Nanoencapsulated Lecitase Ultra and Thermomyces lanuginosus

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Nanoencapsulated Lecitase Ultra and Thermomyces lanuginosus lipase, a comparative structural study. Karen M. Gonçalves, Ivaldo Itabaiana, Vassiliki Papadimitriou, Maria Zoumpanioti, Ivana C. R. Leal, Rodrigo Octavio Mendonça Alves de Souza, Yraima Cordeiro, and Aristotelis Xenakis Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b00826 • Publication Date (Web): 13 Jun 2016 Downloaded from http://pubs.acs.org on June 22, 2016

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Nanoencapsulated LecitaseTM Ultra and Thermomyces lanuginosus lipase, a comparative structural study. Karen M. Gonçalves1, Ivaldo Itabaiana2, Vassiliki Papadimitriou3, Maria Zoumpanioti3, Ivana C. R. Leal1, Rodrigo O.M.A. de Souza2, Yraima Cordeiro1*, Aristotelis Xenakis3* 1

Faculty of Pharmacy, Federal University of Rio de Janeiro, RJ, 21941-902, Brazil; 2Biocatalysis and Organic Synthesis Group, Chemistry Institute, Federal University of Rio de Janeiro, RJ, 21941-909, Brazil; 3Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, Athens, Greece.

KEYWORDS Lipases, reverse micelles, Small Angle X-ray Scattering (SAXS), Fluorescence, Electron Paramagnetic Resonance (EPR), Dynamic Light Scattering (DLS).

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ABSTRACT

Two commercially available and widely used enzymes, the parent Thermomyces lanuginosus lipase (TLL) and the shuffled phospholipase A1 LecitaseTM (Lecitase Ultra), were encapsulated in AOT/isooctane reverse micelles and evaluated regarding their structure and activity. Preparations were also tested as effective biocatalysts. Small-angle X-ray scattering (SAXS), electronic paramagnetic resonance (EPR) and fluorescence spectroscopy were the techniques applied to assess the effects of enzyme incorporation to a reverse micellar nanostructure. SAXS analysis showed that the radius of gyration (Rg) changed from 16 to 38 Å, as the water content (w0) increased. Elongated shapes were most commonly observed than spherical shapes after enzyme encapsulation. EPR studies indicated that enzymes do not participate in the interface, being located in the aqueous center. Fluorescence energy transfer showed that TLL is located in the water core, whereas Lecitase Ultra is closer to the interface. Enzymatic activity towards a standard esterification reaction endured after the enzyme was incorporated into the micelles. The activity of TLL for systems with w0 15 showed the highest conversion yield, 38% in 2 h, while the system with w0 10 showed the highest initial velocity, 0.43 µM/min. This last system had a Rg of 19.3 Å, similar to that of TLL monomer. Lecitase Ultra showed the highest conversion yields in systems with w0 10, 55% in 2 h. However, the initial rate was much lower than that of TLL, suggesting less affinity for the substrates, which is expected since Lecitase Ultra is a phospholipase. In summary, we here used several spectroscopic and scattering techniques to reveal the shape and stability of TTL and Lecitase Ultra encapsulated systems, which allowed the selection of w0 values to provide optimized enzymatic activity.


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INTRODUCTION Biocatalysis in nano-scale environments with restricted water content, such as water-in-oil (w/o) microemulsions or reverse micelles, is one of the approaches introduced to handle enzymes in non-conventional media. Microemulsions are ternary systems composed by mixtures of oil (or non-polar organic solvents), water and surfactants. They present polar and non-polar microdomains separated by a surfactant monolayer which offers the advantage to solubilize or encapsulate both hydrophilic and lipophilic substances 1. This condition allows a broad field of applications especially in biotechnology, solving a long-known challenge: the use of enzymes in nonaqueous medium 2. A variety of enzymes have been used inside reverse micelles, however the protein most adapted to this environment is lipase due to its activation by the lipid/water interface 1. Both the activity and structure of lipases from different sources were studied when hosted in various such microemulsions 1. Here we evaluate the differences between two related commercial enzymes widely applied in industry, namely Thermomyces lanuginosus lipase (TLL) and phospholipase LecitaseTM Ultra encapsulated in AOT/isooctane reverse micelles. TLL, a thermostable enzyme with high activity in the range of 20-50 ºC, is widely applied in lipid hydrolysis, triglycerides modification and in diacyl- and monoacyl-glycerol synthesis

3-5

and was reported to have co-existing monomeric and dimeric stable populations in solution 6. TLL has important applications in industry, being used in powder detergents for washing clothes, since lipase facilitates the removal of grease stains 7. In addition, its characteristics are also advantageous for other applications such as the production of biodiesel, resolution of racemic mixtures, pesticide synthesis, chemo-enzymatic route of drugs and in production of food


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ingredients such as glycerides for margarine and structured lipids with functional properties 5. In such cases, the enzyme is preferably used in immobilized form. On the other hand, Lecitase Ultra is an interesting commercially available enzyme displaying both lipase and phospholipase A1 properties in a single active site 8. It is artificially obtained from the fusion of lipase genes from TLL and phospholipase genes from Fusarium oxysporum, cloned and expressed in relevant fungal strains

9

undergo interfacial activation similarly to lipases

. This shuffled enzyme has been described to 10

. Lecitase Ultra stability is similar to TLL,

being hyper-activated by some surfactants and recognizes different esters 11. This phospholipase, initially developed for degumming process, can be used as an enantioselective biocatalyst in fine chemistry

11

being very useful for the preparation of enantiomerically pure commercially

important intermediates 8. When immobilized, Lecitase Ultra promotes the esterification of food waste streams in monoacylglycerols giving high conversion yields 12. Its catalytic properties can be modified by distinct immobilization protocols 13, increasing number of possible applications. In this work we conducted structural studies of the above mentioned lipase and related phospholipase encapsulated in reverse micelles and evaluated how the size, shape and enzyme location in the micelles can determine the validity of the catalytic system. Using Dynamic Light Scattering (DLS) we obtained information on the size and size distribution of the reverse micelles upon enzyme encapsulation. Small Angle X-Ray Scattering (SAXS) provided information about size, shape and homogeneity. Interfacial properties of the surfactants monolayer in reverse micelles were studied by electron paramagnetic resonance (EPR) spectroscopy using an adequate spin probe. EPR using a polar spin probe was applied to detect changes in the aqueous phase upon enzyme encapsulation.


