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Immobilization and stabilization of a bimolecular aggregate of the lipase from Pseudomonas fluorescens by multipoint covalent attachment
Process Biochemistry 48 (2013) 118–123
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Immobilization and stabilization of a bimolecular aggregate of the lipase from Pseudomonas fluorescens by multipoint covalent attachment Lionete Nunes de Lima a,b , Caio C. Aragon a,c , Cesar Mateo a , Jose M. Palomo a , Raquel L.C. Giordano b , Paulo W. Tardioli b , Jose M. Guisan a,∗ , Gloria Fernandez-Lorente d,∗ a
Instituto de Catalisis, CSIC, 28049 Madrid, Spain Chemical Engineering Department, Federal University of São Carlos, São Carlos, SP, Brazil c Instituto de Química, UNESP-Univ. Estadual Paulista, Departamento de Bioquímica e Tecnologia Química, Araraquara, SP, Brazil d Instituto de Investigación en Ciencias de la Alimentación (CIAL) CSIC-UAM, 28049 Madrid, Spain b
a r t i c l e
i n f o
Article history: Received 27 July 2012 Received in revised form 19 October 2012 Accepted 8 November 2012 Available online 19 November 2012 Keywords: Pseudomonas fluorescens lipase Lipase aggregates Enzyme immobilization and stabilization Improved enantioselectivity
a b s t r a c t The soluble lipase from Pseudomonas fluorescens (PFL) forms bimolecular aggregates in which the hydrophobic active centers of the enzyme monomers are in close contact. This bimolecular aggregate could be immobilized by multipoint covalent linkages on glyoxyl supports at pH 8.5. The monomer of PFL obtained by incubation of the soluble enzyme in the presence of detergent (0.5% TRITON X-100) could not be immobilized under these conditions. The bimolecular aggregate has two amino terminal residues in the same plane. A further incubation of the immobilized derivative under more alkaline conditions (e.g., pH 10.5) allows a further multipoint attachment of lysine (Lys) residues located in the same plane as the amino terminal residues. Monomeric PFL was immobilized at pH 10.5 in the presence of 0.5% TRITON X-100. The properties of both PFL derivatives were compared. In general, the bimolecular derivatives were more active, more selective and more stable both in water and in organic solvents than the monomolecular ones. The bimolecular derivative showed twice the activity and a much higher selectivity (100 versus 20) for the hydrolysis of R,S-2-hydroxy-4-phenylbutyric acid ethyl ester (HPBEt) in aqueous media at pH 5.0 compared to the monomeric derivative. In experiments measuring thermal inactivation at 75 ◦ C, the bimolecular derivative was 5-fold more stable than the monomeric derivative (and 50-fold more stable than a one-point covalently immobilized PFL derivative), and it had a half-life greater than 4 h. In organic solvents (cyclohexane and tert-amyl alcohol), the bimolecular derivative was much more stable and more active than the monomeric derivative in catalyzing the transesterification of olive oil with benzyl alcohol. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Glyoxyl groups are aldehydes with very short chains (Support0–CH2 –CHO). Glyoxyl-activated supports are able to react very rapidly with non-ionized amino groups, but they form very unstable Schiff’s bases, and they are not able to immobilize enzymes via a single enzyme-support attachment [1,2]. Glyoxyl supports are able to immobilize enzymes under alkaline conditions via a multipoint covalent connection between two or more Lys residues on the enzyme surface and very highly activated supports. At pHs lower than 10, glyoxyl supports are not able to immobilize monomeric enzymes that have an unique reactive amino terminus.
∗ Corresponding authors. E-mail addresses: [email protected] (J.M. Guisan), gflorente@ifi.csic.es (G. Fernandez-Lorente). 1359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2012.11.008
However, glyoxyl supports are able to immobilize, at pHs lower than 10, enzymes that have more than one amino terminus. For example, some multimeric enzymes have two or more amino termini in the same plane of the enzyme surface [3], in such cases, a multipoint covalent immobilization can take place. Soluble lipases can form non-covalent bimolecular aggregates [4,5] that are dissociated in the presence of detergents by stabilization of the open form of the enzymes. In these bimolecular aggregates, the two hydrophobic open active centers of the monomers are in close contact one with the other and form stable aggregates with new active centers and sometimes with new catalytic properties [6,7]. These systems have been successfully used for site-specific lipase purification [8,9]. Glyoxyl supports might be able to covalently immobilize those bimolecular aggregates in cases where both amino terminal are on the same planar surface. The aggregates would be immobilized by simultaneous multipoint immobilization of both amino terminal (at pH 8.5). After the first immobilization, the derivatives could be
L.N. de Lima et al. / Process Biochemistry 48 (2013) 118–123
incubated under more alkaline conditions, allowing Lys residues located in the same region as the amino termini to attach to the highly activated support. In contrast, the monomeric form of the lipase can be immobilized and stabilized by multipoint covalent immobilization under alkaline conditions in the presence of moderately high concentrations of detergents. This immobilization occurs via the region of the monomeric lipase with the highest density of Lys residues, which is located on the surface opposite to the active center [4]. In the present manuscript, the multipoint covalent immobilization of lipase from Pseudomonas fluorescens (PFL) bimolecular aggregates was investigated. The properties of the immobilizedstabilized bimolecular aggregates were compared to those of the immobilization-stabilized monomeric enzyme. The stability, activity and enantioselectivity of both types of immobilized derivative were evaluated. Two types of support were used for different immobilizations of PFL. Agarose gels are very useful for work performed in aqueous media and in stirred tank reactors, and Sepabeads resins are very useful for work performed in organic solvents and in any type of reactor system (stirred tank, fixed bed, etc.). Different PFL catalysts were applied to two biotransformations as model reactions: the kinetic resolution of (R,S)-2-hydroxy-4phenylbutyric acid ethyl ester (HPBEt) in aqueous media and the transesterification of olive oil in organic solvents. 2. Materials and methods 2.1. Materials Buffering salts, TRITON X-100 (TX), p-nitrophenyl butyrate (p-NPB), tert-amyl alcohol, cyclohexane, and benzyl alcohol were from Sigma Chemical Co. (St. Louis, USA). Octyl SepharoseTM CL-4B was purchased from GE Healthcare (Uppsala, Sweden). C18-Sepabead resins were kindly donated by Resindion S.R.L. Agarose 6BCL was purchased from Agarose Bead Technologies. The racemic mixture of R,S-2hydroxy-4-phenylbutyric acid ethyl ester (HPBEt) was a kind gift from VITA INVEST SA. The P. Fluorescens lipase (PFL) was purchased from Amano Pharmaceutical Co., Ltd. (Nagoya, Japan). The other reagents and solvents used in this study were of analytical or HPLC grade.
