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Biochemical Engineering Journal 75 (2013) 32– 38

Contents lists available at SciVerse ScienceDirect

Biochemical Engineering Journal

journa l h om epage: www.elsev ier .com/ locate /be j

egular article

inetic study of the colloidal and enzymatic stability of-galactosidase, for designing its encapsulation route through sol–gel

oute assisted by Triton X-100 surfactant

indy Escobar, Claudia Bernal, Monica Mesa ∗

rupo Ciencia de los Materiales, Instituto de Química, FCEN, Universidad de Antioquia. Medellín, Colombia

r t i c l e i n f o

rticle history:eceived 27 November 2012eceived in revised form 11 March 2013ccepted 13 March 2013vailable online 19 March 2013

a b s t r a c t

The kinetic study of the colloidal and enzymatic stability for the �-galactosidase of Bacillus circulans wascarried out in function of the presence of Triton X-100 surfactant, under orbital agitation and varying thepH and temperature. The correlation between the Dynamic Light Scattering and enzyme assay data, sup-ported by z-potential and Differential Scanning Calorimetry analyses, gave insights about the mechanismof the protective role of the surfactant against the enzyme deactivation during its incubation. The bestconditions for preserving the enzymatic activity, under orbital agitation, were: presence of 1 × 10−3M

eywords:-galactosidase deactivationolloidal stabilityiocatalysisol–gel conditions

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 sodiumcations or 4% ethanol. As these conditions also affect the polycondensation of the siliceous species andthe enzyme-silica interactions, these could be considered as primary information for designing and opti-mizing an encapsulation route of �-galactosidase in silica, by a sol–gel process assisted by surfactant.

on-ionic surfactant Triton X-100�-galactosidases (�-gal))

. Introduction

The �-galactosidases (�-gal) from different sources hydrolyze-galactosidic bonds present in lactose or other analogs molecules.hey are very useful enzymes for obtaining delactosed dairy prod-cts, improving the tasting of the ice creams and bioconvertinghe whey into more valuable products [1,2]. They also exhibitransferase activity, attaching galactose to other compounds, forroducing galacto-oligosaccharides [3,4]. The available commercialxtracts of �-gal from Bacillus circulans are constituted by differentsoforms, which determine their physicochemical characteristicsnd enzymatic behavior [2,5,6]. Their intrinsic and operationaltabilities must be warranted for an effective implementation atndustrial level [1,7,8]. Many techniques have been used for main-aining or increasing the enzyme stability, including its chemical

odification, aggregation [9], immobilization to solid matrices10–12] and medium engineering [13,14]. Some reviews discussingbout different stabilization methods have been published alonghe time [15–17]. Some of these techniques involve the use of sur-actants, for example as stabilizing agents during the immobiliza-

ion process [10,18], avoiding the disruption of the vesicular struc-ure of liposomes, in which the enzyme is entrapped [19] and for the

edium engineering obtaining highly active catalysts [13,20]. The

∗ Corresponding author. Tel.: +57 4 2196546, fax: +57 4 2191049.E-mail address: mmesacad@gmail.com (M. Mesa).

369-703X/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.bej.2013.03.010

© 2013 Elsevier B.V. All rights reserved.

sol–gel process, which is the polycondensation of siliceous speciesfrom different silica sources at low temperature, has been also usedfor stabilizing enzymes by encapsulation [21–23].

Here, we studied the correlation between the enzymatic activ-ity and colloidal stability for the �-gal from B. circulans (B. circulans�-gal), during its incubation in the presence of non-ionic TritonX-100 surfactant at different concentrations, varying pH and tem-perature conditions, in order to see the role of this surfactant inthe B. circulans �-gal stability. Taking into account that there arenot reports for the �-gal from B. circulans and Triton X-100 inter-actions at the concentration assayed in this work, we attemptedto rationalize the found results based on DLS, z-potential and DSCmethods. Some experiments were carried out in the presence ofethanol or sodium ions for simulating the reaction system in thesynthesis of siliceous materials by sol–gel route. The results couldprovide the basis for establishing rational strategies for a surfactantassisted sol–gel encapsulation of the B. circulans �-gal in siliceoussupports. The determination of deactivation constants will con-tribute to the knowledge about deactivation kinetic mechanism ofthis enzyme, which is very important for design processes relatedwith its stabilization and industrial implementation.

