A pectin–lipase derivative as alternative copolymer for lipase assay

Journal of Molecular Catalysis B: Enzymatic 102 (2014) 25–32

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A pectin–lipase derivative as alternative copolymer for lipase assay Karla A. Batista ∗ , Luiza L.A. Purcena, Guilherme L. Alves, Kátia F. Fernandes Laboratório de Química de Proteínas, Departamento de Bioquímica e Biologia Molecular, Instituto de Ciências Biológicas – ICB II, Universidade Federal de Goiás, PB 131, Goiânia, GO, Brazil

a r t i c l e

i n f o

Article history: Received 28 June 2013 Received in revised form 8 January 2014 Accepted 10 January 2014 Available online 28 January 2014 Keywords: Arabic gum substitution Immobilization Thermodynamic parameters Kinetic parameters Solanum lycocarpum

a b s t r a c t In this study Arabic gum and free lipase were successfully replaced by a lipase immobilized onto pectin (PECp-lipase) for pNP palmitate hydrolysis. Using a Central Composite Rotatable Design the optimum pH and temperature for free and PECp-lipase reaction were established at pH 8.0, 30–40 ◦ C, and pH 8.0, 40–50 ◦ C, respectively. PECp-lipase maintained 100% of initial activity after 35 days of storage at room temperature. The thermal kinetic parameters (kd and t1/2 ) and Ed evidenced that immobilization provide higher thermal stability to PECp-lipase compared to free enzyme. Thermodynamic parameters (H◦ , S◦ and G◦ ) confirmed the thermal stability acquired by PECp-lipase and indicated that tridimensional structure was preserved. The apparent Michaelis constant estimated for the PECp-lipase (1.15 mM) was not statistically different from the free enzyme (1.09 mM). PECp-lipase represents a faster, single step and, therefore, a very attractive substitute for the lipase standard methodology of pNP palmitate hydrolysis. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Pectins comprise a complex family of heteropolysaccharides that play an important role in plant growth and development [1–3]. Currently, pectins represent one of the most studied polysaccharides as they are target of many applications in the food and pharmaceutical industry, such as their use as thickeners, stabilizers, gelling agents and texture modifiers [1,4,5]. Recently, the pectin extracted from the fruit of Solanum lycocarpum, known as lobeira in Brazil, was characterized as high methoxyl pectin with intrinsic viscosity and molecular weight similar to those observed in the citrus pectin [3]. However, S. lycocarpum pectin presented remarkable features, such as a two-fold higher emulsifying capacity than that presented by citrus pectin. This property allows new applications for S. lycocarpum pectin, making it an important key for many industrial processes. Lipases are enzymes that catalyze the hydrolysis of acylglycerides and other esters at the interface between water and lipidic substrates [6–8]. Considering the hydrophobic nature of the substrate and the hydrophilic nature of lipase, it is mandatory for measurement of lipase activity the use of emulsifying agents that act in the interface between substrate and enzyme [9,10]. The standard colorimetric method for lipolytic activity measurement is based on the p-nitrophenyl palmitate (pNPP) hydrolysis [11–14]. To measure activity by this approach, two solutions are required: the p-nitrophenyl palmitate and Arabic gum solution. These solutions

∗ Corresponding author. Tel.: +55 62 3521 1492; fax: +55 62 3521 1190. E-mail address: [email protected] (K.A. Batista). 1381-1177/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.molcatb.2014.01.010

