Lipids (2015) 50:49–55 DOI 10.1007/s11745-014-3973-9
ORIGINAL ARTICLE
Ionic Liquids Increase the Catalytic Efficiency of a Lipase (Lip1) From an Antarctic Thermophilic Bacterium Patricio A. Muñoz · Daniela N. Correa‑Llantén · Jenny M. Blamey
Received: 30 June 2014 / Accepted: 6 November 2014 / Published online: 27 November 2014 © AOCS 2014
Abstract Lipases catalyze the hydrolysis and synthesis of triglycerides and their reactions are widely used in industry. The use of ionic liquids has been explored in order to improve their catalytic properties. However, the effect of these compounds on kinetic parameters of lipases has been poorly understood. A study of the kinetic parameters of Lip1, the most thermostable lipase from the supernatant of the strain ID17, a thermophilic bacterium isolated from Deception Island, Antarctica, and a member of the genus Geobacillus is presented. Kinetic parameters of Lip1 were modulated by the use of ionic liquids BmimPF6 and BmimBF4. The maximum reaction rate of Lip1 was improved in the presence of both salts. The highest effect was observed when BmimPF6 was added in the reaction mix, resulting in a higher hydrolytic activity and in a modulation of the catalytic efficiency of the enzyme. However, the catalytic efficiency did not change in the presence of BmimBF4. The increase of the reaction rates of Lip1 promoted by these ionic liquids could be related to possible changes in the Lip1 structure. This effect was measured by
quenching of tryptophan fluorescence of the enzyme, when it was incubated with each liquid salt. In conclusion, the hydrolytic activity of Lip1 is modulated by the ionic liquids BmimBF4 and BmimPF6, improving the reaction rate and the catalytic efficiency of this enzyme when BmimPF6 was used. This effect is probably due to changes in the structure of Lip1 induced by the presence of these ionic liquids, stimulating its catalytic activity. Keywords Ionic liquids · Lipase · Kinetic parameters · Thermophilic · Antarctica · Deception Island Abbreviations pNP p-Nitrophenol pNPL p-Nitrophenyl laurate BmimBF4 1-Butyl-3-methylimidazolium tetrafluoroborate BmimPF6 1-Butyl-3-methylimidazolium hexafluorophosphate
Introduction P. A. Muñoz, D. N. Correa-Llantén and J. M. Blamey contributed equally to this work. P. A. Muñoz (*) · D. N. Correa‑Llantén · J. M. Blamey Fundación Científica y Cultural Biociencia, José Domingo Cañas 2280, Ñuñoa, Santiago, Chile e-mail: [email protected] D. N. Correa‑Llantén e-mail: [email protected] J. M. Blamey e-mail: [email protected] J. M. Blamey Universidad de Santiago de Chile, Avenida Libertador Bernardo O’Higgins 3363, Santiago, Chile
Lipases (triacylglycerol hydrolases, EC 3.1.1.3) catalyze the hydrolysis and synthesis of ester bonds of long chain fatty acids [1]. These enzymes have a range of properties widely used in industry, including the ability to catalyze a huge variety of reactions [2], high stereo- and regio-selectivity, the ability to catalyze heterogeneous reactions at water–lipid interfaces [3] and a high biocatalytic potential in aqueous and non-aqueous media [4]. Many industrial processes employing lipases are carried out at temperatures above 50 °C, thus requiring active and resistant enzymes at these temperatures [5]. These kinds of enzymes from thermophiles and hyperthermophiles have
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been described [1, 6–8]. They exhibit improved resistance to conditions imposed by the industry [4]. Thermophilic microorganisms have been isolated from geothermal sites which are distributed worldwide. One of these sites is Deception Island, an active stratovolcano located in the South Shetland Islands, Antarctica. This volcanic island has been very active during the last century: fumarolic emissions, thermal springs and hot soils are the present witnesses of Deception Island volcanic activity [9]. Geothermal anomalies mainly extend from Fumarole Bay to Pendulum Cove. The temperatures of emissions range from 90 to 110 °C at the fumaroles of Fumarole Bay. The soil temperature is highly variable, reaching values between 40 and 60 °C at Whalers Bay, and more than 70 °C at Pendulum Cove [10]. Previous studies indicate the presence of thermophilic bacteria belonging to Geobacillus, Bacillus, Brevibacillus and Thermus genera in samples collected from Deception Island [8, 11]. From these, a thermophilic and lipase-producing bacterium from Geobacillus genus was previously isolated. This bacterium, named ID17, produce four lipases, of which Lip1 is the most thermostable and has the ability to catalyze the thermophilic hydrolysis of ester bonds of long chain fatty acid under alkaline conditions [12]. These properties make Lip1 an excellent candidate for its use in different applications such as an additive in detergent preparations and chemical synthesis. Moreover, the industry has explored the use of ionic liquids in order to improve the catalytic properties of several enzymes. However, the effect of these liquid salts on kinetic parameters of lipases is poorly understood. Ionics liquids have shown themselves to have a huge impact in biocatalysis, since they not only increase the enzymatic activity, but also favor the stability and selectivity of different enzymes without emission of additional volatile or contaminant compounds that can damage the environment [13]. Ionic liquids are liquid organic salts at temperatures below 100 °C [14]. Their unique properties, namely no volatility, no inflammability, a high capacity to dissolve a huge range of substrates and excellent chemical and thermal stability, makes them an attractive environmentally friendly alternative. Due to their physical and chemical properties such as polarity, hydrophobicity and basicity of the hydrogen bond, they can be manipulated through an appropriate selection of the respective cation and anion [15]. They have been recognized as solvents or co-catalysts in a wide variety of applications that include organic and inorganic catalysis, biocatalysis and polymerization [15, 16]. In the last few years, a large number of enzymatic reactions have been carried out in systems that contain ionic liquids, e.g., resolution of 1-phenylethanol, reactions of alcoholysis, perhydrolysis and amoniolysis catalyzed by lipases [17–19]. Although, a wide amount of enzymatic
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reactions have been tested using a variety of ionic liquids, most studies have been focused on measurements of enzymatic activity, without considering the kinetic behavior of the catalysis in ionic liquids. For these reasons, we studied the effect of the ionic liquids 1-butyl-3-methylimidazolium tetrafluoroborate (BmimBF4) and 1-butyl-3-methylimidazolium hexafluorophosphate (BmimPF6) on the kinetic parameters of Lip1, the most thermostable lipase purified from the bacterium ID17. Both ionic liquids have been widely used to improve the activity, stability and enantio selectivity of different bacterial and fungal lipases [20]. In this way, we could determine the effect of these liquid salts on the kinetic behavior of Lip1 in order to establish further applications of this enzyme.
