International Journal of Biological Macromolecules 81 (2015) 560–567
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Role of spacer length in interaction between novel gemini imidazolium surfactants and Rhizopus oryzae lipase Sunita Adak a , Sougata Datta b , Santanu Bhattacharya b , Rintu Banerjee a,∗ a b
Department of Agricultural & Food Engineering, Indian Institute of Technology, Kharagpur 721302, India Department of Organic Chemistry, Indian Institute of Science, Bangalore 560012, India
a r t i c l e
i n f o
Article history: Received 19 May 2015 Received in revised form 6 August 2015 Accepted 23 August 2015 Available online 28 August 2015 Keywords: Lipase Gemini imidazolium surfactant Activity Stability Structural study Interactions
a b s t r a c t An insight into the effects of new ionic liquid-type gemini imidazolium cationic surfactants on the structure and function of the lipases is of prime importance for their potential application. Changes in the activity, stability and structure of Rhizopus oryzae lipase in the presence of novel gemini surfactants, [C16 -3-C16 im]Br2 and [C16 -12-C16 im]Br2 were probed in the present study. Surfactant with shorter spacer length, [C16 -3-C16 im]Br2 was found to be better in improving the hydrolytic activity and thermal stability of the lipase. For both the surfactants, activation was concentration dependent. CD spectroscopy results showed a decrease in ␣-helix and an increase in -sheet content in the presence of these surfactants. A higher structural change observed in presence of [C16 -12-C16 im]Br2 correlated with lower enzyme activity. Isothermal titration calorimetric studies showed the binding to be spontaneous in nature based on sequential two site binding model. The forces involved in binding were found to differ for the two surfactants proving that the spacer length is an important factor which governs the interaction. These surfactants could be used as promising components both in enzyme modification and media engineering for attaining the desired goals in biocatalytic reactions. © 2015 Published by Elsevier B.V.
1. Introduction Surfactants find their application in diverse fields like food, pharmaceutical, cosmetics, detergents and biotechnology. The association of surfactants and proteins has been observed to play an important role, where the associations are very much surfactant/protein dependent. Surfactants affect the protein structure, conformational stability, polarity, solubility and catalytic activity of enzymes [1–4]. Among enzymes, extensive study and application of surfactants has been carried out with lipases. Surfactants change the rate of catalysis, the interfacial properties and enhance substrate availability [5–9]. An insight into the mechanism involved in lipase–surfactant interactions could provide the basis for enzyme activation, stabilization, media designing and help in better utilization of surfactants. In order to better understand the interaction between the lipase and surfactants, numerous studies have been undertaken. Association between lipase and surfactants has been observed at interfaces
∗ Corresponding author at: Microbial Biotechnology and Downstream Processing Laboratory, Department of Agricultural & Food Engineering, Indian Institute of Technology, Kharagpur 721302, India. E-mail address:
[email protected] (R. Banerjee). http://dx.doi.org/10.1016/j.ijbiomac.2015.08.051 0141-8130/© 2015 Published by Elsevier B.V.
and bulk, with subsequent effect on the enzyme structure and biological activity [10,11]. These interactions have been found to be dependent on the type of lipase and molecular structure of surfactants [1]. Rhizomucor miehei lipase was found to bind with cationic surfactants whereas Humicola lanuginose lipase formed complex with anionic surfactant [12]. Thermomyces lanuginosus lipase was well activated by ionic and non-ionic surfactants alike, at different range of concentrations exclusive of the classical interfacial activation phenomenon [13]. Both positive and negative effects of surfactants on activity of Rhizopus sp. lipase have been reported along with changes in the structural organization [14,15]. Although most of the studies have focussed on conventional surfactants, interaction with gemini surfactants has shown far better results [16,17]. Gemini surfactants are made up of two hydrophilic head groups linked by a spacer and two hydrophobic alkyl tails. Ionic liquid-type gemini cationic imidazolium surfactants are new generation of amphiphilic molecules and are superior to conventional surfactants. These novel gemini surfactants with imidazolium head group possess special properties and find potential application in the field of separation science, electrochemistry, material science, petrochemistry, organic synthesis and biocatalysis [18–20]. Apart from tail length, spacer length also plays an important role in modifying the polarity as well as aggregation properties of these surfactants [21]. Gemini surfactants with
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enzyme showed maximum stability under these conditions. Both the enzyme and the surfactants were dissolved in 10 mM phosphate buffer (pH 7) and assays were conducted in triplicate against suitable blanks containing corresponding concentration of surfactant and substrate except enzyme. One unit (IU) of lipase activity equals to the amount of enzyme that releases 1 mole of p-nitrophenol per minute under the assay conditions. 2.4. Study of gemini surfactants induced structural changes in the enzyme
Fig. 1. Structure of gemini surfactants.
