Design of β-galactosidase/silica biocatalysts: Impact of the enzyme properties and immobilization...

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Claudia Bernal

Ligia Sierra

Monica Mesa

Grupo Ciencia de los Materiales,Instituto de Quımica,Universidad de Antioquia,Medellın, Colombia

Research Article

Design of β-galactosidase/silica biocatalysts:Impact of the enzyme properties andimmobilization pathways on their catalyticperformance

The use of heterogeneous biocatalysis in industrial applications is advantageous andthe enzyme stability improvement is a continuous challenge. Therefore, we designedβ-galactosidase heterogeneous biocatalysts by immobilization, involving the sup-port synthesis and enzyme selection (from Bacillus circulans, Kluyveromyces lactis,and Aspergillus oryzae). The underivatized, tailored, macro-mesoporous silica ex-hibited high surface area, offered high enzyme immobilization yields and activity.Its chemical activation with glyoxyl groups bound the enzyme covalently, whichsuppressed lixiviation and conferred higher pH and thermal stability (120-fold thanfor the soluble enzyme), without observable reduction of activity/stability due to thepresence of silica. The best balance between the immobilization yield (68%), activity(48%), and stability was achieved for Bacillus circulans β-galactosidase immobilizedon glyoxyl-activated silica, without using stabilizing agents or modifying the enzyme.The enzyme stabilization after immobilization in glyoxyl-activated silica was similarto that observed in macroporous agarose-glyoxyl support, with the reported mi-crobiological and mechanical advantages of inorganic supports. The whey lactolysisat pH 6.0 and 25◦C by using this catalyst (1 mg ml−1, 290 UI g−1) was still 90%,even after 10 cycles of 10 min, in batch process but it could be also implemented oncontinuous processes at industrial level with similar results.

Keywords: β-Galactosidases immobilization / Enzyme adsorption / Lactolysis / Multipointcovalent attachment / Thermal stabilization

� Additional supporting information may be found in the online version of this article atthe publisher’s web-site

Received: January 2, 2013; revised: March 1, 2013; accepted: April 23, 2013

DOI: 10.1002/elsc.201300001

1 Introduction

The β-galactosidase (β-gal) or lactase (E.C. 3.2.1.23, β-gal) cat-alyzes the hydrolysis of lactose from milk and whey into glucose

Correspondence: Prof. Monica Mesa ([email protected]),Grupo Ciencia de los Materiales, Instituto de Quımica, Universi-dad de Antioquia, 62 St. # 52-55, Medellin, Colombia.

Abbreviations: β-gal, β-galactosidase; GOS, galacto-oligosaccharides;K. lactis, Kluyveromyces lactis; B. circulans, Bacillus circulans; A.oryzae, Aspergillus oryzae; EDA, ethylenediamine; CDI, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; SGx, silica-glyoxyl; AGx, Glyoxyl-activated agarose supports; IY, immobilization yield; Vmax, maximumreaction rate; Km, Michaelis-Menten affinity constant; Kcat, turnovernumber

and galactose [1,2] and exhibits also transferase activity for pro-ducing galacto-oligosaccharides (GOS) [3–5]. Its physicochemi-cal properties and intrinsic stability toward pH and temperaturedepend on its source, extraction, and purification procedures, asit has been reported for example for β-gal from Kluyveromyceslactis (K. lactis) [6, 7], Bacillus circulans (B. circulans) [8], andAspergillus oryzae (A. oryzae) [9, 10]. Even though the first twoenzymes have a similar molecular weight, the dimeric nature ofthe K. lactis β-gal stabilized by magnesium ions, makes this en-zyme very sensible toward deactivation by the presence of othercations in the catalysis medium [11, 12]. The challenges relatedwith their use in industrial applications include improvementson the stability against changes of pH and temperature of thereaction medium (modulated to increasing the GOS production[4,5] or lactose hydrolysis efficiency [2]) and implementation in

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continuous and sequential batch processes [13]. Both objectivescan be achieved by enzyme immobilization on solid supports[1, 14, 15], which contributes to decrease the industrial processcosts.

