Biochemical Engineering Journal 75 (2013) 3238

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Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Regular article

Kinetic study of the colloidal and enzymatic stability of -galactosidase, for designing its encapsulation route through solgel route assisted by Triton X-100 surfactant
Sindy Escobar, Claudia Bernal, Monica Mesa
Grupo Ciencia de los Materiales, Instituto de Qumica, FCEN, Universidad de Antioquia. Medelln, Colombia

a r t i c l e

i n f o

a b s t r a c t
The kinetic study of the colloidal and enzymatic stability for the -galactosidase of Bacillus circulans was carried out in function of the presence of Triton X-100 surfactant, under orbital agitation and varying the pH and temperature. The correlation between the Dynamic Light Scattering and enzyme assay data, supported by z-potential and Differential Scanning Calorimetry analyses, gave insights about the mechanism of the protective role of the surfactant against the enzyme deactivation during its incubation. The best conditions for preserving the enzymatic activity, under orbital agitation, were: presence of 1 103 M Triton X-100, at pH 6.0 and 25 C or 40 C during less than 24 h, even in the presence of 0.1 M sodium cations or 4% ethanol. As these conditions also affect the polycondensation of the siliceous species and the enzyme-silica interactions, these could be considered as primary information for designing and optimizing an encapsulation route of -galactosidase in silica, by a solgel process assisted by surfactant. 2013 Elsevier B.V. All rights reserved.

Article history: Received 27 November 2012 Received in revised form 11 March 2013 Accepted 13 March 2013 Available online 19 March 2013 Keywords: -galactosidase deactivation Colloidal stability Biocatalysis Solgel conditions Non-ionic surfactant Triton X-100 (-galactosidases (-gal))

1. Introduction The -galactosidases (-gal) from different sources hydrolyze -galactosidic bonds present in lactose or other analogs molecules. They are very useful enzymes for obtaining delactosed dairy products, improving the tasting of the ice creams and bioconverting the whey into more valuable products [1,2]. They also exhibit transferase activity, attaching galactose to other compounds, for producing galacto-oligosaccharides [3,4]. The available commercial extracts of -gal from Bacillus circulans are constituted by different isoforms, which determine their physicochemical characteristics and enzymatic behavior [2,5,6]. Their intrinsic and operational stabilities must be warranted for an effective implementation at industrial level [1,7,8]. Many techniques have been used for maintaining or increasing the enzyme stability, including its chemical modication, aggregation [9], immobilization to solid matrices [1012] and medium engineering [13,14]. Some reviews discussing about different stabilization methods have been published along the time [1517]. Some of these techniques involve the use of surfactants, for example as stabilizing agents during the immobilization process [10,18], avoiding the disruption of the vesicular structure of liposomes, in which the enzyme is entrapped [19] and for the medium engineering obtaining highly active catalysts [13,20]. The

solgel process, which is the polycondensation of siliceous species from different silica sources at low temperature, has been also used for stabilizing enzymes by encapsulation [2123]. Here, we studied the correlation between the enzymatic activity and colloidal stability for the -gal from B. circulans (B. circulans -gal), during its incubation in the presence of non-ionic Triton X-100 surfactant at different concentrations, varying pH and temperature conditions, in order to see the role of this surfactant in the B. circulans -gal stability. Taking into account that there are not reports for the -gal from B. circulans and Triton X-100 interactions at the concentration assayed in this work, we attempted to rationalize the found results based on DLS, z-potential and DSC methods. Some experiments were carried out in the presence of ethanol or sodium ions for simulating the reaction system in the synthesis of siliceous materials by solgel route. The results could provide the basis for establishing rational strategies for a surfactant assisted solgel encapsulation of the B. circulans -gal in siliceous supports. The determination of deactivation constants will contribute to the knowledge about deactivation kinetic mechanism of this enzyme, which is very important for design processes related with its stabilization and industrial implementation. 2. Materials and methods 2.1. Preparation of -gal solutions

Corresponding author. Tel.: +57 4 2196546, fax: +57 4 2191049. E-mail address: mmesacad@gmail.com (M. Mesa). 1369-703X/$ see front matter 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2013.03.010

The commercial extract of -gal from B. circulans (BIOLACTASA NTL CONC X2, constituted by 1,4 -galactosidase, I.U.B: 3.2.1.23,

