Siloxane-Based Biocatalytic Films and
`Paints for Use as Reactive Coatings
`
`Young Duk Kim,1 Jonathan S. Dordick,2 Douglas S. Clark1
`
`1DepartmentofChemicalEngineering,UniversityofCalifornia,Berkeley,
`California94720,USA;telephone:510-642-2408;fax:510-643-1228;e-mail:
`clark@cchem.berkeley.edu
`2DepartmentofChemicalEngineering,RensselaerPolytechnicInstitute,
`Troy,NewYork,USA
`
`Received22June2000;accepted8October2000
`
`Abstract: We have developed a new methodology for
`preparing films and paints suitable for use as biocatalytic
`coatings. The hydrolytic enzymes pronase and a-chymo-
`trypsin were immobilized by either sol-gel entrapment or
`by covalent attachment into a polydimethylsiloxane
`(PDMS) matrix and cast into thin films or incorporated
`into an oil-based paint formulation. All of the coatings
`retained enzymatic activity and adhered to several differ-
`ent materials. The enzymatic films and paints also exhib-
`ited higher thermostability than enzyme free in solution
`or covalently attached to the outer surface of PDMS. A
`porous membrane based on a PDMS-immobilized en-
`zyme was also prepared by an immersion precipitation
`process. Protein adsorption measurements showed that
`the enzyme-containing films and paints adsorbed less
`protein than enzyme-free controls, and that protein ad-
`sorption decreased with increasing proteolytic activity of
`the coating. These coatings thus provide the means to
`apply a stable enzymatic surface to a wide range of ma-
`terials, and may be generally useful as biocatalytic paints
`and films. © 2001 John Wiley & Sons, Inc. BiotechnolBioeng
`72: 475–482, 2001
`Keywords: polydimethylsiloxane; immobilized enzyme
`films; biocatalytic paints
`
`INTRODUCTION
`
`Silicone materials have mechanical and physicochemical
`properties intermediate to those of glass and organic resins.
`Distinctive characteristics of silicone materials include good
`thermal and oxidative stability, low-temperature flexibility,
`inertness, and “nonstick” surfaces. The latter property is
`particularly important for fouling-resistant coatings. In this
`connection, polydimethylsiloxane (PDMS), a commercially
`important linear silicone polymer with excellent nonstick
`properties, is being considered as a nontoxic antifouling
`coating for ship hulls to replace current antifouling paints
`containing toxic biocides (Vincent and Bausch, 1997; Wat-
`termann et al., 1997). The unique structure of PDMS im-
`parts a low surface energy due to tightly packed methyl
`
`Correspondence to: D. S. Clark
`Contract grant sponsor: Office of Naval Research, Korea Science and
`Engineering Foundation
`
`© 2001 John Wiley & Sons, Inc.
`
`groups, providing a low glass transition temperature (Tg)
`(Owen, 1988), which may be related to the ability to deter
`fouling by proteins and microorganisms.
`The inertness and high stability of silicone materials have
`also been exploited for enzyme immobilization (Gill and
`Ballesteros, 2000; Gill et al., 1999; Shtelzer and Braun,
`1994; Venon and Gudipati, 1995; Wu et al., 1994). Enzyme
`immobilization generally relies on the sol-gel approach to
`generate silica matrices by acid- or base-catalyzed hydroly-
`sis of hydrolyzable silane compounds such as tetramethyl-
`orthosilicate (TMOS) (Avnir et al., 1994; Dave et al., 1994).
`Whereas high temperatures are required to prepare glass by
`the traditional melting of silica, sol-gel processes involve
`low-temperature hydrolysis of monomeric precursors, and
`are thus highly suitable for the microencapsulation of fragile
`biomolecules. The application of PDMS in enzyme immo-
`bilization has been limited, however, by the extremely hy-
`drophobic nature of PDMS. To date, relatively low-
`molecular-weight PDMS (MW <4200) has been used as a
`minor component for sol-gel encapsulation in aqueous me-
`dia, with the final product being immobilized-enzyme pow-
`ders and hydrogels (Gill and Ballesteros, 1998; Reetz et al.,
`1996). If, on the other hand, PDMS could be incorporated as
`a major component and polymerized, a flexible immobi-
`lized enzyme film should result, stemming from the low
`glass transition temperature of the polymer.
