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Heterofunctional hydrophilic-hydrophobic porous silica as support for multipoint covalent immobilization of lipases: application to lactulose palmitate synthesis Claudia Bernal, Andres Illanes, and Lorena Wilson Langmuir, Just Accepted Manuscript • DOI: 10.1021/la4047512 • Publication Date (Web): 12 Mar 2014 Downloaded from http://pubs.acs.org on March 13, 2014
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Heterofunctional hydrophilic-hydrophobic porous silica as support for multipoint covalent immobilization of lipases: application to lactulose palmitate synthesis Claudia Bernal*, Andres Illanes, Lorena Wilson Escuela de Ingeniería Bioquímica, Pontificia Universidad Católica de Valparaíso, P.O. Box 4059, Valparaíso, Chile KEYWORDS heterofunctional silica; lipase immobilization; Pseudomonas stutzeri; Alcaligenes sp.; sugar ester; lactulose palmitate. ABSTRACT The lipase-catalyzed synthesis of sugar esters, as lactulose palmitate, requires harsh conditions making necessary to immobilize the enzyme. Therefore, a study was conducted to evaluate the effect of different chemical surfaces of hierarchical meso-macroporous silica in the immobilization of two lipases, from Pseudomonas stutzeri (PsL) and Alcaligenes sp. (AsL), which exhibit esterase activity. Porosity and chemical surface of silica supports, before and after functionalization and after immobilization, were characterized by gas adsorption and Fourier transform infrared (FTIR) spectroscopy. PsL and AsL were immobilized in octyl (OS), glyoxyl (GS) and octyl- glyoxyl silica (OGS). Hydrolytic activity, thermal and solvent stability and sugar
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ester synthesis were evaluated with those catalysts. The best support in terms of expressed activity was OS in the case of PsL (100 IU g-1), while OS and OGS were the best for AsL with quite similar expressed activities (60 and 58 IU g-1 respectively). At 60 ºC in aqueous media the more stable biocatalysts were GS-PsL and OGS-AsL (half-lives of 566 and 248 h respectively), showing the advantage of a heterofunctional support in the latter case. Lactulose palmitate synthesis was carried out in
acetone medium (with 4% of equilibrium moisture) at 40ºC
obtaining palmitic acid conversions higher than 20% for all biocatalysts, being the highest those obtained with OGS-AsL and OS-PsL. Therefore, screening of different chemical surfaces on porous silica used as supports for lipase immobilization allowed obtaining active and stable biocatalyst to be employed in the novel synthesis of lactulose palmitate. INTRODUCTION Sugar fatty acid esters are a family of compounds, some of which used as surfactants in food and pharmaceutical products because of their emulsifying and stabilizing properties 1. The main problem in synthesizing sugar fatty acid esters is related to the different functionality of the reactants: the sugar is polar and water soluble while the fatty acid is non-polar and soluble in organic solvents. Moreover, when chemical catalysis is performed the presence of several OH in the sugar molecule may produce several different compounds. The promiscuity of lipases in the recognition of different natural substrates and the regiospecificity in the esterification reaction allows solving the problem of multiple products that will be produced if doing chemical synthesis with this kind of sugar 2. Lipases from Acaligenes sp. (AsL) and Pseudomonas stutzeri (PsL) are two particularly interesting enzymes among the commercially available enzyme toolbox for sugar ester synthesis, both exhibiting high regioselectivity and stability at reaction conditions. AsL has been used for the synthesis of wax esters 34 and resveratrol derivatives 4, in
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both cases attaining conversions higher than 55%. On the other hand, benzoin ester 5 and wood sterols
6-7
have been produced with PsL. In order to use lipases in different reaction media and