shorter spacer have lower critical micelle concentration (CMC) value [21–23] depicting better surface active property than geminis with longer spacer. The spacer length also influences the shape of the micelles and nanoparticles formed as observed by Pal et al. [22] and Datta et al. [23]. A transition in micelle shape from ellipsoid to spheroid has been reported with increasing spacer length. Both short and long chain imidazolium cationic ionic liquids have shown the ability to enhance the catalytic activity of lipases in various biotransformation reactions and in some cases improved stability as well regioselectivity of the enzyme [24–26]. However, the effect of ionic liquid-type gemini cationic imidazolium surfactants on lipases along with its structure–function relationship has not been probed yet. In the present study, the authors have attempted for the first time to find out the effect of novel ionic liquid-type gemini cationic imidazolium surfactants on Rhizopus oryzae lipase (rhL). These surfactants possess two 16 carbon (C16 ) long alkyl tails, each attached to two separate imidazolium rings linked by an alkyl chain spacer. The spacer lengths in [C16 -3-C16 im]Br2 and [C16 -12-C16 im]Br2 are 3 and 12 respectively (Fig. 1). A comparative study based on the changes in the lipase activity, thermal stability, structure and associated binding energetics was conducted to find the influence of spacer length on the surfactant–rhL interaction. 2. Materials and methods 2.1. Materials p-Nitrophenyl acetate was procured from Sigma Aldrich (St. Louis, MO, USA). Novel ionic liquid-type cationic gemini imidazolium surfactants of varying spacer lengths, [C16 -3-C16 im]Br2 and [C16 -12-C16 im]Br2 , were provided by Prof. S. Bhattacharya, Department of Organic Chemistry, Indian Institute of Science, Bangalore (India). All other reagents were of analytical grade. 2.2. Lipase Lipase of R. oryzae NRRL 3562 was produced and further purified by following the procedure of Kumari et al. [27] and Adak and Banerjee [28], respectively.
2.4.1. UV–vis spectroscopy UV–vis spectra of lipase (10 M) in the presence of varying concentration of gemini surfactants (0–80 M) was recorded employing UV-Vis spectrophotometer (Agilent). Quartz cuvettes of 1 cm path length were used for scanning the samples in the range of 250–300 nm against suitable controls (with corresponding amount of surfactant). 2.4.2. Circular dichroism (CD) spectroscopy Far-UV CD spectra of the enzyme (10 M) with gemini surfactants at 0–80 M concentrations were recorded by JASCO J-810 CD Spectropolarimeter. The range of 200–240 nm was scanned with 0.1 nm step resolution and 50 nm s−1 scan speed. All the spectra were recorded at 30 ◦ C in N2 atmosphere using 0.1 cm path length quartz cell. Final spectra, representing average of three scans, were corrected by subtracting the base line recorded for every medium. Prediction of secondary structure was carried out with the standard spectral analysis software provided by the manufacturer, based on the reference CD spectra of distinct proteins given by Yang et al. [30]. 2.5. Binding thermodynamics study by isothermal titration calorimetry (ITC) ITC (VP cal 2000) studies were undertaken to evaluate the interactions involved in binding between the gemini surfactants and enzyme. Both enzyme and surfactants (in 10 mM phosphate buffer, pH 7) were degassed extensively prior to initiating the experiments. Enzyme (10 M) filled in the sample cell was titrated by [C16 -3-C16 im]Br2 and [C16 -12-C16 im]Br2 (500 M) taken in the injector. Total 25 injections of 10 L of surfactant were carried out at 120 s interval and 307 rpm. Heat evolved during each injection was measured by the instrument. The data were analyzed by Origin 8 software supplied along with the instrument after subtracting heat of dilution of the surfactants, obtained separately by injecting into the buffer. 2.6. Thermal stability study 2.6.1. Enzyme activity assay at varying temperatures To determine the effect of the surfactants on the thermal stability of the enzyme, rhL (10 M) was incubated with the gemini surfactants (20 M) for 1 h at 60 and 70 ◦ C. These two temperatures were chosen as lipase showed good stability till 50 ◦ C in an earlier study [28]. After incubation the enzyme activity was measured in triplicate employing pNPA as substrate against suitable controls. Residual activity (%) was calculated based on the activity of lipase without any addition at t0 time of incubation.
