Improvement of Chitosan Derivatization for the Immobilization of

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Improvement of Chitosan Derivatization for the Immobilization of Bacillus circulans β‑Galactosidase and Its Further Application in Galacto-oligosaccharide Synthesis Paulina Urrutia, Claudia Bernal, Lorena Wilson, and Andrés Illanes* School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Avenida Brasil, 2147 Valparaı ́so, Chile S Supporting Information *

ABSTRACT: Chitosan was derivatized by two methodologies to design a robust biocatalyst of immobilized Bacillus circulans βgalactosidase from a low-cost support for its further application in the synthesis of galacto-oligosaccharides (GOS). In the first one, chitosan was derivatized by cross-linking with glutaraldehyde and activated with epichlorohydrin; in the second one, crosslinking and activation were done with epichlorohydrin in a two-step process, favoring first support cross-linking and then support functionalization (C-EPI-EPI). Epoxy groups were hydrolyzed and oxidized, obtaining two supports activated with different aldehyde concentrations (100−250 μmol/g). The expressed activity and stability of the immobilized biocatalysts varied according to the derivatization methodology, showing that both the cross-linking agent and the activation degree are key parameters in the final biocatalyst performance. The best compromise between expressed activity and thermal stability was obtained using C-EPIEPI with 200 μmol of aldehyde groups per gram of support. The immobilization conditions were optimized, obtaining a biocatalyst with 280 IU/g, immobilization yields in terms of activity and protein of 17.3 ± 0.4 and 61.5 ± 3.9%, respectively, and a high thermal stability, with a half-life of 449 times the value of the soluble enzyme. The biocatalyst was applied to the synthesis of GOS in repeated batch operation without affecting the product composition. Four successive batches were required for obtaining a cumulative specific productivity higher than the one obtained with the soluble enzyme. KEYWORDS: chitosan, β-galactosidase, immobilization, galacto-oligosaccharides

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INTRODUCTION β-Galactosidase (β-D-galactoside galactohydrolase, EC 3.2.1.23) is a widely utilized enzyme in the dairy industry mainly due to its ability to hydrolyze lactose from milk.1 A newer and more profitable application of this enzyme is the catalysis of transgalactosylation reactions in which a galactose moiety is transferred to a nucleophilic acceptor other than water, potentially any sugar present in the reaction medium, leading then to the synthesis of oligosaccharides.2−4 When lactose is used as substrate, galacto-oligosaccharides (GOS) are synthesized. GOS are products with a recognized prebiotic capacity composed by two, three, or even more sugar units, of which glucose is the terminal unit and galactose the rest of them.5,6 The synthesis of GOS is a kinetically controlled reaction where the product concentration increases to a maximum value and then decreases as a result of the competition between reactions of hydrolysis and synthesis. The prevalence of synthesis over hydrolysis varies according to the β-galactosidase origin,7,8 initial lactose concentration,9−11 and the water thermodynamic activity.12 The use of soluble or immobilized enzymes may also affect GOS yield, because the diffusional restrictions of the immobilized biocatalyst may limit the maximum product concentration attainable13 and immobilization may alter the enzyme catalytic properties.14 Among the different β-galactosidases that have been utilized in GOS synthesis, the one from Bacillus circulans is often preferred due to the higher GOS yield attained.15,16 Several isoenzymes have been identified in commercial preparations of B. circulans β-galactosidase: Mozaffar et al.17 reported the © XXXX American Chemical Society

presence of two monomeric isoenzymes with molecular weights of 240 and 160 kDa and similar isoelectric points of 4.5; later, Vetere and Paoletti 18 reported the presence of three monomeric isoenzymes (212, 145, and 86 kDa), and Song et al.19 isolated a fourth monomeric isoform, establishing that isoenzymes molecular weights were 189, 154, 135, and 92 kDa. The synthesis of GOS was evaluated with each isoenzyme at 40 °C, pH 6, and 5% of lactose, reporting that maximum GOS concentration varied according to the β-galactosidase isoform used.19 Warmerdam et al.20 evaluated the effect of initial lactose concentration on the synthesis of GOS with the isolated isoenzymes, finding that the increase of lactose concentration resulted in a smaller difference among the GOS yields obtained with each isoform. Biocatalysts with high thermal stability are desirable because high reaction temperature will increase lactose concentration and favor synthesis over hydrolysis. Enzyme immobilization is a mature technology that may allow enzyme stabilization with the additional advantage of obtaining an insoluble biocatalyst that can be easily recovered, extending its lifespan, facilitating the control of the reaction, and improving product quality.21,22 Additionally, the immobilization of enzymes may modify their catalytic properties.23,24 Among the different methods for enzyme immobilization, immobilization by covalent bonding Received: January 22, 2014 Revised: September 4, 2014 Accepted: September 4, 2014

