Protein J (2010) 29:572–582 DOI 10.1007/s10930-010-9287-8
Biophysical Characterization of a Recombinant a-Amylase from Thermophilic Bacillus sp. strain TS-23 Meng-Chun Chi • Tai-Jung Wu • Tzu-Ting Chuang Hsiang-Ling Chen • Huei-Fen Lo • Long-Liu Lin
•
Published online: 10 November 2010 Ó Springer Science+Business Media, LLC 2010
Abstract Environmental variables can significantly influence the folding and stability of a protein molecule. In the present study, the biophysical properties of a truncated Bacillus sp. TS-23 a-amylase (BACDNC) were characterized in detail by glutaraldehyde cross-linking, analytical ultracentrifugation, and various spectroscopic techniques. With cross-linking experiment and analytical ultracentrifuge, we demonstrated that the oligomeric state of BACDNC in solution is monomeric. Far-UV circular dichroism analysis revealed that the secondary structures of BACDNC were significantly altered in the presence of various metal ions and SDS, whereas acetone and ethanol had no detrimental effect on folding of the enzyme. BACDNC was inactive and unstable at extreme pH conditions. Thermal unfolding of the enzyme was found to be highly irreversible. The native enzyme started to unfold beyond *0.2 M guanidine hydrochloride (GdnHCl) and reached an unfolded intermediate, [GdnHCl]0.5, N–U, at 1.14 M. BACDNC was active at the concentrations of urea
Meng-Chun Chi and Tai-Jung Wu contributed equally to this work. M.-C. Chi T.-J. Wu L.-L. Lin (&) Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi County 60004, Taiwan e-mail:
[email protected]
below 6 M, but it experienced an irreversible unfolding by [8 M denaturant. Taken together, this work lays a foundation for the future structural studies with Bacillus sp. TS23 a-amylase, a typical member of glycoside hydrolases family 13. Keywords Amylase Analytical ultracentrifugation Guanidine hydrochloride Urea Fluorescence Circular dichroism Abbreviations GH Glycoside hydrolase SBD Starch-binding domain BACDNC Bacillus sp. TS-23 a-amylase without signal sequence and SBD LB Luria-Bertani Ni2?-NTA Ni2?-nitrilotriacetate IPTG Isopropyl-b-thiogalactopyranoside SDS–PAGE Sodium dodecyl sulfate–polyacrylamide gel electrophoresis AUC Analytical ultracentrifugation GdnHCl Guanidine hydrochloride CD Circular dichroism kmax Wavelength maximum AEW Average emission wavelength BlGGT Bacillus licheniformis c-glutamyltranspeptidase
T.-T. Chuang Department of Aquatic Biosciences, National Chiayi University, 300 Syuefu Road, Chiayi County 60004, Taiwan
1 Introduction
H.-L. Chen H.-F. Lo Department of Food Science and Technology, Hungkuang University, Shalu, Taichung 433, Taiwan
a-Amylase (1,4-a-D-glucan glucanohydrolase, EC 3.2.1.1) is an endo-acting enzyme that catalyzes the hydrolysis of
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a-1,4-glucosidic linkages in starch and related substrates to release maltooligosaccharides and maltodextrins. The enzyme belonging to glycoside hydrolases (GH) family 13 employing an a-retaining mechanism of action [38]. a-Amylases are widely distributed in three domains of life [24]. Microbial a-amylases are the most important enzymes with a wide application in starch industry and food processing [21, 60]. a-Amylase and related amylolytic enzymes from different organisms exhibit similar three-dimensional structures despite differences in their primary structures, with an a/b barrel as a central part (domain A), a Greek key motif as domain C and at least one additional domain [38]. Substrate binding is localized to a cleft between the a/b barrel and domain B, comprising the highest flexibility in size and structure with irregular folding. Domain C not only enables substrate binding but also stabilizes