Materials and Methods Bacterial Strain The thermophilic bacterium ID17 (Accession number GU366067), belonging to the genus Geobacillus isolated by Muñoz et al. [12] obtained from Deception Island (Antarctica), was aerobically grown in a liquid medium, as described. Enzyme Assay Lipase activity was measured spectrophotometrically at 65 °C with p-nitrophenyl laurate (pNPL) as the substrate at 405 nm [1, 12]. Each reaction mixture contained 300 mM pNPL in acetonitrile and 100 mM Tris-HCl. One unit (U) of lipase activity was defined as 1 µmol of p-nitrophenol (Ɛ = 1.82 × 104 M−1 cm−1) released from pNPL per minute. Protein concentration was measured by the Bradford method [21] with a commercial assay kit (BioRad) using BSA as the standard. Enzyme Purification Lipase was purified from ID17 cells at 23 °C. Ten liters of ID17 culture were centrifuged (9,000×g for 10 min) and the supernatant solution was filtered using a 0.45-µm acetate filter (Sartorius Stedim Biotech GmbH) to remove all cells. Extracellular lipases were concentrated by ultrafiltration (PM-10 membrane filter; Amicon). The proteins (10 ml) were dissolved in buffer A [50 mM Tris-HCl (pH 8.0) containing 10 % (vol/vol) glycerol, 2 mM dithiothreitol, and 2 mM sodium dithionite] and loaded onto a column (Pharmacia, C 16/20) of Q-Sepharose Fast Flow (Pharmacia Biotech) equilibrated with buffer A. After the column was washed with the same buffer, the enzyme was
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Table 1 Kinetic constants for the hydrolysis of pNPL by Lip1 in the presence of different concentrations of ionic liquids Condition
VMax (mM min−1)
KM (mM)
kcat (min−1)
Catalytic efficiency (106 M−1 min−1)
Control 60 mM BmimBF4 180 mM BmimBF4 300 mM BmimBF4
24.1 ± 3.4 31.1 ± 4.1 49.8 ± 4.8 37.9 ± 5.2
0.6 ± 0.05 0.6 ± 0.04 1.5 ± 0.10 0.6 ± 0.06
2687.7 ± 379.5 3464.0 ± 457.6 5549.3 ± 535.7 4225.0 ± 580.4
4.2 ± 0.76 5.3 ± 1.14 4.1 ± 0.54 7.0 ± 0.97
600 mM BmimBF4 60 mM BmimPF6 180 mM BmimPF6 300 mM BmimPF6
ND 24.4 ± 3.5 26.7 ± 3.6 95.2 ± 4.2
ND 0.6 ± 0.07 0.5 ± 0.03 1.0 ± 0.05
ND 2720.5 ± 390.6 2974.4 ± 401.8 10622.9 ± 468.8
ND 4.8 ± 0.56 6.3 ± 1.3 11.1 ± 0.94
600 mM BmimPF6
142.9 ± 3.9
0.7 ± 0.06
15934.3 ± 435.3
23.9 ± 0.73
The control represents kinetic parameters in the absence of ionic liquids. Errors correspond to the standard deviation of three independent repetitions ND not determined
eluted with a linear gradient (200 ml) of 0–1 M NaCl in buffer A. Lipase activity started to elute when 0.9 M NaCl was applied to the column. The fractions from Q-Sepharose Fast Flow column containing Lip1 activity were combined and concentrated to a volume of 0.5 ml by ultrafiltration (PM-10 membrane filter; Amicon). The Q-Sepharose column elution was controlled by a Pharmacia FPLC system. Protein concentration was measured by Bradford method [21] with a commercial assay kit (BioRad) using BSA as the standard.
The quenching of Trp in the presence of ionic liquids was analyzed by the Stern–Volmer equation:
F0 /F = 1 + KSV [Q] where, F and F0 are the fluorescence intensity in the presence and absence of an ionic liquid, KSV is the Stern–Volmer quenching constant and [Q] is the ionic liquid concentration [22].
Results Kinetic Parameters of Lip1 Kinetics Parameters of Lip1 The Michaelis–Menten constant (KM) and the maximum velocity (VMax) for the hydrolysis of pNPL were determined. Lipase activity was measured as described above. The kinetic constant of Lip1 for the hydrolysis of pNPL was obtained using different concentrations of substrate ranging from 0 to 6 mM. Apparent KM and VMax were obtained adjusting the double reciprocal plot (Lineweaver– Burk plot).
VMax and KM of Lip1 for the hydrolysis of pNPL were determinated by adjusting double reciprocal plots. A linear regression (r2 = 0.98) was obtained. Apparent KM and VMax values were 0.6 ± 0.05 mM and 24.1 ± 3.4 mM min−1, respectively (Table 1). The kcat was 2687.7 min−1 and the catalytic efficiency was 4.2 × 106 M−1 min−1. Effect of Ionic Liquids on the Kinetic Parameters of Lip1
Ionic Liquid Effect on the Enzymatic Kinetics of Lip1 Ionic liquids BmimBF4 and BmimPF6 were used to determine their effect on the enzymatic parameters of Lip1. Ionic liquid concentrations used ranged between 0 and 600 mM. The lipase concentration was 0.04 mg/ml. Fluorescence Analysis The fluorescence of tryptophan (Trp λexc = 290 nm; λem = 350 nm) was measured using a Synergy® HT MultiMode Microplate Reader. Measurements were performed in the buffer 100 mM Tris-HCl pH 9.0, using 0.04 mg/ml of Lip1 and a concentration of 0–600 mM of the ionic liquid.
