Journal of Colloid and Interface Science 316 (2007) 413–419 www.elsevier.com/locate/jcis
Circular dichroism analysis of penicillin G acylase covalently immobilized on silica nanoparticles Bertolt Kranz a , Jochen Bürck b,∗ , Matthias Franzreb a , Rainer Köster a , Anne S. Ulrich b a Institute for Technical Chemistry, Water Technology and Geotechnology, Forschungszentrum Karlsruhe GmbH, P.O. Box 3640, 76021 Karlsruhe, Germany b Institute for Biological Interfaces, Forschungszentrum Karlsruhe GmbH, P.O. Box 3640, 76021 Karlsruhe, Germany
Received 2 August 2007; accepted 28 August 2007 Available online 31 August 2007
Abstract Circular dichroism (CD) was used to characterize the secondary structure of penicillin G acylase upon covalent immobilization on silica nanoparticles. Covalent immobilization was achieved by functionalizing the silica nanoparticles with glutardialdehyde and coupling to the free NH2 groups of the enzyme (lysine and arginine side chains). The loading of the covalently bound enzyme was increased up to saturation, which was reached at 54.6 mg immobilized enzyme per g silica nanobeads. For structural characterization of the commercially available enzyme its exact molecular mass was determined by mass spectrometry in order to enable precise evaluation of the CD data. The fraction of secondary structure elements of the free and immobilized enzyme were estimated from the respective CD spectra using standard algorithms (CONTINLL, CDSSTR, SELCON3). The fractions obtained by the different algorithms for the free enzyme agreed well with one another and also with data from X-ray diffraction described in the literature. Interestingly, the secondary structure fractions found for the immobilized enzyme were very similar to the free enzyme and nearly constant over all experiments. These results indicate that even a loading of up to 55.8 mg/g (enzyme per silica nanoparticles) causes only slight structural changes. However, the specific activity determined by a kinetic assay decreased by around 60%, when increasing the loading from 14.9 to 55.8 mg/g. Because of the fact that we found no major changes in the secondary structure, diffusion limitation seems to be the main reason for the decline of the specific activity. © 2007 Elsevier Inc. All rights reserved. Keywords: Penicillin G acylase; Silica nanoparticles; Covalent immobilization; Circular dichroism; Secondary structure deconvolution; Specific enzyme activity
1. Introduction Immobilization of enzymes has two main advantages: The possibility of enzyme separation (centrifugation, filtration, magnetic separation) enables a better handling and re-use of the enzyme, and an increased operational and storage stability reduces the costs of enzyme-catalyzed industrial processes. Other improvements are an increased enzyme stability against extremes in pH, temperature and ionic strength. Enzymes can be immobilized by cross-linking, crystallization, inclusion, adsorption or covalent binding. All of these methods provide different benefits and disadvantages, which should be taken into account. Also mass-transfer effects have a great influence on the properties and activity of the immobilized enzyme [1]. * Corresponding author. Fax: +49 7247 82 4842.
E-mail address:
[email protected] (J. Bürck). 0021-9797/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2007.08.062
Busto [2] found a higher thermal stability of immobilized β-glucosidase compared to the free enzyme. While the pH stability was the same, the kinetic properties of the immobilized enzyme decreased. For lipase from Candida antarctica, Cao et al. [3] examined several supports for immobilization and found that even under optimal conditions there was a 25% decrease in conversion after six cycles and a 30% decrease in activity after raising the temperature from 30 to 70 ◦ C. Bryjak et al. [4] immobilized penicillin G acylase on acrylic carriers and demonstrated that the storage stability, the stability at lower pH-values and thermal stability had increased. Circular dichroism (CD) is a well-established spectroscopic technique for analyzing structural properties of proteins on a global scale, and numerous reports on the secondary structure composition and tertiary structure fingerprints of proteins have been published. The fundamentals of CD and examples of applications can be found in the reviews [5–8]. CD has been
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primarily used to study proteins in solution, and well-defined algorithms are available to extract the secondary structure fractions from the experimental spectrum of a structurally unknown protein, based on the characteristic far UV CD spectra of the different secondary structure elements. However, if structural changes of enzymes immobilized on solid particles have to be monitored, strong light scattering in the far UV spectral range and fast sedimentation may lead to considerable difficulties in recording CD spectra free from artifacts due to absorption flattening and differential light scattering [9]. To avoid these problems, several groups have performed CD conformational studies of various enzymes non-covalently immobilized