must be mixed cautiously and gently in order to properly emulsify the substrate, which makes this methodology laborious and time consuming. Therefore, any other methodology employed to reduce the time spent preparing the substrate would provide a more rapid lipolytic reaction. In this matter, the use of immobilized lipase on a material presenting emulsifying capacity, such as the one presented by S. lycocarpum pectin, may be promising and, thus, decreasing the time spent during the reaction preparation. Moreover, previous reports have described improvements in the characteristics and activity of enzymes after immobilization, especially those related to thermal, pH and storage stability. In addition, immobilization processes usually allows the repeated use of enzymes, which may be of financial interest [15–18]. In this sense, in the present study lipase was immobilized onto the pectin extracted from S. lycocarpum and this pectin–lipase system was used to replace Arabic gum in the lipolytic reaction medium. This new system was evaluated regarding to the enzyme stability during storage as well as parameters such as thermal stability, thermodynamics and kinetics. 2. Materials and methods 2.1. Immobilization of lipase onto pectin from S. lycocarpum The pectin from S. lycocarpum (PEC) was extracted as described by Torralbo et al. [3] and used as a support for the lipase immobilization (Thermomyces lanuginosus – Lipolase® , Novozymes, Araucária, Paraná, Brazil). The immobilization was carried out according to Silva-Filho et al. [19]. Briefly, the pectin was acti-

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vated using 0.1 mol L−1 sodium metaperiodate solution (PECp) and the immobilization of lipase was conducted by adding 1 mL of an enzyme solution (488 U), prepared in 0.1 mol L−1 Tris–HCl buffer, pH 8.0–5 mg of PECp. The system was incubated for 30 min at 4 ◦ C under orbital stirring. Then, cold ethanol (92%, v/v) was added to precipitate the PECp-lipase. The precipitate product was washed twice with 0.1 mol L−1 Tris–HCl buffer to remove unbounded enzyme. The scheme representing the mechanism of PECp-lipase production was obtained using the software ChemStetch 11.02 (Advanced Chemistry Development Inc., Toronto, CA). The tridimensional model of lipase was obtained using the software Jmol 13.0.6 (http://www.jmol.org). 2.2. Storage stability The storage stability of the PECp-lipase was evaluated as follows. Samples were dried at 40 ◦ C in an air-forced oven until constant weight. The dry samples were stored in hermetically closed vials at room temperature. The free lipase (solution containing 445.8 U) was also stored at room temperature and at 4 ◦ C. The storage stability of the free lipase and PECp-lipase was determined by measuring their residual activity every 7 days.

where V1 represents the volume (mL) of KOH used in the titration of olive oil after hydrolysis; V2 represents the volume (mL) of KOH used in the titration of olive oil before hydrolysis; M is the molar concentration (mol L−1 ) of KOH; t is the reaction time (min); and m is the amount (mg) of PECp-lipase used. 2.4. Effect of pH and temperature on the enzyme activity The effect of temperature and pH on the activity of the free and immobilized lipase was evaluated by using a Central Composite Rotatable Design (CCRD) 22 associated with Response Surface Methodology (RSM). For the experimental design, the two independent variables were used, both represented in two levels: for temperature, it was used 25 ◦ C (low level) to 75 ◦ C (high level); for the pH, it was used 3 (low level) to 9 (high level). A central point (50 ◦ C; pH 6), with two replicates, was also included for statistical evaluation (at 95% confidence level). Results from CCRD were analyzed using the software Statistica 6.0 (Statsoft Inc., Tulsa, USA, 1997). The adjustment of the experimental data for the independent variables in the RSM was represented by the second-order polynomial equation: y = ˇ0 +

 j

2.3. Determination of enzyme activity In this study, the methodology described by Babu and Rao [13] was employed for measurement of free lipase activity. Briefly, the production of p-nitrophenol (pNP) was monitored by its absorbance at 410 nm. A calibration curve was constructed in order to calculate the enzyme activity using the absorbance of standard pNP solutions. One unit of enzyme (U) was defined as the amount of free or immobilized lipase that releases 1 ␮mol min−1 mL−1 of pNP. In order to evaluate the efficiency of Arabic gum substitution, two assays were carried out as follows: Assay (A): the methodology described by Babu and Rao [13] was employed, but the Arabic gum was replaced by 0.1% of pectin. Lipase was added as free enzyme. Assay (B): the reaction was performed using only PECp-lipase as emulsifying and lipolytic agent, in absence of Arabic gum.