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Finally, steady state fluorescence spectroscopy using enzymes as naturally occurring fluorophores provided information on the location of the enzyme in the microemulsion. Moreover, specific information concerning the topology of the enzyme in relation to the various microphases of the system can be retrieved by applying the fluorescence energy transfer technique 14.


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EXPERIMENTAL SECTION Materials. Enzymes: Lecitase Ultra and Thermomyces lanuginosus lipase were obtained by Novozyme. The surfactant sodium bis-(2-ethylhexyl) sulfosuccinate (AOT) and oleic acid 99% were from Alfa Aesar, spectroscopic grade isooctane from Merck, 3-(4-nitrophenoxy carbonyl)– proxyl and 5-doxyl stearic acid (5-DSA) probes were obtained from Sigma-Aldrich. LTryptophan was supplied by BHD biochemicals. 99% cis-parinaric acid ([9.11.13.15]cis,trans,trans,cis-octadeca-tetraenoic acid or PNA) was obtained from Molecular Probes (Invitrogen). Stock solutions of PNA in AOT microemulsions were prepared and stored at -20 °C. Methods Reverse micelles preparation. A stock solution of AOT/isooctane 0.2 M was prepared and the reverse micelles were formed by the addition of the appropriate amount of enzyme solubilized in 0.2 M Tris/HCl buffer, pH 7.5. The final quantity of enzyme in the systems was ~ 60 µg of protein per milliliter of microemulsion for all w0 values (5 to 20) and for both enzymes. The water content was calculated as w0 (w0 = [H2O]/[AOT]). The systems were prepared until the limit of w0 20, when the systems were completely transparent. The addition of higher water or enzyme content promoted phase separation. The calculated volume fractions for the systems were observed from 0.014 to 0.072 (1.4 to 7.2%), increasing with water addition or w0 ratio. Enzymatic activity tests. For the activity tests, oleic acid (50 mM) and ethanol (100 mM) were added at a stock AOT/isooctane solution. The reactions were performed at 40 °C after enzyme addition, without stirring; the conversion was calculated by the Lowry-Tinsley method 15 using isooctane as a solvent.


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Dynamic light scattering (DLS). The hydrodynamic radius (Rh) of the reverse micelles was evaluated using the DynaPro Nanostar (Wyatt, USA) with a 630 nm laser. In quartz cuvette, 50 µL of the samples were added and maintained at 25 °C. The information was collected three times, being ten cumulants to each. For the reverse micelles systems containing enzymes, the analyses were done at distinct temperatures. The information was collected using ten cumulants acquired every 5 ºC, from 25 to 75 ºC. The size results correspond to the mean of the three measurements including all ten cumulants for each. Population distributions were analyzed by the correlation function graphs. The size distribution function is generated using the regularization algorithm (Dynamics software), which makes no assumptions regarding the number of size populations. In regularization, it is assumed that the underlying distribution of Rh is smooth. The size distribution graph expresses the individual populations identified with their specific polydispersity values. In Table 1, the data was numerically represented as the Rh average from all the populations identified in each w0 system. Small Angle X-Ray Scattering (SAXS). SAXS experiments were performed at the SAXS1 beamline of the Brazilian Synchrotron Light Laboratory (CNPEM, LNLS, Campinas, Brazil). A monochromatic X-ray beam was used (λ = 1.55 Å) with the sample to detector distance set at ~ 974 mm. Scattering curves were recorded within the q range from 0.015 to 0.347 Å-1. q = 4πsinθ/λ is the scattering vector, being 2θ the scattering angle. Scattering data were recorded (each frame collected for 30 s) using a two-dimensional position-sensitive MARCCD detector. The SAXS curves were corrected by buffer (Tris/HCl 0.2 M, pH 7.5) scattering taking into account the sample’s attenuation. Samples monodispersity was confirmed by analysis of the Guinier plots and data were corrected properly and fitted using GNOM

16

, which also provided

the pair-distance distribution functions (p(r)).


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Electron Paramagnetic Resonance (EPR) spectroscopy. EPR spectra were recorded at room temperature using a Bruker EMX spectrometer operating at the X-band. Samples were contained in a WG-813-Q Wilmad Suprasil flat cell. Typical instrument settings were: center field, 0.348 T; scan width, 0.01 T; gain, 2.83x103; time constant, 163 ms; conversion time 5 ms; modulation amplitude, 0.4 mT; frequency, 9.76 GHz. Data collection and analysis were performed using the Bruker WinEPR acquisition and processing program. Two different nitroxide spin probes, 5-doxyl stearic acid (5-DSA) and 3-(4-nitrophenoxy carbonyl)–proxyl (Figure 1), were employed to study different regions of the reverse micellar systems. The chemical structures of the spin probes are shown in Figure 1. Stock solutions of the spin probes were prepared as follows: a) 5-doxyl stearic acid (5-DSA) was added in ethanol to give a final concentration of 10-2 M and b) 3-(4-nitrophenoxi carbonyl)–proxyl was added in 0.2 M Tris/HCl (pH 7.5) buffer to give a 2.6 x 10-2 M solution. To obtain the desired concentration of the spin probes in the AOT microemulsions, two different experimental procedures were followed. In the case of 5-DSA, a certain amount of the stock solution was added in a test tube. After solvent evaporation, 1 mL of each microemulsion was added to yield an overall spin probe concentration of 10-4 M. In the case of the hydrophilic spin probe 3-(4-nitrophenoxy carbonyl)–proxyl, the requisite amount of the stock solution was added in the aqueous phase of the reverse micelles to yield an overall concentration of 2.6 x 10-4 M. Experimental results were analyzed in terms of rotational correlation time (τR) and order parameter (S), as described in detail elsewhere 17,18.


