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Specificity enhancement towards phenolic substrate by immobilization of laccase on surface plasmon resonance sensor chip
Journal of Molecular Catalysis B: Enzymatic 121 (2015) 32–36
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic journal homepage: www.elsevier.com/locate/molcatb
Specificity enhancement towards phenolic substrate by immobilization of laccase on surface plasmon resonance sensor chip Safoura Jabbari, Bahareh Dabirmanesh, Khosro Khajeh ∗ Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 9 April 2015 Received in revised form 5 July 2015 Accepted 31 July 2015 Available online 3 August 2015 Keywords: Laccase Surface plasmon resonance Phenolic compounds Substrate specificity Covalent immobilization
a b s t r a c t In the modern biomedical and environmental technology, development of high performance sensing methods for phenolic compound is a critical issue because of its potential toxicity for human and environment. Laccases are polyphenol oxidases that exhibit broad substrate specificity; they act on both phenolic and non-phenolic compounds. Constructing a selective, sensitive and fast phenolic detecting system is a challenge for modern technologies. In the present study a laccase from Bacillus sp. HR03 was immobilized on the carboxymethyl dextran chip that altered its substrate specificity toward phenolic substrate. Since the opposite side of active site was enrich of Lys, oriented attachment via amine coupling was expected. Atomic force microscopy revealed a uniform distribution of the enzyme over the sensor surface. Surface plasmon resonance demonstrated no interactions toward non phenolic substrate (2,2 azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) and HPLC analysis of the reaction products revealed no peak. Significant conformational changes of the free enzyme toward non-phenolic substrate were detected using fluorescence spectroscopy. Therefore, the inactivation of immobilized enzyme toward non phenolic substrate was due to rigidification. The results of the current study could lead to the utility of laccase in developing a sensitive and specific catalytic detection system of phenolic compound based on surface plasmon resonance. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Laccases (benzenediol:oxygen oxidoreductases, EC 1.10.3.2) belong to the group of blue oxidases and represent the largest subgroup of multicopper oxidases. These enzymes are able to oxidize a broad range of phenolic and non-phenolic substrates, and they can be considered as generalists [1]. In general, laccases oxidize phenolic and non-phenolic compounds such as anilines, aryl diamines, hydroxyindols, benzenethiols, bilirubin, ascorbate and lignin related molecules. Among them, phenol compounds are byproducts of large-scale production such as drugs, dyes, antioxidants, paper pulp and pesticides that cause ecologically undesirable effects [2]. Nonetheless, most phenolic compounds are distinguished by their toxic, noxious, mutagenic and carcinogenic activity [3,4]. Therefore, detection and elimination of hazardous phenolic compounds using laccases has gained attention during recent decades. To date, various biosensors for both environmentally
∗ Corresponding author at: Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, P.O.Box 14115-175, Tehran, Iran. Fax: +98 2182884717. E-mail address: [email protected] (K. Khajeh). http://dx.doi.org/10.1016/j.molcatb.2015.07.016 1381-1177/© 2015 Elsevier B.V. All rights reserved.
important pollutants and clinically relevant metabolites have been developed [5–7]. Although the determination of phenol and its derivative compounds is of environmental greatness [8,9], but in clinical detection system preparation, specific detection of a single substrate in complex mixtures is more critical and requires the use of enzymes with high substrate specificity, which means that laccase with broad range specificity [10], could not be a highly suitable candidate for detection. In fact, in all clinical laccase based phenolic detection system, such as detection of dopamine or catechol, it is necessary to study significant interference like ascorbic acid, as a non-phenolic laccase substrate in real mixture samples [11]. In order to reduce and completely omit the necessity of considering interference in the mixture samples, the enhancement of laccase specificity and activity towards the target metabolites could be the fundamental step in its detection system construction. Recently, to create practical catalysts, few researches reported the narrowing of laccase substrate specificity toward non-phenolic substrate, 2,2 -azino-bis(3-ethylbenzothiazoline-6sulphonic acid) (ABTS), by using mutagenesis [1,10]. In the current study, we have achieved a novel specific laccase towards phenolic compounds by immobilization on the surface plasmon resonance (SPR). Different methodologies have been used for laccase immobilization such as, polypropylene membrane [12], sol–gel matrix of
S. Jabbari et al. / Journal of Molecular Catalysis B: Enzymatic 121 (2015) 32–36
Fig. 1. Side view of the three-dimensional model of laccase from Bacillus HR03 was built using Swiss model utilizing CotA (1GSK) as the template. The model shows that lysine residues (gray sphere) are located far away from the active site (white sphere).