ˇj xj +

 i≺j

ˇij xi xj +



ˇjj xj2 + e

j

where y is the dependent variable to be modeled; ˇ0 , ˇj , ˇij and ˇjj are regression coefficients, xi and xj are independent variables and e is the error. The model was simplified by dropping terms that were not statistically significant (p > 0.01) by ANOVA. 2.5. Thermal behavior and inactivation kinetics of free and immobilized lipase The thermal stability of free and immobilized lipase (PECplipase) was evaluated by measuring the remaining activity of pre-incubated free enzyme or PECp-lipase at different temperatures (25–75 ◦ C) in Tris–HCl buffer solution (0.1 mol L−1 , pH 8.0) for 2 h. After 30 min at 25 ◦ C (temperature equilibration), their activities were established as above described. The percentage of remaining activity was calculated as follows: Remaining activity

The following equation was used to calculate the specific activity (U mg−1 protein) of the free and immobilized lipases: Specific activity =

activity of free or immobilized lipase amount of protein

The amount of protein was determined according to methodology described by Bradford [20], using bovine serum albumin as standard. The hydrolysis of olive oil was carried out according to Soares et al. [21]. Briefly, the substrate was prepared by mixing 2.5 mL of olive oil with 2.5 mL of Arabic gum solution (7% (w/v) in 50 mmol L−1 Tris buffer pH 8.0). Five milliliters of this substrate emulsion was added to the reactor containing 200 mg of PECplipase and the reaction proceeded at 40 ◦ C for 60 min under stirring. The released fatty acid was titrated with 0.02 mol L−1 potassium hydroxide solution. One unit (U) of enzyme activity was defined as the amount of enzyme that produces 1 ␮mol of free fatty acid/min under the assay conditions, calculated according following equation: EU =

(V1 − V2 ) · M · 106 t·m

=

enzyme activity after incubation × 100 enzyme activity at the optimal temperature

Kinetic data analysis of thermal inactivation of enzymes can often be described by the first-order reaction [22–24]: dAt = −kd · At dt where At is the enzyme activity at treatment time t, and kd is the reaction rate constant at the temperature studied. For constant extrinsic/intrinsic factors, in the case of a first-order reaction, the kinetics can be described by the following equation: At = e−kd t A0 where At is the enzyme activity at time t, A0 is the initial enzyme activity, t is the treatment time (h), and kd is the inactivation rate constant at the temperature studied. The inactivation rate constant kd can be estimated by linear regression analysis of the natural logarithm of residual activity versus treatment time.

K.A. Batista et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 25–32

The half-life (t1/2 ) value for lipase thermal denaturation was calculated as:

t1/2 =

ln(2) kd

27

The thermodynamic parameters were calculated from the Arrhenius equation:

ln kd = −

Ed + ln C RT

Fig. 1. Scheme representing the mechanism of PECp-lipase production.

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The change in Ed was obtained from the slope of the Arrhenius plot (log denaturation rate constants (ln kd ) versus reciprocal of the absolute temperature), as described below: slope =

Ed R

Change in the enthalpy (H◦ ) for each temperature was calculated according to: H ◦ = Ed − RT where R is the universal gas constant (8.3145 J mol−1 K−1 ) and T is the absolute temperature. The values of variation in free energy of inactivation (G◦ ) at different temperatures were calculated from the first-order rate constant of the inactivation process by: G◦ = −RT ln

k h d

KT

where h is the Planck constant (6.6262 × 10−34 J s) and K is the Boltzmann constant (1.3806 × 10−23 J K−1 ). Using the equations above, the activation entropy (S◦ ) for lipase can be calculated as: S ◦ =

