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Quillaja bark saponin effects on Kluyveromyces lactis β-galactosidase activity and structure
Food Chemistry 303 (2020) 125388
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Quillaja bark saponin effects on Kluyveromyces lactis β-galactosidase activity and structure
T
Cíntia Tiemi Misugi Kayukawaa, Marco Aurélio Schüler Oliveirab, Elaine Kaspchaka, ⁎ Heloisa Bruna Soligo Sanchukic, Luciana Igarashi-Mafraa, Marcos R. Mafraa, a
Department of Chemical Engineering, Federal University of Paraná (UFPR), Polytechnic Center, Jardim das Américas, 81531-990 Curitiba, PR, Brazil Department of Biochemistry, University of Maringá (UEM), 87020-900 Maringá, PR, Brazil c Department of Biochemistry and Molecular Biology, Federal University of Paraná (UFPR), Polytechnic Center, Jardim das Américas, 81531-990 Curitiba, PR, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Saponin-protein interactions Enzymatic activity Circular dichroism Fluorescence spectroscopy Thermodynamic parameters
Saponins are known for their bioactive and surfactant properties, showing applicability to the food, cosmetic and pharmaceutical industries. This work evaluated the saponins effects on Kluyveromyces lactis β-galactosidase activity and correlated these changes to the protein structure. Enzyme kinetic was evaluated by catalytic assay, protein structure was studied by circular dichroism and fluorescence, and isothermal titration calorimetry was used to evaluate the interactions forces. In vitro enzymatic activity assays indicated an increase in the protein activity due to the saponin–protein interaction. Circular dichroism shows that saponin changes the β-galactosidase secondary structure, favoring its protein-substrate interaction. Besides, changes in protein microenvironment due to the presence of saponin was observed by fluorescence spectroscopy. Isothermal titration calorimetry analyses suggested that saponins increased the affinity of β-galactosidase with the artificial substrate o-nitrophenyl-β-galactoside. The increase in the enzyme activity by saponins, demonstrated here, is important to new products development in food, cosmetic, and pharmaceutical industries.
1. Introduction Saponins are a group of natural glycosides, which are widely distributed in plants and some marine animals (Dyck, Gerbaux, & Flammang, 2010; Sparg, Light, & Staden, 2004; Vincken, Heng, Groot, & Gruppen, 2007). These compounds are formed from a hydrophobic aglycone, denominated sapogenin, linked to one or more hydrophilic sugar moieties through an ether or ester glycosidic linkage, at one or two glycosylation sites (Güçlü-Üstündağ & Mazza, 2007). One of the most common sources of saponins employed in the food, cosmetics, and pharmaceutical industries is the Quillaja bark extract, obtained from the tree Quillaja saponaria Molina (Nord & Kenne, 1999; San Martín & Briones, 1999). Quillaja saponins are formed predominantly by triterpene bisdesmosides that possess sugar chains consisting of glucose, galactose, xylose, rhamnose, arabinose, and glucuronic acid, substituted at the C-3 and C-28 positions of the triterpene aglycone (Kezwon & Wojciechowski, 2014). For many years, saponins were predominantly studied for their detergent properties (Güçlü-Üstündağ & Mazza, 2007). Today, the interest in saponins is primarily due to their bioactive properties. Studies demonstrate that the compounds present immunostimulatory, ⁎
hypocholesterolemic, antitumor, anti-inflammatory, antibacterial, antiviral, antifungal, and antiparasitic activities (El Barky, Hussein, AlmEldeen, Hafez, & Mohamed, 2016; Thompson, 1993). Regarding all these properties, Quillaja extract has great potential applications. For commercial purposes, e.g., the extracts are usually mixed with other compounds, such as proteins (Kezwon & Wojciechowski, 2014). The interaction between saponins and proteins can modify the properties of the protein and the solution. In this context, there are found some studies that evaluate their interaction. Potter, Jimenez-Flores, Pollack, Lone, and Berber-Jimenez (1993) evaluated the interaction quillaja saponin-caseins, and it was concluded that their interaction resulted in high molecular weight complexes that presented positive effects on animal blood lipids. The interactions between different saponins and salt soluble proteins from walleye pollack meat were also evaluated by Tanaka, Fang-I, Ishizaki, and Taguchi (1995) and it was concluded that different saponins could modify the proteins aggregation properties. More recently, Kezwon and Wojciechowski (2014) evaluated the effect of β-lactoglobulin and lysozyme on the surface tension of saponins. The different proteins interact with saponins by electrostatic and hydrophobic forces, causing changes in their surface tension. It was also observed that the interaction depends on the specific sites of the
Corresponding author. E-mail address: [email protected] (M.R. Mafra).
https://doi.org/10.1016/j.foodchem.2019.125388 Received 10 February 2019; Received in revised form 13 August 2019; Accepted 17 August 2019 Available online 19 August 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
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substrate was evaluated by isothermal titration calorimetry (ITC).
proteins with sugar or carboxylic moieties of the saponin molecules. Böttcher, Scampicchio, and Drusch (2016) also evaluated the interactions between saponins and β-lactoglobulin. It was suggested that they could interact probably through hydrogen bonds and/or hydrophobic interactions, and changes in the foam properties of the protein were observed. About the effects of saponins on enzymes, it is known that the compound can inhibit the activity of some digestive enzymes, such as lipase, amylase or glucosidase (Birari & Bhutani, 2007; Ercan & El, 2016). As can be observed, soponin promotes important changes in the properties and activity of several proteins, and its knowledge is of great relevance both for the development of new products as well as for the understanding of its physiological aspects. β-Galactosidase is one of the most important biotechnological enzymes used in the food industry, owing to its ability to hydrolyze the lactose present in milk, which helps to prevent the effects of lactose maldigestion (Gekas & Lopez-Leiva, 1985). The enzyme is a glycoside hydrolase enzyme that catalyzes the hydrolysis of glycosidic bonds, producing monosaccharides from β-galactosides. β-Galactosidase can be utilized in the food industry in the production of lactose-free dairy products, or it can be consumed by the population in pill form when milk products are ingested. In both instances, the reaction occurs in the presence of several other components. It is known that various components can modify the β-galactosidase structure and affect some of the enzyme functional parameters such as Vmax, Km, Kcat, as well as its catalytic activity (Illanes, Altamirano, & Cartagena, 1994). Some of the substances known to decrease the enzyme activity are silver and copper ions (Wutor, Togo, & Pletschke, 2007), tetracycline hydrochloride (Gao, Bi, Zuo, Jia, & Tang, 2017), and copper oxide nanoparticles (Rabbani, Jahir, Ahmad, Yusof, & Hasan, 2014). Examples of substances known to enhance β-galactosidase activity are milk proteins (Greenberg & Mahoney, 1984), ions Na+ and K+ at specific conditions (Reithel & Kim, 1960) and low concentrations of calcium and ferrous ions (Wutor et al., 2007). About the saponin and enzymes interactions, it has been shown that they can interact with each other and change their properties. Bouarab, Melton, Peart, Baulcombe, and Osbourn (2002) verified that the fungal saponin-detoxifying enzyme (tomatinase) could hydrolyze antimicrobial saponins, causing a suppression on plants defenses responses and favoring plants infection. Ishaaya and Birk (1965) reported that soybean saponins interact and can inhibit, to a certain extent, enzymes such as cholinesterase, chymotrypsin, trypsin, and papain. Luyen, Dang, Binh, Hai, and Dat (2018) demonstrated that a triterpenoid saponin isolated from Polyscias fruticosa leaves strongly inhibited the enzymes α-amylase and α-glucosidase. Additionally, it was observed that the saponin also decreased the postprandial blood glucose level in a mouse model. Studies about the interactions of β-galactosidase with saponins are still scarce. Zhang et al. (2015) evaluated