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Figure 1. Chemical structures of (A) 5-doxyl stearic acid and (B) 3-(4-nitrophenoxy carbonyl)– proxyl. Steady state fluorescence spectroscopy. In order to monitor the steady state fluorescence emission spectra, a series of AOT microemulsions was prepared with constant AOT concentration (0.2 M) and different w0 values. The aqueous phase of the system was constituted by Tris-HCl buffer at pH 7.5 containing the enzymes. The measurements were made right after the enzyme addition. The excitation wavelength was 280 nm while the emission was monitored between 300 and 400 nm. All experiments were carried out at 25 °C. The fluorescence emission spectra were monitored using a Jasco FP 6300 fluorometer (Jasco Corp., Japan). Fluorescence energy transfer. Energy transfer was examined by measuring the effect of the concentration of parinaric acid (PNA) on enzyme fluorescence. To obtain the desirable concentrations of PNA, appropriate amounts of stock solutions AOT/isooctane/PNA and AOT/isooctane were previously mixed. This procedure was carried out in a series of microemulsions with w0 values ranging from 5 to 20 and for constant AOT concentration at 0.2 M. The concentration of PNA ranged from 0.9×10−5 to 3.6×10−5 M. The concentration of the lipase was kept constant (0.5 µg/ml of microemulsion). The effect of the PNA concentration on the energy transfer efficiency has been studied for the lipases as well as for the free tryptophan. The energy transfer efficiency (T) between the donor-acceptor pair was determined from the


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reduction of the donor (tryptophan residues or free tryptophan) fluorescence intensity in the presence of different concentrations of acceptor (PNA), as described by Zoumpanioti and coauthors19.


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RESULTS AND DISCUSSION Dynamic Light Scattering A DLS experiment allows the size determination of the reverse micelles, is able to identify populations with distinct size distributions, and can reveal the presence/formation of aggregates. The autocorrelation function represents the laser interaction with the particles in movement inside the solution per time and can reveal the size distribution of the samples, identifying two or more populations with almost the same size. The scattering autocorrelation function of the empty reverse micelles (Figure S1, A) showed an increase in the autocorrelation intensity upon water addition, indicating changes in the size of the micelles. In this case, the addition of buffer resulted in the formation of micelles with higher diameters, as expected. On the other hand, for the reverse micelles containing enzyme, the autocorrelation function indicated the presence at least of two distinct populations. This was clearly observed by two different decay times, for TLL in the system with w0 5 and 10 and for Lecitase Ultra at w0 20 (Figure S1, B and C, respectively). This behavior was not evidenced for the empty reverse micelles (Figure S1), suggesting a size change due to protein addition. Longer decay times as present in the autocorrelation function of these systems (Figure S1, B and C) are indicative of different populations with large species or the presence of aggregates in the sample. This condition could be a consequence of non-incorporating enzymes, suffering aggregation outside the micelle, or of an interaction between droplets. Reverse micelles containing Lecitase Ultra and TLL at w0 8 presented an autocorrelation function with longer decay times indicating the presence of a polydisperse population with higher mean Rh than the micelles with other w0 values (Figure S1). This condition was clearly


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observed in the size distribution graphs (Figure S2, B and C) taking into account the polydispersity values of each observed population. Therefore, the polydispersity of the samples (Table 1) corroborates the presence of two or more populations, indicating a dynamic system that can be composed of both protein-containing micelles and empty ones 20. The enzyme addition induces changes in the size of the micelles as revealed by the reduction in polydispersity values for the w0 15 and 20 systems (Table 1), indicating that this conditions allow better accommodation of the enzymes inside the micelles. Table 1. Mean Rh and polydispersity values corresponding to the hydrodynamic radius of AOT microemulsions for empty systems or systems containing TLL or Lecitase Ultra with different water content (w0)

Hydrodynamic radius, Rh (Å)

Polydispersity (%)

w0

buffer*

TLL

Lecitase Ultra

5

17 ± 2

17 ± 1

15 ± 2

8

21 ± 1

21 ± 2 and 9 ± 1a

18 ± 3

10

23 ± 2

23 ± 1

20 ± 1

15

22 ± 2

23 ± 1

23 ± 2

20

26 ± 1

25 ± 1

24 ± 2 and 15 ± 2a

5

5.9

Multimodal

Multimodal

8

2.8

Multimodal

Multimodal

10

13.0

Multimodal

23.9

15

19.9

18.4

13.0

20

19.8

5.8

9.9

a

*empty systems without protein. Rh values for the two identified populations. Rh values shown were calculated from three independent measurements. The polydispersity values shown were calculated based in all species present


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in the size distribution graphs (regularization analysis) (Figure 2, Fig. S2 and S3). The polydispersity of individual peaks were significantly lower than those shown in the Table 1.

