Improved activity and stability of lipase immobilized in cage-like large pore mesoporous organosilicas

Microporous and Mesoporous Materials 154 (2012) 133–141

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Improved activity and stability of lipase immobilized in cage-like large pore mesoporous organosilicas Zhou Zhou a, Alexandra Inayat b, Wilhelm Schwieger b, Martin Hartmann a,⇑ a b

Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen Catalysis Resource Center (ECRC), Egerlandstraße 3, 91058 Erlangen, Germany Friedrich-Alexander-Universität Erlangen-Nürnberg, Lehrstuhl für Chemische Reaktionstechnik, Egerlandstraße 3, 91058 Erlangen, Germany

a r t i c l e

i n f o

Article history: Available online 20 January 2012 Keywords: Cage-like PMOs Lipase Transesterification IR spectroscopy

a b s t r a c t Lipase from Thermomyces lanuginosus has been immobilized in different mesoporous organosilicas (PMOs) containing ethane and benzene groups with large cage-like pores. The immobilization of lipase as well as the application of these catalysts in hydrolysis and transesterification reactions is studied in detail. The results show that the structural properties of the support employed have a significant influence on the adsorption capacity. In general, materials with high specific surface areas and pore volumes show high adsorption capacity. However, in the case of monolayer adsorption, the hydrophobicity of the surface has a significant impact on the adsorption efficiency due to strong hydrophobic interactions. Furthermore, the transesterification of vinyl propionate with 1-butanol catalyzed by immobilized lipase was studied. The influence of lipase loading, the effect of water content in the reaction mixture and the influence of different solvents were investigated. The results show that lipase immobilized in the most hydrophobic material (LPbenzene) exhibits excellent catalytic performance, which is attributed to the enhanced interfacial activity of lipase due to the interaction between lipase and the hydrophobic surface of the material. In order to further disclose the influence of the hydrophobic surface on the lipase activity, ATR-FTIR spectroscopy was used to study the secondary structure of lipase immobilized on different supports. The results confirm that a structural transition of entrapped lipase to the open-lid conformation takes place on LPbenzene, which shows that the observed increase in enzymatic activity is triggered by interfacial activation of lipase. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction Enzymes exhibit high activities as well as high chemo-, regio-, and stereo-selectivities under mild reaction conditions and have attracted tremendous attention as catalysts in the field of green and sustainable chemistry [1]. However, stability and reusability of enzymatic catalysts are still key issues for their industrial applications [2]. Immobilization of the enzyme onto solid supports such as polymers, ceramics and porous (alumina-)silicas provides an attractive method to overcome these drawbacks [3]. Recently, mesoporous materials have been explored as supports for the immobilization of enzymes [4,5]. Previous studies have shown that adsorption of the enzyme onto mesoporous silicas materials can in selected cases enhance the activity and stability of the enzyme under study [6,7]. Immobilization of enzymes onto mesoporous supports was carried out either by physical adsorption, covalent attachment of the enzyme onto the pore walls, encapsulation/ entrapment of the enzyme into the pore channels or the formation of cross-linked enzyme aggregates in cage-like pore systems [8,9]. ⇑ Corresponding author. Tel.: +49 (0) 9131 85 28792; fax: +49 (0) 9131 85 27421. E-mail address: [email protected] (M. Hartmann). URL: www.ecrc.uni-erlangen.de (M. Hartmann). 1387-1811/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.micromeso.2012.01.003

It is recognized that physical adsorption of enzymes into the pores or channels of the support is the most simple and cost-effective method, thus, it is one of the most commercially viable alternatives [10]. However, the weak interaction between the support and the enzyme is the major drawback in most cases, resulting in enzyme leaching from the support. Several techniques have been developed in order to reduce leaching of the enzyme, e.g. via modification (e.g. silylation) of the support to reduce the size of the pore entrance or by cross-linking of the enzyme inside the pore system. These efforts have been only partially successful when deactivation of the enzyme occurred during the chemical treatment [11,12]. However, some reports suggest that hydrophobization of certain supports may result in reduced leaching and enhanced enzymatic activity [13,14]. Periodic mesoporous organosilicas (PMOs) were developed recently as a novel branch of hybrid ordered mesoporous materials, where the organic fragments are regularly distributed within the framework [15,16]. Compared to pure silica mesoporous materials, PMOs not only maintain a highly ordered structure, high specific surface area and pore volume, but also combine these advantages with tunable hydrophobicity imparted by the organic components in the framework [17]. We have recently described the synthesis of a series of PMOs with large cage-like pores and primarily studies