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Immobilization on glyoxyl-agarose [10] or glyoxyl-sepabeads was carried out by adding 4.2 g of support to 36 mL of a solution at pH 10.5 containing 0.1 M sodium bicarbonate with 0.5% (v/v) of TRITON X-100, and 5 mg/mL of the lipase solution. Three derivatives were prepared with these supports. The first was prepared in bicarbonate buffer at pH 8.5 in the presence of 0.5% TRITON X-100 (TX), where the enzyme is not immobilized. The second was prepared in the same buffer but in the absence of TX, where the enzyme is immobilized as bimolecular aggregate (Gly-bi derivative). The third derivative (Gly-mono) was prepared in buffer at pH 10.5 and 0.5% TX. In all cases, at the reaction end point, 36 mg of sodium borohydride was added. Interfacial adsorption [11–13] of PFL on octyl agarose or octadecyl-Sepabeads (OS or Sep-C18 derivatives) was performed by adding 4.2 g of support to 36 mL of 5 mM sodium phosphate, pH 7.0 containing 5 mg/mL of PFL. In each case, a reference suspension, where the activated support was substituted by inert agarose, was prepared. In all cases, the activity of this reference suspension showed 100% of the activity associated with the supernatant, indicating that no lipase was non-specifically adsorbed to the support. Immobilization yield was defined as the percentage of lipase (determined by the activity on p-NPB) immobilized on the different supports. 2.2.3. Irreversible inactivation of immobilized PFL in the presence of diethyl-p-nitrophenylphosphonate (D-pNP) Aliquots of (0.4 g) of Gly-mono, Gly-bi or OS derivatives were suspended in 5 mL of 25 mM sodium phosphate buffer solution at pH 7 and 25 ◦ C, and 1.45 mM of the inhibitor D-pNP was added to the suspension. The reaction was very gently stirred until the activity of the immobilized enzyme was zero. 2.2.4. Enzymatic synthesis of benzyl oleate A 0.1 g aliquot of equilibrated immobilized lipase was added to 1.42 mL of the substrate solution (1 micromole of benzyl alcohol and 2 mol of olive oil) in 9.94 mL of organic solvent (tert-amyl alcohol or cyclohexane). To ensure anhydrous conditions, 100 mg of dry molecular sieves were also added to the reaction mixture. The progress of the reaction was followed by HPLC. 2.2.4.1. HPLC analysis. Reactants and products were analyzed by RP-HPLC (Spectra Physic SP 100 coupled with a Spectra Physic SP 8450 UV detector) using a reversedphase column (Ultrabase-C18, 250 mm × 4.6 mm, 5 m). Products were eluted at a flow rate of 1.0 mL/min with methanol/water/CH3 COOH (60:40:0.1, by vol.), and the UV absorbance was monitored at 254 nm. In fact, the total area of the chromatogram at 254 nm is constant during the whole course of the condensation. At this wavelength, the oil, the fatty acids and glycerol do not absorb. Reaction rates and synthetic yields could be easily calculated from the pure peak areas corresponding to benzyl alcohol (retention time (RT) of 5 min) and benzyl oleate (RT of 9 min).
2.2. Methods 2.2.1. Determination of lipase activity An activity assay was performed by calculating the initial velocity of the hydrolysis of 0.4 mM p-nitrophenyl butyrate (p-NPB) in 25 mM sodium phosphate at pH 7 and 25 ◦ C, catalyzed by a known amount of PFL. The reaction was followed by measuring, at 348 nm (this wavelength correspond to the isosbestic point of pNP) ( = 5.150 M−1 cm−1 ), the increase with the time in the absorbance, produced by the release of the reaction product p-nitrophenol (pNP). The reaction was performed using a spectrophotometer with a thermostated cell with continuous magnetic stirring. To initialize the reaction, 0.05–0.2 mL (depending of the dilution and activity of the sample) of the soluble lipase (blank or supernatant) or their immobilized preparations (suspension) were added to 2.5 mL of substrate solution. Enzymatic activity was calculated as micromoles of hydrolyzed p-NPB per minute per milligrams of enzyme (IU) under the conditions described above. The calculations were done according to this expression: IU mL−1 solution = (A min
−1
)VT −1 1000ε−1 b−1 Vs −1
IU mL−1 solution: international units per mL of solution expressed in mol of product per minute, (A min−1 ): increment of absorbance per minute, VT : total volume in the cell expressed in mL, ε: molar extinction coefficient, b: width of the cell. Vs : volume of sample in the cell expressed in mL. 2.2.2. PFL immobilization For immobilization of the lipases on the CNBr-Sepharose support, the first step was activation of the support prior to use by its suspension in an acidic aqueous solution (pH 2–3) for 1 h. The activated support was dried by filtration under vacuum. Next, 4.2 g of activated CNBr-Sepharose was added to 36 mL (0.2 mg of lipase/mL corresponding to 100 total IU of p-NPB hydrolysis) of the lipase solution in the presence of 0.5% TRITON X-100 (TX). The mixture was stirred at 4 ◦ C and 250 rpm for 20 min. Afterwards, the solution was removed by filtration, the supported lipase (CNBr-S derivative) was washed twice with 100 mM NaHCO3 at pH 8 and re-suspended in 15 mL of 1 M ethanolamine at pH 8 for 90 min to block the unreacted imidocarbonate reactive groups [2]. The blocked resin was recovered by filtration and washed with abundant distilled water. The immobilization procedure was followed by the enzymatic activity assay described above.
2.2.5. Hydrolysis of 2-hydroxy-4-phenylbutyric acid ethyl ester (HPBEt) Evaluation of the enzyme activity was performed by adding 0.5 g of derivative to 10 mL of 5 mM (R,S) HPBE in 25 mM phosphate buffer at pH 7.5 and 25 ◦ C with mechanical stirring [14]. The pH was maintained by automatic titration, and the enzymatic activity (mol of substrate hydrolyzed per minute per mg of immobilized protein) was determined by reverse-phase HPLC. The stereo specificity of the hydrolysis was analyzed by chiral-HPLC. 2.2.5.1. Analysis by HPLC. The hydrolysis products were analyzed by a HPLC device (Spectra Physic SP 100) coupled to an UV detector (Spectra Physic SP 8450) at 225 nm. The column was a Chiracel OD-R, the mobile phase was a mixture of acetonitrile (40%) and water (60%), and the analyses were performed at a flow rate of 0.5 mL/min. Quantification of the conversion degree was performed by comparison with standard solutions of the racemate and pure esters. Enantioselectivity (E value) was calculated from the enantiomeric excess of the remaining ester and the percent conversion as previously reported [15]. 2.2.6. Thermal inactivation of PFL-agarose derivatives Different immobilized preparations of PFL were incubated in 5 mM sodium phosphate buffer at pH 7 and 75 ◦ C. Samples were periodically withdrawn using a pipette with a cut-tip and under vigorous stirring to ensure a homogeneous biocatalyst suspension, and their remaining activities were determined as described for the p-NPB assay. 2.2.7. Stability of PFL-Sepabeads derivatives Thermal stability: the different PFL-Sepabeads derivatives were incubated in 5 mM sodium phosphate buffer at pH 7 and 65 ◦ C. Samples were periodically withdrawn using a pipette with a cut-tip and under vigorous stirring to ensure a homogeneous biocatalyst suspension, and their remaining activities were determined as described for the p-NPB assay. Stability in organic solvents: the enzyme derivatives were equilibrated with the solvent (tert-amyl alcohol or cyclohexane) at the desired temperature (60 ◦ C). Subsequently, at different times, 0.1 g of catalyst incubated in the inactivation conditions was added to the synthesis reaction solution, and the activity was analyzed using the enzymatic synthesis assay described in the previous section.
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Fig. 1. Immobilization of PFL on glyoxyl-supports at pH 8.5. Squares: PFL in the presence of detergent (0.5% TX); circles: PFL in the absence of detergent.
Fig. 3. Schematic representation of a bimolecular aggregate of PFL (taken from PDB code 3LIP). Light blue: amino terminus; dark blue: lysine residues; red: lipase active centers. (For interpretation of the references to color in the artwork, the reader is referred to the web version of the article.)
Experiments were carried out in triplicate, and the standard error was never greater than 5%.
3. Results and discussion 3.1. Preparation of PFL derivatives 3.1.1. Immobilization of PFL in the presence or absence of detergents on glyoxyl supports at pH 8.5 (agarose gels and Sepabeads supports) In aqueous medium, the imine formed by reaction of aldehyde and primary amine groups is in equilibrium shifted to the unlinked specie, due to this, the immobilization on glyoxyl supports is only produced when more than one reactive amino group can simultaneously react with more than one aldehyde groups on the support [10]. In the presence of 0.5% of TX, the enzyme is inefficiently immobilized at pH 8.5 even after 24 h (Fig. 1), presumably because the enzyme is in monomeric form under these conditions and has only one amino terminus that is unable to be irreversibly attached to glyoxyl-supports (at this pH, lysines are protonated and subsequently they are not reactive and only one amino terminus is unprotonated and reactive) (Fig. 2). However, in the absence of
detergent (bimolecular aggregates as it was previously reported [5], PFL is slowly immobilized at pH 8.5, and after 24 h, all the offered enzyme becomes immobilized on the support. The existence of two amino termini in the same planar surface in the bimolecular aggregate would explain the different behavior of the two forms of PFL (Fig. 3) and hence PFL is able to be immobilized by slow multipoint covalent immobilization (Fig. 2). After immobilization, the derivative can be incubated at pH 10.5 for 24 h to promote the attachment to the support of Lys residues located in the vicinity of the two amino termini (Fig. 2).