the catalytic domain by shielding hydrophobic residues of domain from the solvent [38, 62]. Additionally, the amino acids involved in substrate binding and catalysis are highly conserved among the different enzymes [28]. Among the known a-amylases, about 10% posses the raw-starch-digesting ability owing to the presence of a sequence motif called starch-binding domain (SBD), which is structurally distinct from the catalytic domain [9, 39]. The SBD mediates the interaction between an enzyme and the insoluble substrate [57]. Despite the slight variations in the amino acid sequence and fold, most of SBDs seem to have a similar way of substrate binding [39]. Earlier, the raw-starch-degrading a-amylase from Bacillus sp. strain TS-23 has been cloned and overexpressed in Escherichia coli [32]. The recombinant enzyme could digest raw starch to produce maltopentoase as the main end product [33]. Deletion analysis showed that the complete SBD of the cloned enzyme is not essential for the amylolytic activity but is involved in the binding of granular starch [34]. A truncated Bacillus sp. TS-23 a-amylase (BACDNC) lacking the N-terminal signal sequence and the SBD was also constructed and actively expressed in E. coli M15 [35]. The primary structure of BACDNC comprises 489 amino acid residues corresponding to a molecular mass of 55,181 Da, which is similar to the size of a-amylases from Bacillus stearothermophilus [44] and Bacillus liheniformis [67]. However, its biophysical properties have not been explored before. In the present work, we have focused on the biophysical characterization of BACDNC using analytical ultracentrifugation, circular dichroism (CD), and intrinsic tryptophan fluorescence measurements. Our results provide clues to the oligomeric state, the stability in different environmental conditions, and the chemical-induced unfolding of the protein molecule.
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2 Materials and Methods 2.1 Materials Luria-Bertani (LB) media for bacterial culture were purchased from Difco Laboratories (Detroit, MI, USA). Imidazole, guanidine hydrochloride, glutaraldehyde, ampicillin, and kanamycin were acquired from Sigma– Aldrich Chemical (St. Louis, MO, USA). Ni2?-nitrilotriacetate (Ni2?-NTA) resin was obtained from Qiagen Inc. (Valencia, CA, USA). Protein assay reagents, acrylamide, bis-acrylamide, TEMED, and ammonium persulfate were purchased from Bio-Rad Laboratories (Hercules, CA, USA). All other chemicals were commercial products of analytical or molecular biological grade. 2.2 Expression and Purification of the Recombinant Enzyme The expression and purification of BACDNC was carried out as described previously [8]. The enzyme was overexpressed in Escherichia coli M15 harboring the plasmid pQE-AMYDNC. To purify BACDNC, cells were grown in LB broth with 100 lg/ml ampicillin and 25 lg/ml kanamycin and induced with 0.1 mM isopropyl-b-thiogalactopyranoside (IPTG). Cultures were then grown at 28 °C for 12 h and cells were harvested by centrifugation at 6,0009g for 10 min at 4 °C. The pellet was resuspended in a buffer (pH 7.9) containing 50 mM Tris–HCl, 300 mM NaCl, and 10 mM imidazole. Cells were lysed by sonication (30-s bursts and pulses for 5 min) and cell debris was subsequently removed by centrifugation. Recombinant proteins were purified by affinity chromatography using a Ni2?-NTA agarose column (Qiagen) under native conditions. The collected fractions were pooled and dialyzed against 1,000 volumes of 50 mM Tris–HCl buffer (pH 7.9) with a 10-kDa cutoff membrane to remove salts. The homogeneity of the purified protein was evaluated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). 2.3 Glutaraldehyde Cross-linking The oligomeric state of BACDNC was monitored using the glutaraldehyde cross-linking method [66] modified to include trichloroacetic acid/deoxycholate precipitation. The cross-linked samples were analyzed on a 12% polyacrylamide–SDS gel. 2.4 Analytical Ultracentrifugation Sedimentation velocity was carried out in a BeckmanCoulter XL-A analytical ultracentrifuge. Sample (370 ll)
