryloyl chloride to the lysine residues of chemotrypsin and trypsin,122,

124-125

Free radical

polymerization with a silane containing methacrylate, and subsequent hydrolysis and silane condensation yielded 4-8 nm particles which could be further encapsulated within nanoporous silica.124 The covalent modification of the enzyme led to a 50% reduction in the its initial activity, but didn‘t reduce the substrate binding and resulted in impressive thermal stability at 30°C. The same approach was later used by Yadav et al. to encapsulate carbonic anhydrase within a silane polymer network, which was then hydrolyzed to form a silica shell. A similar a reduction in the initial enzyme activity along with vastly improved stability over 100 days at 30°C was observed.126


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By employing a milder NHS coupling of the polymerisable unit to the enzyme, and by forming the nanogel from acrylamide instead of a silane, Lui, Ouyang and coworkers were able to reduce this initial activity loss and extend the thermal stability imparted by the nanogel. In the case of horseradish peroxidase (HRP), almost no loss in activity was observed as 80% of its activity was retained after 90 min incubation at 65°C.127 The nanogels were highly monodisperse, with shell thicknesses between 2-5 nm. Lipase nanogels showed similar results in buffer solutions,128 and retained 60-80% of their activity in DMSO, even after heating for 3h at 70°C.129The more fragile carbonic anhydrase retained 70% of its activity after encapsulation and the enzyme‘s half-life at 75°C was increased from 3 min (in the case of the free enzyme) to over 90 min.130 These single enzyme nanogels can be readily immobilized on surfaces without harming the enzyme.131Similar particles were also prepared by Hegedus and Nagy on β-xylosidase, endoxylanase and endocellulase, which retained 1120% of their initial activity after creation of the polymer shell and retained 50-60% of this activity after heating for 72h at 50°C.132Yan et al. used the same technique to encapsulate HRP, caspase and superoxide dismutase in SENs and found that they could be used to retain the activity of the enzymes both in vitro and in vivo.133 By using an acid degradable crosslinker, the capsule could be degraded at pH 5.5 without compromising its stability against other enzymes such as trypsin and chemotrypsin. This degradability allowed delivery and release of both enhanced green fluorescent protein and caspase in their active forms to HeLa cells. Nanogel formation can also be used to encapsulate single copies of two or more different enzymes within a nanoparticle, as shown in 2013 by Shi, Chen, Ji and Lu.134 In their work invertase, glucose oxidase, and horseradish peroxidase were bound together using a DNA scaffold, which was removed after formation of the acrylamide nanogel around the enzyme complexes. Co-localization of these enzymes resulted in a more than 10-fold increase in turnover rates for a cascade reaction of sucrose to glucose to hydrogen peroxide to water. As with the single enzyme nanogels, encapsulation improved the thermal stability of the enzymes, which retained more than 70% activity after 1h at 65°C (Figure 8).134



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Figure 8. Enzyme cascades encapsulated within a single enzyme nanogel. Adapted from 134 (Springer Nature, Nat. Nanotech, Biomimetic enzyme nanocomplexes and their use as antidotes and preventive measures for alcohol intoxication, Yang Liu, Juanjuan Du, Ming Yan, Mo Yin Lau, Jay Hu et al. 2013)

Despite these impressive results, modification of the enzyme substrate by covalent attachment of the polymerisable unit can lead to denaturation and deactivation of more sensitive enzymes due to loss of the positive charge on the lysine residues. However, under the right conditions hydrogen bonding of acrylamide to the enzyme substrate can be enough to from a SEN without prior modification of the enzyme. This was first shown by Delaittre and coworkers with glucose oxidase,131 and later shown to be enhanced by performing the nanogelpolymerisation in a concentrated (5% w/w) sugar solution.135 Various sugars have been shown to work, although disaccharides such as sucrose resulted in a greater shell thickness than monosaccharides. Formation of the nanogel in this way results in less activity loss than covalent modification of the enzyme substrate, and enabled the group to form nanogels from a wide variety of enzymes including horseradish peroxidase, β-glucosidase, a lipase (CalB), a laccase (TvL), catalase, glucose oxidase, alcohol oxidase, and an esterase (PfE). Shell thickness could be controlled to between 2-8 nm. As in the immobilization examples cited in the previous section, a tradeoff was observed between activity and stability, although because of the thinner shell, and the individual wrapping of the enzyme, the effect is less pronounced this case. Thicker shells were found to increase thermal stability, but decrease activity, presumably due to a reduction in substrate diffusion (Figure 9). For example, the kcat of a highly cross-linked glucose oxidasenanogel was reduced by slightly more than 50% relative to the unmodified enzyme, but this dense shell enabled 100% retention of activity across a very wide pH range (pH 3-9) and retained >80% of its initial activity after more than 90 min at 65°C.135


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a)

b)

Figure 9.Single enzyme nanogels.Effect of shell thickness on a) activity, and b) stability. Adapted with permission from ref135. Copyright 2017 Royal Society of Chemistry.

