Enzyme Sheathing Enables Nanoscale Solubilization of Biocatalyst

Feb 23, 2008 - Virginia Depp,† Joel L. Kaar,‡ Alan J. Russell,*,‡ and. Bhalchandra S. Lele*,†. ICX-Agentase, 2240 William Pitt Way,. Pittsburg...
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Notes Enzyme Sheathing Enables Nanoscale Solubilization of Biocatalyst and Dramatically Increases Activity in Organic Solvent

Scheme 1. Hydrophobic Interactions between Amphiphilic Comb Polymer and CT for Coating the Enzyme Surface

Virginia Depp,† Joel L. Kaar,‡ Alan J. Russell,*,‡ and Bhalchandra S. Lele*,† ICX-Agentase, 2240 William Pitt Way, Pittsburgh, Pennsylvania 15238, and McGowan Institute for Regenerative Medicine, 100 Technology Drive, University of Pittsburgh, Pittsburgh, Pennsylvania 15219 Received November 19, 2007 Revised Manuscript Received January 14, 2008

Introduction Enzymes, nature’s nanocatalysts, exhibit high stereoselectivity and high turnover rates under mild reaction conditions in predominantly aqueous environments. In organic reaction media these catalytic properties are challenged to a great extent due to insolubility and enzyme deactivation. Over the last two decades, chemists have shown that enzymes can also catalyze reactions in certain organic solvents using lyophilized and stabilized protein powders.1,2 To minimize solvent-induced deactivation, enzymes have been immobilized on a host of supports ranging from macro- and micoscale hydrogels3 and polymer beads4 to nanoscale silica gel particles5,6 and amphiphilc networks supported on glass.7 These efforts have also resulted in significant improvements in the recyclability of the biocatalysts. However, these techniques still produce heterogeneous catalytic systems that are controlled by limitations in the diffusion of substrate and product.8 To conduct more efficient homogeneous catalysis, different techniques have been developed to modify native enzymes and “dissolve” modified enzymes in organic solvents. These approaches include covalent attachment to hydrophilic9 as well as hydrophobic10 polymers, physical mixing with polymers,11 and ion pairing with surfactants.12 These modifications have resulted in “solubilizing” enzymes from very low (0.1–1 mg/ mL) to moderate (10 mg/mL)13 concentrations in nonpolar solvents (such as toluene, chloroform) that are capable of dissolving practically useful quantities of substrates while retaining some enzymatic activity. We note that many of these reports lack characterization of the particle size of enzymes “solubilized” in organic solvents. Conjugation of poly(ethylene glycol) (PEG) to enzymes has been the most popular method for “solubilizing” enzymes in hydrophobic organic solvents. The amphiphilic nature of PEG allows covalent modification of enzymes in aqueous medium and apparent dissolution of lyophilized, pegylated enzyme in organic solvent. However, in reality, rather than being truly solubilized, PEG-modified enzymes in solvents form micrometer scale dispersions of particles ranging in size from 260 to 340 nm.14 PEGylated enzymes therefore lose their nanoscale properties and cannot conduct true homogeneous catalysis in organic * Corresponding authors: [email protected], [email protected]. † ICX-Agentase. ‡ McGowan Institute for Regenerative Medicine.

solvents.14 We have been looking for a more robust way to recover the nanoscale advantages of enzymes in solvents and herein we describe how a sheath of amphiphilic, comb-shaped random copolymer of hydrophobic (2-ethylhexyl methacrylate) and hydrophilic (MPEG-methacrylate) monomers reduces the particle size of modified enzyme in solvent to 5–10 nm at protein concentration of 5 mg/mL. The nanoscale solubilization leads to very dramatic enhancement in the activity of biocatalysts in anhydrous and hydrophobic media. In general, proteins exhibit electrostatic and hydrophobic interactions with polymers and surfactants. Gao and Dublin15 used frontal analysis continuous capillary electrophoresis to

