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Encapsulation of Synthetically Valuable Biocatalysts into Polyelectrolyte Multilayer Systems Lars O. Wiemann,†,‡ Andreas Buthe,†,§ Mathias Klein,† Anne van den Wittenboer,† Lars Da¨hne,| and Marion B. Ansorge-Schumacher*,†,‡ RWTH Aachen UniVersity, Department of Biotechnology, Worringerweg 1, D-52074 Aachen, Germany, and Capsulution Nanoscience AG, Volmerstr. 7b, D-12489 Berlin, Germany ReceiVed September 25, 2008. ReVised Manuscript ReceiVed NoVember 3, 2008 Layer-by-Layer (LbL) technology recently turned out to be a versatile tool for the encapsulation of bioactive entities. In this study, the factual potential of this technology to encapsulate synthetically valuable biocatalysts, that is enzymes and whole cells expressing a specific catalytic activity, was investigated. The biocatalysts were embedded into a polyelectrolyte multilayer system involving poly(allylamine) hydrochloride (PAH) and poly(styrene sulfonate) sodium salt (PSS). The enzymes were adsorbed to CaCO3 or DEAE-cellulose previous to encapsulation. A slight increase (32%) of the catalytic performance was observed for lipase B from Candida antarctica when four layers of polyelectrolytes were applied. On the whole, however, the residual activity of the investigated enzymes after encapsulation was rather low. Similar results were obtained with whole-cell biocatalysts. It was found that the activity decrease can be attributed to mass transfer restrictions as well as direct interactions between polyelectrolytes and catalytically active molecules. Both effects need to be understood in more detail before LbL technology can be advanced to technically efficient biocatalysis.

Introduction Layer-by-layer (LbL) technology is a versatile and simple method to generate surface coatings on template bodies by adsorption of polyelectrolyte layers.1-4 The coating is achieved by placing the template successively in deposition solutions of oppositely charged polyelectrolytes, which spontaneously adsorb to the surface. The surface charge thus inverses with every deposition step. Theoretically, more than 100 deposition cycles are feasible, but usually fewer than 10 are applied. Shell architecture, that is thickness and/or polymer mediated features, vary with type and number of polyelectrolyte layers.5 The large number of possible arrangements of polyelectrolytes and template particles enables a very simple and targeted tuning and modulation of physicochemical shell properties.6 Versatility and simplicity of surface coating with LbL technology have led to the development of a large application field including electrochemistry, nanoengineering, and nanosciences.7-9 Particularly promising is the encapsulation of bioactive target substances such as drugs, lipopolysaccharides, * To whom correspondence should be addressed. Tel: +49-30-314-22127. Fax: +49-30-314-21126. E-mail: [email protected]. † RWTH Aachen University. ‡ Present address: Technical University of Berlin, Inst. of Chemistry, Dept. of Enzyme Technology (TC4), Str. des 17. Juni 124, D-10623 Berlin, Germany. § Present address: Ciba Inc, CH-4002 Basel, K420.2.18, Switzerland. | Capsulution Nanoscience AG. (1) Decher, G. Layered Nanoarchitectures via Directed Assembly of Anionic and Cationic Molecules. In ComprehensiVe Supramolecular Chemistry, Vol. 9, Templating, Self-Assembly and Self-Organization; Sauvage, J.-P., Hosseini, M. W., Eds.; Pergamon Press: Oxford, 1996; 507-528. (2) Knoll, W. Curr. Opin. Colloid Interface Sci. 1996, 1, 137–143. (3) Decher, G.; Eckle, M.; Schmitt, J.; Struth, B. Curr. Opin. Colloid Interface Sci. 1998, 3, 32–39. (4) Sukhorukov, G. B.; Donath, E.; Davis, S.; Lichtenfeld, H.; Caruso, F.; Popov, V. I.; Mo¨hwald, H. Pol. AdV. Technol. 1998, 9, 759–767. (5) Decher, G. Science. 1997, 277, 1232–1237. (6) Shenoy, D. B.; Sukhorukov, G. B. Macromol. Biosci. 2005, 5, 451–458. (7) De´jugnat, C.; Halozan, D.; Sukhorukov, G. B. Macromol. Rapid Commun. 2005, 26, 961–967. (8) Hyde, K.; Rusa, M.; Hinestroza, J. Nanotechnology. 2005, 16, 422–428. (9) Shipway, N.; Katz, E.; Willner, I. Chemphyschem. 2000, 1, 18–52.