the interaction of Lactobacillus bulgaricus β-galactosidase and steroidal saponins. It was reported that the enzyme could glycosylate saponins using lactose as a donor substrate. The results provide a new way to generate compounds with a novel or improved properties. Given the changes caused by saponins on properties, structure, and functionality of different enzymes, further investigations on the topic are required. Due to the importance of β-galactosidase for the pharmaceutical, chemical, and food industry, the enzyme was chosen as an object of this study. Additionally, in the current scenario, where saponins are being studied for their bioactive properties, that can provide new properties to other products, it is important to evaluate their interactions with different enzymes. Knowing that the interaction of saponins with βgalactosidase has not yet been characterized, this work aims to assess the effect of saponins on the enzyme activity, by monitoring the changes in the enzyme kinetic parameters (Vmax, Kcat, and Km). Furthermore, these data were correlated with transformations in the enzyme’s secondary and tertiary structure upon saponin exposure by using circular dichroism and fluorescence spectroscopy. Finally, the effect of saponins on the interaction of β-galactosidase synthetic
2. Materials and methods 2.1. Materials The enzyme β-galactosidase (EC:3.2.1.23) produced by Kluyveromyces lactis was provided by Granolab Granotec company (Araucária, Paraná, Brazil). The β-galactosidase activity was described by the manufacturer as 6500 acid lactase units per gram, and its concentration was determined by the Bradford assay (Bradford, 1976) using bovine serum albumin as the standard, and the concentration obtained was 113.84 mg mL−1 or 0.967 mmol L−1. The enzyme was analyzed by SDS-PAGE previously (data presented in Kayukawa et al., 2018). The results showed that the enzyme, with a molar mass of ~117 KDa, was the predominant protein of the sample. The densitometric analyses of the protein bands in that gel indicated 70% of purity of β-galactosidase. The reagents o-nitrophenyl-β-D-galactopyranoside (ONPG) (> 98% purity; molar mass of 301.25 g mol−1) and 2-nitrophenol (ONP) (98% purity; molar mass of 139.11 g mol−1) were obtained from Sigma–Aldrich (St Louis, Missouri, USA). Concentrated extracted of Quillaja bark saponin (QBS) was obtained from Vetec Sigma-Aldrich (Rio de Janeiro, Brazil). According to the supplier, the concentration of saponin in the extract is < =100% (CAS number: 8047-15-2). A Milli-Q system (Millipore Corp., Billerica, Massachusetts, USA) was used to provide deionized water. 2.2. Methods 2.2.1. Enzyme activity assay The hydrolytic activity of β-galactosidase was evaluated using the synthetic substrate ONPG. The methodology was adapted from Inchaurrondo, Yautorno, and Voget (1994). The enzyme activity assay was performed with samples constituted by 75 μL of 6.37 × 10−3 µmol L−1 β-galactosidase enzyme, 75 μL of 1.5 mmol L−1 artificial substrate ONPG and 775 μL of potassium phosphate buffer pH 6.8, 75 mmol L−1. In the case of saponin samples, the amount of buffer used was 700 μL, and the protein and additive quantities were both 75 μL. The QBS extract concentration was 0.05 mg mL−1, chosen according to preliminary enzymatic assays and ITC assays. After preparing the samples, they were promptly conditioned at 37 °C in the thermostatic bath Dubnoff (304/D, Ethik Technology, Vargem Grande Paulista, São Paulo, Brazil). Sodium carbonate solution (75 µL; 2 mol L−1) was added to each sample to stop the reaction in intervals ranging from 1 to 240 min. The absorbance was measured in a UV–Vis spectrophotometer (UV-1800, Shimadzu Corporation, Kyoto, Japan) at 420 nm. To quantify the enzymatic product formation, an ONP standard curve was constructed, and the results were expressed in mmol L−1 (see Supporting information). The samples enzymatic activity was calculated as follows:
Ea =
A. Vreaction ε . Venz . t
(1) −1
where Ea represents the enzyme catalytic activity in U mL . A is the absorbance measured (420 nm), Vreaction consist of the volumes present in the reaction in mL (buffer solution + ONPG solution + enzyme solution + sodium carbonate solution), ε (4024.66 L mol−1 cm−1) is the extinction coefficient of ONP under the experiment and measured at 420 nm, Venz is the volume of the enzyme extract in mL, and t is the time in minutes. Each of the β-galactosidase unit (U) represents the quantity necessary of the enzyme to release 1 μmol o-nitrophenol per minute under the assay conditions. The kinetic parameters, i.e., the Michaelis–Menten constant (Km), maximum reaction rate (Vmax) and turnover number (kcat) of the effects of the saponin on the β-galactosidase and ONPG interaction, were 2
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interaction of β-galactosidase with ONPG, as well as the saponin presence, were evaluated by ITC (ITC 200, Microcal, Inc., Northampton, Massachusetts, USA). The analysis was performed at 750 rpm and 37 °C. All of the solutions were prepared in the potassium phosphate buffer pH 6.8 (75 mmol L−1). The interactions were evaluated in two different ways: the titration of the protein against ONPG and the protein and saponin against ONPG. To assess the molecular interaction of the enzyme and substrate (ONPG), the syringe was filled with 40 µL of ONPG (1.5 mmol L−1) and the sample cell with 200 µL of β-galactosidase solution (0.85 µmol L−1). Additionally, to verify the effect of the saponin on the reaction, the titration was performed with 0.85 µmol L−1 β-galactosidase solutions in the presence of QBS extract at 0.05 mg mL−1 (cell sample) against solutions of 1.5 mmol L−1 ONPG (syringe). For all ITC assays, the reference cell was filled with 200 µL of ultrapure water. The titration was performed with the first injection of 0.4 µL, followed by 24 successive injections of 1.5 µL. The duration of each injection was 3 s, and the intervals between the injections were 100 s. All the results obtained had the blank assays discounted (see Supplementary material). The one-site model was fitted to the experimental data in order to obtain the thermodynamics parameters (stoichiometry (n), affinity constant (Ka), and binding enthalpy (ΔH)). Microcal Origin 7.0 software (ITC Data Analysis in Origin, Microcal, Northampton, Massachusetts, USA) in this procedure. Finally, the entropy was calculated from Eq. (6).
evaluated. The kinetic parameters were obtained using Origin software 8.6. (Northampton, Massachusetts, USA) and correlating the Michaelis–Menten model (Eq. (2)) to the experimental data.
v=
Vmax . S Km + S
(2)
where v corresponds to the hydrolysis rate (mmol ONP min−1), S is the substrate concentration, Km is given in mmol L−1, and Vmax in mmol ONP min−1. The kcat parameter and the enzyme catalytic efficiency (Eef) were calculated, according to Eqs. (3) and (4), respectively:
kcat =
Vmax Et
(3)
Eef =
kcat Km
(4)
where Et is the enzyme molar concentration in the assay (mol L−1). 2.2.2. Fluorescence spectroscopy assay The interactions between β-galactosidase as a function of the QBS extract concentration were assessed by fluorescence spectroscopy, as described by Soares, Mateus, and De Freitas (2007), and more recently, as used in our previous work (Kayukawa et al., 2018), using the spectropolarimeter UV–Vis (J-815, Jasco Int., Co., Tokyo, Japan) connected to the temperature controller Peltier (PTP-1). The equipment was stabilized at 37 °C, and the purge gas (nitrogen) was fed at a rate of 10 L min−1. All the solutions were prepared in 15 mmol L−1 potassium phosphate buffer pH 6.8. The β-galactosidase concentration was maintained at 0.085 mmol L−1 during the assay, and the QBS extract solution concentrations were 0.49, 0.73, 0.98, 1.47, 2.94, 5.88, and 9.80 mg mL−1. The samples were excited at 295 nm, and the emission measurements were recorded in the 310–370 nm range. Measurements of sample solutions were discounted by blank solutions (without saponin), and the results were expressed by plotting the fluorescence intensity against the wavelength.
ΔS =
θ × 100 × MM C×l×n
(6)
where T is the temperature of the system in Kelvin, and R is the ideal gas constant (8.31 J K−1 mol−1). The binding Gibbs energy (ΔG) was determined, according to Eq. (7):
ΔG = ΔH − T ΔS
(7)
The experiments were carried out in duplicate, and the thermodynamic parameters consisted of the mean of duplicate measurements ± standard deviation.
