Nanotechnology and the Environment - American Chemical Society

Chapter 29

Nano-Biotechnology in Using Enzymes for Environmental Remediation: Single-Enzyme Nanoparticles 1,2

1,3

Jungbae Kim and Jay W. Grate 1

Downloaded by UNIV LAVAL on October 1, 2015 | http://pubs.acs.org Publication Date: December 14, 2004 | doi: 10.1021/bk-2005-0890.ch029

Pacific Northwest National Laboratory, 902 Battelle Boulevard, P.O. Box 999, Richland, WA 99352 [email protected] [email protected] 2

3

We have developed armored single-enzyme nanoparticles (SENs), which dramatically stabilize a protease (α -chymotrypsin, CT) by surrounding each enzyme molecule with a porous composite organic/inorganic shell of less than a few nanometers thick. The armored enzymes show no decrease in CT activity at 30°C for a day while free CT activity is rapidly reduced by orders of magnitude. The armored shell around CT is sufficiently thin and porous that it does not place any serious mass-transfer limitation of substrate. This unique approach will have a great impact in using enzymes in various fields, including environmental remediation.

There is a growing interest in using enzymes for remediation purposes (12). Recent biotechnological advances in enzyme isolation and purification procedures together with recombinant technology have allowed the production of enzymes at less cost (2). The advantages of enzymatic remediation as compared to conventional microbial remediation can be summarized as follows (1) • 220

More harsh operational conditions (contaminant concentration, pH, temperature, and salinity) © 2005 American Chemical Society

In Nanotechnology and the Environment; Karn, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

221 • • • • •


• •

Application to recalcitrant compounds No requirement of nutrients No requirement of biomass acclimation No formation of metabolic by-products Much reduced mass-transfer limitation on contaminants compared to microorganisms Easy-to-control process Effective in small quantity

The organic compounds, generally targeted for enzymatic remediation, are phenols, polyaromatics, dyes, chlorinated compounds, organophosphorous pesticides or nerve agents, and explosives (TNT, trinitrotoluene). Various enzymes can be used for remediation, and representative candidates are peroxidases, laccases, tyrosinases, dehalogenases, and organophosphorous hydrolases. Even though there is more and more recognition of the usefulness of enzymatic remediation, the cost for enzymes is still expensive, especially, due to the poor stability and short lifetime of enzymes in general. Enzyme stabilization can make enzymatic remediation cost-effective by increasing the lifetime of enzymes. In this paper, we report the development of enzyme-polymer composites in nano-meter scale or single-enzyme scale, resulting in a stabilization of enzyme activity. This new approach is distinct from immobilizing enzymes on preformed solids or entrapping them in sol-gels, polymers, or bulk composite structures. Instead, the process begins from the surface of the enzyme molecule, with covalent reactions to anchor, grow, and crosslink a composite silicate shell around each separate enzyme molecule (Fig. 1 ). The detailed synthetic approach is as follows. A vinyl-group functionality is grafted onto the enzyme surface by covalently modifying the amino groups on the enzyme surface with acryloyl chloride. These modified enzymes are solubilized in an organic solvent such as hexane, as reported previously by Dordick et al. (3-7). The solubilized enzymes are mixed with silane monomers containing both vinyl groups and trimethoxysilane groups. Under suitable conditions, free-radical initiated vinyl polymerization yields linear polymers that are covalently bound to the enzyme surface. Careful hydrolysis of the pendant trimethoxysilane groups followed by condensation of the resulting silanols yields a crosslinked composite silicate coating around each separate enzyme molecule. We call these armored, singleenzyme nanoparticles (SENs). We have synthesized SENs containing CT (SEN-CT) using methacryloxypropyltrimethoxysilane (MAPS) as the vinyl monomer. The products were extracted into cold aqueous buffer solution, aged at 4°C overnight for silanol condensation, filtered, and washed. The wet residue was