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33

Residual activity

sodium benzoate, potassium sorbate, glycerin and sodium chloride; presenting 31.3 0.5 mg of protein per mL and 1353 11 IU per mL) was purchased from Biocon espanola S.A. This was dissolved in buffer solution (13.5 U protein solution per 5 mL buffer solution), without further purication, in the presence of different concentrations (01 103 M) of Triton X-100 surfactant (Sigma, USA), at pH 4.5, 6.0 or 9.0 (100 mM acetate, 100 mM phosphate or 100 mM bicarbonate buffer, respectively), at 25 C or 40 C. The 0.1 M sodium chloride or 4%vol ethanol was added in some solutions. 2.2. Analysis of -gal solutions The B. circulans -gal solutions were incubated in the absence of substrate, at the specied conditions in each test, under orbital agitation and they were analyzed periodically during 48 h. The following data were determined: 2.2.1. Enzyme activity The specic activity for the -gal solutions, expressed as mM glucose U1 protein min1 , was measured by visible spectroscopy technique (Perkin Elmer Lambda35 UV/vis spectrophotometer, UV Winlab software), at 25 C and under controlled stirring (Perkin Elmer PTP-1 Peltier Temperature Programer System and Heating by Thermo scientic). In each test, 50 L of enzyme solution was added to a cell containing 1.5 mL of 80 mM lactose in phosphate buffer (100 mM, pH 7) and 0.5 mL of commercial color-developer reagent (Glucose oxidase-peroxidase assay kit by ByoSystems, Spain). The increase of the absorbance at 500 nm and 25 C, corresponding to the release of quinoneimine, was recorded [11]. All enzyme assays were performed in triplicate and the average standard deviations were always less than 2% of the mean. The enzyme deactivation proles were graphed in terms of residual activity (ratio of the specic activity at time t (At ) and zero time (A0 ) of incubation) in order to compare the persistence of enzymatic activity, under different incubation conditions that are also used in solgel processes. 2.2.2. Deactivation kinetic constants The experimental data were adjusted to classical rst-order (Eq. (1)) and series-type deactivation (Eq. (2) and Eq. (3)) kinetic mechanisms. The last one assumes that the deactivation can occurs in different steps, with active intermediaries and active or deactivated nal enzyme states [24,25]. The tting procedure was carried out by iterative non-lineal regression method, using the Solver tool of Microsoft Ofce Excel 2007 software. At /A0 = exp(k1 t)
E E1 E1 k1 k2

1.0

0.8

0M 5 x 10-5 M 2 x 10-4 M 1 x 10-3 M

0.6

0.4

0.2

0.0 0 10 20 30 40 50

Time (h)
Fig. 1. B. circulans -gal decay prole at pH 6.0 and 25 C, in function of the Triton X-100 surfactant concentration (0 M 1 103 M). Each experiment was carried out three times and the bars represent the standard deviation from the mean.

Light Scattering (DLS, Horiba Scientic LB-550 instrument, LB-5503.57 version software) by using the StokesEinstein equation [26]. The polydispersity percentage of these measurements (%Pd) was calculated taking into account the standard deviation (SD) of the hydrodynamic diameter distribution: %Pd = SD 100 D (4)

The z-potential for some selected B. circulans -gal solutions was measured in a Malvern Instruments Zetasizer nanoseries NANOZS equipment, at the specied pH and temperature. 2.2.4. Denaturation temperature This temperature was determined for some selected B. circulans -gal solutions, from Differential Scanning Calorimetry (DSC) thermogram, obtained from 15 C to 350 C at 10 C min1 scan rate (TA instruments DSC Q100-1151 equipment, Universal Analysis 2000 software), under inert atmosphere (50 mL min1 nitrogen) and using hermetic pans. 3. Results and discussion The B. circulans -gal activity at pH 6.0, 25 C and in the absence of Triton X-100 varied during the incubation time (0 M, Fig. 1). The enzyme activity loss or deactivation occurred following a rstorder kinetic mechanism (R2 = 0.9832, Table 1) and the enzyme activity after 48 h of incubation was only 16% of its initial value, as it is usual for non-thermostable monomeric enzymes [24]. The deactivation during the incubation was expected because temperatures higher than 4 C (more frequent storing temperature), orbital agitation and diluted conditions make the protein molecules more prone to aggregation, unfolding or any deleterious change in their native three-dimensional structure [27]. The DLS results for the B. circulans -gal solution during the rst 2 h of incubation showed that the enzyme molecules can be in equilibrium between tight and extended forms, whose average hydrodynamic diameters vary 13 nm or as small agglomerates 30 nm. Species larger than 100 nm appeared after 2 h of incubation (Fig. 2), indicating that the barrier for an effective collision was overcome. Nevertheless, they coexisted with the smaller ones and their size was not stable on the time, which is related with the dynamic nature of the non-covalent aggregation process [28,29]. These time course DLS results were well-correlated with the B. circulans -gal enzymatic behavior during the incubation (Fig. 1), since the larger species (aggregates) are less active than single

(1) (2)

At /A0 = +[(1 + k1 /(k2 k1 ) k2 /(k2 k1 )] exp(k1 t ) [( ) k1 /(k2 k1 )] exp(k2 t )) (3) (h1 );

where k1 and k2 are rst-order deactivation rate constants E, E1 and E2 are specic activities of initial (E) and intermediates (E1 and E2 ) active enzyme states, respectively; and are ratios of specic activities of the intermediate and initial enzyme form, and of the nal state and initial enzyme form, respectively ( = E1 /E and = E2 /E). The half-life times (t1/2 ) were calculated with the model that better t to the experimental results, according to the mean squared error (Er) and correlation factor (R2 ) 2.2.3. Colloidal parameters The hydrodynamic diameter (D) for the species present in the different B. circulans -gal solutions was determined by Dynamic

