Activity and Spatial Distribution of Candida antarctica Lipase B

Article pubs.acs.org/Langmuir

Activity and Spatial Distribution of Candida antarctica Lipase B Immobilized on Macroporous Organic Polymeric Adsorbents Anne V. F. Nielsen,† Pavle Andric,§ Per M. Nielsen,‡ and Lars H. Pedersen*,† †

Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark ‡ Department of Bioenergy Opportunities, Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark § Department of Solid Products Development, Novozymes A/S, Smoermosevej 11, DK-2880 Bagsvaerd, Denmark ABSTRACT: A systematic study of the influence of carrier particle size (500−850 μm) and enzyme load (26 200−66 100 lipase activity units (LU)/g dry carrier) on the content and activity of Candida antarctica lipase B (CALB) immobilized by adsorption onto macroporous poly(methyl methacrylate) (PMM) and polystyrene (PS) carriers was conducted. Furthermore, localization of CALB on the carrier was investigated by light and fluorescence microscopy of freeze microtome sliced catalyst particles. Fluorescence microscopy showed localization of enzyme in an outer rim of 50−85 and 10−20 μm thickness for the PMM and PS catalysts, respectively, whereas no rim was observed in the absence of enzyme. Statistical analyses showed that carrier type was the major effect in determining the activities of the catalysts, with enzyme load being the second most significant effect and particle size also exerting a significant, yet smaller, effect. The PMM catalysts showed higher activities compared to PS catalysts, possibly indicating that the microenvironment interactions of CALB with the PMM are more favorable than with the PS carrier, resulting in a higher specific enzyme activity. Furthermore, smaller particles and higher enzyme load had a positive influence on the activities within the investigated ranges, and the carrier type and enzyme load interaction was statistically significant (p < 0.001).

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INTRODUCTION

methacrylate) particles (Novozym 435) resulted in a 80−100 μm thick loading front.10 This paper describes a systematic study of the effects of carrier particle size and enzyme load on the content and activity of CALB immobilized by adsorption onto macroporous poly(methyl methacrylate) and polystyrene carriers. Furthermore, the distribution and localization of CALB were investigated by light and fluorescence microscopy of freeze microtome sliced catalyst particles.

Immobilization of enzymes to a solid carrier facilitates efficient separation and recovery of the biocatalyst from the reaction mixture, making it attractive to downstream processing and possible to reuse. Most importantly, physical enzyme immobilization has been shown to affect activity and selectivity and to improve stability of the enzyme. In this respect, the microenvironment imposed on the enzyme by the physicochemical properties of the carrier is extremely important, and in the case of lipases, hydrophobic carriers have been shown to increase specific enzyme activity.1,2 Particularly macroporous organic polymeric adsorbents have been shown to be efficient carriers of lipases for immobilization and use as catalysts in a range of production processes including deracemization as well as chiral and regioselective organic synthesis of drug precursors and intermediates for fine chemicals and pharmaceuticals.3−6 Research in immobilization of lipases has included identification of carriers and strategies for optimizing enzyme load, activity, and stability.7−9 The distribution of Candida antarctica lipase B (CALB) covalently immobilized onto epoxy-activated macroporous poly(methyl methacrylate) spherical particles has been studied by infrared microspectroscopy showing that CALB was localized within a loading front of 50 μm thickness.10 Similarly, CALB immobilized on 600 μm diameter spherical poly(methyl © 2014 American Chemical Society

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EXPERIMENTAL SECTION

A liquid formulation of CALB kindly provided by Novozymes A/S, Bagsvaerd, Denmark, was used for the investigation. As estimated by SDS-PAGE, the lipase content was 95% of the protein and the specific activity of the liquid formulation was 20 kLU/g. CALB was immobilized on two different carriers: poly(methyl methacrylate) (PMM) and polystyrene (PS), the basic properties of which are listed in Table 1. Carrier particles were fractionated into particle sizes of 500−600 μm (referred to as the 550 μm fraction), 600−710 μm (655 μm fraction), and 710−850 μm (780 μm fraction), and enzyme load in immobilization solutions was varied with 26 200, 37 200−37 300, and 52 400−66 100 lipase activity units (LU)/g dry carrier. Received: January 3, 2014 Revised: April 15, 2014 Published: April 15, 2014 5429