Agarose-based supports activated with glutaraldehyde, gly-oxyl, or epoxy groups have been widely used due to their highcompatibility and geometric congruence with the enzymes andthe ability for increasing the thermal stability, as a consequenceof the enzyme stiffening after the multipoint covalent attach-ment, without significantly affecting the enzymatic activity afterimmobilization [16–18]. Especially the glyoxyl-activated agarosesupports, in which the aldehyde groups are moderately separatedfrom the gel surface, offer low steric hindrances for reactions withthe nonionized amine groups of the enzyme (in the lysine-richregions) and the multipoint covalent attachment, promoted atalkaline pH, confers higher thermal stability to the enzyme thanin other supports [19–21]. The enzyme multipoint covalent im-mobilization on glyoxyl-activated supports occurs at pH 10.0by the formation of Schiff bases between the aldehyde groupof the support and neutral ε-amine groups of the enzyme andthe subsequent stabilization of the chemical bond by reduction[19–21].

Compared with the agarosed-based supports, the silicas withcontrolled porous structures offer special properties as immo-bilization supports, such as high surface area, thermal and me-chanical stability, ease of handling, ability to withstand highflow rates in continuous reactors, nontoxicity, and they are notattacked by microbes and organic solvents [22]. The tailoring oftheir chemical, porous, and morphological characteristics, forbeing used as enzyme supports, is an active field that offers newopportunities in heterogeneous biocatalysis [23–25]. The poresize of these silicas can be modulated for immobilization of alarge variety of biomolecules. An actual challenge is to enlargetheir pore size for immobilization of enzymes of relative highmolecular weight, as the β-gal evaluated in this work [6–10] orothers with MW>100 kDa. This lead to enhance the enzyme-loading capacity [26], favors the substrate diffusion toward theenzyme active site and also maintain a higher enzyme confor-mational freedom within the pores, which improves the enzymeactivity [27]. The silanol groups in the silica surface are involvedin the enzyme immobilization by adsorption (mainly throughvan-der-Waals forces, hydrogen bonding, electrostatic, and dipo-lar interactions) or can be modified by silylation to activate thesematerials as support for covalent immobilization (for example,silica can be activated with glyoxyl functionalities for promotingthe formation of Schiff bases) [22, 26, 28–31].

There are several reports about the covalent immobilization ofdifferent β-gal in silica gel [32], porous silica [33], or controlledporous glass [2] silylated with aminoalkyloxysilane and activatedwith glutaraldehyde), at pH ∼ 7.0. These siliceous supports ex-hibit higher capacity for the β-gal attachment and higher glucoseproduction compared with a cyano-agarose activated support,as a consequence of the high surface area and higher flow ratesachieved in them [33]. It has also shown that the quantity of an-chored enzyme and its activity depend more on the pore fractionhaving diameter size larger than 15 nm than on the total specificsurface area [34]. The use of glyoxyl-activated silica as immo-bilization support and the effect of its activation degree in thecatalytic behavior and stability for penicillin G acylase have been

studied [35, 36] and a recent publication shows that hierarchi-cal macro-mesoporous glyoxyl-activated silica is responsible forthermal stabilization of β-galactosidase from B. circulans [37].

The objective of this work is to design β-gal/silica biocat-alysts, with improved thermal and pH stability for their use inrepeatedly batch lactolysis processes, by using tailored porous sil-ica (synthesized under conditions for achieving large mesoporesconnected with macroporous, high surface area, and a block-typemorphology), underivatized or activated with glyoxyl groups.The characteristics of this support allow diffusion of β-gal fromthree sources (K. lactis, B. circulans isoenzyme I, and A. oryzae)to the surface of the pores, where its surface chemistry mod-ulates the β-gal/support interactions (adsorption or chemicalbonding), giving different immobilization yield (IY), expressedactivity, thermal, and pH stabilities after the immobilization. Thebiocatalysts obtained in this work are very promising candidatesfor implementation also in lactolysis continuous processes andfor production of GOS in continuous or batch systems, underdifferent pH and temperature conditions.

2 Materials and methods

2.1 Materials

The silica-based supports were synthesized by following the hy-drothermal, calcination, and chemical activation procedures re-ported in recent publications [26,38,39]. Sepharose CL-6B bead(6% agarose, cross-linked with epichlorohydrin, GE Healthcare,Spain) was the organic counterpart. The β-gal from B. circu-lans (BIOLACTASA NTL X2 3000 IU) and A. oryzae (100 000IU) were purchased to Biocom (Barcelona, Spain) and Sigma(Missouri, USA), respectively, β-gal from Kluyveromices lactys(LACTOZYM 300IU) was donated by NOVO (Denmark). Thelactose (Sigma) and glucose oxidase-peroxidase assay kit (fromByosystem, Spain) were used for measuring the catalytic activity.Other analytical grade reactants purchased from Sigma (Mis-souri, USA) were sodium borohydride, ethylenediamine (EDA),1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, glycerin, andglycidol.