2.3. Effect of gemini surfactants on the enzyme activity The enzyme (10 M) activity was determined in the presence of varying concentration of gemini surfactants (0–100 M) following the standard method developed by De Caro et al. [29]. The assay was carried out at 30 ◦ C and pH 7 as according to previous study [28], the
2.6.2. Differential scanning calorimetry (DSC) DSC measurements of lipase with and without gemini surfactants were performed using Pyris Diamond Differential Scanning Calorimeter (Perkin Elmer). Lipase (10 M) was incubated for one hour at pH 7, 30 ◦ C in the presence of gemini surfactants (20 M). An
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Fig. 2. Variation in relative activity of lipase catalyzed hydrolysis of pNPA in presence of (a) [C16 -3-C16 im]Br2 and (b) [C16 -12-C16 im]Br2 at various concentrations (0–100 M). 100% activity corresponds to 48.66 IU/ml of enzyme activity devoid of surfactants. Each value represents mean ± standard deviation (n = 3) activity relative to enzyme activity without surfactants.
aliquot of 20 l of sample was placed in aluminium pans and hermetically sealed before inserting in the DSC machine. Scans were performed in range of 30–80 ◦ C with a scan rate of 1.5 ◦ C/min. The reference sample pan was filled with an equal volume of corresponding control (buffer/buffer with surfactant). 3. Results and discussion Novel imidazolium cationic gemini surfactants could serve as a promising role in the field of biocatalysis due to their unique properties. The authors have attempted to study its effect on the activity and stability of rhL. Investigations were also carried out to understand the mode of structural and thermodynamic interactions between the surfactants and rhL. 3.1. Variation in the hydrolytic efficiency Conventional ionic and non-ionic surfactants depict the ability to enhance the activity of lipases within a particular range of concentration. This range has been found to vary for different surfactants as well as for different lipases [13,31]. Fig. 2 illustrates the effect of different concentrations of surfactants on the enzyme activity. In both the cases hydrolysis increased with gradual rise in the surfactant concentration, attaining the maximum value at a particular concentration. Thereafter, further increase in surfactant concentration decreased the rate of catalysis. Though the pattern of activation in both cases was similar, [C16 -3-C16 im]Br2 enhanced the activity by 97% while [C16 -12-C16 im]Br2 showed an increment of only 18% at the same concentration (20 M).
However, the activity decreased by 10% when [C16 -3-C16 im]Br2 concentration was increased above 30 M compared to control (without [C16 -3-C16 im]Br2 ). Whereas [C16 -12-C16 im]Br2 showed greater reduction in the activity with 40% fall. Thus, among the two gemini surfactants, one with shorter spacer length was found to be more effective in increasing the catalytic activity of the enzyme. Unlike [C16 -12-C16 im]Br2 , [C16 -3-C16 im]Br2 was able to retain about 90% of the enzyme activity even at higher concentrations. Increased activity of lipases has been reported in the presence of imidazolium based ionic liquids during various transesterification processes [24,26]. Different gemini surfactants have also shown similar effects on the lipase activity [16,17]. Surfactant concentration as well as structure is found to be a major contributor in modulation of enzyme activity [32]. Debnath et al. [33] investigated the effect of imidazolium based ionic liquids having varying lengths of side arms (C1 C4 ) on the activity of trypsin. They deduced a negative correlation between increased arm length and enzyme activity. In contradiction to this, Das et al. [34] reported that the activity of interfacially active enzymes increased with concentration (5–40 mM) and length of alkyl chain of monomeric imidazolium surfactant. Difference in the extent of influence of the gemini surfactants on the rhL activity, could be caused by disparity between the spacer lengths. This structural variation could further affect the interaction between the two surfactants and rhL in solution. Such association has been attributed to alter protein structure, conformational stability and catalytic activity [2,35]. Probable binding between rhL and gemini surfactants was investigated by spectroscopy.
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Fig. 3. Variation in the absorbance at 280 nm of lipase vs surfactants (a) [C16 -3-C16 im]Br2 at concentrations (M): 0, 10, 20, 30, 40, 60 and 80; (b) [C16 -12-C16 im]Br2 at concentrations (M): 0, 10, 20, 30, 40, 60 and 80.