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showed that these materials are beneficial for the immobilization of lipase [18]. Lipases are used as biocatalysts for ester hydrolysis, alcoholysis, acidolysis, aminolysis and interesterification in various applications in the food, paper, detergent, textile, leather, cosmetic and pharmaceutical industries [19]. Nowadays, the amount of available commercial lipase has increased steadily. In this work, lipase from Thermomyces lanuginosus (TLL) was immobilized on novel PMOs (ethane or benzene groups) with large cage-like pores by physical adsorption in order to investigate the influence of the surface properties of pure silica materials and different PMOs on the activity of immobilized lipase. Hydrolysis and transesterification reactions were employed in order to evaluate the catalytic activity of these novel catalysts. 2. Experimental section 2.1. Materials Tetraethyl orthosilicate (TEOS), Pluronic triblock copolymer (P123), 4-nitrophenyl palmitate and 1,3,5-trimethylbenzene (TMB) were purchased from Sigma–Aldrich GmbH (Germany), and bis(triethoxysilyl)ethane (BTEE) was obtained from ABCR GmbH & Co. Kg (Germany). 1,4-bis(triethoxysilyl)benzene was synthesized by a one step Grignard reaction and distilled under vacuum as described in the literature [20]. Lipase fromT. lanuginosus (E.C.3.1.1.3), a product of Novozyme Corp. was obtained from Sigma–Aldrich. Lipase immobilized on Immobead 150 from T. lanuginosus (P3000 U/g tributyrin activity) and amorphous Silica Gel 100 (LPS100) were purchased from Sigma–Aldrich. 2.2. Preparation of the PMOs materials The large pore cage-like PMOs were synthesized by using TMB as a swelling agent as described in our previous paper [18]. In a typical preparation, the triblock copolymer P123 (2.0 g, 0.4 mmol) was dissolved in a solution containing 10 mL of concentrated HCl (37 wt.%) and 65 mL of distilled water at 38 °C under stirring for 2 h. After addition of TMB (0.5 g), the mixture was stirred at 38 °C for another 2 h and then BTEE (5.1 g, 14 mmol) was added. After further stirring for 24 h at 38 °C, the reaction mixture was aged at 98 °C for 48 h under static conditions. The mixture was allowed to cool down to room temperature and the white precipitate was filtered off and dried in air for at least 2 days. Afterwards, the template was removed by extraction with acetone and the product was named LPethane. Following the same procedure, PMOs containing benzene moieties (LPbenzene) were synthesized using the bridged-silane precursor 1,4-bis(trimethoxysilyl)benzene, while keeping all other parameters constant. For comparison, a pure silica mesostructured cellular foam material denoted as LPsilica was synthesized employing TEOS as silica source with a TMB:P123 ratio of 0.25. 2.3. Immobilization of lipase A portion of 100 mg of support was added to 10 mL of sodium phosphate buffer (0.1 M, pH 7.2) containing different concentrations of lipase (from T. lanuginosus; Sigma–Aldrich). The mixture was shaken (200 rpm) at 25 °C for 3 h. Finally, the material containing the encapsulated lipase was collected by centrifugation at 8000g for 10 min at room temperature and then washed three times with 10 mL of buffer solution and separated by centrifugation. The initial supernatant and the washing solutions were collected to determine the amount of lipase immobilized on the support using the hydrolysis assay. The protein content was measured by the standard Bradford method. The solid was treated

by two methods prior to its use in the catalytic reaction: (a) The solid catalysts were dried under vacuum at 40 °C and then kept in a closed water-saturated oven overnight. (b) The solid was directly dried under vacuum (30 mbar) at 25 °C overnight and the catalysts contained ca. 30 wt.% of water after this treatment. 2.4. Lipase assay The lipase activity was determined using the well known lipase substrate 4-nitrophenyl palmitate (pNPP) [21]. This choice allowed a reasonable comparison of lipase activity in the hydrolysis of oil and fats. Furthermore the reaction can also be performed in organic media which offers several important advantages for the evaluation of lipase in organic synthesis [22]. The method is based on the hydrolysis of pNP-palmitate by the enzyme, followed by spectrophotometric measurement of the p-nitrophenol release rate at 400 nm. In a typical process, 100 mM of sodium phosphate buffer containing 150 mM of sodium chloride and 0.5% (v/v) Triton X-100 (pH 7.2 at 25 °C) were used as the reaction media. An aliquot of lipase solution was added to 2 mL of buffer solution in a cuvette. After the absorbance at 400 nm reached a constant value, 150 lL of pNNP (10 mM in 2-propanol) substrate solution were added and the increase of the absorbance at 400 nm was recorded for approximately 5 min against a reference with the same volume and amount of buffer and enzyme solution. One unit is defined as the amount of lipase that liberates 1 lmol p-nitrophenol per minute under standard conditions (at 25 °C, pH 7.2, with Triton X-100 as emulsifier and pNPP as substrate). The activity of immobilized lipase was determined by mixing an aliquot of solid with 2 mL of the sodium phosphate buffer (pH 7.2, with Triton X-100 as emulsifier) at 25 °C. The reaction was initiated by adding 150 ll of pNNP (10 mM in 2-propanol) substrate solution and the activity was determined by measuring the increase in absorbance at 400 nm. 2.5. Characterization of the supports and catalysts N2 adsorption/desorption isotherms were measured at 77 K using a Micromeritics ASAP 2010 sorption analyser. The samples were degassed at 120 °C for 12 h under vacuum. The specific surface area was determined by the Brunauer–Emmett–Teller (BET) method and the pore size distribution was derived from the respective isotherm employing the NLDFT formalism. Ar adsorption/desorption was carried out on an Autosorb-1-MP from Quantachrome Instruments. The sample was degassed at 120 °C for 12 h under vacuum and the measurement was performed at 87 K in liquid argon. The pore size distribution was calculated by the NLDFT method. Transmission electron microscopy (TEM) was performed on a CM300-UT instrument (Philips, Amsterdam, Netherlands). Infrared spectra were measured in the range from 1500 to 1700 cm 1 using a JASCO 6000 FTIR spectrometer equipped with an universal ATR sampling accessory. A 80 scan interferogram was recorded at 4 cm 1 resolution with a scan rate of 5 cm 1 (scanning from 4000 to 1000 cm 1) in order to obtain the IR spectrum. In order to analyze the secondary structure of the protein, the second derivative of the spectrum was calculated using the Savitsky– Golay algorithm for a 9 data point window using the spectra analysis software (JASCO). Karl Fischer titration was carried out on a Metrohm 756 KF coulometer, which is also equipped with the 832 KF thermoprep for the measurement of solid materials. 2.6. Transesterification reactions The catalytic tests were carried out in small sealed vials containing 3.0 mL of hexane, a certain amount of water (0–0.22 g) and the immobilized lipase catalyst (10 mg). The mixture was stirred at 500 rpm for 5 min at reaction temperature. The reaction