2. Materials and methods

2.1. Preparation of ˇ-gal solutions

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

Engineering Journal 75 (2013) 32– 38 33

spmiwtpbc

2

oal

2

gtWETt((itwwpcti

2

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E

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2

d

0 10 20 30 40 50

0.0

0.2

0.4

0.6

0.8

1.0 0M

5 x 10-5 M

2 x 10-4 M

1 x 10-3 M

Re

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Time (h)

S. Escobar et al. / Biochemical

odium benzoate, potassium sorbate, glycerin and sodium chloride;resenting 31.3 ± 0.5 mg of protein per mL and 1353 ± 11 IU perL) was purchased from Biocon espanola S.A. This was dissolved

n buffer solution (13.5 U protein solution per 5 mL buffer solution),ithout further purification, in the presence of different concen-

rations (0–1 × 10−3 M) of Triton X-100 surfactant (Sigma, USA), atH 4.5, 6.0 or 9.0 (100 mM acetate, 100 mM phosphate or 100 mMicarbonate buffer, respectively), at 25 ◦C or 40 ◦C. The 0.1 M sodiumhloride or 4%vol ethanol was added in some solutions.

.2. Analysis of ˇ-gal solutions

The B. circulans �-gal solutions were incubated in the absencef substrate, at the specified conditions in each test, under orbitalgitation and they were analyzed periodically during 48 h. The fol-owing data were determined:

.2.1. Enzyme activityThe specific activity for the �-gal solutions, expressed as mM

lucose U−1 protein min−1, was measured by visible spectroscopyechnique (Perkin Elmer Lambda35 UV/vis spectrophotometer, UV

inlab software), at 25 ◦C and under controlled stirring (Perkinlmer PTP-1 Peltier Temperature Programer System and Heating byhermo scientific). In each test, 50 �L of enzyme solution was addedo a cell containing 1.5 mL of 80 mM lactose in phosphate buffer100 mM, pH 7) and 0.5 mL of commercial color-developer reagentGlucose oxidase-peroxidase assay kit by ByoSystems, Spain). Thencrease of the absorbance at 500 nm and 25 ◦C, corresponding tohe release of quinoneimine, was recorded [11]. All enzyme assaysere performed in triplicate and the average standard deviationsere always less than 2% of the mean. The enzyme deactivationrofiles were graphed in terms of residual activity (ratio of the spe-ific activity at time t (At) and zero time (A0) of incubation) in ordero compare the persistence of enzymatic activity, under differentncubation conditions that are also used in sol–gel processes.

.2.2. Deactivation kinetic constantsThe experimental data were adjusted to classical first-order (Eq.

1)) and series-type deactivation (Eq. (2) and Eq. (3)) kinetic mech-nisms. The last one assumes that the deactivation can occurs inifferent steps, with active intermediaries and active or deactivatednal enzyme states [24,25]. The fitting procedure was carried outy iterative non-lineal regression method, using the Solver tool oficrosoft Office Excel 2007 software.

t/A0 = exp(−k1 × t) (1)

k1−→E˛1

k2−→Eˇ1 (2)

t/A0 = ˇ+[(1 + ˛×k1/(k2−k1) − × k2/(k2 − k1)] × exp(−k1 × t)

− [( − ˇ) × k1/(k2 − k1)] × exp(−k2 × t)) (3)

here k1 and k2 are first-order deactivation rate constants (h−1);, E1 and E2 are specific activities of initial (E) and intermediatesE1 and E2) active enzyme states, respectively; and are ratios ofpecific activities of the intermediate and initial enzyme form, andf the final state and initial enzyme form, respectively ( = E1/E and

= E2/E).The half-life times (t1/2) were calculated with the model that

etter fit to the experimental results, according to the meanquared error (Er) and correlation factor (R2)

.2.3. Colloidal parametersThe hydrodynamic diameter (D) for the species present in the

ifferent B. circulans �-gal solutions was determined by Dynamic

Fig. 1. B. circulans �-gal decay profile at pH 6.0 and 25 ◦C, in function of the TritonX-100 surfactant concentration (0 M – 1 × 10−3 M). Each experiment was carried outthree times and the bars represent the standard deviation from the mean.