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Immobilization. 20−50 g carrier size fractions were transferred to 250 mL cubic blue cap flasks, and 30−88 g of CALB solution and 40− 116 g of deionized water were added. During immobilization pH was 4.5, and the flasks were incubated at room temperature at 200 rpm in a shaking incubator for 20 h. After incubation, the immobilization solutions were filtered, and the filtrate was frozen at −20 °C. The catalysts were washed with deionized water and dried to a final dry matter content of 98−100% (w/w). Dry matter content of the carriers was measured gravimetrically (approximately 3 g of wet carrier) by drying overnight at 105 °C. Light and Fluorescence Microscopy of Sliced Catalysts. Novozym 435 (NZ435: CALB immobilized on PMM carrier particles with chemical and physical properties identical to the PMM carrier used in this work, kindly provided by Novozymes A/S), PMM, and PS carriers with and without immobilized enzyme were embedded into a matrix of Sakura Finetek Tissue-Tek OCT (optimal cutting temperature) compound (Sakura Finetek, Torrance, CA, US), cooled overnight at 5 °C, and then frozen to −80 °C for at least 15 h prior to slicing. The cast was removed from the mold and mounted onto a metal plate, which was fastened in a Miles Tissue-Tek II Cryostat freezing microtome (Sakura Finetek) set to a cutting temperature of −20 °C and slice thickness of 30 μm. Slices were transferred to a glass slide at ambient temperature, where the OCT compound melted and left the particle slices available for viewing in the Zeiss AX10 Imager A1 equipped with light sources HBO100 and HAL100 and a camera (CoolSnap cf, Photometric). Exposure times were between 7 and 100 ms, and a Zeiss filter BP 365/12, FT 395, LP 397 was used. Protein Analysis. Protein content of the CALB solution, immobilization solution (after immobilization), carrier materials, and catalysts were measured in duplicate using a FP-528 nitrogen/protein determinator from LECO Corporation (St. Joseph, MI), which was calibrated using EDTA (LECO) as standard and air samples as blanks. For the dry samples, 50 mg was transferred to tin foil cups (LECO), and for the liquid samples, 170 mg was transferred to tin capsules (LECO). The Kjeldahl factor (6.25) was used to convert nitrogen measurements to protein mass. Activity Assays on Immobilized Products. Activity of the catalysts was determined by an esterification process carried out according to Kirk and Christensen11 and Nakaoki et al.7 with the following conditions: molar substrate ratio of lauric acid:propanol was 1:1, reaction was carried out in closed conical flasks at 60 °C and 200 rpm. 10 μL samples were taken after 20 min and transferred to 1 mL of heptane. Reaction progress was measured via quantification of propyl laurate and lauric acid by gas chromatography (n = 4). Activity was reported in propyl laurate units (PLU), where 1 PLU corresponds to the amount of enzyme that produces 1 μmol of propyl laurate under the conditions given above and is given in μmol g catalyst−1 min−1. All catalysts, carriers without enzyme, and the commercial reference, NZ435, were analyzed. Statistical Data Analysis. Analysis of variance (ANOVA) was carried out using JMP 9 software (SAS, Cary, NC). Particle size, carrier type, enzyme load and their first-order interactions were analyzed as fixed effects with protein content and enzyme activity as the response variables. Prediction profiles were generated from the regression model visualizing the dependence of specific activity on the individual immobilization parameters: particle size and enzyme load.

Table 1. Properties of the Two Carrier Materials property specific surface area [m2/g] pore diameter [nm] chemical composition

poly(methyl methacrylate) (PMM)

polystyrene (PS) ≥800

130

26a

15b

aliphatic polymer w/ aromatic cross-linking

methacrylate polymer w/ divinylbenzene cross-linking

a

Most prevalent diameter based on pore volume; pore diameters ranging from 2 to 32 nm. bAverage.

The choice of particle sizes was based on the size distribution of the carriers, and the target enzyme loads were scaled with the average external surface area (πd2) of the different size fractions of particles. An overview of the experiments performed is seen in Table 2.