Kinetic constants of Lip1 for the hydrolisis of pNPL in prescence of BmimBF4 and BmimPF6 were determined. Lip1 reaction rates varied with increasing concentrations of substrate in the presence of different concentrations of ionic liquids (Fig. 1). When a concentration of 180 mM of BmimBF4 was used, VMax and kcat increased two times with respect to the measurements on the standard buffer mixture (Fig. 1a; Table 1). However, at the same concentration of BmimBF4 a slight increase in KM to 1.5 mM was observed, meanwhile the catalytic efficiency (kcat/KM) was not affected (Table 1). At 60 and 300 mM of BmimBF4, a slight increase in VMax and catalytic efficiency was observed,
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without affecting the KM of Lip1. When a concentration of 600 mM of BmimBF4 was used, an inhibitory effect on lipolytic activity of Lip1 was determined and the hydrolysis rate of pNPL acquired a linear behavior. Kinetic parameters at this concentrations could not be determinated. On the other hand, when 300 and 600 mM of BmimPF6 was used, increases in VMax and kcat of ~4 and 6 times, respectively, were measured (Table 1), indicating that these parameters increase with higher concentrations of BmimPF6. The catalytic efficiency showed similar behavior with increasing concentrations of this ionic liquid. However, at 300 mM of BmimPF6 this change was not as evident as when 600 mM was used, showing a slight increase in KM. Effect of Ionic Liquids on the Secondary Structure of Lip1
Fig. 1 Formation rate of pNP at different concentrations of pNPL in the presence of BmimBF4 (a) or BmimPF6 (b). The assay was developed at different concentrations of ionic liquid: 0 (filled circles), 60 (open circles), 180 (filled inverted triangles), 300 (open triangles) and 600 mM (open squares). Error bars represent standard deviations of three replicates
A clear decrease in the fluorescence intensity of Trp over time was observed when Lip1 was incubated in the presence of 60 mM BmimBF4 and 60–180 mM BmimPF6 (Table 2). However, the reverse phenomenon was observed at higher concentrations, where the fluorescence intensity of Trp increased as a function of time. The presence of both ionic liquids, BmimBF4 and BmimPF6, promoted a quenching effect on the Trp fluorescence of Lip1, according to the Stern–Volmer plots (Fig. 2), suggesting the accessibility of these ionic liquids to this amino acid. In the case of BmimBF4 a reduction in the Stern–Volmer constant (KSV) over time was observed (Table 3), indicating a lower accessibility of this ionic liquid to Trp at longer incubation times. When BmimPF6 was used (Table 3), a higher KSV value was obtained, indicating a higher exposure of Trp to this liquid salt when is incubated at different times, promoting a higher quenching effect of Trp fluorescence.
Table 2 Percentage of fluorescence emission of Lip1 in the presence of different concentrations of BmimBF4 and BmimPF6 Condition
0 min
30 min
60 min
Discussion
Control 60 mM BmimBF4 180 mM BmimBF4 300 mM BmimBF4 600 mM BmimBF4 60 mM BmimPF6 180 mM BmimPF6 300 mM BmimPF6
100 ± 3.7 100 ± 7.9 100 ± 7.9 100 ± 2.3 100 ± 7.1 100 ± 12 100 ± 9.8 100 ± 1.7
94 ± 2.9 82.9 ± 4.8 103.1 ± 3.1 129.9 ± 3.2 167.1 ± 3.6 53.2 ± 6.6 72.1 ± 3,7 96.6 ± 4.5
86.7 ± 4.1 47.3 ± 8.1 126.5 ± 7.4 132.6 ± 5.9 271.8 ± 4.1 33.8 ± 8.4 68.9 ± 5.8 94.7 ± 5.7
600 mM BmimPF6
100 ± 6.6
112.3 ± 3.7
117.4 ± 1.7
Lipases are very valuable enzymes in food, pharmaceutical, detergent, textile, craft, leather and cosmetic industries. The main restriction to their applications is their limited thermostability at high temperatures. For this reason, the search for new enzyme sources has huge importance. This work is based on the study of a new lipase purified from a microorganism obtained from Deception Island, a volcano with an important geothermal activity located in the South Shetland Island, Antarctica. Lip1 is a very thermostable enzyme in comparison to commercial and native lipases already available and very active over a wide range of temperatures. Its purification
The control represents Trp fluorescence of Lip1 without ionic liquid. 100 % of fluorescence intensity is the value measured at 0 min. Each error value is the standard deviation of three independent repetitions
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Fig. 2 Stern–Volmer plots of the interaction between BmimBF4 (a) and BmimPF6 (b) with Lip1. The assay was done at different incubation times for each ionic liquid with Lip1: 0 min (filled circles), 30 min (open circles), 60 min (filled inverted triangles) Table 3 Effect of ionic liquids on KSV for Lip1 Ionic liquid BmimBF4
BmimPF6
Time Equation (min)
r2
KSV (M−1)
0
F0/F = 16.54 × [Q] + 1
0.9606 16.54
30
F0/F = 10.04 × [Q] + 1
0.9918 10.04
60
F0/F = 5.99 × [Q] + 1
0.9551
5.99
0
F0/F = 3.26 × [Q] + 1
0.9846
3.26
30
F0/F = 4.75 × [Q] + 1
0.9731
4.75
60
F0/F = 6.59 × [Q] + 1
0.9737
6.59
involves only one chromatographic step, reducing costs and times of this process drastically. These characteristics make Lip1 a good candidate for employment in industry [12]. Ionic liquids have shown themselves to increase the catalytic activity of some enzymes. Moreover, a limited number of studies on the effect of ionic liquids on the kinetic
behavior of enzymes are available. We determined the effect of two ionic liquids on Lip1. Most of the enzymes that are active in ionic liquids are lipases. Ionic liquids, mainly derivatives of methylimidazole, do not inactivate lipases even when they have similar polarity as organic solvents, as methanol or DMF, which are denaturants of proteins. In addition, PF6 and BF4 ions do not have a strong interaction with water, ensuring the optimum hydration of the enzyme [20]. Based on this information, the liquids ionic BmimBF4 and BmimPF6 were selected to determine their effect on the kinetic parameters of Lip1. To elucidate the mechanism of action of the ionic liquid effect on Lip1 catalysis, VMax and KM constants were analyzed in the presence of the ionic liquids. KM was not altered by the presence of these ionic liquids, except at concentrations of 180 mM of BmimBF4 and 300 mM of BmimPF6, where an increase in both values was observed (Table 1). The reaction rate increased in the presence of the ionic liquids (Fig. 1), causing an increase in kcat and catalytic efficiency without affecting the KM in a significant manner. Therefore, the increase in Lip1 activity in the presence of ionic liquids is probably due to changes in the turnover number without affecting KM, indicating high catalytic efficiency (Table 