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S. Escobar et al. / Biochemical Engineering Journal 75 (2013) 3238

Table 1 Deactivation rates at 25 C (k1 and k2 in h1 ), ratios of specic activities of the intermediate and initial enzyme forms ( and ) and half-live (t1/2 in h) values obtained by adjusting the experimental data to a classical rst-order or series-type mechanism. The correlation coefcient (R2 ) and the mean squared error (Er) of the model are shown in each case. Triton X-100 concentration (M) + extra-additives 0 5 105 2 104 1 103 0 1 103 0 1 103 0 + 4% ethanol 1 103 + 4% ethanol pH 6.0 6.0 6.0 6.0 4.5 4.5 9.0 9.0 6.0 6.0 Mechanism First-order Series-type Series-type Series-type Series-type Series-type Series-type Series-type Series-type Series-type k1 0.031 1.341 0.733 0.033 0.151 0.095 0.461 0.177 1.557 0.989 0.677 0.758 0.712 1.440 2.248 1.044 0.597 0.299 0.560 k2 0.018 0.014 0.032 1.902 2.895 0.461 0.032 0 0 0 0 0 0.083 0.118 0.030 0 0 0 t1/2 22.2 17.5 30.7 42.6 6.7 10.8 3.9 13.3 1.9 9.3 R2 0.9832 0.9930 0.9661 0.9726 0.9980 0.9855 0.9934 0.9984 0.9999 0.9930 Er 1.01 0.00 0.02 0.00 0.00 0.01 0.02 0.00 0.00 0.00

-gal molecules, as it was shown before at pH 7.4 and 50 C for -gal from Aspergillus oryzae [29]. The polydispersity percentage of the size (%Pd) for the smallest species increased with incubation time for the B. circulans -gal in the absence of Triton X-100, corroborating their agglomeration (Table 2). The aqueous soluble enzymes are proteins very sensible to the temperature variations and they also exhibit the properties of colloidal dispersions, being susceptible to environmental changes such as pH or the presence of other chemical compounds (surfactants, salts, etc.), which could modify their native threedimensional structure [27,28]. Therefore, the B. circulans -gal stability was studied in function of the presence and concentration of neutral surfactant Triton X-100, under different aqueous buffered conditions, generally used for solgel process. This will help to determine if the surfactant can be used as stabilizing agent of the B. circulans -gal, for its encapsulation in silica by solgel process or its industrial application. 3.1. Effect of the Triton X-100 concentration The B. circulans -gal decay prole at pH 6.0 and 25 C was affected by the presence and concentration of Triton X-100 surfactant (Fig. 1). The -gal preserved 42% of its initial activity after 48 h of incubation in the presence of 1 103 M Triton X-100, which was 2.6-fold higher than in the absence of surfactant (only 16%).

18 16 14

Frequency (%)

12 10 8 6 4 2 0 -2
10 100 1000

0h 1h 2h 3h 5h 24h

Diameter (nm)
Fig. 2. Evolution of the hydrodynamic diameter for B. circulans -gal species in solution, at pH 6.0 and 25 C, in the absence of Triton X-100, determined by DLS.

The adjustment of these enzymatic data to the kinetic models considered in this work [24,25] indicated that the B. circulans -gal deactivation was not homogenous in the presence of surfactant, following a series-type mechanism, in which the intermediate state of the enzyme keeps some activity before to be fully deactivated ( < 1 and = 0, Table 1). The t1/2 values for these samples, calculated with this model (Table 1), were signicantly different at 0.05 signicance level (ANOVA test). According to these t1/2 values (Table 1) and the decay proles (Fig. 1), the increase of the surfactant concentration above its critical micelar concentration (cmc = 2 104 M in phosphate buffer solution, measured by DLS) had a protective or stabilizing effect against the deactivation. This could be related to the formation of new enzyme-surfactant species, which are more stable than the isolate enzyme molecules. This hypothesis was corroborated by DLS, z-potential and DSC experiments. Before showing the results, it is important to highlight that the effect of the non-ionic Triton X-100 surfactant depends on both, surfactant concentration and enzyme type, which dene the kind of interaction that would be present in each case, and therefore its stabilizing or deactivating effect, as it has been shown for other enzymes [15,3033]. The deactivation behavior of the B. circulans -gal in the presence of Triton X-100 surfactant, during the incubation time, could be also associated with the aggregation of -gal molecules, which also include the formation of enzyme-surfactant species, as it was suggested by the series-type deactivation mechanism: Larger species (>100 nm) that accompanied the individual -gal molecules (1230 nm) appeared earlier in the solution where the surfactant is present (less than 1 h of incubation) compared with the results in its absence (Table 2). On the other hand, the B. circulans -gal deactivation proles and t1/2 values in function of the Triton X-100 concentration, suggested that the presence of this surfactant in form of micelles had a higher protective effect on the enzyme activity than when it was as individual molecules in solution or near to cmc. In fact, when the surfactant concentration increases in the aqueous medium, its molecules are less soluble and form micelles that could interact with the -gal through hydrophobic groups of this enzyme. The surfactant micellization would occur as in enzyme-free solution but with the added feature that these micelles may be free in solution or be bound by physical interactions to the enzyme, as occurs in other similar systems [34]. The formed enzymesurfactant aggregates could help to avoid the deactivation of the enzyme, for example during the encapsulation process by solgel methodology as it has been shown in other systems, where the added macromolecule wrap the enzyme molecules protecting them during the invasive polymerization of silanols [35]. The -gal/Triton X-100 interactions also promoted the reorganization of the hydrophobic sites of the enzyme, which could acquire a more favorable folding state that would be responsible of its