In this case, the effect of protein incorporation at low water content (w0 5 and 8) could lead to aggregation between two or more empty droplets (system with buffer, but without protein) to accommodate an enzyme

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. This condition would support the longer decay times observed in

the autocorrelation functions in comparison to the other w0 values (Figure S1, B and C). At w0 10 and 15 with Lecitase Ultra and w0 15 and 20 with TLL (Figure S1, B and C) only one decay time was seen, indicating the presence of one population or of species with very close Rh values. This interpretation is supported by the polydispersity values (Table 1). For TLL, there is a main population, which corresponds to the reverse micelles, and a small group with higher Rh value that can be some contamination from the air or buffer solution (Figure 2). The results also indicate preference for micelle formation with comparable size to the protein as described by some authors

20,21

. The size of TLL monomer is approximately 19 Å6 and the

mean size observed in these systems showed Rh values from 15 to 26 Å (Table 1). The influences of water and enzyme addition in the AOT/isooctane solution is clear since the contributions of AOT molecules in reverse micelles size is around 11 to 12 Å, as described by De and Maitra 23.


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Figure 2. Size distribution graphs obtained from the DLS measurements of samples with w0 = 20. Polydispersity is determined as the width of each peak in the regularization graph. When normalized to the mean size of the peak in the histogram, is shown as percent polydispersity (%Pd). The Rh of the reverse micelles increased with the water addition for all systems studied, however the changes were not significant between w0 10 and 15, which could be explained by the AOT hydration that happens more specifically at w0 values between 10 and 18. At this point, twelve water molecules, in spite of three, are connected with the surfactant 23,24. The microemulsions containing Lecitase Ultra presented lower size than the empty ones in w0 values from 5 to 10. This reduction is probably related to an induction of attractive interactions within the droplets due to the participation of the protein at the interface or, to a minor extent,


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due to the presence of the lipase in the micelle center 21,22. At higher w0 value the enzyme seems not to interfere with the system and the polydispersity was reduced when compared with the empty ones. For the reverse micelles containing TLL, no significant changes in terms of mean size were observed compared to the empty ones, despite the increases in polydispersity and the presence of distinct populations. This fact suggests that the lipase does not interfere significantly with the size of the system, indicating that the TLL is located inside the center 20,21. The sizes of w0 15 and 20 were almost the same observed to the enzyme monomer.

Temperature effect on reverse micelles size Mass transfer inside the system is essential to allow the employment of a certain system in biocatalysis. In order to further understand how different parameters could interfere in the mass transfer inside the systems, we initially evaluated how changes in temperature could interfere in the size of droplets of the enzyme-containing micelles. This was observed by DLS at different w0 values. Some authors

23,25,26

already reported increases in the attractive forces between the

droplets with the increment of temperature. In these cases, there are most enduring collisions. This phenomenon was described as percolation; the interconnection between the droplets being a consequence of Van der Waals forces

21

. Gochman-Hecht and Bianco-Peled

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suggested that

cylindrical structures are formed during this process, increasing the droplets size. The same phenomenon was described by Bardhan and coauthors 27, however it was related to the increase in the water content that promotes higher hydrodynamic radius, a condition clearly shown by the DLS analysis.


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It was proposed that the effects of temperature in these AOT systems are related with the reduction of AOT affinity for the organic phase

28,29

, indicating a transition of this surfactant to

the aqueous phase. Moreover, the dehydration of the polar head of AOT promotes the formation of cylindrical structures and the increase in the temperature can cause the penetration of solvent molecules in the lipophilic (apolar) AOT region 29.


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Figure 3. Temperature-induced changes in Rh values of empty reverse micelles (A) and of micelles containing TLL (B) and Lecitase Ultra (C) observed by DLS. As shown in Figure 3, for both enzymes the size of the reverse micelles increased with temperature. This phenomenon could be related to the formation of cylindrical structures in accordance to the results discussed above

26

. The importance of this event is directly related to


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mass transfer, which could be favored promoting biocatalytic reactions more efficiently. Percolation was already described in reverse micelles systems containing alcohol and was evidenced by the changes in the conductivity 30. Despite the importance of this behavior in mass transfer, it is important to notice that high temperatures applied in these systems can compromise enzymatic stability and modify interactions between the surfactant and the lipase, thus decreasing its activity. Knowledge about the reverse micelles behavior in high temperatures offers other alternatives to promote phase separations besides addition of water. An increase in the temperature of these systems above 80 ºC could be applied in the process of product extraction 21.

Small angle X-ray scattering (SAXS) SAXS measurements were carried out to obtain information about enzyme location in the system, about the sample dispersity/homogeneity, and to calculate the gyration radius (Rg) of reverse micelles under distinct w0 (Figure 4) and to obtain information on the overall shape of the reverse micelles. For all cases, the differences in the scattering profile were evident at low q (Å-1) values (Figure 4) indicating increase in Rg values upon changes in w0. Also, changes in the scattering curve in the cross section between 0.1 and 0.2 Å-1 were observed (Figure 4). In this case, the curve shifts towards the higher q-side upon addition of water showing modifications in the dimension of the reverse micelles and in the transversal section, suggesting more elongated structures in comparison with perfect spheres 29. Although DLS data showed that the systems are polydisperse at some w0 values (Table 1), increases in the radius of gyration (Rg) of the reverse micelles could be clearly observed through


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the Guinier regression, mainly because of a linear behavior at the low q region, for q2 (Å-2) values below 0.1 (Figure S4). Higher water content led to regressions with higher slopes (α), which means higher Rg values (α = Rg2/3) (Figure S4).


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Figure 4. SAXS profile (intensity x q) of the reverse micelles with different water content (w0) without enzymes (A) and with TLL (B) and Lecitase Ultra (C).