1). A similar effect was reported in the hydrolytic activity of lipases from Pseudomonas sp. and Mucor javanicus, which increased significantly in aqueous solutions containing ionic liquids derived from methylimidazole. These compounds were able to interact with charge groups of the enzyme, inducing conformational changes in the lipase structure [23, 24]. These changes in Lip1 behavior could be related to modifications of their secondary structure, probably due to interactions of Lip1 with the ionic liquids under study making the enzyme catalytically more active. Ionic liquids could play an important role in modifying enzymatic activity, probably by direct interaction of their ions with the enzyme or changing the microenvironment surrounding the active site of the enzyme. In fact, it has been described that the effect of ionic liquids on the activity of some enzymes is dependant on its anion [25], as it was seen on Lip1 from ID17, showing a different behavior observed in the presence of ionic liquids with different anions. A possible explanation of this effect is that ionic liquids cause changes to the secondary structure, improving enzymatic activity. In addition, they could stabilize the substrate-enzyme complex, reducing the activation energy of the reaction and increasing the enzymatic activity. Tryptophan fluorescence depends on its surroundings and changes when the proteins have a different conformation. In native protein, Trp residues are located in the core of the protein. When an enzyme is partially folded or unfolded, these residues become exposed to the solvent.
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De Diego et al. [26] have correlated the reduction of Trp fluorescence intensity with the CALB denaturation. The increase in fluorescence intensity was related to the compaction of the enzyme probably being responsible for an increase in the enzymatic activity. Then, a change in the Trp fluorescence intensity indicates possible changes in the protein folding. In this work, Trp fluorescence was used to determine BmimBF4 and BmimBF6 effects on the secondary structure of Lip1 when increasing concentrations of ionic liquids were used (0–600 mM). Changes in the secondary structure of Lip1 in the presence of ionic liquids were evident based on the decrease of fluorescence intensity of Trp, indicating the exposure of this amino acid to polar environments. This kind of structural alteration of Lip1 is in agreement with minor changes in Lip1 catalytic kinetics. At high concentrations of ionic liquid, fluorescence intensity of Trp has a gradual increase as a function of time, suggesting that the protein adopted a new and more active conformation, that allow a higher catalytic efficiency in comparison with the native enzyme structure. This phenomenon has been previously described for the secondary structure of CALB lipase from C. antarctica, where an increase in initial fluorescence intensity was accompanied by a bathochromic shift of λ maximum emission. These changes were associated with structural modifications in the native structure of CALB, which could be responsible for an increase in activity and stability of the enzyme [26]. Fluorescence of Trp is not only affected by polarity. The surrounding microenvironment of amino acids could causes fluorescence quenching. For example, Lys and Tyr quenching is attributed to protons transferred from the excited state, meanwhile Cys and His form non-fluorescent complexes with Trp [27]. A decrease in quenching of Trp is probably due to low access of the quencher to the fluorophore [28]. Our results suggest that in the case of the interaction of Lip1 and ionic liquids BmimBF4 and BmimPF6, both interactions occur in a different manner. The BmimBF4–Lip1 complex promotes possible changes in Lip1 structure, positioning Trp in a less accessible disposition to the ionic liquid (decrease in Ksv as a function of time). However, the BmimPF6–Lip1 complex would cause changes in the enzyme structure that exposes Trp to the ionic liquid, allowing a structure more active in comparison to the control and to the presence of BmimBF4. Not all quenchers of Trp have an effect on the structure and function of a protein. The quenching phenomenon could be associated with dynamic (collisional) or static quenching. In the first case, quenching occurs due to collisions between the fluorophore and the quencher without affecting the structure and biological activity of a protein. Static quenching occurs by the formation
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of a non-fluorescent complex between the fluorophore and the quencher, leading to changes in secondary structure, affecting protein function [29]. Due to the effect of BmimPF6 over VMax and catalytic efficiency of Lip1, it is probable that quenching of Trp occurs due to the mechanism of static quenching, where the ionic liquid forms non-fluorescent complexes with Trp. However, this mechanism must be elucidated by deeper quenching analysis, taking into account the temperature. Dynamic quenching is controlled by diffusion and the quencher must diffuse to the fluorophore during its excited state. Thereby, a high temperature favors quenching collisions due to higher diffusion rates. In static quenching, the temperature will have an opposite effect, it will promote the dissociation of the fluorophore-quencher complex, increasing fluorescence intensity [30]. Lip1 activity can be increased by the presence of ionic liquids, BmimPF6 being the ionic liquid with a higher effect on its enzymatic activity. Sunitha et al. [31] observed the same effect using ionic liquids with a PF6 anion, resulting in higher yields for the methanolysis of sunflower oil, employing C. antarctica lipase (Novozyme 435). These changes were probably associated with changes in the secondary structure of Lip1, leading to a more active enzyme. In this work we demonstrated that Lip1 lipase from ID17 isolate has the ability to increase its catalytic efficiency in the presence of ionic liquids. Hydrolytic activity of Lip1 is modulated by the presence of the ionic liquids BmimBF4 and BmimPF6, favoring the reaction rate and catalytic efficiency of the enzyme. This effect is given by probable changes in the native enzyme structure produced by the ionic liquids; leading to a structural rearrangement of the enzyme that favors the catalysis under the studied conditions. Acknowledgments We would like to thank the Instituto Antártico Chileno for its support. This work was funded by Project RG_10-12 from the Instituto Antártico Chileno.