modulate their activity, selectivity and operational stability, they can be conveniently modified by immobilization 5, 6, 8, 9. One of the purposes of enzyme immobilization is catalyst stabilization and re-utilization under process conditions. Immobilization protocols and supports used can also affect the catalytic properties of the lipase, since the natural substrates of this kind of enzyme are long-chain acyl glycerols, which exhibit poor aqueous solubility, so that an interface is required for adequate catalysis. This requirement is more important for those lipases bearing a “lid” that covers the active site and show interfacial activation mechanism, as is the case of PsL and AsL lipases 10. Thus, considering the interfacial activation mechanism of lipases and the nature of the substrates in sugar ester synthesis (polar sugar and non-polar free fatty acid), it is very important to develop protocols for their immobilization in supports with appropriate chemical surface allowing a significant increase in protein stability and catalyst recovery and reutilization, and favoring catalyst efficiency by promoting good enzyme-substrates interaction. Lipases have been mostly immobilized by hydrophobic interactions with excellent results due to the phenomenon of interfacial activation
8-9, 11
, that allows expressing a higher activity than the soluble enzyme by
the adoption of a more favorable conformation of the lipase in the presence of a hydrophobic micro-environment as provided by the support. On the other hand, covalent multipoint attachment has been tested with lipases
12
, being their main advantages the increase in thermal
stability and stability in organic solvents and the irreversible linkage of the enzyme to the support that prevents leakage during operation. Some authors have studied the influence of the heterofunctionality of the support in the activity and stability of enzymes 13, 14, highlighting their
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advantages with respect to catalyst behavior. Specifically with lipases, Reis et al
15
showed that
the chemical surface of the support influences their hydrolytic or condensation activity. Therefore, lipase immobilization in heterofunctional supports, using hydrophobic adsorption to open the lid and covalent attachment for stabilization, will be beneficial by increasing both reactivity and stability. There is a wide variety of supports amenable for lipase immobilization
16-17
being the porous
silica an excellent support due to offers some unique features due to their high surface area, thermal and mechanical stability, nontoxicity and high resistance to microbial attack and organic solvents. Mesoporous silica have been functionalized with different groups like thiol carboxylic 19, octyl and octadecyl
20
18
,
, amine and epoxide 21; and with heterofunctional groups to
be use as enzyme immobilization 15. The purpose of this work is the exploration of heterofunctional hydrophilic-hydrophobic (octylglyoxyl) silica support for the immobilization of AsL and PsL making the comparison with hydrophilic glyoxyl-silica and hydrophobic octyl-silica carriers and evaluating their expressed activity, thermal stability and catalytic performance in the reaction of synthesis of lactulose palmitate (scheme 1). Lactulose palmitate is an ester that combines the excellent properties of lactulose with one of the most common fatty acids in foodstuffs. Lactulose (4-O-β-Dgalactopyranosyl-D-fructose) is a non-digestible oligosaccharide used as prebiotic in the food industry and in medicine for the treatment of portal systemic encephalopathy and chronic constipation 22. Lactulose palmitate is then an interesting vehicle for this prebiotic delivering also palmitic acid.
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Scheme 1. Palmitate lactulose synthesis: substrates and possible products MATERIALS AND METHODS Materials The following analytical grade reagents were used without further modification. Sodium silicate (25–29% SiO2 and 7.5–9.5% Na2O), ethylacetate (EtAc), sulfuric acid (98%), and sodium periodate were purchased from Merck (Darmstadt, Germany); cetyltrimethylammonium bromide (CTAB), sodium borohydride, glycerin, glycidyloxypropyltrimethoxysilane (GPTMS 99.7%), trimethoxy(octyl)silane (OTMS; 96%), p-nitrophenol (pNP), p-nitrophenyl butyrate (pNPB), acetone and palmitic acid were purchased from Sigma–Aldrich (St. Louis, MO, USA). Lactulose was purchased from Carbosynth (Berkshire, United Kingdom). PsL (50,000 IU per gram of powder), AsL (60,000 IU per gram of powder) commercial extracts and QLC biocatalyst (lipase from Alcaligenes sp. immobilized in diatomaceous earth, 60,000 IU per gram) were kindly donated by Meito-Sangyio Ltd. (Fuchu, Japan). All other reagents and solvents were of the highest available purity and used as purchased. Synthesis and characterization of supports