Figure 1. (a) Characterization of interactions between CT and the copolymer by tryptophan fluorescence spectroscopy (320 nm excitation wavelength): (A) oxidized CT (0.1 mg/mL) + copolymer (1.0 mg/ mL); (B) CT (0.1 mg/mL); (C) copolymer (1.0 mg/mL); (D) CT (0.1 mg/mL) + copolymer (1.0 mg/mL). (b) Tryptophan fluorescence in CT increases upon its interaction with the copolymer (320 nm excitation wavelength): (A) CT (0.1 mg/mL); (B) CT + copolymer (0.4 mg/mL); (C) CT + copolymer (0.9 mg/mL); (D) CT + copolymer (1.4 mg/mL).

10.1021/bm701281x CCC: $40.75  2008 American Chemical Society Published on Web 02/23/2008

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Figure 2. CD spectra of native (A) and copolymer-sheathed CT (B).

Figure 4. Left panel: Photograph showing (A) PEGylated CT and (B) copolymer-sheathed CT in toluene. Protein concentration was 5 mg/ mL in both A and B. Right panel: Graph of light scattering intensity vs particle size for solution B. Figure 3. UV spectra of (A) HRP (1 mg/mL) and (B) HRP (1 mg/mL) + copolymer (10 mg/mL).

demonstrate the significance of hydrophobic interactions by characterizing complex formation between similarly charged proteins and amphiphilic polyelectrolytes. We exploited these hydrophobic interactions by covering the surface of an enzyme with a hydrophobic polymer and thereby increasing the solvation of the enzyme in organic solvents. We envisioned this will enable true dissolution of enzymes and reduction in their particle size in solvents. We selected chymotrypsin (CT) and horseradish peroxidase (HRP) as two model enzymes and synthesized a random copolymer of 30% w/w 2-ethylhexyl methacrylate and 70% w/w MPEG-methacrylate as the amphiphilic copolymer in an enzyme sheath (Scheme 1). CT is a classic model enzyme of known structure that catalyzes alcoholysis reactions in a variety of anhydrous organic solvents. HRP catalyzes commercially relevant polymerizations of polyphenolics in a nonaqueous environment. The choice of copolymer was made to obtain a water-soluble yet sufficiently hydrophobic polymer that would increase the net hydrophobic content within protein-polymer adducts and facilitate their dissolution (particle size reduction) in organic solvent. We first characterized the interaction between CT and the amphiphilic copolymer by fluorescence spectroscopy. CT contains eight tryptophan residues and changes in their microenvironment can easily be monitored by observing differences in the fluorescence spectra of the enzyme. As shown in Figure 1a, all emission spectra exhibit a small peak at 360 nm which is characteristic for Raman scattering by water. CT shows

a broader peak at 396 nm for tryptophan residues (trace B). The copolymer itself has negligible fluorescence (trace C). When CT and the copolymer are mixed together, the emission peak intensity at 396 nm increases greatly, indicating exposure of tryptophan residues in the enzyme to hydrophobic environment provided by the added copolymer (trace D). When the copolymer is added to CT pretreated with N-bromosuccinamide, which oxidizes tryptophan residues in the enzyme, no increase in the fluorescence intensity is observed (trace A). Figure 1b shows fluorescence of tryptophan residues in CT increases gradually with the addition of the copolymer in the buffer solution containing the enzyme. The emission maxima reach saturation at 1:10 enzyme: copolymer w/w ratio (see Supporting Information). These observations are consistent with increased tryptophan fluorescence of bovine serum albumin in the presence of a surfactant16 or a PEG-lipid conjugate.17 A space-filling model of CT shows that there are a number of hydrophobic residues present on the surface of CT to which polymer interaction can occur (gray spheres in Scheme 1). 18 We observed no significant difference in circular dichroism spectra of native and sheathed CT (Figure 2). Thus, it is reasonable to expect that the interaction of tryptophan residues in CT with amphiphilic copolymers can occur without damaging the enzyme’s secondary structure. In the case of HRP also, the circular dichroism (CD) spectra of native and copolymer sheathed enzymes are similar from wavelengths 215 to 245 nm (see Supporting Information). Soret band peaks at 400 nm in UV spectra of native and copolymer sheathed HRP are shown in Figure 3. There is only decrease in the absorbance at 400 nm and no change in Soret band peak position when HRP is