3.1.2. Immobilization of PFL on glyoxyl-supports at pH 10.5 in the presence of detergent (0.5% of TX) Under these conditions (0.5% of TX), the enzyme immobilizes on glyoxyl supports at moderate rates (6 h on glyoxyl agarose and 24 h on glyoxyl-Sepabeads). Each monomer may be immobilized through its region with the highest amount of Lys groups. PFL is not very rich in Lys residues, and glyoxyl-Sepabeads is not very rich in glyoxyl groups.
Fig. 2. Schematic representation of the immobilization of the different lipase populations (bimolecular aggregated or deaggregated lipase at pH 8.5) on glyoxyl supports.
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121
120
Residual acvity (%)
100 80 60 40 20 0
0
5
10
15
20
25
Time, minutes Fig. 5. Irreversible inhibition of PFL derivatives with diethyl-pnitrophenylphosphate.Squares: Gly-mono; circles: Gly-bi; triangles: OS.
3.1.3. Immobilization of the monomeric PFL on CNBr-activated Sepharose The enzyme is immobilized in the presence of detergent (monomeric form) at pH 7.0 at 4 ◦ C for 15 min on CNBr-activated Sepharose. The used immobilization conditions allow producing a PFL derivative to be used as a reference for lipase behavior. In general, immobilization of enzymes under these conditions will occur to just one covalent bond, and these immobilized preparations result in a good model of the enzyme properties in absence of intermolecular phenomena (Fig. 4) [4,10]. Lipases have been shown to exhibit a strong tendency to form lipase–lipase aggregates, making it very difficult to study isolated lipase molecules in soluble form [7,8]. The obtained immobilization yield was 30% of the input protein. As expected, immobilization yields in the used conditions are not very high but the formation of any kind of multipoint covalent attachment is avoided. After immobilization, the detergent is removed, and this derivative is taken as a reference of the behavior of native monomeric PFL without the formation of bimolecular aggregates. 3.1.4. Immobilization of PFL by adsorption on hydrophobic supports (octyl-Sepharose and octadecyl-Sepabeads) The enzyme was immobilized in more than 95% yield on hydrophobic supports, and it becomes hyper-activated: the measured activity is 150% on octyl-Sepharose and 300% on octadecyl-Sepabeads (Table 1). Table 1 Preparation of different immobilized derivatives of PFL. Activated Support
Immobilization yield, %
Recovered Activity, %
Derivative
CNBr-activated Sepharose Octyl-Sepharose Octadecyl-Sepabeads Glyoxyl-bimolecular Glyoxyl-monomeric
30 99 99 98 95
95 150 300 70 75
CNBr-S OS Sep-C18 Gly-bi Gly-mono
Experiments were performed as described in Section 2.
3.2. Modification of immobilized PFL with a covalent irreversible inhibitor Modification of lipases by covalent modification of the catalytic Ser with an irreversible inhibitor (e.g., diethyl p-nitro phenylphosphonate) will be faster when the active center of the lipase is open. In fact, the inactivation of the PFL adsorbed on octyl agarose is much faster than the inactivation of the monomeric derivative in the absence of detergent (Fig. 5). The modification of the bimolecular derivative is also faster than the one of the monomeric derivative, which could be the effect of two open active centers in close contact. 3.3. Thermal inactivation of the PFL-agarose derivatives The bimolecular derivative was 5-fold more stable than the monomeric one and is also more stable than PFL adsorbed on octylSepharose (Fig. 6). These latter derivatives are 10-fold more stable than the one-point-immobilized PFL (CNBr-S). CNBr-S derivative was used as control because soluble enzyme is forming aggregates 120 100
Remaining acvity (%)
Fig. 4. Schematic representation of the different PFL derivatives. CNBr-S: one-point covalently immobilized monomeric derivative Gly-mono: multipoint covalently immobilized monomeric derivative Gly-bi: multipoint covalently immobilized bimolecular aggregate OS: PFL adsorbed on octyl-Sepharose.
The immobilization of lipases on hydrophobic supports by interfacial adsorption [11–13] permits to fix the open conformation on a solid phase (Figure 4) representing a simple and elegance methodology to get very high active lipase catalysts. The hydrophobic nature of the support affected to the hyperactivation [16]. This depended on the hydrophobic nature of the lid (e.g., number of amino acids) or the surrounding area of the active site of a particular lipase.
80 60 40 20 0
0
1
2
3
4
5
Time, hours Fig. 6. Time-course of the thermal inactivation of different PFL-agarose derivatives. Derivatives were incubated at 75 ◦ C and at different times, aliquots were withdrawn and assayed at 25 ◦ C with p-NPB. Circles: Gly-bi; squares: Gly-mono; triangles: OS; empty cycles: CNBr-S.
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Table 2 Enantioselective hydrolysis of (R,S) -2-hydroxy-4-phenylbutyric acid ethyl ester (HPBET) by PFL derivatives.
CNBr-S-PFL Gly-mono-PFL Gly-bi-PFL OS-PFL
0.4 0.4 0.8 1.2
7 20 >100 >100
Reference [6] [6]
Experiments were performed at pH 5.0 as described in Section 2.
and the soluble monomer only can be obtained using detergent in the medium. In these conditions the structure of the soluble enzyme is different since the lid that normally covers the active site is opened changing the structure of the lipase and often their catalytic properties. Hydrophobic adsorption and multipoint covalent attachment seem to have an interesting stabilizing effect. It appears that bimolecular derivatives may be stabilized by two different effects: hydrophobic adsorption of both active centers and multipoint covalent attachment of the bimolecular structure. The new derivative of PFL is 50-fold more stable than the one-pointimmobilized derivative. 3.4. Enantioselective hydrolysis of R,S-2-hydroxy-4-phenylbutyric acid ethyl ester (HPBEt) by PFL derivative The activity of the bimolecular derivative is higher than that of the multipoint-attached and one-point-attached monomeric derivatives and is lower than that of the octyl-Sepharose derivative (Table 2). However, selectivity is similar to that of the octylSepharose derivative (E > 100) and is much higher than that of the one-point-attached derivative (E = 7) and the multipoint-attached monomeric derivative (E = 20). The hydrophobic adsorption of active centers seems to be a key factor for the improvement of enantioselectivity. The bimolecular derivative is more active, more selective and more stable than both monomeric derivatives. Compared to the octyl-Sepharose derivative, the biomolecular derivative was more stable, less active and exhibits a similar very high selectivity. 3.5. Stability of PFL-Sepabeads derivatives Thermal stability: the bimolecular derivative is much more stable than the monomeric one and its stability is slightly lower than the hydrophobic derivative (PFL adsorbed on octadecyl-Sepabeads) (Fig. 7). Multipoint attachment of Sepabeads is less extensive than on agarose because Sepabeads have a low density of glyoxyl groups. Indeed, the immobilization rates on Sepabeads are also much lower. The most relevant stabilizing effect seems to be the stabilization of the open form of PFL. Stability in organic solvents: all derivatives are quite stable in cyclohexane at 60 ◦ C. The monomeric derivative is the least stable (Fig. 8). All derivatives are less stable in a more polar solvent (tertamyl alcohol), and differences between the bimolecular and the monomeric derivatives are higher in such solvents (Fig. 9). 3.6. Enzymatic transesterification of olive oil with benzyl alcohol
100
Remaining acvity (%)
E value
80 60 40 20 0
0
5
10
15
20
25
Time, hours Fig. 7. Time-course of the thermal inactivation of different PFL-Sepabeads derivatives. Derivatives were incubated at 65 ◦ C, pH 7.0, and at different times, aliquots were withdrawn and assayed at 25 ◦ C with p-NPB. Triangles: Sep-C18; Circles: Gly-bi; squares: Gly-mono.
