Integration of Enzymatic Catalysts in a Reactive Distillation Column

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Integration of Enzymatic Catalysts in a Reactive Distillation Column with Structured Packings Rene Heils,*,† Annika Sont,†,§ Paul Bubenheim,‡ Andreas Liese,‡ and Irina Smirnova† †

Thermal Separation Processes, Hamburg University of Technology, Eißendorfer Str. 38, D-21073 Hamburg, Germany Technical Biocatalysis, Hamburg University of Technology, Denickestr. 15, D-21073 Hamburg, Germany

‡

S Supporting Information *

ABSTRACT: The integrated process setup of reaction and separation in reactive distillation can be favorable for enzymecatalyzed reactions that are often equilibrium-limited and/or inhibited by the product of the reaction. However, until now, no appropriate way has described the implementation of the enzymes into distillation columns. In this work, a special type of biocatalytic coating for commercially available structured packings was developed to enable enzymatic reactions in reactive distillation columns. Lipase CALB was immobilized within the silica-gel based coating and showed high long-term stability, i.e., the coatings can be stored for at least 50 days at room temperature without any significant loss of enzyme activity. In addition, the silica-gel coatings were stable in organic media and at increased temperatures. Reactive distillation experiments were carried out in a batch reactive distillation setup comprising of structured A3-500 packings from Montz (Hilden, Germany) coated with the catalytic silica gel. Stepwise removal of the low boiling point product from the distillate shifted the equilibrium to the product side and increased the conversion of the reactants from 60% to 98%, in comparison with the corresponding setup in a standard batch reactor. The stability of the silica-gel coating and loss of the enzyme was investigated during the distillation runs. Overall, the integration of an enzymatic reaction into a reactive distillation column offers new possibilities for the application of biocatalysts in organic synthesis.

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INTRODUCTION The increasing application of biocatalytic reactions in organic synthesis is due to the high regioselectivity, stereoselectivity, or enantioselectivity of enzymes. In contrast to chemical catalysts, enzymes selectively catalyze the conversion of specific enantiomers in a racemic mixture, resulting in a significant reduction of reaction steps in a synthesis route. Examples for enantiopure synthesis can be found in the production of therapeutics such as the antitumor agent epithilone A,1 agrochemicals such as (S)-indanofan,2 and flavors like (−)-menthol.3 An overview of industrially relevant enzymatic reactions can be found in Liese et al.4 Because of thermodynamic and/or kinetic limitations, the final yield of biocatalytic reactions is often low; therefore, purification of the product is both extensive and costly. One possibility to overcome these limitations is the integration of the reaction and separation steps into one operational unit, which is the most widely applied technique for process intensification.5,6 The simultaneous removal of product out of the reaction zone is especially useful for equilibrium-limited reactions to enhance the conversion. Furthermore, it prevents undesirable side reactions and can be useful in case of product inhibition during an enzymatic reaction. There are several examples for integrated processes with lipases, e.g., membrane reactors with lipase immobilized on the membrane,7 membrane reactors with encapsulated lipase in reversed micelles,8 and continuous reaction/separation process within scCO2.9 The first combined setup of an enzymatic reaction and a distillation column consisted of a fixed bed reactor with immobilized lipase that was connected to the bottom of the distillation column.10,11 The conversion of the © 2012 American Chemical Society

esterification or transesterification reaction was enhanced by product removal through azeotropic distillation. For more volatile substrates, the fixed-bed reactor with the lipase was placed between the condenser and the column.12 The first fully integrated setup of a lipase-catalyzed transesterification reaction in a laboratory-scale distillation column was demonstrated by Paiva et al.13 In order to maintain low temperatures for the thermolabile enzyme, a vacuum of ∼15 kPa was applied. The reactants ethyl butyrate and butanol were heated in the bottom of the column and reacted by means of the immobilized lipase in the inverted pear bulbs of the column. The removal of the low-boiling ethanol led to increased final yield of butyl butyrate. However, the extent to which the yield of the integrated process increased, compared to the batch reaction, was not shown. Furthermore, on a larger scale, the introduction of the enzyme in the inverted pear bulbs would lead to low accessibility of the substrates to the catalyst in the packed bed. For this reason, the goal of this work is to find an appropriate method for the integration of enzymes in a reactive distillation (RD) column. For the introduction of catalysts into distillation columns, several configurations are available. An overview is given by Taylor and Krishna.14 Widely used catalytic packings have sandwich structures with catalytic granules pressed between two layers of metal wire gauze, such as Katapak-S from Sulzer (Winterthur, Switzerland) or Katamax from KochGlitsch (Wichita, KS, USA).15−18 To increase the separation Received: Revised: Accepted: Published: 11482