3.2. Surfactants induced structural changes in the enzyme 3.2.1. UV–vis spectroscopy Protein structural stability and dynamics can be monitored by the absorbance spectra of aromatic amino acids (Phe, Tyr and Trp) in the near-UV region [36,37]. Binding between surfactant and protein has been analyzed via absorbance at this wavelength. UV intensity can increase or decrease upon changes in protein conformation [3,38]. Addition of gemini surfactants decreased the absorbance of rhL. A plot of maximum absorbance (280 nm) against concentration of surfactant added has been represented in Fig. 3. Presence of [C16 -3-C16 im]Br2 lowers the absorbance to a lesser extent in the given concentration range compared to [C16 -12C16 im]Br2 . In both the cases change in absorbance intensity indicates probable alteration in the enzyme structure which could be related to changes in rhL’s activity pattern. At low concentration (1–20 M) absorbance decreased to a lesser extent for [C16 -3-C16 im]Br2 and [C16 -12-C16 im]Br2 alike. Enzyme activity was also found to be the highest in this range but with further rise in surfactant concentration, drop in the absorbance as well as activity was observed for both. For [C16 -12-C16 im]Br2 the reduction was found to be greater which corresponds to rhL’s lower activity in its presence which is contrary to [C16 -3-C16 im]Br2 . Similar to results obtained in present study, decrease in absorbance of HSA on addition of sodium perfluorooctanoate has also been reported by Messina et al. [38]. These changes have been linked to conformational changes occurring in HSA due to the surfactant. Significant changes in the absorbance were also reported in -lactoglobulin A upon binding with SDS and Triton X-100, where the pattern differed for the two surfactants [3]. The above studies
suggest addition of surfactant leads to profound structural variations in the studied proteins. To further ascertain alterations in rhL structure and its effect on activity, CD spectroscopic analysis was carried out. 3.2.2. CD spectroscopy CD spectroscopy is a widely used technique for examining protein structure, dynamics and folding. The CD signals of proteins (<250 nm) mainly originate from the –* and n–* transitions of peptide bonds [39,40]. Fig. 4 illustrates the changes induced by the surfactant on the secondary structure of the lipase. In the presence of both the gemini surfactants ␣-helix content decreased and -sheet content increased with rise in surfactant concentration (Table 1). Higher catalytic efficiency of lipases from Burkholderia cepacia, Chromobacterium viscosum, Candida rugosa and T. lanuginosus in water-in-ionic liquid microemulsions has been related to decrease in ␣-helix and increase in -sheet content [31,41]. Decrease in ␣-helix influences the active site of enzyme by stimulating higher tendency for the open conformation which allows easier access to the substrate [42]. Within the concentration range studied here, [C16 -12-C16 im]Br2 caused greater changes in the ␣-helix and -sheet content, which could be a possible reason behind the larger reduction in the enzyme activity compared to that of [C16 -3-C16 im]Br2 . Similarly, interaction between dicationic alkylammonium bromide gemini surfactants with model membranes was found to be dependent on spacer length [43]. Surfactant with long spacer (C10 ) was found to be more effective in membrane destabilization and disruption, leading to drastic loss in membrane integrity even at smaller concentrations. Relative to short spacer (C2 ), long spacer (C10 ) being more hydrophobic bends and
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Fig. 4. CD spectra of lipase at different concentration of (a) [C16 -3-C16 im]Br2 : 10, 20, 40 and 80 M; (b) [C16 -12-C16 im]Br2 : 10, 20, 40 and 80 M.
Table 1 Secondary structure of the Rhizopus oryzae lipase in presence of varying concentration of [C16 -3-C16 im]Br2 and [C16 -12-C16 im]Br2 . Surfactant
Concentration (M)
␣-helix (%)
-sheet (%)
-turn (%)
Random coil (%)
[C16 -3-C16 im]Br2
0 10 20 40 80
14.7 12.4 10.9 8.4 11.9
29.5 34.4 48.2 53.7 55.7
18.0 16.0 8.7 5.7 0.8
37.8 37.2 32.2 32.1 31.5
[C16 -12-C16 im]Br2
10 20 40 80
13.3 8.8 7.3 6.7
33.7 54.1 56.9 50.1
16.2 6.0 2.3 10.4
36.7 31.1 33.5 32.7
embeds itself deep into the bilayer. This structural conformation and positioning based on spacer length contributed to the membrane disrupting effect, which could be applicable in the present study as well. For RNase A gemini with longer spacer had more destabilizing effect monitored by loss of far UV-CD spectral signal. The loss in the helical structure increased modestly with the length of the carbon chain in the spacer [44]. Das et al. [34] also observed changes in the helix content of lipase which was relative to increase in activity in presence of mixed cationic and imidazoilumbased amphiphiles reverse micelles system. Present study shows higher surfactant concentrations cause greater changes in the secondary structure which lowers its activity. At high concentration (80 M) [C16 -3-C16 im]Br2 showed increase in ␣-helix content. Gull et al. [45] reported equivalent conformational changes occurring in serum albumins on interacting with gemini counterpart of CTAB and attributed it as refolding of proteins. With this information further probe into the binding parameters and thermodynamics was carried out to better understand the process.