diglycerysilane and Ca and Cu–alginate beads [13,14], to construct laccase biosensors for phenolic compounds [15], but they did not report any innovation about laccase specificity and activity towards its substrate during immobilization. In this study, we investigated, for the first time the immobilization of laccase (CotA) from Bacillus sp. HR03 [16] on the SPR carboxymethyldextran (CMD) chip and employed surface plasmon resonance (SPR) system as a powerful and sensitive technique to monitor the activity and specificity of the immobilized enzyme. The activity of immobilized laccase was promoted towards its phenolic substrate, syringaldazine (SGZ). Further consideration using fluorescence and HPLC techniques have proved the specificity enhancement of laccase by immobilization. Our results represent a simple and new strategy to construct more effective and specific clinical catalytic detection system of phenolic compounds. 2. Materials and methods 2.1. Materials All the reagents were prepared with chemicals of analytical grade. All chemicals were purchased from Sigma–Aldrich Chemical (USA). The carboxymethyldextran (CMD 200 M), sensor chip and the amine-coupling kit containing N-hydroxysuccinimide (NHS), N-ethyl-N -(3-diethylaminopropyl) carbodiimide (EDC), and ethanolamine hydrochloride were obtained from Xantec Bioanalytics (Germany). Laccase (CotA) from Bacillus sp. HR03 was expressed in Escherichia coli BL21 (DE3) cells in our laboratory [16]. 2.2. Methods 2.2.1. Immobilization of laccase onto CMD chip surface The enzyme was immobilized via its primary amine groups (lysine residues) through EDC/NHS esters [17,18]. The immobilization protocol was designed to obtain an increased binding capacity (Rmax ) of the sensor chip (i.e., with a large amount of covalently bound protein). For this purpose, the activation of the surface carboxyl groups was performed by injecting a 10 min pulse of EDC/NHS. For immobilization, 250 l of laccase (0.2 mg/ml) in 10 mM acetate buffer (pH 4.5) was injected over the activated chip. The remaining active sites of the SPR sensor were blocked
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Fig. 2. Sensogram for covalent immobilization of laccase onto SPR-CMD surface. Carrier solution: 20 mM phosphate buffer (pH 7), flow speed 25 l/min, flow duration: 10 min.
by 1 M ethanolamine (pH 8.5). All of the mentioned operations were followed by rinsing with a phosphate buffer solution. The SPR SR7500DC instrument detects changes in refractive index and measures these changes in microrefractive-index units (RIU). A change in refractive index is proportional to the quantity (mass) of analyte interacting with the surface. Over amine coupling process, the signal up to 10,873 microrefractive-index units (RIU) was achieved by laccase immobilization on the surface. 2.2.2. SPR measurements SPR measurements were performed on an SPR SR7500DC instrument (XanTec bioanalytics GmbH, Germany) equipped with an automatic flow injection system. The interaction between substrates and immobilized laccase was studied by flowing substrate over the sensor surface. After optimizing the pH condition for good binding interaction, 20 M of ABTS in 100 mM phosphate buffer (pH 4) was injected over the laccase immobilized CMD surface for 10 min with a flow rate of 25 l/min at 25 ◦ C [16,19]. Subsequently, the flow-through was collected for the HPLC analysis. 10 M of SGZ (100 mM phosphate buffer, pH 7) was also injected for 5 min with a flow rate of 50 l/min at 25 ◦ C. The collected sample was analyzed by HPLC. A blank control run was performed by injecting the above mentioned buffers. Reference sensorgrams were subtracted from binding sensorgrams using the Scrubber analysis program (Biologic Software Pty. Ltd., Canberra, Australia). 2.2.3. Laccase activity assay Laccase activity was measured as previously described [16]. Briefly, the oxidation of ABTS (2 mM) in 100 mM phosphate buffer (pH 4) was measured by the increase in absorbance at 420 nm ( = 36,000 M−1 cm−1 ) [20]. Oxidation of SGZ in 100 mM phosphate buffer (pH 7) was monitored at 525 nm ( = 65,000 M−1 cm−1 ) [21]. One activity unit (U) was defined as the amount of enzyme that oxidized 1 mol of substrate per minute at 25 ◦ C. 2.2.4. High performance liquid chromatography (HPLC) assay Chromatographic analysis was performed on a reverse-phase high-performance liquid chromatography (HPLC) system (Knauer, Berlin, Germany) using an analytical Inertsil ODS-3 column (In Ertsil, Eindhoven, Netherlands). Samples were filtered through a 0.2-m syringe filter prior to HPLC analysis. A mobile phase composed of 50% methanol and 50% water was used at a low rate of 0.5 ml min−1 . The injection of the samples was performed on an
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S. Jabbari et al. / Journal of Molecular Catalysis B: Enzymatic 121 (2015) 32–36
Fig. 3. Two and three dimensional AFM micrographs of bare (A and B) and immobilized surface (C and D). Scanning area: 500 nm × 500 nm. The height profiles are shown at the bottom. The AFM images along with the height profile indicate a larger height difference on the laccase immobilized surface than on the bare CMD surface.
injector equipped with a 20 l loop. Knauer 2500 UV detector was set at 420 nm for ABTS and 525 nm for SGZ.