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at a protein concentration of 1.5 lg/ll and 50 mM Tris– HCl buffer (400 ll; pH 7.9) solutions were loaded into the double sector centerpiece separately, and built up in a Beckman An-50Ti rotor. Experiments were performed at 20 °C and a rotor speed of 42,000 rpm. Protein sample was monitored by UV absorbance at 280 nm in a continuous mode with a time interval of 8 min and a step size of 0.003 cm. Multiple scans at different time points were analyzed with the SEDFIT program [3, 54]. The sedimentation velocity data were fitted using a two-dimensional distribution with respect to frictional ratio c(s, f/fo) according to Lamm equation [54] (Eq. 1): Z Z a ðr; tÞ ¼ cðs; f =fo Þvðs; Dðs; f =fo Þ; r; tÞdsd ðf =fo Þ ð1Þ with a(r,t) denoting the observed optical signal at radius r and time t; v(s,D,r,t), the solution of the Lamm equation; and D(s, f/fo), the dependence of diffusion coefficient (D) on sedimentation coefficient (s) and frictional ratio (f/fo), where ov 1 o ov ¼ rD sx2 r 2 v ð2Þ ot r or or rffiffiffiffiffiffiffiffiffiffiffiffiffi pffiffiffi 2 kT 1 1 mq pffiffi qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Dðs; f =fo Þ ¼ ð3Þ m 18p s ðgf =fo Þ3 with x denoting angular velocity; k, Boltzmann constant; T, absolute temperature; v; enzyme partial specific volume; g, buffer viscosity; and q, buffer density. All two-dimensional distributions were solved and normalized to a confidence level of p = 0.95 by maximum entropy and a resolution N of 200 with sedimentation coefficients between 0.1 and 20 S. The anhydrous friction ratio is from 1.0 to 2.0 or 3.5 at a resolution of 10. 2.5 Enzymatic Activity Assay The assay mixture contained 250 ll of 1% (w/v) soluble starch in 50 mM Tris–HCl buffer (pH 8.0) and 250 ll of tenfold diluted enzyme solution. The reaction was performed at 60 °C for 10 min and stopped by the addition of 0.5 ml of 3,5-dinitrosalicyclic reagent [41]. The amylolytic activities were obtained from a calibration curve prepared by following the same procedure with D-glucose as the standard. One unit of amylase activity is defined as the amount of enzyme that releases the amount of reducing sugar equivalent to 1 lmol of glucose per min under the assay conditions. 2.6 CD and Spectrofluorimetric Analyses Far UV-CD spectra of BACDNC were acquired on a JASCO model J-815 spectropolarimeter (JASCO Inc.,
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Japan) from 250 to 190 nm in cuvettes at 25 °C using a 1.0-nm bandwidth, 0.1-nm resolution, 0.1-cm path length, 1.0-s response time, and a 100-nm/min scanning speed. The photomultiplier absorbance was always below 600 V in the analyzed region. Each scanning was repeated ten times and an average was reported. Data were corrected for the buffer effect and the results were expressed as molar ellipticity [H] in the units of degrees cm2 decimol-1 according to Eq. 4: ½H ¼
H 10 C l
ð4Þ