Delaittre and coworkers have also investigated the question of purity of single enzyme nanogels in some detail. Using fluorescence burst analysis they were able to show that provided the concentration of enzyme is higher than 10 µM during the polymerization, every enzyme does indeed get encapsulated within a nanogel, and no empty nanogels (without enzyme) form.135At lower concentrations of enzyme this is not necessarily true and mixed populations of encapsulated and non-encapsulated enzymes can be found. It is generally difficult to form an inorganic nanoparticle, such as a MOF, around an enzyme because of the harsh synthetic conditions required to form the particle. However there are certain coordination networks such as zeoliticimidazolate frameworks (ZIFs), which can be synthesized in water under very mild conditions and this allows the formation of the framework around the enzyme in situ by a co-precipitation.50-51The pore size of the framework may be much smaller than the enzyme this can lead to greatly improved thermal and solvent stability. Lyu et al. reported the first example of this in 2014 in which they coprecipitatedcytochrome c with ZIF-8 and ZIF-10.136 Their method required the use of 17 ACS Paragon Plus Environment


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methanol as a solvent and for this reason the enzyme needed to be coated with polyvinylpyrolidone prior to formation of the framework to avoid denaturation. Horseradish peroxidase and lipase from thermomyceslanuginosus were less stable to this method of encapsulation and lost significant activity. Liang et al. later showed that biomolecules could trigger crystallization of ZIF-8 frameworks around themselves in water, avoiding the need for this polymer coating. Successful incorporation of lysozyme, horseradish peroxidase, urease, and tryspin,137 and lipase,138 have been shown by this method without such loss in activity. Enzymes protected in this way were able to show activity after 1h in boiling water (100°C) and even in boiling DMF (153°C), as well as triggered release at pH 500 nm), and contain multiple copies of the enzyme. While they are therefore not single enzyme nanoparticles as in the previous examples, the framework both wraps each enzyme individual 18 ACS Paragon Plus Environment

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and allows high diffusion of substrate through the structure due to its high porosity. In some cases the MOF / ZIF provides comparable stability to the polymer based approaches and in most the well-defined cage structure results in provide stability better than approaches based on mesoporous silica.

Non-covalent assembly of a pre-formed polymer 1.1.1 Covalent conjugation and excipients Single enzymes can also be encapsulated within nanoparticles by assembling a pre-formed polymer around them. One way to do this is to chemically conjugate the nanoparticle to a preformed chain. This has been elegantly shown by Zhuang, Thayymanavan and co-workers who formed a nanoparticle (of around 8-10 nm) around cytochrome c by reaction of its amine residues with a p-nitrophenyl carbonate moiety on the side chain of their polymer.141 Cleavage of a disulfide linkage resulted in disassembly of the structure and release of the native enzyme with near quantitative recovery of its activity.141 In some cases good stabilization can be achieved by simply mixing the enzyme with a preformed polymer. As discussed above, simple glycopolymers such as poly(trehalose) have been shown to provide complete protection against lyophilisation and some thermal protection to horseradish peroxidase and β-galactosidase when chemically conjugated to the enzyme,76but this can also be observed in the absence of any chemical conjugation.142 A range of excipients such as these have been used in the stabilization of proteins, including polyamino saccharides,143 poly(caprolactone)s with pendent sugar functionalities,144 and acrylic acid / C18 copolymers.145 In many cases the excipients used are amphiphilic in nature, and it is possible therefore that at least part of their protective function comes from the entrapment of the enzyme within a nanoparticle. While the nanostructure around the enzyme is not always well characterized, where it is the size indicates that such excipients do yield single enzyme nanoparticles of around 10 nm even in the presence of NaCl.145 As the enzyme in these examples is encapsulated by a purely non-covalent and noncrosslinked interaction, the structure of both the polymer and the enzyme will have a significant effect on the nanoparticle formed. In solution almost all enzymes display patches of both hydrophobicity and hydrophilicity on their surface, as well as a variety of different charges. These patches are typically 1-2 nm in diameter, and 1-2 nm apart but very little work 19 ACS Paragon Plus Environment