3. Results and discussion 3.1. Immobilization of laccase on CMD chip surface
2.2.5. Atomic force microscopy (AFM) studies Surface topology images of the CMD SPR-chip, before and after enzyme immobilization was obtained with an AFM Instrument (Veeco-Autoprobe-CP-research). The AFM imaging was performed in non contact mode at room temperature in air. For all AFM images a resolution of 256 pixels was applied.
2.2.6. Fluorescence spectroscopy The intrinsic fluorescence of the enzyme was measured on a PerkinElmer luminescence spectrometer LS 55. Samples were incubated in the cell for 5 min, and excited at 295 nm then the emission was registered from 300 to 400 nm. The excitation and emission slit were both set to 5 nm.
Previously, we have reported and isolated a laccase in Bacillus HR03 from Iranian microflora [16,22]. The optimum pH, and kinetic parameters (KM and kcat ) for both substrates (ABTS and SGZ) were determined [16]. This enzyme shows 98.2% identity and 98.8% similarity with CotA from Bacillus subtilis (1GSK), the structure of which has been already solved [23]. Three-dimensional model of laccase from Bacillus HR03 was built using Swiss model utilizing CotA (1GSK) as the template (Fig. 1) [23]. Tertiary structure analysis of the enzyme was carried out and showed that nearly 70% of lysine residues were centralized at the opposite side far enough away from the active site region (Fig. 1). Since, here we were focusing on developing a specific, selective and sensitive laccase based catalytic detection system, we decided to immobilize the enzyme via amine groups. The oriented distribution of lysine was assumed
S. Jabbari et al. / Journal of Molecular Catalysis B: Enzymatic 121 (2015) 32–36
to decrease the random coupling of the laccase, and the similarly oriented ligand increases the sensitivity of the sensor significantly. Based on our analysis, laccase (CotA) could be immobilized on the CMD 200 D sensor chip by amine coupling using EDC/NHS [24]. The signal up to 10KRU was achieved by enzyme immobilization to ensure that the low molecular weight of the analyte was not a limiting factor to cause a measurable change in refractive index (Fig. 2) [25,26]. Fig. 3 illustrates the atomic force microscopy (AFM) image of a bare and immobilized CMD surface in two and threedimensional views. The enzyme was homogeneously distributed over the sensor chip. Moreover, the average surface roughness was raised from 0.258 nm to 0.439 nm. Due to the high surface density of immobilized enzyme, we assumed that mass transfer could be a limiting factor through binding. Therefore, three injections with identical substrate (SGZ) concentrations and different flow rates were used to ensure that the mass transport was not the limiting factor during analyte binding [27]. As shown in Fig. 4 SGZ interaction was not affected by the mass transfer limitations. 3.2. Phenolic and non-phenolic substrate interactions with immobilized laccase After EDC/NHS surface activation, and covalent immobilization on carboxymethyl dextran matrix under acidic conditions (10 mM sodium acetate, pH 4.5) (Fig. 2), ABTS (non-phenolic substrate)
35
Fig. 4. Mass transport limitation. 10 M of SGZ was injected in separate three flow rates, 50, 75 and 100 l/min. It shows same response signal in the different flow rates.
in 100 mM phosphate buffer (pH 4) was injected simultaneously over the immobilized laccase and the reference protein, bovine serum albumin (BSA), that is unrelated to the ligand. Fig. 5A illustrates the SPR response between ABTS and the immobilized laccase. The results revealed no interaction between laccase and its non phenolic substrate. To provide direct experimental proof, the product formation of injected ABTS was collected and analyzed by
Fig. 5. (A) Sensorgram of 20 M ABTS with laccase. (B) HPLC chromatogram for ABTS before and after injection on the sensor chip. Absorbance was monitored at 420 nm. (C) Sensogram for 10 M SGZ over the laccase sensor chip. (D) HPLC chromatogram for SGZ before and after injection. Detection was at 525 nm.