where l represents the light path length (cm), C is the molar concentration of protein (mol/l), and H represents the observed ellipticity (mdeg). The unfolding transition curves for BACDNC were obtained by measuring the ellipticity at 222 nm in a 1-mm cell. The temperature was increased with heating rates of 0.5, 1.0 and 4.0 °C/min from 20 to 100 °C and the transition mid point (Tm) was recorded. For refolding experiments, the temperature was decreased by 0.5, 1, and 4 °C/ min and measurements were taken once every min. BACDNC was unfolded with different concentrations of GdnHCl in 20 mM Tris–HCl buffer (pH 8.0) at room temperature. A 12-h incubation was employed in unfolding experiments. Fluorescence spectra of BACDNC were monitored at 30 °C in a Hitachi F-7000 fluorescence spectrophotometer with an excitation wavelength of 280 nm. All spectra were corrected for buffer absorption. The fluorescence emission spectra of protein samples with a concentration of 24 lM were recorded from 300 to 400 nm at a scanning speed of 240 nm/min. The maximal peak of the fluorescence spectrum and the change in fluorescence intensity were used in monitoring the unfolding processes of the enzyme. Both the red shift and the change in fluorescence intensity were analyzed together using the average emission wavelength (AEW) (k) according to Eq. 5 [50]: Pk N i¼k ðFi ki Þ ð5Þ hki ¼ PkN1 i¼k1 ðFi Þ in which Fi is the fluorescence intensity at the specific emission wavelength (ki). 2.7 Unfolding Data Analysis The unfolding data of BACDNC were treated with the following thermodynamic model by global fitting of the data. The two-state unfolding model (Scheme I) was described by Eq. 6 [47]. KNU
N ! U
Biophysical Characterization of a Recombinant a-Amylase from Thermophilic Bacillus sp. strain TS-23
Scheme I
DGðH
2 OÞN!U
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yobs ¼ yN þyU eDGðH OÞN!U mN!U ½GdnHCl or ½Urea 2 RT 1þe
ð6Þ
where yobs is the observed biophysical signal; yN and yU are the calculated signals of the native and unfolded states, respectively. [GdnHCl] and [Urea] are the concentrations of GdnHCl and urea respectively, and DGN–U is the free energy change for the N $ U process. The mN–U is the sensitivity to denaturant concentration.
3 Results and Discussion 3.1 Quaternary Structure of the Purified Enzyme To determine the biophysical properties of BACDNC, the enzyme in the crude extract of IPTG-induced E. coli M15 (pQE-AMYDNC) was purified to near homogeneity using Ni2?-NTA resin. SDS–PAGE analysis revealed that the molecular mass of BACDNC was approximately 54 kDa (Fig. 1). The purification procedure resulted in a protein yield of about 15% and the purified enzyme had a specific activity of 179.2 ± 10.3 U/mg protein. Quaternary structure of proteins has piqued recent interest in the literature as a potential modulation of protein dynamics [1, 2, 4, 19], which are increasingly recognized as being critical for enzyme catalysis. As recombinant DNA techniques have now made many macromolecules available for study on the 10–100 mg level, there has been
Fig. 1 Analysis of the purified BACDNC by SDS–PAGE. The protein samples were electrophoresed on 12% polyacrylamide gel and visualized by Coomassie brilliant blue staining. Lanes M protein size marker; 1 the purified BACDNC; 2 the cross-linked BACDNC; 3 the purified BlGGT; 4 the cross-linked BlGGT
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a renewed interest in understanding of the relationship between molecular structure and biological function. Among the analytical methods for defining macromolecular assemblies, AUC is experiencing a renaissance due largely to the availability of modern computer-based instrumentation and its resulting expanded applications [23, 30]. In this regard, the oligomeric state of BACDNC was examined by AUC. As shown in Fig. 2, the BACDNC molecule sediments at 4.62 ± 0.26 S corresponding to a species with molar mass of 64227.2 ± 5266.7 Da, which is in agreement with the molecular mass calculated from the amino acid sequence of a monomer (55.2 kDa). The excellent matching of the experimental data points and the fitted curve (Fig. 2a), the homogeneous bitmap picture (Fig. 2b), and the randomly distributed residual values (data not shown) all indicate that a highly reliable model for the sedimentation velocity experiments is obtained and the AUC is an excellent biophysical probe for assessing the molecular structure of this enzyme. The self-association of proteins play an important role in the catalytic activity of a variety of enzymes, such as cytidine 5’-triphosphate synthase from E. coli [37], dihydrofolate reductase from Thermotoga maritima [36], and c-cyclodextrin-specific cyclodextrinase from Bacillus clarikii [43]. Crystal structures of Flavobacterium sp. cylodextrinase [15] and Thermoactinomyces vulgaris R-47 a-amylase 1 [25] have shown that these two enzymes exist predominantly as a homodimer. Similar to Flavobacterium sp. and T. vulgaris enzymes, Pyrococcus furiosus a-amylase was determined to be a dimeric enzyme by gel filtration and, however, the quaternary structure does not seem to be critical for the catalytic activity [11]. Gel filtration analysis also showed that the molecular size of a maltogenic amylase from Thermoplasma volcanium appears to be a homodimeric protein [26]. In contrast, the AUC data clearly indicated that the monomer is a predominant form of BACDNC in solution at a protein concentration of 1.5 mg/ml (Fig. 2). Protein oligomerization is reported as a concentrationdependent event and increases logarithmically with increasing concentration [56]. Accordingly, many proteins were reported to have an interconvertible mixture of monomers and oligomers [10, 20, 37]. Although Flavobacterium sp. and T. vulgaris amylases have been described as dimers in the past, it is still not clear whether the GH family 13 enzymes form active monomers or dimers in the solution, or what the physiological role of a possible oligomerization state. At present, our findings cannot directly address these issues but we reported that the recombinant a-amylase, a GH family 13 enzyme, exists in solution as a monomer at a fairly high protein concentration. To confirm the oligomeric state of BACDNC, the enzyme was chemically cross-linked and analyzed by SDS–PAGE. As shown in Fig. 1, the protein band with a
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Fig. 2 Sedimentation velocity experiments of the purified BACDNC. a A typical trace of absorbance at 280 nm of the enzyme during the sedimentation velocity experiment. The symbols are experimental data and the lines are computer-generated results by fitting the experimental data to the Lamm equation with SEDFIT program. b The residuals of the model fitting of the data in panel A. c The continuous sedimentation coefficient distribution of the enzyme at high protein concentration of 1.5 mg/ml. d The continuous molar mass distribution of the enzyme at the indicated concentration
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molecular mass of approximately 54 kDa was observed in the SDS-gel, and, as a control, the large and small subunits of a heterodimeric Bacillus licheniformis c-glutamyltranspeptidase (BlGGT) were successfully cross-linked into a protein species of *63 kDa [7]. These results reflect the fact that the molecular architecture of BACDNC is monomeric in solution. 3.2 Effects of Metal Ions, Chemicals, and Organic Solvents on the BACDNC Structure To analyze the secondary structures of BACDNC, far-UV CD measurements were performed under various conditions. The CD spectrum of BACDNC displays strong peaks of negative ellipticity at 208 and 222 nm, indicative of substantial a-helical contents (Fig. 3). The addition of Mg2?, Ca2?, Cu2?, and Mn2? ions did not lead to any significant structural changes, although Zn2?, Co2?, Fe2?