S. Escobar et al. / Biochemical Engineering Journal 75 (2013) 3238 Table 2 Hydrodynamic diameter (D) and polydispersity percentage (%Pd) for B. circulans -gal species in buffer and buffer-surfactant solutions, at pH 6.0 and 25 C. Time (h) 0 M Triton X-100 D (nm) 0 27.4 %Pd 50.2 5 105 M Triton X-100 D (nm) 27.0 %Pd 36.6 2 104 M Triton X-100 D (nm) 18.0* 361.0 3115.0 21.0 1067.0 5348.0* 823.0 3640.2* 21.1* 318.0 1864.4 4078.3 %Pd 35.7 49.4 54.8 30.1 51.8 15.7 34.3 30.1 31.6 36.6 42.3 19.3 1 103 M Triton X-100 D (nm) 16.0* 475.0 18.4* 1412.2 29.5* 823.3 3640.4 24.0* 1225.0 16.0* 414.0 3202.1 1229.9 824.3 1223.9 %Pd 64.9 45.5 48.2 62.0 34.3 30.1 43.4 45.5 52.7 55.0 32.1 31.1 51.3 29.8

35

13.8

79.4

21.0

53.4

2 3

18.0* 1419.0 3649.3

87.5 31.8 47.2

24 27 29

27.0* 715.0 2426.0 417.3 939.4 1223.5

23.7 27.8 31.2 80.8 50.5 30.7

12.0* 104.0 18.0* 417.3 4152.0 2119.6 5271.2* 939.1 1224.4 1422.5

28.7 23.0 44.7 47.4 38.7 23.4 19.9 33.3 21.9 30.2

1246.3 624.0 2420.0 1415.9

30.5 40.5 31.6 38.4

When there are several species on the solution, the main specie (more frequent value) is marked with (*)

Table 3 Results of z-potential (at pH 6.0 and 25 C) and denaturation temperature, at zero time, for B. circulans -gal solutions in function of the Triton X-100 surfactant concentration. Molar concentration of Triton X-100 0.0 5.0 105 2.0 104 1.0 103 z-potential (mV) 9.8 9.3 13.9 26.8 0.5 1.0 1.2 1.0 Denaturation temperature ( C) 95 75 75 65 0.5 0.5 0.5 0.5 %Pd 50.2 36.6 35.7 64.9

long-term enzymatic stability, as in the case of other enzymes [36], compared with the behavior in the absence or low concentration of surfactant. The variations of the z-potential values in function of the Triton X-100 concentration corroborated the enzymesurfactant interactions and the higher effect of the surfactant when its concentration was above to the cmc value (Table 3) (ANOVA one-way analysis indicated that z-potential values were signicantly different at 0.05 level, except for the differences between the values at 0 M and 5 105 M according to the Tukey test). The reorganization of the hydrophobic sites for the B. circulans -gal was responsible of the denaturalization temperature changes for this enzyme, in function of the surfactant concentration (Table 3). It would be noticed that at zero time, the inverse relation between the %Pd and the denaturation temperature for the B. circulans -gal (Table 3), was also an indication of the conformational changes during the incubation [37]. 3.2. Effect of the pH The deactivation of B. circulans -gal at 25 C, in the absence of surfactant, was faster at pH 4.5 and 9.0 than at pH 6.0 (Fig. 3). This could be related with the enhancing of electrostatic repulsions, ionization of acid or basic groups in the active site and hydrolysis of the peptidic bonds at pH far from 6.0 [16]. All of these phenomena affected the B. circulans -gal native structure, leading to its fast deactivation at pH 4.5 and 9.0. The deactivation was also faster at pH 4.5 and 9.0 than at pH 6.0 in the presence of 1 103 M Triton X-100. However, the stabilizing effect of the surfactant was seen, increasing the t1/2 at these pH values (Table 1). Probably, the Triton X-100 surfactant protected the B. circulans -gal against the acid or base attack by modifying the enzyme conformation or its environment. The effect of the pH for other enzymes in contact with Triton X-100, has been observed, corroborating that it depends on the surfactant concentration and physicochemical characteristics of each enzymatic system [38].