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S. Jabbari et al. / Journal of Molecular Catalysis B: Enzymatic 121 (2015) 32–36
coupling immobilization onto CMD chip surface. The activity and specificity toward SGZ and ABTS were altered due to the conformational flexibility changes provoked by the binding of the enzyme to the SPR chip surface. Specificity enhancement of immobilized laccase towards its phenolic substrate (SGZ) could make this enzyme a promising candidate for constructing an effective catalytic detection system of phenolic compounds for clinical and environmental samples. This ability is being reported for the first time in the literature. Acknowledgements We would like to thank the Research Council of Tarbiat Modares University for the financial support of this investigation References
Fig. 6. Tryptophan fluorescence spectra of laccase in phosphate buffer pH 7 ( ), phosphate buffer pH 7 containing 0.1 M SGZ ( phosphate buffer pH 4 ( ) and phosphate buffer pH 4 containing 0.3 M ABTS ( ).
),
reversed phase high-performance liquid chromatography (HPLC). HPLC chromatogram of product indicated a loss in immobilized enzyme activity towards its non-phenolic substrate, ABTS (Fig. 5B). These results attracted us to investigate the affinity and activity of the immobilized laccase toward its phenolic substrate, SGZ (in 100 mM phosphate buffer pH 7). Fig. 5C and D demonstrates the SPR signal and the HPLC chromatogram of the enzyme product at 525 nm. The immobilized enzyme was catalytically active by repeating the SGZ injection (10 and 5 M) over the chip surface. Unusual behavior of laccase towards ABTS could be interpreted by the unspecific interaction (e.g., electrostatic) of negatively charged ABTS with interfacial area (solid/liquid), such as a few exposed Lys/Arg residues. In addition, intrinsic fluorescence results suggested that conformational changes of free enzyme occur predominantly in the present of ABTS compared to SGZ. For SGZ, no significant changes in the emission intensity were observed upon binding to the enzyme while the presence of ABTS exhibited a relatively large decrease in the fluorescence emission intensity (without a change in the wavelength of maximum emission) (Fig. 6). Therefore, more local or global conformational flexibility is required for an interaction between laccase and ABTS. The rigidity of laccase structure via multi point covalent attachment may have prevented the enzyme conformational changes induced by ABTS [28] which assumed to play a crucial role in the different laccase activity behavior [29]. Researchers reported that immobilization of an enzyme could decrease or improve its activity, specificity or selectivity [28,30]. Previously immobilization of lipases via interfacial activation on hydrophobic supports increased the enzyme specificity towards hydrophobic substrates [31]. 4. Conclusion Overall, this study has demonstrated that the activity of laccase toward its conventional substrates can be affected by amine
[1] N. Gupta, E.T. Farinas, Comb. Chem. High Throughput Screening 12 (2009) 269–274. [2] J.J. Roy, T.E. Abraham, K.S. Abhijith, P.V.S. Kumar, M.S. Thakur, Biosens. Bioelectron. 21 (2005) 206–211. [3] J.C. Gonzalez-rivera, J.E. Preciado, J.F. Osma, M. Stoytcheva, J.F. Osma, Biosensors: Recent Advances in Mathematical Challenges, OmniaScience, Barcelona: Espana, 2014, pp. 111–127. [4] J. Michałowicz, W. Duda, Pol. J. Environ. Stud. 16 (2007) 347–362. [5] S.A.S.S. Gomes, M.J.F. Rebelo, Sensors 3 (2003) 166–175. [6] Y. Li, C. Qin, C. Chen, Y. Fu, M. Ma, Q. Xie, Sens. Actuators B Chem. 168 (2012) 46–53. [7] J. Kulys, R. Vidziunaite, Biosens. Bioelectron. 18 (2002) 319–325. [8] J. Sołoducho, J. Cabaj, J. Biomater. Nanobiotechnol. 04 (2013) 17–27. [9] J. Cabaj, J. Sołoducho, V. Somerset (Eds.), Environmental Biosensors, InTech, 2006, pp. 214–236. [10] N. Gupta, E.T. Farinas, Protein Eng. Des. Sel. 23 (2010) 679–682. [11] D. Quan, W. Shin, Electroanalysis 16 (2004) 1576–1582. [12] S. Georgieva, T. Godjevargova, M. Portaccio, M. Lepore, D.G. Mita, J. Mol. Catal. B Enzym. 55 (2008) 177–184. [13] M.R. Montereali, L.D. Seta, W. Vastarella, R. Pilloton, J. Mol. Catal. B Enzym. 64 (2010) 189–194. [14] K.N. Niladevi, P. Prema, World J. Microb. Biot. 24 (2007) 1215–1222. [15] Y. Li, L. Zhang, M. Li, Z. Pan, D. Li, Chem. Cent. J. 6 (2012) 1–8. [16] M. Mohammadian, M. Fathi-Roudsari, N. Mollania, A. Badoei-Dalfard, K. Khajeh, J. Ind. Microbiol. Biotechnol. 37 (2010) 863–869. [17] M. Puiu, O. Istrate, L. Rotariu, C. Bala, Anal. Biochem. 421 (2012) 587–594. [18] D.J. O’shannessy, M. Brigham-Burke, K. Peck, Anal. Biochem. 