March 29, 2012 July 26, 2012 August 9, 2012 August 9, 2012 dx.doi.org/10.1021/ie300837v | Ind. Eng. Chem. Res. 2012, 51, 11482−11489

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(MTMS:TMOS). The amount of solvent (MeOH) and catalyst (NH4OH) was adjusted for the appropriate gelation time. For the preparation of the gel, TMOS and MTMS were mixed with MeOH and stirred in an ice bath (solution A). In solution B, all water-soluble components were prepared, i.e., NH4OH (25 wt %) as catalyst, PEG, enzyme solution, and additional water. Solution B was then mixed with solution A, thus starting the polymerization reaction. For the so-called adjusted gel of Novak, the molar ratio of the components in the final mixture was as follows (for all given molar ratios, the aqueous enzyme solution is included in the amount of water):

efficiency, catalytic layers were combined with normal rectifying layers in so-called hybrid packings, e.g., Katapak-SP.19 Another approach is to produce packings with a catalytically active coating. Norton ceramic saddles were coated with magnesium acetate and converted to catalytically active magnesium oxide by calcination. The coated saddles were employed in the reactive distillation column and showed increased selectivity and yield, compared to catalytic pockets filled with ionexchange resins.20 Furthermore, a binder-free film of zeolite crystals was used to coat the structured packings.21 For enhanced binding of the zeolites, the structured packings were precoated with a silica layer as a type of primer.22 Zeolitecoated saddles were successfully applied in reactive distillation.23 Mehrabani et al. investigated the coating of aluminum structured packings with a silica layer containing active cationexchange resins.24 In this work, the sol−gel method was used to fix enzymes on conventional structured A3-500 packings from Montz (Hilden, Germany). Sol−gel entrapment is a common technique for immobilization of several enzymes and was described for enzymes such as serine protease,25 glucose oxidase,26 and alkaline phosphatase.27 For application in reactive distillation, lipase CALB was chosen as a model enzyme. Lipases are promising candidates for use as catalysts in organic synthesis, because these enzymes show increased stability and they do not need cofactors or coenzymes.28 In addition, the entrapment of lipase in hydrophobic silica gels showed enhancement of the esterification activity by a factor up to 88, compared to commercial lipase powders (free enzyme) and excellent enzyme stability for the given process conditions as well as during storage.29 The application of the silica-gel coating in the distillation column requires sufficient stability in organic solvents at increased temperatures. An appropriate sol−gel method has been established for coatings on the metallic packing surface. Finally, a reactive distillation column was built up and the coated packings with lipase CALB were tested in a transesterification reaction as a model reaction. Thermal stability of lipase CALB within the coating was investigated, as well as leaching of the enzyme and stability of the coating for repeated use in the reactive distillation column.

1 mol TMOS: 4 mol MTMS: 20 mol MeOH: 17 mol H 2O : 0.4 mol PEG: 0.35 mol NH4OH

The amount of CALB was 5.9 wt % of the total amount of gel solution. For the final gel coating, NH4OH was replaced by NaF and the amount of CALB and water was increased. The gel was prepared similar to the previously described procedure for the adjusted gel of Novak. The molar ratio of the final gel coating was as follows: 1 mol TMOS: 4 mol MTMS: 20 mol MeOH: 30 mol H 2O : 0.4 mol PEG: 0.09 mol NaF