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ORIGINAL ARTICLE
Ionic Liquids Increase the Catalytic Efficiency of a Lipase (Lip1) From an Antarctic Thermophilic Bacterium Patricio A. Muñoz · Daniela N. Correa‑Llantén · Jenny M. Blamey
Received: 30 June 2014 / Accepted: 6 November 2014 / Published online: 27 November 2014 © AOCS 2014
Abstract Lipases catalyze the hydrolysis and synthesis of triglycerides and their reactions are widely used in industry. The use of ionic liquids has been explored in order to improve their catalytic properties. However, the effect of these compounds on kinetic parameters of lipases has been poorly understood. A study of the kinetic parameters of Lip1, the most thermostable lipase from the supernatant of the strain ID17, a thermophilic bacterium isolated from Deception Island, Antarctica, and a member of the genus Geobacillus is presented. Kinetic parameters of Lip1 were modulated by the use of ionic liquids BmimPF6 and BmimBF4. The maximum reaction rate of Lip1 was improved in the presence of both salts. The highest effect was observed when BmimPF6 was added in the reaction mix, resulting in a higher hydrolytic activity and in a modulation of the catalytic efficiency of the enzyme. However, the catalytic efficiency did not change in the presence of BmimBF4. The increase of the reaction rates of Lip1 promoted by these ionic liquids could be related to possible changes in the Lip1 structure. This effect was measured by
quenching of tryptophan fluorescence of the enzyme, when it was incubated with each liquid salt. In conclusion, the hydrolytic activity of Lip1 is modulated by the ionic liquids BmimBF4 and BmimPF6, improving the reaction rate and the catalytic efficiency of this enzyme when BmimPF6 was used. This effect is probably due to changes in the structure of Lip1 induced by the presence of these ionic liquids, stimulating its catalytic activity. Keywords Ionic liquids · Lipase · Kinetic parameters · Thermophilic · Antarctica · Deception Island Abbreviations pNP p-Nitrophenol pNPL p-Nitrophenyl laurate BmimBF4 1-Butyl-3-methylimidazolium tetrafluoroborate BmimPF6 1-Butyl-3-methylimidazolium hexafluorophosphate
Introduction P. A. Muñoz, D. N. Correa-Llantén and J. M. Blamey contributed equally to this work. P. A. Muñoz (*) · D. N. Correa‑Llantén · J. M. Blamey Fundación Científica y Cultural Biociencia, José Domingo Cañas 2280, Ñuñoa, Santiago, Chile e-mail: [email protected] D. N. Correa‑Llantén e-mail: [email protected] J. M. Blamey e-mail: [email protected] J. M. Blamey Universidad de Santiago de Chile, Avenida Libertador Bernardo O’Higgins 3363, Santiago, Chile
Lipases (triacylglycerol hydrolases, EC 3.1.1.3) catalyze the hydrolysis and synthesis of ester bonds of long chain fatty acids [1]. These enzymes have a range of properties widely used in industry, including the ability to catalyze a huge variety of reactions [2], high stereo- and regio-selectivity, the ability to catalyze heterogeneous reactions at water–lipid interfaces [3] and a high biocatalytic potential in aqueous and non-aqueous media [4]. Many industrial processes employing lipases are carried out at temperatures above 50 °C, thus requiring active and resistant enzymes at these temperatures [5]. These kinds of enzymes from thermophiles and hyperthermophiles have
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been described [1, 6–8]. They exhibit improved resistance to conditions imposed by the industry [4]. Thermophilic microorganisms have been isolated from geothermal sites which are distributed worldwide. One of these sites is Deception Island, an active stratovolcano located in the South Shetland Islands, Antarctica. This volcanic island has been very active during the last century: fumarolic emissions, thermal springs and hot soils are the present witnesses of Deception Island volcanic activity [9]. Geothermal anomalies mainly extend from Fumarole Bay to Pendulum Cove. The temperatures of emissions range from 90 to 110 °C at the fumaroles of Fumarole Bay. The soil temperature is highly variable, reaching values between 40 and 60 °C at Whalers Bay, and more than 70 °C at Pendulum Cove [10]. Previous studies indicate the presence of thermophilic bacteria belonging to Geobacillus, Bacillus, Brevibacillus and Thermus genera in samples collected from Deception Island [8, 11]. From these, a thermophilic and lipase-producing bacterium from Geobacillus genus was previously isolated. This bacterium, named ID17, produce four lipases, of which Lip1 is the most thermostable and has the ability to catalyze the thermophilic hydrolysis of ester bonds of long chain fatty acid under alkaline conditions [12]. These properties make Lip1 an excellent candidate for its use in different applications such as an additive in detergent preparations and chemical synthesis. Moreover, the industry has explored the use of ionic liquids in order to improve the catalytic properties of several enzymes. However, the effect of these liquid salts on kinetic parameters of lipases is poorly understood. Ionics liquids have shown themselves to have a huge impact in biocatalysis, since they not only increase the enzymatic activity, but also favor the stability and selectivity of different enzymes without emission of additional volatile or contaminant compounds that can damage the environment [13]. Ionic liquids are liquid organic salts at temperatures below 100 °C [14]. Their unique properties, namely no volatility, no inflammability, a high capacity to dissolve a huge range of substrates and excellent chemical and thermal stability, makes them an attractive environmentally friendly alternative. Due to their physical and chemical properties such as polarity, hydrophobicity and basicity of the hydrogen bond, they can be manipulated through an appropriate selection of the respective cation and anion [15]. They have been recognized as solvents or co-catalysts in a wide variety of applications that include organic and inorganic catalysis, biocatalysis and polymerization [15, 16]. In the last few years, a large number of enzymatic reactions have been carried out in systems that contain ionic liquids, e.g., resolution of 1-phenylethanol, reactions of alcoholysis, perhydrolysis and amoniolysis catalyzed by lipases [17–19]. Although, a wide amount of enzymatic
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reactions have been tested using a variety of ionic liquids, most studies have been focused on measurements of enzymatic activity, without considering the kinetic behavior of the catalysis in ionic liquids. For these reasons, we studied the effect of the ionic liquids 1-butyl-3-methylimidazolium tetrafluoroborate (BmimBF4) and 1-butyl-3-methylimidazolium hexafluorophosphate (BmimPF6) on the kinetic parameters of Lip1, the most thermostable lipase purified from the bacterium ID17. Both ionic liquids have been widely used to improve the activity, stability and enantio selectivity of different bacterial and fungal lipases [20]. In this way, we could determine the effect of these liquid salts on the kinetic behavior of Lip1 in order to establish further applications of this enzyme.