205 (1992) 132–136. [19] C. Fernández-Sáncheza, T. Tzanova, G.M. Gübitzb, A. Cavaco-Paulo, Bioelectrochemistry 58 (2002) 149–156. [20] R.E. Childs, W.G. Bardsley, Biochem. J. 145 (1975) 93–103. [21] J.H. Harkin, J.R. Obst, Experientia 37 (1973) 381–387. [22] A. Badoei-Dalfard, K. Khajeh, M.R. Soudi, H. Naderi-Manesh, B. Ranjbar, R.H. Sajedi, Enzyme Microb. Technol. 39 (2006) 1409–1416. [23] F.J. Enguita, L.O. Martins, A.O. Henrique, M.A. Carrondo, J. Biol. Chem. 278 (2003) 19416–19425. [24] C. Hahnefeld, S. Drewianka, F.W. Herberg, in: J. Decker, U. Reischl (Eds.), Molecular Diagnosis of Infectious Diseases, Humana Press Inc., Totowa, NJ, 2004, pp. 299–320. [25] Protocol for Measuring Small Molecule Interactions Using Biocore: A Practical Guide to Experimental Design and Data Evaluation, Biacore Symposium 2002, Chicago, Illinois, pp. 1–17. [26] J.E. Gestwicki, H.V. Hsieh, J.B. Pitner, Anal. Chem. 73 (2001) 5732–5737. [27] M. Ritzefeld, N. Sewald, J. Amino Acids 2012 (2012) 1–19. [28] R.C. Rodrigues, C. Ortiz, Á. Berenguer-Murcia, R. Torres, R. Fernández-Lafuente, Chem. Soc. Rev. 42 (2013) 6290–6307. [29] F. Secundo, Chem. Soc. Rev. 42 (2013) 6250–6261. [30] C. Mateo, J.M. Palomo, G. Fernandez-Lorente, J.M. Guisan, R. Fernandez-Lafuente, Enzyme Microb. Technol. 40 (2007) 1451–1463. [31] G. Fernández-Lorente, J.M. Palomo, Z. Cabrera, J.M. Guisán, R. Fernández-Lafuente, Enzyme Microb. Technol. 41 (2007) 565–569.
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic journal homepage: www.elsevier.com/locate/molcatb
Specificity enhancement towards phenolic substrate by immobilization of laccase on surface plasmon resonance sensor chip Safoura Jabbari, Bahareh Dabirmanesh, Khosro Khajeh ∗ Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 9 April 2015 Received in revised form 5 July 2015 Accepted 31 July 2015 Available online 3 August 2015 Keywords: Laccase Surface plasmon resonance Phenolic compounds Substrate specificity Covalent immobilization
a b s t r a c t In the modern biomedical and environmental technology, development of high performance sensing methods for phenolic compound is a critical issue because of its potential toxicity for human and environment. Laccases are polyphenol oxidases that exhibit broad substrate specificity; they act on both phenolic and non-phenolic compounds. Constructing a selective, sensitive and fast phenolic detecting system is a challenge for modern technologies. In the present study a laccase from Bacillus sp. HR03 was immobilized on the carboxymethyl dextran chip that altered its substrate specificity toward phenolic substrate. Since the opposite side of active site was enrich of Lys, oriented attachment via amine coupling was expected. Atomic force microscopy revealed a uniform distribution of the enzyme over the sensor surface. Surface plasmon resonance demonstrated no interactions toward non phenolic substrate (2,2 azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) and HPLC analysis of the reaction products revealed no peak. Significant conformational changes of the free enzyme toward non-phenolic substrate were detected using fluorescence spectroscopy. Therefore, the inactivation of immobilized enzyme toward non phenolic substrate was due to rigidification. The results of the current study could lead to the utility of laccase in developing a sensitive and specific catalytic detection system of phenolic compound based on surface plasmon resonance. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Laccases (benzenediol:oxygen oxidoreductases, EC 1.10.3.2) belong to the group of blue oxidases and represent the largest subgroup of multicopper oxidases. These enzymes are able to oxidize a broad range of phenolic and non-phenolic substrates, and they can be considered as generalists [1]. In general, laccases oxidize phenolic and non-phenolic compounds such as anilines, aryl diamines, hydroxyindols, benzenethiols, bilirubin, ascorbate and lignin related molecules. Among them, phenol compounds are byproducts of large-scale production such as drugs, dyes, antioxidants, paper pulp and pesticides that cause ecologically undesirable effects [2]. Nonetheless, most phenolic compounds are distinguished by their toxic, noxious, mutagenic and carcinogenic activity [3,4]. Therefore, detection and elimination of hazardous phenolic compounds using laccases has gained attention during recent decades. To date, various biosensors for both environmentally
∗ Corresponding author at: Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, P.O.Box 14115-175, Tehran, Iran. Fax: +98 2182884717. E-mail address: [email protected] (K. Khajeh). http://dx.doi.org/10.1016/j.molcatb.2015.07.016 1381-1177/© 2015 Elsevier B.V. All rights reserved.