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Silica synthesis Synthesis of silica was carried out from a reaction mixture with the following molar proportion of reagents: SiO2: Na2O: CTAB: EtAc: H2O = 1 : 0.3 : 0.24 : 7.2 : 193. This mixture was heated at 80 ºC for 48 h under quiescent conditions. The solid obtained was calcined at 540 ºC (heating rate: 1.5 ºC/ min) for 3 hours. More details on the procedure of synthesis can be found in Bernal et al 23. Silica derivatization Synthesized silica was chemically modified to obtain three kinds of carriers: hydrophilic with glyoxyl groups, hydrophobic with octyl groups and hydrophilic-hydrophobic with both glyoxyl and octyl groups. Hydrophilic derivatization: For the preparation of glyoxyl silica (GS), 1 g support (activated under vacuum at 200 ºC) was silylated in 30 mL of 10% GPTMS aqueous solution at pH 8.5 and 94ºC for 6 hours under gentle stirring. After filtration, the support was washed with 70:30 (v/v) water/acetone mixture and dried; oxidation proceeded by contacting the support with 0.1 M NaIO4 solution for 2 hours at 25 ºC. Details of the grafting reaction can be found in Bernal et al. 2423. The quantity of glyoxyl groups was determined spectrophotometrically by back titration with NaHCO3/KI. Hydrophobic derivatization: For the preparation of octyl silica (OS), 1 g silica (activated under vacuum at 200 ºC) was silylated in 30 mL of 10% OTMS in toluene solution at reflux for 6 hours under gentle stirring. After filtration, the support was washed with acetone to remove residual toluene and silane, then with abundant water and finally dried at room temperature. Hydrophilic-hydrophobic derivatization: The octyl and glyoxyl co-bonded silica (OGS) particles were prepared by refluxing toluene with a 10% GPTMS-OTMS mixture in a 1:1 proportion. A
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typical derivatization process was: 1 g of dried silica in 30 mL of anhydrous toluene under reflux was mechanically stirred for 6 h at 105 ºC. After filtration, the support was washed with acetone to remove residual toluene and silanes, and then with abundant water. The oxidation process for glyoxyl formation was done in the same way than for the GS support 24. Silica characterization Nitrogen adsorption isotherms of the silica samples were measured using the ASAP2010 instrument (Micromeritics) after activation at 100 ºC for 12 hours. The specific surface area (SBET) was evaluated from the nitrogen adsorption isotherms using the Brunauer–Emmett– Teller (BET) method, and the mesopore volume (VM) was calculated using the Barrett–Joyner– Halenda (BJH) model. Fourier transform infrared (FTIR) spectroscopy (Spectrum One, Perkin Elmer) was used for determining changes on the surface chemistry of porous silica particles before and after the derivatization process. Samples previously dried at 30 ºC under vacuum were mixed with KBr and then compressed to form tablets. The spectra were obtained at room temperature with 32 scans and 4 cm−1 of resolution. Thermogravimetric analyses (TGA) were carried out on TGA Q500 Instrument (TA instruments) for evaluating the organic content after the derivatization process. The samples were heated at a rate of 10 ºC/ min, from room temperature up to 600 ºC under nitrogen atmosphere. Enzyme activity assays The activity of lipases was assayed by following the increase of absorbance at 348 nm produced by the release of pNP by the hydrolysis of 0.4 mM pNPB in 25 mM sodium phosphate buffer at pH 7.0 and 25 °C. One international unit of activity (IU) was defined as the amount of enzyme that hydrolyzes 1 µmol of pNPB per minute under the conditions described above.