and proteins for pharmaceutical use.10-13 On this background, it is quite surprising that only little attention has so far been paid to the application of LbL technology to synthetically valuable biocatalysts. Biocatalysts are biological entities such as enzymes, cell fragments, and whole cells that constantly gain importance in the industrial production of chemicals, particularly building blocks for pharmaceutically active compounds.14 This mainly results from the selectivity of biocatalyzed reactions combined with their sustainability in terms of energy consumption and toxicity.15,16 For technical use, stabilization of the fragile biocatalysts for process requirements such as organic solvents or mechanical stress and preparation for continuous or repetitive use are often mandatory. In this context, the immobilization of biocatalysts by encapsulation in polymeric matrices can be advantageous.17 A major drawback of the currently applied techniques, however, is the restricted transfer of substrate and product molecules into and out of the capsule, respectively. Theoretically, this could be overcome or at least considerably improved by applying tailormade capsules as available by LbL technology. Numerous studies have already demonstrated that enzymes such as horseradish peroxidase, β-glucosidase, glucose oxidase, urease, trypsin,18-24 (10) Volodkin, D. V.; Larionova, N. I.; Sukhorukov, G. B. Biomacromolecules. 2004, 5, 1962–1972. (11) Yu, A.; Liang, Z.; Caruso, F. J. Mater Chem. 2005, 17, 171–175. (12) Xu, J.; Zhao, W.; Luo, X.; Chen, H. Chem. Commun. 2004, 6, 792–794. (13) Caruso, F.; Trau, D.; Mo¨hwald, H.; Renneberg, R. Langmuir. 2000, 16, 1485–1488. (14) Straathof, A. J. J.; Panke, S.; Schmid, A. Curr. Opin. Biotechnol. 2002, 6, 548–556. (15) Schoemaker, H. E.; Mink, D.; Wubbolts, M. G. Science 2003, 299, 1694– 1697. (16) Bommarius, A. S.; Riebel, B. Biocatalysis: Fundamentals and Applications; Wiley-VCH: Weinheim, Germany, 2004; ISBN: 3-527-30344-8. (17) McMorn, P.; Hutchings, G. J. Chem. Soc. ReV. 2003, 33, 108–122. (18) Liang, Z.; Wang, C.; Tong, Z.; Ye, W.; Ye, S. React. Polym. 2005, 63, 85–94. (19) Garbers, E.; Mitlo¨hner, R.; Georgieva, R.; Ba¨umler, H. Macromol. Biosci. 2007, 7, 1243–1249. (20) Gao, C.; Liu, X.; Shen, J.; Mo¨hwald, H. Chem. Commun. 2002, 1928–29. (21) Rao, S. V.; Anderson, K. W.; Bachas, L. G. Biotechnol. Bioeng. 1999, 65, 389–396.

10.1021/la803152c CCC: $40.75  2009 American Chemical Society Published on Web 12/09/2008

Polyelectrolyte Multilayer Systems

and whole cells of Escherichia coli or Saccharomyces cereVisia25,26 can be encapsulated into polyelectrolyte multilayer(PEMU-) systems with good retention of activity. However, these biocatalysts are both simple and robust and hardly represent the enzymatic and whole-cell systems relevant for chemical catalysis. In this study, we evaluate the factual potential of the LbL technology as a method for the immobilization of synthetically valuable enzymes and of recombinant organisms overexpressing a specific biocatalytic activity. The investigated enzymes were lipase B from Candida antarctica (CALB, E.C. 3.1.1.3),27 esterase from Bacillus coagulans (BCE, E.C. 3.1.1.1),30 benzaldehyde lyase from Pseudomonas fluorescens Biovar I (BAL, E.C. 4.1.2.38),31-33 and alcohol dehydrogenase from Lactobacillus breVis (LBADH, E.C. 1.1.1.2),34-37 and thus belonged to the three technically relevant classes of hydrolases (E.C.3), lyases (E.C.4), and oxidoreductases (E.C.1). As whole-cell biocatalysts E. coli strains SG13009 and JM109 overexpressing the BAL or a carbonyl reductase from Candida parapsilosis (CPCR, E.C.1.1.1.1), respectively, were studied. As the encapsulation matrix, a PEMU system of poly(allylamine) hydrochloride (PAH) and poly(styrene sulfonate) sodium salt (PSS)38-40 was chosen.