2.2 Synthesis, chemical modification, andcharacterization of siliceous supports

The experimental details [26,38,39], including those related withthe porous, morphological, and chemical characterization for thebare (S) and silica glyoxyl-activated (SGx##) homemade poroussilica supports used in this work are given in the Supportinginformation.

2.3 Glyoxyl-activated agarose supports (AGx)preparation and characterization

The Sepharose CL-6B, chosen in this work for having similarmacroporosity to the synthesized silica, was activated with gly-cidol and subsequently oxidated with NaIO4. The glyoxyl group

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content was quantified as previously described by Guisan [19].The code for this agarose-glyoxyl sample is “AGx.”

2.4 Hydrodynamic diameter and pI for the enzymes

The enzyme hydrodynamic diameter was measured in aque-ous buffered solutions, at pH 6.0 and 10.0 at 25◦C, by Dy-namic Light Scattering (Horiba instrument). The zeta-potential(z-potential), for enzyme aqueous solutions prepared at differ-ent pH values (controlled by addition of 0.1 M NaOH or 0.1 MCH3COOH), was measured by using a Nanozetasizer Nano ZSMalverninstrument at 25◦C and the pI was determined from thez-potential/pH curve.

2.5 Activity assay

The specific activity for the soluble and immobilized enzymeswas measured by visible spectroscopy (Lambda35 UV/Vis spec-trophotometer), under stirring and 25◦C, following the glucoseproduction at 500 nm in the presence of commercial glucose kitas color-developer reactant [26, 40]. One international unit ofactivity (IU) was defined as the amount of enzyme that produces1 μmol of glucose per min under the described conditions [26].The experiments were made by triplicate and the SDs respectingto the mean value are calculated.

2.6 Immobilization process for preparing the hybridbiocatalysts

In a general procedure, the β-gal solution and exact amount ofsupport were put in contact, under mild orbital stirring, and thespecified conditions in each case. The activity assay for aliquotsof supernatant and hybrid biocatalyst was measured at differenttimes during the immobilization process. In parallel, a refer-ence solution of the free enzyme was incubated under the sameconditions (blank). The IY was calculated according to:

Y = 100 − 100(S/B i) (1)

where Bi and S are specific activities in the blank (at zero time)and in the immobilization supernatant suspension, respectively

Immobilization by adsorption: 1 g freshly calcined silica “S”was dispersed into 5 mL of enzymatic solution (100 IU ofβ-gal solution, prepared in 100 mM phosphate buffer, pH 6.0).This dispersion was left under orbital agitation at 25◦C duringdifferent times in order to follow the adsorption kinetic. Thehybrid material was recovered by filtration and washed severaltimes with 100 mM phosphate buffer solution. The enzyme-loading capacity (mg enzyme per gram of support) was deter-mined by thermogravimetry (TGA, TA-500 instrument), follow-ing the weight loss between 250 and 350◦C, under air flux (theenzyme represents the only source of organic matter that sufferscombustion in the sample).

Immobilization by covalent attachment in SGx and AGx##

supports: It was carried out by mixing 5 mL of buffered en-zyme solution (100 IU of β-gal) per 1 g of support, at pH 10.0(100 mM potassium bicarbonate). The suspension was incu-

bated at 4◦C during different times. In some cases, the mediumalso contains 40% glycerine in order to see if it could help topreserve the enzyme activity during the immobilization, dueto its enzyme stabilizing effect [41, 42] and its effect on theviscosity. Finally, the derivative was reduced by using 4 mg ofNaBH4 per mL of solution, during 30 min at 4◦C, as previousdescribed in glyoxyl-activated agarose-based systems [20,21]. Atthe end, the solid was washed. The loading capacity of glyoxyl-activated samples is determined from the difference between theoffered and final protein concentration (at the end of the immo-bilization process) in the supernatant, measured by the Bradfordmethod.

Some covalent immobilization experiments with the β-galfrom A. oryzae were carried out with the enzyme chemicallymodified by inserting amino groups on the glutamic and as-partic acid residues before the immobilization experimentswith the objective to improve multipoint covalent attachmentas it was described before [43]. The A. oryzae β-gal solution(2.5 mg mL−1) was mixed with 1M EDA solution at pH 4.8 (1:1proportion), then 10 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was added and the solution was stirred by 1.5 h atroom temperature. At the end, the solution was dialized.