A

dx.doi.org/10.1021/jf500351j | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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Figure 1. Possible structures after cross-linking (1) and activation (2) of C-EPI-EPI (a) and C-GA-EPI (b).

usually offers the highest degree of rigidification25 and, consequently, enzyme stabilization, being normally selected when the objective is to improve the enzyme stability and/or when the immobilized biocatalyst will be used in an aqueous media where immobilization by adsorption is in most cases precluded.26 However, the strength of the enzyme−support linkage makes it difficult or impossible to recover the support after enzyme inactivation; therefore, support cost may have a high impact on the economics of the process employing the immobilized biocatalyst. Chitosan is a natural biopolymer outstanding for its relatively low cost, innocuousness, biocompatibility, antimicrobial activity, and the presence of functional groups that may be chemically modified.27,28 It is a linear polysaccharide composed of β-(1−4)-linked D-glucosamine and N-acetyl-D-glucosamine units, which is produced by partial deacetylation of chitin that is obtained from the exoskeletons of crustaceans, particularly from

wastes of the seafood-processing industry.29 The use of chitosan as a carrier for the covalent immobilization of enzymes requires the introduction of functional groups that can react with the amino acid residues of the protein surface. Additionally, polymer chains must be cross-linked to improve the support mechanical resistance and avoid its solubilization in aqueous acidic media due to its cationic nature (pKa of the amino groups ∼ 6.5).27 One typical derivatization method is the cross-linking and activation of chitosan with the bifunctional reagent glutaraldehyde (GA). The chemistry of the reaction between GA and amino groups is not fully understood, and according to the pH, the reaction mechanism may involve Schiff bases, nucleophilic substitution of amino groups, and Michael addition.30,31 The reaction of GA with chitosan allows the cross-linking of different polymer chains and the activation of the support with aldehyde groups that may react with the enzyme through its amino groups (lysine residues and terminal B