`In the present work, we describe a new method for in-
`corporating enzymes into PDMS films under nonaqueous
`conditions, and report the formulation of a siloxane-based
`biocatalytic paint. Specifically, a-chymotrypsin (a-CT) and
`pronase were used to prepare highly stable enzyme-
`containing PDMS films and paints suitable for coating a
`variety of surfaces (e.g., metals and plastics). Moreover, the
`hydrolytic activity of the film had the effect of reducing
`protein adsorption below the relatively low level observed
`with unmodified PDMS. Biocatalytic PDMS films could
`also be cast into porous membranes at room temperature,
`demonstrating their potential for the preparation of biocata-
`lytic filters. Thus, these formulations warrant further con-
`sideration as candidates for environmentally benign coat-
`
`Reactive Surfaces Ltd. LLP
`Ex. 1027 (Rozzell Attachment H)
`Reactive Surfaces Ltd. LLP v. Toyota Motor Corp.
`IPR2016-01914
`
`475
`
`

`

`ings, resins, and paints with biocatalytic and antifouling
`properties.
`
`MATERIALS AND METHODS
`
`Materials
`
`a-Chymotrypsin (a-CT; from bovine pancreas), pronase
`(from Streptomyces griseus), succinyl-Ala-Ala-Pro-Phe-p-
`nitroanilide (suc-AAPF-pNa), L-leucine-p-nitroanilide
`(LpNa), N-acetyl-L-phenylalanine ethyl ester (APEE),
`4-methylumbelliferyl-p-tri-methylammonium cinnamate
`chloride (MUTMAC), bovine serum albumin, polyvinylpyr-
`rolidone (PVP; MW 4 40,000), and azocasein were ob-
`tained from Sigma Chemical Co. (St. Louis, MO). Dimeth-
`yldimethoxysilane was obtained from Fluka (Milwaukee,
`WI) and poly(dimethylsiloxane-block-ethylene oxide) was
`obtained from Gelest, Inc. (Tullytown, PA). Tetramethyl
`orthosilicate (TMOS), methytrimethoxysilane (MTrMOS),
`tetraethyl orthosilicate (TEOS), 3-aminopropyltriethoxysi-
`lane (APTS), polydimethylsiloxane (PDMS), polysulfone,
`glutaraldehyde, chlorinated rubber, iron acetylacetonate,
`rosin, and dibutyltin dilaureate were obtained from Aldrich
`Chemical Co. (Milwaukee, WI) and were used without fur-
`ther purification.
`
`of 15 min to 20 mL phosphate buffer containing 312 mg of
`APTS and 300 mg of enzyme. The resultant mixture was
`dialyzed for 24 h and freeze-dried for 48 h to obtain a
`lyophilized powder. To increase the activity of the immo-
`bilized CT PDMS film, polyvinylpyrrolidone (PVP) was
`added to the aqueous mixture before freeze-drying, yielding
`a final dry preparation of 79% (w/w) PVP. The film-
`formation procedure was the same as that of the sol-gel
`entrapment method.
`
`SurfaceAttachment(Method3)
`
`PDMS films having no immobilized enzyme were prepared
`first from a mixture of 0.21 g of TMOS, 2.47 g of PDMS,
`and 12 mg of dibutyltin dilaureate. The PDMS film was
`then partially hydrolyzed in 3 M HCl for 30 min to provide
`silanol functional groups on the film surface, rinsed with
`deionized water, and dried under vacuum for 12 h. The
`PDMS films were then treated with 2% 3-aminopropyltri-
`ethoxysilane in acetone followed by reaction with 5% glu-
`taraldehyde in 0.4 M phosphate buffer (pH 7.5) for 30 min.
`Enzyme solution (25 U/mL for CT or 4.4 U/mL for pronase)
`was added to the glutaraldehyde-treated films and incubated
`for 1 h at 4°C.
`
`Preparation of Biocatalytic PDMS Films
`
`Membrane Formation
`
`Sol-GelEntrapment(Method1)
`
`Homogeneous sol was typically prepared by sonicating 1.46
`g of TMOS, 1.52 g of MTrMOS, 0.4 g of distilled water,
`and 30 mL of 40 mM HCl for 10 min in a sonication bath
`containing a mixture of ice and water. To the freshly pre-
`pared sol was added 6.5 mL of enzyme solution containing
`300 mg of enzyme in 100 mM phosphate buffer (pH 7.5).