Experimental Section Materials. Poly(styrene sulfonate) sodium salt (PSS, MW 70 kDa) and poly(allylamine) hydrochloride (PAH, MW 70 kDa) were purchased from Aldrich (Germany). BIO-RAD Protein Assay (Bradford reagent) was obtained from Riedel-de Hae¨n (Germany). Diethylaminoethyl-(DEAE)-cellulose (DE52) was obtained from Whatman (UK). All other chemicals were purchased from SigmaAldrich (Germany) and used as obtained. Pure water with a specific resistance higher than 18 MΩ cm2 was used in all experimental steps. 1,2-O-isopropylidenglycerol- (IGP)-butyrate was synthesized according to Bratovska et al.41 Enzymes. Lipase B from Candida antarctica (CALB) (Chirazmye L-2, Roche Diagnostics, Germany) and alcohol dehydrogenase from Lactobacillus breVis (Ju¨lich Chiral Products, Germany) were used as obtained. Recombinant benzaldehydlyase was isolated from (22) Shutava, T. G.; Kommireddy, D. S.; Lvov, Y. M. J. Am. Chem. Soc. 2006, 128, 9926–9934. (23) Stein, E. W.; Volodkin, D. V.; McShane, M. J.; Sukhorukov, G. B. Biomacromolecules 2006, 7, 710–719. (24) Petrov, A. I.; Volodkin, D. V.; Sukhorukov, G. B. Biotechnol. Prog. 2005, 21, 918–925. (25) Neu, B.; Voigt, A.; Mitlo¨hner, R.; Leopratti, S.; Gao, C. Y.; Donath, E.; Kiesewetter, H.; Mo¨hwald, H.; Meiselman, H. J.; Ba¨umler, H. J. Microencapsul. 2001, 18, 385–389. (26) Diaspro, A.; Silvano, D.; Krol, S.; Cavalleri, O.; Gliozzi, A. Langmuir 2002, 18, 5047–5050. (27) Kirk, O.; Christensen, M. W. Org. Process Res. DeV. 2002, 6, 446–451. (28) Haeffner, F.; Norin, T.; Hult, K. Biophys. J. 1998, 74, 1251–1262. (29) Lo´pez-Garcı´a, M.; Alfonso, I.; Gotor, V. Chem.sEur. J. 2004, 10, 3006– 3014. (30) Molinari, F.; Gandolfi, R.; Cavenago, K. S.; Romano, D.; Romano, A. Tetrahedron: Asymmetry 2004, 15, 1945–1947. (31) Gonzales, B.; Vicuna, R. J. Bacteriol. 1989, 171, 2401–2405. (32) Iding, H.; Siegert, K.; Mesch, K.; Pohl, M. Biochim. Biophys. Acta. 1998, 1385, 307–322. (33) Scho¨rken, U.; Sprenger, G. A. Biochim. Biophys. Acta 1998, 1385, 229– 243. (34) Liese, A.; Seelbach, K.; Wandrey, C. Industrial Biotransformations; WileyVCH: Weinheim, Germany, 2000; ISBN 3-527-30094-5. (35) Schlieben, N. H.; Niefind, K.; Mu¨ller, J.; Riebel, B.; Hummel, W.; Schomburg, D. J. Mol. Biol. 2005, 349, 801–813. (36) Niefind, K.; Mu¨ller, J.; Riebel, B.; Hummel, W.; Schomburg, D. J. Mol. Biol. 2003, 327, 317–328. (37) Wollberg, M.; Hummel, W.; Mu¨ller, M. Chem.sEur. J. 2001, 7, 4562– 4571. (38) Antipov, A. A.; Shchukin, D.; Fedutik, Y.; Petrov, A. I.; Sukhorukov, G. B.; Mo¨hwald, H. Colloids Surf., B 2003, 224, 175–183. (39) Phuvanartnuruks, V.; McCarthy, T. J. Macromolecules. 1998, 31, 1906– 1914. (40) Shenoy, D. B.; Antipov, A. A.; Sukhorukov, G. B.; Mo¨hwald, H. Biomacromolecules 2002, 4, 265–272. (41) Batovska, D. I.; Tsubota, S.; Kato, Y.; Asano, Y.; Ubukata, M. Tetrahedron: Asymmetry 2004, 15, 3551–3559.

