NANO LETTERS
Single-Enzyme Nanoparticles Armored by a Nanometer-Scale Organic/Inorganic Network
2003 Vol. 3, No. 9 1219-1222
Jungbae Kim* and Jay W. Grate* Pacific Northwest National Laboratory, 902 Battelle BlVd., P.O. Box 999, Richland, Washington 99352 Received June 13, 2003; Revised Manuscript Received July 26, 2003
ABSTRACT We have developed armored single-enzyme nanoparticles (SENs) that surround each enzyme molecule with a porous composite organic/ inorganic network of less than a few nanometers thick. This approach has significantly stabilized two proteases (r-chymotrypsin, CT, and trypsin, TR), and the armor network around CT is sufficiently thin and porous that it does not place a large mass-transfer limitation on the substrate. These new hybrid enzyme nanostructures offer great potential as a method to stabilize enzymes for various applications.
The specificity of enzymes promises great improvements in various applications such as chemical conversions, biosensing, and bioremediation.1-4 However, the short catalytic lifetimes of enzymes presently limit their usefulness.5 Several approaches have been taken to improve catalytic stability of enzymes: enzyme immobilization, enzyme modification, genetic modification, and medium engineering.6-10 As an innovative way of enzyme stabilization, we have developed a unique form of enzyme-polymer composite of nanometer scale, which we call “single-enzyme nanoparticles (SENs)”. Each enzyme molecule is modified with a porous organic/ inorganic structure (or “armor”) of less than a few nanometers thick. This approach represents a new way to modify and stabilize enzymes, and a new type of nanostructure as well. In experiments with a protease (R-chymotrypsin, CT), the catalytic activity of the armored SEN form of the enzyme was greatly stabilized compared to free CT without imposing a serious mass transfer limitation of substrate. The synthesis of SENs involves enzyme surface modification, vinyl polymer growth from the enzyme surface, and finally an orthogonal polymerization by silanol condensation reactions that cross-link the attached polymer chains (Figure 1) into a network around the enzyme.11 High stability is achieved only after the second orthogonal polymerization step (Figure 2). The reactions are carried out so that crosslinking is largely confined to individual enzyme surfaces, yielding discrete nanoparticles rather than bulk solids (Figure 3). SENs containing CT (SEN-CT) were synthesized by first reacting surface amino groups with acryloyl chloride to yield * Corresponding authors. Jungbae Kim: Phone (509) 376-4621, Fax (509) 376-5106, Email:
[email protected]. Jay W. Grate: Phone (509) 376-4242, Fax (509) 376-5106, Email:
[email protected]. 10.1021/nl034404b CCC: $25.00 Published on Web 08/13/2003
© 2003 American Chemical Society
Figure 1. Schematic (a) and chemistry (b) for the synthesis of single-enzyme nanoparticles.
surface vinyl groups.11 The modified enzyme was solubilized in hexane and mixed with methacryloxypropyltrimethoxysilane (MAPS), a vinyl monomer with pendant trimethoxysilane groups. After free-radical initiated vinyl polymerization, the products were extracted into cold aqueous buffer solution, aged at 4 °C at least 3 days for silanol condensation, filtered through a syringe filter with a 100 nm pore size, and washed on a membrane with a 10,000 Dalton molecular weight cutoff. The yields of enzyme activity in the form of SENs were 38-73%.11
Figure 2. Stability of SEN-CT after aging (9), SEN-CT before aging (b), and free CT (4). Aging represents the completion of second silicate polymerization, consisting of hydrolysis and crosslinking of pendent trimethoxysilane groups.
High resolution transmission electron microscopy (TEM) images confirmed the presence of individual nanoparticles with a contrasting outer structure (Figure 3a). The seemingly hollow centers of the nanoparticles, which result from the transparency of the core protein to the electron beam, are 4 to 8 nm across, which matches the size and shape of CT (4 × 3 × 8 nm). The dark image surrounding the transparent core is the composite cross-linked polymer; the presence of silicon was confirmed by energy-dispersive X-ray analysis in the TEM instrument.