`The resulting hydrogel was dried under vacuum for 24 h and
`crushed in a mortar to a fine powder of immobilized en-
`zyme. The typical procedure for film formation was as fol-
`lows: 1 mL of the aforementioned sol mixture containing
`enzyme was emulsified with 3 mL of 45% (w/w) PDMS
`solution in tetrahydrofuran (THF) by adding poly(dimeth-
`ylsiloxane-block-ethylene oxide). The resulting mixture was
`stirred with 2 mg of dibutyltin dilaureate for 3 min and
`spread over the inner surface of a polypropylene plate, after
`which it was incubated at room temperature for at least 24
`h to allow film formation by polymerization of the PDMS.
`The final film thickness was ca. 50 mm for all samples, as
`determined by the use of calipers.
`
`CovalentAttachment(Method2)
`
`Ten milliliters of 1.25 wt% glutaraldehyde solution in 0.4 M
`phosphate buffer (pH 7.5) was added dropwise over a period
`
`A homogeneous reaction mixture was prepared by sonicat-
`ing and vortexing 12 mg of immobilized pronase powder
`(method 2), 0.02 g of TMOS, 0.2 g of PDMS, 4 mL of THF,
`and 20 mg of dibutyltin dilaureate. The mixture was stirred
`for 30 min to promote polymerization of PDMS, after which
`0.5 g of polysulfone and 1 mL of N-methylpyrrolidone were
`added to prepare a casting solution. The casting solution
`was spread onto a glass plate and immersed into a water
`bath containing 10% (v/v) THF. This process induced a
`liquid–liquid phase separation of the casting solution, re-
`sulting in the formation of a porous membrane.
`
`Formulation of Biocatalytic Paint
`
`The following components were combined and mixed with
`a mechanical stirrer for 2 h at room temperature: 0.5 g of
`PDMS; 0.5 g of chlorinated rubber; 50 mL of dimethyldi-
`methoxysilane; 20 mg of powdered silane-functionalized
`a-CT (by Method 2); 50 mL of poly(dimethylsiloxane-
`block-ethylene oxide); 38 mg of iron acetylacetonate dis-
`solved in 0.3 mL of THF; 0.5 g of rosin; and 1.5 mL of
`xylene. Using iron acetylacetonate as the polymerization
`catalyst produced a red-orange paint; alternatively, replac-
`ing iron acetylacetonate with the more toxic dibutyltin di-
`laureate, with and without cuprous oxide, produced green
`and pale yellow paints, respectively. Protein adsorption was
`
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`measured using poly(ethylene terephthate) films (6 cm ×
`6 cm) painted on both sides with the biocatalytic paints.
`
`determined by collecting initial rate data at 25°C using a
`substrate concentration of 0.5 mM.
`
`Active-Site Titration
`The concentration of active a-CT was determined by active-
`site titration with MUTMAC. Fluorescence measurements
`were carried out in 1-cm cuvettes in a FluoroMax-2 spec-
`trofluorimeter (Jobin Yvon-Spex Instruments, Edison, NJ).
`The samples were excited at 356 nm, and emission was
`followed at 451 nm. A baseline was established by adding
`250 mL of 0.2 mM MUTMAC solution in water to 3.0 mL
`of potassium phosphate buffer (pH 7.3, 1 M NaCl). For the
`a-CT-containing PDMS films and paint, 1-cm2 sections
`were inserted into the cuvette and stirred for 30 s, at which
`point the fluorescence of liberated 4-methylumbelliferone
`was measured, corresponding to the concentration of func-
`tional active sites.
`
`Enzyme Activity Assays
`Assays of pronase and a-CT were performed using LpNa
`and suc-AAPF-pNa as substrates, respectively, in Tris-HCl
`buffer (0.1 M, pH 7.8) at 25°C. The substrate concentrations
`ranged from 0.25 to 4.0 mM for LpNa and from 0.125 to 2.0
`mM for suc-AAPF-pNa. The product of each hydrolysis
`reaction, p-nitroanilide, was measured spectrophotometri-
`cally at 405 nm using a Beckman DU-6 UV-Vis spectro-
`photometer (Beckman Instruments Inc., Palo Alto, CA).
`Film and paint samples were assayed by placing a small (ca.
`0.5-cm2) patch in the cuvette, which settled to the bottom,
`out of the light path. The kinetic parameters Vmax and Km
`were obtained by fitting initial rate data to the Michaelis–
`Menten equation through Eadie–Hofstee plots. All plots
`were linear, indicating the absence of diffusional limitations
`(Blanch and Clark, 1995), except for the case of pronase
`immobilized by method 1, which showed slight curvature.
