Nanomaterials as Matrices for Enzyme Immobilization

Abstract

Nanomaterials constitute novel and interesting matrices for enzyme immobilization. While their high surface to volume ratio is an obvious advantage, their Brownian motion can impact the behavior of enzymes immobilized on these matrices. Carbon nanotubes, superparamagnetic nanoparticles, and mesoporous materials constitute some important classes of matrices. Such immobilized enzyme systems have been used in both aqueous and low water media for biocatalysis and resolution of racemates. This overview examines the behavior of enzymes immobilized on nanomaterials and discusses the results reported with such biocatalyst preparations.

Keywords::

• superparamagnetic nanoparticles
• carbon nanotubes
• enzyme immobilization
• enantioselective biocatalysis
• enzyme catalysis in low water media
• quantum dots
• mesoporous materials
• protein refolding

Enzymes as biocatalysts have always been considered superior to chemical catalysts. The high catalytic efficiency coupled with chemoselectivity, regioselectivity and stereoselectivity are rightly perceived as highly desirable properties for applications at the industrial level. The following relatively recent developments have further added to their potential as catalysts useful in biotechnological processes. The use of different reaction media is possible with enzymes. Examples of such media are nearly anhydrous organic solvents, aqueous-organic cosolvent mixtures, aqueous-organic two phase systems, reverse micelles, ionic liquids, gaseous state and solid phase [1–8].

• Metagenomics promises that if one preserves enough, we are likely to get an efficient enzyme for a chosen reaction [9,10].

• Recombinant DNA-based technologies like site directed mutagenesis and directed evolution make it possible to innovate as far as substrate specificity, catalytic efficiency and even stability towards harsher environments are concerned for a given enzyme.

Biological promiscuity has been revealed as a new, valuable property of enzymes. Using the same active site, the enzyme is able to also catalyze unexpected reactions. For example, lipases have been shown to catalyze a host of C-C bond formation reactions [11,12].

Added to the ever-increasing usefulness of these biocatalysts is the fact that these are considered to be an important factor in designing “greener” alternatives to existing practice in chemical industries. Hence the new words like white biotechnology and green biotechnology have been coined to highlight this trend in biotechnological practices.

Immobilized enzymes have been the preferred form in which these biocatalysts are used in bioreactor designs for bioconversions and biotransformations [13,14]. Generally, micron-size particles have been most frequently used as carriers or matrices for enzyme immobilization. A matter of concern has been that the volume occupied by the carriers/ matrices is far more than needed for the enzyme molecules. Desirability of reducing bioreactor sizes was the one important motivation for the early work on enzyme aggregates (where no carriers are used) [15]. A more recent version of such enzyme aggregates is crosslinked enzyme aggregates (CLEA) [16–18]. With the advent of nanotechnology and emphasis on miniaturization, it was natural that nanomaterials captured the attention of the people working with enzyme immobilization. One reason that has been consistently given is that nanoparticles offer high volume/ size ratio. The implication is that use of nanomaterials will help in reducing the size of bioreactors. This is because it should be possible to load higher amount of biocatalyst per unit weight of the carrier.

It is sometime mistakenly believed that recombinant DNA-based technologies have reduced the importance of enzyme immobilization. On the other hand, all the developments listed above in the beginning of this review have enlarged the scope of the technique and created increased opportunities and challenges in the design of immobilized biocatalysts. Use of nanomaterials as matrices is yet another dimension. The present article is aimed at reviewing the growing interfaces created by cross-fertilization of several areas with a focus on enzymes immobilized on nanomaterials.

ADVANTAGES OF ENZYME IMMOBILIZATION

First of all, it is pertinent to briefly recall the reasons why immobilized enzyme in general is the preferred form for using these biocatalysts.

Stabilization

The stability of the correctly folded enzymes (as compared to unfolded structure) is in the range of ΔΔG of 5–20 Kcal/mol [19]. This is the reason for the correctly perceived notion that enzymes are more fragile catalysts as compared to chemical catalysts. In the pre-recombinant DNA era, adding stabilizers, chemical modification [19–22] and immobilization on solid supports were the only techniques available for further stabilization. Contrary to popular notion, immobilization does not always lead to significant stabilization [23]. It should be added that both storage stability and operational stability can be achieved by immobilization [14].