H ◦ − G◦ T

2.6. Kinetics parameters Kinetic parameters for the hydrolytic activity of the free (Km /Vmax ) and immobilized lipase (Km.app /Vmax.app ) were determined using pNP palmitate as the substrate in the concentrations ranges from 0.1 to 15 mmol L−1 , at pH 8.0 and 40 ◦ C, under orbital stirring. Equivalent amounts of free and immobilized enzyme (400 U) were used in the assay. The initial reaction rate was recorded measuring the absorbance of the p-nitrophenol produced. Data from linear range of Michaelis–Menten plot were used to construct the double-reciprocal plot of Lineweaver–Burk (reciprocal of V0 versus reciprocal of pNP palmitate concentration). The values of Vmax /Vmax.app can be deduced from the reciprocal of the intercept of the straight line on the ordinate and the values of Km /Km.app either from the negative reciprocal of the intercept on the abscissa, using 1 the Michaelis–Menten equation: V1 = V Km·[S] + Vmax where V is the max initial reactive rate, [S] is the initial substrate concentration, Vmax is the maximum reaction rate attained at infinite initial substrate concentration and Km is the Michaelis–Menten constant.

surface suggests that the N O bound may occur between lipase and PECp through arginine or lysine with the same probability. The achievement of a high yield bioactive material containing immobilized lipase allows several potential applications for this PECp-lipase system. One of these applications also explore the high emulsifying property presented by pectin that enables it to substitute Arabic gum in lipase assay [25]. The substitution of Arabic gum by pectin (Assay A) resulted in a reduction of 38.2% on the lipase activity (276.1 ± 6.6 U mg−1 protein). However, using the system containing immobilized lipase (Assay B), the activity obtained (400.9 ± 8.9 U mg−1 protein) was very close to standard methodology (445.8 ± 5.3 U mg−1 protein), corresponding to 89.9% of efficiency. As reported by Silva-Filho et al. [19], the lipase immobilization via adsorption onto pectin resulted in a system with lower catalytic activity than those obtained via covalent linkage. This finding was confirmed in this study, since free lipase probably adsorbed to pectin (Assay A) resulting in a less active system. The random nature of the pectin–lipase interactions occurring in the adsorption process may lead to an immobilized lipase with active site inaccessible to substrate. The covalent linkage was directed to specific groups in the lateral chain of lipase which are probably far from the active site resulting in a system without steric hindrance (Fig. 1). An additional advantage of the immobilization through covalent bound is the stability of the linkage. Considering the possibility of N O bound formation the typical value of G for this linkage is around 250 kJ mol−1 [26]. Compared to the standard methodology, the PECp-lipase is a single step and faster methodology. The substrate solution is prepared by dissolving pNP palmitate in a mixture of isopropanol, Triton X100 and Tris–HCl buffer (0.1 mol L−1 pH 8.0). The reaction starts by the simple addition of substrate solution to the PECp-lipase.

3. Results and discussion 3.1. Immobilization In a previous report, Silva-Filho et al. [19] showed that S. lycocarpum pectin can be modified by glutaraldehyde or metaperiodate activation. These pectins derivative was used for lipase immobilization and the best efficiency was obtained using metaperiodate as activator. The route for obtaining the PECp-lipase is depicted in Fig. 1. As showed in Fig. 1, the metaperiodate pectin derivative has a reactive carbonyl group in the polysaccharide chain. This carbonyl group is especially reactive to imidazole or amino groups such as those present at lateral chain of arginine and lysine residues in the proteic structure of lipase. The soft conditions of the immobilization procedure favor the reaction between the nitrogen from imidazole or amino groups and the oxygen from the carbonyl group (N O bound). The tridimensional structure of the T. lanuginosus lipase obtained from RCSB Protein Data Base (RCSB: O59952), shows the presence of 16 arginine and 14 lysine residues on the surface of the protein (Fig. 2). The availability of these amino acids at enzyme’s

Fig. 2. Tridimensional structure of lipase from Thermomyces lanuginosus evidencing the (a) arginine and (b) lysine residues present in the protein.