Light Scattering (DLS, Horiba Scientific LB-550 instrument, LB-550-3.57 version software) by using the Stokes–Einstein equation [26].The polydispersity percentage of these measurements (%Pd) wascalculated taking into account the standard deviation (SD) of thehydrodynamic diameter distribution:

%Pd = SDD

× 100 (4)

The z-potential for some selected B. circulans �-gal solutions wasmeasured in a Malvern Instruments Zetasizer nanoseries NANOZSequipment, at the specified pH and temperature.

2.2.4. Denaturation temperatureThis temperature was determined for some selected B. circu-

lans �-gal solutions, from Differential Scanning Calorimetry (DSC)thermogram, obtained from 15 ◦C to 350 ◦C at 10 ◦C min−1 scanrate (TA instruments DSC Q100-1151 equipment, Universal Analy-sis 2000 software), under inert atmosphere (50 mL min−1 nitrogen)and using hermetic pans.

3. Results and discussion

The B. circulans �-gal activity at pH 6.0, 25 ◦C and in the absenceof Triton X-100 varied during the incubation time (0 M, Fig. 1).The enzyme activity loss or deactivation occurred following a first-order kinetic mechanism (R2 = 0.9832, Table 1) and the enzymeactivity after 48 h of incubation was only 16% of its initial value,as it is usual for non-thermostable monomeric enzymes [24]. Thedeactivation during the incubation was expected because tempera-tures higher than 4 ◦C (more frequent storing temperature), orbitalagitation and diluted conditions make the protein molecules moreprone to aggregation, unfolding or any deleterious change in theirnative three-dimensional structure [27].

The DLS results for the B. circulans �-gal solution during thefirst 2 h of incubation showed that the enzyme molecules can bein equilibrium between tight and extended forms, whose averagehydrodynamic diameters vary ∼13 nm or as small agglomerates∼30 nm. Species larger than 100 nm appeared after 2 h of incuba-tion (Fig. 2), indicating that the barrier for an effective collisionwas overcome. Nevertheless, they coexisted with the smaller onesand 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

34 S. Escobar et al. / Biochemical Engineering Journal 75 (2013) 32– 38

Table 1Deactivation rates at 25 ◦C (k1 and k2 in h−1), ratios of specific activities of the intermediate and initial enzyme forms ( and ˇ) and half-live (t1/2 in h) values obtained byadjusting the experimental data to a classical first-order or series-type mechanism. The correlation coefficient (R2) and the mean squared error (Er) of the model are shownin each case.

Triton X-100 concentration(M) + extra-additives

pH Mechanism k1 k2 t1/2 R2 Er

0 6.0 First-order 0.031 – – – 22.2 0.9832 1.015 × 10−5 6.0 Series-type 1.341 0.677 0.018 ∼0 17.5 0.9930 0.002 × 10−4 6.0 Series-type 0.733 0.758 0.014 ∼0 30.7 0.9661 0.021 × 10−3 6.0 Series-type 0.033 0.712 0.032 ∼0 42.6 0.9726 0.000 4.5 Series-type 0.151 1.440 1.902 0.083 6.7 0.9980 0.001 × 10−3 4.5 Series-type 0.095 2.248 2.895 0.118 10.8 0.9855 0.010 9.0 Series-type 0.461 1.044 0.461 0.030 3.9 0.9934 0.021 × 10−3 9.0 Series-type 0.177 0.597 0.032 ∼0 13.3 0.9984 0.00

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3

af4w

Fs

0 + 4% ethanol 6.0 Series-type 1.557

1 × 10−3 + 4% ethanol 6.0 Series-type 0.989

-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 smallestpecies increased with incubation time for the B. circulans �-galn the absence of Triton X-100, corroborating their agglomerationTable 2).

The aqueous soluble enzymes are proteins very sensible tohe temperature variations and they also exhibit the proper-ies of colloidal dispersions, being susceptible to environmentalhanges such as pH or the presence of other chemical compoundssurfactants, salts, etc.), which could modify their native three-imensional structure [27,28]. Therefore, the B. circulans �-galtability was studied in function of the presence and concentra-ion of neutral surfactant Triton X-100, under different aqueousuffered conditions, generally used for sol–gel process. This willelp to determine if the surfactant can be used as stabilizing agentf the B. circulans �-gal, for its encapsulation in silica by sol–gelrocess or its industrial application.