`From the y-intercept of the Eadie–Hofstee plot, the Thiele
`modulus, f, was determined to be ca. 1.5 (Blanch and
`Clark). However, the reported Vmax/Km value was deter-
`mined from the linear portion of the plot and therefore re-
`flects the intrinsic kinetics. The intrinsic catalytic efficien-
`cies (kcat/Km) were obtained by normalizing Vmax/Km by the
`concentration of active enzyme determined in active-site
`titration measurements. The reproducibility of kinetic pa-
`rameters was verified from two independent sets of rate
`data. Based on the uncertainty of the least-squares fitting,
`the individual kinetic parameters had relative errors ranging
`from 0.25% to 13.6%, with the majority being less than
`10%.
`
`Thermal Stability
`Samples were immersed in Tris-HCl buffer (pH 7.8) and
`incubated in a shaker at 75°C (50°C for a-CT). Periodically,
`samples were removed from the incubation solution, rinsed
`with buffer at room temperature, and assayed for residual
`activity as described previously. Remaining activity was
`
`Protein Adsorption
`
`Bovine serum albumin (BSA) was chosen as a model pro-
`tein for protein adsorption studies. Experiments were car-
`ried out by immersing a sample film of 6 cm2 in a propylene
`centrifuge tube containing 2 mL of 20 mg/mL protein so-
`lution in 50 mM phosphate buffer (pH 7.0). Tubes were
`shaken at 30°C for 24 h at 250 rpm. The film was then
`removed from the tubes, and the remaining protein solution
`was vortexed. The protein concentration remaining in solu-
`tion was determined according to the amino acid analysis
`procedure described by Sears and Clark (1993).
`
`RESULTS AND DISCUSSION
`
`Preparation of Immobilized Enzyme PDMS Films
`
`Three different methods were used to immobilize CT and
`pronase in a PDMS matrix. Figure 1 summarizes the prepa-
`ration of immobilized enzyme PDMS films by methods 1
`and 2. Sol-gel entrapment, the first step of method 1, is a
`convenient way to synthesize a host matrix and entrap en-
`zymes or other proteins while retaining their functional
`characteristics (Braun et al., 1990; Ellerby et al., 1992). To
`this end, enzymes were entrapped in sol-gel particles, the
`particles dispersed in a solution of PDMS in THF, and the
`PDMS cured via a room-temperature vulcanizing process
`that involved condensing silanol-terminated PDMS with the
`silane crosslinker TMOS, catalyzed by dibutyltin dilaureate.
`The covalent attachment approach (method 2) produced a
`material in which the chemically modified enzyme is cova-
`lently bound to the PDMS matrix. This method requires the
`initial chemical treatment of enzyme with 3-aminopropyl-
`triethoxysilane (APTS) to provide a chemical functionality
`reactive with PDMS. The modified enzyme was then incor-
`porated into polydimethylsiloxane by a condensation reac-
`tion between silanol end groups in PDMS and ethoxy
`groups in APTS. Figure 1 shows that both methods 1 and 2
`produced homogeneous, immobilized enzyme films. Both
`preparations were suitable for coating various materials fol-
`lowing pretreatment of the surface with epoxy adhesive
`(e.g., steel, neoprene, and PVC), as shown in Figure 2. A
`third method involving direct surface attachment (method 3)
`is a more conventional way to immobilize enzymes onto a
`silica support, and was employed for the sake of compari-
`son. In this case, the surface of a pre-made PDMS film was
`activated with 3 M HCl and derivatized with APTS, fol-
`lowed by covalent attachment of enzyme using glutaralde-
`hyde. Thus, this method requires that film formation pre-
`cedes enzyme attachment, and does not produce a biocata-
`lytic composite suitable for coating different surfaces.
`Methods 1 and 2 are also suitable for preparing porous
`immobilized enzyme membranes. To demonstrate this con-
`
`KIM, DORDICK, AND CLARK: BIOCATALYTIC FILMS AND PAINTS
`
`477
`
`

`

`Figure 1.
`
`Incorporation of enzyme into polydimethylsiloxane polymers via sol-gel entrapment (method 1) and covalent attachment (method 2).
`
`cept, an immersion precipitation process (Kim et al., 1999a,
`1999b; Mulder, 1991; Vadalia et al., 1994) was employed to
`prepare membranes from immobilized enzyme PDMS. In
`this process, a homogeneous PDMS solution in THF is con-
`tacted with water (the nonsolvent), and subsequent ex-
`change of THF and water across the interface effects liquid–
`liquid phase separation resulting in the porous membrane.
`To prevent the pore structure from collapsing during drying,
`polysulfone was added to the PDMS as a reinforcing agent.