K.A. Batista et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 25–32

The use of PECp-lipase for olive oil hydrolysis was tested and the results showed that the immobilized lipase was able to hydrolyze this substrate producing considerable amounts of fatty acid. Each cycle of hydrolysis produced about 2.65 ␮mol of fatty acid per milligram of PECp-Lipase per minute of reaction. The absence of steric hindrance and the preserved capacity of hydrolysis of its natural substrate was another important result of this work, indicating that this material can be used in several different applications. 3.2. Storage stability Storage stability is one of the most important criteria for the application of an enzyme on a commercial scale. The higher storage stability of PECp-lipase has already been showed in a previous report [19]. In that study, free and PECp-lipase retained 25.7% and 85.4% of the initial activity, respectively, after 11 weeks stored at 4 ◦ C in the presence of ethanol. In this study, the storage of free lipase at room temperature resulted in an inactive enzyme after 7 days. Additionally, free enzyme showed decreasing activity when stored at 4 ◦ C, as can be seen in Fig. 3. The stability of the PECp-lipase system was clearly demonstrated in the storage tests. First of all, the activity of PECp-lipase remaining unchanged after drying process at 40 ◦ C for 4–6 h. The dry PECp-lipase stored at room temperature was very stable, preserving 100% of the initial activity after 35 days (Fig. 3).

29

Table 1 Experimental design and lipase activity according to the CCRD 22 . Variable level

Enzyme activity (U mg−1 protein)

Run

pH X1

Temperature (◦ C) X2

Free lipase

PECp-lipase

1 2 3 4 5 6 7 8 9 (C) 10 (C)

(−) 7.0 (−) 7.0 (+) 9.0 (+) 9.0 (−) 7.0 (+) 9.0 (0) 8.0 (0) 8.0 (0) 8.0 (0) 8.0

(−) 30 (+) 50 (−) 30 (+) 50 (0) 40 (0) 40 (−) 30 (+) 50 (0) 40 (0) 40

145.17 120.17 355.17 35.17 123.50 171.83 366.83 153.50 316.83 316.28

251.83 90.17 291.83 220.17 196.83 265.17 453.50 265.17 535.17 551.84

where X1 and X2 denote pH and temperature, respectively. The value of r2 (0.93) indicates that experimental data can be explained successfully by the experimental model. Using the equation above, a three dimensional plot was drawn. Fig. 4b shows a well-defined region of optimum values for the reaction conditions. The response surface showed a maximum activity for free lipase when the reaction was carried out at pH 8.0 and 30 ◦ C. Otherwise, the results of the multivariate analysis evidenced that all variables affected significantly (p < 0.01) the activity of

3.3. Effect of pH and temperature In order to determine the activity-pH and activity-temperature profiles for free and PECp-lipase, a Central Composite Rotatable Design (CCRD) was used. Table 1 shows the factors investigated in the CCRD, as well as the coded and decoded levels, and the means of lipolytic activity for free and PECp-lipase. The multivariate analysis for activity of the free lipase (Fig. 4a) showed that the quadratic term for temperature (X2 ) had no effect (p > 0.01) on this response while the linear and quadratic terms for pH (X1 ) and the interaction factor significantly affected the lipase activity (p < 0.01). The regression analysis showed an adequate fit of experimental values to the second-order polynomial model as a function of the significant factors. Consequently, the mathematical model describing the correlation between the response and the variables is presented as follows: Activity (U) = 2403.89X1 − 129.99X12 + 49.69X2 − 7.37X1 · X2 − 10250.39

Fig. 3. Storage stability of ( ) free and ( ) PECp-lipase. Free lipase stored as solution (445.8 U) at 4 ◦ C and PECp-lipase as powder (400.9 U) at room temperature.

Fig. 4. Pareto chart (a) and response surface plot (b) for free lipase activity as function of pH and temperature.