The radius of gyration (Rg) represents the second moment of the distance distribution of the particle around the center

31

and can be calculated by the pair-distance distribution function

(p(r)). The p(r) function is obtained when the scattering profile is submitted to an indirect Fourier transformation (IFT) calculated with the software GNOM

16

. The graphic of p(r) displays

information about the structure in real space 32, the particle shape 33, and it provides Rg values of the scattering particle. As described by Gochman-Hecht and Bianco-Peled

20

, the AOT reverse micelles tend to be

spheres, changing their shape to cylinders at high water content (w0>70) for empty micelles or for those containing small quantities of protein with high w0. However, in our study, both empty and reverse micelles with enzymes Lecitase Ultra and TLL seem to be more elongated, as observed in the scattering curves (Figure 4). This elongated shape was already observed by Hopkins and coworkers

34

that investigated the

transition in geometry by the addition of hydrotropes in AOT reverse micelles. The authors observed that the addition of hydrotropes with higher water solubility promoted transition of the reverse micelles from a cylindrical to an elongated ellipsoid shape in the AOT/heptane/water systems, converting to spheres only in high values of w0 (>30) 34. The normalized p(r) function reveals similar bell-shaped profiles (Figure 5), indicating that changes in the reverse micelles occurred in the size (Rg), but not in the shape of the droplets when the w0 changed.


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Figure 5. Graphic of the normalized pair-distance distribution function (p(r)) of the empty systems (A), or loaded with Lecitase Ultra (B) and TLL (C).

The Rg values of the reverse micelles at different water contents obtained from the p(r) were compared in the presence and in the absence of the enzymes. As observed in Table 2, the Rg of the systems increased upon water addition. In the cases of w0 5 and 8, there was no significant change between Rg obtained for empty and loaded with enzymes micelles, which could be


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explained by the equilibrium between droplets with and without protein that could happen after the combination of n droplets to accommodate the protein 20-22. The differences between the Rg were more pronounced at w0 20 when the systems containing protein were compared with the empty ones, which were larger. To explain this, Gochman-Hecht and Bianco-Peled20 showed that the number of micelles formed is not constant, being higher in the presence of protein. In agreement with the authors, since more micelles are formed and the AOT concentration was not altered, the reverse micelles tend to be smaller with the enzyme insertion. Besides, the polydispersion observed by DLS might be due to the presence of two types of droplets, with and without the hosted protein. Moreover, the fraction of smaller micelles containing protein is probably higher than that of the larger ones without protein.

Table 1. Radius of gyration values of reverse micelles measured by SAXS Rg from GNOM (Å)

Rg from Guinier (Å)

w0

buffer*

TLL

Lecitase Ultra

buffer*

TLL

Lecitase Ultra

5

16.20 ± 0.00

17.00 ± 0.00

16.10 ± 0.00

16.09

16.15

17.08

8

18.40 ± 0.00

18.30 ± 0.00

18.50 ± 0.00

19.74

19.10

16.21

10

19.30 ± 0.00

18.80 ± 0.00

18.80 ± 0.00

23.58

20.27

22.89

15

25.40 ± 0.00

24.30 ± 0.00

24.50 ± 0.00

30.88

30.02

27.92

20

38.70 ± 0.04

32.50 ± 0.01

31.10 ± 0.01

39.71

34.55

32.64

*empty system

The ratio between radius of gyration and hydrodynamic radius values (ρ = Rg/Rh) indicates the shape/compactness of the droplets (Table 3). A ratio value of 0.775 corresponds to solid spheres and higher values suggest cylindrical or elongated systems

35,36

. In this study, according

to our results of the p(r) analyses, the systems seem to be an ellipse.


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Table 2. Ratio between the gyration and hydrodynamic radius of the reverse micelles measured by DLS and SAXS Rg/Rh (ρ) w0

buffer*

TLL

Lecitase Ultra

5

0.95

1.00

1.15

8

0.88

0.80

1.03

10

0.88

0.82

0.94

15

1.10

1.06

1.07

20

1.49

1.30

1.41

*empty system

The micelles did not present a spherical profile (Table 2) even in the systems without protein (Table 3, buffer). This affirmative can be reinforced by the values of the ratio Dmax/2 that were different from the Rg values obtained from the SAXS measurements of the reverse micelles. Besides, the Dmax/Rg ratio was higher than 2 for all systems (Table S1)33, indicating that the systems are elongated, with either prolate or oblate shapes

33

. If the micelles were symmetrical

spheres, the Dmax/Rg ratio would be 2. TLL insertion promoted reduction in the ρ value, meaning an increase in the curvature of the droplets that tend to be less elongated than the empty systems. On the contrary, Lecitase Ultra caused a formation of more elongated droplets, suggesting a more extended structure than TLL.


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EPR measurements EPR spectroscopy using the spin-probing technique was employed in the present study to evaluate the properties of both the surfactants monolayer and the aqueous cores of AOT microemulsions, at increasing w0 values, in the presence and in the absence of lipases. The 5-DSA is a lipid spin probe with an amphiphilic character which upon solubilisation in micellar systems is preferably localized at the interfaces intercalating within the surfactant molecules. EPR data from the corresponding spectra (Figure S5) were analyzed to obtain information regarding molecular motion and lipid order in the membranes under investigation 37. The effect of the water content, as expressed by the w0 values, on the rotational correlation times, τR, and order parameters, S, of 5-DSA in ΑΟΤ/isooctane microemulsions is presented in Table 3. It is obvious that, as long as the w0 values increase from 5 to 15, both the τR and S values of 5-DSA increase. More specifically, the increase of the aqueous phase caused an increase to the rotational correlation time of the spin probe, from 1.19 ns to 3.21 ns, and an increase of the order parameter, from 0.17 to 0.20.