3.3. Thermodynamics of gemini surfactants and lipase interaction Binding affinity and energetics of the interaction was measured by ITC. Fig. 5(a) and (b) represent the binding isotherm of [C16 -3C16 im]Br2 and [C16 -12-C16 im]Br2 with rhL, respectively. Integrated plot of the amount of heat liberated per injection as a function of molar ratio of surfactant to rhL were further predicted by fitting into different binding models using in-built Origin 8 software. Injection of buffer into lipase gave negligible heat effects, hence were not considered for subtraction. The data appears to best fit at two sites sequential binding model with lowest 2 . Generally binding between surfactant and protein follows multiple binding pattern, majorly divided into four steps. Initial step of binding occurs at low concentration of surfactants where surfactants behave as ligands and show specific binding. This is followed by non-cooperative, cooperative and saturated binding steps. Micelle formation takes place after all the binding sites on the protein are saturated with surfactants [46–48]. In present study two steps of binding were observed, where for both the surfactants the first binding shows higher affinity than the second (Table 2). [C16 -3-C16 im]Br2 binds with higher affinity to rhL as depicted by the higher K1 value compared to [C16 -12-C16 im]Br2 , whereas [C16 12-C16 im]Br2 shows greater affinity for the second site. Binding process was found to be spontaneous for the two sites in both the surfactants at given temperature. Binding mechanism was elucidated from the enthalpic and entropic contributions to the Gibbs free energy. For [C16 -3-C16 im]Br2 negative value of H indicates binding process to be exothermic. H1 having value less than 60 kcal mol−1 indicates involvement of non-covalent interactions [49]. Electrostatic interactions between rhL and surfactant could be one of the forces involved as rhL is negatively charged at given pH (pI 6.5 as reported by Adak and Banerjee [28]). This shows involvement of surfactant monomer binding with high specificity
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Fig. 5. ITC study of lipase with gemini surfactants (a) [C16 -3-C16 im]Br2 and (b) [C16 -12-C16 im]Br2 . Integrated heat responses obtained from titration profile with the solid line representing best fit to sequential two site binding model.
Table 2 Thermodynamic parameters of gemini surfactants-lipase binding reaction at 30 ◦ C and pH 7.
K1 (M−1 ) K2 (M−1 ) H 1(kcal mol−1 ) H 2(kcal mol−1 ) S1(cal mol−1 K−1 ) S2 (cal mol−1 K−1 ) G1 (kcal mol−1 ) G2 (kcal mol−1 )
[C16 -3-C16 im]Br2
[C16 -12-C16 im]Br2
(1.47 ± 0.32) × 105 (3.5 ± 0.12) × 103 −10.3 ± 1.32 −127.7 ± 3.61 −10.3 −405 −7.18 −4.99
(2.05 ± 0.58) × 104 (1.14 ± 0.9) × 104 −24.34 ± 0.57 50.41 ± 0.11 −60.6 185 −5.98 −5.65
which is exothermic in nature and driven by electrostatic interactions. Similar observations have been reported for lysozyme and myoglobin with different surfactants [46,47]. It was elucidated that at this stage surfactants are found to activate lipases and also confer stability to protein [11]. Second site binding involves large negative enthalpic change. Such an effect has been observed during the stacking interaction between aromatic amino acids in Escherichia coli SSB protein and nucleic acid bases in ssDNA [50]. Occurrence of – stacking interactions between the imidazolium rings of [C12 -s-C12 im]Br2 and aromatic ring of amino acids of BSA has been reported by Zhou et al. [4]. The entropy was found to