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Biocatalysts preparation and characterization AsL and PsL were immobilized in three supports with different chemical surface. The immobilization conditions were changed according to the support in order to favor enzymesupport interaction: for GS, immobilization was carried out at pH 10 (100 mM bicarbonate buffer) to favor the formation of Schiff bases between aldehyde groups of the support and εamino groups of lysine residues in the protein surface
25
, while the immobilization in OS was
done at pH 7 (25 mM phosphate buffer). The enzyme stability at these conditions was reported in a previous work
26
. For the heterofunctional support a combination of these methodologies was
conducted with the aim of obtaining an enzyme orientation similar to the one attained with OS, but also involving covalent bonding. Immobilization was carried out according to the chemical surface of each support, offering 160 IU and 200 IU of PsL and AsL per gram of support respectively. Details of the immobilization procedures for every support are described below. Immobilization in hydrophilic glyoxyl silica: 1 g of GS support was contacted with an enzyme solution (40 mL) containing 160 IU and 200 IU of PsL and AsL per gram of support respectively, prepared in 100 mM sodium bicarbonate buffer pH 10 at 25 ºC. The suspension was incubated under gentle stirring until the activity in the supernatant was either zero or constant. Afterwards, the biocatalysts were reduced with 1 mg of sodium borohydride per mL for 30 minutes, as previous described
25
, then thoroughly washed with water and dried at 25 ºC. PsL
and AsL immobilized in GL are referred as GS-PsL and GS-AsL respectively. Immobilization in hydrophobic octyl silica: 1 g of OS support was contacted with an enzyme solution (40 mL) containing 160 IU and 200 IU of PsL and AsL per gram of support respectively, prepared in 25 mM potassium phosphate buffer pH 7 at 25 ºC. The suspension was incubated under gentle stirring until the activity in the supernatant was either zero or constant.
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Then, the biocatalysts were filtered, thoroughly washed with water, and dried at 25ºC. PsL and AsL immobilized in OL are referred as OS-PsL and OS-AsL respectively. Hydrophilic-hidrophobic derivatization: 1 g of OS support was contacted with an enzyme solution (40 mL) containing 160 IU and 200 IU of PsL and AsL per gram of support respectively, prepared in 25 mM potassium phosphate buffer pH 7 at 25 ºC. The suspension was incubated under gentle stirring until the activity in the supernatant was either zero or constant. Then, the biocatalysts were filtered, thoroughly washed with water, re-suspend in 100 mM sodium bicarbonate buffer pH 10 (40 mL) and gently stirred at 25 ºC for 1 hour. Afterwards, the biocatalysts were reduced with 1 mg/mL of sodium borohydride for 30 minutes, as previous described
25
, and then thoroughly washed with water and dried to 25 ºC. PsL and AsL
immobilized in OGS are referred as OGS-PsL and OGS-AsL respectively. Immobilization in hydrophilic-hydrophobic support (OGS) was done in two steps: first at pH 7 to favor enzyme adsorption (similarly than with the OS support) and then at pH 10 to allow Schiff bases formation between the enzyme and the support (similarly than with the GS support). For every biocatalyst, immobilized protein was calculated by the difference of protein content in the supernatant at the beginning and at the end of immobilization. Protein was determined by Bradford methodology 27. Immobilization yield in terms of protein (IYp) and activity (IYa) were calculated according to Eqs. 1 and 2, respectively Eq. 1
100
Eq. 2
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where PO is the offered protein, PL is the loaded protein in the biocatalyst, AO is the offered activity and A is the expressed activity in the immobilized enzyme. Stability of biocatalysts Stability of the biocatalysts and the soluble lipases was evaluated under two non-reactive conditions: in 25 mM sodium phosphate buffer at pH 7.0 and at 60 ºC and in 100% acetone at 40 ºC without the addition of surfactant or activity stabilizers in both cases. Aliquots were withdrawn periodically under stirring in order to have a homogeneous biocatalyst suspension. The residual activity of the enzymes was measured as described previously, and half-life (period of time at which the enzyme have lost one half of its initial activity) was determined. Thermal stabilization by immobilization was quantified in terms of the stabilization factor (SF), defined as the ratio of the half-life of the immobilized enzyme and the soluble counterpart. Lactulose palmitate synthesis Lactulose palmitate was synthesized by esterification of lactulose (5 mM) with palmitic acid (2.5 mM) in acetone at 40 ºC, at a 10 mL scale. The reaction mixture contained 15 mg/mL of biocatalyst. The conversion results were compared with the commercial extracts of every lipase and the commercial biocatalyst QLC, using the same enzyme proportion in every case. Substrates and products of reactions were determined by HPLC using C-18 column and UV-Vis spectrophotometer detector (JASCO model AS-2089). Separation was achieved by eluting with acetonitrile:water mixtures as mobile phase at a flow-rate of 1 mL/min with the following gradient program: 60:40 (v/v) for 6 minutes and then 95:5 (v/v) for 18 minutes. Detection was performed in a UV detector at 270 nm and the monoester of lactulose palmitate was analyzed by HPLC-MS (Model 2020, Shimadzu, Kyoto, Japan) with photodiode detector (SPD M 20A) and mass analyzer (MS 2020) with electrospray ionization (ESI) system.
