Catalase (CAT) is a common enzyme found in nearly all aerobic organisms. It catalyzes the decomposition of hydrogen peroxide to water and oxygen [34]. CAT was used by Balabushevich et al. [35] as a model protein of high molecular weight for encapsulation. Polyelectrolyte microspheres were obtained by alternating adsorption of dextran sulphate and protamine on melamine formaldehyde (MF) particles followed by partial hydrolysis of the MF core. In fact, the main difference between the fabrication of hollow capsules and microspheres prepared by this method employing MF cores is the core hydrolysis step (Figure 2). While the complete hydrolysis of the MF core produces hollow capsules, during the slow partial hydrolysis of MF core under mild conditions, the newly formed and positively charged amino groups interact with polyanionic structures of the first layer of the microcapsule shell resulting in the redistribution of membrane PEs and formation of a homogeneous, weakly cross‐linked, charged gelatinous matrix inside the microspheres [36].
CAT was encapsulated in the microspheres by simple incubation and mixing for sufficient time. The loaded microspheres were collected by centrifugation and washed. Finally, an additional layer of dextran sulphate was added to protect the enzyme adsorbed at the surface of the positive protamine layer. CAT was entrapped in microparticles at high efficiencies (70‐100%), depending on its original concentration in the incubation medium. The specific activity observed was dependent on the amount of protein entrapped in the microspheres. The authors reported that electrostatic and hydrophobic interactions were responsible for the interaction of the protein with the microspheres’ gelatinous core. Therefore, encapsulation and release of proteins from the proposed LbL system can be controlled via adjustment of pH, ionic strength, and temperature of the incubation medium [35].
Figure 2: Scheme of production of microspheres and hollow microcapsules using commercially available melamine formaldehyde particles and alternating adsorption of polyelectrolytes.35 “Source: Biochemistry (Mosc) 69, (7), 2004, 763‐9, Encapsulation of catalase in polyelectrolyte microspheres composed of melamine formaldehyde, dextran sulfate, and protamine, Balabushevich, N. G.; Zimina, E. P.; Larionova, N. I, Figure 1, © 2004 MAIK “Nauka/Interperiodica”. Reproduced with kind permission from Springer Science and Business Media.”
A different approach was adopted by Caruso et al. [37], where CAT crystals themselves were used as templates to deposit alternating layers of PSS/PAH to form the polymer capsule encapsulating the enzyme in the core at very high efficiency. The method takes advantage of the fact that the enzyme is present as a crystalline suspension in water at pH 5‐6 and may therefore be treated as a colloidal particle. It should be noted that enzyme crystal templating, however, presents several
challenges that do not apply when templating, e.g., latex particles. First, the crystals are formed only under strictly defined conditions. Therefore, suitable conditions that facilitate polymer multilayer deposition on the crystal surface and do not destroy the enzyme crystal morphology (i.e.
to avoid its solubilization) need to be determined. Second, the permeability of the polymer capsule walls must be such that it permits encapsulation of the enzyme. In addition, since the primary usefulness of enzymes is their biological function, their activity must be preserved during encapsulation.
Practically, CAT crystals were separated from solubilized protein by washing and centrifugation several times with potassium acetate buffer of pH 5 at 4 °C. Chilled solutions were used to avoid significant solubilization of the enzyme crystals. CAT crystals exhibited a positive surface charge in water at pH 5 (+20 mV), as determined by electrophoretic mobility measurements. This positive charge at the surface of the crystals in principle makes them suitably charged templates for the deposition of polyelectrolyte layers of PSS and PAH. The successful deposition was proven by the reversal of the surface charge after each cycle of deposition, which is a characteristic of polyelectrolyte multilayer growth on colloidal templates [37].
To investigate the effect of the encapsulation process on the activity of CAT, the activity was measured after solubilization of CAT (by changing pH) and release from the polymer capsules. A recovered specific activity of 97% was obtained, compared with 100% for the uncoated CAT. This shows that the polymer multilayer coating of the CAT crystals proceeded without causing any significant loss of enzyme activity. Another important point was the ability of the capsule wall to protect the enzyme against proteolytic activity. As shown in Figure 3, solubilized, uncoated CAT (curves d and e) was inactivated by protease to more than 90% during an incubation time of 100 min. In contrast, no measurable loss in enzyme activity was observed for the polymer‐encapsulated (solubilized) CAT within 100 min under the same conditions (curves b and c). These results clearly demonstrate that a thin polymer coating of four layers (thickness of about 8 nm) is sufficient to prevent proteolysis of polymer encapsulated CAT [37]. These findings are consistent with the observation that proteins of molecular sizes greater than approximately 5 nm do not penetrate polyelectrolyte multilayer films [38]. Compared to traditional LbL methods, where solubilized charged enzyme molecules are deposited among the layers, encapsulated enzyme crystals display an up to 50‐fold increased biocatalytic activity, thus making them attractive candidates for various biotechnological applications [39].
Figure 3: Stability of (a, d, e) solution‐solubilized catalase and (b, c) polymer‐multilayer encapsulated (solubilized) catalase with respect to proteolysis: (a) solution‐solubilized catalase crystals, no protease incubation (control); (b) [(PSS/PAH)2]‐coated (four layers) catalase, protease incubation; (c) [(PSS/PAH)4]‐
coated (eight layers) catalase, protease incubation; (d) and (e) repeat experiments for solubilized catalase, protease incubation. Proteolysis of the catalase was determined by measuring the decrease in the catalase enzyme activity [37] “Reprinted with permission from reference 37. Copyright 2000 American Chemical Society.”