2.7 Lixiviation test

The heterogenous biocatalyst was put in contact with 0.1 Mphosphate buffer (pH 7.0) and 0.1 M KCl solution, under or-bital agitation and the enzymatic activity of the supernatant wasmeasured at settled times.

2.8 Determination of the pH and temperature ofenzyme maximal activity

The activity for each prepared heterogeneous biocatalyst wasmeasured, as described under activity assays but at differentpH values (pH 4.5–9.0 modulated in appropriated buffered so-lutions: acetate buffer for pH 4.5–6.0 and phosphate for pH6.5–9.0) and at different temperatures (25–55◦C) and the con-ditions for maximal activity were determined. The activity mea-surements were carried out under high substrate concentration(lactose, 80 mM).

2.9 Inactivation assays for determining the enzymestabilization

The heterogenoeus biocatalysts were incubated under differentpH (4.5, 6.0 and 9.0) and temperature (25 and 55◦C) conditionsin the absence of substrate. The activity for aliquots of thesesamples was determined periodically and the residual activitywas calculated every time respecting to the initial activity. Thestabilizing factor is defined as the ratio of the half-life times forthe heterogenous biocatalysts and the soluble enzyme, under thesame incubation conditions.



2.10 Kinetic parameters

The maximum reaction rate (Vmax) of the enzymatic reaction, theMichaelis–Menten affinity constant (Km) and turnover number(Kcat) were determined from the Lineweaver Burk plot, assuminga steady state [44]. The data were obtained at 25◦C and pH7.0, for lactose concentrations between 5–80 mM. The lactoseconversion was measured by UV-Vis spectroscopy (Lambda35UV/Vis spectrophotometer).

2.11 Whey lactolysis catalyzed by different β-galpreparations

It was made in order to evaluate the Bc-SGx1.0 catalytic behaviorin the whey matrix (5% lactose), at pH 6.5 and 4C or 25◦C.The heterogeneous biocatalyst (290 IU per gram of support)was dispersed in the whey (1 mg mL−1) and some repeatedlactolysis cycles of 10 min each one, were carried out. The lactoseconversion (expressed as the percentage of hydrolyzed lactose)and the biocatalyst residual activity (measured as described inthe activity assay) were determined after each cycle.

2.12 Statistics

All experiments are done by triplicate and the SD is calculatedaccording to:

σx= ±√(0.5�(xi − x)2) (2)

where xi is the individual value for every measure and x is theaverage value in the experiment.

3 Results and discussion

3.1 β-Gal immobilization by adsorption on bare silica(S) support

The hierarchical porosity of the silica, synthesized in this workfrom a cheap source of silica (sodium silicate), was tailored forhaving mesopores with diameter higher than 15 nm connectedwith macropores and surface area equal to 457 m2 g−1 [38]. Thesecharacteristics allow its use as support for immobilizing highmolecular weight proteins, such us the β-gal evaluated in thiswork (MW>100 kDa, Supporting information, Table 1). Theimmobilization conditions by adsorption were selected takinginto account the pH and temperature range of stability for thethree enzymes [6–10].

3.1.1 Adsorption kinetics and loading capacity at pH 6.0The enzyme adsorption equilibrium is reached after 6 h of theimmobilization process for B. circulans and 7 h for K. lactisβ-gal, at pH 6.0 (Fig. 1). The adsorption rate (Fig. 1) andload capacity (determined by TGA, after 10 h of immobiliza-tion) in the bare silica are higher for β-gal from B. circulans(70.1 mg g−1 support) than for K. lactis β-gal (35.5 mg g−1

Figure 1. Immobilization kinetics for β-galactosidase from B. cir-culans (square) and K. lactis (triangle) in siliceous support byadsorption (full) and by covalent attachment in SGx6.0 support(empty). The bars stand for the SD between the three indepen-dent repeated experiments.

support) at pH 6.0 and room temperature. These results are ex-plained by z-potential values (Supporting information, Fig. 3),which suggest a stronger electrostatic attraction between B. cir-culans β-gal (z-potential = −25.0 mV) and silica (z-potential =−11.0 mV) than in the case of K. lactis β-gal (z-potential =−12.5mV). These results corroborate the role of the electrostatic in-teractions on the immobilization process, as it was shown beforefor K. lactis β-gal in bare silica [26].