Table 2. Overview of Experimentsa carrier type

particle size [μm]

enzyme load [LU/g dry carrier]

PS PS PS PS PS PMM PMM PMM PMM PMM PMM PMM PMM PMM PMM PMM PMM PMM PMM

550 550 655 780 780 550 550 550 655 655 655 655 655 655 655 780 780 780 780

26 192 65 935 66 132 26 191 65 733 26 199 37 280 52 376 26 214 37 207 37 210 37 223 37 302 54 731 54 809 26 187 37 227 37 277 54 784

a Particle sizes are given as mean values, i.e., 550 μm covers the 500− 600 μm fraction, 655 μm the 600−710 μm fraction, and 780 μm the 710−850 μm fraction. The enzyme load (enzyme activity in immobilization solution) is given per g dry carrier.

Particle Size Distribution and Fractionation. Polystyrene (PS) macroporous beads were washed with deionized water prior to use and dried at 30 °C together with the poly(methyl methacrylate) (PMM) beads. Particle size distributions of both carrier materials were determined (n = 4) by laser diffraction (low angle light scattering) using a Mastersizer MS2000 instrument with a Scirocco 2000S module (Malvern Instruments, UK) using the following procedure: 10 g of carrier; measurement time: 5 s; snaps: 5000; background time: 12 s; snaps: 12 000; dispersive pressure: 0.25 bar; vibration feed rate: 50% of maximum vibration. The PMM carrier was additionally analyzed on an EasySieve System: Vibratory Sieve Shaker AS 200 control and EasySieve Comfort software from Retsch, Germany; XS 6002S scale from Mettler Toledo, Switzerland; sieves mesh sizes: 250, 300, 355, 425, 500, 600, 710, 850, 1000, 1180, 1400, 1700, and 2000 μm. Particles were loaded onto the sieves, which were subjected to a 3 min shaking program, after which the masses of fractions on the different sieves were determined gravimetrically. For particle size fractionation, sieving was carried out using the following mesh sizes 850, 710, 600, and 500 μm, and a 3 min shaking program with the equipment described above.

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RESULTS AND DISCUSSION Immobilization Procedure. The degree of immobilization ranged between 24% and 65% evaluated as the fraction of initial protein recovered in the solid fraction with averages and standard deviations for PMM and PS carriers being 52 ± 5% and 49 ± 18%, respectively. The remaining lipase activity in the immobilization solution was observed to be higher for the PS than for the PMM carriers. No systematic correlations between the degree of immobilization and particle size or enzyme load were observed. 5430

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Fluorescence Microscopy of Sliced Particles. Carrier Particles. The commercial reference, NZ435, PMM, and PS particles with and without immobilized enzyme were cut into 30 μm thick slices and investigated by light and fluorescence microscopy. In Figure 1, light and fluorescence microscopy images of sliced PMM particles are shown.

with regard to enzyme loading. Most likely, the observation was a result of the former, as these fully fluorescent slices were generally obtained from smaller particles. For some of the images, two rims of different intensities surrounding a darker core were observed, as vaguely demonstrated in Figure 3b. The two layers observed seemed

Figure 1. Sliced PMM particles without enzyme viewed by (a) light microscopy and (b) fluorescence microscopy. The scale bar corresponds to 100 μm.

Figure 3. Sliced PMM catalyst (655 μm; 55 000 LU/g) viewed by (a) light microscopy and (b) fluorescence microscopy. The scale bar corresponds to 100 μm.

slightly visible even in the light microscope (Figure 3a, bottom left), but only for some of the particles, whereas other particles appeared homogeneous throughout the entire radius (not shown). These observations may be caused by variation in particle density creating physical barriers within the carrier particles limiting enzyme penetration, or it could be merely a question of diffusion limitation, which may be overcome by increasing the incubation time of the carriers in the enzyme solution. Experimental Catalysts. Looking at the PMM catalysts of this study (see Figure 3a,b), the observations were similar to NZ435. Thus, loading fronts of 50−85 μm were observed as well as slices that were fluorescent throughout the whole cross section. The PS catalysts looked quite different from the PMM catalysts in the microscope, and the PS carrier without enzyme seemed to have higher fluorescent background signal (homogeneous throughout particle; not shown). However, as for the PMM catalysts, a fluorescent rim was present in the PS catalysts, although these rims were generally thinner than the fronts observed for the PMM catalysts with about 10−20 μm visible rim of enzyme (Figure 4).

Commercial Reference. Figure 2 shows images of the commercial reference NZ435 corresponding to the images of the empty PMM carrier in Figure 1.

Figure 2. Sliced PMM catalyst (commercial product, NZ435) viewed by (a) light microscopy and (b) fluorescence microscopy. The scale bar corresponds to 100 μm.