The adjustment of the experimental data to the kinetic models [24,25] indicated that at extreme pH (4.5 and 9.0) the deactivation followed a series-type deactivation mechanism more than the classical one followed at pH 6.0 (Table 1). This corroborated that the deactivation at these extreme pH values is the consequence of a more complex process, which can involve active enzyme intermediates ( different to 0 at pH 4.5 and pH 9.0) but the nal enzyme state was almost fully inactivated ( values tend to very small values near to zero, Table 1). The presence of intermediate enzyme states at extreme pH values (4.5 and 9.0) could be related with different phenomenon: (i) variations on the hydrogen bonding, Van der Waals and electrostatic interactions at non-neutral pH values, that affect the tertiary structure of the proteins [16]; (ii) the

1.0 0.8

Residual activity

pH 6.0 (0M) pH 6.0 (1x10-3M) pH 4.5 (0M) pH 4.5 (1x10-3M) pH 9.0 (0M) pH 9.0 (1x10-3M)

0.6 0.4 0.2 0.0 0 10 20 30 40 50

Time (h)
Fig. 3. Effect of the pH on the -gal enzymatic activity at 25 C, in the absence (empty symbols) or 1 103 M (full symbols) Triton X-100. ( , ) pH 4.5; ( , ) pH 6.0; ( , ) pH 9.0. Each experiment was carried out three times and the bars represent the standard deviation from the mean. In some cases the symbols are bigger than the bars.

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S. Escobar et al. / Biochemical Engineering Journal 75 (2013) 3238

1.2 1.0

25 (0M) 25 (1 x 10-3 M) 40 (0M) 40 (1 x 10-3 M)

1.10 1.05 1.00

0.1M Na (0M) 0.1M Na (1x10-3M) 0M Na (0M) 0M Na (1x10-3M)

Residual activity

Residual activity
0 10 20 30 40 50

0.8 0.6 0.4 0.2

0.95 0.90 0.85 0.80 0.75

0.0

0.70 0 2 4 6 8

Time (h)
Fig. 4. Effect of the incubation temperature on the B. circulans -gal activity, at pH 6.0, in function of the presence of surfactant (0 M and 1 103 M Triton X-100). Each experiment was carried out three times and the bars represent the standard deviation from the mean. In some cases the symbols are bigger than the bars.

Time (h)
Fig. 5. Effect of the presence of 0.1 M sodium cations on the B. circulans -gal activity, at pH 6.0 and 25 C, in function of the presence of surfactant (0 M and 1 103 M Triton X-100). Each experiment was carried out three times and the bars represent the standard deviation from the mean. In some cases the symbols are bigger than the bars.

disulde shufing, specially at high pH where the thiol-disulde exchange is favored, causing conformational changes that affect the enzyme activity [39,40] and (iii) the alteration of the oxidation stage in the active site of the enzyme [41].

interactions, rendering the enzyme behavior similar in the presence and in the absence of surfactant at 40 C.