120
Remaining acvity (%)
Specific activity (IU/mg)
100 80 60 40 20 0
0
50
100
150
200
Time, hours Fig. 8. Time-course of the inactivation of different PFL-Sepabeads derivatives incubated in cyclohexane. Derivatives were incubated in 100% of cyclohexane and 60 ◦ C, and at different times, aliquots were withdrawn and assayed at 25 ◦ C through the reaction of transesterification of olive oil with benzyl alcohol. Triangles: Sep-C18; Circles: Gly-bi; squares: Gly-mono.
120 100
Remaining acvity (%)
Derivative
80 60 40 20 0
0
50
100
Time, hours This model reaction is very easily performed in organic solvents (e.g., in cyclohexane), and it is very easily followed by HPLC-UV. Again, the bimolecular derivative is much more active than the monomeric one (Fig. 10). The most active derivative is the one that was PFL-adsorbed on C18-Sepabeads (data not shown). The best synthetic yields, 85% of benzyl alcohol converted into benzyl oleate, were obtained with different derivatives of PFL. The reaction
Fig. 9. Time-course of the inactivation of different PFL-Sepabeads derivatives incubated in tert-amyl alcohol. Derivatives were incubated in 100% of tert-amyl alcohol and 60 ◦ C, and at different times, aliquots were withdrawn and assayed at 25 ◦ C through the reaction of transesterification of olive oil with benzyl alcohol. Triangles: Sep-C18; Circles: Gly-bi; squares: Gly-mono.
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CSD2007-00063 FUN-C-FOOD (CICYT) and ALIBIRD S2009/AGR1469. We gratefully recognize the Spanish Ministry of Science and Innovation for the “Ramón y Cajal” contract for Dr. FernandezLorente. We also thank the Brazilian agencies CAPES and CNPq for the scholarship of Lionete N. Lima.
80
Synthec yield (%)
123
70 60 50 40
References
30 20 10 0
0
20
40
60
80
Time, hours Fig. 10. Time-course of the enzymatic synthesis of benzyl oleate via the transesterification of olive oil with benzyl alcohol. The reaction was performed at 25 ◦ C in tert-amyl alcohol. Experimental conditions are described in Section 2. Experiments were carried out as described in Section 2. Circles: Gly-bi; squares: Gly-mono.
time required to achieve the maximum yield depends on each catalyst. 4. Conclusions The immobilization and stabilization of bimolecular aggregates of PFL was only possible because of the special features of glyoxylsupports: (1) they immobilize enzymes that have two amino termini at pHs lower than 10 by a multipoint covalent immobilization involving only the amino terminus, and (2) they are able to promote an additional multipoint covalent attachment at pHs greater than 10 involving Lys residues on same region of the enzyme surface. The derivatives of the bimolecular aggregates are more stable than derivatives of the monomeric enzyme with respect to thermal and solvent inactivation (from 5 to 15-fold). In addition, the derivatives of the bimolecular aggregates are also more active in the hydrolysis of HPBEt (from 2 to 6-fold). Finally, the derivatives of the bimolecular aggregates are much more enantioselective than the monomeric derivatives (from E > 100 to E = 20). Additionally, in the derivatives of the bimolecular aggregates, the open form of the enzyme appears to be much more stabilized than in the monomeric ones (in equilibrium between the closed and the open form). Good catalyst engineering strongly improves the performance of immobilized enzyme derivatives. Acknowledgments This work was sponsored by the Spanish Ministry of Science and Innovation(project AGL-2009-07526), Consolider INGENIO 2010
[1] Mateo C, Palomo JM, Fuentes M, Betancor L, Grazu V, López-Gallego F, et al. Glyoxyl agarose: a fully inert and hydrophilic support for immobilization and high stabilization of proteins. Enzyme Microb Technol 2006;39: 274–80. [2] Mateo C, Abian O, Bernedo M, Cuenca E, Fuentes M, Fernandez-Lorente G, et al. Some special features of glyoxyl supports to immobilize proteins. Enzyme Microb Technol 2005;37:456–62. [3] Bolivar JM, Rocha-Martin J, Mateo C, Cava F, Berenguer J, Vega D, et al. Purification and stabilization of a glutamate dehygrogenase from Thermus thermophilus via oriented multisubunit plus multipoint covalent immobilization. J Mol Catal B: Enzym 2009;58:158–63. [4] Palomo JM, Fuentes M, Fernández-Lorente G, Mateo C, Guisan JM, FernándezLafuente R. General trend of lipase to self-assemble giving bimolecular aggregates greatly modifies the enzyme functionality. Biomacromolecules 2003;4:1–6. [5] Fernández-Lorente G, Palomo JM, Fuentes M, Mateo C, Guisán JM, FernándezLafuente R. Self-assembly of Pseudomonas fluorescens lipase into bimolecular aggregates dramatically affects functional properties. Biotechnol Bioeng 2003;82:232–7. [6] Palomo JM, Ortiz C, Fernandez-Lorente G, Fuentes M, Guisan JM, FernandezLafuente R. Lipase–lipase interactions as a new tool to immobilize and modulate the lipase properties. Enzyme Microb Technol 2005;37:456–62. [7] Wilson L, Palomo JM, Fernaˇındez-Lorente G, Illanes A, Guisaˇın JM, FernaˇındezLafuente R. Effect of lipase–lipase interactions in the activity, stability and specificity of a lipase from Alcaligenes sp. Enzyme Microb Technol 2006;39:259–64. [8] Palomo JM, Ortiz C, Fuentes M, Fernández-Lorente G, Guisán JM, FernándezLafuente R. Use of immobilized lipases for lipase purification via specific lipase–lipase interactions. J Chromatogr A 2004;1038:267–73. [9] Volpato G, Filice M, Ayub MAZ, Guisan JM, Palomo JM. Single-step purification of different lipases from Staphylococcus warneri. J Chromatogr A 2010;1217:473–8. [10] Guisan JM. Aldehyde gels as activated support for immobilization stabilization of enzymes. Enzyme Microb Technol 1988;10:375–8. [11] Sabuquillo P, Reina J, Fernández-Lorente G, Guisán JM, Fernández-Lafuente R. Interfacial affinity chromatography of lipases: separation of different fractions by selective adsorption on supports activated with hydrophobic groups. Biochem Biophys Acta 1998;1388:337–48. [12] Bastida A, Sabuquillo P, Armisen P, Fernández-Lafuente R, Huguet J, Guisán JM. A single step purification, immobilization and hyperactivation of lipases via interfacial adsorption on strongly hydrophobic supports. Biotechnol Bioeng 1998;58:486–93. [13] Fernández-Lafuente R, Armisen P, Sabuquillo P, Fernández-Lorente G, Guisán JM. Immobilization of lipases by selective adsorption on hydrophobic supports. Chem Phys Lipids 1998;93:185–97. [14] Fernández-Lorente G, Terreni M, Mateo C, Bastida A, Fernández-Lafuente R, Dalmases P, et al. Modulation of lipase properties in macro-aqueous systems by controlled enzyme immobilization: enantioselective hydrolysis of a chiral ester by immobilized Pseudomonas lipase. Enzyme Microb Technol 2001;28:389–96. [15] Holmberg E, Hult K. Temperature as an enantioselective parameter in enzymatic resolutions of racemic mixtures. Biotechnol Lett 1991;43:323–6. [16] Fernández-Lorente G, Cabrera Z, Godoy C, Fernandez-Lafuente R, Palomo JM, Guisan JM. Interfacially activated lipases against hydrophobic supports: effect of the support nature on the biocatalytic properties. Process Biochem 2008;43:1061–7.