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and Ni2? ions caused substantial changes in the spectrum (Fig. 3a). Mn2?, Co2?, and Fe2? ions have a stimulatory effect on the BACDNC activity and, in contrast, Zn2? and Cu2? ions exhibit an inhibitory effect on the activity [33]. Additionally, a significant alteration in the CD spectrum of the enzyme was observed in the presence of 15 mM EDTA (Fig. 3a). Based on the above-mentioned facts, it is difficult to conclude the effects of metal ions and EDTA on the secondary structures of BACDNC. Possibly, the structural changes caused by the tested ions alter the local environment of the active site and, as a consequence, readjust the hydrolytic capability of the enzyme. SDS, an anionic surfactant, has been demonstrated to unfold and inactivate creatine kinase [65] and aminoacylase [22]. Many microbial a-amylases were also inhibited by SDS [6, 27, 55]. As shown in Fig. 3b, high concentration of SDS had marked affect on the secondary structures of BACDNC. This might be the reason for the abolishment of amylolytic
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Wavelength (nm) Fig. 3 Influence of environmental variables on CD spectra of BACDNC. a Effects of metal ions and EDTA. b Effects of SDS and organic solvents. The enzyme was incubated with the desired environmental variable for 30 min at room temperature and monitored the changes in the secondary structures by far-UV CD
activity in the presence of 10% SDS (data not shown). In contrast, no significant alteration in the CD spectra was observed with the addition of 5–10% acetone and 10–20% ethanol, reflecting that BACDNC is stable in the presence of organic solvents. Organic-solvent-tolerant a-amylases appear to be quite attractive for commercial applications such as bioremediation of industrial wastewaters contaminated with organic solvents. The organic-solvent-tolerant characteristic has been reported in a-amylases from Haloarcula sp. strain S-1 [17] and Nesterentonia sp. strain F [55]. 3.3 pH-induced Changes in Activity and Structure of BACDNC BACDNC was found to retain [83% of the enzymatic activity in the pH range 6.1–9.0 (Fig. 4a). Irreversible inactivation occurred when the enzyme was pre-incubated
Fig. 4 Effects of pH on the enzymatic activity (a) and fluorescence spectra (b) of BACDNC. The amylase activity was assayed in 50 mM buffer (pH 3–7), 50 mM Tris–HCl buffer (pH 7–9), 50 mM NaHCO3–NaOH (pH 7–11), and 50 mM KCl–NaOH buffer (pH 12–13). The results are the mean of three individual experiments
at pH \ 4.0 or pH [ 10.0 for 30 min. BACDNC stability was examined by measuring intrinsic tryptophan fluorescence as a function of pH, following incubation of the samples at 25 °C. Maximum stability for the BACDNC structure was observed at pH 8.0. Shift in the fluorescence intensity of the enzyme occurred at both acidic and basic pH conditions (Fig. 4b), indicating that BACDNC was unfolded under such conditions. Structural perturbations of sorghum a-amylase at extreme pH conditions have been presumed mainly due to the disruption of electrostatic interactions [51]. 3.4 Thermal Unfolding of BACDNC Thermal denaturation of BACDNC was followed by monitoring the ellipticity at 222 nm under constant heating rates. Figure 5 shows the transition curves obtained with BACDNC solutions at heating rates of 0.5, 1.0 and 4.0 °C/
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min. As compared with the heating rates of 1.0 and 4.0 °C/ min, the thermal transition for 0.5 °C/min appeared at lower temperature. This resembles a common result for transitions under kinetic control [14, 52, 58]. Also, the lower heating rate resulted in larger changes in the far-UV CD transition, probably indicating aggregation of the enzyme. Aggregation of the protein sample was confirmed by light scattering (data not shown). The impact of aggregation and heating rates on the apparent transition temperatures has also been described for several cases [18, 31, 48, 64]. To further explore if the unfolding reaction was reversible or not, the sample was heated at a constant heating rate. After the thermal denaturation transitions went to completion, the protein solutions were cooled down to 30 °C at the same scan speed. Figure 5 shows the cooling curve obtained with BACDNC solution at a heating rate of 0.5 °C/min. It is clear that the secondary structures of the native polypeptide were not recovered after the sample was cooled down to the indicated temperature. Similarly, no detectable recovery in the secondary structures was observed for other heating rates (data not shown). These results indicate that thermal denaturation of BACDNC is highly irreversible. The irreversible process seems to be the more common case for most multidomain proteins. As a typical representative for a medium-sized multidomain protein (*60 kDa), a-amylases in nearly all cases unfold irreversible [12, 16, 59, 63]. 3.5 Molecular Properties of BACDNC Associated with GdnHCl- and Urea-induced Unfolding The effect of GdnHCl- and urea-induced changes on the structural and functional properties of BACDNC was