Z. Zhou et al. / Microporous and Mesoporous Materials 154 (2012) 133–141

was started by adding an equimolecular mixture of vinyl propionate (1.0 mmol) and 1-butanol (1.0 mmol). At regular time intervals, 20 ll aliquots were withdrawn and analyzed by a Varian gas chromatograph equipped with a CP-sil5 fused silica column using decane as the internal standard. The conversions of the other alcohols were performed under the same reaction conditions, with the exception of the 1-phenylethanol, where the reaction was carried out using 5 equivalents of vinyl propionate. 3. Results and discussion 3.1. Characterization of the PMOs Three cage-like mesoporous materials with different framework composition have been explored for their potential in the immobilization of lipase: the native hydrophilic cage-like mesoporous silica (LPsilica), a relatively hydrophobic cage-like PMO with ethane groups in the framework (LPethane) and a more hydrophobic benzene containing PMO (LPbenzene). The synthesis of these hierarchical materials with large cage-like pores was realized by using P123 as template and 1,3,5-trimethylbenzene (TMB) as pore expander and the weight ratio of TMB to P123 was adjusted to 0.25. The detailed structural characterization of the materials under study was reported in our previous publication [18] and some important textural physicochemical parameters are listed in Table 1. Here, in addition results from nitrogen and argon adsorption experiments at 77.4 and 87.3 K, respectively, on LPethane are given in Fig. 1 and for comparison some characteristic TEM images are shown in Fig. 2. Both isotherms clearly reveal capillary condensation accompanied by a very wide hysteresis loop (Fig. 1a), which indicates the presence of cage-like spheroidal pores connected by narrow windows [23–25]. The nitrogen desorption isotherm of LPethane exhibits a relatively sharp capillary evaporation step taking place at the relative pressures of 0.45–0.49, which is close to the lower limit (P/P0  0.42) of hysteresis behavior for N2 at 77.4 K (Fig. 1a) [26]. In this case, the evaporation pressure may not correlate to the size of the entrance of ink-bottle-type pores and the information obtained from the desorption branch may not reflect the real entrance size of the material. Therefore, in order to identify whether desorption from the pore body occurs via percolation or cavitation and to evaluate the real entrance size, argon adsorption experiments were conducted (Fig. 1a). The argon adsorption isotherm is quite similar to the nitrogen adsorption isotherm and the equilibrium capillary condensation occurs at high relative pressure, which proves the presence of large cage-like pores. The argon desorption isotherm shows that spontaneous evaporation starts at a relative pressure of ca. 0.6 and closes at P/P0 = 0.42. The absence of a sharp step related to the cavitation transition (spontaneous nucleation of a bubble) at the pressure limit of argon (P/ P0  0.38), indicates that the percolation mechanism is in effect for LPethane and that evaporation happens at the pressure of equiTable 1 Textural properties of the supports employed in this study.

a

Sample

DCage (nm)a

Analysis gas

Dp (nm)b

dw (nm)c

SBET (m2/g)d

Vp (cm3/g)

LPsilica LPethane

25.0 9.0

LPbenzene

50.0

N2 N2 Ar N2

20.2 9.1 8.5 7.0/60

7.5 5.3 4.6 5.1

680 918 969 557

1.1 1.4 1.6 0.8

Cage diameter estimated from the TEM images. b Cage diameter calculated by the NLDFT method from the result of adsorption isotherm. c Window diameter calculated by the NLDFT method from the result of desorption isotherm. d Calculated employing the BET theory.