Langmuir, Vol. 25, No. 1, 2009 619 Escherichia coli SG13009 [pBALhis6] according to Iding et al.42 Carboxylesterase from Bacillus coagulans (BCE) was purified from the wild-type strain B. coagulans NCIMB 9365 as follows: Cells were grown on medium containing IPG/IPG-butyrate. BCE was purified from the crude extract by heating to 65 °C for 1 h as described by Romano et al.43 and subsequent two-step precipitation with 20% and 30% w/w PEG 4000, respectively. In the first step, most of the undesired proteins were precipitated and eliminated by centrifugation, and in the second step, BCE was selectively precipitated. The enzyme was freeze-dried for storage. Whole-Cell Biocatalysts. For preparation of whole-cell biocatalysts E. coli strains SG13009 [pRep4] containing plasmid pBALhis6 and JM109 containing plasmid pKK223-3-CPCR were used. Cells were grown in LB medium at 37 °C until midlog phase and protein expression was induced by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Cells were harvested after further incubation for 5 h at 28-30 °C and stored in 50 mM potassium phosphate buffer (pH 7) at 4 °C. Protein expression was verified by SDS-PAGE according to La¨mmli.44 Activity Assays. CALB activity was determined for the esterification of caprylic acid and 1-octanol (50 mM each) in n-hexane at room temperature. Reactions were performed under permanent stirring in screw-capped glass vessels containing 5 mL of substrate solution and 10-20 mg of catalytically active particles. Samples of 50 µL were taken for GC analysis every 10 min. BCE activity was determined for the stereoselective hydrolysis of racemic IPG-butyrate (10 mM) in 0.1 mM phosphate buffer (pH 6.8) at 45 °C. Reactions had a volume of 1 mL (1.5 mL reaction tubes) and were permanently shaken in an overhead shaker. Samples of 100 µL were taken after 3, 5, and 7 h and extracted with ethylacetate prior to GC analysis. Molar conversion and E-values were calculated according to Chen et al.45 BAL activity and activity of whole cells expressing BAL were determined for the formation of benzoin from 50 or 40 mM benzaldehyde respectively in 50 mM potassium phosphate buffer (pH 7.4 and 7.0, respectively) containing 2.5 mM MgCl2 and 0.15 mM thiamine diphosphate (ThDP) at 30 °C. Samples were extracted with 2-octanone before analysis. LBADH activity was determined for the oxidation of 2-octanone (20 mM) to 2-octanol in n-hexane at 30 °C as described by Eckstein et al.46 Activity of whole cells expressing CPCR was determined for the synthesis of 2-butanol from 2-butanone (40 mM) in 50 mM potassium phosphate (pH 6.8) containing 2.8% (v/v) isopropanol. All activities were calculated from initial product formation rates quantified by GC. One unit of enzyme activity (U) is defined as the amount of enzyme that catalyzes the conversion of 1 µmol of the respective substrate per minute under the given conditions. The specific enzyme activity is expressed as units per mg of total protein (U/mgprotein). GC Analysis. The quantification of products from the biocatalyzed reactions was accomplished as summarized in Table 1. All readings were carried out with a flame ionization detector. The sample volume was 1-2 µL. All measurements were performed in triplicate. Typical retention times were 6.1 min for benzoin, 6.15 min for 2-octanol, 2.06 min for 2-butanol, 5.96 min for octyloctanoate, and 54.1 or 54.6 min for (S)- and (R)-IPG-butyrate, respectively. Preparation of Calcium Carbonate Particles. Polydisperse calcium carbonate particles were prepared by mixing supersaturated equimolar (0.33 M) salt solutions of Na2CO3 and CaCl2 at room temperature under vigorous stirring.47,48 Formation of particles (42) Iding, H.; Du¨nnwald, T.; Greiner, L.; Liese, A.; Mu¨ller, M.; Siegert, P.; Gro¨tzinger, J.; Demir, A. S.; Pohl, M. Chem.sEur. J. 2000, 6, 1483–1495. (43) Romano, D.; Falcioni, F.; Mora, D.; Molinari, F.; Buthe, A.; AnsorgeSchumacher, M. Tetrahedron: Asymmetry 2005, 16, 841–845. (44) Laemmli, U. K. Nature 1970, 227, 680–685. (45) Chen, C. S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. J. Am. Chem. Soc. 1982, 104, 7294–7299. (46) Eckstein, M.; Filho, M. V.; Liese, A.; Kragl, U. Chem. Commun. 2004, 1084–1085. (47) Volodkin, D. V.; Petrov, A. I.; Prevot, M.; Sukhorukov, G. B. Langmuir 2004, 20, 3398–3406. (48) Antipov, A. A.; Sukhorukov, G. B. AdV. Colloid Interface Sci. 2004, 111, 49–61.

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Wiemann et al. Table 1. Operational Settings for GC Analysis

detection of

GC and column type

a

injector/detector

carrier gas/settings

temperature program start: 60 °C gold: 1 min gradient: 40 °C/min end temp.: 280 °C start: 60 °C hold: 1 min gradient: 35 °C/min end temp.: 200 °C hold: 3 min start: 40 °C hold: 1 min gradient: 20 °C/min end temp.: 135 °C hold: 1 min start: 75 °C gradient: 0.7 °C/min stop:100 °C gradient: 10 °C/min stop: 120 °C gradient 1 °C/min end temp.: 135 °C hold 5 min start: 70 °C hold: 1 min gradient: 30 °C/min end temp.: 200 °C

benzoin

Varian 3400CW column A*

290 °C

helium head pressure: 25 psi

octyloctanoate

HP 5980 column B*

220 °C

nitrogen head pressure: 80 psi

2-octanol 2-octanone

HP 5980 column B*

220 °C

nitrogen head pressure: 50 psi

(R)-/(S)-IPG-butyrate (R)-/(S)-IPG

HP 5980 column C*

220 °C

helium head pressure: 70 psi

2-butanol

HP 5980 column B*

195 °C/ 220 °C

helium head pressure: 50 psi

a *A: polar capillary column (FS-Supreme-5, CS-Chromatographie Service, Langerwehe); *B: polar capillary column (FFAP-CW, CS-Chromatographie Service, Langerwehe); *C: chiral column (FS-cyclodex beta-I/P CS-Chromatographie Service, Langerwehe).