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studied. Time-dependent changes in the structural properties and amylolytic activity of this enzyme in 0–6 M GdnHCl and 0–12 M urea were monitored to standardize the incubation time required to achieve equilibrium. Under the conditions studied, the changes occurred within maximum of 4 and 12 h for GdnHCl and urea respectively, with no further alterations in the values obtained up to 24 h (data not shown). These observations suggest that a minimum time of 4 h (GdnHCl) and 12 h (urea) is sufficient to achieve the equilibrium. Enzymatic activity can be regarded as the most sensitive probe to study the changes in enzyme conformation during various treatments, because it reflects subtle readjustments at the active site, enabling very small conformational variations of an enzyme structure to be detected. The effect of increasing concentrations of GdnHCl on the enzymatic activity of BACDNC is summarized in Fig. 6a. The enzyme treated with GdnHCl below 0.6 M retained [82% of the amylase activity. An increase in concentration up to 1 M resulted in 39.2% of the activity remaining, whereas the enzyme was completely inactivated after 1.4 M GdnHCl treatment. These results reveal that the GndHCl concentration below 0.6 M induces minor changes, probably in and around the active site. GdnHCl-induced unfolding of BACDNC was performed to explore the effect of this denaturant on the secondary structures of the protein. The effect of increasing GdnHCl concentrations on the ellipticity of BACDNC at 222 nm is illustrated in Fig. 6b. Obviously, the secondary structures of BACDNC were very sensitive to GdnHCl treatment. A large decrease in the negative ellipticity was observed at GdnHCl concentrations between 0.5 and 2 M, indicating a significant disruption in the secondary structures of the enzyme under these conditions. By fitting with Eq. 6, BACDNC showed [GdnHCl]0.5, N–U of 1.14 M, corresponding to a free energy change of 1.99 kcal/mol for the N?U process. Fluorescence spectra provide a sensitive means to characterize proteins and their conformation. The spectrum is determined chiefly by the polarity of the environment of the tryptophan and tyrosine residues and by their specific interactions [49]. In this regard, AEW that reports on the changes in both fluorescence wavelength and intensity was used to calculate the thermodynamic parameters of the unfolding process. For BACDNC, the AEW values in the absence of GdnHCl and at GdnHCl concentrations [4.5 M were 335.2 and 348.1 nm, respectively. The transition was occurred at 3.29 M GdnHCl and a plateau region existed from 4.5 to 6 M (Fig. 6c). Treatment of BACDNC with 6 M GdnHCl, the tryptophan emission kmax of 348.5 nm was observed. Normally exposed tryptophan in the unfolded protein shows emission kmax between 340 and 356 nm [29], indicating that incubation of BACDNC with a higher
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Fig. 7 Urea-induced denaturation of BACDNC. a Change in enzymatic activity of BACDNC with increasing concentrations of urea. The value corresponding to the native enzyme was taken as 100%. b Urea-induced changes in the secondary structures of BACDNC as monitored by the negative ellipticity of the sample at 222 nm. c Ureainduced changes in the AEW value of BACDNC
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concentration of GdnHCl leads to significant unfolding of the protein molecule. Although urea and GdnHCl are believed to have a similar mode of action [47], GdnHCl is a monovalent salt that has both ionic and chaotropic effects [42] but urea has only chaotropic effect. Thus, urea is an ideal control agent for distinguishing between the ionic and chaotropic effects of GdnHCl. The effect of increasing concentration of urea on the enzymatic activity of BACDNC was therefore investigated. As shown in Fig. 7a, more than 90% of the BACDNC activity was retained at the concentration of urea up to 2 M. A sharp decrease in amylase activity was observed when urea concentrations were set between 3 and 7 M, and the activity was abolished completely at concentrations greater than 9 M. The effect of increasing urea concentrations on the secondary structures of BACDNC was studied by monitoring changes in ellipticity at 222 nm. As shown in Fig. 7b, only a slight decrease (approximately 8%) in the negative ellipticity occurred when the urea concentration was increased to 3.2 M. However, a rapid decrease in the negative ellipticity was observed at urea concentrations