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BP-100 Ag columns (300 mm × 7.8 mm) for carbohydrate analysis (Benson Polymerics, Reno, NV, USA).41 Support Preparation. Chitosan particles were produced as previously reported.39 Chitosan was solubilized in acetic acid solution (3% v/v) to achieve a concentration of 1.5% (w/v) and stirred for 12 h. This solution was fed by gravity and sprayed into 1 M NaOH in 40% (v/v) ethanol solution under stirring. The particles were recovered and washed with distilled water and phosphate buffer (0.05 M, pH 7.0). The particles were derivatized by two different methodologies, and the possible chemistry involved after cross-linking and activation of chitosan is presented in Figure 1. Cross-Linking with Glutaraldehyde and Activation with Epichlorohydrin (C-GA-EPI). Chitosan particles were cross-linked with GA as previously reported,39,42 modifying the quantity of GA utilized. To favor the polymer cross-linking, 1 mol of GA was added per 4 equiv mol of chitosan amino groups, assuming 75% deacetylation. The imines formed by the reaction of GA with the amino groups of chitosan were reduced to secondary amines with NaBH4 to form stable bonds. Finally, the cross-linked chitosan was activated with excess EPI to favor chitosan activation. A typical cross-linking and activation process was performed as follows: 1 g of chitosan particles was suspended in 10 mL of a solution of 0.78 g/L of GA prepared in 5 mM phosphate buffer, pH 7, and maintained under stirring for 30 min at 4 °C. The cross-linked particles were washed with distilled water. For the reduction of imines, 1 g of cross-linked particles was suspended in 10 mL of a solution of 3.8 g/L of NaBH4 in 0.1 M bicarbonate buffer, pH 10, and maintained under stirring for 2 h at 25 °C. Afterward, the particles were washed with distilled water. The support was activated by suspending 1 g of support in 6 mL of an aqueous solution containing 40 g/L of NaOH, 37% (v/v) acetone, and 3.8 g/L of NaBH4 at 4 °C. Then, EPI was added to reach a final concentration of 185 g/L. The mixture was stirred at 25 °C for 18 h, and the support was carefully washed with 20% (v/v) aqueous acetone solution, with distilled water, and finally with 50 mM phosphate buffer, pH 7.0. The support was finally contacted with a 0.5 M H2SO4 solution during 1 h to hydrolyze all unreacted epoxy groups, and then the diol groups were oxidized during 2 h with an aqueous solution containing NaIO4 (a ratio of 1 g of support per 10 mL of solution was used in both steps). Because 1 mol of NaIO4 oxidizes 1 equiv mol of diol, the activation degree (moles of aldehyde groups per gram of support) was modified by varying the concentration of NaIO4 utilized during the oxidation step (10−25 mM). Cross-Linking and Activation with Epichlorohydrin (C-EPI-EPI). Chitosan particles were derivatized in a two-step process with EPI as previously reported.24 For support cross-linking, 1 mol of EPI was contacted per 4 equiv mol of primary hydroxyl groups of chitosan, assuming a 75% deacetylation. For this process, the chitosan particles (1 g) were resuspended in 2 mL of mixture with 4.8 g/L of EPI and 2.7 g/L of NaOH at 45 °C during 2 h. The cross-linked support was then activated with EPI following the same procedure as the activation of C-GA-EPI. For both supports the quantification of aldehyde groups was done spectrophotometrically by back-titration with NaHCO3/ KI.38 Enzyme Immobilization. The enzyme was immobilized in C-GAEPI and C-EPI-EPI contacting 10 mL of the enzyme solution per 1 g of support at 25 °C. The β-galactosidase solution was prepared in 0.1 M bicarbonate buffer, pH 10, with 20% (v/v) of glycerol, which was used to avoid enzyme inactivation during immobilization. Finally, to obtain stable bonds between the enzyme and the support, the Schiff bases formed were reduced by adding NaBH4 to reach a final concentration of 1 mg/mL. The suspension was maintained under stirring during 30 min at 25 °C, and the biocatalyst was thoroughly washed with distilled water. Immobilization was followed by measuring the enzyme activity in the suspension and in the supernatant. The activity of the suspension was determined according to the same methodology utilized for the soluble enzyme. Immobilization yield in terms of the bound protein (IYP) was defined as the mass ratio of immobilized protein (difference between contacted protein and unbound protein in the supernatant) to contacted protein. Immobilization yield in terms of expressed

amino group)32,33 and eventually with other surface functional groups (thiols, phenols, and imidazoles).30 Epichlorohydrin (EPI) has been also utilized in the derivatization of chitosan because it may react with amino and hydroxyl groups of the polymer, cross-linking different chitosan chains and activating the support with epoxide groups. The reaction occurs mainly through the chitosan hydroxyl groups when high temperature and alkaline media are utilized.34,35 Because part of the residual epoxy groups may be spontaneously hydrolyzed, a heterofunctional support is obtained, presenting epoxide, diols, and amino groups. The residual epoxy groups may be also hydrolyzed, and diols could be then oxidized with periodate to aldehyde groups that form Schiff bases with the ε-amino group of protein lysine residues. To avoid the opening of the chitosan pyranosidic rings due to the oxidation of vicinal diols with periodate,36 a limited quantity of periodate should be added and chitosan chains should be previously cross-linked. This final step of support modification presents the advantage of obtaining a more homogeneous support, and aldehydes may favor a more intense multipoint covalent attachment than when epoxy groups are utilized because the instability of Schiff bases should favor immobilization through the enzyme surface area with the highest density of lysine residues.37 It has been reported that according to the methodology utilized for chitosan derivatization, the immobilization yield and thermal stability of the biocatalyst may be modified.33,38,39 These results may be related to the modification of chitosan functional groups, the activation degree, and/or the modification of the structure of the support by varying the crosslinking process. The objective of this investigation was to design a methodology for chitosan derivatization that offers the best compromise of expressed activity and thermal stability of an immobilized biocatalyst of B. circulans β-galactosidase for its further application in GOS synthesis in repeated batch operation.