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Immobilization and stabilization of a bimolecular aggregate of the lipase from Pseudomonas fluorescens by multipoint covalent attachment Lionete Nunes de Lima a,b , Caio C. Aragon a,c , Cesar Mateo a , Jose M. Palomo a , Raquel L.C. Giordano b , Paulo W. Tardioli b , Jose M. Guisan a,∗ , Gloria Fernandez-Lorente d,∗ a
Instituto de Catalisis, CSIC, 28049 Madrid, Spain Chemical Engineering Department, Federal University of São Carlos, São Carlos, SP, Brazil c Instituto de Química, UNESP-Univ. Estadual Paulista, Departamento de Bioquímica e Tecnologia Química, Araraquara, SP, Brazil d Instituto de Investigación en Ciencias de la Alimentación (CIAL) CSIC-UAM, 28049 Madrid, Spain b
a r t i c l e
i n f o
Article history: Received 27 July 2012 Received in revised form 19 October 2012 Accepted 8 November 2012 Available online 19 November 2012 Keywords: Pseudomonas fluorescens lipase Lipase aggregates Enzyme immobilization and stabilization Improved enantioselectivity
a b s t r a c t The soluble lipase from Pseudomonas fluorescens (PFL) forms bimolecular aggregates in which the hydrophobic active centers of the enzyme monomers are in close contact. This bimolecular aggregate could be immobilized by multipoint covalent linkages on glyoxyl supports at pH 8.5. The monomer of PFL obtained by incubation of the soluble enzyme in the presence of detergent (0.5% TRITON X-100) could not be immobilized under these conditions. The bimolecular aggregate has two amino terminal residues in the same plane. A further incubation of the immobilized derivative under more alkaline conditions (e.g., pH 10.5) allows a further multipoint attachment of lysine (Lys) residues located in the same plane as the amino terminal residues. Monomeric PFL was immobilized at pH 10.5 in the presence of 0.5% TRITON X-100. The properties of both PFL derivatives were compared. In general, the bimolecular derivatives were more active, more selective and more stable both in water and in organic solvents than the monomolecular ones. The bimolecular derivative showed twice the activity and a much higher selectivity (100 versus 20) for the hydrolysis of R,S-2-hydroxy-4-phenylbutyric acid ethyl ester (HPBEt) in aqueous media at pH 5.0 compared to the monomeric derivative. In experiments measuring thermal inactivation at 75 ◦ C, the bimolecular derivative was 5-fold more stable than the monomeric derivative (and 50-fold more stable than a one-point covalently immobilized PFL derivative), and it had a half-life greater than 4 h. In organic solvents (cyclohexane and tert-amyl alcohol), the bimolecular derivative was much more stable and more active than the monomeric derivative in catalyzing the transesterification of olive oil with benzyl alcohol. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Glyoxyl groups are aldehydes with very short chains (Support0–CH2 –CHO). Glyoxyl-activated supports are able to react very rapidly with non-ionized amino groups, but they form very unstable Schiff’s bases, and they are not able to immobilize enzymes via a single enzyme-support attachment [1,2]. Glyoxyl supports are able to immobilize enzymes under alkaline conditions via a multipoint covalent connection between two or more Lys residues on the enzyme surface and very highly activated supports. At pHs lower than 10, glyoxyl supports are not able to immobilize monomeric enzymes that have an unique reactive amino terminus.
∗ Corresponding authors. E-mail addresses: [email protected] (J.M. Guisan), gflorente@ifi.csic.es (G. Fernandez-Lorente). 1359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2012.11.008
However, glyoxyl supports are able to immobilize, at pHs lower than 10, enzymes that have more than one amino terminus. For example, some multimeric enzymes have two or more amino termini in the same plane of the enzyme surface [3], in such cases, a multipoint covalent immobilization can take place. Soluble lipases can form non-covalent bimolecular aggregates [4,5] that are dissociated in the presence of detergents by stabilization of the open form of the enzymes. In these bimolecular aggregates, the two hydrophobic open active centers of the monomers are in close contact one with the other and form stable aggregates with new active centers and sometimes with new catalytic properties [6,7]. These systems have been successfully used for site-specific lipase purification [8,9]. Glyoxyl supports might be able to covalently immobilize those bimolecular aggregates in cases where both amino terminal are on the same planar surface. The aggregates would be immobilized by simultaneous multipoint immobilization of both amino terminal (at pH 8.5). After the first immobilization, the derivatives could be
L.N. de Lima et al. / Process Biochemistry 48 (2013) 118–123
incubated under more alkaline conditions, allowing Lys residues located in the same region as the amino termini to attach to the highly activated support. In contrast, the monomeric form of the lipase can be immobilized and stabilized by multipoint covalent immobilization under alkaline conditions in the presence of moderately high concentrations of detergents. This immobilization occurs via the region of the monomeric lipase with the highest density of Lys residues, which is located on the surface opposite to the active center [4]. In the present manuscript, the multipoint covalent immobilization of lipase from Pseudomonas fluorescens (PFL) bimolecular aggregates was investigated. The properties of the immobilizedstabilized bimolecular aggregates were compared to those of the immobilization-stabilized monomeric enzyme. The stability, activity and enantioselectivity of both types of immobilized derivative were evaluated. Two types of support were used for different immobilizations of PFL. Agarose gels are very useful for work performed in aqueous media and in stirred tank reactors, and Sepabeads resins are very useful for work performed in organic solvents and in any type of reactor system (stirred tank, fixed bed, etc.). Different PFL catalysts were applied to two biotransformations as model reactions: the kinetic resolution of (R,S)-2-hydroxy-4phenylbutyric acid ethyl ester (HPBEt) in aqueous media and the transesterification of olive oil in organic solvents. 2. Materials and methods 2.1. Materials Buffering salts, TRITON X-100 (TX), p-nitrophenyl butyrate (p-NPB), tert-amyl alcohol, cyclohexane, and benzyl alcohol were from Sigma Chemical Co. (St. Louis, USA). Octyl SepharoseTM CL-4B was purchased from GE Healthcare (Uppsala, Sweden). C18-Sepabead resins were kindly donated by Resindion S.R.L. Agarose 6BCL was purchased from Agarose Bead Technologies. The racemic mixture of R,S-2hydroxy-4-phenylbutyric acid ethyl ester (HPBEt) was a kind gift from VITA INVEST SA. The P. Fluorescens lipase (PFL) was purchased from Amano Pharmaceutical Co., Ltd. (Nagoya, Japan). The other reagents and solvents used in this study were of analytical or HPLC grade.