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has explored wrapping enzymes by systematically designing materials to interact with these patches.146 Xu and coworkers have recently argued that fully water soluble copolymers with the right balance of hydrophilic and hydrophobic units can be very effective at wrapping around the surface of enzymes through interaction with these patches without disrupting their folding. The group designed a range of random copolymers containing the hydrophilic monomers oligo(ethylene glycol)methacrylate and 3-sulfopropylmethacrylate and the hydrophobic monomers 2-ethylhexyl methacrylate and methyl methacrylate (Figure 11).146 Simple mixing of their polymer with enzymes such as horseradish peroxidase and glucose oxidase resulted in the formation of a polymer shell that enabled the enzyme to be dissolved in organic solvents such as toluene and chloroform with only 20% activity loss (in the case of HRP) and 50% loss (in the case of GOx) over 24h compared to the unmodified enzyme. The nanoparticles formed by the process were found to be 50-60 nm suggesting the presence of more than one enzyme per particle. However, because the interaction is non-covalent, relying only on hydrophilic/hydrophobic interactions, no modification of the enzyme substrate is necessary making the method highly generalizable. a)

c)

b)

d)

Figure 11.Course grain models showing a) HRP and a random heteropolymer, and b) the expected assembly upon mixing at the point circled in (d). c) Surface coverage as a function of adsorption strength and d) as a function of MMA composition in the polymer. Adapted from 146 (The American Association for the Advancement of Science, Science, Random heteropolymers preserve protein function in foreign environments, Brian Panganiban, BaofuQiao, Tao Jiang, et al. 2018)


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1.1.2 Polyion complex micelles and liquid proteins Of course, hydrophobicity is not the only non-covalent interaction that can be used to assemble pre-formed polymers around an enzyme, and there has been a significant body of work devoted to the use of charge for this purpose.147Kataoka and co-workers have pioneered the co-assembly of oppositely charged hydrophilic block copolymers into polyion complex micelles (PICs).148-150 This technique can also be used to assemble a charged block copolymer around proteins with a net opposite charge.151Upon mixing, the charged block of the polymer will bind to the protein, and the neutral block, such as polyethylene glycol,148 polyacrylamide,152or poly(N-(2-hydroxypropyl) methacrylamide),153 will stabilize the particle. The protein can be pre-modified with in order to vary its charge where the charge on the native protein is close to neutral.154 A range of different chargedpolymer blocks have been investigatedincluding the cationic polymers polylysine,155 poly(aminoalkylaspartamide), 154

and poly (aminoalkyl methacrylate),156 which all interact strongly with anionic proteins; as

well as the anionic polymers acidic polyacrylates,157 and poly(aspartate),158 which interact with cationic proteins. Typically multiple biomolecules are incorporated per nanoparticle, although it is possible to vary this by tuning the polymer size, block ratios, preparation method, and the ratio of polymer to protein.147While most of the work in this field has been performed with proteins, PICs from lysozyme,151, 157glucose oxidase,159 and catalase,160 have been prepared. The groups of Perrimen and Mann have prepared a range of polyion complex micelles from ferratin,12myoglobin,13glucose oxidase,161 as well as lipases from rhizomucormiehei and thermomyces lanuginosus,162and a PEG surfactant, which they term ‗liquid proteins‘. In their work the enzyme is first canonizedthrough the reaction of its peripheral carboxylic acids with a diamine, and then assembled with an anionic PEG based surfactant (Figure 12). The discreet single enzyme nanoparticles formed are 2-3 nm larger than the native enzyme in all cases and readily soluble in both water and dichloromethane.12 After removal of the water, at room temperature the conjugate crystallizes into a melt, thus the term ‗liquid protein‘ although

upon

reintroduction

of

solvent

the

melt

will

dissolve

to

form

a

11

nanoparticle. Remarkably enzymes protected by this method are able to retain their structure and almost 100% of their activity in the total absence of water and at temperatures of up to 150°C. This challenges the long held assumption that a hydration layer around the periphery of the enzyme is required for it to retain its activity.



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Figure 12.Schematic and TEM image showing cationic modification of ferratin and subsequent encapsulation with an anionic PEG surfactant to form a liquid protein. Adapted with permission from ref12. Copyright 2009 John Wiley and Sons.