2.2.3. Circular dichroism (CD) assay The evaluation of the β-galactosidase secondary and tertiary structure by CD was performed using a CD spectropolarimeter (J-815, Jasco Int., Co), as described by Rabbani et al. (2014). The conditions of the purge gas, as well as the concentrations and pH of the solutions employed, were the same as those described in our previous work (Kayukawa et al., 2018). The saponin effect on the protein was observed by the addition of the QBS extract solutions at 0.1, 0.01, and 0.001 mg mL−1 to the protein solutions. The tertiary structure was obtained by near UV CD spectra assay (Kayukawa et al., 2018). The protein solutions were prepared in the potassium phosphate buffer (15 mmol L−1; pH 6.8) in the concentration of 8.50 µmol L−1, and the saponin effect on the system was obtained with the inclusion of QBS extract at 5 and 10 µg mL−1. Measurements were performed in a range of 250–350 nm, and a blank solution spectrum (without the saponin and protein) was subtracted to obtain the CD spectra. The protein samples secondary and tertiary structure were investigated using the molar mean residue ellipticity (θ MRE), that were calculated using Eq. (5):
θMRE =
ΔH − RlnK a T
2.2.5. Statistical analyses One-way analysis of variance (ANOVA) was applied to all experimental data, which were expressed as the means of duplicates. Mean values were evaluated by Tukey’s test at the 95% significance level, with the aid of Microsoft Excel 2010 action supplement (Redmond, Washington, USA). 3. Results and discussion 3.1. Influence of saponin on the enzymatic activity of β-galactosidase The β-galactosidase catalytic activity was achieved by ONPG assay using the enzyme in the concentration of 6.37 × 10−3 µmol L−1 at 30 min of the reaction. The enzymatic activity value obtained for the sample was 0.04746 U mL−1. The assay was also performed in the presence of the saponin (QBS extract) at various concentrations (30 min of reaction), and at that specific reaction time, no changes in the enzymatic activity were induced by the saponin (Fig. 1a). To explore the effects of the saponin on the enzyme, the kinetic of the reaction catalyzed by β-galactosidase was evaluated, using ONPG as a substrate in the presence or absence of saponin. The analyses were done at the QBS extract concentration of 0.05 mg mL−1. Fig. 1b displays the correlation of the Michaelis–Menten equation (Eq. (2)) to the experimental kinetic data, as well as the kinetic parameters (Km, kcat). The kcat value for the control sample (without saponin) was 209.105 ± 9.680 min−1, and the Km value for the same sample was 34.09 ± 6.980 mmol L−1. Thus, the enzyme catalytic efficiency was 6133.915 ± 1579.294 M−1 min−1. By adding QBS extract at
(5)
where θ correspond to the ellipticity degree (°); M(M) is the molar mass (kDa), C is the concentration (mg mL−1), l is the optical path length (cm), and n is the number of β-galactosidase residues. The mean residue ellipticity (θ MRE) is given in ° cm2 dmol−1 (Corrêa & Ramos, 2009). 2.2.4. Isothermal titration calorimetry (ITC) assay The energy and thermodynamic parameters resulted by the 3
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Fig. 1. Saponin effect on β-galactosidase enzymatic activity evaluated by UV–Vis. (a) Effect of different concentrations of QBS extract on the β-galactosidase (6.37 × 10−3 µmol L−1) enzymatic activity at 30 min of reaction. (b) Kinect of the reaction of β-galactosidase and ONPG in the absence (black line) or presence of QBS extract 0.05 mg mL−1 (red line). Kinetic parameters were obtained from the adjustment of the Michaelis-Menten equation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
0.05 mg mL−1 to the system, the reaction kinetic profile and the kinetic parameters changed (Fig. 1b). For the sample with saponin, the reaction occurred slightly faster than the control. Analyzing the kinetic parameters, the QBS extract did not significantly affect the kcat values compared to the control (189.922 ± 13.565 min−1), but the Km parameter decreased to 15.445 ± 1.450 mmol L−1. Hence, the QBS extract presence increased the catalytic efficiency, which shifted to 12296.697 ± 1274.736 M−1 min−1, showing it increases the enzyme activity, which is important for food, chemical and pharmaceutical industries for increase catalytic activity of enzymes of interest. Another surfactant known to present a positive effect on β-galactosidase activity is Triton X-100, and it is reported that this surfactant increased the thermal stability of the enzyme (Soto et al., 2017).
emission peak at 336 nm, regarding the tryptophan residues of β-galactosidase. Besides, a consistent decline in the fluorescence intensity of β-galactosidase occurred with an increase of QBS extract concentration. This decrease in the fluorescence intensity is related to changes in the protein microenvironment near the tryptophan residues and is correlated to changes in protein conformation (Lakowicz, 1999). It can also be observed changes in the wavelength of maximum emission (λmax), red shifts, with the QBS extract addition. These are indicatives that as the QBS extract concentration increase, the accessibility of tryptophan residues to the solvent also increases. The red shifts also occurred for the pancreatic lipase interaction with botulin and ginsenoside saponins, and it was related to the conformational changes on the enzyme tertiary structure (Yu, Dong, Wang, & Liu, 2017). Another compound that caused changes in β-galactosidase conformation (tertiary structure) is Triton X-100 (Soto et al., 2017). The substance, like the saponins, has surfactant properties, and it was concluded that their interaction helped the enzyme stability. The β-galactosidase tertiary structure was evaluated by CD spectra in the near UV region (Fig. 2b). The control sample (native protein) exhibits a positive peak at 275 nm and signals around 265–290 nm, that
3.2. Influence of saponin (QBS extract) on the β-galactosidase tertiary structure Fig. 2a shows the fluorescence emission curve obtained at λex = 295 nm for β-galactosidase at pH 6.8, with and without the addition of saponin (QBS extract). As can be seen, the protein had a strong
Fig. 2. Fluorescence spectra evaluating the impact of saponin on the tertiary structure of β-galactosidase solutions. (a) Emission spectra of β-galactosidase 0.425 µmol L−1 in the presence of QBS extract at concentrations of 0.125–4.500 mg mL−1. Fluorescence emissions intensity were recorded at λex = 295 nm, and the λem maximum occurred at 336 nm. (c) Near UV CD spectra of β-galactosidase 8.50 µmol L−1 in the presence of QBS extract at concentrations of 5 µg mL−1 and 10 µg mL−1. 4
Food Chemistry 303 (2020) 125388
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Fig. 3. β-Galactosidase 1.74 µmol L−1 secondary structure in the presence of saponin. (a) UV CD spectrum of β-galactosidase with different QBS extract concentrations varying of 0 mg.mL−1 (control) and 3 µg mL−1. Percentage of β-galactosidase secondary structures in the presence of 0 mg mL−1 (control) and 3 µg mL−1 of QBS extract.
the order of 105 L mol−1) and the molar ratio (n) of ONPG:protein (122:1) indicated a moderate affinity between β-galactosidase and ONPG. Additionally, one can see in Fig. 4c that this interaction is governed mainly by enthalpic contributions (ΔH = −16300 J mol−1 and TΔS = 1345 J mol−1) and presents ΔG = −41580 J mol−1. Enthalpic contributions may be related to the hydrolysis of ONPG by the enzyme, hence be associated with the enzyme activity (Kayukawa et al., 2018). Fig. 4b presents the influences of 0.05 mg mL−1 QBS extract on the binding of ONPG with β-galactosidase. Under this condition, the saponin positively impacted the protein interactions with ONPG. The addition of QBS extract caused an increase in the ΔH, TΔS, and ΔG parameters, indicating changes in the interaction mechanism. The increment in the enthalpic contributions (absolute value), from −16300 ± 395.7 (initial) to −41580 ± 1345 kJ mol−1, demonstrated modifications on the interactions of the enzyme with ONPG. The entropic contribution (TΔS) changed from 2223.7 ± 18 (control) to −300.81 ± 22 kJ mol−1, implying the saponin decreased the hydrophobic interaction contributions to the reaction, compared to the control sample. Also, the ΔG values decreased with the addition of saponin (QBS extract), indicating that the spontaneity of the protein–ONPG interaction increased. The Ka value was also increased, which means a higher affinity of the protein to the substrate, and the molecular interaction reached the saturation earlier in the sample with QBS extract when compared to the control sample (without saponin), indicating that saponin promoted an increase in the reaction affinity. ITC results demonstrated that QBS extract changed the molecular interaction mechanism of β-galactosidase and ONPG and increased the protein’s affinity for the substrate. These results complement the kinetic data, since changes in the mechanism and affinity of the protein may also modify its catalytic activity. The ITC assays allow concluding that the observed changes in the kinetic analysis (Fig. 1b) is probably a consequence of the higher affinity of the enzyme to the substrate when QBS extract is present. The results of fluorescence and circular dichroism analysis supports ITC results since changes in the protein secondary and tertiary structure can modify the enzyme interaction mechanism. Although the bioactive properties of the saponin are already known by the scientific community, the results presented here show, for the first time, the increase of β-galactosidase catalytic activity by the presence of QBS extract. However, further structural and functional studies must be performed to elucidate the exact saponin effects on proteins under a range of experimental conditions (concentrations, ionic strength, pH, temperature, interactions with other substances) to assess
are characteristics of the protein aromatic amino acids residues. The presence of QBS extract at a concentration of 5 µg mL−1 modifies the curve, suggesting that changes occurred on the protein tertiary structure. When the QBS extract concentration increases to 10 µg mL−1, the changes became more evident, with a clear enhancement in intensity on the overall curve and a maxima peak at 265 nm. The increase on the signal can be related to an increase on the enzyme stability, since the decrease of the signal is known to be characteristic of a loss on the protein stability (Kayukawa et al., 2018; Yu, Yang, & Labahn, 2017). Therefore, the saponin changed β-galactosidase microenvironment, increasing the accessibility of tryptophan residues to the solvent. 3.3. Influence of saponin (QBS extract) on the β-galactosidase secondary structure The saponin (QBS extract) effects on the β-galactosidase secondary structure were evaluated by CD. The specters of the β-galactosidase without (control) and with saponin are shown in Fig. 3a and revealed a maximum peak at 195 nm and a minimum peak at 215 nm, characteristic of proteins with rich β-sheet contents (Corrêa & Ramos, 2009). These results are consistent with previous CD analysis of K. lactis βgalactosidase (Kayukawa et al., 2018; Tello-Solís et al., 2005). Fig. 3b shows the secondary structure composition of the samples. The β-galactosidase control sample contained 22.6% α-helix, 29% β-sheet, 20.9% β-turn, and 27.5% random coil. Fig. 3 presents the changes caused by 3 µg mL−1 of QBS extract on the protein secondary structure. It can be seen that θ MRE values at 195 nm increased and the region of 215–230 nm, decreased, hence a modification on the protein secondary structure content to 35.5% αhelix and 25.5% β-sheet, 17.2% β-turn, and 21.5% random coil (Fig. 3b). The increase of α-helix content on Kluyveromyces lactis β-galactosidase structure can be related to a favorable interaction between the enzyme and its substrate, as observed in Tello-Solís et al. (2005). 3.4. Influence of saponin (QBS extract) on the interactions and thermodynamic parameters on the binding of β-galactosidase with ONPG The interactions of β-galactosidase and ONPG and how they are modified by saponin (QBS extract) were studied by ITC are presented in Fig. 4. The experimental data were fitted using the one-site binding model to assess the thermodynamic parameters of the interaction (Table 1). Fig. 4 confirms the binding of β-galactosidase to ONPG at 37 °C, since a sigmoidal-shaped thermogram was obtained. The value of Ka (in 5
Food Chemistry 303 (2020) 125388
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Fig. 4. ITC curves evaluating the effect of saponin on the binding of β-galactosidase with ONPG. Each injection of 1.5 µL is represented by the peaks with 100 s of intervals. (a) The syringe is filled with 0.85 µmol L−1 β-galactosidase and the cell with 1.5 mmol L−1 ONPG solution (control sample) (b) The syringe is filled with 0.85 µmol L−1 β-galactosidase + QBS extract 0.05 mg mL−1, (c) thermodynamic profile of ONPG interacting with β-galactosidase control and in with QBS extract 0.05 mg mL−1. Solid lines are the best fit of the data using the one-site model; symbols are experimental data, n is stoichiometry, Ka is the association constant, ΔH is enthalpy change, ΔS is entropy change and ΔG is the Gibbs free energy.
