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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers
Effect of surfactant structure on the superactivity of Candida rugosa lipase Francesco Gabriele, Nicoletta Spreti, Tiziana Del Giacco, Raimondo Germani, and Matteo Tiecco Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b02255 • Publication Date (Web): 28 Aug 2018 Downloaded from http://pubs.acs.org on September 2, 2018
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Langmuir
Effect of surfactant structure on the superactivity of Candida rugosa lipase
Francesco Gabrielea, Nicoletta Spretia*, Tiziana Del Giaccob, Raimondo Germanib, Matteo Tieccob
a
Department of Physical and Chemical Sciences, University of L'Aquila, Via Vetoio, I-67100 Coppito,
L'Aquila, Italy. b
CEMIN, Centre of Excellence on Nanostructured Innovative Materials, Department of Chemistry,
Biology and Biotechnology, University of Perugia, Via Elce di Sotto 8, I- 06123 Perugia, Italy
*Corresponding author: Department of Physical and Chemical Sciences, University of L'Aquila, Via Vetoio, I-67100 Coppito, L'Aquila, Italy. E-mail address:
[email protected] 1 ACS Paragon Plus Environment
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ABSTRACT
In this work, we present the effects of ionic and zwitterionic surfactants on the hydrolytic activity of Candida rugosa lipase (CRL), one of the most important and widely used microbial lipases. A series of amine N-oxide surfactants was studied to explore the relationship between their molecular structures and their effect on catalytic properties of CRL. These zwitterionic amphiphiles are known for their ability to form aggregates that can increase their size thanks to a sphere-rod transition, without any additive. Enzyme activity seemed to be improved by morphological changes of micelles from spherical to rod-like, and the structure of the monomers played a crucial role in this transition. In fact, all the amine oxides investigated provoked superactivation, but the CRL activity increased by lengthening the alkyl chain of N-oxide surfactants, while it decreased in presence of bulky head groups. Superactivity was mainly due to an increase of kcat (0.57 s-1 in buffer, 0.80-1.99 s-1 in surfactant solutions) and, in some cases, to a decrease in KM (2×10-3 M in buffer, 1.08-4.28×10-3 M in surfactant solutions). Micelles seemed to play a dual role: superactivity occurred at surfactant concentrations higher than their CMC, but, on the other hand, micelles subtracted the substrate from the bulk, making it not available for the catalysis.
Keywords: Candida rugosa lipase (CRL); superactivity; amine N-oxide surfactants; sphere-rod transition
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INTRODUCTION
Lipases (triacylglycerol acyl hydrolases, E.C. 3.1.1.3) are a class of enzymes, that catalyze the hydrolysis of long chain triglycerides. They are ubiquitous enzymes belonging to the group of serine hydrolases, which do not need any cofactor for their catalytic action. Lipases constitute one of the most important group of biocatalysts for biotechnological applications because they can catalyze different reaction types including hydrolysis, transesterification, alcoholysis, acidolysis, esterification, and aminolysis1. In fact, lipases are endowed with a very wide substrate specificity that could lead to a potential boundless application of these enzymes. Many reviews report various industrial applications of lipase in different topics such as pharmaceuticals, foods, cosmetics, paper, detergents, perfumery and textile2–7. Lipases are ubiquitous in nature and they are produced by various plants, animals, and microorganisms. However, for the production of industrial enzymes, microorganisms are the most preferred source, since microbial lipases are able to remain active under extreme conditions of temperature and pH and in organic solvents8–11. One of the most important lipases from a commercial point of view belongs to yeasts: Candida rugosa lipase (CRL) is a hydrophobic glycoprotein with a molecular weight of 60 kDa with 4.2% neutral sugars. This yeast produces and secretes mixture of lipase isoenzymes in various proportions. Two isoenzymes, initially called LipA and Lip B, were identified, purified and characterized12; nowadays, a family of seven lipase genes have been described (Lip1-Lip7), and five of them have been fully biochemical characterized13. Commercial CRL samples are a mixture of several isoenzymes with different catalytic properties and distinct differences in substrate specificity14–16. CRL is a member of the α/β hydrolase fold family; the active site contains the catalytic triad, Ser 209 His 449 – Glu 341, common to all serine hydrolases. Access to the active site may be shielded by a mobile element, the lid or flap, whose position closed or open determines the enzyme in an inactive or 3 ACS Paragon Plus Environment