Recovery and Reusability

Economic considerations make it desirable to use the enzyme repeatedly in batch reactor cycles or for a longer time period if the reactor (with appropriate design) is run continuously. This virtue is operational stability and reusability originates from that. Recovery after use may be distinguished from reusability and refers to the ease with which the immobilized enzymes can be removed/separated from the reaction components for reuse! Appropriate protocol before reuse or cleaning-in-place operations has to be thoughtfully designed to achieve optimum reusability. When solid supports are used, centrifugation or membranes are obvious choices. Use of smart carriers allows one to develop stimuli-sensitive immobilized enzymes, which can function as homogeneous catalysts but separate out conveniently from the reaction mixture just like heterogeneous catalysts [24–27]. Use of magnetic carriers allows easy separation for enzymes immobilized on solid supports by use of magnetic fields [28–31]. It is here that magnetic nanocarriers have proved an especially interesting and valuable option. This will be discussed later on in more detail.

Flexibility of Bioreactor Design

Immobilization of enzymes allows one to choose among batch, packed bed, or fluidized bed column reactors [32].

Porous micron-sized particles (like those of agarose, dextran, cellulose, or their composite materials) have been most frequently used carriers for immobilization [13,14]. Very early it was realized that the internal surface of the beads in such cases provided much larger surface area than the outside surface. As the dimensions of the enzymes/proteins are of nanodimensions and much smaller than the pores of such beads, a larger amount of enzyme invariably was immobilized inside the beads. This refers to the situation when adsorption or covalent coupling is used for the immobilization. In the case of entrapment/encapsulation, the enzyme molecules are inside the beads. The problem may not be so acute when low molecular weight substrates are used. However, macromolecular substrates like polysaccharides, proteins, and nucleic acids as substrates do not work well; these big molecules can not freely access the enzyme molecules in the interior of the bead. In kinetics of immobilized enzymes including determination of apparent K_m and V_max in such cases, assessment of these mass transfer constraints is fairly well understood and has been dealt with adequately at a number of places [33,34].

In low water media, the situation is somewhat different and often misunderstood. The earliest (and still most commonly preferred by organic chemists) form in which enzymes were used was lyophilized powders. In low water media, these suspended particles have a mass transfer constraint as these are not freely dispersed in the media. One of the popular ways is to “immobilize” these on celite. As pointed out by Halling [35], this is more of a case of celite working as an additive and helping in “spreading out” the enzyme molecules.

No matter what the mechanism, in this case (unlike lyophilized powder, which acts more like enzyme aggregates) the catalytic surface area is much larger and higher initial rates are observed. It is very common to state that the immobilization has stabilized the enzyme against denaturation because of the organic solvent (in case the low water medium is the “nearly anhydrous organic solvents”). It is unfortunately difficult to settle the issue. It looks more likely that the so-called “stabilization” is just the reduction of mass transfer constraint.

ADVANTAGES OF NANOSIZED CARRIERS

So why the increasing interest in the use of nanosized matrices in recent years?

• All the advantages of immobilized enzymes on micron-sized particles are inherited when nanomaterials are used as solid supports. Also, just as adsorption, covalent coupling, entrapment and encapsulation have been used with solid supports of conventional sizes [13–14, 36], similar strategies are all useful for immobilization on nanomaterials as well. Some important new consequences arise when the size of the carrier approaches nanodimensions. Mostly, these all work out in the favor of using nanosized materials.

• Large surface area: volume ratio:- This is the most obvious one and has been referred to earlier in this review. As pointed out by Wang [37], for the same diameter, nanofibers offer two-third of the surface: volume ratio over (nearly spherical) particles. However, these are easier to prepare, easier to handle, and offer larger flexibility in rector design.

• Mass transfer constraints:- Both porous and nonporous nanocarriers are known. As the virtue of larger surface area (in the interior) for the porous micron-sized particles is taken care of by large surface: volume ratio for nanosized particles, nonporous nanosized materials offer very attractive loading capacity for the enzymes/proteins. When enzymes/proteins are immobilized on a surface (by adsorption or covalent binding), the surviving biological activity of the protein depends upon how “crowded” the surface is. Initially, the value of their activity (sometimes called immobilization efficiency) increases as number of enzyme molecules are added on a given surface (this is because if there are few enzyme molecules on a large surface, thermodynamic considerations lead to enzyme molecules making attempts to maximize contact and thereby undergo conformational distortion). After reaching an optimum value, the activity starts decreasing as enzyme density is increased further (this is because as the surface become crowded with too many protein molecules; there is again conformational deactivation) [38]. Thus, it is very helpful to have a large surface area for maximizing enzyme loading. Non-porous nanomaterials allow high loading with very little mass transfer limitation.