The requirement to covalently modify the surface via cationisation is perhaps a disadvantage of this approach, which may limit its viability with very sensitive enzymes. That said, in the all of the examples studied to date both the structure andhigh activity of the enzymes have been retained following this treatment. In the case of glucose oxidase similar turnover numbers were observed after both cationisation and subsequent encapsulation within the surfactant. A slight increase in the kM from 30 mM (free GOx) to 34 mM (cationisedGOx) to 37nM (encapsulated GOx) was observed which indicates a reduction in substrate binding but this effect was surprisingly small given the dramatic modification to the enzyme surface. 161 The preservation of the enzyme structure and activity of the enzymes in the complete absence of water runs counter to the prevailing wisdom that a hydration layer is necessary for enzymatic activity, and suggests that close binding of an active polymer layer may actually promote rather than detract from stability in single enzyme nanoparticles.11


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Table 1.Summary of selected single enzyme nanoparticles produced by polymerisation around the enzyme. Method

Single Enzyme Nanogel (SEN)

Covalent modification of enzyme?

Coating

Enzyme

Particle size

Initial activity (relative to free enzyme)

Stability comments

Acryloyl chloride

Crosslinked silane

Chemotrypsin Trypsin Carbonic anhydrase

NHS acrylate

Acrylamide nanogel

NHS acrylate

4-8 nm 4-8 nm 70-80 nm

50%

Thermal stability at 30°C

124-126

HRP

12-17 nm

100%

80% after 90 min at 65°C in buffer

127

Acrylamide nanogel

Lipase

40 nm

100%

60-80% after 3h at 70°C in DMSO

128-129

NHS acrylate

Acrylamide nanogel

Carbonic anhydrase

9-18 nm

70%

t1/2 from 3 min to >90 min at 75°C

130

NHS acrylate

Acrylamide nanogel (± degradable cross-linker)

HRP Caspase Superoxide dismutase

12-16 nm 12 nm 20 nm

Not quantified

Activity seen in vitro, and stable against trypsin at pH 7.4

NHS acrylate

Acrylamide nanogel

Cellulases

4-10 nm

11-20%

50-60% of activity after 72h at 50°C

132

-

Acrylamide nanogel

Invertase + GOx + HRP

27-33 nm

10-fold increase in turnover rate

>70% after 1h at 65°C

134

GOx, HRP, Catalase β-glucosidase, CALB, TV laccase, Alcohol oxidase

10-20 nm

50-100% (depending on shell thickness)

~100% at pH 3-9 (GOx) 75% after 40 min at 65°C (GOx)

131, 135

136

-

ZIF in situ synthesis

Acrylamide nanogel

Ref

122,

PVP

ZIF-8 and ZIF-10 ZIF-8 ZIF-8

Cytochrome c HRP Lipase

300 nm

Up to 10x 28% 88%

-

-

ZIF-8

Lysozyme, HRP, Urease, Tryspin, Lipase, GOx

0.5-3 µm

-

Some activity after 1h in boiling water or boiling DMF

-

ZIF-90

Catalase

~1 µm

3%

No degradation by proteinase K

133

137-138, 140 139


Journal of the American Chemical Society 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Page 24 of 37

Table 2.Summary of selected single enzyme nanoparticles produced by Method

Coating

Enzyme

Particle size

Initial activity (relative to free enzyme)

Stability comments

Ref

Tb mesoMOF (3.9 & 4.7 nm cage)

Cytochrome c

N/A

N/A

N/A

78

-

PCN-333 MOF (3.4 & 5.5 nm cage)

Cytochrome c HRP

> 5 µm

kcat30%, kM 50% kcat50%, kM 30%

Some activity of HRP in THF

79

-

PCN-888 (5.0 & 6.2 nm cage)

GOx + HRP

~100%

~100% after 3h @ 37°C with trypsin

80

-

Zr PCN-128y MOF (4.4 nm channel)

Organophosphorus acid anhydrolase

10 x 1 µm

~50%

80% after 30 min at 70°C

81

Crosslinking

Carbamate

Crosslinked PEG brush

Cytochrome C

8-10 nm

< 50%

100% recovery after release from nanoparticle

141

Excipients

-

Acrylate copolymers

HRP, GOx

50-60 nm

100%

50-80% in toluene and CHCl3.