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active conformation. The interaction with a hydrophobic phase can cause the opening of the lid making the active site accessible. As many other lipases, CRL is a versatile biocatalyst and numerous strategies have been adopted for its use in non-conventional media17. It is widely used for the catalysis in organic solvents in a free-form18,19 or in a surfactant-coated enzyme form both in organic solvents20,21 and in organic-aqueous two-phase systems22. Reverse micelles and w/o microemulsions were also used for CRL-catalyzed reactions23–25 as well as ionic liquids26–28. Moreover, CRL was immobilized on a variety of supports, such as silica29, magnetic poly(glycidyl methacrylate-methyl methacrylate) beads30, hybrid epoxy-silica polymer films31, calcium alginate gel32, montmorillonite33, titania powder34, celite35, polypropylene hollow fiber membrane modified with phospholipid analogous polymers36 and exfoliated graphene oxide37. Only a few studies were devoted to the effect of surfactant structures on the catalytic properties of CRL in aqueous solution38–40. This topic is relevant considering the different effects that could be obtained by changing the molecular structures of the amphiphiles41 and notwithstanding the importance of CRL for technical applications (such as detergency, cosmetics and drug delivery). Anionic and nonionic surfactants38 and polyethylene glycols39 interact with the isoforms of CRL in a different way. Lipase B was more sensitive than Lipase A to the presence of sodium dodecyl sulfate (SDS) and Triton X-100 and its deactivation was rapid. Polyethylene glycols activated one of the two selected isoforms of CRL and this activation is the consequence of the shift of the equilibrium between the open and the closed forms of the lid to the open form. Goswami et al.40 reported the effects of different cationic, anionic and nonionic surfactants on castor oil hydrolysis catalyzed by CRL: in this work, the nonionic surfactant Span 80 resulted the most suitable because it did not denature the enzyme and it acted as stabilizer for water/oil emulsions, promoting the substrate hydrolysis. The presence of amphiphiles might be responsible for the conformational changes of the protein that induce enzyme activation42. In fact, lipases having a lid, like CRL, showed interfacial activation; 4 ACS Paragon Plus Environment
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surfactants can induce those conformational changes in enzyme that open the lid and give access to its active site. On the other hand, surfactants can interact with the enzyme, and such interactions can have various consequences on protein structure and activity, depending on the nature of the surfactant and on its concentration. The effect of surfactants on enzymatic performances in aqueous solutions was widely investigated and studied in literature43–47: both hydrophobic and electrostatic interactions can occur between protein and surfactant. In this paper, the effect of differently structured surfactants (anionic, cationic, and zwitterionic) on the catalytic properties of CRL in aqueous media is reported. Structures and acronyms of all the additives employed in this paper are reported in Scheme 1. CH3 CH3 (CH2)10 CH2 OSO3
-
+
+
CH3 (CH2)14 CH2 N
Na
CH3 Br-
CH3
SDS
CTABr
CH3 +
CH3 +
-
CH3 (CH2)10 CH2 N CH2 CH2 CH2 SO3-
CH3 (CH2)10 CH2 N CH2 COO CH3
CH3
CB1-12 CH3 (CH2)m +
-
CH3 (CH2)n CH2 N O
(CH2)m CH3
n=10 m=0 n=12 m=0 n=14 m=0 n=12 m=1 n=12 m=2
SB3-12
AOMe-12 AOMe-14 AOMe-16 AOEt-14 AOPr-14
CH3 CH3 (CH2)10 CH2 O
+
CH2 N OCH3
pDoAO
Scheme 1 – Structure of the surfactants used in this work.
The hydrolysis of p-nitrophenyl acetate (pNPA) as probe reaction was investigated. The dependence of reaction rates on surfactant concentrations was studied and kinetic parameters were determined to 5 ACS Paragon Plus Environment
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better understand the effects of the different surfactants on CRL catalytic properties. Correlation between enzyme superactivity and the hydrophobicity of amine N-oxide surfactants was attempted to better understand the role of the surfactant structure on the enzyme properties.