Materials and Methods Bacterial Strain The thermophilic bacterium ID17 (Accession number GU366067), belonging to the genus Geobacillus isolated by Muñoz et al. [12] obtained from Deception Island (Antarctica), was aerobically grown in a liquid medium, as described. Enzyme Assay Lipase activity was measured spectrophotometrically at 65 °C with p-nitrophenyl laurate (pNPL) as the substrate at 405 nm [1, 12]. Each reaction mixture contained 300 mM pNPL in acetonitrile and 100 mM Tris-HCl. One unit (U) of lipase activity was defined as 1 µmol of p-nitrophenol (Ɛ = 1.82 × 104 M−1 cm−1) released from pNPL per minute. Protein concentration was measured by the Bradford method [21] with a commercial assay kit (BioRad) using BSA as the standard. Enzyme Purification Lipase was purified from ID17 cells at 23 °C. Ten liters of ID17 culture were centrifuged (9,000×g for 10 min) and the supernatant solution was filtered using a 0.45-µm acetate filter (Sartorius Stedim Biotech GmbH) to remove all cells. Extracellular lipases were concentrated by ultrafiltration (PM-10 membrane filter; Amicon). The proteins (10 ml) were dissolved in buffer A [50 mM Tris-HCl (pH 8.0) containing 10 % (vol/vol) glycerol, 2 mM dithiothreitol, and 2 mM sodium dithionite] and loaded onto a column (Pharmacia, C 16/20) of Q-Sepharose Fast Flow (Pharmacia Biotech) equilibrated with buffer A. After the column was washed with the same buffer, the enzyme was
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Table 1 Kinetic constants for the hydrolysis of pNPL by Lip1 in the presence of different concentrations of ionic liquids Condition
VMax (mM min−1)
KM (mM)
kcat (min−1)
Catalytic efficiency (106 M−1 min−1)
Control 60 mM BmimBF4 180 mM BmimBF4 300 mM BmimBF4
24.1 ± 3.4 31.1 ± 4.1 49.8 ± 4.8 37.9 ± 5.2
0.6 ± 0.05 0.6 ± 0.04 1.5 ± 0.10 0.6 ± 0.06
2687.7 ± 379.5 3464.0 ± 457.6 5549.3 ± 535.7 4225.0 ± 580.4
4.2 ± 0.76 5.3 ± 1.14 4.1 ± 0.54 7.0 ± 0.97
600 mM BmimBF4 60 mM BmimPF6 180 mM BmimPF6 300 mM BmimPF6
ND 24.4 ± 3.5 26.7 ± 3.6 95.2 ± 4.2
ND 0.6 ± 0.07 0.5 ± 0.03 1.0 ± 0.05
ND 2720.5 ± 390.6 2974.4 ± 401.8 10622.9 ± 468.8
ND 4.8 ± 0.56 6.3 ± 1.3 11.1 ± 0.94
600 mM BmimPF6
142.9 ± 3.9
0.7 ± 0.06
15934.3 ± 435.3
23.9 ± 0.73
The control represents kinetic parameters in the absence of ionic liquids. Errors correspond to the standard deviation of three independent repetitions ND not determined
eluted with a linear gradient (200 ml) of 0–1 M NaCl in buffer A. Lipase activity started to elute when 0.9 M NaCl was applied to the column. The fractions from Q-Sepharose Fast Flow column containing Lip1 activity were combined and concentrated to a volume of 0.5 ml by ultrafiltration (PM-10 membrane filter; Amicon). The Q-Sepharose column elution was controlled by a Pharmacia FPLC system. Protein concentration was measured by Bradford method [21] with a commercial assay kit (BioRad) using BSA as the standard.
The quenching of Trp in the presence of ionic liquids was analyzed by the Stern–Volmer equation:
F0 /F = 1 + KSV [Q] where, F and F0 are the fluorescence intensity in the presence and absence of an ionic liquid, KSV is the Stern–Volmer quenching constant and [Q] is the ionic liquid concentration [22].
Results Kinetic Parameters of Lip1 Kinetics Parameters of Lip1 The Michaelis–Menten constant (KM) and the maximum velocity (VMax) for the hydrolysis of pNPL were determined. Lipase activity was measured as described above. The kinetic constant of Lip1 for the hydrolysis of pNPL was obtained using different concentrations of substrate ranging from 0 to 6 mM. Apparent KM and VMax were obtained adjusting the double reciprocal plot (Lineweaver– Burk plot).
VMax and KM of Lip1 for the hydrolysis of pNPL were determinated by adjusting double reciprocal plots. A linear regression (r2 = 0.98) was obtained. Apparent KM and VMax values were 0.6 ± 0.05 mM and 24.1 ± 3.4 mM min−1, respectively (Table 1). The kcat was 2687.7 min−1 and the catalytic efficiency was 4.2 × 106 M−1 min−1. Effect of Ionic Liquids on the Kinetic Parameters of Lip1
Ionic Liquid Effect on the Enzymatic Kinetics of Lip1 Ionic liquids BmimBF4 and BmimPF6 were used to determine their effect on the enzymatic parameters of Lip1. Ionic liquid concentrations used ranged between 0 and 600 mM. The lipase concentration was 0.04 mg/ml. Fluorescence Analysis The fluorescence of tryptophan (Trp λexc = 290 nm; λem = 350 nm) was measured using a Synergy® HT MultiMode Microplate Reader. Measurements were performed in the buffer 100 mM Tris-HCl pH 9.0, using 0.04 mg/ml of Lip1 and a concentration of 0–600 mM of the ionic liquid.