Table 2. Properties of Native Enzymes,52-54 Assembling Process, and Enzyme-PEMU Capsules (CaCO3/PAH/Enzyme/PAH/PSS/PAH) property molecular weight [kDa] isoelectric point (IEP) [pH] pH of deposition solution cofactor dependency protein loading amount determined by Bradford [mgprotein/gimmobilizate] specific activity of native enzyme [U/mgprotein] specific activity of enzyme-PEMU capsules [U/mgprotein] a

CALB 33 6 8 8.8 ( 1.5 1.9 ( 0.26 2.5 ( 0.19

BAL

LBADH

4 subunits, 58 each 4.6 7.4 Mg2+ and ThDP 41.7 ( 2.4 10.9 ( 0.3 0.22 ( 0.017

4 subunits, 26.6 each 5.75 7 Mg2+ and NADPH+H+ 18.2 ( 1.44 a

Four millimolar product after 24 h.

followed rules of colloidal aggregation as described elsewhere.10,49 The obtained microparticles were filtered using a fluted filter, rinsed with pure water, dried overnight at 50 °C, and stored in a dry state to prevent recrystallization. Formation of Polyelectrolyte-Enzyme Multilayers on CaCO3 Particles. CaCO3 particles (10% w/v) were mixed with 1 mL of a deposition solution containing 2 mg PAH or PSS per mL respectively and 0.5 M NaCl in water at pH 6.5. The suspension was gently shaken for 20 min, centrifuged, and the supernatant was removed. Enzymes (1 mgenzyme/mL aqueous solution) were deposited at pH values that guaranteed positive overall charges (CALB at pH 8, BAL at pH 7.4, and LBADH at pH 7). pH values were set with NaOH or HCl. The solutions for BAL and LBADH deposition were enriched with 0.15 mM ThDP and MgCl2 or 1 mM NADPH+H+, respectively. Alternating LbL deposition of polyelectrolytes and enzymes was carried out by repeating these steps in sequence. Enzyme loadings were determined spectrophotometrically at 595 nm using Bradford reagent for each deposition step (Table 2). All particles were shortly lyophilyzed to strip away redundant surface water and thus prevent agglomeration in organic solvents. Formation of Microencapsulated Whole-Cell Biocatalysts. Prior to deposition of polyelectrolytes, recombinant cells of E. coli were washed in 50 mM sodium acetate buffer at pH 5.6. The first (49) Sukhorukov, G. B.; Volodkin, D. V.; Gu¨nther, A. M.; Petrov, A. I.; Shenoy, D. B.; Mo¨hwald, H. J. Mater. Chem. 2004, 14, 2073–2081.

layer of PAH (1 mg/mL) was deposited by incubating 1% (w/w) cells in 50 mM sodium acetate buffer (pH 4.6) for 20 min. All following layers of PAH and PSS were assembled in 50 mM sodium acetate buffer (pH 5.6) containing 0.2 M NaCl. The encapsulated cells were separated by centrifugation at 10 000 × g for 5 min and washed three times with water. Capsule shells consisted of 4-8 alternating polyelectrolyte layers. Formation of a Polyelectrolyte-Enzyme Multilayer System on DEAE-Cellulose. DEAE-cellulose (10% w/v) was suspended and equilibrated in 0.1 M phosphate buffer (pH 6.8). It was then separated by centrifugation. Purified BCE was dissolved in phosphate buffer (0.1 M, pH 6.8) at a concentration of 4.6 mg/mL, and 1 mL of this solution was added to 200 mg of the pre-equilibrated DEAE-cellulose. Immobilization was performed at room temperature on an overhead shaker for 7 h. Immobilizates were washed three times with phosphate buffer (0.1 M, pH 6.8), and the protein (50) Wiemann, L. O.; Buthe, A.; Klein, A.; Ansorge-Schumacher, M. B. Biotechnol. J. 2008, 3, 403–409. (51) Lourenco, J. M. C.; Ribeiro, P. A.; Bothelho do Rego, A. M.; Fernandes, F. M. B.; Moutinho, A. M. C.; Raposo, M. Langmuir 2004, 20, 8103–8109. (52) Kirk, O.; Christensen, M. W. Org. Process Res. DeV. 2002, 6, 446–451. (53) Janzen, E. Die Benzaldehydlyase aus Pseudomas fluoreszens. Biochemische Charakterisierung und die Untersuchung von Struktur-Functionsbeziehungen. Ph.D. Thesis, Heinrich-Heine-Universita¨t, Du¨sseldorf, 2002. (54) Riebel, B. Biochemische und molekularbiologische Charakterisierung neuer mikrobieller NAD(P)-abha¨ngiger Alkoholdehydrogenasen. Ph.D. Thesis, Heinrich-Heine-Universita¨t, Du¨sseldorf, 1996.