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MATERIALS AND METHODS

Materials. A commercial preparation of β-galactosidase from B. circulans (Biolactasa-NTL CONC X2) in the form of a liquid solution was purchased from BIOCON (Barcelona, Spain), presenting 31.3 ± 0.5 mg of protein per mL and 1353 ± 11 IU per mL (determination of enzyme activity is in section Analysis). o-Nitrophenyl-β-D-galactopyranoside (o-NPG) was from Carbosynth Limited (Bershire, UK). Lactose monohydrate was from Quimatic S.A. (Santiago, Chile). Sodium metaperiodate and glycerol were purchased from Merck (Darmstadt, Germany). EPI, GA, sodium borohydride, o-nitrophenol (o-NP), and chitosan (≥75% deacetylated; MW 190,000−375,000; batch 061M0046 V) were from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of the highest available purity and used as purchased. Analysis. Enzymatic activity of β-galactosidase was assessed spectrophotometrically by measuring the production of o-NP at 420 nm. The reaction was initiated by adding 50 μL of β-galactosidase solution or suspension to 2 mL of 45 mM o-NPG solution, using a temperature-controlled cell with constant magnetic stirring. One international unit of β-galactosidase activity (IU) was defined as the amount of enzyme producing 1 μmol of o-NP per minute from a 45 mM o-NPG solution in 0.1 M citrate−phosphate buffer, pH 6, at 25 °C. The extinction molar coefficient of o-NP under the assay conditions was 560 M−1 cm−1. Protein concentration was determined according to the Bradford methodology, using bovine serum albumin as standard.40 Lactose and products of GOS synthesis (galactose, glucose, and GOS) were measured as previously reported, using a Jasco RI 2031 HPLC delivery system, provided with a refractive index detector, an isocratic pump (Jasco PU2080), an autosampler (Jasco AS 2055), and C



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Figure 2. Effect of the activation degree of C-GA-EPI (gray bars) and C-EPI-EPI (black bars) on the performance of immobilized B. circulans βgalactosidase: (a) immobilization yield in terms of expressed activity (IYA); (b) immobilization yield in terms of protein (IYP); (c) stability factor (SF); (d) maximum catalytic potential (MCP). Immobilization conditions: 0.1 M citrate−phosphate buffer, pH 10, 20% glycerol, and 25 °C. Inactivation conditions: 60 °C and 0.1 M citrate−phosphate buffer, pH 6. After selecting the chitosan derivatization methodology, the contacted protein and immobilization time were optimized using a central composite rotatable design of two variables at two levels with five replicas at the central point. Galacto-oligosaccharide Synthesis. GOS synthesis with the soluble and immobilized β-galactosidase was carried out using 50% (w/w) lactose solution at pH 6 (0.1 M citrate−phosphate buffer) and 60 °C. β-Galactosidase activity in the reaction mixture was adjusted to 40 IU/g lactose, as previously reported.41 To evaluate the impact of reusing the immobilized enzyme on GOS synthesis and measure the biocatalyst thermal stability under reactive conditions, sequential batches of GOS synthesis were carried out. In this case, the ratio of biocatalyst mass to lactose mass was maintained constant and equal to the one used in the first batch. The following parameters were used for evaluating GOS synthesis: Yield (YGOS): maximum GOS mass (MGOS) per unit mass of initial lactose mass (ML0)

activity (IYA) was defined as the ratio of the activity expressed by the immobilized biocatalyst to the contacted activity. Selection of the Methodology of Chitosan Derivatization and Optimization of Immobilization Conditions. To select a methodology of chitosan derivatization, an enzymatic solution of 1.3 mg protein/mL was immobilized in C-GA-EPI and C-EPI-EPI with different activation degrees for 10 h, under the conditions previously mentioned. The performance of the biocatalysts was evaluated in terms of their immobilization yield and thermal stability. The thermal stability was evaluated through the stability factor (SF), defined as the ratio of the half-lives of immobilized and soluble biocatalyst at 60 °C and pH 6 (0.1 M citrate−phosphate buffer). Half-life was calculated after modeling biocatalyst inactivation kinetics by a biphasic mechanism:

⎡ ⎡ AI k1 ⎤ k1 ⎤ = ⎢1 + α × ⎥ × exp(− k1 × t ) − ⎢α × ⎥ AI0 ⎣ k 2 − k1 ⎦ k 2 − k1 ⎦ ⎣ × exp(− k 2 × t )