From the images in Figures 1 and 2, it is clear that the fluorescence signal from NZ435 is not present in the PMM particles without enzyme, implying that the presence of enzyme caused the fluorescence signal, possibly as result of light scattering from the enzyme. Other studies investigating the enzyme distribution on NZ435 gave observations very similar to the present investigation. Mei et al.12 used infrared microspectroscopy to investigate the enzyme localization on the particles and obtained an image of a particle with an apparent rim of enzyme protein along the outer perimeter of the particle of approximately 75 μm thickness. Wiemann and co-workers visualized an apparent enzyme protein rim of 20−30 μm thickness on NZ435 using light microscopy of toluene-soaked particles.13 These observations obtained by very different methods all point to the localization of the enzyme protein in an outer rim of the PMM particle. Importantly, our investigations proved the absence of a fluorescent rim in the PMM particles without enzyme, indicating that the observed fluorescent signal primarily was caused by the presence of enzyme. For the particles in Figure 2b, the rims of protein were about 70 μm thick on average and similar thicknesses were seen for the other images of NZ435. Some catalyst slices were fluorescent all the way through (see Figure 2b). Most likely, this is a result of either the slice being cut closer to the surface and therefore containing enzyme throughout the entire diameter or a difference between particles

Figure 4. Sliced PS catalyst (550 μm; 66 100 LU/g) viewed by fluorescence microscopy. The scale bar corresponds to 100 μm.

The present study showed no correlations between the thickness of the loading front and the particle size. In both the fluorescence and light microscopy images, occasional visible droplets caused by air in the microscopy oil covering the particle slices were observed. Influence of Immobilization Parameters on Protein Content of Catalysts. Based on nitrogen measurements, the amount of protein in the catalysts was determined. A positive correlation between enzyme load and adsorbed amount of 5431

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protein was observed (see Figure 5). This was expected up to a certain concentration at the point of saturation. A statistical

Table 3. Theoretical enzyme loads (enzyme mass/carrier mass) on PS and PMM carriers based on monolayer and geometrical assumptions on CALB occupation of carrier surfacea assumptions enzyme located:

throughout particle [g/g]

rim [g/g]

carrier

circularb

squarec

circular

square

PS 550 μm PS 655 μm PS 780 μm PMM 550 μm PMM 655 μm PMM 780 μm

3.49

2.19

0.57

0.36

2.04 1.79 1.56 0.33 0.29 0.25

1.28 1.13 0.98 0.21 0.18 0.16

a

Assumptions include the area occupied by CALB on the carrier: Circular with a diameter corresponding to the average CALB diameter14 or rectangular with an area corresponding to the largest dimensions of CALB15 and the physical localization of enzyme in the particles: All the way through the particle or in a rim near the external surface of the particles (70 μm thickness for PMM and 20 μm for PS). Values are given as mass of enzyme per mass of carrier. b Corresponding to 4.36 mg enzyme/m2 carrier surface area. c Corresponding to 2.74 mg enzyme/m2 carrier surface area.

Figure 5. Measured protein % (w/w) as a function of initial protein content in the immobilization solution on dry carrier basis. Protein measurements have been corrected for dry matter and carrier protein content. The estimated lipase content of the total protein in the immobilization solution was 95%.

transport conditions were expected, as the pore size of the PMM carrier was even smaller (average 15 nm), whereas the PS carrier pore sizes were in the same range (26 nm) as in the experiment by Chen et al.16 Consequently, it is concluded that saturation (here given by the theoretical monolayer loads calculated in Table 3) of the carrier with enzyme might not have been achieved with 20 h of incubation as used in the present investigation. In principle, this means that the carrier surface would have allowed space for the CALB molecules to be adsorbed and positioned without aggregation. With immobilization of CALB on PS resins varying in particle sizes (diameters of 35−600 μm) and pore sizes (30− 100 nm), using the same immobilization procedure as in their previous study,16 Chen and co-workers17 achieved enzyme loadings ranging from 0.079 to 0.087 g/g. These were similar to what was obtained in the present investigation (0.044−0.10 g/ g) with a PS carrier having smaller pore sizes and with a lower pH during immobilization. The differences in pH during immobilization between the present study and the investigations by Chen et al.16,17 on the acidic and the alkaline side of the pI= 6.0 of CALB,11 respectively, did not seem influence the protein load of the catalysts, indicating that the protein adsorbed equally well in the pH range investigated. Given that the resins used by Chen et al.17 had lower specific surface areas, these minor differences could be attributed to the larger pore sizes, allowing more enzyme to penetrate into the particles in their experiment. In their experiments, Chen and co-workers observed faster adsorption for the PS resins (24 h before saturation for the large particles with diameters of 350−600 μm) compared to the PMM resins, explained by the relatively more hydrophobic properties of the former. The higher capacity of the PS resins for CALB was confirmed by Scatchard analysis, showing a maximum loading of 0.141 g/g carrier corresponding to 0.707 mg/m2 for the 35 μm particles.16,17 Influence of Immobilization Parameters on Specific Catalyst Activity. Specific activities of the catalysts were evaluated by formation of propyl laurate ester from propanol and lauric acid at 60 °C. Two types of specific activities are reported: based on the mass of the final catalyst (catalyst-based,