3.1.2 Comparison of the immobilization efficiency byadsorption for the different β-gal

The adsorption–desorption of β-gal from B. circulans on thelarge surface area of the bare silica led to IY and expressed ac-tivity higher than 80% at pH 6.0 after 10 h of immobilizationprocess (Bc-S sample, Table 1). The IY and expressed activity forthe β-gal from K. lactis are lower than for B. circulans β-gal (Table1). The silica pore dimensions, which are higher than the aver-age hydrodynamic diameter for these β-gal (at 25◦C and pH 6.0,Fig. 2A), favors their diffusion into the mesopores, enhancedby the presence of macropores. However, the weaker K. lactisβ-gal-support interactions (based on the z-potential values, asit is shown above), affecting the adsorption–desorption equilib-rium, are responsible of the lower IY for this enzyme.

The immobilization by adsorption does not confer high ther-mal stabilization at 55◦C neither for K. lactis nor for B. cir-culans β-galactosidases (stabilization factors for Kl-S and Bc-S biocatalyst, Table 1), because the adsorption does not pro-mote the enzyme stiffening. The immobilization experiments atpH 4.5 with β-gal from A. oryzae, which is the smallest enzymemolecule tested in this work, exhibiting an pI equal to 3.6, led toa low quality biocatalyst by the adsorption pathway. The inter-actions between the negative charged evaluated β-gal and baresilica support are so weak, that the lixiviation is extensive dur-ing the use of these biocatalysts and the half- life time of these



Table 1. Immobilization results for β-galactosidases from B. circulans (Bc), K. lactis (Kl), and A. oryzae (Ao) in bare (S) and glyoxyl-activatedsiliceous (SGx) and agarose-based (AGx) supports. The values are measured after 2 h or 10 h of immobilization by covalent or adsorptionpathway, respectivelya)

Immobilization Expressed Lixiviation Optimum Optimum Stabilization factoryield (%) activity (%) (%) pH temperature (◦C) at 55◦C and pH 6.0

Bc-S 81 ± 5 91 ± 7 71 6.0 ± 0.5 45 ± 1 2Bc-SGx6.0 66 ± 6 40 ± 6 0 6.0 ± 0.5 45 ± 1 120Bc-AGx 26 ± 5 60 ± 5 0 6.0 ± 0.5 45 ± 1 130Kl-S 40 ± 5 82 ± 6 22 6.5 ± 0.5 37 ± 1 4Kl-SGx6.0 30 ± 6 1 ± 5 0 n.d. n.d. n.d.Kl-SGx6.0 + MgCl2 3M 24 ± 5 10 0 6.5 ± 0.5 37 ± 1 50Kl-SGx6.0 + gly 60 ± 8 11 ± 6 0 6.5 ± 0.5 37 ± 1 128Ao-SGx6.0 20 ± 7 15 ± 5 0 4.5 ± 0.5 50 ± 1 n.d.Ao*-SGx6.0 54 ± 6 62 ± 5 0 4.5 ± 0.5 50 ± 1 25Ao-AGx 15 ± 7 35 ± 6 0 4.5 ± 0.5 50 ± 1 11

a)Adsorption is carried out with bare silica (S) and covalent pathway is on the glyoxyl-activated support (SGx6.0 and AGx).*The chemical surface of this enzyme is modified with EDA before its immobilization.n.d.: non-determined.The experiments are carried out by triplicate and the SDs are calculated.

Figure 2. Hydrodynamic diameter for the β-galacatosidses fromdifferent sources obtained by DLS at pH 6.0 (A) and 10.0 (B).

heterogeneous biocatalysts, compared the free enzyme, is notimproved (Table 1).

On the other hand, the temperature and pH values for themaximum enzyme activity of the β-gal/silica biocatalysts do notchange respective to the values for the free enzyme (Table 1).These results indicate insignificant irreversible changes on theenzymes 3D structure due to the weak enzyme-support inter-actions, even though the protein adsorption on a solid sur-face causes conformational reorientations for some enzymes,depending on the immobilization conditions [45, 46].