.1. Effect of the Triton X-100 concentration

The B. circulans �-gal decay profile at pH 6.0 and 25 ◦C wasffected by the presence and concentration of Triton X-100 sur-

actant (Fig. 1). The �-gal preserved 42% of its initial activity after8 h of incubation in the presence of 1 × 10−3 M Triton X-100, whichas 2.6-fold higher than in the absence of surfactant (only 16%).

10 100 1000

-2

0

2

4

6

8

10

12

14

16

18 0h

1h

2h

3h

5h

24 h

Fre

qu

en

cy (

%)

Diameter (nm)

ig. 2. Evolution of the hydrodynamic diameter for B. circulans �-gal species inolution, at pH 6.0 and 25 ◦C, in the absence of Triton X-100, determined by DLS.

0.299 ∼0 ∼0 1.9 0.9999 0.000.560 ∼0 ∼0 9.3 0.9930 0.00

The adjustment of these enzymatic data to the kinetic modelsconsidered in this work [24,25] indicated that the B. circulans �-galdeactivation was not homogenous in the presence of surfactant, fol-lowing a series-type mechanism, in which the intermediate state ofthe enzyme keeps some activity before to be fully deactivated ( < 1and = 0, Table 1). The t1/2 values for these samples, calculated withthis model (Table 1), were significantly different at 0.05 significancelevel (ANOVA test). According to these t1/2 values (Table 1) andthe decay profiles (Fig. 1), the increase of the surfactant concen-tration above its critical micelar concentration (cmc = 2 × 10−4 Min phosphate buffer solution, measured by DLS) had a protectiveor stabilizing effect against the deactivation. This could be relatedto the formation of new enzyme-surfactant species, which aremore stable than the isolate enzyme molecules. This hypothesiswas corroborated by DLS, z-potential and DSC experiments. Beforeshowing the results, it is important to highlight that the effect of thenon-ionic Triton X-100 surfactant depends on both, surfactant con-centration and enzyme type, which define the kind of interactionthat would be present in each case, and therefore its stabilizingor deactivating effect, as it has been shown for other enzymes[15,30–33].

The deactivation behavior of the B. circulans �-gal in the pres-ence of Triton X-100 surfactant, during the incubation time, couldbe also associated with the aggregation of �-gal molecules, whichalso include the formation of enzyme-surfactant species, as itwas suggested by the series-type deactivation mechanism: Largerspecies (>100 nm) that accompanied the individual �-gal molecules(∼12–30 nm) appeared earlier in the solution where the surfactantis present (less than 1 h of incubation) compared with the resultsin its absence (Table 2).

On the other hand, the B. circulans �-gal deactivation profilesand t1/2 values in function of the Triton X-100 concentration, sug-gested that the presence of this surfactant in form of micelles hada higher protective effect on the enzyme activity than when it wasas individual molecules in solution or near to cmc. In fact, whenthe surfactant concentration increases in the aqueous medium, itsmolecules are less soluble and form micelles that could interactwith the �-gal through hydrophobic groups of this enzyme. Thesurfactant micellization would occur as in enzyme-free solution butwith the added feature that these micelles may be free in solution orbe bound by physical interactions to the enzyme, as occurs in othersimilar systems [34]. The formed enzyme–surfactant aggregatescould help to avoid the deactivation of the enzyme, for exampleduring the encapsulation process by sol–gel methodology as it hasbeen shown in other systems, where the added macromoleculewrap the enzyme molecules protecting them during the invasive

polymerization of silanols [35].