• Mobility:- When enzymes are immobilized on micron-sized carriers, shaking or stirring is required in a batch reactor. However, some very novel findings from the research group of Prof. Wang suggest [37] that well-dispersed nanoparticles show Brownian motion. According to the Stokes-Einstein equation, the mobility and diffusitivity of the nanoparticles have to be smaller than those of free enzymes, owing to their relatively larger size. “This mobility difference may point to a transitional region between the homogeneous catalysis with free enzyme and heterogeneous catalysis with immobilized enzyme” [37].

It has been shown that, as predicted by Stokes-Einstein equation and collision theory, this Brownian motion occurs and may be responsible for high activities obtained when enzymes are immobilized on nanoparticles. Furthermore, “Interestingly, it was found that the reaction catalyzed by enzymes can also derive the motion of nanoparticles (i.e. improve their mobility)” [37].

Let us now consider specific kinds of nanomaterials that generate some additional features because of their special properties.

Superparamagnetic Nanoparticles

Nanoparticles as such have two disadvantages when used as carriers of enzymes. Firstly, these particles often form clumps and sonication with ultrasonic waves has to be carried out for temporary dispersion. Secondly, because their size, separation by either centrifugation (ultracentrifugation is generally required) or membranes (nanofiltration is costly) is often not easy. Superparamagnetism solves both of these problems. Superparamagnetism consists of a material becoming magnetic only in the presence of a magnetic field. Such particles disperse easily in solution and can be recovered by use of a simple magnet. Luckily, magnetic particles less than about 30 nm show superparamagnetism [39]. Magnetite nanoparticles (Fe₃O₄) have been most extensively used as superparamagnetic supports [40,41].

The immobilization of enzymes on Fe₃O₄ particles has mostly followed either of the following approaches (Table 1(a)).

Table 1. shows the immobilization of enzymes on different nanomaterials and their application.