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librium evaporation of argon from the cage entrance [27]. Consequently, reliable information about the size of the narrow mesopore entrance connecting the larger cages can be obtained from the desorption branch. The pore size distributions calculated from the desorption branches of nitrogen and argon isotherms are in the same range (from 4.3 to 7.5 nm) and differ only slightly at the center position, which corresponds to 5.3 and 4.6 nm, respectively (Fig. 1b). The pore size distributions of LPethane derived from the adsorption branch of the respective nitrogen and argon isotherms exhibit maxima both in the micro- and mesopore range. The micropores are probably intrawall pores, which are formed due to the interpenetration of PEO segments of PEO–PPO–PEO type triblock copolymers into the silica matrix [28]. Both mesopore size distributions calculated from the adsorption branch of the argon and nitrogen isotherm center at ca. 8.8 nm (Fig. 1c), which is consistent with the cage diameter (ca. 9 nm) observed in the TEM images (Fig. 2a, b and Table 1). 3.2. Adsorption of lipase Lipase from T. lanuginosus (TLL) has been adsorbed on selected PMOs from buffer solution. Physical adsorption is the simplest immobilization method which favors the adsorption of the enzyme in an energetically preferred location and in a favorable orientation due to the absence of fixed chemical bonds between the enzyme and the support [3,29]. Adsorption studies were performed in order to determine the optimum concentration of lipase solution for batch adsorption and the adsorption capacity of the materials under study (Fig. 3a). While a pseudo Langmuir-type isotherm is observed for TLL adsorption at LPbenzene, S-type isotherms are recorded for LPsilica and LPethane indicating a weaker interaction between lipase and the support compared to LPbenzene. The observed high capacity for protein adsorption reflects the high specific surface area and pore volume of the supports under study. LPethane possesses both higher specific surface area and pore volume as compared to LPsilica. Thus, it exhibits a higher adsorption capacity than LPsilica (Fig. 3a and Table 2). LPbenzene has the lowest specific surface area and pore volume of the three materials under study, thus its loading capacity is reduced at higher lipase concentration compared to the other two supports and close to its saturation capacity (Fig. 3a). Although LPbenzene has a lower capacity as compared to the other two materials, it is worth to note that at low concentration the hydrophobic material (LPbenzene) shows the highest loading (Fig. 3a) as well as the highest immobilization efficiency (up to 100%) (Table 2). It is anticipated that the dominant driving forces for protein adsorption in this case are hydrophobic interactions. At low lipase concentration, the surface of the material is not completely covered by enzyme molecules, thus the chance of lipase anchoring via hydrophobic interaction with the surface is higher than at high concentrations, where multilayer adsorption occurs instantly. Therefore, the most hydrophobic material (LPbenzene) initially exhibits higher adsorption efficiency as compared to the other two materials (55% for LPsilica and 76% for LPethane) at a low lipase concentration (Table 2). This is in line with a study by Al-Duri and Young showing that lipase clusters are formed on the surface of a hydrophobic support which results in an increase in lipase loading [30]. However, inactive complexes are formed that would not enhance the biocatalytic activity of the system. One important structural feature of LPbenzene is that the material is composed of versicular nano-particles with sizes in the range of 50–80 nm (Fig. 2c and d). The surface area of this material derived from N2 sorption amounts to ca. 557 m2/g. A geometrical calculation of the outer surface area was performed by assuming uniform spherical particles with estimated diameter and wall thickness from TEM and results in ca. 190 m2/g, when the intersection area

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b

1800

1200

•

Argon at 87.3 K Nitrogen at 77.4 K

0.18 0.15 0.12

3

3

Volume (cm /g)

1500

dV/dlog(D) / (cm /g/nm)

a

900 600 300 0 0.0

0.09 0.06 0.03 0.00

0.2

0.4

0.6

0.8

0

1.0

3

6

p/p0

12

15

0.035 0.030 0.025

3

dV/dlog(D) / (cm /g/nm)

c

9

dp / nm

0.020 0.015 0.010 0.005 0.000

0

5

10

15

20

25

30

35

dp / nm Fig. 1. (a) Nitrogen and argon adsorption–desorption isotherms on LPethane (filled symbols: adsorption branch; unfilled symbols: desorption branch). (b) NLDFT pore size distribution calculated from desorption branch. (c) NLDFT pore size distribution calculated from the adsorption branch.

between the particles is not excluded. This indicates the outer surface of these small particles is not negligible and might contribute to enzyme adsorption. Therefore, an immobilization experiment was carried out by using LPbenzene where the pores are filled with template as support under the same conditions. Here, a maximum adsorption of lipase of 6.35 U/g is determined. This result implies that the outer surface of particles only contributes to a minor extent to lipase immobilization and most of adsorbed lipase is located inside the pores. This conclusion is further supported by the similar zeta potential of the parent material ( 25.2 mV) and the catalyst after immobilization of lipase ( 26.4 mV, 50 U/g). The hydrolytic activity of immobilized lipase was tested by hydrolysis of 4-nitrophenyl palmitate (pNPP) in buffer solution. In Fig. 3b, the activity of immobilized lipase on different materials is plotted as a function of loading. At low loading, LPethane exhibits a higher activity (ca. 250 U/g) as compared to the silica support or LPbenzene. It has been suggested that on hydrophobic supports a monolayer of adsorbed lipase is formed where the enzyme is in an active conformation and, thus, interfacial activation of lipase occurs in this case [30]. Moreover, substrate diffusion may be enhanced in this material compared to the pure silica support due to its higher hydrophobicity. With increasing lipase loading, the activity of LPethane declines and finally reaches a plateau at ca. 120 U/g, which is lower than that of LPsilica. Here, multilayer adsorption of lipase might occur resulting in aggregation of enzyme molecules. In this case, the majority of the enzyme molecules are surrounded by other lipases and, thus, the contact with the surface of the support is reduced, which will weaken the influence of interfacial activation on the lipase activity. In addition, diffusion of the