3.4. Effect of the presence of ethanol or sodium chloride 3.3. Effect of temperature The enzyme stability for B. circulans -gal at 40 C was less affected than at 25 C, during the incubation time at pH 6.0, independent of the presence of Triton X-100 (Fig. 4). This is an unexpected result, because the most common phenomenon is that the enzyme stability decreases with temperature, because the stability is clearly related to molecular stiffening. However, the activation at 40 C for two isoforms of B. circulans -gal [2] and for -gal produced by pure and mixed cultures of Streptococcus thermophilus and Lactobacillus delbrueckii ssp bulgaricus [42] was reported before. This behavior has been attributed to conformational changes in the active site of other enzymes, in the absence of surfactant [42,43], which is also corroborated here by the variability on the stability during the initial stages of heating (Fig. 4). Although the enzyme is less stable at 25 C than at 45 C, working at 25 C could be very advantageous for the B. circulans -gal encapsulation into a silica network, through solgel process. The synthesis of siliceous materials at room temperature allows controlling the enzymesilica interactions as well as enzyme-silica-surfactant ones and the polycondensation of siliceous species, which is very important for having an efcient encapsulation in terms of quantity of encapsulated enzyme. The mean value of the enzyme activity at 40 C was 96.9 7.6 in the absence of surfactant and 99.4 3.9 in the presence of 1 103 M Triton X-100. The two samples t-Student test for these data showed that there was no signicant difference at 0.05 level in the enzyme decay proles at these two levels of surfactant concentration, contrary to what was observed at 25 C, where the surfactant had a protective effect. In this case, Triton X-100 acts as positive modulator probably due to the rigidity of the enzyme molecules and aggregates caused by its presence [16]. The changes on the surfactant micellization and cmc; enzyme aggregation and Brownian movement of the all molecules in the system with the increase of the temperature, could weaken the enzyme-surfactant The tetraethylorthosilicate and sodium silicate are common sources of silica, among other alcoxysilanes and alkylalcoxysilanes, used in the synthesis of siliceous materials by solgel process. These two sources produce ethanol or sodium ions, respectively during the reaction [21,44] and they could have effects on the enzyme, which will be studied here as examples, taking common experimental values for each one. The effect of the sodium cations during the rst 3 h of incubation for B. circulans -gal solution at pH 6 and 25 C in the absence of Triton X-100 surfactant was not signicant (Fig. 5), as it was tested by two-samples t-Students test, at 0.05 level, for the mean residual activity (0.898 0.088 and 0.910 0.063 in the absence and presence of 0.1 M NaCl, respectively). Similarly to the surfactant effect, the positive or negative effect of a salt is highly dependent on its concentration and the enzyme type, under specic incubation conditions, because the interactions for each enzymesalt pair and the changes in water organization are different [45,46]. However, a synergic effect on the enzyme stability was seen when the sodium cations and Triton X-100 were present in the incubation medium at the same pH and temperature conditions, reinforcing the protective effect of this surfactant on the B. circulans -gal stability (Fig. 5). The two samples t-Student test for the mean residual activities for B. circulans incubated at pH 6.0, 25 C in presence of 0.1 M NaCl (0.910 0.063 in the absence of surfactant and 1.010 0.059 with 1 103 M Triton X-100) showed a signicantly difference at 0.05 level in function of the presence of Triton X-100. The presence of 4% ethanol affected negatively the enzyme stability of B. circulans -gal incubated at pH 6.0 and 25 C (Fig. 6), as it had been observed for other enzymes, because its presence disturbs the hydrogen bonding and hydrophobic interactions, involved in the enzyme stabilization [47,48]. However this effect was lower in the presence of Triton X-100 surfactant (Fig. 6), which increased the t1/2 (Table 1), corroborating that the surfactant in form of micelles interacted with the enzyme, inhibiting the action of the alcohol.

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0% Eth (1x10-3M) 0% Eth (0M) 4% Eth (0M) 1.0 0.9 4% Eth (1x10-3M)