on nano-sized hydrophilic silica or hydrophobic Teflon particles, where light scattering is minimal and fast sedimentation is absent [10–13]. It was also attempted to circumvent the problem by acquiring CD spectra of bovine serum albumin in solution before and after being in contact with sorbents separated by centrifugation [14]. Assuming fast exchange between the adsorbed and dissolved states of the enzyme CD analysis indicated a pronounced conformational change after the adsorption/ desorption process for polystyrene particles but not for silica. To get usable CD spectra Vermeer and Norde [15] proposed to insert thin Teflon-coated quartz plates with protein adsorbed onto the Teflon layer into a quartz cell containing a buffer solution. However, the spectra of the Teflon-adsorbed IgG presented in their paper are rather noisy at wavelengths 200 nm, and both spectra show strong deviations from the α-chymotrypsin spectrum given, e.g., in Celej et al. [18]. We have chosen penicillin G acylase, which was covalently immobilized on silica nanoparticles to avoid the spectral artifacts described above. For characterizing the effects of immobilization on the enzyme performance in addition to CD structural analysis, the specific enzyme activity and the loading of the solid support have to be specified [19]. Therefore, we have determined the loading of the silica nanoparticles based on the UV absorption at 280 nm caused by the Trp and Tyr residues of penicillin G acylase [20]. The specific activity of the free and immobilized penicillin G acylase was assessed by the method described by Kasche et al. [21], because of the fact that the
particles did not disturb the measurements. This is a direct spectrophotometric technique based on a colorimetric activity test using 6-nitro-3-phenylacetamido benzoic acid (NIPAB). Penicillin G acylase (pdb entry 1h2g) is an industrially important enzyme for the production of β-lactam antibiotics [22], and it belongs to the N-terminal nucleophile aminohydrolase (Ntn) family [23]. These enzymes cleave amide or ester bonds with the catalytic nucleophile oxygen or sulfur. As common for these enzymes, the active site of penicillin G acylase is created by autocatalytic maturation. The CD spectrum of free penicillin G acylase was first determined by Lindsay and Pain [24], which was taken in this work as reference. Covalent immobilization of penicillin G acylase on solid supports was accomplished by several working groups [25,26] and resulted in improvements similar to those described above. In our study, covalent immobilization was achieved by functionalizing the silica nanoparticles with glutardialdehyde and coupling to the free NH2 groups of the enzyme (lysine and arginine side chains). The loading of the particles was successively increased up to saturation to find out whether tight enzyme packing on the surface of the particles would affect the secondary structure and specific activity of the enzyme. 2. Materials and methods 2.1. Enzyme and chemicals Penicillin G acylase from E. coli (EC. 3.5.1.11) and 6-nitro3-phenylacetamido benzoic acid were purchased from Sigma– Aldrich, sequencing grade modified trypsin was purchased from Promega (Mannheim, Germany). The glutardialdehyde solution (reaction quality) was from VWR International. All other chemicals purchased from VWR International were of analytical quality and used without further purification. 2.2. Silica nanoparticles Silica nanoparticles (Ludox® -HS 40) were a kind donation from Grace Davison (Columbia, Maryland, USA). They have a size of 12 nm and a specific surface area of 198–258 m2 /g. The specific gravity is 1.292–1.312, and the zeta potential at the working pH of 7.5 is −52 mV. The particles are transparent and form a stable colloidal suspension in water, which has a higher viscosity than water. 2.3. Immobilization procedure A scheme of the immobilization reactions is shown in Fig. 1. The silica nanoparticles were functionalized with glutardialdehyde solution (1.0 M, pH 3–4) for 1 h at room temperature. Afterwards they were washed five times with water and separated by ultracentrifugation at 30,000 rpm for 90 min at 25 ◦ C. The covalent immobilization of penicillin G acylase was performed for 1 h at 25 ◦ C in a thermo mixer in 0.2 M phosphate buffer of pH 7.5 with ratios of 40–400 mg active enzyme per g silica nanobeads. In the next step the covalently immobilized enzyme was washed five times with 0.1 and 0.01 M phosphate
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Fig. 1. Reaction scheme for covalent enzyme immobilization on silica nanoparticles using glutardialdehyde activation.
buffer of pH 7.5, using again ultracentrifugation procedure to retrieve the particles. The enzyme activity of the supernatants was controlled and it was confirmed that the last one showed no activity, to be sure that all adsorbed enzyme molecules had been removed and only covalently bound enzyme remained on the particles.
neurotensin, Glu-fibrinopeptide-B) to generate peptide mass fingerprints which were used in data base search (GPS-Explorer Software, Applied Biosystems, MASCOT search, NCBI Data base) for identification.