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Immobilization on glyoxyl-agarose [10] or glyoxyl-sepabeads was carried out by adding 4.2 g of support to 36 mL of a solution at pH 10.5 containing 0.1 M sodium bicarbonate with 0.5% (v/v) of TRITON X-100, and 5 mg/mL of the lipase solution. Three derivatives were prepared with these supports. The first was prepared in bicarbonate buffer at pH 8.5 in the presence of 0.5% TRITON X-100 (TX), where the enzyme is not immobilized. The second was prepared in the same buffer but in the absence of TX, where the enzyme is immobilized as bimolecular aggregate (Gly-bi derivative). The third derivative (Gly-mono) was prepared in buffer at pH 10.5 and 0.5% TX. In all cases, at the reaction end point, 36 mg of sodium borohydride was added. Interfacial adsorption [11–13] of PFL on octyl agarose or octadecyl-Sepabeads (OS or Sep-C18 derivatives) was performed by adding 4.2 g of support to 36 mL of 5 mM sodium phosphate, pH 7.0 containing 5 mg/mL of PFL. In each case, a reference suspension, where the activated support was substituted by inert agarose, was prepared. In all cases, the activity of this reference suspension showed 100% of the activity associated with the supernatant, indicating that no lipase was non-specifically adsorbed to the support. Immobilization yield was defined as the percentage of lipase (determined by the activity on p-NPB) immobilized on the different supports. 2.2.3. Irreversible inactivation of immobilized PFL in the presence of diethyl-p-nitrophenylphosphonate (D-pNP) Aliquots of (0.4 g) of Gly-mono, Gly-bi or OS derivatives were suspended in 5 mL of 25 mM sodium phosphate buffer solution at pH 7 and 25 ◦ C, and 1.45 mM of the inhibitor D-pNP was added to the suspension. The reaction was very gently stirred until the activity of the immobilized enzyme was zero. 2.2.4. Enzymatic synthesis of benzyl oleate A 0.1 g aliquot of equilibrated immobilized lipase was added to 1.42 mL of the substrate solution (1 micromole of benzyl alcohol and 2 mol of olive oil) in 9.94 mL of organic solvent (tert-amyl alcohol or cyclohexane). To ensure anhydrous conditions, 100 mg of dry molecular sieves were also added to the reaction mixture. The progress of the reaction was followed by HPLC. 2.2.4.1. HPLC analysis. Reactants and products were analyzed by RP-HPLC (Spectra Physic SP 100 coupled with a Spectra Physic SP 8450 UV detector) using a reversedphase column (Ultrabase-C18, 250 mm × 4.6 mm, 5 m). Products were eluted at a flow rate of 1.0 mL/min with methanol/water/CH3 COOH (60:40:0.1, by vol.), and the UV absorbance was monitored at 254 nm. In fact, the total area of the chromatogram at 254 nm is constant during the whole course of the condensation. At this wavelength, the oil, the fatty acids and glycerol do not absorb. Reaction rates and synthetic yields could be easily calculated from the pure peak areas corresponding to benzyl alcohol (retention time (RT) of 5 min) and benzyl oleate (RT of 9 min).
2.2. Methods 2.2.1. Determination of lipase activity An activity assay was performed by calculating the initial velocity of the hydrolysis of 0.4 mM p-nitrophenyl butyrate (p-NPB) in 25 mM sodium phosphate at pH 7 and 25 ◦ C, catalyzed by a known amount of PFL. The reaction was followed by measuring, at 348 nm (this wavelength correspond to the isosbestic point of pNP) ( = 5.150 M−1 cm−1 ), the increase with the time in the absorbance, produced by the release of the reaction product p-nitrophenol (pNP). The reaction was performed using a spectrophotometer with a thermostated cell with continuous magnetic stirring. To initialize the reaction, 0.05–0.2 mL (depending of the dilution and activity of the sample) of the soluble lipase (blank or supernatant) or their immobilized preparations (suspension) were added to 2.5 mL of substrate solution. Enzymatic activity was calculated as micromoles of hydrolyzed p-NPB per minute per milligrams of enzyme (IU) under the conditions described above. The calculations were done according to this expression: IU mL−1 solution = (A min
−1
)VT −1 1000ε−1 b−1 Vs −1
IU mL−1 solution: international units per mL of solution expressed in mol of product per minute, (A min−1 ): increment of absorbance per minute, VT : total volume in the cell expressed in mL, ε: molar extinction coefficient, b: width of the cell. Vs : volume of sample in the cell expressed in mL. 2.2.2. PFL immobilization For immobilization of the lipases on the CNBr-Sepharose support, the first step was activation of the support prior to use by its suspension in an acidic aqueous solution (pH 2–3) for 1 h. The activated support was dried by filtration under vacuum. Next, 4.2 g of activated CNBr-Sepharose was added to 36 mL (0.2 mg of lipase/mL corresponding to 100 total IU of p-NPB hydrolysis) of the lipase solution in the presence of 0.5% TRITON X-100 (TX). The mixture was stirred at 4 ◦ C and 250 rpm for 20 min. Afterwards, the solution was removed by filtration, the supported lipase (CNBr-S derivative) was washed twice with 100 mM NaHCO3 at pH 8 and re-suspended in 15 mL of 1 M ethanolamine at pH 8 for 90 min to block the unreacted imidocarbonate reactive groups [2]. The blocked resin was recovered by filtration and washed with abundant distilled water. The immobilization procedure was followed by the enzymatic activity assay described above.
2.2.5. Hydrolysis of 2-hydroxy-4-phenylbutyric acid ethyl ester (HPBEt) Evaluation of the enzyme activity was performed by adding 0.5 g of derivative to 10 mL of 5 mM (R,S) HPBE in 25 mM phosphate buffer at pH 7.5 and 25 ◦ C with mechanical stirring [14]. The pH was maintained by automatic titration, and the enzymatic activity (mol of substrate hydrolyzed per minute per mg of immobilized protein) was determined by reverse-phase HPLC. The stereo specificity of the hydrolysis was analyzed by chiral-HPLC. 2.2.5.1. Analysis by HPLC. The hydrolysis products were analyzed by a HPLC device (Spectra Physic SP 100) coupled to an UV detector (Spectra Physic SP 8450) at 225 nm. The column was a Chiracel OD-R, the mobile phase was a mixture of acetonitrile (40%) and water (60%), and the analyses were performed at a flow rate of 0.5 mL/min. Quantification of the conversion degree was performed by comparison with standard solutions of the racemate and pure esters. Enantioselectivity (E value) was calculated from the enantiomeric excess of the remaining ester and the percent conversion as previously reported [15]. 2.2.6. Thermal inactivation of PFL-agarose derivatives Different immobilized preparations of PFL were incubated in 5 mM sodium phosphate buffer at pH 7 and 75 ◦ C. Samples were periodically withdrawn using a pipette with a cut-tip and under vigorous stirring to ensure a homogeneous biocatalyst suspension, and their remaining activities were determined as described for the p-NPB assay. 2.2.7. Stability of PFL-Sepabeads derivatives Thermal stability: the different PFL-Sepabeads derivatives were incubated in 5 mM sodium phosphate buffer at pH 7 and 65 ◦ C. Samples were periodically withdrawn using a pipette with a cut-tip and under vigorous stirring to ensure a homogeneous biocatalyst suspension, and their remaining activities were determined as described for the p-NPB assay. Stability in organic solvents: the enzyme derivatives were equilibrated with the solvent (tert-amyl alcohol or cyclohexane) at the desired temperature (60 ◦ C). Subsequently, at different times, 0.1 g of catalyst incubated in the inactivation conditions was added to the synthesis reaction solution, and the activity was analyzed using the enzymatic synthesis assay described in the previous section.
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Fig. 1. Immobilization of PFL on glyoxyl-supports at pH 8.5. Squares: PFL in the presence of detergent (0.5% TX); circles: PFL in the absence of detergent.
Fig. 3. Schematic representation of a bimolecular aggregate of PFL (taken from PDB code 3LIP). Light blue: amino terminus; dark blue: lysine residues; red: lipase active centers. (For interpretation of the references to color in the artwork, the reader is referred to the web version of the article.)
Experiments were carried out in triplicate, and the standard error was never greater than 5%.
3. Results and discussion 3.1. Preparation of PFL derivatives 3.1.1. Immobilization of PFL in the presence or absence of detergents on glyoxyl supports at pH 8.5 (agarose gels and Sepabeads supports) In aqueous medium, the imine formed by reaction of aldehyde and primary amine groups is in equilibrium shifted to the unlinked specie, due to this, the immobilization on glyoxyl supports is only produced when more than one reactive amino group can simultaneously react with more than one aldehyde groups on the support [10]. In the presence of 0.5% of TX, the enzyme is inefficiently immobilized at pH 8.5 even after 24 h (Fig. 1), presumably because the enzyme is in monomeric form under these conditions and has only one amino terminus that is unable to be irreversibly attached to glyoxyl-supports (at this pH, lysines are protonated and subsequently they are not reactive and only one amino terminus is unprotonated and reactive) (Fig. 2). However, in the absence of
detergent (bimolecular aggregates as it was previously reported [5], PFL is slowly immobilized at pH 8.5, and after 24 h, all the offered enzyme becomes immobilized on the support. The existence of two amino termini in the same planar surface in the bimolecular aggregate would explain the different behavior of the two forms of PFL (Fig. 3) and hence PFL is able to be immobilized by slow multipoint covalent immobilization (Fig. 2). After immobilization, the derivative can be incubated at pH 10.5 for 24 h to promote the attachment to the support of Lys residues located in the vicinity of the two amino termini (Fig. 2).
