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Production and characterization of a new cyclodextrin glycosyltransferase from Bacillus firmus isolated from Brazilian soil
Process Biochemistry 42 (2007) 1384–1390 www.elsevier.com/locate/procbio
Production and characterization of a new cyclodextrin glycosyltransferase from Bacillus firmus isolated from Brazilian soil Cristiane Moriwaki a, Glauciane L. Costa a, Rubia Pazzetto a, Gisella M. Zanin b, Fla´vio F. Moraes b, Ma´rcia Portilho a, Graciette Matioli a,* a
Pharmacy and Pharmacology Department, State University of Maringa´, Av. Colombo, 5790 - 87020-900 Maringa´, PR, Brazil b Chemical Engineering Department, State University of Maringa´, Av. Colombo, 5790 - 87020-900 Maringa´, PR, Brazil Received 30 June 2006; received in revised form 3 May 2007; accepted 11 July 2007
Abstract A new CGTase was obtained from Bacillus firmus, strain 7B, isolated from oat soil culture, using a high alkaline pH medium containing 1% Na2CO3. The enzyme was characterized in soluble form, for pH 5–11, temperature from 30 to 85 8C, using a 1% maltodextrin substrate solution and appropriate buffers. It produced mainly b-CD and the cell-free supernatant had a precipitating activity measured by the trichloroethylene method that is a 100-fold greater than that of the enzyme of Bacillus firmus, strain 37, previously studied by our group. The molecular weight of the pure protein was measured as 56,230 Da with SDS-PAGE. The optimum temperature for the enzyme activity was 50 8C and it was most active at pH 6.0. Thermal deactivation was noticeable above 65 8C and the enzyme was highly stable below 60 8C. The influence of substrate or product concentration on the initial rate of CD production was studied and the kinetic parameters were determined. The enzyme showed cyclization activity on different raw and hydrolyzed starches and hydrolyzed cornstarch gave the highest activity. # 2007 Elsevier Ltd. All rights reserved. Keywords: Cyclodextrin glycosyltransferase; CGTase; Cyclodextrin; Alkalophylic bacillus; Activation energy; Deactivation energy
1. Introduction One of the areas of importance in biotechnology and bioengineering is the phenomenon of molecular complexation, which is useful in selecting, separation and solubilization of various bio-molecules. Cyclodextrins (CDs) are advantageous molecular complexation agents [1]. CDs are cyclic oligosaccharides consisting of six (a-CD), seven (b-CD), eight (g-CD) or more glucopyranosyl units linked by a-(1,4) bonds. They are also known as cycloamylose, cyclomaltose and Schardinger dextrins [2]. They are produced by reacting liquefied starch with the enzyme cyclodextrin glycosyltransferase (CGTase). Usually, a mixture of CDs is formed and their concentration ratios depend on the enzyme source. Under normal conditions, b-CD is produced in a greater amount, in some cases a-CD, but g-CD is seldom produced in high yields. Depending on which CD a-, b- or g-CD is the main product, the enzyme is called an a-, b- or g-CGTase,
* Corresponding author. Tel.: +55 44 3261 4301; fax: +55 44 3261 4119. E-mail address: [email protected] (G. Matioli). 1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2007.07.007
respectively [3–5]. The CGTases from most microorganisms are extracellular enzymes [6]. The CD molecules have a unique structure with a hydrophobic cavity and a hydrophilic surface. Due to this feature, they can form inclusion complexes with a wide variety of solid, liquid and gaseous compounds [2,7]. In the complexes, a guest molecule is held within the cavity of the CD host molecule. Complex formation is a dimensional, geometrically limited fit, between host cavity and guest molecule [1]. Generally, hydrophobic molecules or even hydrophilic ones have greater affinity for the CD cavity when they are in a water solution [8]. Moreover, the molecular encapsulation changes the physical and chemical properties of the included molecules. Accordingly, the use of CDs is largely increasing in the pharmaceutical, agricultural, chemical, cosmetic and food industries [4–6]. In addition to the common use of CDs, they are used in separation science because they allow discriminating between positional isomers, functional groups, homologues and enantiomers. This property makes them a useful agent for a wide variety of separations [1]. The CGTase is usually a monomeric enzyme, with a molecular weight of the order of 74,500 Da that presents a