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Fig. 6. Graphic representation of the thermal stability of ( PECp-lipase after incubation for 2 h in each temperature.

) free and (

)

to optimize this system using the classical and largely disseminated univariate design approach would not lead to the optimal reaction condition. In the univariate design, the factors are evaluated separately, and thus, the combined effect of the interactions would not be clearly exposed. Other important observation was the maintenance of the optimum reaction pH in both systems (Figs. 4 and 5). This result indicates an absence of partition effect of the support in the microenvironment of enzyme catalysis. However, the optimum temperature was different for free and immobilized lipase. PECplipase presented a broad range of high activities around 40 ◦ C, while free enzyme was restricted around 30 ◦ C. 3.4. Thermal behavior and inactivation kinetics of free and immobilized lipase Fig. 5. Pareto chart (a) and response surface plot (b) for PECp-lipase activity as function of pH and temperature.

PECp-lipase. As can been seen in Pareto chart (Fig. 5a), the linear and quadratic terms of temperature and the quadratic term of pH negatively affected the enzyme activity. On the other hand, the effect of the linear term of pH and the interaction between pH and temperature positively affected the response. The regression analysis showed an adequate fit of the experimental values to the second-order polynomial model as function of significant factors (r2 = 0.91). The mathematical model is represented in the following equation: Activity (U) = 3425.91X1 − 217.26X12 + 49.11X2 − 0.89X22 + 2.25X1 · X2 − 14148.40 where X1 and X2 denote pH and temperature, respectively. The fitness was expressed by the r2 value, which indicates that 91% of the variability in the response can be explained by the model. This suggested that the model represented accurately the data in the experimental region. The effect of inter-relations and interactions of the independent variables (pH and temperature) on the PECp-lipase activity are depicted in Fig. 5b. As can be observed, the higher PECp-lipase activity was obtained in pH ranging from 7.6 to 8.4 and temperatures from 30 to 48 ◦ C. In addition, the optimum pH-activity and temperature-activity profile were found in pH 8.0 and 40 ◦ C. The significant effect of interaction between pH and temperature observed in the response for free (−9.01) and immobilized lipase (4.59) is a very important finding. It indicates that attempts

Thermal stability is an important parameter to be analyzed in order to determine the application for an immobilized enzyme. The chemical nature of the support is the main factor affecting thermal stability of an immobilized enzyme, since the support chemical composition determines its thermal properties such as thermal conduction, thermal convection, and thermal conductivity [27–29]. In this sense, thermal stability of an immobilized enzyme directly depends of the support thermal features, which influences the microenvironment where catalytic reaction occurs [18]. As can be seen in Fig. 6, free lipase and PECp-lipase showed the same pattern of thermal stability. However, while the free enzyme showed good stability in the range from 30 ◦ C to 40 ◦ C, PECp-lipase presented the same stability from 40 ◦ C to 50 ◦ C. Although the activity of both decreased proportionally, PECp-lipase preserved 91% of its original activity after 2 h incubation at 50 ◦ C whereas free enzyme preserved only 48%. There are at least three factors that can be associated to the shift observed in the maximum activity of PECp-lipase: (i) the interactions between enzyme and support, (ii) the different amount and thermal properties of the polysaccharides present in the reaction medium (Arabic gum or pectin). In the standard methodology (Assay A), free lipase is adsorbed in the Arabic gum. In this kind of immobilization, the weak forces involved in the immobilization are broken when the temperature of the reaction medium is increased, resulting in an enzyme with preserved mobility. Otherwise, in the reaction with pectin (Assay B), the lipase is covalently bonded to the polysaccharide. In this sense, the restricted movements of the covalently bounded enzyme will require more energy to reach the maximum activity than the adsorbed counterpart (Table 3).