3.1.2. Immobilization of PFL on glyoxyl-supports at pH 10.5 in the presence of detergent (0.5% of TX) Under these conditions (0.5% of TX), the enzyme immobilizes on glyoxyl supports at moderate rates (6 h on glyoxyl agarose and 24 h on glyoxyl-Sepabeads). Each monomer may be immobilized through its region with the highest amount of Lys groups. PFL is not very rich in Lys residues, and glyoxyl-Sepabeads is not very rich in glyoxyl groups.
Fig. 2. Schematic representation of the immobilization of the different lipase populations (bimolecular aggregated or deaggregated lipase at pH 8.5) on glyoxyl supports.
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121
120
Residual acvity (%)
100 80 60 40 20 0
0
5
10
15
20
25
Time, minutes Fig. 5. Irreversible inhibition of PFL derivatives with diethyl-pnitrophenylphosphate.Squares: Gly-mono; circles: Gly-bi; triangles: OS.
3.1.3. Immobilization of the monomeric PFL on CNBr-activated Sepharose The enzyme is immobilized in the presence of detergent (monomeric form) at pH 7.0 at 4 ◦ C for 15 min on CNBr-activated Sepharose. The used immobilization conditions allow producing a PFL derivative to be used as a reference for lipase behavior. In general, immobilization of enzymes under these conditions will occur to just one covalent bond, and these immobilized preparations result in a good model of the enzyme properties in absence of intermolecular phenomena (Fig. 4) [4,10]. Lipases have been shown to exhibit a strong tendency to form lipase–lipase aggregates, making it very difficult to study isolated lipase molecules in soluble form [7,8]. The obtained immobilization yield was 30% of the input protein. As expected, immobilization yields in the used conditions are not very high but the formation of any kind of multipoint covalent attachment is avoided. After immobilization, the detergent is removed, and this derivative is taken as a reference of the behavior of native monomeric PFL without the formation of bimolecular aggregates. 3.1.4. Immobilization of PFL by adsorption on hydrophobic supports (octyl-Sepharose and octadecyl-Sepabeads) The enzyme was immobilized in more than 95% yield on hydrophobic supports, and it becomes hyper-activated: the measured activity is 150% on octyl-Sepharose and 300% on octadecyl-Sepabeads (Table 1). Table 1 Preparation of different immobilized derivatives of PFL. Activated Support
Immobilization yield, %
Recovered Activity, %
Derivative
CNBr-activated Sepharose Octyl-Sepharose Octadecyl-Sepabeads Glyoxyl-bimolecular Glyoxyl-monomeric
30 99 99 98 95
95 150 300 70 75
CNBr-S OS Sep-C18 Gly-bi Gly-mono
Experiments were performed as described in Section 2.
3.2. Modification of immobilized PFL with a covalent irreversible inhibitor Modification of lipases by covalent modification of the catalytic Ser with an irreversible inhibitor (e.g., diethyl p-nitro phenylphosphonate) will be faster when the active center of the lipase is open. In fact, the inactivation of the PFL adsorbed on octyl agarose is much faster than the inactivation of the monomeric derivative in the absence of detergent (Fig. 5). The modification of the bimolecular derivative is also faster than the one of the monomeric derivative, which could be the effect of two open active centers in close contact. 3.3. Thermal inactivation of the PFL-agarose derivatives The bimolecular derivative was 5-fold more stable than the monomeric one and is also more stable than PFL adsorbed on octylSepharose (Fig. 6). These latter derivatives are 10-fold more stable than the one-point-immobilized PFL (CNBr-S). CNBr-S derivative was used as control because soluble enzyme is forming aggregates 120 100
Remaining acvity (%)
Fig. 4. Schematic representation of the different PFL derivatives. CNBr-S: one-point covalently immobilized monomeric derivative Gly-mono: multipoint covalently immobilized monomeric derivative Gly-bi: multipoint covalently immobilized bimolecular aggregate OS: PFL adsorbed on octyl-Sepharose.
The immobilization of lipases on hydrophobic supports by interfacial adsorption [11–13] permits to fix the open conformation on a solid phase (Figure 4) representing a simple and elegance methodology to get very high active lipase catalysts. The hydrophobic nature of the support affected to the hyperactivation [16]. This depended on the hydrophobic nature of the lid (e.g., number of amino acids) or the surrounding area of the active site of a particular lipase.
80 60 40 20 0
0
1
2
3
4
5
Time, hours Fig. 6. Time-course of the thermal inactivation of different PFL-agarose derivatives. Derivatives were incubated at 75 ◦ C and at different times, aliquots were withdrawn and assayed at 25 ◦ C with p-NPB. Circles: Gly-bi; squares: Gly-mono; triangles: OS; empty cycles: CNBr-S.
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Table 2 Enantioselective hydrolysis of (R,S) -2-hydroxy-4-phenylbutyric acid ethyl ester (HPBET) by PFL derivatives.
CNBr-S-PFL Gly-mono-PFL Gly-bi-PFL OS-PFL
0.4 0.4 0.8 1.2
7 20 >100 >100
Reference [6] [6]
Experiments were performed at pH 5.0 as described in Section 2.
and the soluble monomer only can be obtained using detergent in the medium. In these conditions the structure of the soluble enzyme is different since the lid that normally covers the active site is opened changing the structure of the lipase and often their catalytic properties. Hydrophobic adsorption and multipoint covalent attachment seem to have an interesting stabilizing effect. It appears that bimolecular derivatives may be stabilized by two different effects: hydrophobic adsorption of both active centers and multipoint covalent attachment of the bimolecular structure. The new derivative of PFL is 50-fold more stable than the one-pointimmobilized derivative. 3.4. Enantioselective hydrolysis of R,S-2-hydroxy-4-phenylbutyric acid ethyl ester (HPBEt) by PFL derivative The activity of the bimolecular derivative is higher than that of the multipoint-attached and one-point-attached monomeric derivatives and is lower than that of the octyl-Sepharose derivative (Table 2). However, selectivity is similar to that of the octylSepharose derivative (E > 100) and is much higher than that of the one-point-attached derivative (E = 7) and the multipoint-attached monomeric derivative (E = 20). The hydrophobic adsorption of active centers seems to be a key factor for the improvement of enantioselectivity. The bimolecular derivative is more active, more selective and more stable than both monomeric derivatives. Compared to the octyl-Sepharose derivative, the biomolecular derivative was more stable, less active and exhibits a similar very high selectivity. 3.5. Stability of PFL-Sepabeads derivatives Thermal stability: the bimolecular derivative is much more stable than the monomeric one and its stability is slightly lower than the hydrophobic derivative (PFL adsorbed on octadecyl-Sepabeads) (Fig. 7). Multipoint attachment of Sepabeads is less extensive than on agarose because Sepabeads have a low density of glyoxyl groups. Indeed, the immobilization rates on Sepabeads are also much lower. The most relevant stabilizing effect seems to be the stabilization of the open form of PFL. Stability in organic solvents: all derivatives are quite stable in cyclohexane at 60 ◦ C. The monomeric derivative is the least stable (Fig. 8). All derivatives are less stable in a more polar solvent (tertamyl alcohol), and differences between the bimolecular and the monomeric derivatives are higher in such solvents (Fig. 9). 3.6. Enzymatic transesterification of olive oil with benzyl alcohol
100
Remaining acvity (%)
E value
80 60 40 20 0
0
5
10
15
20
25
Time, hours Fig. 7. Time-course of the thermal inactivation of different PFL-Sepabeads derivatives. Derivatives were incubated at 65 ◦ C, pH 7.0, and at different times, aliquots were withdrawn and assayed at 25 ◦ C with p-NPB. Triangles: Sep-C18; Circles: Gly-bi; squares: Gly-mono.