The thickness of network can be controlled by changing the synthetic parameters during the vinyl group polymerization. For example, the replacement of MAPS with the silicate monomer containing less reactive vinyl group such as VTMS (vinyltrimethoxysilane) resulted in a thinner network (Figure 3b) since the length of linear polymer from the vinyl group polymerization must be shortened. On the other hand, the addition of cross-linker (TMA, trimethylpropane trimethacrylate) during the vinyl group polymerization resulted in a thicker and denser network, which started to cover the hollow images of CT molecules under TEM observation (Figure 3c). Aggregates of the nanoparticles made with the vinyl cross-linker have also been observed (Figure 3d). In some images, we have found nanoparticles with sizes corresponding to two protein molecules or two nanoparticles end to end. The CT enzymes armored as SENs exhibited impressive catalytic stability compared to free CT. They did not show any decrease in CT activity in a buffer solution at 30 °C for 4 days while free CT is inactivated very rapidly by autolysis under the same conditions (Figure 2). In an extended experiment, SEN-CT started to show about 5-10% decrease of CT activity after ten-day incubation at 30 °C, indicating that the half-life of CT activity in the form of SENs could reach up to 143 days. The storage stability of the SEN-CT in buffer solution was also impressive. A sample of this solution showed negligible decrease in CT activity over five months when stored in the refrigerator (4 °C). As an extension, we have also synthesized SEN-TR (single-enzyme nanoparticles containing TR). The stability of TR activity in the form of SEN-TR was also increased significantly, as shown in Figure 4. The catalytic stability of protease CT in the form of SENCT indicates that the activity is not lost by denaturation within the nanostructure, nor is the activity lost by denatur-
Figure 3. TEM images of SEN-CT. (a) SEN-CT, synthesized via the standard protocol with MAPS.11 (b) SEN-CT with thin coating could be synthesized by performing the vinyl group polymerization with less reactive VTMS (vinyltrimethoxysilane) instead of MAPS (methacryloxypropyltrimethoxysilane). (c) SEN-CT with thick coating could be prepared by adding the cross-linker (TMA, trimethylpropane trimethacrylate) during the vinyl group polymerization. (d) Aggregate of SEN-CT prepared using TMA as observed on the TEM grid. The scale bars represent 20 nm except in d (100 nm). 1220
Nano Lett., Vol. 3, No. 9, 2003
Figure 4. Stability of SEN-TR (O) and free TR (4). SEN-TR was synthesized by the standard protocol for SEN-CT.11 Table 1. Activity of Free CT and SEN-CTa sample
kcat (s-1)
Km (µM)
kcat/Km (× 105 M-1 s-1)
free CT SEN-CT
29.9 ( 0.7 13.8 ( 0.6
38.9 ( 2.7 40.2 ( 4.6
7.70 ( 0.06 3.44 ( 0.04
a The CT activity was determined by the hydrolysis of TP (1.6-160 µM) in an aqueous buffer (10 mM phosphate, pH 7.8) at room temperature (22 °C). Kinetic constants were obtained by using software (Enzyme Kinetics Pro from ChemSW, Farifield, CA) that performs nonlinear regression based on the least-square method. The active site concentrations were determined by the MUTMAC assay19,20 while BCA assay (Pierce, Rockford, IL) was used to determine the protein content in the samples.21