substrates may play a more dominant role [31]. The dimension of the entrance size of LPethane is ca. 4.6 nm (Fig. 1c and Table 1), which is in the same range as the size of the enzyme (3.5  4.5  5.0 nm). Therefore, the reduced activity is probably due to the limitation of substrates and product diffusion in the highly occupied pore system of LPethane. In contrast, diffusion limitations are absent in the case of LPsilica, which posseses a larger cage size (ca. 20.0 nm) and a higher entrance diameter (ca. 7.5 nm). A steady increase of activity is observed with increased loading on LPsilica (Fig. 3b) until a maximum activity of 214 U/g (Table 2) is reached. The most hydrophobic material, LPbenzene shows a fast rise in activity with increasing loading and finally reaches the highest activity (ca. 300 U/g) at a loading of 150 mgprotein/gsupport (Fig. 3b and Table 2). The overall result is supported by the conclusions drawn by other researchers [31–33]: a low apparent activity is observed at low surface coverage, a maximum apparent activity occurs at an intermediate coverage, and again a low activity is found at a high or multi-layer surface coverage. These results show that the apparent activity of immobilized lipase is balanced by the amount adsorbed and the diffusion of the substrates and products. It is worth to note that the most hydrophobic material, LPbenzene exhibits the highest activity in the entire loading range studied (Fig. 3b), which indicates that lipases on this support are in a more hydrophobic environment resulting in an interfacial activation of lipase promoting the activity. Moreover, because of the large cage size, mass transfer limitations are probably absent even at relatively high loading. Thus, LPbenzene exhibits high activity even at high TLL loading. Furthermore, compared to free lipase, all catalysts containing immobilized lipases show higher specific activity (Table 2), indicative of activation upon immobilization.

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Fig. 2. TEM images of LPethane (a, b) and LPbenzene (c, d).

b

a 2000

250

1500 1000 500 0

350 300

Activity / (U/g)

Amount adsorbed / (U/g)

2500

200 150 100 50

0

5

10

15

20

25

Equilibrium concentration of lipase / (U/mL)

0

0

100

200

300

400

Amount adsorbed / (mg protein/g support)

Fig. 3. (a) Amount of lipase adsorbed as function of the equilibrium concentration of lipase. (b) Influence of amount adsorbed on the activity of immobilized TLL. (j) LPsilica, (s) LPethane and (N) LPbenzene.

3.3. Transesterification reaction The immobilized lipase was tested as a catalyst for the transesterification of vinyl propionate with 1-butanol at a molar ratio of 1:1 using hexane as solvent. When the immobilized lipase is dried under high vacuum at 40 °C, no catalytic activity is found, while after saturation with water, the catalysts show improved activity. This indicates that the presence of water is essential for the reaction. It is known that for lipases from T. lanuginosus a certain amount of water is required to maintain their active conformation [34]. Thus, the transesterification of vinyl propionate and 1-butanol was carried out in the presence of 1.0 wt.% of water. Initially, catalysts with loadings representing the highest hydro-

lytic activity, which are 300 mg/g for LPsilica, 20 mg/g for LPethane and 150 mg/g for LPbenzene (Fig. 3b) were tested at 40 °C in hexane (Table 3). After 24 h of reaction, LPbenzene yields 77% of butyl propionate, while the yield is 66% and 62% over LPethane and LPsilica, respectively. However, the rather high loadings of 300 mg/g for LPsilica and 150 mg/g for LPbenzene indicate low immobilization efficiencies, which are only ca. 20% and ca. 50%, respectively. Thus, for more reliable comparison, around 50 mg/g of lipase were loaded onto the three supports and further tested under the reaction conditions described above (Table 3). Decreasing the loading of lipase on LPbenzene and LPsilica causes a slight increase in the yield compared to the higher loadings. On the contrary, increasing the lipase loading on LPethane from 20 to 50 mg/g results in a 10% decrease in

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Table 2 Hydrolytic activity of free lipase and lipase immobilized on different supports. Catalyst

Free 5.5 LPsilica LPethane LPbenzene

Highest loading (U/g)a lipase 1919 2515 1588

Highest immobilization efficiency (%)

Highest activity (U/g)

Highest specific activity (U/mg protein)

–

–

–

55 76 100

214 264 307

15.0b 33.6c 13.7d

a Reaction conditions: in buffer solution (pH 7.2, c = 40 U/mL) at 25 °C after 3 h of incubation. b The loading amounts to 10 U/g. c The loading amounts to 16 U/g. d The loading amounts to 21 U/g.