(1)

YGOS =

AI/AI0 represents the residual activity at time t, α is the specific activity ratio of the intermediate species with respect to the native enzyme species, and k1 and k2 are the transition rate constants from the native to the intermediate and from the intermediate to the final enzyme species, respectively. To consider both the activity of the immobilized biocatalyst and its thermal stability, the lumped parameter maximum catalytic potential (MCP) was also utilized for selecting a chitosan derivatization methodology.39,41 MCP integrates the variation of expressed activity (AI) along biocatalyst inactivation (eq 2), considering the time of biocatalyst utilization (tf) as the one at which residual activity is 35%. MCP =

∫0

tf

A I dt

MGOS × 100 ML0

(3)

Volumetric productivity (π): total GOS mass per unit of reaction volume (V) and unit of reaction time (t). For the purposes of this work, π was evaluated at the time when maximum GOS concentration was achieved.

π=

MGOS V×t

(4)

Cumulative specific productivity (πC): total GOS mass obtained over the whole operation of sequential batches (n batches) per unit mass of biocatalyst protein (MBP) and unit of reaction time. For the purposes of this work, total GOS mass of each batch was evaluated at the moment when maximum GOS concentration was achieved.

(2) D



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Table 1. Inactivation Parameters of B. circulans βGalactosidase Immobilized in Different Chitosan Supports, Determined According to a Series Biphasic Model (Equation 1)a

batchn

πC =

∑batch1 MGOS MBP ×

batchn ∑batch1 t

(5)

In the case of the immobilized enzyme, πC is expressed in terms of the mass of protein that was contacted with the support for biocatalyst immobilization. MB represents the mass of biocatalyst utilized in the first batch of GOS synthesis.

support C-GA-EPI100 C-GA-EPI150 C-GA-EPI200 C-GA-EPI250 C-EPI-EPI100 C-EPI-EPI150 C-EPI-EPI200 C-EPI-EPI250

batchn

πC =

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∑batch1 MGOS batchn

MB × Pc × ∑batch1 t

(6)

RESULTS AND DISCUSSION Selection of Chitosan Derivatization Method. Chitosan particles were derivatized either using glutaraldehyde for crosslinking and epichlorohydrin for support activation (C-GA-EPI) or else using epichlorohydrin for both cross-linking and activation (C-EPI-EPI). The derivatization of chitosan with epichlorohydrin was done in a two-step process because it was previously reported39 that this methodology allows a higher degree of activation than when chitosan is derivatized in just one step.38 This difference may be related to the fact that the first step of cross-linking should produce some steric hindrances that limit the additional cross-linking of chitosan chains when a high concentration of epichlorohydrin is contacted during the second step of derivatization, favoring the support activation. The characterization of C-GA-EPI is presented as Supporting Information. In the case of C-EPI-EPI, its physical and chemical characterization was reported previously,39 showing that the used methodology allows the modification of the amino and hydroxyl groups of chitosan. In the case of C-GA-EPI, the modification was tested with FTIR, showing that the GA and EPI groups have reacted with the chitosan matrix and new aldehyde groups were introduced as reflected by the best resolution bands related with the vibration of CN of amine and CO of aldehydes (1570 and 1650 cm−1, respectively) (Figure SM3, Supporting Information). The concentration of aldehyde groups of C-GA-EPI and CEPI-EPI was modified by varying the concentration of NaIO4 utilized for the oxidation of the support. After oxidation, all NaIO4 was reacted, obtaining supports with activation degrees in the range from 100 to 250 μmol of aldehyde per gram. The effect of chitosan derivatization method over IYA, IYP, SF, and MCP of the immobilized enzyme is presented in Figure 2. The inactivation kinetics of each biocatalyst was modeled by a biphasic mechanism (eq 1), and the values of the corresponding inactivation parameters are summarized in Table 1. The activation degree of both supports affected the biocatalysts’ expressed activity; however, immobilized protein varied significantly only in the case of C-EPI-EPI. In general, CGA-EPI produced the higher values of IYA and IYP independent of the aldehyde concentration in the support. With both supports IYP was higher than IYA, which is explained by the immobilization of proteins different from the β-galactosidase (the commercial enzyme preparation is not pure) and also by the presence of diffusional restrictions and/or inactivation of the catalyst due to the immobilization process. The stability (SF) of the biocatalysts immobilized in both supports also varied according to the activation degree. The maximum values of IYA and SF were obtained when the C-GA-EPI and C-EPIEPI were activated with 150 and 200 μmol of aldehyde per gram of support, respectively. The increase in the concentration

k1 (h−1)

k2 (h−1)