3.2 β-gal immobilization by covalent attachment inglyoxyl-activated silica supports (SGx)

The derivatization of the silica for obtaining the glyoxyl-activatedSGx6.0 support produced a slight decrease of the surface areaand pore volume of the support up to 350 m2 g−1 and0.57 cm3 g−1, respectively, and made the pore size distributionnarrower but without affecting the average cavity size (Support-ing information, Fig. 1A), which indicates that the material stillpreserves the qualities for being used as immobilization supportfor the chosen β-gal because the pore dimensions fit well withthe enzyme diameter [37]. The results in Table 1 (optimum pHand optimum temperature) show the covalent immobilizationof the evaluated β-gal in this glyoxyl-activated silica supportsoccurred without affecting the optimum pH and temperaturefor their maximal enzyme activity.

3.2.1 Comparison of the immobilization efficiency byadsorption and covalent attachment

The covalent immobilization of β-gal from B. circulans andK. lactis on SGx6.0 support, from aqueous buffered solution atpH 10.0, leads to lower IYs than their respective adsorption onthe bare silica at pH 6.0 (Bc-SGx6.0 versus Bc-S and Kl-SGx6.0

versus Kl-S samples, Table 1). This trend is also observed for theexpressed activities (Bc-SGx6.0 versus Bc-S and Kl-SGx6.0 versus



Kl-S samples, Table 1) and for the loading capacity (for examplefor β-gal from B. circulans is 70.1 and 22.0 mg protein per gramof support by adsorption and covalent immobilization, respec-tively). Aggregation or dissociation of the enzyme is excluded ascause of this apparently lower loading capacity, as it is shownby DLS for the buffered solutions of the enzyme at pH 10.0(Fig. 2B). The fact that some glyoxyl groups are not exposed andavailable for reacting with the enzymes, due to the porous silicacharacteristics that cause internal diffusion restrictions, appearsto be responsible for these results.

3.2.2 Comparison of the immobilization efficiency bycovalent attachment for the three evaluated β-gal

The IY varies according to the following order: Bc-SGx6.0

>Kl-SGx6.0>Ao-SGx6.0 in buffered solutions at pH 10.0. The3D distribution of lysine residues, which are involved in theimmobilization by covalent bonding in glyoxyl-activated sup-ports [19–21], is different for each enzyme, which couldexplain these differences. Although β-gal from A. oryzaehas an appreciable lysine content (5% total amino acid,http://www.uniprot.org/uniprot/B7VU80), it is probable thattheir distribution does not favor the immobilization process,resulting in low IY. This is corroborated when the surface ofβ-gal from A. oryzae is modified by amination of their glutamicand aspartic acid residues with EDA, which increases the IY up54% (compare samples Ao-SGx6.0 and Ao*-SGx6.0, Table 1). Thechemical modification of soluble enzymes before their immo-bilization has been reported already as complementary tool forimproving the multipoint covalent attachment [43, 47], whichallows obtaining more loaded and active heterogeneous biocat-alysts.

The expressed activity for the β-gal from K. lactis, covalentlyimmobilized on SGx6.0 support at pH 10.0 from buffered aque-ous solution (Kl-SGx6.0 sample Table 1), is very low comparedwith the values for β-gal from B. circulans or A. oryzae im-mobilized under the same conditions (Bc-SGx6.0 and Ao-SGx6.0

samples, Table 1). The strong deactivation of K. lactis β-gal islowered when the immobilization experiments are carried outat the same temperature and pH but in the presence of MgCl2

or 40% glycerine (Table 1). These results indicate that both, (i)the magnesium–sodium ionic exchange and (ii) the enzyme dis-sociation [48], contribute to decrease the enzyme activity. Theionic exchange occurs between the Mg2+ that stabilizes the K.lactis β-gal structure [11, 12] and Na+ ions present in the silicastructure. Despite the protective effect of the glycerine againstthis deactivation process [41,42] and the increase of the mediumviscosity, which hampers the diffusion of sodium cations fromthe silica structure, a further increase of glycerine concentra-tion is not practical because it also affects the transport of theenzyme molecules toward the pores during the immobilizationprocess. The immobilization of K. lactisβ-gal in glyoxyl-activatedagarose (sodium-free support) without deactivation [49] is an-other proof that the sodium ions, present in the silica structure,are involved in the enzyme deactivation.