yield. This result is in line with the hydrolytic activity and further proves the formation of inactive lipase clusters or the presence of mass transfer limitations after loading 50 mg/g of lipase onto LPethane. While the same observation is found in the hydrolysis reaction, such an effect is not observed for LPsilica and LPbenzene, which posseses larger cage and entrance sizes. It is worth to note that lipase immobilized on LPbenzene exhibits higher activity in both cases, indicating that the hydrophobic surface may result in higher lipase activity, which is probably due to the existence of interfacial activation resulting in more lipase molecules in their active conformation. Moreover, the transfer of organic substrates may be easier in this hydrophobic material compared to the other two more hydrophilic materials. Two units of free lipase in solution (corresponding to the total amount of lipase on the solid support) were also tested in this reaction and resulted in a butyl propionate yield of 51%, which is lower compared to the other catalysts. This result is in line with the results from the hydrolysis reaction and further proves that TLL immobilization into these cage-like mesoporous materials induces an increase in activity. The advantage of a cage-like mesoporous structure is evident by comparison with the results of lipase immobilized on commercially available Silica Gel 100 (Silica 100). After 24 h of reaction, a yield of 53% (Table 3) is found, which is similar to the result obtained for free lipase and is lower than that of other cage-type mesoporous materials. Thus, higher product yields are observed for immobilized lipase in these cage-type materials probably due to contributions of surface structure (such as curvature) and the surface chemistry (such as hydrophobic surface containing organic groups) [35], resulting in some interfacial activation.

Table 3 Butyl propionate yield in the transesterification of vinyl propionate with 1-butanol (1:1 M ratio) for different catalysts.a

O O

O

Lipase OH

Hexane

O

O

Catalysta

Loading (mg/g)b

Yield (%)c

LPsilica

300 50 20 48 150d 52 2 Ue 51

62 66 66 56 77 80 51 53

LPethane LPbenzene Free lipase Silica100

a Reaction in hexane at 40 °C under stirring (500 rpm) in the presence 1.0 wt.% of water. b The immobilized lipase with highest hydrolytic activity. c After 24 h of reaction. d The immobilized lipase with relatively high immobilization efficiency. e Equal amount of free lipase (2 U) as used for immobilized lipase.

The three materials containing ca. 50 mg/g of lipase are compared in Fig. 4a. For LPsilica the initial reaction is fast until the yield reaches 66%. The activity, however, is completely lost after the 1st catalyst recycle. Thus, a leaching test was carried out by filtration of the catalyst after a reaction time of 30 min. The filtrate was stirred for additional 10 h at 40 °C, but the product yield did not increase further implying that lipase was trapped in the material and only negligible leaching occured. Therefore, the observed loss in lipase activity might be due to product inhibition under the prevailing reaction conditions. It is known that pure silica surfaces contain more silanol groups compared to PMOs [36,37]. Thus, a relatively strong H-bonding may be present between the products and the catalyst surface, which hinders the release of products from the support [38]. The BP yield over LPethane amounts to only 56% in the 1st run, but drops to 30% after the first recycle. For LPbenzene as support, the highest yield (80%) is observed, which is only reduced to 73% in the second run (Fig. 4a). Thus, the hydrophobicity of the support also plays an important role for the recyclability of the catalyst. In addition to product inhibition also leaching might contribute to the observed decrease in activity for the PMOs, but again the leaching test did not show a significant amount of lipase released into the solution. Nevertheless, a loss of catalyst during the recycling experiments may also provoke the observed loss in activity. As mentioned above, the presence of water is essential for preserving the activity of TLL. Here, the water content was adjusted by drying the catalysts under vacuum (P  30 mbar) at room temperature over night. The water content of the solid catalyst after drying was measured by Karl Fischer titration and amounted to 28, 35 and 24 wt.% for LPsilica, LPethane and LPbenzene, respectively. These catalysts were tested in the same reaction at 40 °C in the presence of ca. 0.1 wt.% of water (Fig. 4b). The product yields are 37% and 59%, when LPsilica and LPethane are used as catalysts, respectively, and the reaction rates are also somewhat lower. On the contrary, the reaction rate of LPbenzene is remarkably enhanced and a product yield of almost 100% is observed. This result suggests that lipase immobilized on the benzene-containing PMO requires a smaller amount of water in order to maintain its active conformation or promote the interfacial effect between the hydrophobic and hydrophilic phase, which further proves that the interaction of lipase with the hydrophobic surface of LPbenzene may result in opening of the lipase lid and stabilization of its active conformation. In order to further study the influence of water on the catalytic activity of different hydrophobic materials, the reaction was performed in the presence of different amounts of water using LPsilica and LPbenzene as catalyst (Fig. 5a). With increasing water content, the final ester yield steady decreases in the case of LPbenzene, while for LPsilica the maximal yield is observed for a water content of 2.2%. Thus, it is shown that a relatively large amount of water is required to stabilize the active conformation of lipase in LPsilica. However, at the same time, the subsequent hydrolysis reaction is fast in the presence of large quantities of water and, thus, a maximum butyl propionate yield of 68% is achieved at 100% vinyl propionate conversion. The transesterification of vinyl propionate and 1-butanol over LPbenzene is studied in different solvents (Fig. 6a). The highest yield (100%) is found for n-hexane followed by another non-polar solvent, namely toluene (91%). The BP yield decreases in the order of increased solvent polarity (CH2Cl2 > THF > Aceton > CH3CN). This result is probably attributed to the different solubility of the substrates. In addition, a non-polar solvent may form an interphase layer between the immobilized lipase, the substrates and the solvent, which might be beneficial for the catalytic performance of lipase. The results of the recycling experiments of immobilized lipase in LPbenzene in the transesterification reaction under study are shown in Fig. 6b. The activity of immobilized lipase on LPbenzene