α

R2

1.35 0.77 0.94 1.37 1.14 1.83 0.63 2.54

0.03 0.02 0.03 0.05 0.03 0.03 0.02 0.03

0.28 0.29 0.37 0.28 0.53 0.60 0.58 0.63

0.998 0.988 0.999 0.996 0.996 0.999 0.996 0.993

a 2

R , coefficient of determination.

of aldehyde groups in the support should allow the immobilization of a higher proportion of the contacted enzyme and favor multipoint interaction between the protein and the support, which may explain the increase of expressed activity and stability until 150 and 200 μmol of aldehyde per gram of CGA-EPI and C-EPI-EPI, respectively. On the other hand, the decrease of IYA in the supports with a higher activation degree may be explained by the formation of covalent bonds close to the active site or by an excessive stiffening of the protein, reducing mobility below the minimum level required for catalysis. In the case of SF, an excessive stiffening of the protein may expose areas more prone to denaturation, explaining the lower thermal stability of the biocatalysts. In general, independent of the activation degree, best results were obtained with C-GA-EPI in terms of IYA: in the case of C-GA-EPI150 IYA was almost twice that obtained with C-EPI-EPI150. The opposite effect was observed for SF, a better performance being obtained with the β-galactosidase immobilized in C-EPIEPI, irrespective of the activation degree. The highest stability was obtained with C-EPI-EPI200, for which the SF was 4 times the value reported for the biocatalyst of B. circulans βgalactosidase immobilized in glyoxyl-agarose under optimized conditions and 7 times the value reported for the enzyme immobilized in silica support.41,43 Because C-GA-EPI and CEPI-EPI were activated using the same methodology, the different performance obtained with both supports may be explained by a difference in support structure as a consequence of their cross-linking process. When the combined effect of activity and stability was assessed using MCP as lumped parameter, the support C-EPI-EPI200 offered the best performance due to the prevalence of the effect of thermal stabilization. This support was selected for the further optimization of protein loading and immobilization time. The leaching of β-galactosidase from the derivatized supports was not evaluated in this work because Bernal et al.44 showed that the covalent immobilization of this enzyme in a support activated with aldehyde groups avoids the leaching of the enzyme. Optimization of Immobilization Conditions. Biocatalyst specific activity and thermal stability are important parameters for the industrial application of immobilized enzymes because the first one will define the quantity of catalyst required for a specific reaction and the latter will determine its lifespan. Biocatalyst specific activity varies according to the quantity of enzyme contacted during the immobilization process, whereas it has been reported that the thermal stability of an immobilized enzyme may be improved through the increase of immobilizaE



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tion time.45 To set the range of contacted protein to be considered in the optimization of immobilization conditions, the maximum protein loading capacity of C-EPI-EPI was assessed (Figure 3), obtaining a maximum of 23.8 mg of

Figure 4. Thermal inactivation at 60 °C and pH 6 (0.1 M citrate− phosphate buffer) of the soluble enzyme (closed circles) and derivatives of β-galactosidase immobilized in C-EPI-EPI200 at maximum protein loading capacity (solid symbols) and at 10% of that maximum (open symbols) at different immobilization times: (rhombuses) tmin; (squares) tmin + 48 h. tmin is the minimum time required to obtain a constant activity in the supernatant.