EXPERIMENTAL SECTION Materials Lipase type VII from Candida rugosa and p-nitrophenyl acetate (pNPA) were purchased from SigmaAldrich. Enzyme and substrate were used with no further purification. The commercial grade surfactants, SDS, CTABr, and SB3-12 were also supplied by Sigma-Aldrich and purified by recrystallization in acetone/methanol or ethyl acetate/methanol mixtures. CB1-12 and the amine N-oxides were prepared in our laboratories starting from the correspondent tertiary amines. CB1-12 was synthesized by the reaction of N,N-dimethyldodecylamine with the sodium chloroacetate to reflux in acetonitrile-ethanol mixture for 48 hours. For the N-oxides, the tertiary amines were dissolved in ethanol, then 1.5 eq of H2O2 (30% water solution) were added; the mixtures were stirred at 70°C for 6-8 hours. All surfactants were purified as previously described48–50. The purities of all the surfactants were verified via surface tension measurements with a Sigma 700 Force Tensiometer with a platinum ring (diameter 1.9 cm).
CRL catalytic assay The CRL activity measurements were carried out spectrophotometrically at 25.0 ± 0.1 °C, following the increase in the absorbance at 348 nm, which corresponds to the isosbestic point of p-nitrophenol/pnitrophenoxide (pNP), taking the molar extinction coefficient as 5400 M-1 cm-1. Measurements were performed with a Shimadzu UV-160A UV-VIS spectrophotometer equipped with a thermostated cell (3 mL volume and 1-cm pathlength). 6 ACS Paragon Plus Environment
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CRL activity assay mixture was prepared in 0.01 M phosphate buffer at pH 7.0; the enzyme concentration was always 0.05 mg/ml (0.83 µM). The substrate pNPA was prepared in CH3CN:H2O (50:50 v/v) mixtures and its concentration in the assay mixture was 1×10-3 M (5% CH3CN). The reaction starts by enzyme addition from a stock solution (0.2 mg/ml) to a thermostated solution of substrate in pure buffer or at different surfactant concentrations. The linear increase of absorbance at 348 nm due to pNP formation was then recorded as a function of time for 300 seconds; reaction rate of CRL, defined as moles of pNP formed per unit of time, was calculated from the slope of the initial linear curve of pNP concentration vs. time. When autohydrolysis of pNPA (pNP formation in the presence of surfactants but without enzyme in the cuvette) was detected, reaction rates were calculated by subtracting the reaction rates in the absence of enzyme at the enzyme-catalyzed hydrolysis rates in surfactant solution. Kinetic parameters kcat and KM in pure buffer and in presence of surfactants were obtained from the linear regression analysis of the double reciprocal Lineweaver-Burk plots in a range of substrate concentration between 1×10-4 M and 2.0 × 10-3 M. Regression coefficient was always higher than 0.99. All sets of experiments were reproduced at least three times and the differences between duplicates in each experiment were always below 5%.
RESULTS AND DISCUSSION Effect of cationic and anionic surfactants on CRL activity The effect of the anionic sodium dodecyl sulfate (SDS) and of the cationic cetyltrimethylammonium bromide (CTABr) were investigated to evaluate the effect of the head group surfactant charge on CRL activity. In fact, it is well known that the surfactant head group has a crucial role in protein-surfactant interactions because of strong ionic interactions between the surfactant polar groups and specific charged sites on the protein surface. 7 ACS Paragon Plus Environment
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Figure 1 shows profiles of enzyme activity in the presence of SDS and CTABr as a function of surfactant concentration. In this plot, as in the following ones, the enzyme activity is reported as the ratio between the reaction rate in the surfactant solution and in pure buffer (rsurfactant/rbuffer).
2.0 1.8 1.6 1.4
rsurfactant/rbuffer
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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1.2 1.0 0.8 0.6 0.4 0.2 0.0 0.000
0.002
0.004
0.006
0.008
0.010
[surfactant], M
Figure 1 – Effect of surfactant concentration on CRL activity (as rsurfactant/rbuffer) in 0.01 M phosphate buffer, pH 7.0 at 25.0 °C; [pNPA] = 1×10-3 M, [CRL] = 0.05 mg/ml (0.83 µM). () SDS, () CTABr.