`Figure 2a shows the porous structure of a membrane pre-
`pared with immobilized pronase PDMS and polysulfone.
`The pore size can be controlled by adjusting process pa-
`rameters such as polymer concentration, molecular weight
`of the PDMS, and the choice of solvent/nonsolvent pair
`(Reuveres and Smolders, 1987; Reuvers et al., 1987).
`
`Preparation of Biocatalytic Paint
`
`A PDMS-based enzyme-containing paint was formulated to
`demonstrate the feasibility of a biocatalytic paint with po-
`tential for application as a fouling-resistant coating. Con-
`ventional antifouling paints contain toxic agents such as
`cuprous oxide or tributyltin, which are widely used as bio-
`cides. In lieu of such compounds, our formulation contained
`silanol-functionalized enzyme (method 2) for biocatalytic
`activity. The PDMS was polymerized with iron acetylace-
`tonate, which acted as both a polymerization catalyst and
`red-orange pigment (Fig. 2d). Once air-dried, the painted
`surface was smooth and firm to the touch. Because the
`enzyme was covalently attached to the PDMS, there was no
`
`apparent release of enzyme from the final formulation (data
`not shown).
`
`Active-Site Concentration and
`Catalytic Properties
`
`Active-site titration in aqueous buffer, performed with the
`fluorogenic a-CT substrate MUTMAC, revealed that the
`percentage of active and accessible immobilized a-CT
`ranged from 1.8% of the immobilized enzyme for the paint
`to 13% for method 2 (Table I). The higher active enzyme
`content obtained in method 2 suggests that the former con-
`tains a more porous network than the latter. Indeed, it is
`expected that a paint would consist of a dense packing of
`polymer chains, thereby resulting in reduced enzyme acces-
`sibility. In both cases, samples were also prepared using
`polyvinylpyrrolidone (PVP) as a lyoprotectant during freeze
`drying, which resulted in much more active formulations.
`Polyethylene-glycol, KCl, and soybean trypsin inhibitor
`were also used as possible lyoprotectants but none of these
`produced significant activation (data not shown).
`The a-CT preparations were assayed in aqueous buffer
`for the hydrolysis of the chromogenic substrate suc-AAPF-
`pNa. In general, Km increased upon immobilization and kcat
`decreased. Thus, the catalytic efficiency, kcat/Km, decreased
`upon immobilization, with method 1 yielding the highest
`value. The exact cause(s) of the substantial reduction in
`kcat/Km remain to be determined; however, possibilities in-
`clude conformational changes to the enzyme induced by
`local crowding of enzyme molecules and/or unfavorable
`
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`Figure 2. Various formulations of biocatalytic films and paints. (a) Scanning electron micrograph of pronase-containing PDMS membrane prepared using
`the immersion precipitation process (cross-section, 950-fold magnification). (b) Pronase-containing PDMS films (from left: method 2, method 1, and porous
`membrane). (c) a-CT-containing PDMS films coated onto a steel plate and a neoprene tube. (d) PDMS-based biocatalytic paints, prepared with (from left)
`cuprous oxide and dibutyltin dilaureate, iron acetylacetonate, and dibutyltin dilaureate.
`
`interactions between the enzyme and the surrounding hy-
`drophobic matrix, and, in the case of method 2, partial de-
`naturation resulting from the formation of covalent bonds
`between the enzyme and PDMS during polymerization.
`Pronase, an unusually nonspecific protease, was also for-
`mulated into paint and films. Pronase films and paint were
`all active for the hydrolysis of LpNa (Table II), as reflected
`by the values of Vmax/Km for the immobilized enzyme (kcat/
`Km could not be calculated because no active-site titrating
`agent is available for pronase). Interestingly, method 2
`yielded the higher value of Vmax/Km for immobilized pro-
`nase, in strong contrast to the results for kcat/Km of immo-
`
`bilized a-CT. Moreover, including PVP during the freeze-
`drying step had no effect on the activity of pronase films
`prepared by method 2 (data not shown). The pronase-
`containing paint had activity comparable to that of the films.
`In addition, the membrane prepared by immersion precipi-
`tation showed considerable activity, illustrating that bio-
`catalytic membranes can be prepared at room temperature
`using the appropriate solvent/nonsolvent system.
`
`Thermal Stability of Films and Paints
`Figure 3 shows the thermal stability of the biocatalytic
`PDMS films and paints determined by the initial reaction
`
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`
`479
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`

`Table I. Activity of chymotrypsin PDMS films for hydrolysis of
`succinyl-Ala-Ala-Pro-Phe-p-nitroanilide.