Nature of the particle	Enzyme	Conjugation method	Applications/comments	Reference No.
(a) Superparamagnetic nanoparticles
Fe3O4 particles encapsulated in anhydrous and porous silica coating of 3.5 nm thickness.	Lactamase	Covalent coupling	Easy recovery, 99.5 % loss in enzyme activity.	42
γ-Fe2O3	Candida rugosa lipase	Covalent coupling with two different approaches.	46 % loss in hydrolytic activity of lipases. Improved storage stability.	43
Maghemite (iron oxide)	Candida rugosa lipase	Covalent coupling with 3-aminopropyltriethoxysilane and glutaraldehyde chemistry	High stereoselectivity in kinetic resolution of racemic carboxylates. Improved long-term stability.	44
Iron oxide in “siliceous mesocellular foam”	Pseudomonas cepacia lipase	Noncovalent coupling	Kinetic resolution of (±)-1-phenyl-ethanol. Seven times reusable.	45
Glycidyl methacrylate coated iron oxide nanoparticles	Chloroperoxidase (CPO)	Covalent coupling	Enhancement in the stability of nanobiocatalyst, with no loss of activity after recycling 11 times.	46
Silica coated magnetic nanoparticles	Alpha-chymotrypsin and lipase	Covalent immobilization of crosslinked enzyme clusters	Highly active and stable under harsh shaking for more than 15 days.	47
Magnetite (Mag NP)	Candida antarctica lipase	Immobilization via γ-aminopropyltriethoxysilane (APTS)	Easy recovery, reusable. Highly enantioselective for secondary alcohols.	48
Nature of silica	Enzyme entrapped	Type of immobilization	Comments	Reference No.
(b) Mesoporous Materials
Single enzyme nanoparticles (SENs)	alpha-chymotrypsin and trypsin	Covalent linkage of polymer to the surface of enzymes	Due to thin porous polymer network (1 nm), no mass transfer constraints, high stability and activity in organic solvents.	54
Biomodal mesoporous silica	Several enzymes with different molecular weights like catalase, protease, peroxidase and cytochrome C	Immobilization by adsorption followed by assembling an organic/inorganic nanocomposite shell on the particle surface	High enzymatic activity, stability and protection from proteolysis.	55
Nanoporous silica gel glass	alpha-chymotrypsin	Covalent binding	Half-life of entrapped enzyme was 1000 times higher than that of native alpha-chymotrypsin in organic solvents.	56
Type of carbon nanotube	Enzyme	Conjugation method	Applications/comments	Reference No.
c) Carbon nanotubes
Multi-walled carbon nanotubes	Soybean peroxidase, Subtilisin Carlsberg, Candida antartica lipase B (CALB) and Tyrosinase	Attachment via carbodiimide chemistry	Water soluble carbon nanotube enzyme conjugates. Low mass transfer resistance, high activity, stability and reusability.	60
Multi-walled carbon nanotubes	Candida rugosa lipase	Adsorption	Immobilization led to 2.2 and 14 fold increase in transesterification activity in n-hexane and [Bmim] [PF6] respectively. Also showed high enantioselectivity as determined by kinetic resolution of (±) 1-phenylethanol in [Bmim] [PF6].	61
Multi-walled carbon nanotubes	Xylanase	Adsorption	Refolding of 8 M urea denatured xylanase was done. 55 % activity could be regained with denatured form of purified form of xylanase and 92 % with commercial preparation of xylanase in 8 M urea.	62
Single-walled carbon nanotube	Soybean peroxidase, subtilisin Carlsberg, trypsin, proteinase K, and horseradish peroxidase	Adsorption	Stabilized proteins at elevated temperatures and in organic solvents to a greater extent than conventional flat supports.	63
Multi-walled Carbon nanotubes were solubilized via functionalization with poly(propionylethylenimine-co-ethylenimine).		Immobilization via diimide activated amidation reaction	The method for efficient preparation of solubilized carbon nanotubes was developed.	64
Type of nanoparticle	Enzyme	Conjugation method	Applications/comments	Reference No.
d) Some other less used nanomaterials
Polystyrene nanoparticles	Alpha-chymotrypsin	Covalent attachment	Effect of particle mobility on the catalytic efficiency of enzyme was studied.	69
Modified (by grafting with carboxylic surfactant) zirconia nanoparticles.	Lipases from Candida rugosa and Pseudomonas cepacia	Immobilization of enzymes by adsorption	Both lipases gave significantly higher activity and enantioselectivity compared with crude lipase powders. Also, these nanohybrid could be reused for eight cycles	70
Titanium oxide	Alpha-chymotrypsin, RNase A, Papain, Lysozyme, Subtilisin A, GFPuv, Pseudomonas cepacia lipase	Adsorption	Refolding of heat denatured enzymes carrying positive charges was carried out.	71
Silica nanoparticles	Chicken egg lysozyme	Adsorption	Effect of nanoparticle size on structure and enzymatic activity was studied.	72

1. Coating of the particles with other materials and covalent coupling of the enzymes after surface functionalization of the coated particles has been frequently carried out. Covalent coupling invariably results in some loss of enzyme activity. In such cases, there is trade-off between storage/operational stability and biological activity [42,43]. Some available information about the enzyme should be kept in mind while designing the coupling chemistry. For example, in some cases [43,44,49], Candida rugosa lipase has been linked via its amino groups. It is well known that, in the case of Candida rugosa lipase, amino groups are essential for biological activity [50,51]. Also, if the coating material is porous, some of the enzyme molecules would be immobilized inside this porous coating. Such a situation would lead to the well-known mass transfer limitations [42].

2. Adsorption of the enzyme by noncovalent interactions with or without coating the particles has also been reported. This approach is gentler and enzymes generally retain higher biological activity. Zhang et al. [45] grafted iron oxide molecules to “silicaceous mesocellular foam” and tried adsorbing Pseudomonas cepacia lipase either directly or after hydrophobization with octyl groups (Figure 1). The latter expectedly was found to be a better support. This work demonstrates the usefulness of such biocatalyst preparations for kinetic resolution of racemates. The work of Wang et al. [46] illustrates the usefulness of thick shell around the particles in achieving biocatalysts with high recyclability.

3. Bioaffinity immobilization via fusion tags has also been reported [52]. The fusion tags had specific affinity for either iron oxide or the silica coat on the particles.