Experimental data reported in Figure 1 showed that the CRL activity strongly depends on the charge of the surfactant head group. In fact, in the presence of SDS, the relative activity showed a bell-shaped trend as the additive concentration increased. Reaction rate reached a maximum at [SDS] = 2×10-3 M, with an enzyme activation of 1.72, and then decreased, but still remaining higher than the value in buffer. On the contrary, CTABr showed a strong deactivating effect and CRL lost more than 70% of its activity already at a concentration of 5×10-4 M, a value below the critical micelle concentration (CMC), i.e. 8.7×10-4 M; then, it remained almost constant as the surfactant concentration increased. The negative effect of CTABr could be explained considering that CRL have an isoelectric point located at pH 4.65, since its surface is characterized by the presence of 31 acidic and 18 basic amino acid 8 ACS Paragon Plus Environment
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residues51. In our experimental conditions (pH 7.0), lipase has an overall negative charge and the cationic head groups of CTABr can establish strong electrostatic interactions with the protein. This could lead to enzyme deactivation. In literature, it is reported that the activity of Rhizomucor miehei lipase is much lower in the presence of a cationic surfactant than in anionic or nonionic one52. Moreover, positively charged surfactants form a complex with the enzyme in solution at pH values both above and slightly below the isoelectric point of the lipase53 and the complex formation is due to both electrostatic and hydrophobic interactions. Nevertheless, the reduced enzyme activity may not be due to conformational changes of the protein, but to the binding of the surfactant with one or more lipase binding sites close to the active site.
Effect of zwitterionic surfactants on CRL activity: Carboxy- and sulfobetaines An interesting category of surfactants, widely used in cosmetic for hair and skin care and as biological carriers for hydrophobic drugs, is the zwitterionic one. This class is characterized by the presence of both a positive and a negative charge, that renders the molecule overall neutrally-charged at neutral pH. Some types of zwitterions are susceptible to pH changes in a solution and may become completely cationic or anionic in acidic or basic environments. The positively-charged portion in these molecules is typically a quaternized ammonium ion, while the negatively-charged portion can be a sulfate, a carboxylate, or a sulfonate. Two betaines having the same alkyl chain, but that differ for both the type of anionic group and for the intercharge
distance
(dodecyldimethylammonium
methanecarboxylate
(CB1-12)
and
dodecyldimethylammonium propanesulfonate (SB3-12)), were studied. The effect of betaine concentration on CRL activity is reported in Figure 2 (the enzyme activity is reported as the ratio between the reaction rate in the surfactant solution and in pure buffer, rsurfactant/rbuffer). 9 ACS Paragon Plus Environment
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2.5
2.0
rsurfactant/rbuffer
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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1.5
1.0
0.5
0.0 0.00
0.02
0.04
0.06
0.08
0.10
[surfactant], M
Figure 2 – Effect of surfactant concentration on CRL activity (as rsurfactant/rbuffer) in 0.01 M phosphate buffer, pH 7.0 at 25.0 °C; [pNPA] = 1×10-3 M, [CRL] = 0.05 mg/ml (0.83 µM). () CB1-12, () SB3-12.
As it is clearly showed in Figure 2, there was a positive effect of both CB1-12 and SB3-12 on enzyme activity with a similar behaviour. In fact, CRL activity showed a bell-shaped trend, a two-fold enzyme superactivity occured at the same surfactant concentration, equal to 5×10-3 M, and it was rapidly weakened at betaine concentrations higher than 5×10-2 M. Therefore, in this case, the nature of betaine head group appeared to have no effect on the interaction between the additive and the enzyme.
Effect of zwitterionic surfactants on CRL activity: amine oxide Amine oxides are a particularly interesting class of zwitterionic surfactants because of their small but highly polar head group and the ability to protonate it by varying the pH, leading to cationic species. The pKa of amine oxide surfactants in bulk solution is about 5, therefore in pure water a mixture of neutral and ionic amine oxide molecules are expected to be present50.