References
[1] Q. Husain, -Galactosidases and their potential applications: a review, Crit. Rev. Biotechnol. 30 (2010) 4162. [2] A. Vetere, S. Paoletti, Separation and characterization of three -galactosidases from Bacillus circulans, Biochim. Biophys. Acta 1380 (1998) 223231. [3] B. Rodriguez-Colinas, A. Poveda, J. Jimenez-Barbero, A.O. Ballesteros, F.J. Plou, Galacto-oligosaccharide synthesis from lactose solution or skim milk using the -galactosidase from Bacillus circulans, J. Agric. Food Chem. 60 (2012) 63916398. [4] T. Palai, S. Mitra, P.K. Bhattacharya, Kinetics and design relation for enzymatic conversion of lactose into galacto-oligosaccharides using commercial grade galactosidase, J. Biosci. Bioeng. 114 (2012) 418423. [5] J. Nelms, I. G. Fotheringham, Two new <beta>-d-galactosidases from Bacillus circulans: a common sequence homology surrounding the putative active site region in several <beta>-glycosidase families, Uniprot database (entry Q45093 encodes a protein of 690 AA; entry O31341 encodes a protein of 586 AA). [6] J. Song, K. Abe, H. Imanaka, K. Imamura, M. Minoda, S. Yamaguchi, K. Nakanishi, Causes of the production of multiple forms of <beta>-galactosidase by Bacillus circulans, Biosci. Biotechnol. Biochem. 75 (2011) 268278. [7] M.L. Richmond, J.I. Gray, C.M. Stine, Beta-Galactosidase., Review of recent research related to technological application, nutritional concerns and immobilization, J. Dairy Sci. 64 (1981) 17591771. [8] C. Mateo, J.M. Palomo, G. Fernandez-Lorente, J.M. Guisan, R. FernandezLafuente, Improvement of enzyme activity, stability and selectivity via immobilization techniques, Enzyme Microb. Technol. 40 (2007) 14511463. [9] M. Wang, W. Qi, Q. Yu, R. Su, Z. He, Cross-linking enzyme aggregates in the macropores of silica gel: a practical and efcient method for enzyme stabilization, Biochem. Eng. J. 52 (2010) 168174. [10] A.I. Batal, K.S. Atia, M. Eid, Stabilization of -amylase by using anionic surfactant during the immobilization process, Radiat. Phys. Chem. 74 (2005) 96101. [11] C.A. Guidini, J. Fischer, M.M.D. Resende, V.L. Cardoso, E.J. Ribeiro, Immobilization of Aspergillus oryzae -galactosidase in ion exchange resins by combined ionic-binding method and cross-linking, Biochem. Eng. J. 52 (2010) 137143. [12] F.F. Freitas, L.D.S. Marquez, G.P. Ribeiro, G.C. Brando, V.L. Cardoso, E.J. Ribeiro, A comparison of the kinetic properties of free and immobilized Aspergillus oryzae -galactosidase, Biochem. Eng. J. 5859 (58) (2011) 3340. [13] C.A. Godoy, G. Fernndez-Lorente, B. Rivas, M. Filice, J.M. Guisan, J.M. Palomo, Medium engineering on modied Geobacillus thermocatenulatus lipase to prepare highly active catalysts, J. Mol. Catal. B-Enzym. 70 (2011) 144148. [14] E. Jahangiri, R. Agharafeie, H-J. Kaiser, Y. Tahmasbi, R.L. Legge, K. Haghbeen, Medium engineering to enhance mushroom tyrosinase stability, Biochem. Eng. J. 60 (2012) 99105. [15] P.V. Iyer, L. Ananthanarayan, Enzyme stability and stabilization aqueous and non-aqueous environment, Process Biochem. 43 (2008) 10191032. [16] J. Ge, D. Lu, Z. Liu, Z. Liu, Recent advances in nanostructured biocatalysts, Biochem. Eng. J. 44 (2009) 5359. [17] C.. Fgin, Enzyme stabilization recent experimental progress, Enzyme Microb. Technol. 33 (2003) 137149. [18] M. Goto, C. Hatanaka, M. Goto, Immobilization of surfactantlipase complexes and their high heat resistance in organic media, Biochem. Eng. J. 24 (2005) 9194. [19] M.C. Annesini, C.M. Braguglia, C.M. Memoli, L.G. Palermiti, S. Di Sario, G. Mossa, Surfactant controlled activity of -galactosidase in liposomes, Eur. J. Pharm. Sci. 4 (1996) S137. [20] J. Wang, D. Lu, Y. Lin, Z. Liu, CTAB assists the refolding of native and recombinant lysozyme, Biochem. Eng. J. 24 (2005) 269277. [21] R.B. Bhatia, C.J. Brinker, A.K. Gupta, A.K. Singh, Aqueous solgel process for protein encapsulation, Chem. Mater. 12 (2000) 24342441. [22] K. Han, Z. Wu, J. Lee, I-S. Ahn, J.W. Park, B.R. Min, K. Lee, Activity of glucose oxidase entrapped in mesoporous gels, Biochem. Eng. J. 22 (2005) 161166. [23] V. Nichele, M. Signoretto, E. Ghedini, -Galactosidase entrapment in silica gel matrices for a more effective treatment of lactose intolerance, J. Mol. Catal. B-Enzym. 71 (2011) 1015. [24] J.P. Henlye, A. Sadana, Categorization of enzyme deactivations using a seriestype mechanism, Enzyme Microb. Technol. 7 (1985) 5060. [25] J.P. Henlye, A. Sadana, Deactivation theory, Biotech. Bioeng. 28 (1986) 12771285. [26] C. Washington, Particle Size Analysis in Pharmaceutics and Other Industries, Ellis Horwood, New York, 1992. [27] A. Illanes, Enzyme Biocatalysis, Principles and Applications, Springer Netherlands, Netherlands, 2008. [28] H-L. Liu, W-J. Chen, S-N. Chou, Mechanisms of aggregation of alpha- and betaamylases in aqueous dispersions, Colloids Surf. B 28 (2003) 215225. [29] S. Yoshioka, Y. Aso, K. Izutsu, T. Terao, Aggregates formed during storage of betagalactosidase in solution and in the freeze-dried state, Pharm. Res. 10 (1993) 687691. [30] Y. Zhanga, Z. Zeng, G. Zeng, X. Liud, Z. Liua, M. Chena, L. Liua, J. Li, G. Xie, Effect of Triton X-100 on the removal of aqueous phenol by laccase analyzed with a combined approach of experiments and molecular docking, Colloids Surf. B 97 (2012) 712. [31] L. Huizhou, Y. Weijing, C. Jiayong, Effects of surfactants on emulsication and secondary structure of lysozyme in aqueous solutions, Biochem. Eng. J. 2 (1998) 187196.

Residual activity

0.8 0.7 0.6 0.5 0.4 0.3 0 2 4 6 8

Time (h)
Fig. 6. Effect of the presence of 4% ethanol on the B. circulans -gal activity at pH 6.0 and 25 C in function of the presence of surfactant (0 M and 1 103 M Triton X-100). Each experiment was carried out three times and the bars represent the standard deviation from the mean. In some cases the symbols are bigger than the bars.