important pollutants and clinically relevant metabolites have been developed [5–7]. Although the determination of phenol and its derivative compounds is of environmental greatness [8,9], but in clinical detection system preparation, specific detection of a single substrate in complex mixtures is more critical and requires the use of enzymes with high substrate specificity, which means that laccase with broad range specificity [10], could not be a highly suitable candidate for detection. In fact, in all clinical laccase based phenolic detection system, such as detection of dopamine or catechol, it is necessary to study significant interference like ascorbic acid, as a non-phenolic laccase substrate in real mixture samples [11]. In order to reduce and completely omit the necessity of considering interference in the mixture samples, the enhancement of laccase specificity and activity towards the target metabolites could be the fundamental step in its detection system construction. Recently, to create practical catalysts, few researches reported the narrowing of laccase substrate specificity toward non-phenolic substrate, 2,2 -azino-bis(3-ethylbenzothiazoline-6sulphonic acid) (ABTS), by using mutagenesis [1,10]. In the current study, we have achieved a novel specific laccase towards phenolic compounds by immobilization on the surface plasmon resonance (SPR). Different methodologies have been used for laccase immobilization such as, polypropylene membrane [12], sol–gel matrix of
S. Jabbari et al. / Journal of Molecular Catalysis B: Enzymatic 121 (2015) 32–36
Fig. 1. Side view of the three-dimensional model of laccase from Bacillus HR03 was built using Swiss model utilizing CotA (1GSK) as the template. The model shows that lysine residues (gray sphere) are located far away from the active site (white sphere).
diglycerysilane and Ca and Cu–alginate beads [13,14], to construct laccase biosensors for phenolic compounds [15], but they did not report any innovation about laccase specificity and activity towards its substrate during immobilization. In this study, we investigated, for the first time the immobilization of laccase (CotA) from Bacillus sp. HR03 [16] on the SPR carboxymethyldextran (CMD) chip and employed surface plasmon resonance (SPR) system as a powerful and sensitive technique to monitor the activity and specificity of the immobilized enzyme. The activity of immobilized laccase was promoted towards its phenolic substrate, syringaldazine (SGZ). Further consideration using fluorescence and HPLC techniques have proved the specificity enhancement of laccase by immobilization. Our results represent a simple and new strategy to construct more effective and specific clinical catalytic detection system of phenolic compounds. 2. Materials and methods 2.1. Materials All the reagents were prepared with chemicals of analytical grade. All chemicals were purchased from Sigma–Aldrich Chemical (USA). The carboxymethyldextran (CMD 200 M), sensor chip and the amine-coupling kit containing N-hydroxysuccinimide (NHS), N-ethyl-N -(3-diethylaminopropyl) carbodiimide (EDC), and ethanolamine hydrochloride were obtained from Xantec Bioanalytics (Germany). Laccase (CotA) from Bacillus sp. HR03 was expressed in Escherichia coli BL21 (DE3) cells in our laboratory [16]. 2.2. Methods 2.2.1. Immobilization of laccase onto CMD chip surface The enzyme was immobilized via its primary amine groups (lysine residues) through EDC/NHS esters [17,18]. The immobilization protocol was designed to obtain an increased binding capacity (Rmax ) of the sensor chip (i.e., with a large amount of covalently bound protein). For this purpose, the activation of the surface carboxyl groups was performed by injecting a 10 min pulse of EDC/NHS. For immobilization, 250 l of laccase (0.2 mg/ml) in 10 mM acetate buffer (pH 4.5) was injected over the activated chip. The remaining active sites of the SPR sensor were blocked
33
Fig. 2. Sensogram for covalent immobilization of laccase onto SPR-CMD surface. Carrier solution: 20 mM phosphate buffer (pH 7), flow speed 25 l/min, flow duration: 10 min.