The amount of CALB was 10.5 wt % of the total amount of gel solution. The CALB solution from Codexis contains 10.9 mg/mL protein, determined by quantitative amino acid analysis. Accordingly, the final loading of the gel coating with enzyme (protein) was 2.9 mg per g of dried silica gel. Because of the exothermic hydrolysis reaction, the reaction needs to be stirred on ice for the first 4 min, followed by further stirring at room temperature. The coating procedure contained several repetitions of immersion and drying, i.e., the packing was completely immersed into the gel solution for 20 s, followed by 20 s of drying at air (room temperature). The procedure was repeated until the mother solution solidified (7−9 min). The gel coating was then dried at room temperature until the weight was constant (∼2−3 days). Alternatively, CALB was immobilized in silica gel using the original procedure provided by Reetz et al.:29 the alkoxy silanes TMOS and PTMS were mixed and stirred at room temperature (solution A), without using additional solvent. Solution B consisted of sodium fluoride, PVA, enzyme solution, and water. Solutions A and B were vigorously mixed to prevent phase separation. The molar ratio of the components was

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EXPERIMENTAL SECTION Materials. CALB enzyme solution was provided by Codexis (Redwood City, CA, USA) and Novozyme (Bagsvaerd, Denmark). Ethyl butyrate (EtBu), butyl butyrate (BuBu), polyethylene glycol (PEG, MW 400) and poly(vinyl alcohol) (PVA) were purchased from Merck (Darmstadt, Germany); 1butanol (BuOH), ethanol (EtOH), methanol (MeOH), acetonitrile, isoamylalcohol, and sodium hydroxide were obtained from Carl Roth (Karlsruhe, Germany); tetramethyl orthosilicate (TMOS), methyl trimethoxysilane (MTMS), and ammonium hydroxide (NH4OH) were obtained from Fluka (Buchs, Switzerland); and propyl trimethoxysilane (PTMS) and sodium fluoride and acetone were purchased from Prolabo (East Grinstead, U.K.). All chemicals were pro analysis grade and used without further purification. Immobilization. To coat the Montz A3-500 packing, the enzyme lipase CALB was immobilized by entrapment in a silica gel matrix. The composition of the gel coating was achieved by a combination of the method of Novak and Knez30 and Reetz et al.29 First, the pure TMOS in the Novak method was replaced by a MTMS/TMOS mixture with a molar ratio of 4:1

1 mol TMOS: 5 mol PTMS: 53 mol H 2O: 0.18 mol PVA : 0.10 mol NaF The amount of enzyme was 21.9 wt % of the total amount of gel solution. Accordingly, an enzyme loading of 6.1 mg per g of dried silica gel was obtained. The gel solution was stirred at room temperature. The heat of reaction for this polymerization reaction is low, so that no additional cooling was required. After 1−3 min, the gel solidified in the beaker and was stored for at least 12 h at 4 °C for aging. Subsequently, the gel was dried at room temperature until constant weight was achieved. The dried gel was comminuted in a mortar and classified into 0.7− 1.4 mm particles. Reactive Distillation: Setup and Reaction Conditions. The coated packings were tested in a batch distillation depicted in Figure 1. The distillation setup consisted of a Normag (Ilmenau, Germany) glass column with an inner diameter of 45 11483