4. A promising approach is to place enzyme aggregates inside superparamagnetic nanoparticles [47,53].

Figure 1. Preparation procedure for the magnetically separable biocatalysts. (1) Fe(NO₃)₃·9H₂O, methanol; propionic acid; calcination; (2) octyltrimethoxylsilane, toluene, 120 °C for 24 h; (3) 0.2 g of magnetic foam, 20 mL of 50 mg/mL, 6 h at rt; (4) 0.2 g of hydrophobic magnetic foam, 20 mL of 50 mg/mL, 6 h at rt. (Reproduced from [45] with the permission of Copyright Clearance Center's Rightslink service, Elsevier (http://s100.copyright.com/CustomerAdmin/PLF.jsp?lID=2010051_1274898046362).

Mesoporous Materials (Table 1(b))

We have mentioned that nanosized nonporous particles show surface to volume advantage. However, nanoporous or mesoporous materials as carriers for enzyme immobilization have also generated considerable interest. Mesoporous silica particles in this regard have attracted considerable interest. To understand the special significance of nanosized space around enzyme molecule(s), let us look back at entrapment of enzymes in polyacrylamide gels. Klibanov [57] pointed out that much of this stabilization observed for entrapped enzymes originates in part of the polypeptides non-covalently interacting with the gel molecules. So, what is normally considered entrapment is more like adsorption inside porous materials. A large amount of data on protein stability accumulated in different contexts makes it clear that whatever establishes enough contacts with a polypeptide chain, it prevents the unfolding of the polypeptide. For almost all deactivation mechanisms operating under various denaturing conditions, the starting point is unfolding of the polypeptide chain [19,58]. It is easy to visualize that when the space or pore dimensions around the enzyme molecule(s) are considerably reduced, this confining space leads to multiple contacts between enzyme molecule(s) and the nanopores of the mesoporous materials. Again, Wang [37] has quoted some impressive results on the stabilization of α-chymotrypsin in both mesoporous silica and mesoporous glass. One such impressive stabilization is in non-aqueous solvents when the half-life of the chymotrypsin increased 1000 times. Furthermore, a tantalizing observation is that hard shells around enzymes lead to better stabilization than soft gels. If true, this is going to phase out the long era of soft gels as immobilization matrices. Some other results from Wang's own laboratory outline how a coenzyme NAD(H) on a flexible arm could shuttle between two enzymes placed in pores of 30 nm and 100 nm diameters. Cofactors regeneration was thus possible. The importance of this strategy lies in the fact that cost of coenzymes limits the wider utility of many dehydrogenases in stereoselective synthesis of many valuable compounds [59]. Hence many strategies have been attempted for enzyme regeneration.

Kim and Grate [54] developed “armored single enzyme nanoparticles.” In this approach, essentially a single enzyme molecule was modified by a porous organic/ inorganic “armor” of less than few nanometer thickness (Table 1(b)). The results with two proteases (chymotrypsin and trypsin) showed that considerable stabilization was possible and the thin porous armor did not create “large mass-transfer limitation.” Wang and Caruso [55], on the other hand, encapsulated enzymes in mesoporous silica spheres. The bimodal mesoporous silica (BMS) of 2–4 μm particle size had smaller pores (2–3 nm) and larger pores (10–40 nm). Their results show that these BMS spheres had much higher enzymes-loading capacity as compared to mesoporous materials with only small pores of ≈ 2nm. The BMS sphere was encapsulated within a nanocomposite shell. The latter strategy resulted in prevention of enzyme leakage and protection from proteases.

Carbon Nanotubes as Immobilization Matrices

Both single walled carbon nanotubes (SWNT) and multi-walled carbon nanotubes (MWNT) have been used for immobilization of protein/enzyme (Table 1(c)). Most of the early work pertained to use of SWNT-protein bioconjugates in biosensor design [65–68]. Later work, mostly carried out with MWNT, used these nanomaterials for crafting nanobiocatalysts [60,64]. The solubilization of MWNTs has been achieved by sonication [64]. Asuri et al. [60] described a protocol for obtaining water-soluble MWNT-emzyme conjugates by covalent coupling. Unfortunately, considerable loss in enzyme activity for various enzymes was reported [60]. Shah et al. [61] immobilized Candida rugosa lipase on MWNT by adsorption with retention of 97% of the activity (Figure 2). The immobilized enzyme showed 2.2- and 14-fold increase in initial rates of transesterification activity in nearly anhydrous hexane and water immiscible ionic liquid [Bmim] [PF₆], respectively, as compared to lyophilized powders. It is presumed that the interaction with the hydrophobic surface of the nanotubes led to the opening of the molecular lid and activated the enzyme. The immobilized lipase also showed high enantioselectivity in resolution of (±) 1-phenylethanol in [Bmim] [PF₆].