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Moreover, surfactants possessing smaller head group area are favored to form cylindrical rod-like micelles, and their mean aggregation number is sensitive to the total surfactant concentration. Therefore, they can form long flexible aggregates that exhibit viscoelastic properties and this feature depends on monomer structure. For these reasons, several amine oxide surfactants with different alkyl chain length and head group size were chosen: dodecyldimethylamine N-oxide (AOMe-12), tetradecyldimethylamine
N-oxide
(AOMe-14),
esadecyldimethylamine
N-oxide
(AOMe-16),
tetradecyldiethylamine N-oxide (AOEt-14) and tetradecyldipropylamine N-oxide (AOPr-14). These surfactants have a very similar molecular structure and the effect of slight structural changes on the hydrolysis rate of p-NPA catalyzed by CRL was therefore evaluated. In this case, unlike the other additives considered in this work, amine oxide surfactants showed a moderate nucleophilic power. Then, relative reaction rates (rsurfactant/rbuffer) were calculated by subtracting the reaction rates in the absence of enzyme at the enzyme-catalyzed hydrolysis rates in surfactant solution (rsurfactant) and the obtained data was divided by the reaction rate in pure buffer. In Figures 3 the effect of hydrocarbon chain length of amine oxide surfactants on CRL activity (as rsurfactant/rbuffer) is reported.
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4.5 4.0 3.5 3.0
rsurfactant/rbuffer
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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2.5 2.0 1.5 1.0 0.5 0.0 0.00
0.02
0.04
0.06
0.08
0.10
[surfactant], M
Figure 3 – Effect of surfactant concentration on CRL activity (as rsurfactant/rbuffer) in 0.01 M phosphate buffer, pH 7.0 at 25.0 °C; [pNPA] = 1×10-3 M, [CRL] = 0.05 mg/ml (0.83 µM). () AOMe-12, () AOMe-14, () AOMe-16.
As with the other zwitterionic surfactants, a rapid increase in the relative activity at low amine oxide concentrations was observed, with higher activation with longer alkyl chains. In fact, the effect of AOMe-12 was very similar to those observed with CB1-12 and SB3-12, with a maximum at a concentration of 5 ×10-3 M, at which the enzymatic activity was twice compared to that in pure buffer. The chain elongation caused a further increase in the relative activity and the superactivity reached 3.6 in 5×10-3 M AOMe-14 and 4.2 in 2×10-3 M AOMe-16. At higher surfactant concentrations, a decrease in enzyme activity was observed, but in the presence of 0.1 M AOMe-14 the enzyme activity remained higher than that in pure buffer, (rsurfactant/rbuffer = 1.5), while with AOMe-12 a deactivation was observed for concentrations greater than 0.05 M. As regards AOMe-16, an increase in the viscosity of the solution was attained at 6×10-3 M and an accurate and reproducible measurement of the reaction rate was not allowed.
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For these reasons, the effect of head group size was evaluated with amine oxide surfactants having a tetradecyl hydrocarbon chain, and the effect of AOEt-14 and AOPr-14 on CRL activity was compared with that obtained with AOMe-14. Results are shown in Figure 4, where the enzyme activity is again reported as rsurfactant/rbuffer.
4.0 3.5 3.0 2.5
rsurfactant/rbuffer
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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2.0 1.5 1.0 0.5 0.0 0.00
0.02
0.04
0.06
0.08
0.10
[surfactant], M
Figure 4 – Effect of surfactant concentration on CRL activity (as rsurfactant/rbuffer) in 0.01 M phosphate buffer, pH 7.0 at 25.0 °C; [pNPA] = 1×10-3 M, [CRL] = 0.05 mg/ml (0.83 µM). () AOMe-14, () AOEt-14, () AOPr-14.