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min for synthetic air. Samples were prediluted in 1:50 ratio and injected with a split ratio of 1:30. Isoamyl alcohol was used as an internal standard. CALB Activity Test. The activity of the immobilized CALB was tested for the transesterification reaction of EtBu with BuOH. The coated packing was immersed in an equimolar mixture of BuOH and EtBu stirred at 400 rpm at 60 °C. To avoid evaporation losses, the flask was locked up with a cap. Samples were directly mixed with acetonitrile on a vortex and centrifuged for 1.5 min at maximum speed to remove coating particles and stop the reaction. The product formation over time (enzyme activity) was analyzed by means of GC analysis. For the repeated activity tests, the coated packing material was washed in EtBu for 15 min and dried under the fume hood until constant weight was achieved, to avoid prewetting effects with reactants or products. Determination of Total Nitrogen via the Kjeldahl Method. Since the enzyme is considered to be the only source for nitrogen in the entire system, leaching of enzyme into the distillation bottom was determined by analysis of total nitrogen. Prior to analysis, the organic products were removed with a rotary evaporator and the total nitrogen was analyzed in the solid residue according to the Kjeldahl method. This method is described elsewhere.31 The total nitrogen analysis was carried out by the Central Analytical Lab of TU Hamburg−Harburg. Amino Acid Analysis. The amount of enzyme in the original enzyme solutions was determined by quantitative amino acid analysis. The proteins were hydrolyzed in 6 N HCl for 16 h at 115 °C to obtain the monomeric amino acids. The amino acids were then separated by HPLC. Derivatization with ophthaldialdehyde (OPA) enabled the detection of the amino acids with a fluorescence detector. The amino acid analysis was carried out by the Central Analytical Lab of TU Hamburg− Harburg. BET Analysis and Scattering Electron Microscopy (SEM). Pore size distribution and specific surface area of the silica gels with entrapped enzymes were determined by nitrogen adsorption and desorption using the BJH and BET methods, respectively.32 A defined amount of silica gel (150−250 mg) was degassed for 24 h at 40 °C. The analysis was carried out in Nova 3000e from Quantachrome (Odelzhausen, Germany). Microscopic analysis was carried out using scattering electron microscopy (SEM) (Model JSM-840, JEOL, Tokyo, Japan) with a Si(Li) detector.

Figure 1. Flow sheet of the reactive distillation setup.

mm and height of 960 mm connected to a 4-L round-bottom flask as a bottom vessel. The flow of air through a pipet into the bottom vessel avoids a delay in boiling. The reflux rate was adjusted by a magnetic valve with time switch control. The column was operated under vacuum (11 kPa). The vacuum pump was connected to the top of the cooler, as well as to the reflux valve. A second pump with a lower vacuum (1 kPa) was employed in the setup for taking samples from both the bottom and the distillate. The column was equipped with 12 Montz A3500 packings with a total height of 720 mm. To maximize the enzyme loading, all 12 packings were coated with the enzymesilica gel. No additional rectifying sections were included in the column. Alternatively, enzymes can be immobilized in silica gel granulate (using the method by Reetz et al.;29 see above). The granulate was filled into a metal tube with wire gauze on each side, which was then placed in the column, instead of the second lowest packing. For the distillation experiments, the reactants 1-butanol (BuOH, 30% mol/mol) and ethyl butyrate (EtBu, 70% mol/ mol) were placed in the bottom vessel and heated in a water bath to 60 °C. The time measurement started when the bottom began to boil. Samples were taken from both the bottom and the distillate. The volume of the removed samples was determined to monitor the reaction volume in the column. In all of the experiments, the column holdup was estimated to be 5% of the total reaction volume. The composition of the holdup was calculated by the average composition of the distillate and the bottom. Analytical Methods. Gas Chromatography. Both reactants and products were analyzed by gas chromatography (GC). For GC analysis, a capillary column with polyethylene glycol coating CW-20M-CB (CS Chromatographie, Langerwehe, Germany) with a length of 60 m, ID = 0.32 mm, and film thickness of 0.5 μm was used. The oven temperature was held at 100 °C for 11 min, then increased to 120 °C (4 °C/min) and held constant for 3 min. Samples were detected by flame ionization detector (FID) (Tdetector = 250 °C, Tinjector = 180 °C). Gas flow was equal to 30 mL/min for H2 and N2 and 400 mL/

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RESULTS AND DISCUSSION Development of the Biocatalytic Silica-Gel Coatings. In order to introduce lipase CALB into the column, structured packings were coated with the silica gel that served as an immobilization matrix for the enzyme. The structural properties of the gel were heavily dependent on the parameters of the sol−gel reaction, which is affected by several factors, such as water content, type of catalyst, type of silane precursors, additives (e.g., PEG), temperature, and pH of the sol. Because of the exothermal hydrolysis in the sol−gel reaction, cooling of the sol solution was necessary to prevent thermal deactivation of the CALB. Without cooling, the local temperature increased to 56 °C and the lipase CALB was completely deactivated. Of course, other conditions during the sol−gel reaction (such as extreme pH, the organic solvent, and mechanical stresses of the stirring) might also deactivate the enzyme. The sol was stirred in an ice bath for 4 min (Tmax = 43 °C). After 4 min, the temperature of the sol dropped below 30 °C and the solution 11484