analysis of variance of the influence of particle size, enzyme load, and carrier type showed (model p-value = 0.0004) that only enzyme load had a clear effect on protein content of the final catalysts (n = 1 for protein determination). Theoretical Enzyme Loads. For comparison of theoretical and measured protein loads, calculations were made based on the following assumptions regarding carrier geometry and obtaining a theoretical enzyme monolayer: (1) Shape of occupation area of CALB on carrier surface: (a) circular with a diameter corresponding to the mean diameter of CALB (4 nm) as in calculations by Torres-Salas and co-workers;14 (b) square with sides of 5 and 4 nm corresponding to the two largest dimensions of CALB as in calculations by Wannerberger and Arnebrant.15 (2) Occupation of internal pores in the carrier materials: (a) enzymes are distributed throughout the particles; (b) enzymes are located in a layer close to the external surface of the carrier particles as indicated by the microscopy carried out in this study. This layer is assumed to be 70 μm thick for PMM and 20 μm thick for PS particles, as estimated from the microscopy. The results of the calculations are seen in Table 3. For any set of assumptions the theoretical loads were, on average, up to 1 order of magnitude higher than the observed protein contents in the experiments of the present investigation (0.037−0.10 g/g). In experiments conducted by Chen and co-workers16 with immobilization of CALB on PMM resins (pH during immobilization was 7.8 and the enzyme load was 10% (protein mass/carrier mass)) with particle sizes ranging from 35 to 710 μm and average pore diameter of 25 nm enzyme loadings of 0.051−0.057 g/g were achieved, which were in the same range as the present investigation (0.037−0.080 g/g). Using Scatchard analysis, Chen et al.16 observed a maximum loading of 0.0718 g/g carrier, corresponding to 0.144 mg/m2 for the 35 μm particles. For the larger particles (560−710 μm), they did not achieve saturation until after 48 h of immobilization, which was related to the small pore size of the carrier, causing diffusional limitations at the core of the particles. Therefore, in the present investigation, similar mass 5432

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CB) or based on the measured protein content of the catalysts (protein-based, PB). The catalysts showed specific CB activities in a range similar to the commercial product NZ435. A tendency that larger particles had lower specific CB activity and a clear tendency that higher enzyme loads resulted in higher specific CB activities was observed. A theoretical evaluation of the effect of particle size on the specific CB activity using the Frössling correlation showed a 70% activity increase due to a reduction in particle diameter from 780 to 550 μm. However, the experiments showed a 10% increase, indicating that mass transport did not limit the process substantially. The main conclusion of the corresponding ANOVA of the influence of particle size, enzyme load, and carrier type on specific CB activity was that carrier type (p < 0.0001), enzyme load (p < 0.0001), interaction between carrier type and enzyme load (p < 0.001) and particle size (p < 0.002) were significant. Decreasing specific CB activity with increasing particle size was also found by Chen et al.16 for PMM catalysts, whereas they found no correlation between specific CB activity and particle size for PS catalysts,17 which was explained by differences in accessible surface area for the two carrier materials. With regard to the carrier effect, the average (± standard deviation) predicted specific CB activity for the PS catalysts was 3453 ± 442 and 7748 ± 232 μmol propyl laurate g catalyst−1 min−1 for the PMM catalysts, both evaluated at a particle size of 655 μm and an enzyme load of 35 000 LU/g dry carrier. The significance of the carrier−enzyme load interaction effect was particularly interesting, indicating that the specific PB activities of CALB immobilized on PS and PMM were influenced differently by increased enzyme loads. Prediction profiles for the influence of particle size and enzyme load on specific CB activity are shown in Figures 6 and 7.