3.2.3 Lixiviation, thermal, and pH stabilityIn spite of the lower IY and expressed activity, the covalent im-mobilization on SGx## support confers higher thermal stability

Figure 3. Half-life time for soluble and immobilizedβ-galactosidase from (A) A. oryzae at 55◦C and pH 4.5(black), 25◦C and pH 4.5 (gray), 25◦C and pH 9.0 (pattern), and(B) B. circulans at 55◦C and pH 6.0 (black), 25◦C and pH 4.5(gray), 25◦C and pH 9.0 (pattern). The bars stand for the SDbetween the three independent experiments.

at 55◦C, at least for β-gal from B. circulans and K. lactis (the lat-ter immobilized in the presence of glycerine and MgCl2), and itcompletely suppresses the lixiviation as it is expected, comparedwith the adsorption on the bare silica (Table 1).

The surface chemical modification of the A. oryzae β-gal,increasing its stiffening and avoiding the denaturation duringheating [43,44,48,49], has only low impact on its thermal stabil-ity (Ao*-SGx6.0 sample, Table 1) compared with the results forother two β-gal. In fact, the amination of the free enzyme makesit more labile (Supporting information, Fig. 4) as this processalters the charge on the protein surface with consequences for itsstability and activity.

The improvements of the B. circulans and A. oryzae β-gal half-life under extreme pH and temperature conditions are similarto those achieved by their immobilization in agarose-glyoxylactivated (AGx) support under similar conditions in (Fig. 3).

3.2.4 Effect of chemical surface characteristics of thesupport on the B. circulans β-gal covalentimmobilization

The concentration of glyoxyl groups on the SGx material canbe modulated by varying the NaIO4 concentration used for the



Figure 4. Immobilization results for β-galactosidase from B. cir-culans in function of the support surface chemical characteristics.IY: immobilization yield, EA: expressed activity and SF: stabiliza-tion factor at 55◦C at pH 6.0. The bars stand for the SD betweenthe three independent experiments.

oxidative treatment (Supporting information, Table 2). The IY,expressed activity, and thermal stability at 55◦C for the Bc-SGx##

biocatalysts vary as a function of this glyoxyl content on thesupports (Fig. 4). The best balance between these parameters isobtained when the silica modified with 1.0 mmol glyoxyl g−1

is used as support. The enzyme stiffening caused by covalentattachment in the supports with different activation degree ex-plains the best thermal stability of the B. circulans β-gal in thissupport [37]. Multipoint attachment and stiffness increase asthe glyoxyl content increases, improving IY and thermal stabil-ity (S-Gx1.0). However, when this is so high that the stiffness isexcessive, this leads to a distortion of the enzyme 3D structureand to restricted conformational changes reducing the catalyticeffect.

3.3 Kinetic parameters for soluble and covalentlyimmobilized B. circulans β-gal

Soluble and immobilized β-gal from B. circulans both followthe Michaelis–Menten kinetic model but with different param-eter values. The Lineweaver–Burk (double reciprocal) plot forthe immobilized enzymes shows an inflection point around 30mM lactose (Supporting information, Fig. 6), which indicatesthat internal diffusion restrictions for substrate are present inthese biocatalysts as a normal consequence of the immobiliza-tion in solid supports. Therefore, apparent kinetic parametersKm and Vmax are estimated (Table 2). In both supports, the valueof Vmax decreases slightly compared with the soluble enzyme.Conformational changes of the enzyme during the immobiliza-tion process, restrictions on the substrate transport and stericconstraints for biomolecular reactions slow down the reactionrate [2,10]. The increase in the Km and the slight changes on theKcat and Vmax values, compared with those for the soluble β-galare due to the stiffening of the immobilized enzyme and thedistortion of its active site [44]. Besides that, all the immobilizedβ-gal molecules are not available for catalysis. Taking into ac-

Table 2. Kinetic parameters for soluble and hybrid B. circulansbiocatalysts

Biocatalyst Vmax Km Kcat

(mmol min−1) (mM)

Bc-soluble 3.5 ± 0.4 13.0 ± 0.5 3.9 ± 0.2Bc-AGx 3.1 ± 0.5 13.6 ± 0.5 3.2 ± 0.1Bc-SGx1.0 2.1 ± 0.4 39.4 ± 0.5 2.8 ± 0.2

The experiments are carried out by triplicate and the SDs are calculated.

count the morphological and porous properties of the silica, it isprobable that some active enzyme molecules immobilized in themost internal surface of the silica will not be available for catal-ysis process. The enzyme load in the support (∼22 mg proteinper gram support) and the intricate porous structure of the sil-ica support, which affect the substrate diffusion rate/conversionvelocity rate ratio, could also contribute to the diffusionrestrictions.