be unfavourable for binding at both the sites. This suggests the involvement of hydrogen bonding between the two molecules [51]. In case of [C16 -12-C16 im]Br2 binding to first site follows the same pattern as found in [C16 -3-C16 im]Br2 . Second site binding involves unfavourable enthalpic and favourable entropic changes which suggest the presence of hydrophobic interactions between the two molecules. Presence of a longer spacer could be a probable cause behind the difference in binding pattern of the surfactants. Binding of cationic surfactant with cellulase also follows two sites binding model, where the first and second site binding is exothermic and endothermic respectively. Electrostatic and hydrophobic interaction act as the driving force for the association which was found to be dependent on the chain length and charge density of the surfactant [48]. Interactions between brilliant red and lysozyme showed a similar two site binding pattern [49]. First site binding was observed to cause lowering of S which in turn increased the order of the enzyme structure and second binding led to increased entropy and unfolding of enzyme. Different factors which cause such unfavourable entropy include exposure of hydrophobic surfaces to solvent and decrease in conformational motion in proteins [52]. In the current study CD spectral data illustrates exposure of hydrophobic active site as well as increase in -sheets which have been linked to increased rigidity of lipases in imidazoliun ionic liquid emulsions [31]. These structural changes in presence of
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Table 3 Effect of gemini surfactants on thermal stability of lipase in terms of residual activity (%).a Samples
60 ◦ C
70 ◦ C
Lipase Lipase + [C16 -3-C16 im]Br2 (20 M) Lipase + [C16 -12-C16 im]Br2 (20 M)
44 ± 2.16 67.59 ± 3.74 48.12 ± 2.31
10 ± 0.33 30.08 ± 3.13 6.77 ± 2.85
a 100% activity corresponds to 48.66 IU/ml of lipase activity at t0 . Each value represents mean ± standard deviation (n = 3) residual activity after 1 h incubation at respective temperatures relative to t0 enzyme activity.
Fig. 6. DSC study of lipase unfolding represented as plot of heat flow vs temperature.
imidazolium ionic liquids have also improved the stability of lipases. To further probe into this, stability at high temperatures was checked. 3.4. Thermal stability Thermal stability studies were carried out at 60 and 70 ◦ C. After one hour of incubation with and without gemini surfactants at respective temperatures the hydrolytic activity of lipase was measured. Higher residual activity was observed in lipase with [C16 -3-C16 im]Br2 at both the temperatures compared to [C16 12-C16 im]Br2 and lipase alone (Table 3). This suggests improved thermal stability of lipase in the presence of [C16 -3-C16 im]Br2 . DSC thermogram (Fig. 6) showed an endothermic peak at 57.5 ◦ C for rhL. This is defined as protein deactivation temperature (Td ) and corresponds to lipase folding/unfolding transition [53]. Influence of surfactants on enzyme stability was inferred from the shift in the transition peak. Greater shift in enzyme Td was observed in the presence of [C16 -3-C16 im]Br2 (61 ◦ C) as compared to [C16 -12-C16 im]Br2 (58.5 ◦ C). Increase in deactivation temperature corresponds to higher stability of rhL in presence of [C16 -3-C16 im]Br2. Similar effect on protein stability in terms of Td peak shift was observed by Noel and Combs [53] in Rhizomucor miehei lipase in presence of sorbitol. A different genre of quaternary ammonium based gemini surfactants showed thermal stabilization and activation in RNase A below CMC by preferential binding [44]. One with shorter spacer was found to interact more efficiently with the protein. Increase in thermal stability has also been observed for -lactoglobulin upon binding with SDS monomers which reduces aggregation [54]. Diego et al. [55] reported enhancement in Candida antartica lipase B activity and stability in terms of residual activity in the presence of short chain imidazolium based ionic liquids.