Figure 3. Loading capacity of C-EPI-EPI200 expressed in terms of B. circulans β-galactosidase protein content.

immobilized protein per gram of support. This maximum loading capacity was 45 and 20% lower than the values reported for the immobilization of B. circulans β-galactosidase in glyoxylagarose41 and acrylic resins,46 respectively. Such a difference may be due to the lower total surface area. The range of immobilization time considered for the optimization of the immobilization process was established after evaluating the stability of the biocatalysts at maximum protein loading and at 10% of that maximum. Two different immobilization times were considered: the minimum time required for obtaining a constant activity in the supernatant (tmin) and tmin plus 24 h. As shown in Figure 4, thermal stability of the biocatalyst loaded with the maximum quantity of protein was independent of immobilization time, in contrast to the biocatalyst loaded with a lower quantity of protein. This effect may be explained by a difference in the multipoint interaction between the enzyme and support due to the nonhomogeneous surface of the support, obtaining higher interaction intensity when the enzyme is first linked to the most reactive areas and a lower interaction intensity when the enzyme is immobilized in the less reactive areas. This effect was also reported for glyoxylagarose.41 Considering the interaction observed between the quantity of protein contacted and the immobilization time, both variables were optimized by a central composite design, using MCP as response parameter (Figure 5). The response surface was modeled by a second-order polynomial equation (eq 7), obtaining the maximum value of MPC when 25.8 mg/g was contacted for tmin plus 14.1 h (total immobilization time = 38.1 h).

Figure 5. Optimization of B. circulans β-galactosidase immobilization in C-EPI-EPI200, considering additional immobilization time (tadd) and contacted protein as variables and maximum catalytic potential (MCP) as objective function. Immobilization conditions: 0.1 M citrate− phosphate buffer, pH 10, 20% glycerol, and 25 °C.

tally, obtaining a MCP of 367,032 μmol/g (4.5% lower than the predicted value). The optimized biocatalyst expressed 280 ± 7 IU/g, with an IYA of 17.3 ± 0.4% and an IYP of 61.5 ± 3.9%. The expressed activity was 53% lower than the value reported for the enzyme immobilized under optimized conditions in glyoxyl-agarose.41 However, the SF obtained with the enzyme immobilized in chitosan was 450% higher than the corresponding value obtained with the agarose biocatalyst.41 Additionally, the use of chitosan presents an economic advantage, because one of the limiting factors in the use of agarose is its high cost.47 Galacto-oligosaccharide Synthesis. The optimized biocatalyst was utilized in the synthesis of GOS in repeated batch operation. A comparison of YGOS and π values obtained in one batch of GOS synthesis catalyzed by the soluble and immobilized β-galactosidase is presented in Table 2. The

MCP = Pc × 35010.8 + tadd × 17136.5 − tadd × Pc × 127.9 − Pc 2 × 640.5 − tadd 2 × 491.4 − 190011 (7)

The ANOVA test was performed, obtaining a p value of 0.0005, an R2 value of 0.9366, and an adjusted R value of 0.8913. The optimized conditions were validated experimenF



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Table 2. Comparison of Synthesis of GOS with Soluble βGalactosidase and β-Galactosidase Immobilized in C-EPIEPI200a

a

parameter

soluble β-gal

β-gal/C-EPI-EPI200

YGOS (%) π (gGOS/h·L)

38.3 462.2

39.1 473.7

YGOS, GOS yield; π, volumetric productivity.

production of GOS was not affected by the immobilization of the enzyme, as reflected by the lack of a significant variation in YGOS and π. These results indicate that diffusional restrictions in the immobilized enzyme are not significant and the immobilization process did not produce conformational changes or steric hindrances that affect the activity of the biocatalyst. In contrast, Huerta et al.42 and Sheu et al.48 reported that the synthesis of GOS with Aspergillus oryzae βgalactosidase immobilized in chitosan derivatized with glutaraldehyde resulted in a lower YGOS than the one obtained with the soluble enzyme, a result explained by the presence of diffusional restrictions. The insignificant effect of diffusional restrictions in the biocatalyst of B. circulans β-galactosidase immobilized in C-EPI-EPI200 may be explained by the use of a lower enzyme loading and/or by differences in the support size and structure. Considering that several isoenzymes of B. circulans β-galactosidase have been identified in the commercial soluble enzyme preparation,18−20 the absence of variation in YGOS and π also indicates that the relative activity of each isoform was not significantly altered upon immobilization. The profile of GOS obtained with β-galactosidase immobilized in CEPI-EPI200 was similar to the one obtained with the soluble enzyme (Figure 6). This pattern remained unchanged along 10 repeated batches, as may be observed in Figure 7.