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sequence of amino acids which reveals a structural similarity to the enzyme a-amylase. Besides the cyclization reaction, which forms the CDs, CGTase catalyzes the coupling reaction and the disproportionation of linear maltodextrins [9,10], which opens the ring structure and exchanges segments of maltodextrin linear chains, respectively. Since the discovery of CGTase from B. macerans in 1903, the production of the CGTase enzyme has been studied in several lineages of bacteria, such as B. elaterium, B macerans, Klebsiella pneumoniae, and B. stearothermophillus [3,11]. Bacillus species constitute the major producers of industrial enzymes. Various applications (e.g., in detergents, pulp and paper industry) have prompted the isolation of strains from a variety of alkaline environments, as a source of enzymes with suitable activities. More than 10 alkalophylic Bacillus species have been identified so far [12]. The enzymes obtained from these microorganisms present different properties, such as thermal stability, optimal pH, molecular weight, and capacity for the formation of CDs [11]. Enzymes having the capability of producing predominantly a particular type of CD can decrease downstream purification costs and hence are commercially valuable [4]. We have isolated a microorganism, which produces a new CGTase that makes predominantly b-CD from starch. Its free cell culture supernatant dextrinizing activity was greater than that of the enzyme of Bacillus firmus, strain 37, previously studied by our group. In this paper, we report the production and properties of this new enzyme from an alkalophylic bacillus. 2. Materials and methods 2.1. Enzyme production and purification CGTase enzyme was obtained from Bacillus firmus, strain 7B, which was isolated from a Brazilian soil of oat culture. An alkaline medium, pH 10.3, with the following composition was used for CGTase production: 2.0% (w/v) soluble starch; 0.5% (w/v) polypeptone; 0.5% (w/v) yeast extract; 0.1% (w/v) K2HPO4; 0.02% (w/v) MgSO47H2O; 1.0% (w/v) Na2CO3. The microorganism was inoculated in 5 L of a liquid medium and the cultivation proceeded at 37 8C for 5 days, with stirring at 150 rpm. Thus, the cells and insoluble material were removed by centrifugation at 8800 g for 15 min at 4 8C. The cell-free supernatant was mixed with ammonium sulfate (80% saturation) and left in a refrigerator overnight. A second centrifugation at 8800 g for 30 min was conducted and the precipitate was dissolved in 10 mM Tris–HCl buffer, pH 8.0. The enzyme was further purified by biospecific affinity column chromatography using Sepharose 6B gel and b-CD as ligand [13,14]. Protein concentration was determined by the method of Bradford [15], using bovine serum albumin as standard. The molecular mass of CGTase was estimated under denaturing conditions by electrophoresis. SDS-PAGE was performed on 12.5% polyacrilamide gel [16]. The following standards protein molecular weight markers (SDS7B2 kit-Sigma) were used: triosephosphate isomerase (26,600 Da), fumarase (48,500 Da), pyruvate kinase (58,000 Da), lactic dehydrogenase (84,000 Da), lactoferrin (90,000 Da), b-galactosidase (116,000 Da) and a2-macroglobulin (180,000 Da). Protein bands were visualized by Coomassie blue dye.