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Figure 1. Incorporation of enzymes into a PEMU system based on spherical template particles. For simplification of illustration, it is neglected that both electrolytes and enzymes penetrate the complete porous structure of the carrier instead of forming a closed outer layer as could be deducted from the scheme. a) Coating of a negatively charged template (e.g., CaCO3) with positively charged polyelectrolyte, e.g. PAH (blue); b) assembling of positively charged enzymes (yellow); c) and e) coating with positively charged PAH; d) coating with negatively charged polyelectrolyte, e.g. PSS (red); and f) final coating structure with one enzyme layer (CaCO3/PAH/enzyme/PAH/PSS/PAH).

concentration in the washing solution was measured to calculate the protein loading of the immobilizates. For the coating with polyelectrolyte layers (PAH/PSS), aliquots (20 mg) of BCE/ DEAE-cellulose were twice washed with water, suspended in 200 µL water, and added to 1 mL PE solution (2 mg/mL, 0.2 M NaCl in 0.05 M Na acetate buffer, pH 6). Incubation was carried out on an overhead shaker for 30 min at room temperature. After every coating step, the particles were separated from the supernatant by centrifugation, washed with water (three times), and resuspended in 200 µL water. The first layer always consisted of PAH. Colorimetric Quantification of PSS Loading Amounts. The colorimetric quantification of PSS loadings amounts was carried out as described by Wiemann et al.50 The absorbencies were measured at 695 nm with a UV-vis recording Spectrometer UV-160A, Kyoto (Japan).

Results and Discussion Biological entities such as enzymes and whole cells are characterized by typical, pH-dependent surface charges. Thus, they behave very similar to polyelectrolytes and can easily be complexed with oppositely charged molecules. The cell surfaces used in this work were slightly negative as determined by Zeta potential measurements, and the surface itself is large enough to serve as a template for the formation of a complete capsule. In contrast, the surface loading of enzymes can be switched by changing the pH of the surrounding solution, and solid templates are required to obtain particles with a proper size for technical handling. The enzymes can then be incorporated as a first or intermediate capsule layer. In this study, colloid CaCO3 particles were used as standard templates for enzyme encapsulation because their overall suitability had previously been demonstrated by Volodkin et al.,10,47 and the generation of these particles is cheap, fast, and easily attainable. The average particle size is 5 ( 2 µm, and the total surface area is 8.8 m2/g, that is, a high loading of polyelectrolytes and enzyme can be achieved. Additionally, DEAE- (diethylaminoethyl-) cellulose was used as a template for the encapsulation of esterase from Bacillus coagulans (BCE) because of the observed qualities of this carrier with regard to catalytic performance. Polycationic poly(allylamine) hydrochlo-

ride (PAH) and polyanionic poly(styrene) sulfonate (PSS) were chosen as polyelectrolyte layers for their excellent deposition properties and current importance in LbL technology.51 4.1. PEMU-EmbeddedEnzymesonCaCO3 ColloidParticles. CaCO3 particles have an overall surface charge of -12 mV,10,47 and PAH was therefore deposited as the priming layer. This was followed by a layer of enzyme and three more layers of PEMU to prevent enzyme loss and obtain protection against detrimental effects of the environment. The overall process is illustrated in Figure 1, relevant properties of the investigated enzymes CALB, BAL, and LBADH, and typical conditions of particle formation are summarized in Table 2. The same constitution (CaCO3/PAH/ enzyme/PAH/PSS/PAH) was chosen for all PEMU-enzyme systems in this work to ensure comparability. The loading with PSS ranged from 5-12 µg/mg for each deposition step, and the loading with enzyme was in the range of 9-40 µg/mg (Table 2). The large deviations derive from the differences in molecule size, structure, conformation, and composition of surface charges of the deposited enzymes and can therefore be explained by the complexity of forces contributing to the self assembly of enzymes. Primarily, these are electrostatic attractions between surfaces but van der Waals forces, hydrophilic-hydrophobic interactions, and entropic processes can also play a role.55 The same forces are most likely affecting polyelectrolyte loadings, in particular of PE layers deposited onto the adsorbed enzyme in the subsequent deposition step. The catalytic performance of the three enzymes after incorporation in the PEMU system on CaCO3 colloid particles was investigated with regard to the typical, synthetically important reactions depicted in Scheme 1. For comparison of native and encapsulated enzymes, the specific activities were determined, which are related to the total enzyme content (load) in the reaction. The specific activity of CALB in the standard PEMU systems increased slightly by 32% compared to the native enzyme. This is not exceptionally surprising because immobilization of lipases often improves the dispersion of these enzymes in organic solvents (55) Constantine, C. A.; Mello, S. V.; Duport, A.; Cao, X., Jr.; Oliviera, O. N.; Strixino, F. T.; Pereira, E. C.; Cheng, T.; Defrank, J. J.; Leblanc, R. M. J. Am. Chem. Soc. 2003, 125, 1805–1809.