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was stirred at room temperature, since no thermal denaturation effect of the enzyme was expected. The composition of the silica gel was adjusted to ensure sufficient attachment and stability on the stainless steel packings. Because of gelation times of MTMS) might enhance interaction with the hydrophobic structures of the enzyme, leading to fixation of the lipase in the activated openlid conformation and better accessibility of the enzyme. However, gel coatings with PTMS instead of MTMS were

Table 1. Enzyme Activity and Enzyme Loading of the SilicaGel Coating and the Silica-Gel Particles Prepared According to Reetz et al.29 with Different Silane Precursorsa initial rate

gel sample gel coating (TMOS/MTMS) Reetz gel (TMOS/MTMS) Reetz gel (TMOS/PTMS)

number of experiments

enzyme loading [mg/ggel]

U/ggel

U/mgcat

2

2.9

305 ± 76

104 ± 26

7

6.1

263 ± 34

43 ± 6

25

6.1

369 ± 15

61 ± 2

a

Activity test: transesterification of ethyl butyrate (EtBu, 50 mol %) with butanol (BuOH, 50 mol %), CALB (Codexis), T = 60 °C, p = 1013 mbar, 400 rpm.

In order to determine the long-term stability, the coated packings were stored in dry condition at three different temperatures (4, 20, and 60 °C) and the activity was regularly determined. Figure 2 shows the relative activity as a function of the storage time. Obviously, no significant loss of activity was observed, even after 50 days of storage at 4 °C and at 20 °C. During storage at 60 °C, the relative activity decreases to 58% after 50 days. However, after the initial loss the relative enzyme activity remains rather stable between day 15 (76%) and day 43 (63%). 11485

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composition of the distillate was 96.5% (mol/mol) EtOH and 3.5% (mol/mol) EtBu, which corresponds approximately to the composition of azeotrope 1 predicted by the NRTL model. Since this azeotrope mainly consists of the low-boiling-point products, separation of the products in the quaternary mixture is still feasible and the reactants mainly remain within the column. The final distillate concentration that was reported for the same reaction system in Paiva et al., consisting of 50% (mol/mol) BuOH, 35% (mol/mol) EtBu, and the remaining 15% (mol/mol) of BuBu and EtOH,13 is not evident here. Reactive Distillation with Biocatalytic Packings. In order to carry out the reaction in the column, the entire set of the wire gauze packings (720 mm) was coated with silica gel containing the lipase CALB as described above. Because of the inhibiting effect of BuOH on lipase CALB34 and our own results (not shown here), the BuOH concentration in the bottom was reduced from 50% (mol/mol) to 30% (mol/mol). Stepwise product removal was chosen because of the slow reaction rate. The ethanol formation was too low to be removed continuously with a technically reasonable reflux. At certain time intervals, samples were taken from the bottom and the distillate and analyzed via GC analysis. The reactive distillation was carried out with two CALB preparations with different protein concentrations (Codexis, 2.9 mg protein/g dried gel and Novozyme, 2.0 g protein/g dried gel). The total amount of gel coating for the entire set of packings was 36.1− 39.6 g. The average mole fractions for all reaction components in the bottom and the distillate are depicted in Figures 5a and 5b. For the first 3.5 h, the distillation was operated at total reflux and EtOH becomes enriched in the distillate (see Figure 5a). In the first withdrawal period (shaded fields in the figure), ∼15% of the distillate stream was removed from the column for 30 min and the EtOH concentration decreases to 30% (mol/mol). The reflux then was again switched to total reflux to enrich the product again. The removal of EtOH in the distillate was repeated twice in this manner. During total reflux periods, the mole fraction of EtOH increased and decreased when a reflux ratio was set again. In the distillation bottom, the ester BuBu becomes enriched to 68% (mol/mol) after 9 h (see Figure 5b). The substrate concentration of BuOH and EtBu in the bottom decreased accordingly. After 7.5 h, the BuOH is completely converted. To exclude the concentration effects in the bottom, the amount of BuBu formed per mg of enzyme (in units mol/ mgcat) is plotted as a function of time in Figure 6. The productivity (in units of mmol BuBu/(h·mgcat)) indicates that (1) lipase CALB is active within the packing coating under reaction conditions and (2) the removal of EtOH prevents the reverse reaction and enhances the formation of BuBu and EtOH.