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Z. Zhou et al. / Microporous and Mesoporous Materials 154 (2012) 133–141

a

b

100

80

60

Yield / %

Yield / %

80

100

40 20

60 40 20

0

0 0

5

10 15 Reaction time / h

20

0

25

5

10

15

20

25

Reaction Time / h

Fig. 4. (a) Transesterification reaction of vinyl propionate and 1-butanol (1:1 M ratio) catalyzed by lipase immobilized on different supports in hexane using 10 mg of immobilized lipase (50 mg/g) at 40 °C under stirring (500 rpm) with a water content of 1.0 wt.%, (solid lines) first run, (dash lines) second run; and (b) with a water content of 0.1 wt.%. (j) LPsilica, (d) LPethane and (N) LPbenzene.

100

BP Yield (%)

80 60 40 20 0

0.1

0.5

1.0 2.2 3.2 4 Water content (wt%)

5.8

9.3

Fig. 5. Butyl propionate (BP) yield as function of the water content in the transesterification of vinyl propionate and 1-butanol over lipase immobilized on LPsilica (dark bar) and LPbenzene (light bar) in hexane using 10 mg of immobilized lipase at 40 °C under stirring (500 rpm).

conditions (Fig. 6c). LPbenzene shows an initial activity of 1.8 mmol/min/g (calculated from the yield after 30 min reaction), while the same amount of IMB150 exhibits a slower reaction rate with a initial activity of 0.45 mmol/min/g. This result further suggests that benzene-containing PMOs are a potential support for the immobilization of lipase. Lipase immobilized on LPbenzene was subsequently tested in the transesterification with different alcohols (ethanol, butanol, 1-phenylethanol) by using vinyl propionate or vinyl acetate as acyl donor in hexane at 40 °C. The immobilized lipase shows higher activity for all these substrates than the free lipase (Table 4). Furthermore, the longer chain alcohol n-butanol gives a higher yield as compared to ethanol with the same acyl donor (vinyl propionate). This finding is tentatively assigned to the different solubility of the substrates in hexane. The same trend is observed when different acyl donors are used, the product yield is higher when vinyl propionate is used as donor compared to vinyl acetate. 3.4. IR studies

decreases with the number of recycling runs from almost 100% in the first run to 65% in the fourth run. The decrease in activity of LPbenzene is much lower compared to that of LPsilica, which exhibits almost no activity in the second run (Fig. 4a). These results confirm that the hydrophobic surface of LPbenzene enhances the retention of the TLL during the reaction. A commercial available immobilized lipase (from T. lanuginosus) immobilized on acrylic polymer beads (Immobead 150) was also tested under the same reaction

It is well known that the activity of an enzyme is strongly influenced by its secondary structure as well as its complete threedimensional structure [39]. The changes in the external force field, the pH of the environment and chemical and physical properties of the support will affect the secondary structure of the protein and the resulting enzymatic activity. Infrared spectroscopy is a well established technique for conformational analysis of proteins

b

C

100

1.8

80

40

0.6 0.3 0.0

4

Run

0

3

15

2

B

1

IM

ne

ex H

ue

2C H

To l

F TH C

A

ce

to

N 3C H C

0.9

e

0 an e

0 l2

20

n

20

1.2

en

40

60

nz

60

1.5

be

BP Yield (%)

80 BP Yield (%)

Activity / (mmol/min/g)

100

LP

a

Fig. 6. (a) Solvent effect on the transesterification reaction between vinyl propionate and 1-butanol (1:1 M ratio) over lipase immobilized on LPbenzene with a water content of 0.1 wt.%. (b) Recycling experiments of lipase immobilized in LPbenzene (same conditions as above). (c) Activity of lipase immobilized on LPbenzene and commercial available immobilized lipase on Immobead 150 (IMB150), calculated from the yield at a reaction time of 30 min.

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Table 4 Transesterification reaction with free lipase and immobilized lipase in LPbenzene as catalysts.a

O O Catalyst

R1

R 2OH Substrate

O

Lipase

R2

Hexane

O

O

R1

Time (h)

Conv. (%)

Yield (%)

Free lipase LPbenzene Free lipase LPbenzene

24a 24 24 24

67 85 21 40

58 76 19 36

Free lipase

24

58

54

LPbenzene

24

87

90

a The transesterification reaction was carried out at 1:1 M ratio of alcohol/vinyl propionate (except 1-phenylethanol: vinyl propionate = 1:5) over lipase immobilized on LPbenzene in hexane using 10 mg of immobilized lipase at 40 °C under stirring (500 rpm).