Here again, differences in the surfactant head group did not change the relative rate trends. With both additives, in fact, the profiles showed a bell shape, even if the maximum activation was observed at different concentrations. In particular, in the presence of AOEt-14, the maximum of activity (2.7-fold) was obtained at additive concentration of 5×10-3 M, while in 2×10-3 M AOPr-14 enzyme activity was 2.3 times higher than in buffer. Anyway, enzyme activity lowered as the head group size increase. All the amine oxides used in this work showed a superactivation of lipase and the concentration at which the maximum activity occurred was greater than the corresponding CMC. It therefore seemed 13 ACS Paragon Plus Environment
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evident that, in order to best perform their activating action, they must be in the form of micellar aggregates. The reduction of activity observed at high surfactant concentrations could be attributed to substrate subtraction by micellar aggregates. Therefore, if on one hand micelles were required to increase the activity of lipase, on the other they decreased the amount of substrate available for the enzymatic reaction. The collected results indicated that slight changes on the monomer structure can significantly affect enzyme-surfactant interactions and consequently enzyme activity. We therefore thought to correlate the hydrophobicity of the surfactant with the induced superactivity. The hydrophobicity of surfactants can be evaluated by their both critical micelle concentration value (CMC) and partition coefficient (logP). The tendency of amphiphilic molecules to form micelles in aqueous solution is a consequence of the hydrophobic effect and then CMC decreases with increase in hydrophobicity of surfactant molecules. The logarithm of the 1-octanol/water partition coefficient (logP) is a well-known measure of molecular hydrophobicity and several useful computational methods for estimating logP values of organic compounds were developed. Here, the Ghose-Crippen-Viswanadhan approach (AlogP), one the most widely methods of predicting partition coefficient54,55, was used to calculate the partition coefficient of the amine oxide surfactants. For each compound, logP was calculated by using the software Dragon56 as the sum of the number of all atoms, multiplied by their corresponding hydrophobicity constants. The relative hydrolysis rate catalyzed by CRL, at the surfactant concentration at which superactivity is obtained, was therefore correlated with the hydrophobicity of surfactants (Table 1).
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Table 1 – Correlation between surfactant hydrophobicity and enzyme superactivity.
a
Amine oxide
CMC, Ma
AlogP
rsurfactant/rbuffer
AOMe-12
1.2 ×10-3
3.047
2.05
AOMe-14
1.4×10-4
3.960
3.60
AOMe-16
2.5×10-5
4.872
4.21
AOMe-14
1.4×10-4
3.960
3.60
AOEt-14
1.1×10-4
4.657
2.71
AOPr-14
5.4×10-5
5.705
2.33
in water at 25 °C.
As expected, both CMC and AlogP values seemed to indicate that hydrophobicity increases with increasing the length of the surfactant chain and the size of the head group. However, this increase showed opposite effects on the enzyme activity. Indeed, CRL superactivity doubled passing from AOMe-12 to AOMe-16; on the other hand, it decreased as the head group size increases, being 3.60 and 2.33 in AOMe-14 and AOPr-14, respectively. To explain these results, we hypothesized that the shape of colloidal aggregates affected lipase structure and activity and the morphological changes from spherical to rod-like micelles improved its catalytic properties. As already mentioned, amine oxide surfactants exist as either a nonionic or a cationic species and short-range attractive interactions (hydrogen bonds) between deprotonated and protonated forms remarkably affects the structure of the aggregate. Indeed, intermolecular hydrogen bonding could decrease the area of the head group and the dimers thus formed behave like double chain amphiphiles57. Sphere-rod transition of surfactant micelles is clearly affected by the hydrocarbon chain
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length and the longer the chain, the greater the tendency to form rod-like structures. Therefore, AOMe12 forms spherical aggregates, while AOMe-14 and AOMe-16 are favored to form rod-like micelles. A bulky head group increases the distance between polar head groups, thus weakening the hydrogen bond between the monomers; consequently, it can be hypothesized that, even in the presence of a long alkyl chain, AOEt-14 and AOPr-14 prefer spherical aggregates. Other experimental evidences confirm that an increase in the alkyl residues of the head group can influence the ability of morphological growth from spherical micelles towards rod-like micelles. In the case of cationic surfactants of the family of cetyltrialkyl ammonium bromide (CTRABr; R = methyl, ethyl, n-propyl, n-butyl) the gelling induced by the trans-o-methoxycinnamate (trans-OMCNa) anion is strongly reduced by the increase in alkyl residues (R). In fact, while the CTABr / trans-OMCNa system forms worm-like micelles, the substitution of the methyl groups with the other alkyl groups produces an immediate reduction in the viscosity of the solution58. Similar observations emerged with zwitterionic surfactants of the p-dodecyloxybenzyldialkylamine Noxide family (pDoRAO; R = methyl, ethyl, n-propyl, n-butyl). While the surfactant p-DoAO (R = methyl) forms viscous aqueous solutions already at 0.01M concentration, the substitution of the methyl groups with more voluminous groups drastically reduces the viscosity of the solution at the same surfactant concentration (unpublished results).