Figure 7. Prediction profiles of the statistical analysis of specific CB activity. 655 μm particles. Dashed lines indicate 95% confidence intervals (CI).

Figure 8. Influence of protein content (measured as mass % protein of the catalysts) on the specific PB activities for PMM and PS carrier. Note that the two carriers are depicted on separate axes. The estimated lipase content of the protein concentrate was 95%.

loads were clearly not supported by our experimental observations. In contrast to these findings, Chen and co-workers16 observed for PMM resins with pore sizes of 25 nm and particle sizes of 35 μm that the specific PB activity increased with increasing enzyme content. It was shown that increased enzyme loading resulted in an increased fraction of active sites being exposed to the substrate and hypothesized that this could be an effect of aggregation, leading to favorable enzyme− enzyme interactions, or shorter distance between the individual enzyme molecules, somehow inducing more favorable interactions with the carrier surface. In the present investigation, however, the observed decrease in specific PB activity may reflect the difference in mass transfer properties, as the particles in the experiment by Chen et al.16 were smaller and had larger pores, both factors reducing mass transfer limitations. Thus, in the present investigation, the decrease in PB specific activity observed at higher loading for PMM seemed to be caused by mass transfer limitations. For PS catalysts, the correlation between the specific PB activity and the protein loading was not as clear as for the PMM catalysts (see Figure 8). For PS particles, Chen et al.17 found that the fraction of exposed active sites was higher on PS compared to PMM resins, indicating a favorable environment for CALB activity in the PS carrier. Because of this apparently

Figure 6. Prediction profile of the statistical analysis of specific CB enzyme activity. PMM catalyst, 35 000 LU/g. Dashed lines indicate 95% confidence intervals.

For PMM catalysts, a clear tendency that the average specific PB activity decreased with higher protein content was observed (see Figure 8). In agreement with the linear correlation between protein content and enzyme load (Figure 5), a corresponding decrease in the specific PB activity with increasing enzyme load was observed for the PMM catalysts. This may be caused by the formation of multiple enzyme layers with increased loading blocking active site exposure to the substrate partly or completely or by pore blocking or substrate mass transfer limitations. With reference to this speculation, our theoretical calculations on monolayer enzyme 5433

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(5) Jaeger, K.-E.; Ransac, S.; Dijkstra, B. W.; Colson, C.; van Heuvel, M.; Misset, O. Bacterial lipases. FEMS Microbiol. Rev. 1994, 15, 29−63. (6) Ritthitham, S.; Wimmer, R.; Pedersen, L. H. Polar co-solvents in tertiary alcohols effect initial reaction rates and regio-isomeric ratio ranging from 1.2 to 2.2 in a lipase catalysed synthesis of 6-O- and 6′O-stearoyl sucrose. Process Biochem. 2011, 46, 931−935. (7) Nakaoki, T.; Mei, Y.; Miller, L. M.; Kumar, A.; Kalra, B.; Miller, M. E.; Kirk, O.; Christensen, M.; Gross, R. A. Candida antarctica lipase B catalyzed polymerization of lactones: Effects of immobilization matrices on polymerization kinetics & molecular weight. Ind. Biotechnol. 2005, 1, 126−134. (8) Christensen, M. W.; Andersen, L.; Husum, T. L.; Kirk, O. Industrial lipase immobilization. Eur. J. Lipid Sci. Technol. 2003, 105, 318−321. (9) Arroyo, M.; Sánchez-Montero, J. M.; Sinisterra, J. V. Thermal stabilization of immobilized lipase B from Candida antarctica on different supports: Effect of water activity on enzymatic activity in organic media. Enzyme Microb. Technol. 1999, 24, 3−12. (10) Chen, B.; Hu, J.; Miller, E. M.; Xie, W.; Cai, M.; Gross, R. A. Candida antarctica lipase B chemically immobilized on epoxy-activated micro- and nanobeads: catalysts for polyester synthesis. Biomacromolecules 2008, 9, 463−471. (11) Kirk, O.; Christensen, M. W. Lipases from Candida antarctica: Unique biocatalysts from a unique origin. Org. Process Res. Dev. 2002, 6, 446−451. (12) Mei, Y.; Miller, L.; Gao, W.; Gross, R. A. Imaging the distribution and secondary structure of immobilized enzymes using infrared microspectroscopy. Biomacromolecules 2003, 4, 70−74. (13) Wiemann, L. O.; Nieguth, R.; Eckstein, M.; Naumann, M.; Thum, O.; Ansorge-Schumacher, M. B. Composite particles of novozyme 435 and silicone: Advancing technical applicability of macroporous enzyme carriers. ChemCatChem 2009, 1, 455−462. (14) Torres-Salas, P.; del Monte-Martinez, A.; Cutiño-Avila, B.; Rodriguez-Colinas, B.; Alcalde, M.; Ballesteros, A. O.; Plou, F. J. Immobilized biocatalysts: Novel approaches and tools for binding enzymes to supports. Adv. Mater. 2011, 23, 5275−5282. (15) Wannerberger, K.; Arnebrant, T. Comparison of the adsorption and activity of lipases from Humicola lanuginosa and Candida antarctica on solid surfaces. Langmuir 1997, 13, 3488−3493. (16) Chen, B.; Miller, M. E.; Miller, L.; Maikner, J. J.; Gross, R. A. Effects of macroporous resin size on Candida antarctica lipase B adsorption, fraction of active molecules, and catalytic activity for polyester synthesis. Langmuir 2007, 23, 1381−1387. (17) Chen, B.; Miller, M. E.; Gross, R. A. Effects of porous polystyrene resin parameters on Candida antarctica lipase B adsorption, distribution, and polyester synthesis activity. Langmuir 2007, 23, 6467−6474.