Declaration of Competing Interest
Table 1 Thermodynamic parameters of the interaction between β-galactosidase with ONPG (control) and in the presence of QBS extract 0.05 mg mL−1. Thermodynamic parameters
β-Galactosidase (control)
β-Galactosidase with QBS extract 0.05 mg mL−1
n Ka (mol−1 L) ΔH (J mol−1) TΔS (J mol−1) ΔG (J mol−1)
122 ± 1.84 7.78 × 105 ± 2.00 × 105 −16300 ± 395.7 2223.7 ± 18 −18523.7 ± 320
71.7 ± 1.22 3.76 × 106 ± 1.78 × 106 −41580 ± 1345 −300.81 ± 22 −41279.19 ± 1220
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments Funding: This work was supported by CAPES (Coordination for the Improvement of Higher Education Personnel), Graduate Program in Food Engineering (Federal University of Paraná, Curitiba, Brazil), Granolab Granotec, and INCT-UFPR (National Institute of Science and Technology on Biological Nitrogen Fixation). M. R. Mafra, and L. Igarashi-Mafra is grateful to the Brazilian National Council for Scientific and Technological Development (CNPq – Grant 310182/2018-2, 308517/2018-0, respectively). The authors also thank Thiago Atsushi Takashina for the contribution.
their suitability in future applications.
4. Conclusion It was verified an increase in β-galactosidase activity due to the saponin presence, with a clear decrease on the Michaelis-Menten parameter (Km) of the enzyme and increase on the enzyme efficacy (Eef). The fluorescence spectroscopy data suggested that the QBS extract changes the protein microenvironment, and the changes on the overall protein conformation were followed by near CD assays. By mean of CD analyzes, it can be seen that QBS extract changes the β-galactosidase secondary structure, increase its α-helix content, favoring its proteinsubstrate interaction. The changes on the molecular interaction mechanism of β-galactosidase and ONPG was shown by the ITC measurements, in which one can see that the QBS extract increases the affinity of the protein-substrate, as well as the molecular interaction spontaneity. This behavior is in agreement with the changes observed in the enzyme kinetics. This study may help to understand better the effects that the QBS extract (saponin source) causes on the β-galactosidase structure and catalytic activity. The extract presents some aspects that can be important to the food and pharmaceutical industry. Nevertheless, the extract may have negative effects on other proteins in the digestive system. Therefore, the interactions of QBS extract with other proteins should also be studied.
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.125388. References Birari, R. B., & Bhutani, K. K. (2007). Pancreatic lipase inhibitors from natural sources: Unexplored potential. Drug Discovery Today, 12, 879–889. https://doi.org/10.1016/j. drudis.2007.07.024. Böttcher, S., Scampicchio, M., & Drusch, S. (2016). Mixtures of saponins and beta-lactoglobulin differ from classical protein/surfactant-systems at the air-water interface. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 506, 765–773. https://doi.org/10.1016/j.colsurfa.2016.07.057. Bouarab, K., Melton, R., Peart, J., Baulcombe, D., & Osbourn, A. (2002). A saponin-detoxifying enzyme mediates suppression of plant defences. Nature, 418, 889–892. https://doi.org/10.1038/nature00950. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1–2), 248–254. https://doi.org/10.1016/0003-2697(76)90527-3. Corrêa, D. H. A., & Ramos, C. H. I. (2009). The use of circular dichroism spectroscopy to study protein folding, form and function. African Journal of Biochemistry Research, 3(5), 164–173. https://doi.org/10.1038/nprot.2006.204. Dyck, S. Van, Gerbaux, P., & Flammang, P. (2010). Qualitative and quantitative saponin contents in five sea cucumbers from the Indian ocean. Marine Drugs, 8, 173–189. https://doi.org/10.3390/md8010173.
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nanoparticles on the conformation and activity of β-galactosidase. Colloids and Surfaces B: Biointerfaces, 123, 96–105. Reithel, F. J., & Kim, J. C. (1960). Studies of β-galactosidase isolated from Escherichia coli ML 308. I. The effect of some ions on enzymic activity. Archives of Biochemistry and Biophysics, 90(2), 271–277. https://doi.org/10.1016/0003-9861(60)90579-8. San Martín, R., & Briones, R. (1999). Industrial uses and sustainable supply of Quillaja saponaria (Rosaceae) saponins. Economic Botany, 53(3), 302–311. https://doi.org/10. 1007/BF02866642. Soares, S., Mateus, N., & De Freitas, V. (2007). Interaction of different polyphenols with bovine serum albumin (BSA) and human salivary α-amylase (HSA) by fluorescence quenching. Journal of Agricultural and Food Chemistry, 55(16), 6726–6735. https:// doi.org/10.1021/jf070905x. Soto, D., Escobar, S., Guzmán, F., Cárdenas, C., Bernal, C., & Mesa, M. (2017). Structureactivity relationships on the study of β-galactosidase folding/unfolding due to interactions with immobilization additives: Triton X-100 and ethanol. International Journal of Biological Macromolecules, 96, 87–92. https://doi.org/10.1016/j.ijbiomac. 2016.12.026. Sparg, S. G., Light, M. E., & Staden, J. Van (2004). Biological activities and distribution of plant saponins. Journal of Ethnopharmacology, 94, 219–243. https://doi.org/10.1016/ j.jep.2004.05.016. Tanaka, M., Fang-I, L., Ishizaki, S., & Taguchi, T. (1995). Interaction of saponins with salt soluble proteins from walleye pollack meat. Fisheries Sciences, 61(2), 373–374. https://doi.org/10.2331/fishsci.61.373. Tello-Solís, S. R., Jiménez-Guzmân, J., Sarabia-Leos, C., Gómez-Ruíz, L., Cruz-Guerrero, A. E., Rodríguez-Serrano, G. M., & García-Garibay, M. (2005). Determination of the secondary structure of Kluyveromyces lactis β-galactosidase by circular dichroism and its structure-activity relationship as a function of the pH. Journal of Agricultural and Food Chemistry, 53(26), 10200–10204. https://doi.org/10.1021/jf051480m. Thompson, L. U. (1993). Potential health benefits and problems associated with antinutrients in foods. Food Research International, 26(2), 131–149. https://doi.org/10. 1016/0963-9969(93)90069-U. Vincken, J., Heng, L., Groot, A. De, & Gruppen, H. (2007). Saponins, classification and occurrence in the plant kingdom. Phytochemistry, 68, 275–297. https://doi.org/10. 1016/j.phytochem.2006.10.008. Wutor, V. C., Togo, C. A., & Pletschke, B. I. (2007). The effect of physico-chemical parameters and chemical compounds on the activity of β-d-galactosidase (B-GAL), a marker enzyme for indicator microorganisms in water. Chemosphere, 68(4), 622–627. https://doi.org/10.1016/j.chemosphere.2007.02.050. Yu, H., Dong, S., Wang, L., & Liu, Y. (2017). The effect of triterpenoid saponins on pancreatic lipase in vitro: Activity, conformation, kinetics, thermodynamics and morphology. Biochemical Engineering Journal, 125, 1–9. https://doi.org/10.1016/j. bej.2017.05.010. Yu, K., Yang, G., & Labahn, J. (2017). High-efficient production and biophysical characterisation of nicastrin and its interaction with APPC100. Scientific Reports, 7(1), 1–9. https://doi.org/10.1038/srep44297. Zhang, J., Lu, L., Lu, L., Zhao, Y., Kang, L., Pang, X., ... Ma, B. (2015). Galactosylation of steroidal saponins by β-galactosidase from Lactobacillus bulgaricus L3. Glycoconjugate Journal, 33(1), 53–62. https://doi.org/10.1007/s10719-015-9632-4.