[40] and has recently been used to study the secondary structure of adsorbed lipase confined in porous materials [35]. Using the ATR technique, protein absorption bands can be obtained at low concentration with a good signal-to-noise ratio [41]. Fig. 7a shows the amide I and amide II bands of the spectrum of lipase immobilized on different supports. The amide I band at approximately 1600–1700 cm 1 mainly arises from C@O stretching vibration (83%) coupled with an out-of-phase C–N stretching and C–C–N deformation of the peptide backbone [42]. The amide II band centers around 1550 cm 1 due to in-plane N–H bending and C–N

a

b

Amide I 1900

stretching vibrations. Lipase immobilized in LPsilica and LPethane exhibits similar amide bands. On the contrary, LPbenzene exhibits a shift of the amide I band to higher wave number and a relatively strong amide II absorbance implying a different conformation of lipase in this material. The amide I band is more conformational sensitive due to the frequency of the C@O stretching mode depending on the strength of hydrogen bonding interaction involving the amide group and dipole–dipole interaction between the carboxyl groups. Thus, it is particularly relevant to detailed protein analysis [43]. Therefore, to further investigate the influence of different supports on the activity of immobilized lipase, the second derivative of the amide I spectra is calculated for a detailed analysis in the region of wave numbers between 1590 and 1700 cm 1 as shown in Fig. 7b–d. There are three main secondary structure elements observed in the following frequency intervals: a-helical structures at 1650–1660 cm 1, organized b-sheet structures at 1620–1650 cm 1 and disordered structures (including b-turns, loose b-strands, and polyproline II structures) at 1660– 1690 cm 1. The lower wave number components in the spectral range of 1620–1610 cm 1 are attributed to strongly hydrogen bonded b-sheet structures [44]. The second-derivative of the spectra in the amide I region of LPsilica and LPethane are similar to the free lipase in water, except the loss of relative intensity of the band around 1620 and 1610 cm 1, which can be assigned to intermolecular hydrogen bonded b-sheets. This result indicates that the proteins are less aggregated due to the immobilization on the large pore materials developed in this study. Compared to free lipase, LPbenzene supported lipase is characterized by a reduced content of intermolecular hydrogen bonded b-sheet, which is similar to LPsilica and LPethane, because the lipase is located inside the pore of the support without large aggregation domains. Furthermore, for LPbenzene, the intensity of the bands in the spectral range of

1800

Amide II

1700

1600

1500

1700

1680

c

1700

1660

1640

1620

1600

Wavenumber (cm-1)

Wavenumber (cm -1)

d

1680

1660

1640

1620

Wavenumber cm-1

1600

1700

1680

1660

1640

1620

1600

Wavenumber (cm-1)

Fig. 7. (a) ATR-FTIR spectra in the amides I and II region of immobilized lipase. (b) Second derivative of amide I band for (dash line) free lipase and (solid line) LPsilica. (c) Second derivative of amide I band for (dash line) free lipase and (solid line) LPethane. (d) Second derivative of amide I band for (dash line) free lipase and (solid line) LPbenzene.

Z. Zhou et al. / Microporous and Mesoporous Materials 154 (2012) 133–141

organized b-sheet is decreased. The band around 1644 cm 1 is almost completely lost, while the component bands increase in the area of disordered structure (1660–1690 cm 1). This phenomenon is consistent with the report by Misiunas et al. [45]: Incorporation of lipase (TLL) into a hydrophobic phase triggers the transformation of a part of b-sheet structures into less ordered elements, which can be explained by a structural transition of the entrapped protein to the open-lid or the enzymatic active conformation. Therefore, the IR experiments performed here further prove that lipase immobilized into the most hydrophobic benzene-containing PMO induces the transformation into an active conformation which is in line with the catalytic tests. 4. Conclusions In this work, lipase (TLL) has been immobilized in PMOs with large cage-like pore and an extended study was carried out to investigate the influence of the nature of the support on the immobilization efficiency of lipase as well as the resulting catalytic activity in hydrolysis and transesterification reactions. The structural properties of the support have a significant influence on lipase immobilization as well as the catalytic performance. Materials possessing higher surface area and pore volume exhibit higher adsorption capacity for enzymes. In the case of monolayer adsorption, the higher hydrophobicity of the novel PMOs under study results in better adsorption properties compared to silica, which are related to dominant hydrophobic interaction between the surface of the support and the lipase molecules. Furthermore, the hydrophobicity of the support also triggers higher catalytic activity in the transesterification reaction. The best results are obtained with LPbenzene as support when n-hexane is used as solvent and the water content is adjusted to 0.1 wt.%. In summary, the results show that PMOs with large cage-like pore are promising candidates for lipase immobilization for biocatalytic applications. Acknowledgements The authors gratefully acknowledge the financial support of the Cluster of Excellence ‘Engineering of Advanced Materials’ at the University of Erlangen-Nuremberg, which is funded by the German Research Foundation (DFG) within the framework of its ‘Excellence Initiative’. References [1] M. Held, A. Schmid, J.B. Van Beilen, B. Witholt, Pure Appl. Chem. 72 (2000) 1337–1343. [2] V.V. Mozhaev, M.V. Sergeeva, A.B. Belova, Y.L. Khmelnitsky, Biotechnol. Bioeng. 35 (1990) 653–659.

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