Figure 3. BET surface and pore volume of the gel coating with entrapped lipase compared with the Reetz gels. The Reetz gel was prepared with TMOS and MTMS or TMOS and PTMS.

not possible, because of the extremely long gelation time for the PTMS gel (>24 h). Figure 4 shows SEM micrographs of the coating and the gels according to Reetz et al.29 The surface of the gel coating appears more flat than the surfaces of both Reetz gels. Of course, these micrographs can only give a general idea of the gel morphology. The thickness of the coating was ∼0.25 mm, determined at the edge of the packing material under a normal light microscope. Behavior of the Quaternary Reaction System in the Column without Catalysts. To investigate the separation efficiency and the formation of azeotropes, a quaternary mixture of all reaction components was treated in the distillation column with uncoated packings. The column was operated under total reflux for 3 h. The quaternary mixture consisted of 22.2% (mol/mol) EtBu, 22.1% (mol/mol) BuOH, 22.3% (mol/mol) BuBu, and 33.4% (mol/mol) EtOH. As expected, the products of the transesterification reaction BuBu and EtOH are easily separated in the distillation column. The low-boiling-point product (EtOH) is enriched in the distillate (>95% (mol/mol)), whereas the high-boiling-point product remains in the distillation bottom. For the reaction system, two homogeneous binary azeotropes were predicted by the NonRandom Two-Liquid (NRTL) model, i.e., azeotrope 1 with 98.5% (mol/mol) EtOH and 1.5% (mol/mol) EtBu and azeotrope 2 with 82.4% (mol/mol) EtBu and 17.6 (mol/mol) BuOH, both at p = 113 mbar. The residue curve map for the ternary mixture EtBu/BuOH/EtOH including the azeotropes can be found in the Supporting Information. The average

Figure 4. SEM micrographs of silica gels with entrapped lipase CALB: (a) gel coating, (b) Reetz gel with TMOS/MTMS, and (c) Reetz gel with TMOS/PTMS. 11486

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Figure 6. Formation of BuBu [mol/gkat] in batch reactive distillation plotted over time. The removal of EtOH between 3.5−4.0 h and 5.5− 6.0 h enhances the BuBu formation. The productivity of BuBu is given for the time period before and after first removal. CALB solution from Codexis was used here.

Figure 7. Comparison of (▲) the catalytic coating and (●) a packed bed of gel granulate with entrapped lipase, concerning the productivity of BuBu in a reactive distillation column. Figure 5. Mole fraction plotted over time for all reaction components (EtBu, BuOH, BuBu, and EtOH) (a) in the distillate and (b) in the bottom. The shaded regions represent the time periods for the removal of distillate at given reflux ratios. The reactive distillation was performed using two CALB preparations (Codexis and Novozyme).

Furthermore, the product formation in the batch reactive distillation was compared with the same reaction carried out in a standard batch setup without product removal. The enzyme was immobilized in silica gel as for the coating, mechanically pulverized, and put directly into a stirred vessel. Figure 8 indicates that, in the stirred vessel setup reaction, equilibrium is reached after 7 h, whereas in the reactive distillation setup, BuOH was converted up to 98% through the removal of EtOH. Thus, the conversion of BuOH was significantly increased by the withdrawal of EtOH. Stability of Silica-Gel Coating and CALB under Reaction Conditions. The installation of catalytic packings in reactive distillation requires long-term stability of the catalyst to avoid frequent catalyst change and process shutdown. Therefore, the stability of the silica-gel coating and the enzyme under process conditions was studied in repeated batch experiments. The stability of the silica-gel coating was investigated by determining the weight before and after the reactive distillation experiment, whereas each run lasted for 6−9 h. (see Figure 9). The major weight loss occurs after the first run, because of the less strongly bound parts of the gel that are washed off (24 wt %; all weight