`
`Preparation
`
`Soluble enzyme
`Method 1
`Method 2
`Method 2a
`Method 2b
`Method 3
`Paint
`Paintb
`
`Enzyme
`loading
`(wt%)
`
`100
`5.6
`4.2
`2.9
`0.9
`0.30
`1.3
`0.18
`
`Active site
`conc. (%)
`
`95
`2.4
`3.9
`4.5
`13
`8.4
`1.8
`10
`
`Km
`(mM)
`
`0.049
`0.17
`2.2
`1.7
`0.76
`1.2
`2.1
`1.7
`
`kcat
`(s−1)
`
`12
`1.2
`0.070
`0.060
`1.0
`0.48
`0.025
`0.65
`
`kcat/Km
`(s−1 M−1)
`
`2.5 × 105
`7.1 × 103
`32
`34
`1.3 × 103
`4.0 × 102
`12
`3.7 × 102
`
`Method 1, sol-gel entrapment; method 2, covalent attachment; method 3,
`surface attachment.
`aFilm was solution cast in tetrahydrofuran.
`bFilm and paint were prepared with PVP-treated enzyme powder.
`
`rates measured after incubation at 75°C for pronase activity
`and at 55°C for a-CT activity. In each case, the paint ex-
`hibited the most stable enzymatic activity, with a particu-
`larly large enhancement observed for pronase. For each
`film, method 2 gave the more stable preparation; for ex-
`ample, immobilized pronase retained ca. 77% of its original
`activity after 3 h while soluble pronase was completely
`inactivated. After 24 h, the immobilized pronase film re-
`tained 55% of its original activity (data not shown). The
`higher stability of films prepared by methods 1 and 2 com-
`pared to surface attachment (method 3) suggests that the
`enhanced stability is a consequence of the surrounding
`PDMS matrix. The pronase film also exhibited good storage
`stability, losing ca. 8% of its original activity upon shaking
`at 250 rpm at room temperature (pH 7.8) for 8 days (data not
`shown).
`
`Protein Adsorption
`
`Protein adsorption occurs widely in natural and man-made
`systems, and has had a great impact on many medical, en-
`vironmental, and biotechnological processes. Protein ad-
`sorption is particularly problematic in the biofouling of food
`
`Table II. Activity of pronase PDMS films for hydrolysis of L-Leu–
`p-nitroanilide.
`
`Preparation
`
`Soluble enzyme
`Method 1
`Method 2
`Method 2a
`Method 2 (membrane)b
`Method 3
`Paint
`
`Enzyme
`loading
`(wt%)
`
`100
`3.7
`4.1
`1.3
`1.6
`0.2
`1.2
`
`Km
`(mM)
`
`0.62
`2.0
`1.9
`1.3
`1.7
`0.85
`2.4
`
`Vmax/Km
`(l/min)
`
`0.21
`0.0032
`0.054
`0.015
`0.0042
`0.0020
`0.0082
`
`aFilm was solution cast in tetrahydrofuran.
`bMembrane was formed in THF/water system.
`
`Figure 3. Thermal stability of CT activity at 55°C (A) and pronase
`activity at 75°C (B). (s) soluble enzyme; (d) PDMS film by method 1;
`(j) PDMS film by method 2; (m) immobilized enzyme by method 3; (l)
`PDMS-based paint.
`
`processing equipment, ship hulls, medical implants, ultra-
`filtration membranes, and many other devices (Anderson et
`al., 1995; Lacasse et al., 1998; Mullick et al., 1998). Several
`factors are known to influence protein adsorption, such as
`conformational entropy of the protein, hydrophobic interac-
`tions, and electrostatic interactions (Chaiken et al., 1988;
`Norde, 1996). Whatever the mechanism of the process, pro-
`teins adsorb at the surface of most materials.
`To investigate the effect of immobilized pronase on pro-
`tein adsorption to PDMS films, bovine serum albumin
`(BSA) was used as a model protein. As shown in Figure 4,
`the amount of BSA adsorbed on the immobilized pronase
`PDMS films clearly decreased as the relative proteolytic
`activity increased (normalized by the activity of 1 mg/mL of
`soluble pronase). Control experiments revealed that PDMS
`without enzyme (C1 and C2 in Fig. 4) and PDMS films
`containing immobilized BSA (to determine whether an im-
`mobilized nonenzymatic protein influences adsorption)
`showed no substantial difference in the amount of adsorbed
`protein (data not shown).