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between 3.2 and 4.5 M. By fitting with Eq. 6, BACDNC showed [urea]0.5, N–U of 3.78 M, corresponding to a free energy change of 7.76 kcal/mol for the N?U process. The urea-induced unfolding of BACDNC was also demonstrated by tryptophan fluorescence studies at increasing its concentrations. As shown in Fig. 7c, the fluorescence signal of urea-induced BACDNC followed a monophasic process and the enzyme started to unfold at 3.1 M denaturant with [urea]0.5, N–U of 5.49 M. Low concentration of urea did not induce the change in the AEW value, whereas the tryptophan residues in the protein were highly exposed to the buffer environment at values above 8 M, allowing us to consider the protein completely unfolded. Taken together, BACDNC is stable against the unfolding action of urea but not against GdnHCl. It has been reported that GdnHCl is *2 times more effective as denaturant than urea in the unfolding of proteins [61]. Consistently, GdnHCl is *3 times more effective than urea in the unfolding of BACDNC. It has been shown that [urea]0.5, N–U reveals the net stability of the protein contributed by hydrophobic and electrostatic interactions [42].
Fig. 8 Intrinsic tryptophan fluorescence (a) and far-UV CD spectra (b) of native, denatured, GdnHCl- and urea-induced BACDNC
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Biophysical Characterization of a Recombinant a-Amylase from Thermophilic Bacillus sp. strain TS-23
Similar observations have been reported for sorghum [51] and B. licheniformis a-amylases [13, 46]. Although the exact molecular mechanism of the denaturing action of GdnHCl and urea has not been clearly defined [40, 53], it was presumed that both denaturant molecules unfold proteins by solubilizing the non-polar parts along with the polar groups in the side chains of the protein molecules [45]. The unfolding of BACDNC might follow the same path with both denaturants; however, more studies are required to elucidate their different effects on the unfolding process of BACDNC. 3.6 Irreversibility of the Unfolding Processes Experiments were also performed to study the refolding of GdnHCl- and urea-denatured BACDNC. The enzyme was firstly incubated with 6 M GdnHCl or 8 M urea for 12 h at 4 °C, and these samples were then diluted hundredfold with 50 mM Tris–HCl buffer (pH 8.0) and stirred continuously for overnight. The refolding extent of the enzyme was judged by the recovery of CD and fluorescence spectra. As shown in Fig. 8, the kmax of the renatured sample was found to be 337.2 nm in contrast to 336.5 nm of the native protein and 348 nm of the GdnHCl-denatured protein. This phenomenon was also observed in the ureatreated BACDNC. Consistently, the CD spectrum of the renatured protein failed to coincide with that of the native enzyme (Fig. 8). These results indicate that the refolding of BACDNC could not yield the exact native structure. Irreversibility of GdnHCl- and urea-induced unfolding processes has also been reported in Vibrio cholerae Lys type transcription regulator [5] and Brugia malayi hexokinase [68].
4 Conclusion In summary, biophysical studies of BACDNC provide more information for the inherent quaternary structure of the GH family 13 enzymes and contribute to a better understanding of their enzymatic properties, especially a fuller appreciation of the structure–activity relationship at the molecular level. Additionally, the ability of enzymes to remain active in the presence of organic solvents has received a great deal of attention over the past two decades. There are numerous advantages of using enzymes in organic solvents, such as the increased solubility of nonpolar substrates and the elimination of microbial contamination in the reaction mixture. In this respect, the organic-solvent-tolerant characteristic of BACDNC enables the enzyme suitable for biotechnological processes where the organic solvents are present.
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Acknowledgments The authors are grateful to Dr. Hui-Chih Hung for providing the necessary facilities to carry out the AUC experiment. This work was supported by a research grant (NSC 97-2628-B415-001-MY3) from National Science Council of Taiwan.
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