120
Remaining acvity (%)
Specific activity (IU/mg)
100 80 60 40 20 0
0
50
100
150
200
Time, hours Fig. 8. Time-course of the inactivation of different PFL-Sepabeads derivatives incubated in cyclohexane. Derivatives were incubated in 100% of cyclohexane and 60 ◦ C, and at different times, aliquots were withdrawn and assayed at 25 ◦ C through the reaction of transesterification of olive oil with benzyl alcohol. Triangles: Sep-C18; Circles: Gly-bi; squares: Gly-mono.
120 100
Remaining acvity (%)
Derivative
80 60 40 20 0
0
50
100
Time, hours This model reaction is very easily performed in organic solvents (e.g., in cyclohexane), and it is very easily followed by HPLC-UV. Again, the bimolecular derivative is much more active than the monomeric one (Fig. 10). The most active derivative is the one that was PFL-adsorbed on C18-Sepabeads (data not shown). The best synthetic yields, 85% of benzyl alcohol converted into benzyl oleate, were obtained with different derivatives of PFL. The reaction
Fig. 9. Time-course of the inactivation of different PFL-Sepabeads derivatives incubated in tert-amyl alcohol. Derivatives were incubated in 100% of tert-amyl alcohol and 60 ◦ C, and at different times, aliquots were withdrawn and assayed at 25 ◦ C through the reaction of transesterification of olive oil with benzyl alcohol. Triangles: Sep-C18; Circles: Gly-bi; squares: Gly-mono.
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CSD2007-00063 FUN-C-FOOD (CICYT) and ALIBIRD S2009/AGR1469. We gratefully recognize the Spanish Ministry of Science and Innovation for the “Ramón y Cajal” contract for Dr. FernandezLorente. We also thank the Brazilian agencies CAPES and CNPq for the scholarship of Lionete N. Lima.
80
Synthec yield (%)
123
70 60 50 40
References
30 20 10 0
0
20
40
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Time, hours Fig. 10. Time-course of the enzymatic synthesis of benzyl oleate via the transesterification of olive oil with benzyl alcohol. The reaction was performed at 25 ◦ C in tert-amyl alcohol. Experimental conditions are described in Section 2. Experiments were carried out as described in Section 2. Circles: Gly-bi; squares: Gly-mono.
time required to achieve the maximum yield depends on each catalyst. 4. Conclusions The immobilization and stabilization of bimolecular aggregates of PFL was only possible because of the special features of glyoxylsupports: (1) they immobilize enzymes that have two amino termini at pHs lower than 10 by a multipoint covalent immobilization involving only the amino terminus, and (2) they are able to promote an additional multipoint covalent attachment at pHs greater than 10 involving Lys residues on same region of the enzyme surface. The derivatives of the bimolecular aggregates are more stable than derivatives of the monomeric enzyme with respect to thermal and solvent inactivation (from 5 to 15-fold). In addition, the derivatives of the bimolecular aggregates are also more active in the hydrolysis of HPBEt (from 2 to 6-fold). Finally, the derivatives of the bimolecular aggregates are much more enantioselective than the monomeric derivatives (from E > 100 to E = 20). Additionally, in the derivatives of the bimolecular aggregates, the open form of the enzyme appears to be much more stabilized than in the monomeric ones (in equilibrium between the closed and the open form). Good catalyst engineering strongly improves the performance of immobilized enzyme derivatives. Acknowledgments This work was sponsored by the Spanish Ministry of Science and Innovation(project AGL-2009-07526), Consolider INGENIO 2010
[1] Mateo C, Palomo JM, Fuentes M, Betancor L, Grazu V, López-Gallego F, et al. Glyoxyl agarose: a fully inert and hydrophilic support for immobilization and high stabilization of proteins. Enzyme Microb Technol 2006;39: 274–80. [2] Mateo C, Abian O, Bernedo M, Cuenca E, Fuentes M, Fernandez-Lorente G, et al. Some special features of glyoxyl supports to immobilize proteins. Enzyme Microb Technol 2005;37:456–62. [3] Bolivar JM, Rocha-Martin J, Mateo C, Cava F, Berenguer J, Vega D, et al. Purification and stabilization of a glutamate dehygrogenase from Thermus thermophilus via oriented multisubunit plus multipoint covalent immobilization. J Mol Catal B: Enzym 2009;58:158–63. [4] Palomo JM, Fuentes M, Fernández-Lorente G, Mateo C, Guisan JM, FernándezLafuente R. General trend of lipase to self-assemble giving bimolecular aggregates greatly modifies the enzyme functionality. Biomacromolecules 2003;4:1–6. [5] Fernández-Lorente G, Palomo JM, Fuentes M, Mateo C, Guisán JM, FernándezLafuente R. Self-assembly of Pseudomonas fluorescens lipase into bimolecular aggregates dramatically affects functional properties. Biotechnol Bioeng 2003;82:232–7. [6] Palomo JM, Ortiz C, Fernandez-Lorente G, Fuentes M, Guisan JM, FernandezLafuente R. Lipase–lipase interactions as a new tool to immobilize and modulate the lipase properties. Enzyme Microb Technol 2005;37:456–62. [7] Wilson L, Palomo JM, Fernaˇındez-Lorente G, Illanes A, Guisaˇın JM, FernaˇındezLafuente R. Effect of lipase–lipase interactions in the activity, stability and specificity of a lipase from Alcaligenes sp. Enzyme Microb Technol 2006;39:259–64. [8] Palomo JM, Ortiz C, Fuentes M, Fernández-Lorente G, Guisán JM, FernándezLafuente R. Use of immobilized lipases for lipase purification via specific lipase–lipase interactions. J Chromatogr A 2004;1038:267–73. [9] Volpato G, Filice M, Ayub MAZ, Guisan JM, Palomo JM. Single-step purification of different lipases from Staphylococcus warneri. J Chromatogr A 2010;1217:473–8. [10] Guisan JM. Aldehyde gels as activated support for immobilization stabilization of enzymes. Enzyme Microb Technol 1988;10:375–8. [11] Sabuquillo P, Reina J, Fernández-Lorente G, Guisán JM, Fernández-Lafuente R. Interfacial affinity chromatography of lipases: separation of different fractions by selective adsorption on supports activated with hydrophobic groups. Biochem Biophys Acta 1998;1388:337–48. [12] Bastida A, Sabuquillo P, Armisen P, Fernández-Lafuente R, Huguet J, Guisán JM. A single step purification, immobilization and hyperactivation of lipases via interfacial adsorption on strongly hydrophobic supports. Biotechnol Bioeng 1998;58:486–93. [13] Fernández-Lafuente R, Armisen P, Sabuquillo P, Fernández-Lorente G, Guisán JM. Immobilization of lipases by selective adsorption on hydrophobic supports. Chem Phys Lipids 1998;93:185–97. [14] Fernández-Lorente G, Terreni M, Mateo C, Bastida A, Fernández-Lafuente R, Dalmases P, et al. Modulation of lipase properties in macro-aqueous systems by controlled enzyme immobilization: enantioselective hydrolysis of a chiral ester by immobilized Pseudomonas lipase. Enzyme Microb Technol 2001;28:389–96. [15] Holmberg E, Hult K. Temperature as an enantioselective parameter in enzymatic resolutions of racemic mixtures. Biotechnol Lett 1991;43:323–6. [16] Fernández-Lorente G, Cabrera Z, Godoy C, Fernandez-Lafuente R, Palomo JM, Guisan JM. Interfacially activated lipases against hydrophobic supports: effect of the support nature on the biocatalytic properties. Process Biochem 2008;43:1061–7.