Corresponding to this observation they also detected similar changes in structure of enzyme as obtained in the present study. 4. Conclusion Novel ionic liquid-type cationic gemini imidazolium surfactants differing in spacer length, [C16 -3-C16 im]Br2 and [C16 -12-C16 im]Br2 were found to enhance the hydrolytic activity of rhL. Surfactant with shorter spacer showed better effect and increased the activity up to 97% whereas surfactant with longer spacer gave a rise of 18% at a concentration of 20 M. Similarly the enzyme showed improved thermal stability in the presence of [C16 -3-C16 im]Br2 as depicted by the shift in Td to 61 ◦ C. Structural study revealed all these changes to be associated with conformational changes occurring in the enzyme in presence of surfactants. Considerable fall in absorbance at 280 nm and ␣-helix content along with rise in -sheet content in rhL was observed in the presence of both the surfactants. These changes were found to be greater for [C16 -12-C16 im]Br2 . ITC studies show that the surfactant binding followed sequential two site binding model. [C16 -3-C16 im]Br2 had higher binding affinity compared to [C16 -12-C16 im]Br2 for the first site. The interacting forces were found to differ for the surfactants which could be due to the difference in their structure. The obtained results depict functional and structural stabilizing effect of surfactant with short spacer. These could be effectively used for coating or encapsulating lipases for performing different biotransformations efficiently. Further study with intermediate spacer length surfactants could illustrate a wider view of the underlying mechanism and help in designing newer surfactants as well as their use in diverse fields such as in protein folding as potential chaperones. Acknowledgement Ms Sunita Adak gratefully acknowledges the financial support provided by CSIR, New Delhi, by granting her the Senior Research Fellowship. References [1] R.C. Lu, A.N. Cao, L.H. Lai, J.X. Xiao, Colloids Surf. B 64 (2008) 98–103. [2] A.W. Sonesson, H. Blom, K. Hassler, U.M. Elofsson, T.H. Callisen, J. Widengren, H. Brismar, J. Colloid Interface Sci. 317 (2008) 449–457. [3] A. Taheri-Kafrani, A.K. Bordbar, S.H.A. Mousavi, T. Haertle, J. Agric. Food Chem. 56 (2008) 7528–7534. [4] T. Zhou, M. Ao, G. Xu, T. Liu, J. Zhang, J. Colloid Interface Sci. 389 (2013) 175–181. [5] A. Aloulou, J.A. Rodriguez, S. Fernandez, D.V. Oosterhout, D. Puccinelli, F. Carriere, Biochim. Biophys. Acta 1761 (2006) 995–1013. [6] P. Reis, K. Holmberg, H. Watzke, M.E. Leser, R. Miller, Adv. Colloid Interface Sci. 147–148 (2009) 237–250. [7] H.J. Hsieh, G.R. Nair, W.T. Wu, J. Agric. Food Chem. 54 (2006) 5777–5781. [8] C.M.L. Carvalho, J.M.S. Cabral, Biochimie 82 (2000) 1063–1085. [9] M. Zoumpanioti, H. Stamatis, A. Xenakis, Biotechnol. Adv. 28 (2010) 395–406. [10] V. Delorme, R. Dhouib, S. Canaan, F. Fotiadu, F. Carriere, J.F. Cavalier, Pharm. Res. 28 (2011) 1831–1842. [11] D. Otzen, Biochim. Biophys. Acta 1814 (2011) 562–591. [12] B. Folmer, K. Holmberg, M. Svensson, Langmuir 13 (1997) 5864–5869. [13] J.E. Mogensen, P. Sehgal, D.E. Otzen, Biochemistry 44 (2005) 1719–1730. [14] E.D. Brown, R.Y. Yada, A.G. Marangoni, Biochim. Biophys. Acta 1161 (1993) 66–72. [15] J.C. Diaz, J. Cordova, J. Baratti, F. Carriere, A. Abousalham, Mol. Biotechnol. 35 (2007) 205–214. [16] Y. Mine, K. Fukunaga, N. Maruoka, K. Nakao, Y. Sugimura, J. Biosci. Bioeng. 90 (6) (2000) 631–636. [17] Y. Mine, K. Fukunaga, K.I. Samejima, M. Yoshimoto, K. Nakao, Y. Sugimura, J. Biosci. Bioeng. 96 (6) (2003) 525–528. [18] V. Pino, M. German-hernandez, A. Martin-Perez, J.L. Anderson, Sep. Sci. Technol. 47 (2012) 264–276. [19] M. Ao, G. Xu, Y. Zhu, Y. Bai, J. Colloid Interface Sci. 326 (2008) 490–495. [20] H. Zhang, K. Li, H. Liang, J. Wang, Colloids Surf. A: Physicochem. Eng. Aspects (2008) 75–81. [21] M. Ao, G. Xu, J. Pang, T. Zhao, Langmuir 25 (17) (2009) 9721–9727. [22] A. Pal, S. Datta, V.K. Aswal, S. Bhattacharya, J. Phys. Chem. B 116 (2012) 13239–13247.
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