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Figure 5. HRP-catalyzed indaniline dye formation reaction in toluene containing 3 M N,N-dimethylene-p-phenylenediamine, 3 M phenol, 0.6 mM tert-butyl hydroperoxide in decane: blue diamonds, copolymer sheathed enzyme; red squares, PEGylated enzyme; green triangles, native enzyme. Biocatalyst (protein) concentration in all reactions was 7.5 µM.

sheathed by the copolymer. A similar trend in UV spectra of the complex between a nonionic surfactant and HRP is reported by Kamiya et al.19 We used a 1:10 w/w ratio of enzyme to copolymer to sheath CT and HRP in poly(2-ethylhexyl methacrylate-co-MPEG methacrylate) (Mw ) 64300; Mw/Mn ) 5.5). Enzymes were dissolved in the copolymer solution and stirred for 2 h. Enzyme-polymer containing solutions were concentrated using 30 or 50 kDa MWCO centrifugal ultrafiltration tubes to ensure removal of unsheathed CT (MW 25 kDa) and HRP (MW 44 kDa). The powders obtained after lyophilization retained 8–10% w/w protein. Sheathed CT-containing powder dissolved readily in toluene to up to neat protein concentrations of 5 mg/mL. Thus, enzyme sheathing with the amphiphilic copolymer resulted in at least a 5-fold increase in its solubility over that achieved by PEGylation. Similar trends in solvent solubility were obtained for sheathed and native HRP. Dynamic light scattering analysis performed on toluene solutions of sheathed CT (5 mg/mL) showed that its particle size was markedly smaller (5–10 nm, Figure 4) than that reported for PEGylated subtilisin dissolved in toluene or benzene (260–340 nm), a protease of similar molecular weight.14 Our own characterization of PEGylated chymotrypsin revealed that the enzyme’s particles size is in actuality much greater (∼1 µm) in toluene at protein concentration 5 mg/mL (see Supporting Information). Thus, sheathing the enzyme enables true solubilization versus only partial dissolution via PEGylation. We measured the catalytic activity of CT and HRP in toluene using fluorescence- and colorimetry-based assays. In a fluorescence-based transesterification assay,20 sheathed CT was 7-fold more active (0.35 nmol/min/mg) over that of the PEGylated enzyme (0.05 nmol/min/mg). An initial lag phase was observed in the activity of both enzymes. HRP activity in toluene was measured using a coupled reaction between phenol and N,Ndimethylene-p-phenylene diamine, which forms an indaniline dye.7 Formation of dark purple dye was monitored by measuring the increase in the absorbance of the reaction at 546 nm. Specific catalytic activities were obtained from linear slopes. The specific activities of sheathed and PEGylated HRP were 2433 U and 66 U activity per milligram of “solubilized” protein. As shown in Figure 5, sheathed HRP had a 36-fold higher specific catalytic activity in toluene than that of PEGylated HRP. These results, when combined with the dynamic light scattering data, imply that sheathed CT and HRP act as homogeneous catalysts in organic solvent.