El Barky, A. R., Hussein, S. A., Alm-Eldeen, A. A., Hafez, Y. A., & Mohamed, T. M. (2016). Anti-diabetic activity of Holothuria thomasi saponin. Biomedicine & Pharmacotherapy, 84, 1472–1487. https://doi.org/10.1016/j.biopha.2016.10.002. Ercan, P., & El, S. N. (2016). Inhibitory effects of chickpea and Tribulus terrestris on lipase, α-amylase and α-glucosidase. Food Chemistry, 205, 163–169. https://doi.org/ 10.1016/j.foodchem.2016.03.012. Gao, X., Bi, H., Zuo, H., Jia, J., & Tang, L. (2017). Interaction of residue tetracycline hydrochloride in milk with β-galactosidase protein by multi-spectrum methods and molecular docking. Journal of Molecular Structure, 1141, 382–389. https://doi.org/ 10.1016/j.molstruc.2017.03.096. Gekas, V., & Lopez-Leiva, M. (1985). Hydrolysis of lactose: A literature review. Process Biochemistry, 20, 2–12. Greenberg, N. A., & Mahoney, R. R. (1984). The activity of lactase (Streptococcus thermophilus) in milk and sweet whey. Food Chemistry, 15, 307–313. https://doi.org/10. 1016/0308-8146(84)90114-6. Güçlü-Üstündağ, O., & Mazza, G. (2007). Saponins: Properties, applications and processing saponins: Properties, applications. Critical Reviews in Food Science and Nutrition, 47, 37–41. https://doi.org/10.1080/10408390600698197. Illanes, A., Altamirano, C., & Cartagena, O. (1994). Enzyme reactor performance under thermal inactivation. Advances in Bioprocess Engineering, 467–472. https://doi.org/10. 1007/978-94-017-0641-4_64. Inchaurrondo, V. A., Yautorno, O. M., & Voget, C. E. (1994). Yeast growth and β-galactosidase production during aerobic batch cultures in lactose-limited synthetic medium. Process Biochemistry, 29, 47–54. https://doi.org/10.1016/0032-9592(94) 80058-8. Ishaaya, I., & Birk, Y. (1965). Soybean saponins. IV. The effect of proteins on the inhibitory activity of soybean saponins on certain enzymes. Journal of Food Science, 30, 118–120. https://doi.org/10.1111/j.1365-2621.1965.tb00273.x. Kayukawa, C. T. M., de Oliveira, M. A. S., Kaspchak, E., Sanchuki, H. B. S., Igarashi-Mafra, L., & Rogério Mafra, M. (2018). Effect of tannic acid on the structure and activity of Kluyveromyces lactis β-galactosidase. Food Chemistry. https://doi.org/10.1016/J. FOODCHEM.2018.09.107. Kezwon, A., & Wojciechowski, K. (2014). Interaction of Quillaja bark saponins with foodrelevant proteins. Advances in Colloid and Interface Science, 209, 185–195. https://doi. org/10.1016/j.cis.2014.04.005. Lakowicz, J. R. (1999). Principles of fluorescence spectroscopy(2nd ed.). New York: Kluwer Academic/Plenum Publishers. https://doi.org/10.1016/j.jelechem.2009.09.018. Luyen, N. T., Dang, N. H., Binh, P. T. X., Hai, N. T., & Dat, N. T. (2018). Hypoglycemic property of triterpenoid saponin PFS isolated from Polyscias fruticosa leaves. Annals of the Brazilian Academy of Sciences, 90, 2881–2886. Nord, L. I., & Kenne, L. (1999). Separation and structural analysis of saponins in a bark extract from Quillaja saponaria Molina. Carbohydrate Research, 320, 70–81. https:// doi.org/10.1016/S0008-6215(99)00134-2. Potter, S. M., Jimenez-Flores, R., Pollack, J. A., Lone, T.a., & Berber-Jimenez, M. D. (1993). Protein-saponin interaction and its influence on blood lipids. Journal of Agricultural and Food Chemistry, 41(8), 1287–1291. https://doi.org/10.1021/ jf00032a023. Rabbani, G., Jahir, M., Ahmad, A., Yusof, M., & Hasan, R. (2014). Effect of copper oxide
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Quillaja bark saponin effects on Kluyveromyces lactis β-galactosidase activity and structure
T
Cíntia Tiemi Misugi Kayukawaa, Marco Aurélio Schüler Oliveirab, Elaine Kaspchaka, ⁎ Heloisa Bruna Soligo Sanchukic, Luciana Igarashi-Mafraa, Marcos R. Mafraa, a
Department of Chemical Engineering, Federal University of Paraná (UFPR), Polytechnic Center, Jardim das Américas, 81531-990 Curitiba, PR, Brazil Department of Biochemistry, University of Maringá (UEM), 87020-900 Maringá, PR, Brazil c Department of Biochemistry and Molecular Biology, Federal University of Paraná (UFPR), Polytechnic Center, Jardim das Américas, 81531-990 Curitiba, PR, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Saponin-protein interactions Enzymatic activity Circular dichroism Fluorescence spectroscopy Thermodynamic parameters
Saponins are known for their bioactive and surfactant properties, showing applicability to the food, cosmetic and pharmaceutical industries. This work evaluated the saponins effects on Kluyveromyces lactis β-galactosidase activity and correlated these changes to the protein structure. Enzyme kinetic was evaluated by catalytic assay, protein structure was studied by circular dichroism and fluorescence, and isothermal titration calorimetry was used to evaluate the interactions forces. In vitro enzymatic activity assays indicated an increase in the protein activity due to the saponin–protein interaction. Circular dichroism shows that saponin changes the β-galactosidase secondary structure, favoring its protein-substrate interaction. Besides, changes in protein microenvironment due to the presence of saponin was observed by fluorescence spectroscopy. Isothermal titration calorimetry analyses suggested that saponins increased the affinity of β-galactosidase with the artificial substrate o-nitrophenyl-β-galactoside. The increase in the enzyme activity by saponins, demonstrated here, is important to new products development in food, cosmetic, and pharmaceutical industries.
1. Introduction Saponins are a group of natural glycosides, which are widely distributed in plants and some marine animals (Dyck, Gerbaux, & Flammang, 2010; Sparg, Light, & Staden, 2004; Vincken, Heng, Groot, & Gruppen, 2007). These compounds are formed from a hydrophobic aglycone, denominated sapogenin, linked to one or more hydrophilic sugar moieties through an ether or ester glycosidic linkage, at one or two glycosylation sites (Güçlü-Üstündağ & Mazza, 2007). One of the most common sources of saponins employed in the food, cosmetics, and pharmaceutical industries is the Quillaja bark extract, obtained from the tree Quillaja saponaria Molina (Nord & Kenne, 1999; San Martín & Briones, 1999). Quillaja saponins are formed predominantly by triterpene bisdesmosides that possess sugar chains consisting of glucose, galactose, xylose, rhamnose, arabinose, and glucuronic acid, substituted at the C-3 and C-28 positions of the triterpene aglycone (Kezwon & Wojciechowski, 2014). For many years, saponins were predominantly studied for their detergent properties (Güçlü-Üstündağ & Mazza, 2007). Today, the interest in saponins is primarily due to their bioactive properties. Studies demonstrate that the compounds present immunostimulatory, ⁎
hypocholesterolemic, antitumor, anti-inflammatory, antibacterial, antiviral, antifungal, and antiparasitic activities (El Barky, Hussein, AlmEldeen, Hafez, & Mohamed, 2016; Thompson, 1993). Regarding all these properties, Quillaja extract has great potential applications. For commercial purposes, e.g., the extracts are usually mixed with other compounds, such as proteins (Kezwon & Wojciechowski, 2014). The interaction between saponins and proteins can modify the properties of the protein and the solution. In this context, there are found some studies that evaluate their interaction. Potter, Jimenez-Flores, Pollack, Lone, and Berber-Jimenez (1993) evaluated the interaction quillaja saponin-caseins, and it was concluded that their interaction resulted in high molecular weight complexes that presented positive effects on animal blood lipids. The interactions between different saponins and salt soluble proteins from walleye pollack meat were also evaluated by Tanaka, Fang-I, Ishizaki, and Taguchi (1995) and it was concluded that different saponins could modify the proteins aggregation properties. More recently, Kezwon and Wojciechowski (2014) evaluated the effect of β-lactoglobulin and lysozyme on the surface tension of saponins. The different proteins interact with saponins by electrostatic and hydrophobic forces, causing changes in their surface tension. It was also observed that the interaction depends on the specific sites of the
Corresponding author. E-mail address: [email protected] (M.R. Mafra).