The performance of the catalytic coatings was compared with a packed bed of gel granulate with entrapped CALB (analogous to usual reactive distillation). The gel granulate was filled in a container (as described in the Experimental Section) and placed in the second-lowest position of the column. The rest of the column was filled with the uncoated structured A3-500 packings. The overall amount of enzyme was the same in both cases. Figure 7 shows that the enzyme fixed in the column in this way leads to a lower yield of BuBu, in comparison to the catalytic packings. The increased fluid resistance of the fixed bed probably causes an unequal fluid distribution and an increased flow at the column walls. In addition, the accessibility of the enzyme is limited by the diffusion of reactants into the bed. The use of structured packings with catalyst pockets such as Katapak from Sulzer (Winterthur, Switzerland) is more likely to improve the productivity and is part of future work. 11487

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CONCLUSION This work presents the first successful integration of a biocatalytic reaction into a reactive distillation column with structured wire gauze packings. The lipase CALB was immobilized in a newly developed silica-gel matrix that was applied as a stable coating onto commercially available Montz A3-500 packings. The newly developed coating exhibits a high specific surface area and good thermal stability. The lipase CALB entrapped in the silica-gel coating showed increased activity (104 ± 26 U/mgcat) compared to the standard Reetz gel adopted from the literature (43 ± 6 U/mgcat). The application of the catalytic silica-gel coating in a batch reactive distillation setup has shown that (1) the enzyme CALB was active under reaction conditions in the reactive distillation column and (2) the removal of EtOH increased the conversion of butanol beyond the equilibrium concentration to 98%. The productivity of the catalytic coatings (in units of mmol BuBu/ (h·mgcat) enzyme) was higher than that of a packed bed of gel granulate applied under the same conditions. After the second run in the reactive distillation setup, no significant loss of the coating occurred. According to nitrogen analysis, enzyme loss was 3.9−4.6 wt % for the first two runs in the column. Overall, the newly developed silica-gel coating enabled the first application in reactive distillation packing column and, therefore, offers a promising alternative for biocatalysts to be used in thermal integrated processes such as reactive distillation.

Figure 8. Comparison of the butanol conversion in reactive distillation (RD) and in a stirred vessel setup (batch). The removal of EtOH significantly increases the conversion in reactive distillation. The percentage indicates the fraction of the distillate that is withdrawn from the column within the gray shaded time period. Batch: T = 60 °C, p = 1013 mbar, V = 60 mL, 400 rpm, 1.4 g of silica gel. Reactive distillation (RD): T = 60 °C, p = 110 mbar, V = 1500 mL, 37.9 g of silica gel.

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ASSOCIATED CONTENT

S Supporting Information *

Residue curve map for the ternary system EtBu−BuOH−EtOH including azeotropes (Figure S1), Figure 2 with error bars (Figure S2) and Figures 5a and 5b with error bars (Figure S3). This information is available free of charge via the Internet at http://pubs.acs.org/.

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Figure 9. Stability of silica-gel coating and leaching of enzyme. The weight of the coating was determined before each experiment and related to original weight of the coating. The stability was tested for 6− 9 h in a reactive distillation (RD) setup and for 30 min in a stirred vessel setup.

AUTHOR INFORMATION

Corresponding Author

*Tel.: +49 40 42878 4392. Fax: +49 40 42878 4072. E-mail: [email protected]. Present Address §

ttz Bremerhaven, Am Lunedeich 12, D-27572 Bremerhaven, Germany.

losses correspond to the initial weight of the freshly produced coating). The residual coating remains rather stable on the packing. For run 2, the weight loss was 5.6 wt %. After runs 3 and 4, almost no weight loss was determined (