Notes

To probe the specific nature of the enzyme-copolymer interactions, we performed mild oxidative denaturation of CT and HRP using N-bromosuccinamide21 prior to sheathing with the copolymer. Sheathing of the oxidized enzyme yielded only 0.1–0.5% w/w enzyme incorporation into the lyophilized powder. Analysis of denatured enzymes by CD spectroscopy show significant conformational changes in the denatured enzyme which might affect their ability to interact and associate with the copolymer. Preservation of catalytic activities in sheathed enzymes reported in this technique indicates hydrophobic interactions of the amphiphilic copolymer occur mainly at the surface of the enzymes used. We believe this sheathing with an amphiphilic comb polymer helps organic solvent to solvate the enzymes efficiently and reduce the particle size of dissolved enzyme to 5–10 nm, which represents a 25–50-fold reduction in particles size observed for PEGylated enzymes. Small molecular weight substrates are accessible to enzymes, soluble, immobilized, or wrapped in polymers and surfactants. However, their reaction rates vary depending upon the diffusion of the substrate and the surface area of the biocatalyst being studied. Our results indicate drastic reduction in particle size of sheathed CT and HRP altered these parameters and increased their catalytic activity in organic solvents compared to PEGylated enzymes. In summary, we have presented a new approach to nanoscale dissolution of biocatalysts in organic solvents for conducting efficient enzymatic catalysis. Enzyme sheathing could become an important tool in realizing potential of nonaqueous homogeneous enzymatic catalysis in industrially and pharmaceutically important reactions. Acknowledgment. We thank the US Army Research Office for financial support of this work (Grant W911NF-06-C-0068). We are also grateful to Dr. Michael Cascio from the Department of Molecular Genetics and Biochemistry at the University of Pittsburgh for assistance with CD analysis. Supporting Information Available. Experimental details and characterization data. This material is available free of charge via the Internet at http://pubs.acs.org.

References and Notes (1) Klibanov, A. M. Nature 2001, 409, 241–246. (2) Schmid, A.; Dordick, J. S.; Hauer, B.; Kiener, A.; Wubbolts, M.; Witholt, B. Nature 2001, 409, 258–268. (3) Ansorge-Schumacher, M. B.; Doumeche, B.; Metrangolo, D.; Hartmeier, W. MinerVa Biotechnol. 2000, 12, 265–359. (4) May, S. W. Curr. Opin. Biotechnol. 1997, 8, 181–186. (5) Gill, I.; Ballestros, A. Trends Biotechnol. 2000, 18, 282–296. (6) Reetz, M. T. AdV. Mater. 1997, 9, 943–954. (7) Bruns, N.; Tiller, J. C. Nano Lett. 2005, 5, 45–48. (8) Kamat, S.; Beckman, E. J.; Russell, A. J. Enzyme Microb. Technol. 1992, 14, 265–271. (9) Castro, G. R.; Knubovets, T. Crit. ReV. Biotechnol. 2003, 23, 195– 231. (10) Zhu, G.; Wang, P. J. Am. Chem. Soc. 2004, 126, 11132–11133. (11) Maruyama, T.; Kotani, T.; Yamamura, H.; Kamiya, N.; Goto, M. Org. Biomol. Chem. 2004, 2, 524–527. (12) Paradkar, V. M.; Dordick, J. S. J. Am. Chem. Soc. 1994, 116, 5009– 5010. (13) Kwon, O. H.; Imanishi, Y.; Ito, Y. Biotechnol. Bioeng. 1999, 66, 265– 270. (14) Khan, S. A.; Halling, P. J.; Bosley, J. A.; Clark, A. H.; Peilow, A. D.; Pelan, E. G.; Rowlands, D. W. Enzyme Microb. Technol. 1992, 14, 96–100. (15) Gao, J.; Dublin, P. L. Biopolymers 1999, 49, 185–193. (16) Wu, D.; Xu, G.; Sun, Y.; Zhang, H.; Mao, H.; Feng, Y. Biomacromolecules 2007, 8, 708–712. (17) Castelletto, V.; Krysmann, M.; Kelarakis, A.; Paula Jauregi, P. Biomacromolecules 2007, 8, 2244–2249.

Notes (18) Fischer, N. O.; McIntosh, C. M.; Simard, J. M.; Rotello, C. M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5018–5023. (19) Kamiya, N.; Okazaki, S.-y.; Goto, M. Biotechnol. Tech. 1997, 11, 375– 378.

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(20) Han, M. S.; Jung, S. O.; Kim, M.-J.; Kim, D. H. J. Org. Chem. 2004, 69, 2853–2855. (21) Holmgren, A. J. Biol. Chem. 1973, 248, 4106–4111.

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