Figure 2. TEM images of: (a) MWNTs; (b) Candida rugosa lipase CRL absorbed on MWNTs. The diameters of the nanotubes without enzyme were 20 ± 5 nm and with enzyme 30 ± 5 nm (the diameter values represent the average of 10 TEM images in each case). Reproduced from [61] with the permission of BioMed Central (http://creativecommons.org/licenses/by/2.0).

Some Other, Less Used Nanomaterials

There are some other nanomaterials that either happen to draw less attention or show promise but need to be more carefully looked into (Table 1(d)). We had earlier referred to nanofibers. Herricks et al. [73] described the use of polynanofibers produced by electrospinning as carriers of enzymes. Earlier, Jia et al. [69] had also described similar systems. Hutten et al. [74] described superparamagnetic Co and FeCo nanocrystals affinity materials. There is no reason why these should not work well as matrices for enzyme immobilization. Thus, SiO₂ and iron oxides are not the only metal oxides that can be used to form nanoparticles for use as carriers for enzyme immobilization.

Quantum Dot-protein Conjugates

Quantum dots are highly luminescent colloidal semiconductor nanocrystals. These have some extremely useful properties as fluorophores and are being extensively used in-vivo for imaging and in-vitro for bioanalytical purposes [75,76]. In both applications, their utility is as fluorescent labels. These flurophores score over others in having high quantum yield, high stability towards light and chemicals. The emission wavelength can be tuned within range (decided by chemical composition) by changing the size of the quantum dot. Their broad excitation spectra allow “multiplexing” as a single wavelength can excite different quantum dots with different emission characteristics. Core quantum dots used in biology generally consist of CdSe with a few monolayers of ZnS or CdS. Even these “core-shell” quantum dots are hydrophobic. Their surface functionalization is required for formation of stable colloidal solutions in aqueous media and for attaching other molecules. While bioconjugates of quantum dots with proteins have not been used in biocatalysis, a list of illustrative examples of cellular components and proteins labeled with quantum dots can be found in the review by Medintz et al. [75]. An interesting and somewhat relevant example is of a quantum dot surrounded by about 10 maltose binding protein molecules. Data with these system shows that it is possible to self assemble proteins on a quantum dot surface; more important, the protein molecules assume homogenous orientation. Here, quantum dot can function as nanoscaffold for proteins. A FRET nanosensor for maltose could be developed [75]. It is not unlikely that we will see some applications of conjugates of protein/enzymes with quantum dots in biocatalysis in near future.

Nanomaterials as Facilitators of Protein Refolding

Many recombinant proteins overexpressed in E.coli end up forming inactive solid particles called inclusion bodies. A vast literature on recovery of biological activity of such preparations exists. The common steps are their solubilization in denaturant solutions followed by refolding. A large number of chemical compounds and polymers have been described as useful additives that facilitate protein refolding [77,78]. In this respect, it may be interesting to note that Shah and Gupta [62] showed that MWNTs could be used for simultaneous refolding, purification, and immobilization of xylanase from its urea denatured solutions. FTIR spectroscopy showed that α-helical content of xylanase decreased from 17% to 14%, β-sheet content increased from 53% to 61% and β-turns decreased from 20% to 15% upon immobilization. Both refolded xylanase and native xylanase had similar secondary structure content upon immobilization on MWNTs.

Recently, modified gold nanoparticles [79] and simple TiO₂ nanoparticles [71] have been also shown to facilitate refolding of thermally denatured enzymes (Figure 3). Presumably, electrostatic interactions played a strong role in the particles supporting refolding pathway in preference to competing aggregation pathway.

Figure 3. TiO₂ nanoparticles as synthetic chaperones. Thermally denatured basic proteins interacted with TiO₂ nanoparticles. This interaction prevented protein aggregation and facilitated correct refolding of the protein molecules. Reproduced from [71] with the permission of Royal Society of Chemistry, London.

Self-assembly and Bionanofabrication

For quite some time the area of bioanalysis has been moving into two clear directions: 1) high throughput screening mode; 2) to be able to work with small amounts/volumes of analyte samples and reagents. For both purposes, it is vital to be able to deposit a large amount of biological activity on a small surface. The early development in this regard can be traced back to oriented immobilization [80]. The strategy was to be able to keep “active site” of the biocatalyst accessible and away from the matrix. In this regard, the homogeneous orientation of proteins on the quantum dots mentioned earlier is a very valuable observation. The second development was to create self-assembling layers of proteins sandwiched by other polymers (which show adequate binding to the protein). This sort of affinity layering [81,82] means going vertical and resembles the concept of multistoried buildings, which become necessary as the available surface become limited (Figure 4). In recent years, there has been a growing interface between nanomaterials, microelectronics, and biology. Molecular recognition mediated through noncovalent interactions imparts the property of self-assembly to biological macromolecules.