K.A. Batista et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 25–32

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Table 2 Thermal denaturation kinetic parameters for free and PECp-lipase. T (◦ C)

40 50 60 70

kd (h−1 )

t1/2 (h)

Free

PECp-lipase

Free

PECp-lipase

0.16 0.48 0.86 1.35

0.13 0.17 0.55 0.84

2.52 1.42 0.84 0.40

2.74 2.45 1.28 0.87

Furthermore, the amount of Arabic gum (1 mg mL−1 ) or pectin (5 mg mL−1 ) used in the reaction is different. Considering that Arabic gum and pectin have different thermal properties [28,30], the differences observed in the thermal stability curve may be ascribed to the chemical and thermal properties of the support material. There are many reports of enzyme stabilization after immobilization in the literature [16,31–33]. Immobilization reduces the movement of the molecules and thermal vibration resultant from heating process [18]. In this context, supports with low thermal conduction such as polysaccharides are a good choice for enzyme immobilization, since they frequently preserve enzyme three-dimensional structure and improve the system stability [6]. 3.5. Thermal inactivation kinetics of free and immobilized lipase The thermal denaturation constant (kd ) was determined for both systems to better understand the influence of immobilization on the thermal stability. The thermal denaturation constant gives the rate of enzyme denaturation under pre-defined incubation conditions. In addition, kd directly influence the half-life (t1/2 ) of an enzyme submitted to thermal treatment [22]. Table 2 shows thermal denaturation kinetic parameters for free and immobilized lipase. The results evidenced that as incubation temperatures increase, the values of kd proportionally increase, whereas t1/2 decrease for free and PECp-lipase. Despite kd of both decreased with increasing temperature, the values of kd for PECplipase are consistently lower than those of free lipase. The values of kd for PECp-lipase were 20% (at 40 ◦ C) to 38% (at 70 ◦ C) lower than kd for free lipase, resulting in a half-life 2-fold higher at 70 ◦ C. The lower values of kd for PECp-lipase indicate that the covalent immobilization of lipase onto pectin resulted in an enzyme strongly bonded to the support. This kind of attachment frequently results in a reduced conformational flexibility of the tridimensional protein structure, which consequently became less susceptible to unfolding [17]. Conversely, immobilization via adsorption is frequently less effective in thermal stabilization due to the nature of the weak forces involved in the process. The difference in the thermal behavior of free and PECp-lipase was also evidenced by the thermal inactivation energy (Ed ). Thermal inactivation energy is the minimum energy that must be acquired before protein unfolding takes place [34]. The value of Ed for free and PECp-lipase was determined to be 62.47 kJ mol−1 and 66.25 kJ mol−1 . The inactivation energy (Ed ) has a direct relationship with the enthalpy (H◦ ) and entropy (S◦ ), two opposite parameters related to the stability of the system. Considering the tridimensional proteic structure, these two parameters determine the structural disorder, such as non-covalent bonds broken and unfolding rate [22,27]. Table 3 shows that the higher temperature, the lower is the values of H◦ , for both free and PECp-lipase. However, the values of H◦ for PECp-lipase were higher than for free lipase regardless the temperature, probably as consequence of the higher stability of the immobilized enzyme. Moreover, the values for S◦ reinforce the improved stability of PECp-lipase. If the success of an immobilization procedure is evaluated by the stability it confers to the immobilized enzyme, the integrity of the

Fig. 7. Lineweaver–Burk plot for free (a) and PECp-lipase (b).