The �-gal/Triton X-100 interactions also promoted the reorgani-zation of the hydrophobic sites of the enzyme, which could acquirea more favorable folding state that would be responsible of its

S. Escobar et al. / Biochemical Engineering Journal 75 (2013) 32– 38 35

Table 2Hydrodynamic 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 5 × 10−5 M Triton X-100 2 × 10−4 M Triton X-100 1 × 10−3 M Triton X-100

D (nm) %Pd D (nm) %Pd D (nm) %Pd D (nm) %Pd

0 27.4 50.2 27.0 36.6 18.0*361.03115.0

35.749.454.8

16.0*475.0

64.945.5

1 13.8 79.4 21.0 53.4 21.01067.05348.0*

30.151.815.7

18.4*1412.2

48.262.0

2 18.0*1419.0

87.531.8

12.0*104.0

28.723.0

823.03640.2*

34.330.1

29.5*823.3

34.330.1

3 3649.3 47.2 18.0*417.34152.0

44.747.438.7

21.1*318.01864.4

31.636.642.3

3640.424.0*1225.0

43.445.5

5 27.0*715.02426.0

23.727.831.2

2119.65271.2*

23.419.9

4078.3 19.3 16.0*414.03202.1

52.755.032.1

24 417.3 80.8 939.1 33.3 1246.3 30.5 1229.9 31.127 939.4 50.5 1224.4 21.9 624.0

2420.040.531.6

824.3 51.3

29 1223.5 30.7 1422.5 30.2 1415.9 38.4 1223.9 29.8

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

Table 3Results 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 z-potential (mV) Denaturation temperature (◦C) %Pd

0.0 −9.8 ± 0.5 95 ± 0.5 50.2−5

lcsTicae0obintwi

3

oTitad

ievttfcp

states at extreme pH values (4.5 and 9.0) could be related with dif-ferent phenomenon: (i) variations on the hydrogen bonding, Vander Waals and electrostatic interactions at non-neutral pH val-ues, that affect the tertiary structure of the proteins [16]; (ii) the

0 10 20 30 40 500.0

0.2

0.4

0.6

0.8

1.0

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)

Re

sid

ua

l a

ctivity

Time (h)

5.0 × 10 −9.3 ± 1.0

2.0 × 10−4 −13.9 ± 1.2

1.0 × 10−3 −26.8 ± 1.0

ong-term enzymatic stability, as in the case of other enzymes [36],ompared with the behavior in the absence or low concentration ofurfactant. The variations of the z-potential values in function of theriton X-100 concentration corroborated the enzyme–surfactantnteractions and the higher effect of the surfactant when its con-entration was above to the cmc value (Table 3) (ANOVA one-waynalysis indicated that z-potential values were significantly differ-nt at 0.05 level, except for the differences between the values at

M and 5 × 10−5 M according to the Tukey test). The reorganizationf the hydrophobic sites for the B. circulans �-gal was responsi-le of the denaturalization temperature changes for this enzyme,

n function of the surfactant concentration (Table 3). It would beoticed that at zero time, the inverse relation between the %Pd andhe denaturation temperature for the B. circulans �-gal (Table 3),as also an indication of the conformational changes during the

ncubation [37].

.2. Effect of the pH

The deactivation of B. circulans �-gal at 25 ◦C, in the absencef surfactant, was faster at pH 4.5 and 9.0 than at pH 6.0 (Fig. 3).his could be related with the enhancing of electrostatic repulsions,onization of acid or basic groups in the active site and hydrolysis ofhe peptidic bonds at pH far from 6.0 [16]. All of these phenomenaffected the B. circulans �-gal native structure, leading to its fasteactivation at pH 4.5 and 9.0.

The deactivation was also faster at pH 4.5 and 9.0 than at pH 6.0n the presence of 1 × 10−3 M Triton X-100. However, the stabilizingffect of the surfactant was seen, increasing the t1/2 at these pHalues (Table 1). Probably, the Triton X-100 surfactant protectedhe B. circulans �-gal against the acid or base attack by modifying

he enzyme conformation or its environment. The effect of the pHor other enzymes in contact with Triton X-100, has been observed,orroborating that it depends on the surfactant concentration andhysicochemical characteristics of each enzymatic system [38].

75 ± 0.5 36.675 ± 0.5 35.765 ± 0.5 64.9

The adjustment of the experimental data to the kinetic models[24,25] indicated that at extreme pH (4.5 and 9.0) the deactiva-tion followed a series-type deactivation mechanism more than theclassical one followed at pH 6.0 (Table 1). This corroborated thatthe deactivation at these extreme pH values is the consequence ofa more complex process, which can involve active enzyme inter-mediates ( different to 0 at pH 4.5 and pH 9.0) but the final enzymestate was almost fully inactivated ( values tend to very small val-ues near to zero, Table 1). The presence of intermediate enzyme

Fig. 3. Effect of the pH on the �-gal enzymatic activity at 25 ◦C, in the absence (emptysymbols) or 1 × 10−3 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 thestandard deviation from the mean. In some cases the symbols are bigger than thebars.