Due to the interesting advantages of encapsulating enzyme crystals, such as high enzyme loading, preserved bioactivity of the encapsulated enzyme, the ability of the semipermeable PE coating to prevent the solubilized enzyme from leakage while simultaneously permitting the diffusion of small (substrate) molecules for enzyme reaction, this idea was taken a step forward by Jin et al. [39], where a mixed approach was adopted. CAT microcrystals were first encapsulated by the alternate adsorption of PSS and PAH on their surface, yielding an extremely high loading of active enzyme in the polyelectrolyte multilayer capsule. Then, multilayer films were constructed on planar surfaces (quartz crystal microbalance (QCM) electrodes or quartz slides) by LbL deposition of the polyelectrolyte‐coated CAT crystals and oppositely charged polyelectrolyte.
Moreover, this mixed approach was adopted by Yu et al. [40], where LbL encapsulated CAT microcrystals were assembled onto gold electrodes by sequential deposition with oppositely charged PEs, utilizing electrostatic interactions to form thin enzyme films for biosensing of H2O2. In
addition to the aforementioned advantages of this design, the authors found that the PE layers encapsulating the enzyme effectively increase the surface charge density of the enzyme microcrystals, rendering them suitably charged components for the construction of biofunctional thin films. The PSS/PAH encapsulated CAT was shown to retain biological as well as electrochemical activity. Direct electron transfer between CAT molecules and the gold electrode was achieved without the aid of any electron mediator. As a H2O2 biosensor, films consisting of one layer of the encapsulated CAT displayed considerably higher (~5‐fold) and more stable electrocatalytic responses to the reduction of H2O2 than did corresponding films made of a single layer of non‐
encapsulated CAT or solubilized CAT. An increase in either the number of “precursor” PE layers between the gold electrodes and the CAT microcrystal layers in the film or the number of PE layers encapsulating the CAT microcrystals was found to decrease the electrocatalytic activity of the electrode. At low precursor PE layer numbers (~2) and encapsulating PE layers (~4), the current response was proportional to the H2O2 concentration in the range of 3.0 x 10‐6 to 1.0 x 10‐2 M. The overall electroactivity of the multilayer film increased for the first two layers of encapsulated CAT, after which a plateau was observed. This was attributed to the increasing difficulty of electron transfer and substrate diffusion limitations. Using immobilized PE‐encapsulated enzyme microcrystals for biosensing was shown to provide a versatile method to prepare films of high concentrations and tailored activities of enzymes.
More recently, another novel and versatile approach for the preparation of multilayers was introduced, where CAT was first encapsulated in small gold nanoparticles (CAT‐AuNPs), and then electrostatically assembled with anionic and cationic PEs on colloidal silica particles [41]. CAT‐AuNPs were synthesized directly from CAT stabilized gold suspensions and the diameter of the obtained particles was about 9 ± 3.5 nm. Since the pI of CAT is 5.6 [37], CAT‐AuNPs were positively charged at pH 3 and were assembled with the anionic polymer PSS. However, when the pH of the CAT‐AuNPs solution was changed from 3 to 9, charge reversal took place and the anionic CAT‐AuNPs were bound electrostatically to cationic PAH. As shown in Figure 4, an interesting feature of CAT‐AuNPs is that the pH dependent electrostatic properties of CAT‐AuNPs can control the structure of the hybrid nanocomposite, transforming them from well dispersed small nanoparticles at pH9 (due to repulsion forces between the similarly charged particles) to agglomerated colloidal particles with a diameter of about 50 nm or network‐structured composites, depending on the initial concentration of gold precursor [41].
Figure 4: Schematic and TEM images for the preparation of CAT‐AuNP with (a) dispersed, (b) colloidal and (c) network structures [41]. "Source: S. Kim, J. Park, J. Cho, Layer‐by‐layer assembled multilayers using catalase‐
encapsulated gold nanoparticles. Nanotechnology 21(37) (2010): 375702. (doi:10.1088/0957‐
4484/21/37/375702) © IOP Publishing. Reproduced by permission of IOP Publishing. All rights reserved”
In addition, this structural transformation had a significant effect on the surface morphology of CAT‐Au nanocomposite becoming rougher with fibrillary structures, especially in case of network‐
structured CAT‐AuNP. It was found that the total adsorbed amount of (PE/CAT‐AuNP network)5 multilayers was about 5.7 times higher than for (PAH/CAT)5 multilayers at the same solution concentration. Consequently, the higher CAT adsorption led to higher catalytic activity toward H2O2.Considering using these CAT‐AuNPs to coat an electrode surface, the rugged and fibrillary structure of PE/CAT‐AuNP colloids and PE/CAT‐AuNP network multilayers has an increased surface
area, and as a result, increases the area of contact between the probe molecules and the CAT as well as the effective electron transfer rate. Therefore, this structural morphology may assist in increasing electrochemical sensitivity, which may be beneficial to a variety of biocatalytic applications [41].