tridimensional structure is another important parameter expected to be preserved [35]. The stability of the tridimensional structure may be measured by the Gibbs energy. Hydrophobic and electrostatic interactions, hydrogen bonds and disulfide bridges are the forces that stabilize the protein structure under thermal treatment [22]. The values of G◦ for free and PECp-lipase were very close (Table 3), confirming the preservation of the internal interactions in the protein structure. 3.6. Kinetic parameters Kinetic parameters, Vmax /Vmax.app and Km /Km.app , figure among the most important criteria for evaluation of Arabic gum substitution by pectin. Vmax defines the highest possible velocity when the enzyme is saturated with substrate. Km is defined as the substrate concentration that gives a reaction velocity of ½ Vmax [36,37]. In the case of lipase reaction, the heterogeneous catalysis for both free and PECp-lipase occurs in different microenvironment resultant of the chemical properties of Arabic gum and pectin. In this sense, to be a good substitute, PECp-lipase must present kinetic parameters comparable to those observed for free lipase assay. Fig. 7 depicts the double reciprocal plot and kinetic parameters, Km /Km.app and Vmax /Vmax.app for free and immobilized lipase. As can be seen, the values of Km /Km.app and Vmax /Vmax.app were very close, with no statistical differences (p < 0.05). Two relevant aspects must be pointed out: first of all, considering that lipase reaction occurs in a heterogeneous environment, the similar values of kinetic parameters indicates that substrate partitioning was similar in the presence of Arabic gum or pectin. Second, the immobilization via covalent bounding did not affect the internal interactions of the tridimensional structure or active

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Table 3 Thermodynamic parameters of free and PECp-lipase at different temperatures. T (◦ C)

40 50 60 70

H◦ (kJ mol−1 )

S◦ (J mol−1 K−1 )

G◦ (kJ mol−1 )

Free

PECp-lipase

Free

PECp-lipase

Free

PECp-lipase

59.86 59.78 59.70 59.61

63.64 63.56 63.48 63.39

−118.23 −115.51 −116.74 −118.76

−107.93 −112.32 −110.56 −111.70

96.87 97.09 98.57 100.35

97.43 99.84 100.30 101.71

site architecture of lipase after immobilization onto pectin, since the immobilized enzyme maintained the affinity (Km.app ) for pNP palmitate. 4. Conclusions In this work a lipase was successfully immobilized onto to pectin from S. lycocarpum, resulting in a system with improved properties. Apparently, pectin performed an important role as support, preserving the internal interactions and the tridimensional protein structure. This protective role was also demonstrated by the improvement of the thermodynamic behavior of the PECp-lipase. Furthermore, the PECp-lipase presented the same ability to recognize the pNP palmitate substrate, evidenced by unchanged kinetic parameters Km /Km.app and Vmax /Vmax.app . Finally, PECp-lipase can successfully replace Arabic gum in the lipase assay, resulting in a single step, faster and efficient methodology for pNP palmitate hydrolysis. Acknowledgments Karla A. Batista thanks CAPES for fellowship support. Guilherme L. Alves thanks CNPq for fellowship support. Luiza L.A. Purcena thanks FAPEGO for fellowship support. References [1] L. Liu, M.L. Fishman, J. Kost, K.B. Hicks, Biomaterials 24 (2003) 3333–3343. [2] L. Monfregola, V. Bugatti, P. Amodeo, S.D. Luca, V. Vittoria, Biomacromolecules 12 (2011) 2311–2318. [3] D.F. Torralbo, K.A. Batista, M.C.B. Di-Medeiros, K.F. Fernandes, Food Hydrocolloids 27 (2012) 378–383. [4] S. Tripathi, G.K. Mehrotra, P.K. Dutta, Carbohydrate Polymers 79 (2010) 711–716. [5] J. Leroux, V. Langendorff, G. Schick, V. Vaishnav, J. Mazoyer, Food Hydrocolloids 17 (2003) 455–462. [6] M. Bellusci, I. Francolini, A. Martinelli, L. D’Ilario, A. Piozzi, Biomacromolecules 13 (2012) 805–813. [7] D.H. Zhang, L.X. Yuwen, C. Li, Y.Q. Li, Bioresource Technology 124 (2012) 233–236. [8] C. Calgaroto, R.P. Scherer, S. Calgaroto, J.V. Oliveira, D. Oliveira, S.B.C. Pergher, Applied Catalysis A: General 394 (2011) 101–104. [9] M. Kapoor, M.N. Gupta, Process Biochemistry 47 (2012) 555–569.

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