ation followed by proteolysis. By contrast, free CT undergoes autolysis by a mechanism entailing rate-limiting denaturation followed by irreversible proteolysis of the denatured form.12 Stabilization of the SEN-CT against denaturation likely results from multiple covalent attachment points within the nanostructure.13-14 The catalytic stability noted above and detailed kinetic constants for SEN-CT and free CT were measured by the hydrolysis of N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide (TP) in a buffer solution (10 mM phosphate buffer, pH 7.8). Kinetic constants (kcat, Km, and kcat/Km) were obtained via nonlinear regression based on the least-square method (Table 1). The catalytic efficiency (kcat/Km) of CT in SEN form was decreased by about half, and it was due to reduced kcat. The reduced kcat can be interpreted as the reduced enzyme flexibility for catalysis, due to the multipoint attachment between each CT molecule and the armor network.15 However, the apparent binding constant (Km) of SEN-CT was almost the same as that of free CT. This suggests that the nanoscale “armor” did not cause a large mass-transfer limitation for the substrate (TP).16 Kim et al.17 have produced biocatalytic silicates by a seemingly similar approach using two orthogonal polymerNano Lett., Vol. 3, No. 9, 2003
ization steps. However, the final products were bulky solid composites of micrometer scale (20-250 µm) that were collected on a glass filter unit with maximal pore size 1015 µm. Moreover, the catalytic efficiency (kcat/Km) of the enzyme in these bulk composites was lowered by a factor of 10, indicating a serious limitation on mass-transfer of the substrate through the solid matrix.17 Similarly, biocatalytic plastics isolated as micrometer scale solids are subject to significant mass transfer limitations in an aqueous buffer.18 In general, mass-transfer effects are a fundamental issue in enzyme immobilization methods.16 The enzyme-polymer composites of single-enzyme scale significantly reduced the mass-transfer limitation compared to these other enzyme stabilization methods. We have further demonstrated that SEN-CT can be immobilized in mesoporous silica with an average pore size of 29 nm, and that this provides a much more stable immobilized enzyme system than the native enzyme immobilized by either adsorption or covalent bonding in the same material. For example, when incubated at 22 °C and 250 rpm, the half-life of CT adsorbed in mesoporous silica was 12 h, and the half-life of CT covalently bound to the mesoporous silica was 3.3 days. By contrast, over the 12day experiment, the activity of the SEN-CT in mesoporous silica declined by only a few %. Thus, even if the enzyme is ultimately intended to be immobilized on a micron or larger scale substrate, protection as a single-enzyme nanoparticle can be advantageous for stability. Furthermore, we found that SEN-CT immobilized in mesoporous silica was more stable than free SEN-CT. This hierarchical approach to immobilized enzyme architectures separates the stabilization approach from the immobilization step, so that the larger scale immobilization can then be designed with emphasis on surface area, porosity, and other factors. In summary, we have developed a new form of stabilized enzymes using CT and TR as the initial examples. Individual enzyme molecules are stabilized within a hybrid organic/ inorganic network of nanometer scale thickness. The structure is sufficiently porous to allow substrates as large as (at least) a tetrapeptide to access the active site. A key result is that stabilization of the activity was achieved with minimal substrate mass-transfer limitation compared to enzymes entrapped in larger scale materials. The development of stabilized enzymes as soluble individual enzyme particles also provides the opportunity to further process them into other forms. The SENs can be deposited as films, immobilized on solid supports, or potentially linked with other nanoparticles or molecules as part of multifunctional nanoassemblies. We are optimistic that other enzymes that are compatible with the described synthetic procedure, or with other syntheses designed to modify individual enzymes with networked surface structures, may also be stabilized as singleenzyme nanoparticles. This study has demonstrated the desirable characteristics of armored single-enzyme nanoparticles, namely stabilization of the activity, reduced mass transfer limitation relative to larger scale immobilizations, 1221