`Although it appears that protein adsorption was slightly
`higher for the film prepared by method 1 than for unmodi-
`fied PDMS, control experiments revealed that some sol-gel
`particles were released into solution during the adsorption
`process. Thus, the amount of protein adsorbed for method 1
`was adjusted by subtracting the amount of protein measured
`in the released sol-gel particles, leading to a slightly larger
`
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`These findings indicate that it may be possible to reduce
`surface fouling of solid materials by treating the surface
`with a PDMS film or paint containing immobilized enzymes
`that degrade the fouling biomolecules. Such treatment may
`be useful to reduce fouling by proteins and even bacteria in
`a wide range of applications (e.g., as antifouling paints and
`filters) and as protective coatings for medical devices.
`
`References
`
`Anderson JM, Ziats NP, Azeez A, Brunstedt MR, Stack S, Bonfield TL.
`1995. Protein adsorption and macrophase activation on polydimethyl-
`siloxane and silicone rubber. J Biomater Sci Polym Ed 7:159–169.
`Avnir D, Braun S, Lev O, Ottolenghi M. 1994. Enzymes and other proteins
`entrapped in sol-gel materials. Chem Mater 6:1605–1614.
`Blanch HW, Clark DS. 1995. Biochemical engineering. New York: Marcel
`Dekker.
`Braun S, Rappoport S, Zusman R, Avnir D, Ottolenghi M. 1990. Bio-
`chemically active sol-gel glasses—The trapping of enzymes. Mater
`Lett 10:1–5.
`Chaiken IM, Shai Y, Fassina G, Caliceti P. 1988. Current development of
`analytical affinity chromatography: Design and biotechnological uses
`of molecular recognition surfaces. Die Makromol Chem Macromol
`Symp 17:269–279.
`Dave BC, Dunn B, Valentine JS, Zink JI. 1994. Sol-gel encapsulation
`methods for biosensors. Anal Chem 66:A1120–A1127.
`Ellerby LM, Nishida CR, Nishida F, Yamanaka SA, Dunn B, Valentine JS,
`Zink JI. 1992. Encapsulation of proteins in transparent porous silicate
`glasses prepared by the sol-gel method. Science 255:1113–1115.
`Gill I, Ballesteros A. 1998. Encapsulation of biologicals within silicate,
`siloxane, and hybrid sol-gel polymers: An efficient and generic ap-
`proach. J Am Chem Soc 120:8587–8598.
`Gill I, Ballesteros A. 2000. Bioencapsulation within synthetic polymers
`(Part 1): Sol-gel encapsulated biologicals. Tibtech 18:282–296.
`Gill I, Pastor E, Ballesteros A. 1999. Lipase–silicone biocomposites: Ef-
`ficient and versatile immobilized biocatalysts. J Am Chem Soc 121:
`9487–9496.
`Kim YD, Kim JY, Lee HK, Kim SC. 1999a. Formation of polyurethane
`membranes by immersion precipitation. I. Liquid–liquid phase sepa-
`ration in a polyurethane/DMF/water system. J Appl Polym Sci 73:
`2377–2384.
`Kim YD, Kim JY, Lee HK, Kim SC. 1999b. Formation of polyurethane
`membranes by immersion precipitation. II. Morphology formation. J
`Appl Polym Sci 74:2124–2132.
`Lacasse FX, Filion MC, Philips NC. 1998. Influence of surface properties
`at biodegradable microsphere surfaces: Effects on plasma protein ad-
`sorption and phagocytosis. Pharm Res 15:312–317.
`Mulder MHV. 1991. Basic principles of membrane technology. Amster-
`dam: Elsevier.
`Mullick A, Griffith CM, Flickinger MC. 1998. Expanded and packed bed
`albumin adsorption on fluoride modified zirconia. Biotechnol Bioeng
`60:333–340.
`Norde W. 1994. Protein adsorption at solid surfaces: A thermodynamic
`approach. Pure Appl Chem 66:491–496.
`Norde W. 1996. Driving forces for protein adsorption at solid surfaces.
`Macromol Symp 103:5–18.
`Owen MJ. 1988. Low surface energy organic polymers. Comm Inorg
`Chem 7:195–213.
`Reetz MT, Zonta A, Simpelkamp J. 1996. Efficient immobilization of
`lipases by entrapment in hydrophobic sol-gel materials. Biotechnol
`Bioeng 49:527–534.