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Table 4. Rotational correlation times, τR, and order parameters, S, of 5-DSA in ΑΟΤ/isooctane microemulsions in the presence and absence of lipases. τR (ns)

S

w0

buffer

TLL

Lecitase Ultra

buffer

TLL

Lecitase Ultra

5

1.19

1.17

1.14

0.17

0.17

0.17

8

4.33

4.11

4.28

0.23

0.23

0.23

10

1.74

1.64

1.64

0.17

0.17

0.23

15

3.21

2.86

2.97

0.20

0.20

0.20

20

n.d.

n.d.

n.d.

0.46

0.50

0.47

* n.d., non-determined.

Increases of rotational correlation time reflect a more hindered motion of the probe in the interface. The greater the amount of water added to the system, the less curved the AOT monolayer of the swelled reverse micelles, thus hindering the mobility of the probe. Similar behaviour of the surfactants monolayer upon water addition has been also reported for w/o microemulsions based on AOT 38,39, lecithin 39 and Tween 40 40. When the lipases were added in the aqueous cores of the AOT microemulsions, there was a small reduction in the τR values (Table 4) indicating increases in the curvature of the AOT layer as a protein effect. A possible explanation for this effect could be either lipase location in the aqueous centre of the reverse micelles or the presence of two populations of micelles, protein containing and empty ones. Concerning the order parameter values, S, shown in Table 4, an almost negligible effect was observed in the presence of lipases thus excluding the involvement of the proteins in the surfactants monolayer. At w0 20, τR values were further increased (τR > 3.2 ns) indicating


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transition from the “fast” to the “slow’ motion region. For the “slow motion” region the determination of τR values is difficult and inaccurate. In the last case, the order parameter S is a much better approach to the immobilization degree of the spin probe in the membrane. Interestingly, a decrease in the membrane’s flexibility was observed upon lipase addition. This effect was more profound in the case of TLL. The significant differences in the parameters at w0 8 could be a consequence of the existence of rotational isomerism of AOT. As described before 23, a transformation of rotamers from gauche to trans form starts in the AOT exactly at this water content (w0 8) and is completed in w0 close to 13. This conformational change could interfere in the spin probe mobility when start at w0 8 interfering in both τR, empty and with the lipases incorporated. However, because the marker was not covalently bound to the enzyme, these conclusions are general and do not allow an assessment and a more detailed study of the location of the protein. Furthermore, the proximity of lipases to the interface could provide a binding of 5-DSA with the active site, since its chemical structure corresponds to a natural substrate for these enzymes. To monitor molecular dynamics of the aqueous cores of the AOT reverse micelles, the hydrophilic spin probe 3-(4-nitrophenoxy carbonyl)–proxyl) was used and the calculated τR parameters from the corresponding EPR spectra (Figure S6) are shown in Table 5. With increasing the water content of the microemulsions from w0 5 to 20 the τR values were decreased from 0.82 to 0.44 ns. The observed increase in the mobility of the spin probe could be explained by the appearance of free water molecules at higher w0 values41.


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Table 5. Rotational correlation times, τR, of 3-(4-nitrophenoxy carbonyl)–proxyl) in ΑΟΤ/isooctane microemulsions. τR (ns) w0

buffer

TLL

Lecitase Ultra

5

0.82

0.82

0.96

8

0.67

0.70

0.72

10

0.62

0.65

0.64

15

0.53

0.49

0.54

20

0.44

0.43

0.46

In the presence of both lipases, τR values were increased for w0 ≤ 10 indicating their location in the aqueous cores thus hindering free motion of the spin probe. The observed phenomenon was less intense at w0 15 and 20 probably due to the emergence of more free space for the spin probe to rotate. To summarize, experimental results from the EPR measurements of free and lipase loaded AOT microemulsions using an amphiphilic and a hydrophilic spin probe, indicate that both enzymes tend to be located in the aqueous centre of the reverse micelles not participating in the surfactants monolayer. However, it is not ensured that the lipases do not interact with the polar heads of the AOT molecules, mainly due to the fact that this surfactant is ionic, capable of inducing interactions with charged amino acid residues of the enzymes.


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Steady state fluorescence spectroscopy The fluorescence emission of proteins is due to the aromatic amino acids in their molecule; tyrosine, tryptophan and phenylalanine. By excitation of the proteins in 280–295 nm, the fluorescence is mainly due to the tryptophan residues even at high tyrosine/tryptophan ratios 14. Consequently, the main role of the donor molecule is played by the tryptophan residues 42. TLL contains four tryptophan residues with three of them located close to the surface of the molecule. More specifically, two of the tryptophan residues (Trp89, Trp221) are highly exposed to the environment, one (Trp117) almost buried and one (Trp260) is highly exposed in monomers but relatively buried in dimers of the protein (RCSB Protein Data Bank, code 4GWL). This makes TLL an adequate probe for fluorescence studies. On the other hand, Lecitase Ultra is a protein-engineered phospholipase A1 manufactured and marketed by Novozymes, Denmark, and there are not any specific structural data available. However, the manufacturers did not describe changes in the tryptophan amino acids in the process of developing this enzyme from TLL 43. Hence, both enzymes could be used for the fluorescence studies. The emission spectra of both enzymes were recorded in a series of AOT microemulsions with constant surfactant concentration (0.2 M) and w0 values raged from 5 to 20. The emission maxima of the spectra for both enzymes were constant upon water increase, being 321 nm for TLL and 322 nm for Lecitase Ultra. The relative values obtained in the buffer used, were 328 nm and 332 nm, respectively, showing that in both enzymes Trp residues are located in nonpolar regions of the proteins (λ ≈ 330 nm). Emission of Trp is sensitive to the microenvironment of the fluorophore. The Trp residues in the proteins can be divided into three discrete spectral classes 44, depending on whether they are located in nonpolar regions of the protein (λ ≈ 330 nm), completely exposed to water (λ ≈ 350


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nm) or in an environment of intermediate polarity (λ ≈ 340 nm). The increase of the I350/I330 fluorescence intensity ratio, thus, suggests a more hydrophilic environment for Trp. The values of I350/I330 for w0 values ranging from 5 to 20 are presented, for both enzymes (Table 6). Although the spectra maxima did not change (being 321 nm for TLL and 322 nm for Lecitase Ultra), as can be seen (Table 6), the intensity ratio slightly increases as the water content of the microemulsion increases. Therefore, the Trp residues of the proteins are located in a more polar microenvironment when there is more water in the system. This is more pronounced in the case of Lecitase Ultra.