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Figure 2. Influence of PE layers added to the standard enzyme-PEMU capsules (CaCO3/PAH/enzyme/PAH/PSS/PAH) on the specific activity (U/mgprotein) of CALB. Scheme 1. Schemes of Investigated Reactions (1) CALB-Catalyzed Esterification in n-Hexane, (2) BAL-Catalyzed Condensation in 50 mM Potassium Phosphate Buffer Containing 0.15 mM Thiamin Diphosphate (ThDP), (3) LBADH-Catalyzed Reduction in n-Hexane46

and thus the access for dissolved substrates.56 On the contrary, the obtained activation is rather low with regard to the values reported for alternative methods, for example, the immobilization in silicone elastomers,57 the adsorption and incorporation to silicone biocomposites,58 or the entrapment in hydrophobic sol gels,56 and thus implies a considerable impact of the polyelectrolyte capsule on the mass transfer of substrates and/or products. In fact, this was confirmed by an activity decrease of about 10% per PE layer added to the standard PEMU system (Figure 2). The catalytic efficiency of encapsulated CALB could not be enhanced by increasing the number of enzyme layers in the PEMU system (data not shown), which implies that inner layers of enzyme do not participate in the reaction. This could either be due to a restricted transfer of substrates to these layers or result from the already complete consumption of substrates by the outmost enzyme layer. In any case, it is obviously not expedient for biocatalytic purposes to deposit more than one layer of CALB in a PEMU system. In contrast, it would be worth considering (56) Reetz, T.; Zonta, A.; Simpelkamp, J. Biotechnol. Bioeng. 1996, 49, 527– 534. (57) Buthe, A.; Kapitain, A.; Hartmeier, W.; Ansorge-Schumacher, M. B. J. Mol. Catal. B: Enzym. 2005, 35, 93–99. (58) Gill, I.; Pastor, E.; Ballesteros, A. J. Am. Chem. Soc. 1999, 121, 9487– 9493.

possibilities to decrease the number of deposited enzyme layers in the investigated system to avoid waste of catalytic capacity of the principally high-priced biocatalyst. BAL encapsulated in the standard PEMU system retained only about 2% of its native specific activity. An activity decrease of roughly 50% was observed as a result of the deposition of every additional PE layer on the enzyme. This was considerably more than the activity loss observed for the addition of PE layers to the standard PEMU-CALB system and indicated that here the polyelectrolytes affected not only the mass transfer. The exceptionally high loading of 42 mgprotein/gimmobilizate achieved on the CaCO3 particles with a single PAH layer (Table 2) indicated a very strong interaction of BAL and polyelectrolyte, which could induce conformational changes, reduce flexibility, or block the entrance to the catalytic site of the enzyme. Additionally, it would be conceivable that a direct interaction of primary amino functions in PAH-NH2 with free aldehyde groups (CHdO) of substrate molecules within the PEMU system leads to the formation of azomethines (CHdN), thus building up an additional diffusion barrier that apparently contributes to a hampering of mass transfer. A final explanation for the observed severe activity loss of BAL after encapsulation in a PEMU system can at the moment not be offered. However, an activity loss of about 50%

Polyelectrolyte Multilayer Systems Scheme 2. BCE-Catalyzed Enantioselective Hydrolysis of Racemic 1,2-O-Isopropylideneglycerolesters (IPG: 1,2-O-Isopropylidenglycerol)