https://doi.org/10.1016/j.foodchem.2019.125388 Received 10 February 2019; Received in revised form 13 August 2019; Accepted 17 August 2019 Available online 19 August 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
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substrate was evaluated by isothermal titration calorimetry (ITC).
proteins with sugar or carboxylic moieties of the saponin molecules. Böttcher, Scampicchio, and Drusch (2016) also evaluated the interactions between saponins and β-lactoglobulin. It was suggested that they could interact probably through hydrogen bonds and/or hydrophobic interactions, and changes in the foam properties of the protein were observed. About the effects of saponins on enzymes, it is known that the compound can inhibit the activity of some digestive enzymes, such as lipase, amylase or glucosidase (Birari & Bhutani, 2007; Ercan & El, 2016). As can be observed, soponin promotes important changes in the properties and activity of several proteins, and its knowledge is of great relevance both for the development of new products as well as for the understanding of its physiological aspects. β-Galactosidase is one of the most important biotechnological enzymes used in the food industry, owing to its ability to hydrolyze the lactose present in milk, which helps to prevent the effects of lactose maldigestion (Gekas & Lopez-Leiva, 1985). The enzyme is a glycoside hydrolase enzyme that catalyzes the hydrolysis of glycosidic bonds, producing monosaccharides from β-galactosides. β-Galactosidase can be utilized in the food industry in the production of lactose-free dairy products, or it can be consumed by the population in pill form when milk products are ingested. In both instances, the reaction occurs in the presence of several other components. It is known that various components can modify the β-galactosidase structure and affect some of the enzyme functional parameters such as Vmax, Km, Kcat, as well as its catalytic activity (Illanes, Altamirano, & Cartagena, 1994). Some of the substances known to decrease the enzyme activity are silver and copper ions (Wutor, Togo, & Pletschke, 2007), tetracycline hydrochloride (Gao, Bi, Zuo, Jia, & Tang, 2017), and copper oxide nanoparticles (Rabbani, Jahir, Ahmad, Yusof, & Hasan, 2014). Examples of substances known to enhance β-galactosidase activity are milk proteins (Greenberg & Mahoney, 1984), ions Na+ and K+ at specific conditions (Reithel & Kim, 1960) and low concentrations of calcium and ferrous ions (Wutor et al., 2007). About the saponin and enzymes interactions, it has been shown that they can interact with each other and change their properties. Bouarab, Melton, Peart, Baulcombe, and Osbourn (2002) verified that the fungal saponin-detoxifying enzyme (tomatinase) could hydrolyze antimicrobial saponins, causing a suppression on plants defenses responses and favoring plants infection. Ishaaya and Birk (1965) reported that soybean saponins interact and can inhibit, to a certain extent, enzymes such as cholinesterase, chymotrypsin, trypsin, and papain. Luyen, Dang, Binh, Hai, and Dat (2018) demonstrated that a triterpenoid saponin isolated from Polyscias fruticosa leaves strongly inhibited the enzymes α-amylase and α-glucosidase. Additionally, it was observed that the saponin also decreased the postprandial blood glucose level in a mouse model. Studies about the interactions of β-galactosidase with saponins are still scarce. Zhang et al. (2015) evaluated the interaction of Lactobacillus bulgaricus β-galactosidase and steroidal saponins. It was reported that the enzyme could glycosylate saponins using lactose as a donor substrate. The results provide a new way to generate compounds with a novel or improved properties. Given the changes caused by saponins on properties, structure, and functionality of different enzymes, further investigations on the topic are required. Due to the importance of β-galactosidase for the pharmaceutical, chemical, and food industry, the enzyme was chosen as an object of this study. Additionally, in the current scenario, where saponins are being studied for their bioactive properties, that can provide new properties to other products, it is important to evaluate their interactions with different enzymes. Knowing that the interaction of saponins with βgalactosidase has not yet been characterized, this work aims to assess the effect of saponins on the enzyme activity, by monitoring the changes in the enzyme kinetic parameters (Vmax, Kcat, and Km). Furthermore, these data were correlated with transformations in the enzyme’s secondary and tertiary structure upon saponin exposure by using circular dichroism and fluorescence spectroscopy. Finally, the effect of saponins on the interaction of β-galactosidase synthetic
2. Materials and methods 2.1. Materials The enzyme β-galactosidase (EC:3.2.1.23) produced by Kluyveromyces lactis was provided by Granolab Granotec company (Araucária, Paraná, Brazil). The β-galactosidase activity was described by the manufacturer as 6500 acid lactase units per gram, and its concentration was determined by the Bradford assay (Bradford, 1976) using bovine serum albumin as the standard, and the concentration obtained was 113.84 mg mL−1 or 0.967 mmol L−1. The enzyme was analyzed by SDS-PAGE previously (data presented in Kayukawa et al., 2018). The results showed that the enzyme, with a molar mass of ~117 KDa, was the predominant protein of the sample. The densitometric analyses of the protein bands in that gel indicated 70% of purity of β-galactosidase. The reagents o-nitrophenyl-β-D-galactopyranoside (ONPG) (> 98% purity; molar mass of 301.25 g mol−1) and 2-nitrophenol (ONP) (98% purity; molar mass of 139.11 g mol−1) were obtained from Sigma–Aldrich (St Louis, Missouri, USA). Concentrated extracted of Quillaja bark saponin (QBS) was obtained from Vetec Sigma-Aldrich (Rio de Janeiro, Brazil). According to the supplier, the concentration of saponin in the extract is < =100% (CAS number: 8047-15-2). A Milli-Q system (Millipore Corp., Billerica, Massachusetts, USA) was used to provide deionized water. 2.2. Methods 2.2.1. Enzyme activity assay The hydrolytic activity of β-galactosidase was evaluated using the synthetic substrate ONPG. The methodology was adapted from Inchaurrondo, Yautorno, and Voget (1994). The enzyme activity assay was performed with samples constituted by 75 μL of 6.37 × 10−3 µmol L−1 β-galactosidase enzyme, 75 μL of 1.5 mmol L−1 artificial substrate ONPG and 775 μL of potassium phosphate buffer pH 6.8, 75 mmol L−1. In the case of saponin samples, the amount of buffer used was 700 μL, and the protein and additive quantities were both 75 μL. The QBS extract concentration was 0.05 mg mL−1, chosen according to preliminary enzymatic assays and ITC assays. After preparing the samples, they were promptly conditioned at 37 °C in the thermostatic bath Dubnoff (304/D, Ethik Technology, Vargem Grande Paulista, São Paulo, Brazil). Sodium carbonate solution (75 µL; 2 mol L−1) was added to each sample to stop the reaction in intervals ranging from 1 to 240 min. The absorbance was measured in a UV–Vis spectrophotometer (UV-1800, Shimadzu Corporation, Kyoto, Japan) at 420 nm. To quantify the enzymatic product formation, an ONP standard curve was constructed, and the results were expressed in mmol L−1 (see Supporting information). The samples enzymatic activity was calculated as follows:
Ea =
A. Vreaction ε . Venz . t
(1) −1
where Ea represents the enzyme catalytic activity in U mL . A is the absorbance measured (420 nm), Vreaction consist of the volumes present in the reaction in mL (buffer solution + ONPG solution + enzyme solution + sodium carbonate solution), ε (4024.66 L mol−1 cm−1) is the extinction coefficient of ONP under the experiment and measured at 420 nm, Venz is the volume of the enzyme extract in mL, and t is the time in minutes. Each of the β-galactosidase unit (U) represents the quantity necessary of the enzyme to release 1 μmol o-nitrophenol per minute under the assay conditions. The kinetic parameters, i.e., the Michaelis–Menten constant (Km), maximum reaction rate (Vmax) and turnover number (kcat) of the effects of the saponin on the β-galactosidase and ONPG interaction, were 2
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interaction of β-galactosidase with ONPG, as well as the saponin presence, were evaluated by ITC (ITC 200, Microcal, Inc., Northampton, Massachusetts, USA). The analysis was performed at 750 rpm and 37 °C. All of the solutions were prepared in the potassium phosphate buffer pH 6.8 (75 mmol L−1). The interactions were evaluated in two different ways: the titration of the protein against ONPG and the protein and saponin against ONPG. To assess the molecular interaction of the enzyme and substrate (ONPG), the syringe was filled with 40 µL of ONPG (1.5 mmol L−1) and the sample cell with 200 µL of β-galactosidase solution (0.85 µmol L−1). Additionally, to verify the effect of the saponin on the reaction, the titration was performed with 0.85 µmol L−1 β-galactosidase solutions in the presence of QBS extract at 0.05 mg mL−1 (cell sample) against solutions of 1.5 mmol L−1 ONPG (syringe). For all ITC assays, the reference cell was filled with 200 µL of ultrapure water. The titration was performed with the first injection of 0.4 µL, followed by 24 successive injections of 1.5 µL. The duration of each injection was 3 s, and the intervals between the injections were 100 s. All the results obtained had the blank assays discounted (see Supplementary material). The one-site model was fitted to the experimental data in order to obtain the thermodynamics parameters (stoichiometry (n), affinity constant (Ka), and binding enthalpy (ΔH)). Microcal Origin 7.0 software (ITC Data Analysis in Origin, Microcal, Northampton, Massachusetts, USA) in this procedure. Finally, the entropy was calculated from Eq. (6).