and processability into additional forms including hierarchical architectures. Acknowledgment. We thank A. Dohnalkova and C. Wang for the electron microscopy. This work was supported by Battelle Memorial Institute, U.S. Department of Energy (DOE) LDRD funds administered by the Pacific Northwest National Laboratory, and the DOE Office of Biological and Environmental Research under the Environmental Management Science Program. The research was performed at the W. R. Wiley Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the U.S. Department of Energy’s Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory. PNNL is operated for the Department of Energy by Battelle. References (1) Koeller, K. M.; Wong, C.-H. Nature 2001, 409, 232-240. (2) Schmid, A.; Dordick, J. S.; Hauer, B.; Kiener, A.; Wubbolts, M.; Witholts, B. Nature 2001, 409, 258-268. (3) Guilbault, G. Analytical Uses of Immobilized Enzymes; Marcel Dekker: New York, 1984. (4) Dura´n, N.; Esposito, E. Appl. Catal., B 2000, 28, 83-99. (5) Burton, S. G.; Cowan, D. A.; Woodley, J. M. Nature Biotechnol. 2002, 20, 37-45. (6) Tischer, W.; Wedekind, F. In Biocatalysis - from DiscoVery to Application; Fessner, W. D., Ed.; Springer-Verlag: Berlin, 1999; Vol. 200, pp 95-126. (7) Livage, J.; Coradin, T.; Roux, C. J. Phys.: Condes. Matter 2001, 13, R673-R691. (8) DeSantis, G.; Jones, J. B. Curr. Opin. Biotechnol. 1999, 10, 324330. (9) Govardhan, C. P. Curr. Opin. Biotechnol. 1999, 10, 331-335.
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(10) Mozhaev, V. V. Trends Biotechnol. 1993, 11, 88-95. (11) 3 mg of CT, acryloylated and solubilized in 1.5 mL hexane, was mixed with MAPS (297 µL) and 2,2′-azobis-(2,4-dimethylvaleronitrile). UV light (365 nm) initiated vinyl polymerization. After overnight vinyl polymerization, the hexane was repeatedly extracted with phosphate buffer (200 mM phosphate buffer, pH 8.0) and the combined extracts were aged in the refrigerator. The solution was filtered, and the product was washed as described in the text to remove unreacted reagents and the autolytic products of CT. (12) Jaswal, S. S.; Sohl, J. L.; Davis, J. H.; Agard, D. A. Nature 2002, 415, 343-346. (13) Mozhaev, V. V.; Melik-Nubarov, N. S.; Sergeeva, M. V.; Siksnis, V.; Martinek, K. Biocatalysis 1990, 3, 179-187. (14) Wang, P.; Dai, S.; Waezsada, S. D.; Tsao, A. Y.; Davison, B. H. Biotechnol. Bioeng. 2001, 74, 249-255. (15) Eldin, M. S. M.; Schoe¨n, C. G. P. H.; Janssen, A. E. M.; Mita, D. G.; Tramper, J. J. Mol. Catal., B 2000, 10, 445-451. (16) Biochemical Engineering; Blanch, H. W., Clark, D. S., Eds.; Marcel Dekker: New York, 1997. (17) Kim, J.; Delio, R.; Dordick, J. S. Biotechnol. Prog. 2002, 18, 551555. (18) Wang, P.; Sergeeva, M. V.; Lim, L.; Dordick, J. S. Nature Biotechnol. 1997, 15, 789-793. (19) Gabel, D. FEBS Lett. 1974, 49, 280-281. (20) 100 µL of free CT or SEN-CT solution in various concentrations (10-1000 µg/mL) was mixed with 2 mL of 25 µg/mL MUTMAC solution (0.1 M sodium phosphate, pH 7.8). The fluorescence emission at 450 nm (excitation at 360 nm) was measured using PTI fluorescence system (Lawrenceville, NJ) after the emission intensity reached the plateau. Solution of 4-methylumbelliferone was used as the standard. Tests with the out-of-bottle CT powder showed that 52.4 wt % was active by the active site titration. On the other hand, SEN-CT resulted in 41.2 wt % active CT, indicating that about 20% of the initially active CT was inactivated during the synthesis. (21) Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C. Anal. Biochem. 1985, 150, 76-85.
NL034404B
Nano Lett., Vol. 3, No. 9, 2003