was also observed when free BAL and PAH or PSS were dissolved in an aqueous system, where mass-transfer restrictions can only play a minor role. Standard PEMU systems embedding LBADH did not reveal any catalytic activity at all. This was also observed when the number of layers covering the enzyme was reduced to one and can therefore not result from mass transfer restrictions. The explanation probably lies in the special catalytic mechanism of LBADH, which requires Mg2+ ions and freely diffusible NADPH+H+ as essential cofactors. It was ensured that these components were substituted in every step of PEMU preparation, but due to their positive charges it can be assumed that they were complexed by the anionic PAH layers below and above the enzyme and thus were unavailable for the enzymatic reaction. 4.2. PEMU-Embedded BCE on DEAE-Cellulose. BCE adsorbed to DEAE-(diethylaminoethyl-)cellulose catalyzes the kinetic resolution of 1,2-O-isopropylidenglycerolesters (Scheme 2). The reaction system yields (S)-1,2-O-isopropylideneglycerol (IPG), a valuable chiral synthon for the production of β-blockers, prostaglandins, or leucotrienes,59 with an enantiomeric excess (ee) of 95%,60 but due to the weak binding forces between biocatalyst and support the enzyme is easily desorbed and deactivated. Overlaying the adsorbed enzyme with polyelectrolytes could offer the remedy for this problem. Consequently, two, three, and four layers of alternating PAH and PSS were deposited on the immobilized BCE. In all PEMU systems, a decrease of the specific activity of BCE was observed. With two layers of PE, the residual activity was 13.8 ( 1.5%; with four layers, it decreased to 7.5 ( 0.85%. On the background of the formerly observed decrease of catalytic activity, most likely a result of mass transfer restrictions exceeded by the polyelectrolyte layers, this was not unexpected. However, with the catalytic activity decreased also the enantioselectivity of the reaction. With two layers of PE it was 34%; with four layers, it was 31%. According to Xiu et al.,61 who related a decrease in enantioselectivity of kinetic resolutions with insufficient substrate concentrations, this would be a further effect of mass transfer restrictions exerted by the PE layers. It is also possible, however, that direct interactions between enzyme and polyelectrolytes or desorption of enzyme during the consecutive coating steps contribute to the selectivity loss of the investigated reaction. 4.3. LbL-Coated Whole-Cell Biocatalysts. A direct interaction of polyelectrolytes with encapsulated enzymes or essential cofactors can be ruled out when the enzymes are not extracted from the organisms expressing them, that is the whole cells are incorporated in the PEMU system as biocatalysts. Therefore, two recombinant microbial hosts expressing BAL or CPCR, an (59) Dro¨ge, M. J.; Bos, R.; Woerdenbag, H. J.; Quaxm, W. J. J. Sep. Sci. 2003, 26, 771–776. (60) Molinari, F.; Brenna, O.; Valenti, M.; Aragozzini, F. Enzyme Microb. Technol. 1996, 19, 551–556. (61) Xiu, G.; Jiang, L.; Li, P. Ind. Eng. Chem. Res. 2000, 39, 4054–4062.

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NADH+H+-dependent reductase similar to LBADH, were investigated for their catalytic performance in PEMU systems. The PEMU systems consisted of an initial layer of PAH adsorbed to the negatively charged surface of the cells followed by two to four PAH/PSS double layers. For comparison of catalytic activities, the concentrations of native and encapsulated cells were set to an OD600 of 1 in all reactions. For regeneration of the cofactor NADH+H+, isopropanol was added to the cells expressing CPCR following the protocol of Matsuyama et al.62 The adsorption of two PAH/PSS double layers on whole cells containing BAL reduced their catalytic activity to 0.01% of the initial value (1 U/mLcell suspension) and was thus just detectable. With each deposition of a further pair of polyelectrolytes, this already negligible activity was again decreased about 2.5 fold. Whole cells expressing CPCR did not retain detectable activity after encapsulation in any PEMU system, whereas the native cells revealed an activity around 0.2 U/mLcell suspension. It is rather improbable that in these systems the catalytically active enzymes or cofactors and the PE layers get into a vicinity close enough to allow direct interaction as discussed for the PEMU-enzyme systems. Therefore, the severe activity losses must solely be attributed to the mass transfer restrictions implied by the PEMU. The different impact of this restriction on the catalytic activity of isolated enzymes or the whole-cell biocatalyst can probably be explained by differences in the density of layers deposited on the proteins or cell walls of the investigated systems.

Conclusions Despite the reported beneficial effects of PEMU systems on the long-term stability of enzymes,18-24 the results obtained in this study do not indicate simple applicability to the stabilization of sensitive biocatalysts in chemical synthesis, as a dramatic decrease of the catalytic performance of almost all investigated enzymes and whole-cell systems was observed. The cause of this decrease could not be elucidated in all detail, but it became obvious that several parameters contribute, including mass transfer limitation and direct interactions between polyelectrolytes and catalytic molecules. These should be thoroughly understood before an application of PEMUs to the encapsulation of catalytically active molecules and enzymes can be reconsidered. In view of the almost unlimited multitude of possible PEMU-biocatalyst combinations, it can still be expected that favorable combinations exist. An advisable approach to the successful identification of these combinations would probably include a rational preselection of suitable polyelectrolyte pairs combined with the development of effective screening systems. Preselection should be based on all available knowledge on the complex interactions between polyelectrolytes, biocatalyst, and reactants with regard to activity/productivity and stability. Thus, the effort put into the identification of the most suitable pair of polyelectrolytes for a certain biocatalyst in a certain reaction system should be minimized. A facilitation of mass transfer through the PEMU system by deposition of bigger or tailored PE molecules of changing topology (e.g., globular) or by addition of spacer molecules might be considered. Acknowledgment. The authors thank Dr. Andreas Voigt and Barbara Baude (Capsulution NanoScience AG, Berlin) for their excellent support and introduction into the LbL technology. LA803152C (62) Matsuyama, A.; Yamamoto, H.; Kobayashi, Y. Org. Process Res. DeV. 2002, 6, 558–561.