36 S. Escobar et al. / Biochemical Engineering Journal 75 (2013) 32– 38

0 10 20 30 40 50

0.0

0.2

0.4

0.6

0.8

1.0

1.2

25 (0M)

25 (1 x 10-3 M)

40 (0M)

40 (1 x 10-3 M)

Re

sid

ua

l a

ctivity

Time (h)

Fig. 4. Effect of the incubation temperature on the B. circulans �-gal activity, at pH6.0, in function of the presence of surfactant (0 M and 1 × 10−3 M Triton X-100).Ed

dets

3

aipeiadaTawdlaimsphl

i1dicsamoBi

0 2 4 6 8

0.70

0.75

0.80

0.85

0.90

0.95

1.00

1.05

1.10

0.1M Na (0M)

0.1M Na (1x10-3M)

0M Na (0M)

0M Na (1x10-3M)

Re

sid

ua

l a

ctivity

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 × 10−3 MTriton X-100). Each experiment was carried out three times and the bars represent

ach experiment was carried out three times and the bars represent the standard

eviation from the mean. In some cases the symbols are bigger than the bars.

isulfide shuffling, specially at high pH where the thiol-disulfidexchange is favored, causing conformational changes that affecthe enzyme activity [39,40] and (iii) the alteration of the oxidationtage in the active site of the enzyme [41].

.3. Effect of temperature

The enzyme stability for B. circulans �-gal at 40 ◦C was lessffected than at 25 ◦C, during the incubation time at pH 6.0,ndependent of the presence of Triton X-100 (Fig. 4). This is an unex-ected result, because the most common phenomenon is that thenzyme stability decreases with temperature, because the stabilitys clearly related to molecular stiffening. However, the activationt 40 ◦C for two isoforms of B. circulans �-gal [2] and for �-gal pro-uced by pure and mixed cultures of Streptococcus thermophilusnd Lactobacillus delbrueckii ssp bulgaricus [42] was reported before.his behavior has been attributed to conformational changes in thective site of other enzymes, in the absence of surfactant [42,43],hich is also corroborated here by the variability on the stabilityuring the initial stages of heating (Fig. 4). Although the enzyme is

ess stable at 25 ◦C than at 45 ◦C, working at 25 ◦C could be verydvantageous for the B. circulans �-gal encapsulation into a sil-ca network, through sol–gel process. The synthesis of siliceous

aterials at room temperature allows controlling the enzyme-ilica interactions as well as enzyme-silica-surfactant ones and theolycondensation of siliceous species, which is very important foraving an efficient encapsulation in terms of quantity of encapsu-

ated enzyme.The mean value of the enzyme activity at 40 ◦C was 96.9 ± 7.6

n the absence of surfactant and 99.4 ± 3.9 in the presence of × 10−3 M Triton X-100. The two samples t-Student test for theseata showed that there was no significant difference at 0.05 level

n the enzyme decay profiles at these two levels of surfactant con-entration, contrary to what was observed at 25 ◦C, where theurfactant had a protective effect. In this case, Triton X-100 actss positive modulator probably due to the rigidity of the enzymeolecules and aggregates caused by its presence [16]. The changes

n the surfactant micellization and cmc; enzyme aggregation andrownian movement of the all molecules in the system with the

ncrease of the temperature, could weaken the enzyme-surfactant

the standard deviation from the mean. In some cases the symbols are bigger thanthe bars.

interactions, rendering the enzyme behavior similar in the presenceand in the absence of surfactant at 40 ◦C.

3.4. Effect of the presence of ethanol or sodium chloride

The tetraethylorthosilicate and sodium silicate are commonsources of silica, among other alcoxysilanes and alkylalcoxysilanes,used in the synthesis of siliceous materials by sol–gel process. Thesetwo sources produce ethanol or sodium ions, respectively duringthe reaction [21,44] and they could have effects on the enzyme,which will be studied here as examples, taking common experi-mental values for each one.