Kinetic constants of Lip1 for the hydrolisis of pNPL in prescence of BmimBF4 and BmimPF6 were determined. Lip1 reaction rates varied with increasing concentrations of substrate in the presence of different concentrations of ionic liquids (Fig. 1). When a concentration of 180 mM of BmimBF4 was used, VMax and kcat increased two times with respect to the measurements on the standard buffer mixture (Fig. 1a; Table 1). However, at the same concentration of BmimBF4 a slight increase in KM to 1.5 mM was observed, meanwhile the catalytic efficiency (kcat/KM) was not affected (Table 1). At 60 and 300 mM of BmimBF4, a slight increase in VMax and catalytic efficiency was observed,
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without affecting the KM of Lip1. When a concentration of 600 mM of BmimBF4 was used, an inhibitory effect on lipolytic activity of Lip1 was determined and the hydrolysis rate of pNPL acquired a linear behavior. Kinetic parameters at this concentrations could not be determinated. On the other hand, when 300 and 600 mM of BmimPF6 was used, increases in VMax and kcat of ~4 and 6 times, respectively, were measured (Table 1), indicating that these parameters increase with higher concentrations of BmimPF6. The catalytic efficiency showed similar behavior with increasing concentrations of this ionic liquid. However, at 300 mM of BmimPF6 this change was not as evident as when 600 mM was used, showing a slight increase in KM. Effect of Ionic Liquids on the Secondary Structure of Lip1
Fig. 1 Formation rate of pNP at different concentrations of pNPL in the presence of BmimBF4 (a) or BmimPF6 (b). The assay was developed at different concentrations of ionic liquid: 0 (filled circles), 60 (open circles), 180 (filled inverted triangles), 300 (open triangles) and 600 mM (open squares). Error bars represent standard deviations of three replicates
A clear decrease in the fluorescence intensity of Trp over time was observed when Lip1 was incubated in the presence of 60 mM BmimBF4 and 60–180 mM BmimPF6 (Table 2). However, the reverse phenomenon was observed at higher concentrations, where the fluorescence intensity of Trp increased as a function of time. The presence of both ionic liquids, BmimBF4 and BmimPF6, promoted a quenching effect on the Trp fluorescence of Lip1, according to the Stern–Volmer plots (Fig. 2), suggesting the accessibility of these ionic liquids to this amino acid. In the case of BmimBF4 a reduction in the Stern–Volmer constant (KSV) over time was observed (Table 3), indicating a lower accessibility of this ionic liquid to Trp at longer incubation times. When BmimPF6 was used (Table 3), a higher KSV value was obtained, indicating a higher exposure of Trp to this liquid salt when is incubated at different times, promoting a higher quenching effect of Trp fluorescence.
Table 2 Percentage of fluorescence emission of Lip1 in the presence of different concentrations of BmimBF4 and BmimPF6 Condition
0 min
30 min
60 min
Discussion
Control 60 mM BmimBF4 180 mM BmimBF4 300 mM BmimBF4 600 mM BmimBF4 60 mM BmimPF6 180 mM BmimPF6 300 mM BmimPF6
100 ± 3.7 100 ± 7.9 100 ± 7.9 100 ± 2.3 100 ± 7.1 100 ± 12 100 ± 9.8 100 ± 1.7
94 ± 2.9 82.9 ± 4.8 103.1 ± 3.1 129.9 ± 3.2 167.1 ± 3.6 53.2 ± 6.6 72.1 ± 3,7 96.6 ± 4.5
86.7 ± 4.1 47.3 ± 8.1 126.5 ± 7.4 132.6 ± 5.9 271.8 ± 4.1 33.8 ± 8.4 68.9 ± 5.8 94.7 ± 5.7
600 mM BmimPF6
100 ± 6.6
112.3 ± 3.7
117.4 ± 1.7
Lipases are very valuable enzymes in food, pharmaceutical, detergent, textile, craft, leather and cosmetic industries. The main restriction to their applications is their limited thermostability at high temperatures. For this reason, the search for new enzyme sources has huge importance. This work is based on the study of a new lipase purified from a microorganism obtained from Deception Island, a volcano with an important geothermal activity located in the South Shetland Island, Antarctica. Lip1 is a very thermostable enzyme in comparison to commercial and native lipases already available and very active over a wide range of temperatures. Its purification
The control represents Trp fluorescence of Lip1 without ionic liquid. 100 % of fluorescence intensity is the value measured at 0 min. Each error value is the standard deviation of three independent repetitions
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Fig. 2 Stern–Volmer plots of the interaction between BmimBF4 (a) and BmimPF6 (b) with Lip1. The assay was done at different incubation times for each ionic liquid with Lip1: 0 min (filled circles), 30 min (open circles), 60 min (filled inverted triangles) Table 3 Effect of ionic liquids on KSV for Lip1 Ionic liquid BmimBF4
BmimPF6
Time Equation (min)
r2
KSV (M−1)
0
F0/F = 16.54 × [Q] + 1
0.9606 16.54
30
F0/F = 10.04 × [Q] + 1
0.9918 10.04
60
F0/F = 5.99 × [Q] + 1
0.9551
5.99
0
F0/F = 3.26 × [Q] + 1
0.9846
3.26
30
F0/F = 4.75 × [Q] + 1
0.9731
4.75
60
F0/F = 6.59 × [Q] + 1
0.9737
6.59
involves only one chromatographic step, reducing costs and times of this process drastically. These characteristics make Lip1 a good candidate for employment in industry [12]. Ionic liquids have shown themselves to increase the catalytic activity of some enzymes. Moreover, a limited number of studies on the effect of ionic liquids on the kinetic
behavior of enzymes are available. We determined the effect of two ionic liquids on Lip1. Most of the enzymes that are active in ionic liquids are lipases. Ionic liquids, mainly derivatives of methylimidazole, do not inactivate lipases even when they have similar polarity as organic solvents, as methanol or DMF, which are denaturants of proteins. In addition, PF6 and BF4 ions do not have a strong interaction with water, ensuring the optimum hydration of the enzyme [20]. Based on this information, the liquids ionic BmimBF4 and BmimPF6 were selected to determine their effect on the kinetic parameters of Lip1. To elucidate the mechanism of action of the ionic liquid effect on Lip1 catalysis, VMax and KM constants were analyzed in the presence of the ionic liquids. KM was not altered by the presence of these ionic liquids, except at concentrations of 180 mM of BmimBF4 and 300 mM of BmimPF6, where an increase in both values was observed (Table 1). The reaction rate increased in the presence of the ionic liquids (Fig. 1), causing an increase in kcat and catalytic efficiency without affecting the KM in a significant manner. Therefore, the increase in Lip1 activity in the presence of ionic liquids is probably due to changes in the turnover number without affecting KM, indicating high catalytic efficiency (Table 1). A similar effect was