Effect of p-dodecyloxybenzyldimethylamine oxide (pDoAO) on CRL activity As previously stated, the amine oxide surfactants give rise to aggregates sensitive to the increase in concentration, since they lead to an increase in the size due to the sphere-rod transition, without adding any type of additive. The zwitterionic surfactant p-dodecyloxybenzyldimethylamine N-oxide (pDoAO) has the ability to form highly viscous solutions at relatively low concentrations. This behavior could be due to strong 16 ACS Paragon Plus Environment
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interactions among the tails and its phenyl group plays an important role in the aggregation by π-π stacking. It has been reported that small micelles grow in length (wormlike or threadlike micelles) as pDoAO concentration increases and, at high surfactant concentrations, large aggregates and smaller micelles coexist. Moreover, worm-like micelles are present already at low surfactant concentrations, and the entanglement starts at higher concentrations, when the system gets viscoelastic59. The introduction of an aromatic residue in the hydrophobic moiety induces a large decrease of the CMC with respect to AOMe-12 (1.6×10-5 M vs. 1.2 ×10-3 M) and it is very similar to that of AOMe-16 (2.5×10-5 M), indicating its high tendency to form colloidal aggregates. The effect of pDoAO concentration on CRL activity (as rpDoAO/rbuffer) was investigated and results are showed in Figure 5. 3.0
2.5
2.0
rpDOAO/vbuffer
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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1.5
1.0
0.5
0.0 0.000
0.002
0.004
0.006
0.008
0.010
[pDoAO], M
Figure 5 – Effect of pDoAO concentration on CRL activity (as rpDoAO/rbuffer) in 0.01 M phosphate buffer, pH 7.0 at 25.0 °C; [pNPA] = 1×10-3 M, [CRL] = 0.05 mg/ml (0.83 µM).
Even in the presence of pDoAO, CRL activity showed a bell-shaped trend, and the maximum of activity (2.7-fold) was attained at a concentration of 5 ×10-3 M. At surfactant concentration higher 8 17 ACS Paragon Plus Environment
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×10-3 M, the solution became too viscous and it is no longer possible to obtain reproducible measurements of the reaction rate.
Determination of kinetic parameters To attain a deeper understanding of the activating effect produced by amine oxides, the kinetic parameters of CRL were determined. Measurements were performed at the additive concentrations that induced the maximum of superactivity. All data points obeyed to Michaelis-Menten kinetics and could be correlated in the Lineweaver-Burk plot for an estimation of the kinetic parameters, reported in Table 2.
Table 2 - Effect of amine oxide surfactants on kinetic parameters of CRL in 0.01 M phosphate buffer solution, pH 7.0 at 25.0 °C. [CRL] = 0.05 mg/mL (0.83 µM).