evaluated. The kinetic parameters were obtained using Origin software 8.6. (Northampton, Massachusetts, USA) and correlating the Michaelis–Menten model (Eq. (2)) to the experimental data.
v=
Vmax . S Km + S
(2)
where v corresponds to the hydrolysis rate (mmol ONP min−1), S is the substrate concentration, Km is given in mmol L−1, and Vmax in mmol ONP min−1. The kcat parameter and the enzyme catalytic efficiency (Eef) were calculated, according to Eqs. (3) and (4), respectively:
kcat =
Vmax Et
(3)
Eef =
kcat Km
(4)
where Et is the enzyme molar concentration in the assay (mol L−1). 2.2.2. Fluorescence spectroscopy assay The interactions between β-galactosidase as a function of the QBS extract concentration were assessed by fluorescence spectroscopy, as described by Soares, Mateus, and De Freitas (2007), and more recently, as used in our previous work (Kayukawa et al., 2018), using the spectropolarimeter UV–Vis (J-815, Jasco Int., Co., Tokyo, Japan) connected to the temperature controller Peltier (PTP-1). The equipment was stabilized at 37 °C, and the purge gas (nitrogen) was fed at a rate of 10 L min−1. All the solutions were prepared in 15 mmol L−1 potassium phosphate buffer pH 6.8. The β-galactosidase concentration was maintained at 0.085 mmol L−1 during the assay, and the QBS extract solution concentrations were 0.49, 0.73, 0.98, 1.47, 2.94, 5.88, and 9.80 mg mL−1. The samples were excited at 295 nm, and the emission measurements were recorded in the 310–370 nm range. Measurements of sample solutions were discounted by blank solutions (without saponin), and the results were expressed by plotting the fluorescence intensity against the wavelength.
ΔS =
θ × 100 × MM C×l×n
(6)
where T is the temperature of the system in Kelvin, and R is the ideal gas constant (8.31 J K−1 mol−1). The binding Gibbs energy (ΔG) was determined, according to Eq. (7):
ΔG = ΔH − T ΔS
(7)
The experiments were carried out in duplicate, and the thermodynamic parameters consisted of the mean of duplicate measurements ± standard deviation.
2.2.3. Circular dichroism (CD) assay The evaluation of the β-galactosidase secondary and tertiary structure by CD was performed using a CD spectropolarimeter (J-815, Jasco Int., Co), as described by Rabbani et al. (2014). The conditions of the purge gas, as well as the concentrations and pH of the solutions employed, were the same as those described in our previous work (Kayukawa et al., 2018). The saponin effect on the protein was observed by the addition of the QBS extract solutions at 0.1, 0.01, and 0.001 mg mL−1 to the protein solutions. The tertiary structure was obtained by near UV CD spectra assay (Kayukawa et al., 2018). The protein solutions were prepared in the potassium phosphate buffer (15 mmol L−1; pH 6.8) in the concentration of 8.50 µmol L−1, and the saponin effect on the system was obtained with the inclusion of QBS extract at 5 and 10 µg mL−1. Measurements were performed in a range of 250–350 nm, and a blank solution spectrum (without the saponin and protein) was subtracted to obtain the CD spectra. The protein samples secondary and tertiary structure were investigated using the molar mean residue ellipticity (θ MRE), that were calculated using Eq. (5):
θMRE =
ΔH − RlnK a T
2.2.5. Statistical analyses One-way analysis of variance (ANOVA) was applied to all experimental data, which were expressed as the means of duplicates. Mean values were evaluated by Tukey’s test at the 95% significance level, with the aid of Microsoft Excel 2010 action supplement (Redmond, Washington, USA). 3. Results and discussion 3.1. Influence of saponin on the enzymatic activity of β-galactosidase The β-galactosidase catalytic activity was achieved by ONPG assay using the enzyme in the concentration of 6.37 × 10−3 µmol L−1 at 30 min of the reaction. The enzymatic activity value obtained for the sample was 0.04746 U mL−1. The assay was also performed in the presence of the saponin (QBS extract) at various concentrations (30 min of reaction), and at that specific reaction time, no changes in the enzymatic activity were induced by the saponin (Fig. 1a). To explore the effects of the saponin on the enzyme, the kinetic of the reaction catalyzed by β-galactosidase was evaluated, using ONPG as a substrate in the presence or absence of saponin. The analyses were done at the QBS extract concentration of 0.05 mg mL−1. Fig. 1b displays the correlation of the Michaelis–Menten equation (Eq. (2)) to the experimental kinetic data, as well as the kinetic parameters (Km, kcat). The kcat value for the control sample (without saponin) was 209.105 ± 9.680 min−1, and the Km value for the same sample was 34.09 ± 6.980 mmol L−1. Thus, the enzyme catalytic efficiency was 6133.915 ± 1579.294 M−1 min−1. By adding QBS extract at
(5)
where θ correspond to the ellipticity degree (°); M(M) is the molar mass (kDa), C is the concentration (mg mL−1), l is the optical path length (cm), and n is the number of β-galactosidase residues. The mean residue ellipticity (θ MRE) is given in ° cm2 dmol−1 (Corrêa & Ramos, 2009). 2.2.4. Isothermal titration calorimetry (ITC) assay The energy and thermodynamic parameters resulted by the 3
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Fig. 1. Saponin effect on β-galactosidase enzymatic activity evaluated by UV–Vis. (a) Effect of different concentrations of QBS extract on the β-galactosidase (6.37 × 10−3 µmol L−1) enzymatic activity at 30 min of reaction. (b) Kinect of the reaction of β-galactosidase and ONPG in the absence (black line) or presence of QBS extract 0.05 mg mL−1 (red line). Kinetic parameters were obtained from the adjustment of the Michaelis-Menten equation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
0.05 mg mL−1 to the system, the reaction kinetic profile and the kinetic parameters changed (Fig. 1b). For the sample with saponin, the reaction occurred slightly faster than the control. Analyzing the kinetic parameters, the QBS extract did not significantly affect the kcat values compared to the control (189.922 ± 13.565 min−1), but the Km parameter decreased to 15.445 ± 1.450 mmol L−1. Hence, the QBS extract presence increased the catalytic efficiency, which shifted to 12296.697 ± 1274.736 M−1 min−1, showing it increases the enzyme activity, which is important for food, chemical and pharmaceutical industries for increase catalytic activity of enzymes of interest. Another surfactant known to present a positive effect on β-galactosidase activity is Triton X-100, and it is reported that this surfactant increased the thermal stability of the enzyme (Soto et al., 2017).
emission peak at 336 nm, regarding the tryptophan residues of β-galactosidase. Besides, a consistent decline in the fluorescence intensity of β-galactosidase occurred with an increase of QBS extract concentration. This decrease in the fluorescence intensity is related to changes in the protein microenvironment near the tryptophan residues and is correlated to changes in protein conformation (Lakowicz, 1999). It can also be observed changes in the wavelength of maximum emission (λmax), red shifts, with the QBS extract addition. These are indicatives that as the QBS extract concentration increase, the accessibility of tryptophan residues to the solvent also increases. The red shifts also occurred for the pancreatic lipase interaction with botulin and ginsenoside saponins, and it was related to the conformational changes on the enzyme tertiary structure (Yu, Dong, Wang, & Liu, 2017). Another compound that caused changes in β-galactosidase conformation (tertiary structure) is Triton X-100 (Soto et al., 2017). The substance, like the saponins, has surfactant properties, and it was concluded that their interaction helped the enzyme stability. The β-galactosidase tertiary structure was evaluated by CD spectra in the near UV region (Fig. 2b). The control sample (native protein) exhibits a positive peak at 275 nm and signals around 265–290 nm, that
3.2. Influence of saponin (QBS extract) on the β-galactosidase tertiary structure Fig. 2a shows the fluorescence emission curve obtained at λex = 295 nm for β-galactosidase at pH 6.8, with and without the addition of saponin (QBS extract). As can be seen, the protein had a strong
Fig. 2. Fluorescence spectra evaluating the impact of saponin on the tertiary structure of β-galactosidase solutions. (a) Emission spectra of β-galactosidase 0.425 µmol L−1 in the presence of QBS extract at concentrations of 0.125–4.500 mg mL−1. Fluorescence emissions intensity were recorded at λex = 295 nm, and the λem maximum occurred at 336 nm. (c) Near UV CD spectra of β-galactosidase 8.50 µmol L−1 in the presence of QBS extract at concentrations of 5 µg mL−1 and 10 µg mL−1. 4
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Fig. 3. β-Galactosidase 1.74 µmol L−1 secondary structure in the presence of saponin. (a) UV CD spectrum of β-galactosidase with different QBS extract concentrations varying of 0 mg.mL−1 (control) and 3 µg mL−1. Percentage of β-galactosidase secondary structures in the presence of 0 mg mL−1 (control) and 3 µg mL−1 of QBS extract.