reported in the hydrolytic activity of lipases from Pseudomonas sp. and Mucor javanicus, which increased significantly in aqueous solutions containing ionic liquids derived from methylimidazole. These compounds were able to interact with charge groups of the enzyme, inducing conformational changes in the lipase structure [23, 24]. These changes in Lip1 behavior could be related to modifications of their secondary structure, probably due to interactions of Lip1 with the ionic liquids under study making the enzyme catalytically more active. Ionic liquids could play an important role in modifying enzymatic activity, probably by direct interaction of their ions with the enzyme or changing the microenvironment surrounding the active site of the enzyme. In fact, it has been described that the effect of ionic liquids on the activity of some enzymes is dependant on its anion [25], as it was seen on Lip1 from ID17, showing a different behavior observed in the presence of ionic liquids with different anions. A possible explanation of this effect is that ionic liquids cause changes to the secondary structure, improving enzymatic activity. In addition, they could stabilize the substrate-enzyme complex, reducing the activation energy of the reaction and increasing the enzymatic activity. Tryptophan fluorescence depends on its surroundings and changes when the proteins have a different conformation. In native protein, Trp residues are located in the core of the protein. When an enzyme is partially folded or unfolded, these residues become exposed to the solvent.
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De Diego et al. [26] have correlated the reduction of Trp fluorescence intensity with the CALB denaturation. The increase in fluorescence intensity was related to the compaction of the enzyme probably being responsible for an increase in the enzymatic activity. Then, a change in the Trp fluorescence intensity indicates possible changes in the protein folding. In this work, Trp fluorescence was used to determine BmimBF4 and BmimBF6 effects on the secondary structure of Lip1 when increasing concentrations of ionic liquids were used (0–600 mM). Changes in the secondary structure of Lip1 in the presence of ionic liquids were evident based on the decrease of fluorescence intensity of Trp, indicating the exposure of this amino acid to polar environments. This kind of structural alteration of Lip1 is in agreement with minor changes in Lip1 catalytic kinetics. At high concentrations of ionic liquid, fluorescence intensity of Trp has a gradual increase as a function of time, suggesting that the protein adopted a new and more active conformation, that allow a higher catalytic efficiency in comparison with the native enzyme structure. This phenomenon has been previously described for the secondary structure of CALB lipase from C. antarctica, where an increase in initial fluorescence intensity was accompanied by a bathochromic shift of λ maximum emission. These changes were associated with structural modifications in the native structure of CALB, which could be responsible for an increase in activity and stability of the enzyme [26]. Fluorescence of Trp is not only affected by polarity. The surrounding microenvironment of amino acids could causes fluorescence quenching. For example, Lys and Tyr quenching is attributed to protons transferred from the excited state, meanwhile Cys and His form non-fluorescent complexes with Trp [27]. A decrease in quenching of Trp is probably due to low access of the quencher to the fluorophore [28]. Our results suggest that in the case of the interaction of Lip1 and ionic liquids BmimBF4 and BmimPF6, both interactions occur in a different manner. The BmimBF4–Lip1 complex promotes possible changes in Lip1 structure, positioning Trp in a less accessible disposition to the ionic liquid (decrease in Ksv as a function of time). However, the BmimPF6–Lip1 complex would cause changes in the enzyme structure that exposes Trp to the ionic liquid, allowing a structure more active in comparison to the control and to the presence of BmimBF4. Not all quenchers of Trp have an effect on the structure and function of a protein. The quenching phenomenon could be associated with dynamic (collisional) or static quenching. In the first case, quenching occurs due to collisions between the fluorophore and the quencher without affecting the structure and biological activity of a protein. Static quenching occurs by the formation
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of a non-fluorescent complex between the fluorophore and the quencher, leading to changes in secondary structure, affecting protein function [29]. Due to the effect of BmimPF6 over VMax and catalytic efficiency of Lip1, it is probable that quenching of Trp occurs due to the mechanism of static quenching, where the ionic liquid forms non-fluorescent complexes with Trp. However, this mechanism must be elucidated by deeper quenching analysis, taking into account the temperature. Dynamic quenching is controlled by diffusion and the quencher must diffuse to the fluorophore during its excited state. Thereby, a high temperature favors quenching collisions due to higher diffusion rates. In static quenching, the temperature will have an opposite effect, it will promote the dissociation of the fluorophore-quencher complex, increasing fluorescence intensity [30]. Lip1 activity can be increased by the presence of ionic liquids, BmimPF6 being the ionic liquid with a higher effect on its enzymatic activity. Sunitha et al. [31] observed the same effect using ionic liquids with a PF6 anion, resulting in higher yields for the methanolysis of sunflower oil, employing C. antarctica lipase (Novozyme 435). These changes were probably associated with changes in the secondary structure of Lip1, leading to a more active enzyme. In this work we demonstrated that Lip1 lipase from ID17 isolate has the ability to increase its catalytic efficiency in the presence of ionic liquids. Hydrolytic activity of Lip1 is modulated by the presence of the ionic liquids BmimBF4 and BmimPF6, favoring the reaction rate and catalytic efficiency of the enzyme. This effect is given by probable changes in the native enzyme structure produced by the ionic liquids; leading to a structural rearrangement of the enzyme that favors the catalysis under the studied conditions. Acknowledgments We would like to thank the Instituto Antártico Chileno for its support. This work was funded by Project RG_10-12 from the Instituto Antártico Chileno.
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