4. Conclusions The correlation between the enzymatic activity and colloidal stability data (obtained by Dynamic Light Scattering and zpotential), supported by thermodynamic results (denaturation temperature obtained by Differential Scanning Calorimetry) may help to explain the deactivation behavior for the -galactosidase from B. circulans, which varies in function of the Triton X-100 surfactant concentration. A protective effect against the enzyme deactivation during the incubation time, under different conditions (temperature, pH, sodium and ethanol presence), was observed when the surfactant concentration was higher than the critical micelle concentration, due to the hydrophobic interactions between -galactosidase molecules and Triton X-100 in form of micelles. The study of the effect of the pH, temperature and presence of sodium cations or ethanol, in function of the presence or absence of Triton X-100 led to establish the preliminary conditions for encapsulating -galactosidase from B. circulans, by a solgel process assisted by surfactant: 1 103 M Triton X-100, at pH 6.0 and 25 C or 40 C during less than 24 h, from a sodium silicate or tetraethylortosilicate that did not exceed the liberation of 0.1 M sodium cations or 4% ethanol, respectively to the reaction medium during the silica polycondensation. Because each enzyme must be studied as a unique system, the results obtained by Dynamic Light Scattering, z-potential determination, Differential Scanning Calorimetry combined with the activity assays provide indispensable tool for design of enzyme encapsulation by solgel process.

Acknowledgements We thank Colciencias for the nancial support, through the 111552128274 Project Evaluacin y optimizacin de la hidrolisis enzimtica heterognea de la lactosa del suero lcteo como un paso previo a la fermentacin para la produccin de etanol and Universidad de Antioquia through Estrategia para la Sostenibilidad de los Grupos de Investigacin 20132014

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S. Escobar et al. / Biochemical Engineering Journal 75 (2013) 3238 [41] O. Misset, Stability of industrial enzymes, in: van den TweelW, A. Harder, R. Buitelaar (Eds.), Stability and Stabilization of Enzymes, Elsevier, Amsterdam, 1993, pp. 111131. [42] F.I. Ustok, C. Tari, S. Harsa, Biochemical and thermal properties of galactosidase enzymes produced by artisanal yoghurt cultures, Food Chem. 119 (2010) 11141120. [43] J.N. Rodriguez-Lopez, L.G. Fenoll, J. Tudela, C. Devece, D. Sanchez-Hernandez, E. de los Reyes, Thermal inactivation of mushroom polyphenoloxidase employing 2450 MHz microwave radiation, J. Agric. Food Chem. 47 (1999) 30283035. [44] B. Naik, N.N. Ghosh, A review on chemical methodologies for preparation of mesoporous silica and alumina based materials, Recent Pat. Nanotechnol. 3 (2009) 213214. [45] S. Yoshioka, Y. Aso, K-I. Izutsu, T. Terao, The effect of salts on the stability of -galactosidase in aqueous solution, as related to the water mobility, Pharm. Res. 10 (1993) 14841487. [46] R.C. Dickson, L.R. Dickson, J.S. Markin, Purication and properties of an inducible -galactosidase isolated from the yeast Kluyveromyces lactis, J. Bacteriol. 137 (1979) 5161. [47] O. Miyawaki, M. Tatsuno, Thermodynamic analysis of alcohol effect on thermal stability of proteins, J. Biosci. Bioeng. 111 (2011) 198203. [48] A.M. Baptista, C.M. Soares, Analyzing the molecular basis of enzyme stability in ethanol/water mixtures using molecular dynamics simulations, J. Chem. Inf. Model. 52 (2012) 465473.

[32] G. Savelli, N. Spreti, P.D. Proo, Enzyme activity and stability control by amphiphilic self-organizing systems in aqueous solutions, Curr. Opin. Colloid Interface Sci. 5 (2000) 111117. [33] S-H. Yoon, J.F. Robyt, Activation and stabilization of 10 starch-degrading enzymes by TritonX-100 polyethylene glycols, and polyvinyl alcohols, Enzyme Microb. Technol. 37 (2005) 556562. [34] K. Esumi, M. Ueno, StructurePerformance Relationships in Surfactants, 2nd ed., Marcel Dekker Inc., New York, 1997. [35] B. Dunn, J.M. Miller, B.C. Dave, J.S. Valentine, J.I. Zink, Strategies for encapsulating biomolecules in solgel matrices, Acta Mater. 46 (1998) 737741. [36] J-I. Guanglei, Z. Haibo, H. Feng, H. Xirong, Effects of nonionic surfactant Triton X-100 on the laccase-catalyzed conversion of bisphenol A, J. Environ. Sci. 21 (2009) 14861490. [37] K. Shiba, T. Niidome, E. Katoh, H. Xiang, L. Han, T. Mori, Y. Katayama, Polydispersity as a parameter for indicating the thermal stability of proteins by dynamic light scattering, Anal. Sci. 26 (2010) 659663. [38] S.W. Leung, J.C.K. Lai, Differential effects of anionic surfactants on activities of GDH, LDH, and MDH, Biochem. Eng. J. 25 (2005) 7988. [39] H.F. Gilbert, Molecular, Cellular aspects of thiol-disulde exchange, Adv. Enzymol. 63 (1990) 69172. [40] H.F. Gilbert, Thiol/disulde exchange equilibria and disulde bond stability, Methods Enzymol. 251 (1995) 828.