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Synthesis conversion into lactulose palmitate (CLP) was defined with respect to the limiting substrate and calculated by the disappearance of palmitic acid according to Eq. 3:
− 100
Eq. 3
where Mi and Mf are the initial and final mass of palmitic acid respectively. Biocatalyst Reuse Biocatalyst stability was tested under operation conditions in sequential batch reactor operation: 100 % acetone (4% equilibrium moisture), 40ºC and lactulose: palmitic acid molar ratio1:1. Each batch had a duration of 48 hours. After this time, one aliquot was taken and analyzed by HPLC as previously described in order to determine conversion. Between cycles the reacted medium was filtered out and the biocatalyst retained, washed with acetone and dried at 25ºC to remove residual substrate, products and solvent, prior of using it in the subsequent batch. RESULTS AND DISCUSSION Synthesis and derivatization of mesoporous hydrophilic-hydrophobic silica was studied because it is a promising matrix for industrial biocatalysts to be used in reactions of organic synthesis: it has a high protein loading capacity and is a potential stabilizer due to the multipoint covalent attachment of the enzyme to it. On the other hand, PsL and AsL were selected as a proof of concept, due to their differences in structure and interfacial activation mechanism facilitating the immobilization process by hydrophobic interactions. Synthesis and characterization of supports Conditions worked in this study allowed for the synthesis of materials with hierarchical porosity, block morphologies and high content of silanol groups 24, 28. The nitrogen capillary condensation, observed in the isotherms at high relative pressures (Figure 1a) indicates the presence of large mesopores with diameters between 15 and 40 nm that after derivatization remained in the same
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range (Figure 1b); furthermore, synthesized porous silica exhibited high surface area and pore volume even after chemical modification (Table 1). The mild changes in these values can be due to the fact that surface chemistry modification affects the interaction between adsorbent and adsorbate (N2-silica) and also the SBET measurement, pore diameter and pore volume. Furthermore, the grafting of silanol groups with other groups (glyoxyl or octyl) may cause a decrease of pore diameter and consequently a lower pore volume.
a.
b.
Figure 1.
Characteristics of the porous silica supports: (a) nitrogen adsorption–desorption
isotherm for bare silica and (b) pore size distribution for bare silica (full square), glyoxyl-silica (full triangle), octyl-silica (empty circle) and octyl-glyoxyl silica (empty triangle).
Table 1. Characteristics of the different porous silica supports.
Material Bare Silica GS OS
% glyoxyl
% octyl
Character
A (m2 g−1)a
D range (nm)b
V (cm3 g1 c )
0
0
----
618
4-64 (24)
1.07
100 0
0 100
aldehydes (µmol g-1) --
Hydrophilic 456 4-37 (15) 1.08 1010 Hydrophobic 349 4-100 (22) 0.88 --Hydrophilic 482 OGS 50 50 457 4-41 (14) 1.06 Hydrophobic a A: surface area BET. b D: pore diameter range (values in parenthesis correspond to the average). c V: pore volume
Thermogravimetric analysis shows a similar organic content for the three functionalized silica (Figure S1 in Supplementary Information) indicating that silanes are in the silica material and all
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have similar content of organic material. The grafting process with GPTMS was verified by back titration with NaHCO3/KI, obtaining 1010 and 482 µmol per gram of silica for GS and OGS supports, respectively. The covalent bonding between grafted reactive and silanol groups of silica was previously established by Bernal et al
24
, while the covalent bonding of OTMS to
silica, with the methodology used in this work, was proved with
29
Si nmr by Garcia et al
29
.