2.4. Activity measurements
The concentration of penicillin G acylase dissolved in phosphate buffer solution and immobilized on silica nanoparticles in colloidal solution was determined based on the absorbance of the protein at 280 nm under denaturing conditions according to the method described by Edelhoch [27]. The corresponding protein solution was dissolved in 6 M guanidine hydrochloride in 0.02 M phosphate buffer of pH 6.5. The absorption spectrum in the range of the aromatic bands due to Trp and Tyr was recorded in the range from 400–240 nm using a quartz glass micro-cuvette with 1 cm optical path length. The blank solution for the UV absorption measurement was the corresponding phosphate buffer or silica nanoparticle colloidal solution without enzyme dissolved in 6 M guanidine hydrochloride/0.02 M phosphate buffer. From the flat baseline of the protein UV spectra at wavelengths around 310 nm it was ascertained that light scattering due to the presence of the silica nanoparticles was negligible and scattering correction was not necessary. The concentration of penicillin G acylase present in solution or in the colloidal suspension (when immobilized on silica nanoparticles) was determined from the absorbance at 280 nm using a molar extinction coefficient of 199,000 l mol−1 cm−1 which was calculated as described by Gill and von Hippel [20] (penicillin G acylase has 28 tryptophan and 31 tyrosine residues and no cystein). From the enzyme concentration and the weighed amount of silica nanoparticles before immobilization the loading could be calculated.
The activity of the free and of the immobilized enzyme was measured with 6-nitro-3-phenylacetamidobenzoic acid (NIPAB) according to the method described by Kasche et al. [21]. The activity of the enzyme was determined as follows. The spectrometer was balanced with 0.2 M phosphate buffer of pH 7.5, containing 5 mM NIPAB at 25 ◦ C, and after the addition of the enzyme solution the reaction started. The reaction kinetics were monitored over a period of 60 s. The optical density of the hydrolytic product 5-amino-2-nitrobenzoic acid was monitored at 380 nm. 2.5. Gel electrophoresis and mass spectrometry Penicillin G acylase was purified using SDS–polyacrylamide gel electrophoresis (Mini Protean 3, BioRad, USA), starting from a concentration of 1 µg/µl. The solution was diluted 1:1 with sample buffer: 150 mM Tris (pH 6.8), 120 g/l SDS, 30% (v/v) glycerol, 15% (v/v) β-mercaptoethanol, 120 mg/l Coomassie Brilliant Blue R-250, and boiled 5 min. Sample quantities: 5 µg (12.5% resolving gel, 5% stacking gel) in working buffer (145 g/l glycin, 29 g/l Tris base, 10 g/l SDS). After electrophoresis (170 V, 400 mA, 1 h) the gel was fixed with 200 g/l trichloroacetic acid and stained with 1 g/l Coomassie Brilliant Blue R-250. Penicillin G acylase-characteristic bands (molecular weight approx. 23 and 63 kDa, according to LMWSDS marker kit (molecular-weight-marker) from GE Healthcare (München, Germany)) were cut out and digested with trypsin solution of 25 ng/µl. The peptide solutions were mixed in a ratio of 1:1 with α-cyano-4-hydroxy-cinnamonic acid (saturated solution in 0.1% (v/v) trifluoroacetic acid: acetonitrile, ratio 70:30) and applied to the MALDI target and air-dried. Spotted samples were analyzed in a MALDI-TOF MS/MS unit (ABI 4700 Proteomics Explorer, Applied Biosystems, Nd-YAG laser 355 nm/200 Hz), measuring mode: reflector positive ion mode, 1000 shots/spectrum, external calibration using a mixture of standard peptides (des-Arg-bradykinin, angiotensin,
2.6. UV absorption
2.7. Circular dichroism CD spectra were recorded using a J-810 spectropolarimeter (Jasco Co., Tokyo, Japan). The instrument was routinely calibrated with a 0.06% (w/v) aqueous solution of ammonium D -10-(+)-camphor sulfonate at 290.5 nm. The spectra were scanned between 260 and 180 nm at 0.1 nm intervals. Three repeat scans at a scan rate of 10 nm min−1 , 4 s response time and 1 nm bandwidth were averaged for each sample and its respective blank. Samples containing immobilized penicillin G acylase were prepared by using an appropriate aliquot of the
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freshly prepared enzyme-loaded silica particles in a 2 ml vial, addition of 10 mM phosphate buffer of pH 7.5, and vortexing the particles to get a clear colloidal suspension. CD spectra of the free and immobilized penicillin G acylase were collected in buffer, using rectangular quartz glass cuvettes with 0.1 cm optical path length. The blank control for the free enzyme solution was a 10 mM phosphate buffer and for the immobilized enzyme an appropriate suspension of unloaded silica particles in buffer. The averaged blank spectrum was subtracted from the averaged sample spectrum to get the corrected lineshape. All spectra were recorded at 20 ◦ C using a water thermostated rectangular cell holder. CD spectra were smoothened by the adaptive smoothing method, which is part of the Jasco Spectra Analysis software. Secondary structure analysis was performed using the CONTINLL, CDSSTR and SELCON3 algorithms provided by DICHROWEB [28,29]. The quality of the fit between experimental and back-calculated spectrum corresponding to the derived secondary structure element fractions was assessed from the normalized root mean square deviation (NRMSD), with a value