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Table 6. I350/I330 fluorescence intensity ratio values for TLL and Lecitase Ultra in AOT microemulsions with different w0 I350/I330 w0 TLL

Lecitase Ultra

5

0.789

0.789

8

0.804

0.839

10

0.810

0.838

15

0.812

0.891

20

0.833

0.888

Fluorescence energy transfer Fluorescence energy transfer is primarily a result of dipole–dipole interactions between a donor– acceptor pair and depends on the distance between these two molecules when located in confined media 14. PNA, a conjugated molecule considered to be an excellent fluorophore 42, can be used as an acceptor to the energy emission of the Trp residues. Comparing the absorption spectrum of parinaric acid (PNA), and the emission spectra of the enzymes, a considerable overlap occurred (data not shown), suggesting that fluorescence energy transfer between the pair PNA and tryptophan residues of the enzymes is feasible 19,45. Such fluorescence energy transfer between PNA and the Trps of TLL and Lecitase Ultra in AOT microemulsions was examined by measuring the fluorescence of both enzymes in the absence and in the presence of various PNA concentrations under constant w0 values. The results showed that as the concentration of PNA increases the fluorescence of the enzymes decreases. This can be attributed to Förster type energy transfer as already shown for systems in


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homogeneous media

42

. The energy transfer efficiency was calculated using different PNA

concentrations for selected w0 values. This can allow assessing the distance between the donor and the acceptor pair. Table 7 shows the values of the determined parameters used for the calculation of the Förster distance, R0, as well as the calculated donor–acceptor distance, r. In classic reverse micellar systems, like AOT microemulsions, the addition of water leads to swelling of the reverse micellar cores, and thus to higher distances (r) 28. For TLL, the distance r appears to slightly increase upon w0 increase for low water contents (w0 5 to 10) (Table 7). However, when w0 increases beyond this point the increase of the donoracceptor distance is much more pronounced. This can be attributed to the swelling of the micelles that contain the enzyme. It seems that for low water contents (w0 value up to 10) the lipase is restricted within the micelles, while for higher w0 values increase of the size of the micelles occurs with the appearance of free water around the TLL molecule. This indicates that, if the water content of the system is adequate, TLL can be preferably located inside the micelles. On the other hand, in the case of Lecitase Ultra the distance r shows only slight changes upon water content increase, in the whole range of w0. This could be attributed to the location of Trp residues buried in hydrophobic regions of the protein, hence, inaccessible by water. However, this could seem being in contrast to the results of the steady state fluorescence experiments that lead to the conclusion that Trp residues of Lecitase Ultra are located in a more polar microenvironment when there is more water in the system. A plausible explanation could be that the results of the energy transfer reflect the location of the enzyme closer to the interface.


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Table 7. Calculation of the donor-acceptor distance, r, from the energy transfer efficiency, T, measured for TLL and Lecitase Ultra in AOT microemulsion.

TLL

Lecitase Ultra

a

W0

Φd a

Ja (M-1 cm3)

R0 (Å)

T

r (Å)

5

0.092

19.5 x 10-13

54

0.51

54

8

0.079

17.6 x 10-13

52

0.43

55

10

0.087

16.6 x 10-13

52

0.40

56

15

0.106

18.5 x 10-13

56

0.40

60

20

0.078

17.3 x 10-13

52

0.18

70

5

0.088

18.4 x 10-13

54

0.42

57

8

0.083

17.7 x 10-13

53

0.43

55

10

0.092

17.2 x 10-13

54

0.44

56

15

0.095

16.9 x 10-13

54

0.30

62

20

0.071

15.6 x 10-13

52

0.35

58

J is the spectral overlap; Φd is the quantum yield14

Activity of the encapsulated enzymes Most of attempts to study the catalytic activity of Lecitase Ultra have been performed toward the application of phospholipase activity of this enzyme for degumming of vegetable oils and modifying of phospholipids46,47. However, there are studies revealing its lipase activity in organic synthesis48,49. Therefore, in this study the activity of both encapsulated enzymes was tested by following the esterification reaction between ethanol and oleic acid. It was proved that both enzymes retained their catalytic activity for all w0 values tested, as can be seen in Figure 6.


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Figure 6. Conversion of the esterification of oleic acid (50 mM) with ethanol (100 mM) at 40 °C, after 2 h, for different w0 values for TLL (■) and Lecitase Ultra (▲). In the case of Lecitase Ultra, the results referring to w0 20 are not presented because the hydrolysis of the ester starts after 30 minutes of reaction, leading to low conversion yield (< 11%). At w0 5 the inverse hydrolytic reaction starts with the conversion decreasing to less than 10% in 4 h (data not shown), probably to maintain the system stable as a consequence of the low water quantity, representing a system with limited applications. The curve observed in Figure 6 for TLL is in agreement to the typical bell-shaped curve observed in related studies based on esterification reactions catalyzed by lipases in AOT microemulsions1. As can be seen, the maximum catalytic activity was observed for w0 values of 10 and 15, respectively, giving conversions of 55% and 38%. For each one of the tested enzymes, this probably corresponds to a micellar size comparable to that of the protein, which is


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claimed by some authors to be an optimum condition for enzyme activity