3.4 Lactolysis in whey by using B. circulans β-galbiocatalysts

Homogeneous and heterogeneous lactolysis of whey were carriedout by using the soluble B. circulans β-gal and Bc-SGx1.0 hybridbiocatalysts, respectively. In both cases, the lactose conversion is95% in 15 min at 25◦C and 98% in 4 h at 4◦C (Fig. 5A). Thelactose conversion is lower in heterogeneous catalysis, at 4◦C,during the first hour. This is related to the diffusion restrictionsimposed by the support to the substrate and products transport.

The Bc-SGx1.0 hybrid catalyst maintains 90% of its initialactivity after 10 cycles of lactolysis in batch reactions (Fig. 5B).The time for each cycle is 20 min at 25◦C. Under these conditionsthe whey lactose is completely hydrolyzed. The half-life time ofthis Bc-SGx1.0 biocatalyst is 1465 min under continuous catalysisconditions.

4 Concluding remarks

This study shows the design of heterogeneous β-galactosidasebiocatalyst based on silica, which are highly active at different pHand temperature conditions with a long life time, opening newpossibilities for improving the effectivity of lactolysis and GOSproduction. The design involves the synthesis of porous silicaand selection of the best β-gal. Immobilization by adsorption orchemical bonding is possible depending on the silica chemistry.

Synthesizing glyoxyl-activated silica with tailored porous andchemical characteristics gives the opportunity to overcome thechallenge related with the immobilization of the high molecularweight molecules (such as the evaluated β-galactosidases). Thesilica activation with glyoxyl groups avoids enzyme deactivationseen when other inorganic supports are used.

The screening of different β-galactosidades, under similar im-mobilization conditions, allows to conclude that tailored porousglyoxyl-activated silica support synthesized here is a very versa-tile material for stabilizing enzymes of molecular weight higher



Figure 5. (A) Whey hydrolysis at 4◦C with soluble (�) and Bc-SGx1.0 (�) hybrid catalyst. (B) Activity cycles for Bc-SGx1.0 hybridcatalyst at 25◦C in batch reactions. The bars represent the SDbetween the three independent experiments.

Practical application

The authors describe the properties of the β-gala-ctosidase/silica heterogeneous biocalysts, which can beimplemented for heterogeneous lactolysis on milk andwhey. The latter is important for avoiding pollution prob-lems related with the whey disposal and for producingother compounds, such as galacto-oligosacharides, whichare useful for the chemical and pharmaceutical indus-try and its use as biosensors. These biocatalysts can beused in batch or continuous flow reactors. Furthermore,the authors show methodologies for monitoring the im-mobilization process and for characterizing the heteroge-neous biocatalysts, which can be implemented for otherenzyme—inorganic supports combinations. This studyprovides better strategies for obtaining highly stable β-galactosidase/silica biocalysts.

than 100 kDa like β-galactosidase from B. circulans and A. oryzaeby multipoint covalent attachment.

Especially for β-gal from B. circulans, the immobilization pro-cedure is very simple and does not require special conditions suchas amination of the free enzyme or addition of stabilizing agentslike glycerine or salts to the immobilization medium. This lowersthe down-stream processing and makes it viable for industrialapplication. This heterogeneous biocatalyst shows 66% IY, it ishighly active and 120-fold more stable than soluble enzyme. Itsbiocatalytic properties are comparable with those achieved inthe agarose-activated support, but with the advantages of me-chanical and microbiological resistance and high surface area,including the possibility of regeneration of the silica support bysimple pyrolysis.

Since the immobilization process is simple, the silica supporthas low cost and the immobilized enzyme shows good perfor-mance, the new heterogeneous biocatalysts described here canbe used in lactolysis of acid or sweet whey and oligo-saccharidesproduction, in continuous or batch process, without increasingthe costs.

We thank Colciencias for the financial support through the project111552128274 “Evaluacion y optimizacion de la hidrolisis en-zimatica heterogenea de la lactosa del suero lacteo como un pasoprevio a la fermentacion para la produccion de etanol” and theCOLCIENCIAS—Municipio de Rionegro and Universidad de An-tioquia for the posgraduate scholarship “Becas doctorales 2007 enel convenio de cooperacion 030-2006” and to Antioquia Univer-sity through “Estrategia para la Sostenibilidad de los Grupos deInvestigacion 2013–2014.”

The authors have declared no conflict of interest.

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