Furthermore, in this work, chemical surface modification was tested by FTIR analysis; the main bands for silica materials are shown in Figure 2. The analysis of spectra revealed that the broad band centered at around 3470-3380 cm−1 corresponds to the overlapping of the O-H stretching bands of hydrogen-bonded water molecules (H-O-H···H) and SiO-H stretching of surface silanols hydrogen-bonded to molecular water (SiO-H···H2O). Besides, siliceous structure exhibited a characteristic band at 1104 cm−1 which is attributed to the stretching vibration of Si–O–Si bonds, and a band at 799 cm−1 which is assigned to the vibration of Si–O–Si bonds (Figure 2a) band near 1630 cm-1 can be due to water physisorbed onto the silica structure
31
30
; the
. After
modification, the region between 1600-3000 cm−1 is indicative of the presence of organic matter in the support according to the derivatization process. The presence of aldehyde groups in the GS support is revealed by the band at 1700 cm-1 (the C=O stretching vibration), while the bands at 2942 cm-1 and 2867 cm-1 (the C-H symmetric and antisymmetric stretching vibrations of -CH3 and -CH2 groups) evidence the octyl content in the OS carrier (Figure 2b)
30, 32-35
. On another
hand, with OGS support presence of both bands corresponding to carbonyl and aliphatic groups in the FTIR spectra for the hydrophilic-hydrophobic silica reveals the heterofunctional functionalization (Figure 2b). Biocatalysts preparation and characterization
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Lipases immobilization was initially verified by changes in FTIR spectra for the OGS support. The appearance of an extra band on the FTIR spectrum corresponding to functional groups of the enzyme, (Figure 2c) like the one at 1547 cm-1, revealed the presence of aminated groups through bending vibration of N-H groups due to Schiff base formation between lipase and support, and/or R-groups of some amino acids.
a.
b.
c.
Figure 2. FTIR spectra for bare silica (a), modified silica (b), and biocatalyst obtained with lipase from Pseudomonas stuzeri immobilized in octyl-silica (c) PsL was immobilized with good yields of activity expression (in all cases were higher than 40%), being GSPsL the best catalyst obtained (Table 2). These differences can be due to the enzyme orientation onto the carrier and the protein selectivity resulting from the enzyme-support interactions. In the case of GS, the enzyme is immobilized through the lysine-rich zone and if this zone is very close to the active site, catalysis will be hampered, as probably happened with
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PsL. On the other hand, with OS the interfacial activation mechanism of lipases took place, the enzyme being immobilized with an open configuration of its active site favoring a high expression of activity even though protein immobilization yield was low (Table 2). Furthermore, since the lipase has a hydrophobic pocket that can interact easier with the octyl groups of OS, in situ purification is possible resulting in a higher expressed specific activity. With OGS, the immobilization by combined hydrophobic interactions (similar to OS support) and covalent bond formation affected the expressed activity, generating a biocatalyst (OGSPsL) with lower activity than OSPsL; this is probably because the active site is rigidified when the covalent bonds between enzyme and support are formed, reducing the required mobility for activity expression. However, the high selectivity achieved with this support (only proteins with hydrophobic pockets are immobilized) allowed obtaining a biocatalyst with higher expressed activity and specific activity than GSPsL (Table 2). Very few works have been published about immobilization of PsL and only non-covalent immobilization has been reported
6, 36
being the
activities reported lower or similar than the ones obtained in this work with OS-PsL. Table 2. Parameters of immobilization of Pseudomonas stutzeri and Alcaligens sp.lipases in different porous silica supports. Biocatalyst
YIp (%)
SAP YIa (%) SAB (IU mg−1 (IU g−1 support) protein) Biocatalyst from Pseoudomonas stuzeri lipase GS-PsL 61.1 21.3 40.4 64 OGS-PsL 16.0 77.3 55.9 87 OS-PsL 17.7 88.7 64 100 Biocatalyst from Alcaligenes sp. lipase GS-AsL