the order of 105 L mol−1) and the molar ratio (n) of ONPG:protein (122:1) indicated a moderate affinity between β-galactosidase and ONPG. Additionally, one can see in Fig. 4c that this interaction is governed mainly by enthalpic contributions (ΔH = −16300 J mol−1 and TΔS = 1345 J mol−1) and presents ΔG = −41580 J mol−1. Enthalpic contributions may be related to the hydrolysis of ONPG by the enzyme, hence be associated with the enzyme activity (Kayukawa et al., 2018). Fig. 4b presents the influences of 0.05 mg mL−1 QBS extract on the binding of ONPG with β-galactosidase. Under this condition, the saponin positively impacted the protein interactions with ONPG. The addition of QBS extract caused an increase in the ΔH, TΔS, and ΔG parameters, indicating changes in the interaction mechanism. The increment in the enthalpic contributions (absolute value), from −16300 ± 395.7 (initial) to −41580 ± 1345 kJ mol−1, demonstrated modifications on the interactions of the enzyme with ONPG. The entropic contribution (TΔS) changed from 2223.7 ± 18 (control) to −300.81 ± 22 kJ mol−1, implying the saponin decreased the hydrophobic interaction contributions to the reaction, compared to the control sample. Also, the ΔG values decreased with the addition of saponin (QBS extract), indicating that the spontaneity of the protein–ONPG interaction increased. The Ka value was also increased, which means a higher affinity of the protein to the substrate, and the molecular interaction reached the saturation earlier in the sample with QBS extract when compared to the control sample (without saponin), indicating that saponin promoted an increase in the reaction affinity. ITC results demonstrated that QBS extract changed the molecular interaction mechanism of β-galactosidase and ONPG and increased the protein’s affinity for the substrate. These results complement the kinetic data, since changes in the mechanism and affinity of the protein may also modify its catalytic activity. The ITC assays allow concluding that the observed changes in the kinetic analysis (Fig. 1b) is probably a consequence of the higher affinity of the enzyme to the substrate when QBS extract is present. The results of fluorescence and circular dichroism analysis supports ITC results since changes in the protein secondary and tertiary structure can modify the enzyme interaction mechanism. Although the bioactive properties of the saponin are already known by the scientific community, the results presented here show, for the first time, the increase of β-galactosidase catalytic activity by the presence of QBS extract. However, further structural and functional studies must be performed to elucidate the exact saponin effects on proteins under a range of experimental conditions (concentrations, ionic strength, pH, temperature, interactions with other substances) to assess
are characteristics of the protein aromatic amino acids residues. The presence of QBS extract at a concentration of 5 µg mL−1 modifies the curve, suggesting that changes occurred on the protein tertiary structure. When the QBS extract concentration increases to 10 µg mL−1, the changes became more evident, with a clear enhancement in intensity on the overall curve and a maxima peak at 265 nm. The increase on the signal can be related to an increase on the enzyme stability, since the decrease of the signal is known to be characteristic of a loss on the protein stability (Kayukawa et al., 2018; Yu, Yang, & Labahn, 2017). Therefore, the saponin changed β-galactosidase microenvironment, increasing the accessibility of tryptophan residues to the solvent. 3.3. Influence of saponin (QBS extract) on the β-galactosidase secondary structure The saponin (QBS extract) effects on the β-galactosidase secondary structure were evaluated by CD. The specters of the β-galactosidase without (control) and with saponin are shown in Fig. 3a and revealed a maximum peak at 195 nm and a minimum peak at 215 nm, characteristic of proteins with rich β-sheet contents (Corrêa & Ramos, 2009). These results are consistent with previous CD analysis of K. lactis βgalactosidase (Kayukawa et al., 2018; Tello-Solís et al., 2005). Fig. 3b shows the secondary structure composition of the samples. The β-galactosidase control sample contained 22.6% α-helix, 29% β-sheet, 20.9% β-turn, and 27.5% random coil. Fig. 3 presents the changes caused by 3 µg mL−1 of QBS extract on the protein secondary structure. It can be seen that θ MRE values at 195 nm increased and the region of 215–230 nm, decreased, hence a modification on the protein secondary structure content to 35.5% αhelix and 25.5% β-sheet, 17.2% β-turn, and 21.5% random coil (Fig. 3b). The increase of α-helix content on Kluyveromyces lactis β-galactosidase structure can be related to a favorable interaction between the enzyme and its substrate, as observed in Tello-Solís et al. (2005). 3.4. Influence of saponin (QBS extract) on the interactions and thermodynamic parameters on the binding of β-galactosidase with ONPG The interactions of β-galactosidase and ONPG and how they are modified by saponin (QBS extract) were studied by ITC are presented in Fig. 4. The experimental data were fitted using the one-site binding model to assess the thermodynamic parameters of the interaction (Table 1). Fig. 4 confirms the binding of β-galactosidase to ONPG at 37 °C, since a sigmoidal-shaped thermogram was obtained. The value of Ka (in 5
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Fig. 4. ITC curves evaluating the effect of saponin on the binding of β-galactosidase with ONPG. Each injection of 1.5 µL is represented by the peaks with 100 s of intervals. (a) The syringe is filled with 0.85 µmol L−1 β-galactosidase and the cell with 1.5 mmol L−1 ONPG solution (control sample) (b) The syringe is filled with 0.85 µmol L−1 β-galactosidase + QBS extract 0.05 mg mL−1, (c) thermodynamic profile of ONPG interacting with β-galactosidase control and in with QBS extract 0.05 mg mL−1. Solid lines are the best fit of the data using the one-site model; symbols are experimental data, n is stoichiometry, Ka is the association constant, ΔH is enthalpy change, ΔS is entropy change and ΔG is the Gibbs free energy.
Declaration of Competing Interest
Table 1 Thermodynamic parameters of the interaction between β-galactosidase with ONPG (control) and in the presence of QBS extract 0.05 mg mL−1. Thermodynamic parameters
β-Galactosidase (control)
β-Galactosidase with QBS extract 0.05 mg mL−1
n Ka (mol−1 L) ΔH (J mol−1) TΔS (J mol−1) ΔG (J mol−1)
122 ± 1.84 7.78 × 105 ± 2.00 × 105 −16300 ± 395.7 2223.7 ± 18 −18523.7 ± 320
71.7 ± 1.22 3.76 × 106 ± 1.78 × 106 −41580 ± 1345 −300.81 ± 22 −41279.19 ± 1220
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments Funding: This work was supported by CAPES (Coordination for the Improvement of Higher Education Personnel), Graduate Program in Food Engineering (Federal University of Paraná, Curitiba, Brazil), Granolab Granotec, and INCT-UFPR (National Institute of Science and Technology on Biological Nitrogen Fixation). M. R. Mafra, and L. Igarashi-Mafra is grateful to the Brazilian National Council for Scientific and Technological Development (CNPq – Grant 310182/2018-2, 308517/2018-0, respectively). The authors also thank Thiago Atsushi Takashina for the contribution.
their suitability in future applications.
4. Conclusion It was verified an increase in β-galactosidase activity due to the saponin presence, with a clear decrease on the Michaelis-Menten parameter (Km) of the enzyme and increase on the enzyme efficacy (Eef). The fluorescence spectroscopy data suggested that the QBS extract changes the protein microenvironment, and the changes on the overall protein conformation were followed by near CD assays. By mean of CD analyzes, it can be seen that QBS extract changes the β-galactosidase secondary structure, increase its α-helix content, favoring its proteinsubstrate interaction. The changes on the molecular interaction mechanism of β-galactosidase and ONPG was shown by the ITC measurements, in which one can see that the QBS extract increases the affinity of the protein-substrate, as well as the molecular interaction spontaneity. This behavior is in agreement with the changes observed in the enzyme kinetics. This study may help to understand better the effects that the QBS extract (saponin source) causes on the β-galactosidase structure and catalytic activity. The extract presents some aspects that can be important to the food and pharmaceutical industry. Nevertheless, the extract may have negative effects on other proteins in the digestive system. Therefore, the interactions of QBS extract with other proteins should also be studied.
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