by 1 M ethanolamine (pH 8.5). All of the mentioned operations were followed by rinsing with a phosphate buffer solution. The SPR SR7500DC instrument detects changes in refractive index and measures these changes in microrefractive-index units (RIU). A change in refractive index is proportional to the quantity (mass) of analyte interacting with the surface. Over amine coupling process, the signal up to 10,873 microrefractive-index units (RIU) was achieved by laccase immobilization on the surface. 2.2.2. SPR measurements SPR measurements were performed on an SPR SR7500DC instrument (XanTec bioanalytics GmbH, Germany) equipped with an automatic flow injection system. The interaction between substrates and immobilized laccase was studied by flowing substrate over the sensor surface. After optimizing the pH condition for good binding interaction, 20 M of ABTS in 100 mM phosphate buffer (pH 4) was injected over the laccase immobilized CMD surface for 10 min with a flow rate of 25 l/min at 25 ◦ C [16,19]. Subsequently, the flow-through was collected for the HPLC analysis. 10 M of SGZ (100 mM phosphate buffer, pH 7) was also injected for 5 min with a flow rate of 50 l/min at 25 ◦ C. The collected sample was analyzed by HPLC. A blank control run was performed by injecting the above mentioned buffers. Reference sensorgrams were subtracted from binding sensorgrams using the Scrubber analysis program (Biologic Software Pty. Ltd., Canberra, Australia). 2.2.3. Laccase activity assay Laccase activity was measured as previously described [16]. Briefly, the oxidation of ABTS (2 mM) in 100 mM phosphate buffer (pH 4) was measured by the increase in absorbance at 420 nm ( = 36,000 M−1 cm−1 ) [20]. Oxidation of SGZ in 100 mM phosphate buffer (pH 7) was monitored at 525 nm ( = 65,000 M−1 cm−1 ) [21]. One activity unit (U) was defined as the amount of enzyme that oxidized 1 mol of substrate per minute at 25 ◦ C. 2.2.4. High performance liquid chromatography (HPLC) assay Chromatographic analysis was performed on a reverse-phase high-performance liquid chromatography (HPLC) system (Knauer, Berlin, Germany) using an analytical Inertsil ODS-3 column (In Ertsil, Eindhoven, Netherlands). Samples were filtered through a 0.2-m syringe filter prior to HPLC analysis. A mobile phase composed of 50% methanol and 50% water was used at a low rate of 0.5 ml min−1 . The injection of the samples was performed on an
34
S. Jabbari et al. / Journal of Molecular Catalysis B: Enzymatic 121 (2015) 32–36
Fig. 3. Two and three dimensional AFM micrographs of bare (A and B) and immobilized surface (C and D). Scanning area: 500 nm × 500 nm. The height profiles are shown at the bottom. The AFM images along with the height profile indicate a larger height difference on the laccase immobilized surface than on the bare CMD surface.
injector equipped with a 20 l loop. Knauer 2500 UV detector was set at 420 nm for ABTS and 525 nm for SGZ.
3. Results and discussion 3.1. Immobilization of laccase on CMD chip surface
2.2.5. Atomic force microscopy (AFM) studies Surface topology images of the CMD SPR-chip, before and after enzyme immobilization was obtained with an AFM Instrument (Veeco-Autoprobe-CP-research). The AFM imaging was performed in non contact mode at room temperature in air. For all AFM images a resolution of 256 pixels was applied.
2.2.6. Fluorescence spectroscopy The intrinsic fluorescence of the enzyme was measured on a PerkinElmer luminescence spectrometer LS 55. Samples were incubated in the cell for 5 min, and excited at 295 nm then the emission was registered from 300 to 400 nm. The excitation and emission slit were both set to 5 nm.