Figure 7. Product distribution of GOS synthesized by the βgalactosidase from B. circulans immobilized in C-EPI-EPI200 along batches in repeated batch operation: (black bars) trisaccharides; (gray bars) tetrasaccharides; (white bars) penta- and hexasaccharides. Reaction conditions: 50% (w/w) initial lactose concentration, 0.1 M citrate−phosphate buffer, pH 6, and 60 °C. Biocatalyst added in the first batch was adjusted to 40 IU/g initial lactose, and for the subsequent batches the ratio of biocatalyst mass to lactose mass was maintained constant and equal to the one used in the first batch.

To make a fair comparison between the soluble and immobilized β-galactosidase, the parameters cumulative specific productivity (πc) and total mass of GOS per mass of contacted protein (RGOS) were evaluated. The expression of each parameter in terms of the contacted protein takes into consideration the immobilization yield; therefore, both the soluble and immobilized enzymes are compared considering

Figure 6. Profiles of GOS synthesized by B. circulans β-galactosidase: (a) total GOS; (b) trisaccharides; (c) tetrasaccharides; (d) penta- and hexasaccharides; (solid symbols) soluble enzyme; (open symbols) enzyme immobilized under optimized conditions in C-EPI-EPI200. Reaction conditions: 50% (w/w) initial lactose concentration, 0.1 M citrate−phosphate buffer, pH 6, 60 °C, and 40 IU/g initial lactose. G



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biocatalyst hydrolytic activity was lost after 10 successive batches.

the total mass of protein expended for the production of GOS. The values of πc along 10 successive batches are presented in Figure 8a. The πC of the first batch of synthesis was lower for

Figure 9. Comparison of thermal stability of β-galactosidase immobilized under optimized conditions in C-EPI-EPI200 under nonreactive (circles) and reactive (squares) conditions. Nonreactive conditions: 0.1 M citrate−phosphate buffer, pH 6, 60 °C. Reactive conditions: 50% (w/w) initial lactose concentration, 0.1 M citrate− phosphate buffer, pH 6, 60 °C.

In conclusion, the cross-linking agent utilized for chitosan derivatization and the activation degree of the support had a high impact on the performance of the immobilized B. circulans β-galactosidase. The best compromise between expressed activity and thermal stability was obtained with the enzyme immobilized in cross-linked chitosan and activated with epichlorohydrin at a degree of activation of 200 μmol of aldehydes per gram of support. The optimization of immobilization conditions (protein load and immobilization time) increased biocatalyst expressed activity and thermal stability. The optimized biocatalyst was applied to the synthesis of GOS, without affecting the product profile obtained with the soluble enzyme. Four successive batches were required for obtaining a cumulative specific productivity higher than the one obtained with the soluble enzyme. The appropriate design of a low-cost support, such as chitosan, is a good strategy for the development of a highly stable biocatalyst adequate for GOS synthesis.

Figure 8. Comparison of soluble β-galactosidase (gray bars) and βgalactosidase immobilized under optimized conditions in C-EPI-EPI200 (black bars): (a) cumulative GOS productivity in terms of contacted protein (πc); (b) accumulated mass of GOS per unit mass of contacted protein (RGOS). Reaction conditions: 50% (w/w) initial lactose concentration, 0.1 M citrate−phosphate buffer, pH 6, and 60 °C. Biocatalyst added in the first batch was adjusted to 40 IU/g initial lactose, and for the subsequent batches the ratio of biocatalyst mass to lactose mass was maintained constant and equal to the one used in the first batch.

the immobilized enzyme due to the loss of activity during immobilization; however, the reutilization of the biocatalyst compensates for the loss of activity, requiring only four sequential batches to surpass the πC of the soluble enzyme. After 10 successive batches, the πC of the immobilized enzyme was 2.9 times the value obtained with the soluble enzyme. This value is lower than the πC reported for the enzyme immobilized in glyoxyl-agarose41 due to the lower expressed activity of the biocatalyst. The advantage associated with the high thermal stability of the enzyme immobilized in C-EPI-EPI200 should be observed with a higher number of batches or extending the time of each batch. Values of RGOS are presented in Figure 8b, observing that after 10 batches almost 1 kg of GOS per gram of contacted protein is produced when the immobilized biocatalyst is utilized. The thermal stability of the immobilized enzyme was compared under reactive and nonreactive conditions (Figure 9), observing a positive modulation by the reaction media. Under reactive conditions,

Improvement of Chitosan Derivatization for the Immobilization of

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