146

14-18 nm 50 nm 14-55 nm

100% N/A 90-100%

N/A N/A Improved therapeutic efficacy

159

PEG-pAA

GOx Lysosyme Catalase Ferratin, Myoglobin GOx, Lipases (RML & TLL)

8.0 nm 4.8 nm 8.9 nm 6.0 nm

MOF cage inclusion

Polyion complex / Liquid proteins

Covalent modification of enzyme?

-

Diamine cationisation

Anionic PEO surfactant

151, 157 160 11-13,

~100%

100% at up to 150°C

161-162


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Perspectives and outlook While stabilization of enzymes on surfaces or in matrices is awell-established technique, the more controlled wrapping of individual enzymes is an emerging field. In all such single enzyme nanoparticles the polymer is tightly packed around the enzyme isolating the enzyme from the environment. Although we have described a range of different approaches -SENs, excipients, liquid proteins, PICs - the outcomes are often similar as the enzyme is surrounded by a more or less thin layer of polymer that regulates the movement of the enzyme and the diffusion of the substrate through the material barrier. The difference between these techniques lies in the contact point between polymer and enzyme, which can vary from covalent bonds, to electrostatic interactions and hydrogen bonding. So far, the improvement in thermal and solvent stability achieved by wrapping up enzymes is impressive, highlighting the importance of having a flexible but thin layer molded around the enzyme. Typically a trade-off is observed – thicker layers result in more stable enzymes, but with a greater loss in activity relative to the native enzyme. SENs have successfully been prepared using a range of common enzymes such as catalase, glucose oxidaseand lipase, which are often already reasonably stable by nature. This proof of concept opens the door to the preparation of SENs of less stable enzymes. At this point in time it remains unclear if all the techniques – ranging from PIC micelles to in-situ polymerization - are equally suitable in preserving the structural integrity of fragile enzymes. It is also interesting to note that most investigations focus on the method of wrapping, more than on the chemical constitution of the ―wrapping paper‖, which may be either be a thin polymer or inorganic layer. It is conceivable that the polymer itself will have substantial influences on the stability of the enzyme as various functional groups will affect polarity, thus polymer hydration, as well as the strength of binding between polymer and proteins. A good example is the replacement of simple hydrophilic groups by trehalose, which has been found to substantially enhance stability in a number of cases. Systematic studies with building blocks displaying different non-covalent interactions optimized for each enzyme can potentially result in a tailor-made layer around the enzyme maximizing the enzyme stability. The central question lies in finding whichtypes of intermolecular forces or covalent bonds are best at enabling this and this will involve exploring a much broader range of polymer chemistry than has been investigated to date.



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The advantage of SENs lies in the unique ability to tailor the design of the material environment around each enzyme and provide not just high activity and good stability, but also both tunable and switchable activity. Because the diffusion of substrate is governed by the nature of the protecting layer only and not by the contact between two enzymes, substrate diffusion can be tunedby adjusting the layer thickness and porosity. One unexplored aspect of this is the interaction of the substrate with the material layer. Diffusion of the substrate to the enzyme has only been discussed in terms of physical barriers such as thickness and mesh size, but as in membrane science, the chemical composition of the material layer will play a role in determining the rate of diffusion. This also opens up new opportunities to create switchable enzymes by encapsulating them with switchable polymers, responsive to an external stimulus. This has been shown recently with polymersome encapsulated enzymes,84 but is yet to be tested on SENs. As the size of SENs is significantly smaller than that of polymersomes and the materials is in direct contact with the enzyme, it is conceivable that the on-off switching process could be faster and more robust. We believe the design of SENs to be an emerging field which offers ample opportunities to customize enzymes towards the given application. Reactions at high temperatures and in organic solvents can be made possible as the tight fitting material layer can stabilize the three-dimensional conformation of the enzyme while also altering the overall solubility, which is now determined by the nature of the outer layer. This layer can now be tuned to accommodate the special setting in which the enzyme will be used as it might be necessary to control the flow of the substrate and turn the enzyme on and off in order to regulate the rate of enzymatic reaction.

Acknowledgement RC and MHS would like to thank the Australian Research Council (ARC) for funding (DP170101191 and DE170100315) Authors e-mail: [email protected](Robert Chapman), [email protected] (Martina Stenzel)


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All wrapped up: Stabilisation of enzymes within single enzyme nanoparticles Robert Chapman, Martina H Stenzel


Stabilization of enzymes within single enzyme nanoparticles

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