Previously, we have reported and isolated a laccase in Bacillus HR03 from Iranian microflora [16,22]. The optimum pH, and kinetic parameters (KM and kcat ) for both substrates (ABTS and SGZ) were determined [16]. This enzyme shows 98.2% identity and 98.8% similarity with CotA from Bacillus subtilis (1GSK), the structure of which has been already solved [23]. Three-dimensional model of laccase from Bacillus HR03 was built using Swiss model utilizing CotA (1GSK) as the template (Fig. 1) [23]. Tertiary structure analysis of the enzyme was carried out and showed that nearly 70% of lysine residues were centralized at the opposite side far enough away from the active site region (Fig. 1). Since, here we were focusing on developing a specific, selective and sensitive laccase based catalytic detection system, we decided to immobilize the enzyme via amine groups. The oriented distribution of lysine was assumed
S. Jabbari et al. / Journal of Molecular Catalysis B: Enzymatic 121 (2015) 32–36
to decrease the random coupling of the laccase, and the similarly oriented ligand increases the sensitivity of the sensor significantly. Based on our analysis, laccase (CotA) could be immobilized on the CMD 200 D sensor chip by amine coupling using EDC/NHS [24]. The signal up to 10KRU was achieved by enzyme immobilization to ensure that the low molecular weight of the analyte was not a limiting factor to cause a measurable change in refractive index (Fig. 2) [25,26]. Fig. 3 illustrates the atomic force microscopy (AFM) image of a bare and immobilized CMD surface in two and threedimensional views. The enzyme was homogeneously distributed over the sensor chip. Moreover, the average surface roughness was raised from 0.258 nm to 0.439 nm. Due to the high surface density of immobilized enzyme, we assumed that mass transfer could be a limiting factor through binding. Therefore, three injections with identical substrate (SGZ) concentrations and different flow rates were used to ensure that the mass transport was not the limiting factor during analyte binding [27]. As shown in Fig. 4 SGZ interaction was not affected by the mass transfer limitations. 3.2. Phenolic and non-phenolic substrate interactions with immobilized laccase After EDC/NHS surface activation, and covalent immobilization on carboxymethyl dextran matrix under acidic conditions (10 mM sodium acetate, pH 4.5) (Fig. 2), ABTS (non-phenolic substrate)
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Fig. 4. Mass transport limitation. 10 M of SGZ was injected in separate three flow rates, 50, 75 and 100 l/min. It shows same response signal in the different flow rates.
in 100 mM phosphate buffer (pH 4) was injected simultaneously over the immobilized laccase and the reference protein, bovine serum albumin (BSA), that is unrelated to the ligand. Fig. 5A illustrates the SPR response between ABTS and the immobilized laccase. The results revealed no interaction between laccase and its non phenolic substrate. To provide direct experimental proof, the product formation of injected ABTS was collected and analyzed by
Fig. 5. (A) Sensorgram of 20 M ABTS with laccase. (B) HPLC chromatogram for ABTS before and after injection on the sensor chip. Absorbance was monitored at 420 nm. (C) Sensogram for 10 M SGZ over the laccase sensor chip. (D) HPLC chromatogram for SGZ before and after injection. Detection was at 525 nm.
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coupling immobilization onto CMD chip surface. The activity and specificity toward SGZ and ABTS were altered due to the conformational flexibility changes provoked by the binding of the enzyme to the SPR chip surface. Specificity enhancement of immobilized laccase towards its phenolic substrate (SGZ) could make this enzyme a promising candidate for constructing an effective catalytic detection system of phenolic compounds for clinical and environmental samples. This ability is being reported for the first time in the literature. Acknowledgements We would like to thank the Research Council of Tarbiat Modares University for the financial support of this investigation References
Fig. 6. Tryptophan fluorescence spectra of laccase in phosphate buffer pH 7 ( ), phosphate buffer pH 7 containing 0.1 M SGZ ( phosphate buffer pH 4 ( ) and phosphate buffer pH 4 containing 0.3 M ABTS ( ).
),
reversed phase high-performance liquid chromatography (HPLC). HPLC chromatogram of product indicated a loss in immobilized enzyme activity towards its non-phenolic substrate, ABTS (Fig. 5B). These results attracted us to investigate the affinity and activity of the immobilized laccase toward its phenolic substrate, SGZ (in 100 mM phosphate buffer pH 7). Fig. 5C and D demonstrates the SPR signal and the HPLC chromatogram of the enzyme product at 525 nm. The immobilized enzyme was catalytically active by repeating the SGZ injection (10 and 5 M) over the chip surface. Unusual behavior of laccase towards ABTS could be interpreted by the unspecific interaction (e.g., electrostatic) of negatively charged ABTS with interfacial area (solid/liquid), such as a few exposed Lys/Arg residues. In addition, intrinsic fluorescence results suggested that conformational changes of free enzyme occur predominantly in the present of ABTS compared to SGZ. For SGZ, no significant changes in the emission intensity were observed upon binding to the enzyme while the presence of ABTS exhibited a relatively large decrease in the fluorescence emission intensity (without a change in the wavelength of maximum emission) (Fig. 6). Therefore, more local or global conformational flexibility is required for an interaction between laccase and ABTS. The rigidity of laccase structure via multi point covalent attachment may have prevented the enzyme conformational changes induced by ABTS [28] which assumed to play a crucial role in the different laccase activity behavior [29]. Researchers reported that immobilization of an enzyme could decrease or improve its activity, specificity or selectivity [28,30]. Previously immobilization of lipases via interfacial activation on hydrophobic supports increased the enzyme specificity towards hydrophobic substrates [31]. 4. Conclusion Overall, this study has demonstrated that the activity of laccase toward its conventional substrates can be affected by amine
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