lower affinity of the PMM resin for CALB, it was discussed if the enzyme had an increased tendency to diffuse through the carrier and occupy a larger part of the particle compared to the PS particles, which is in agreement with our observations by microscopy showing a thinner loading front on PS compared to PMM particles. However, in our experiments, the specific PB activity was higher for PMM particles compared to PS particles, indicating either that the PMM catalyst selectively adsorbs CALB over other potential proteins in the CALB formulation or that possibly the mass transfer issues have been different in the two studies, as the activity in this study was measured using lauric acid as a substrate, whereas Chen et al.16,17 used the slightly smaller molecule ε-caprolactone, which was then polymerized.

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CONCLUSIONS Fluorescence microscopy showed localization of CALB enzyme in an outer rim of the PMM and PS catalysts. The thicknesses of these rims were 50−85 and 10−20 μm for the PMM and PS catalysts, respectively. The investigation proved the absence of such a rim in the carrier particles without enzyme, confirming that the observed fluorescent signal primarily was caused by the presence of enzyme. No correlations were found between enzyme load, particle size, and thickness of enzyme rim. Activity analyses showed that the carrier type had the major influence on the observed enzyme activities of the catalysts with the PMM carrier supporting increased specific activities. Higher enzyme loads resulted in higher specific catalyst-based (CB) activities, and finally, smaller particles seemed to increase the specific CB activities of the catalysts.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected] (L.H.P.). Present Address

A.V.F.N.: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Soeltofts Plads 229, 2800 Kgs. Lyngby, Denmark. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Solid Products Development and Research and Development departments of Novozymes A/S in Bagsvaerd, Denmark, for providing materials, lab facilities, and aid in the experimental planning and analysis work, Birger Stjernholm Madsen (Novozymes, Bagsvaerd) for help with the statistical analyses, and Poul Larsen (Aalborg University) for helping with the microscopy work.

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REFERENCES

(1) Noda, S.; Kamiya, N.; Goto, M.; Nakashio, F. Enzymatic polymerization catalyzed by surfactant-coated lipases in organic media. Biotechnol. Lett. 1997, 19, 307−309. (2) Kumar, A.; Gross, R. A. Candida antartica lipase B catalyzed polycaprolactone synthesis: Effects of organic media and temperature. Biomacromolecules 2000, 1, 133−138. (3) Anderson, E. M.; Larsson, K. M.; Kirk, O. One biocatalyst - many applications: The use of Candida antarctica B-lipase in organic synthesis. Biocatal. Biotransform. 1998, 16, 181−204. (4) Schmid, R. D.; Verger, R. Lipases: Interfacial enzymes with attractive applications. Angew. Chem., Int. Ed. 1998, 37, 1608−1633. 5434

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