Figure 4. Affinity layering by bioaffinity immobilization. The lectin concanavalin A (Con A) was bound to its affinity matrix Sephadex G-100. Horseradish Peroxidase (HRP) is a glycoenzyme with known affinity for the lectin and hence was immobilized on Con A-sephadex G-100 by bioaffinity immobilization. In subsequent steps (in which Con A acted as the affinity ligand), alternate layers of Con A and HRP were created. This “going vertical” of affinity layering is a promising strategy for depositing large amount of biological activity on a small surface (for further details about this work, please see reference [82]).

Besides, biological affinity (responsible for key biological processes such as biocatalysis, DNA replication, transcription and translation, drug receptor and hormone-receptor interaction) inherently has specificity and spatial precision and because of this biology has a lot to contribute to fabrication.

Wu and Payne [83] have illustrated how biocatalysis can be used for fabrication. In these examples, the basic strategy is to initiate chemical crosslinking, which in one case illustrates the potential of thermobiolithography. More relevant to the topic of the present review, Wu and Payne [83] also describe the use of enzymes such as phospholipase A2, protease and endonucleases, which were used along with an AFM tip.

Kim et al. [84] has recently reviewed self-assembly of nanomaterial linked enzymes on oil-water interfaces. While lipase are well-known examples that function on such interfaces, even with other enzymes, such biphasic and multiphasic systems facilitate biocatalysis [4,85].

A good recent example is provided by Zhu and Wang [86], which consists of using a conjugate of β-galactosidase with polymer for carrying out galactosylation at the organic-water interface.

Sheldon [87] has discussed the microchannel bioreactors. Microchannel reactors (essentially microfluidic devices) show rapid mass and heat transfer and also provide large surface area to volume ratios. The work by Maeda's group forms a membrane consisting of enzyme aggregates (CLEAs) on the inner walls of the microchannels [88,89]. The early work on immobilizing enzyme on carbon nanofiber-coated monolith also appears promising [87].

CONCLUSION

While this review has focused on biocatalysis, nanomaterials have also been used extensively for bioseparation and in designs of biosensors [90,91] as well. The immobilization of an enzyme/ a protein on a nanocarrier is merely one aspect of the broader contexts of: (a) being able to deposit or place large amount of biological activity on a small surface area; (b) creating hybrid assemblies. The former context is very important in the areas of miniaturizing sensors and reactors. For these designs the interface of bionanotechnology with principles of microfluidics is required. As pointed out by Somorjai et al. [92], biochemists need to acquire great understanding of surface chemistry. For example, surface curvature effects needs to be more extensively studied [72]. The issue of hybrid assemblies is relevant for biofabrication [83]. Greater experience with putting diverse structure together is bound to pay rich dividends. While it would be obviously useful to learn from our vast experience with micron-sized materials, we have to have lateral vision so as not to miss out on novel features. The property of Brownian motion and its consequences discussed by Wang [37] and mentioned earlier in this article is one such illustrative example. Connecting enzyme active site to the matrix by nanowires described by Xiao et al. [93] has wider implications. Grunes et al. [94] have discussed the role that nanomaterials as such play in chemical catalysis. So, nanomaterials may not remain just carriers in the future design of nanobiocatalysts. Also, it should be more widely appreciated that the dynamic layer of proteins is going to be adsorbed to any surface introduced into the blood stream [95]. If these materials are of nanodimensions, these can reach subcellular locations. Hence the implications of interactions of protein with nanocarriers go beyond biocatalysis. With the advert of molecular biology, we started tinkering with basic cellular machinery. The fast developing, rich interface of nonbiological nanostructures with biological structures is very tempting in term of promises (like in the area of drug delivery or medical sciences), but needs to be viewed with caution [95]. We patiently need to gain more experience with protein-nanomaterials interactions. Nevertheless, this short review hopefully conveys the fact that with this alliance with nanomaterials, “times are a changing” in biocatalysis (with due apology to Bob Dylan).

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.