50,51

. In general, bell

shaped curves are observed for enzymatic activity towards w0 changes, where lower w0 leads to lower activity that increases with intermediate w0 values, but decreases again with the addition of more water. In case of lower w0 values the low activity may be explained by the fact that a large fraction of the enzyme could be exposed to the organic solvent

51

. On the other hand, a

microemulsion with high water content could promote fluctuations in the enzyme structure making it difficult to direct the active site at the interface

51

or just because the enzyme in

aqueous environment is more vulnerable to structural changes. Apart from conversion yields, in order to compare the activity of the enzymes encapsulated in these systems, the initial rate was calculated for each one of them in distinct water content (w0). This approach is important as it eliminates effects such as inhibition of substrate or product. TLL presented higher initial rates than Lecitase Ultra, indicating a higher affinity of this lipase to oleic acid (Table 8). The microemulsion with w0 10 seems to be the most adequate system for TLL, however, the system with w0 15 was more appropriate to apply Lecitase Ultra, corroborating the previously obtained results. In the case of Lecitase Ultra it was not possible to calculate the velocity for the systems with w0 5 and 20 by the Lowry-Tinsley method because an ester hydrolysis was observed after a few minutes.


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Table 8. Initial rates of the esterification reaction of ethanol and oleic acid catalyzed by the enzymes encapsulated in reverse micelles with different water content. Initial rate (µmol/min) w0 TLL

Lecitase Ultra

5

0.27

n.c.

8

0.34

0.20

10

0.43

0.10

15

0.27

0.24

20

0.26

n.c.

Condition: oleic acid at 50 mM and ethanol (1:2) at 40 °C. n.c. - non calculated

The studied reaction was applied in a way to monitor the availability of the enzymes inside the reverse micelles and determine their activity following changes in the w0 values. Other substrates and reaction conditions can be applied using these microemulsions promoting better results with high conversions. These systems can also be immobilized in an organogel to obtain very good conversion yields enabling also the recycling of the biocatalyst 19,52.


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CONCLUSIONS The reverse micelles were formed and presented Rg values between 16 and 38 Å and Rh values between 16 and 25 Å, with distinct polydispersity that indicates a possible presence of aggregates or the interaction between droplets at w0 values lower than 10. The Rg/Rh ratio (SAXS and DLS data comparison) indicated that elongated droplets with an ellipsoidal form are present. Fluorescence energy transfer strongly suggests that TLL is located in the water core, whereas Lecitase Ultra is closer to the interface. In any case, they are both not interfering with the interface as EPR studies indicate that they rather seem to be located in the aqueous core of the reverse micelles. Temperature increase can lead to larger mean Rh values, a phenomenon described as percolation. By increasing the chance of contact between droplets mass transfer is favored, and this is an essential condition for application of these microemulsion systems in biocatalysis. The esterification activity of the enzymes inside the reverse micelles was observed and the lipase presented good results in conversions, showing that the enzymes did not lose activity upon incorporation into the micelles. Activity was higher when the Rg of the reverse micelles were closer to the Rg of the enzyme. This might be due to better accommodation of the enzyme inside the microemulsion, a result supported by the polydispersity of these systems. Therefore, showing that the enzymes TLL and Lecitase Ultra did not lose activity after incorporation into the micelles, these systems are feasible and can be applied to other esterification reactions.


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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the http://pubs.acs.org.” The Supporting Information contains Table S1 with calculated ratio Dmax/2 and Dmáx/Rg; Figure S1 shows the autocorrelation functions obtained by DLS analysis of all reverse micelles; Figures S2 and S3 show the size distribution graphs obtained from the DLS measurements at w0 8 and 5; Figure S4 includes the Guinier regression of appreciated q values of the scattering curves of the empty reverse micelles, with TLL and with Lecitase Ultra; Figure S5 shows EPR spectra of 5-DSA in empty and loaded AOT microemulsions; Figure S6 shows EPR spectra of 3(4-nitrophenoxy carbonyl)–proxyl) in empty and loaded AOT microemulsions.

AUTHOR INFORMATION Corresponding Authors *Yraima Cordeiro, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, RJ, Brazil, 21941-902, Av Carlos Chagas Filho 373, bloco B subsolo S17. Phone 55 21 22609192 ext 210. Email: [email protected]; *Aristotelis Xenakis, Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, 48, Vassileos Constantinou Ave., Athens, 11635, Greece. Email: [email protected]


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ACKNOWLEDGMENT We thank Dr. Julia Clarke (UFRJ) for careful revision of the manuscript. This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); from the Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem (INBEB); Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). This work has also been supported by the Brazilian Synchrotron Light Laboratory (LNLS) under proposals SAXS110913, SAXS1-12664 and SAXS1-15970.


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REFERENCES (1) (2) (3)

(4) (5)

(6)

(7) (8)

(9) (10)

(11)

(12)

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Table of Contents Graphic


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Figure 1. Chemical structures of (A) 5-doxyl stearic acid and (B) 3-(4-nitrophenoxy carbonyl)–proxyl. 47x17mm (300 x 300 DPI)


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Figure 2 159x129mm (96 x 96 DPI)


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Figure 3 180x388mm (300 x 300 DPI)


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Figure 4 177x445mm (300 x 300 DPI)


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Figure 5. Graphic of the normalized pair-distance distribution function (p(r)) of the empty systems (A), or loaded with Lecitase Ultra (B) and TLL (C). 149x324mm (300 x 300 DPI)


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Figure 6. Conversion of the esterification of oleic acid (50 mM) with ethanol (100 mM) at 40 °C, after 2 h, for different w0 values for TLL (■) and Lecitase Ultra (▲). 97x89mm (300 x 300 DPI)


Nanoencapsulated Lecitase Ultra and Thermomyces lanuginosus

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