103 KM, M
kcat, s-1
kcat(surfactant)/kcat(buffer)
Pure buffer
2.08
0.57
−
AOMe-12
1.04
0.80
1.40
AOMe-14
1.42
1.52
2.67
AOMe-16
1.75
1.99
3.49
AOMe-14
1.42
1.52
2.67
AOEt-14
2.83
1.89
3.32
AOPr-14
4.28
1.91
3.35
pDoAO
1.13
1.02
1.79
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Regardless of the surfactant used, it is evident that enzyme activation was mainly due to an increase in the turnover number (kcat) and, in some cases, to an increase in the enzyme-substrate affinity (lowering of KM). The addition of surfactant to the buffer increased the hydrophobicity of the reaction microenvironment and this greater lipophilicity may explain the observed effects on enzyme activity. In fact, the first step in CRL catalysis involves the nucleophilic attack of the hydroxyl group of Ser 209 to the carbonyl carbon on the ester substrate to form an enzyme-substrate tetrahedral intermediate and a more hydrophobic microenvironment enhances the nucleophilicity of the catalytic serine residue. Moreover, the enzyme interaction with a hydrophobic phase can cause the opening of the lid, making the active site accessible. As regards the enzyme-substrate affinity, both the lengthening of the alkyl chain and the increase of the head group size enhanced KM value. In the presence of alkyldimethylamine oxide, the enzyme affinity for the substrate was always higher than in pure buffer, even if the lengthening of the chain produced an increase in KM. This trend could be explained with the different tendency of surfactants to form micelles. In fact, as previously reported in Table 1, the longer the alkyl chain the lower the CMC and then, at the same surfactant concentration, the higher the number of micellar aggregates. Micelles seemed to have a dual role: on one hand, they activate the enzyme, on the other they could partially subtract the substrate making it not available for catalysis. It is therefore evident that in AOMe-16 solution (CMC = 2.5 × 10-5 M) the amount of free substrate could be much lower than that in AOMe12 solutions (CMC = 1.2 × 10-3 M), with a consequent increase in KM. Kinetic parameters measured at 5 × 10-2 M AOMe-14 confirmed this hypothesis: kcat remains unchanged if compared to the value at 5 × 10-3 M concentration, while KM is almost doubled, being 2.6 × 10-3 M vs. 1.42 × 10-3 M, due to a greater subtraction of the substrate by micellar aggregates. With surfactants with the same chain length, i.e. tetradecyl, the enzyme superactivity decreased by increasing bulk hydrophobicity of alkyl head groups, following the series methyl < ethyl < n-propyl; 19 ACS Paragon Plus Environment
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this was due to the increase in KM, which in AOPr-14 doubles compared to the buffer. Therefore, the lowering of enzyme superactivity with the increase of the head group size, shown in Figure 4, can be ascribed to a real low affinity of CRL for the substrate. Indeed, in this case, the difference between the CMC of AOMe-14 and AOPr-14 was much smaller (≈ 2.5 times), if compared to that between AOMe12 and AOMe-16 (≈ 500 times) and then the amount of free substrate available for the reaction should be very similar. It was evident that slight changes on the molecular structure of the surfactant can greatly influence CRL catalytic properties. Finally, the activation induced by pDoAO was due both to an increase in enzyme-substrate affinity and to a higher turnover number, since KM and kcat were approximately half and twice of those obtained in buffer. It was therefore evident that the presence of the aromatic residue in the hydrophobic chain did not significantly influence the catalytic properties of CRL.
CONCLUSIONS The present study showed that the activity of lipase from Candida rugosa can be significantly modulated by selecting suitable additives with certain structural features. In fact, the enzyme hydrolytic activity was enhanced in the presence of all the zwitterionic surfactants selected, and the effect of several amine N-oxide showed that slight changes on the monomer structure significantly affected enzyme-surfactant interactions, and consequently enzyme superactivity. In particular, enzyme activity increased by lengthening the alkyl chain of the surfactant and it lowered as the head group size increased. These results were interpreted assuming that the hydrolytic activity of lipase was improved by the sphere-rod transition of N-oxide micelles. Amine N-oxides can, indeed form rod-like structures and this feature depends on monomer structure: the longer the chain, the greater the tendency to form cylindrical aggregates; on the other hand, increasing the distance between the polar head groups, as in the presence of bulky head group, the monomers are expected to form spherical aggregates. 20 ACS Paragon Plus Environment
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Kinetic parameters showed that the enzyme activation was mainly due to an increase in the turnover number, probably due to a more hydrophobic microenvironment; this enhanced the nucleophilicity of the catalytic serine residue and could have caused the opening of the lid, making the active site accessible to the substrate. The decrease of enzyme-substrate affinity by lengthening the alkyl chain could be attributed to substrate subtraction by micellar aggregates, while its reduction increasing the surfactant head group size could be ascribed to a real low affinity of lipase for the substrate, since the amount of free substrate did not significantly change. More experiments are currently in progress to better understand the role of amine N-oxide molecules in controlling the activity of lipase from Candida rugosa and to further clarify the mechanism of superactivation of the enzyme achieved.
Acknowledgments. This work was supported by the Ministry of Education, Universities and Research (MIUR): project Smart Cities and Communities and Social Innovation on Cultural Heritage (SCN_00520). We are also grateful to Prof. Angelo Antonio D’Archivio for the calculation of the molecular descriptors by means of Dragon Software.
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