`Reuvers AJ, Smolders CA. 1987. Formation of membranes by means of
`immersion precipitation. 2. The mechanism of formation of mem-
`branes prepared from the system cellulose acetate–acetone–water. J
`Membr Sci 34:67–86.
`Reuvers AJ, Van den Berg JWA, Smolders CA. 1987. Formation of mem-
`
`Figure 4. Proteolytic activity and adsorption of BSA onto the immobi-
`lized pronase films. The error bars correspond to the standard deviations of
`at least four measurements performed with two different preparations. The
`sample size for measuring proteolytic activity was 0.5 cm2, and the activity
`was normalized by the activity of 1 mg/mL of soluble pronase in 0.2 mM
`azocasein solution. C1, PDMS control; C2, PDMS control containing sol-
`gel particles without enzyme; PR1, film by method 1 (pronase 4 0.7 wt%);
`PR2, film by method 2 (pronase 4 0.9 wt%); PR3, film by method 2
`(pronase 4 1.1 wt%, solution cast in THF); CT1, film by method 2
`(chymotrypsin 4 0.5 wt%, solution cast in THF); CT2, film by method 2
`(PVP-treated chymotrypsin 4 0.3 wt%, solution cast in THF).
`
`experimental error in the final calculation of adsorbed pro-
`tein. It is therefore reasonable to conclude that, within ex-
`perimental error, the amount of protein adsorbed for method
`1 did not exceed that for the PDMS control. On the other
`hand, no particles were released from films prepared by
`method 2. Finally, the important role of protease activity in
`PDMS coatings in reducing protein adsorption was further
`confirmed by the PVP-activated a-CT film (CT2), which
`showed less protein adsorption than the unmodified a-CT
`film (CT1).
`The effect of enzyme activity on protein adsorption was
`also evident for the poly(ethylene terephthalate) (PET) film
`coated with biocatalytic paint. After 42-h incubation, the
`amount of BSA adsorbed to the uncoated PET film was 950
`± 57 ng/cm2, compared with only 350 ± 18 ng/cm2 for the
`film coated with a-CT-containing paint.
`A possible explanation for the reduced adsorptoin of pro-
`tein to the immobilized enzyme PDMS films and paints
`involves a change in entropy due to degradation of poten-
`tially adsorbed protein. In fact, the molecular weight of
`potentially adsorbing protein had a substantial effect on ad-
`sorption. Control experiments showed that pretreatment of
`BSA with concentrated HCl decreased the amount of ad-
`sorbed protein to 40% of that of intact BSA. Partial hydro-
`lysis of the BSA into smaller peptides was confirmed by
`sodium dodecylsulfate-electrophoresis (data not shown). A
`breakdown of protein structure due to enzymatic cleavage
`of peptide bonds by protease in the PDMS film should
`increase the conformational entropy of the adsorbing pro-
`tein. An increase in conformational entropy will decrease
`the Gibbs free energy for adsorption (Norde, 1994, 1996),
`thus reducing the likelihood that the protein will adsorb to
`the surface.
`
`KIM, DORDICK, AND CLARK: BIOCATALYTIC FILMS AND PAINTS
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`branes by means of immersion precipitation. 1. A model to describe
`mass transfer during immersion precipitation. J Membr Sci 34:45–65.
`Sears PS, Clark DS. 1993. Comparison of soluble and immobilized trypsin
`kinetics: Implications for peptide synthesis. Biotechnol Bioeng 42:
`118–124.
`Shtelzer S, Braun S. 1994. An optical biosensor based on glucose oxidase
`immobilized in sol-gel silicate matrix. Biotechnol Appl Biochem 19:
`293–305.
`Vadalia HC, Lee HK, Myerson AS, Levon K. 1994. Thermally induced
`phase separation in ternary crystallizable polymer solutions. J Membr
`Sci 89:37–50.
`
`Venon DL, Gudipati E. 1995. Entrapment of enzymes using organo-
`functionalized polysiloxane copolymers. Biochim Biophys Acta 1250:
`117–125.
`Vincent HL, Bausch GG. 1997. Silicone fouling release coatings. Naval
`Res Rev XLIX:39–45.
`Wattermann B, Berger HD, Sonnichsen H, Willemsen P. 1997. Perfor-
`mance and effectiveness of non-stick coatings in seawater. Biofouling
`11:101–118.
`Wu S, Lin J, Chan SI. 1994. Oxidation of dibenzothiophene catalyzed by
`heme-containing enzymes encapsulated in sol-gel glass. Appl Bio-
`chem Biotechnol 47:11–20.
`
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