222 resuspended in buffer and stored in the refrigerator. The yields of enzyme activity in the form of SENs were 38-73%. High resolution transmission electron microscopy (TEM) images confirmed the presence of individual enzyme particles with a composite shell (Fig. 2). The seemingly hollow center of the nanoparticle, which results from the transparency of the core protein to the electron beam, matches the size and shape of the CT (8). On the other hand, the dark image surrounding the CT is the armored shell, and the presence of silicon was confirmed by energy dispersive x-ray analysis in the TEM instrument. The size of a few particles is bigger than that of single enzyme molecule, due to the co-immobilization of a few enzyme molecules in a single particle. 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 a day while free CT is inactivated very rapidly by autolysis under the same conditions (Fig. 3). The storage stability of the SEN-CT in buffer solution was also impressive. After three-months of storage in the refrigerator (4°C), 82% of the SEN-CT activity remained in solution as determined by measurements on solution aliquots. However, it was noted that a transparent layer of SEN-CT was built up on the inner surface of the glass vial. This immobilized layer exhibited considerable enzyme activity after removal of the solution. Thus, the apparent decrease in solution enzyme activity may be accounted for, at least in part, by immobilization on the vial surface rather than by enzyme degradation. The activities of 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 (kc , K , and kc /K ) obtained via nonlinear regression based on the least squares method are given in Table 1. The catalytic efficiency (k^t/Kn,) of CT in SEN form was decreased by about half, and it was due to reduced k^. However, the apparent binding constant (K ) of SEN-CT was almost the same as that of free CT. This suggests that the armored shell did not cause any significant mass-transfer limitation for the substrate (TP). In summary, we have developed a new form of stabilized enzyme using CT as the initial example. Dramatic activity stabilization was achieved with minimal substrate mass-transfer limitation. The development of stabilized enzymes as soluble individual enzyme particles provides the opportunity to further process these new nanomaterials, in contrast to enzymes entrapped in bulk solids. The SENs can potentially be immobilized on solid supports, assembled with other nanoparticles or molecules as part of multifunctional nanomaterials, and their surfaces can be modified by silane reagents. The SEN provides a stabilized enzyme form that can penetrate and be immobilized within nanostructured or nanoporous substrates. These new hybrid enzyme nanostructures offer great at

m

at

m

m



223

Fig. 1. Schematic for SEN synthesis. The SEN synthesis proceeded via a threestep process: thefirststep is to modify the enzyme surface and solubilize the modified enzyme into a hydrophobic solvent (such as hexane); the second step i vinyl-group polymerization in hexane, forming linear polymers grafted to the enzyme surface; and, thefinalstep is the hydrolysis and condensation of pendant alkoxysilanes, crosslinking the grafted polymers andforming the armored shell around the enzyme molecule.

Fig. 2. TEM image of SENs containing CT (SEN-CT). The sample was extensively washed by distilled water on a membranefilter(MWCO I OK) for the best contrast between the hollow image of CTand the dark image of armored shell. The hollow images of most particles have a dimension of CT molecule (4 χ 3.5 χ 8 nm) (8). The scale bar represents 20 nm. In Nanotechnology and the Environment; Karn, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.


224

Fig. 3. Stability of SEN-CT (Ο) andfree CT(X). Residual activity was determined by the hydrolysis of TP in aqueous buffer (10 mM sodium phosphate, pH 7.8) after incubation at 30°C, and the relative activity is defined by the ratio of residual activity at each time point to the initial activity of each sample. All the incubations were done in plastic tubes since SEN-CT can become immobilized on the inner surface of glass vials.

Table 1. Activity ofFree CT and SEN-CT.*

5

Sample

kc*(s')

Κ,„(μΜ)

Freed

30

39

7.7

SEN-CT

14

40

3.4

SEN-CT/Free CT

0.47

1.0

0.44

kcat/K

m

1

(x i 0 NT's" )

a

The CT activity was determined by the hydrolysis of TP (1.6-160 μΜ) 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 assay. In Nanotechnology and the Environment; Karn, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

225 potential to be used as prepared or processed into other forms for various applications such as bioremediation, biosensors, detergents, and enzymatic synthesis in pharmaceutical and food industries.


Acknowledgements We thank A . Dohnalkova and C. Wang for the electron microscopy. Our work on single enzyme nanoparticles has been funded 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 Pacific Northwest National Laboratory is a multiprogram national laboratory operated for the U.S. DOE by Battelle Memorial Institute.

References 1. N. Durán, E. Esposito, Appl. Catal., Β 28, 83 (2000). 2. J. Karam, J. A. Nicell, J. Chem.Technol.Biotechnol. 69, 242 (1997). 3. P. Wang, M . V. Sergeeva, L. Lim, J. S. Dordick, NatureBiotechnol.15, 789 (1997). 4. J. S. Dordick, S. J. Novick, M . Sergeeva, Chem. Ind. 1, 17 (January 1998). 5. S. J. Novick, J. S. Dordick, Chem. Mater. 10, 955 (1998). 6. J. Kim, R. Delio, J. S. Dordick, Biotechnol. Prog. 18, 551 (2002). 7. V. M . Paradkar, J. S. Dordick, J. Am. Chem. Soc. 116, 5009 (1994). 8. H. Tsukada, D. M . Blow, J.Mol.Biol.184, 703 (1985).


Nanotechnology and the Environment - American Chemical Society

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