The effect of the sodium cations during the first 3 h of incubationfor B. circulans �-gal solution at pH 6 and 25 ◦C in the absence ofTriton X-100 surfactant was not significant (Fig. 5), as it was testedby two-samples t-Student’s test, at 0.05 level, for the mean resid-ual activity (0.898 ± 0.088 and 0.910 ± 0.063 in the absence andpresence of 0.1 M NaCl, respectively). Similarly to the surfactanteffect, the positive or negative effect of a salt is highly dependenton its concentration and the enzyme type, under specific incubationconditions, because the interactions for each enzyme–salt pair andthe changes in water organization are different [45,46]. However, asynergic effect on the enzyme stability was seen when the sodiumcations and Triton X-100 were present in the incubation mediumat the same pH and temperature conditions, reinforcing the protec-tive effect of this surfactant on the B. circulans �-gal stability (Fig. 5).The two samples t-Student test for the mean residual activities forB. 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 with1 × 10−3 M Triton X-100) showed a significantly difference at 0.05level in function of the presence of Triton X-100.

The presence of 4% ethanol affected negatively the enzyme sta-bility of B. circulans �-gal incubated at pH 6.0 and 25 ◦C (Fig. 6), as ithad been observed for other enzymes, because its presence disturbsthe hydrogen bonding and hydrophobic interactions, involved inthe enzyme stabilization [47,48]. However this effect was lower in

the presence of Triton X-100 surfactant (Fig. 6), which increased thet1/2 (Table 1), corroborating that the surfactant in form of micellesinteracted with the enzyme, inhibiting the action of the alcohol.

S. Escobar et al. / Biochemical Engin

0 2 4 6 8

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0% Eth (1x10-3M)

0% Eth (0M)

4% Eth (0M)

4% Eth (1x10-3M)

Re

sid

ua

l a

ctivity

Time (h)

Fig. 6. Effect of the presence of 4% ethanol on the B. circulans �-gal activity at pH6.0 and 25 ◦C in function of the presence of surfactant (0 M and 1 × 10−3 M TritonX-100). Each experiment was carried out three times and the bars represent thesb

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combined approach of experiments and molecular docking, Colloids Surf. B 97

tandard deviation from the mean. In some cases the symbols are bigger than thears.

. Conclusions

The correlation between the enzymatic activity and colloidaltability data (obtained by Dynamic Light Scattering and z-otential), supported by thermodynamic results (denaturationemperature obtained by Differential Scanning Calorimetry) mayelp to explain the deactivation behavior for the �-galactosidase

rom B. circulans, which varies in function of the Triton X-100urfactant concentration. A protective effect against the enzymeeactivation during the incubation time, under different conditionstemperature, pH, sodium and ethanol presence), was observedhen the surfactant concentration was higher than the criti-

al micelle concentration, due to the hydrophobic interactionsetween �-galactosidase molecules and Triton X-100 in form oficelles.The study of the effect of the pH, temperature and presence of

odium cations or ethanol, in function of the presence or absence ofriton X-100 led to establish the preliminary conditions for encap-ulating �-galactosidase from B. circulans, by a sol–gel processssisted by surfactant:

1 × 10−3 M Triton X-100, at pH 6.0 and 25 ◦C or 40 ◦C during lesshan 24 h, from a sodium silicate or tetraethylortosilicate that didot exceed the liberation of 0.1 M sodium cations or 4% ethanol,espectively to the reaction medium during the silica polyconden-ation.

Because each enzyme must be studied as a unique system, theesults obtained by Dynamic Light Scattering, z-potential deter-ination, Differential Scanning Calorimetry combined with the

ctivity assays provide indispensable tool for design of enzymencapsulation by sol–gel process.

cknowledgements

We thank Colciencias for the financial support, through the11552128274 Project “Evaluación y optimización de la hidrolisisnzimática heterogénea de la lactosa del suero lácteo como un paso

revio a la fermentación para la producción de etanol” and Univer-idad de Antioquia through “Estrategia para la Sostenibilidad de losrupos de Investigación 2013–2014”

[

eering Journal 75 (2013) 32– 38 37

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