2.2. CGTase activity assay CGTase activity (or cyclization activity) was measured as a function of the b-CD production rate. The b-CD produced in the assay was determined by the method of dye extinction, i.e. color fading at 550 nm that happens
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after complexation of phenolphthalein with b-CD [17]. One unit of activity (U) corresponds to the amount of CGTase that liberates 1 mmol of b-CD/min at the reaction conditions. The activity assay conditions consisted of a substrate solution containing 1% (w/v) maltodextrin (D.E. 10) in 50 mM Tris–HCl buffer and 5 mM CaCl2, pH 8.0, at 50 8C. For several different tests in this work, pH 8.0 was chosen, because the enzyme was more stable at this pH. The diluted buffer solutions of enzyme and substrate were separately heated at 50 8C. In six test tubes, 1 mL of substrate solution was placed and soon after 1 mL of enzyme solution was added. The tube was agitated and it was incubated for 30 min at 50 8C, and one tube was removed every 5 min. CGTase was inactivated by heating the tube in boiling water for 10 min [13,14]. The reaction time and enzyme dilution were selected by allowing a linear relationship between the concentration of the formed CD and time, seeking to reduce the effect of product inhibition and reagent depletion, according to the criteria established by the method of the initial velocities [18]. The control was a reaction time of 0 min, and in this case, the CGTase was deactivated first by boiling and then, the substrate solution was added. In the same conditions as above, the CGTase activity was also measured replacing maltodextrin by cornstarch, cassava starch and potato starch raw and partially hydrolyzed up to D.E. 10. The preparation of hydrolyzed starch solutions followed the methodology of Lima et al. [19] using a-amylase. CGTase activity in the culture medium was also assayed by the method of successive dilutions of the supernatant after centrifugation and then, reaction of the diluted samples with starch. The amount of CD produced in this case was assayed through precipitation with TCE (trichloroethylene), i.e., the CD-TCE method [20]. For the assay of the CGTase activity for producing different CDs, a 1:50 dilution of the stock enzyme was used. The concentration of different CDs produced was determined by HPLC under the following conditions: aminopropylsilane column, acetonitrile and filtered distilled water (65:35) as the mobile phase, flow rate of 0.7 mL/min and refractive index detector. The coupling activity was determined by measuring the disappearance of bCD in the presence of glucose. Appropriately diluted enzyme was incubated with a mixture of 1% (w/v) b-CD and 1% (w/v) glucose in 50 mM Tris–HCl buffer and 5 mM CaCl2, pH 8.0, at 50 8C for 30 min. The concentration of b-CD was determined by the phenolphthalein colorimetric method [17]. One unit of coupling activity is defined as the amount of enzyme that can convert 1 mmol of b-CD/min. The disproportionation activity was determined by measuring conversion of maltose to larger maltooligosacharides oligomers. Appropriately diluted enzyme was incubated with a mixture of 1% (w/v) maltose in 50 mM Tris– HCl buffer and 5 mM CaCl2, pH 8.0, at 50 8C for 10 min. The conversion of maltose to others malto-oligosaccharides was estimated by the HPLC method above described. One unit of disproportionation activity is defined as the amount of enzyme that can convert 1 mmol of maltose/min.
2.3. Effect of temperature and pH on CGTase stability 2.3.1. CGTase activity as function of temperature and pH The enzyme activity at different temperatures and pH was determined as given in Section 2.2. The only difference being that, instead of the usual value of 50 8C and pH 8.0, the temperature and pH were set to different desired values (pH 5–10 and temperature 35–70 8C). 2.3.2. Residual CGTase activity as function of temperature and reaction time CGTase residual activity was determined by the method of initial velocities [18], at the temperatures from 35 to 85 8C with an interval of 5 8C. A solution of 10% (w/v) maltodextrin, pH 8.0, in 50 mM Tris–HCl buffer and 5 mM CaCl2, was prepared to dilute the enzyme. For each selected temperature, the diluted enzyme solution was incubated for 240 min. Every 40 min, a sample of 1 mL was taken, diluted 1:50, and added to six test tubes containing 1 mL of a 10% substrate solution, and the residual activity was determined at 50 8C. The substrate solution 10% (w/v) was prepared in 50 mM Tris–HCl buffer and 5 mM CaCl2, pH 8.0. The CDs produced from the residual activity test were determined by the colorimetric methods, according to the section on CGTase activity assay (Section 2.2).
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Table 1 Specific enzyme activity of the new CGTase for different substrates Substrate
Activity (U/mg, 1% substrate, pH 8.0, 50 8C)
Maltodextrin
79.8
Cornstarch Raw
Hydrolyzed
Raw
Hydrolyzed
Raw
97.1
0
90.9
6.4
90.3
12.3
2.4. Influence of substrate concentration on CGTase activity The substrate solutions were made with maltodextrin (D.E. 10) (average mol. wt. of 1672 kD; Fluka Chemie AG, Buchs, Switzerland). The influence of substrate concentration was measured in relation to the production of b-CD, and this allowed determining the occurrence of substrate inhibition. The test was carried out at 50 8C and pH 8.0. The substrate solutions were prepared in the following concentrations: 0.05; 0.1; 0.25; 0.5; 1.0; 1.5; 2.5; 5.0; 7.5; 10.0; 12.5; 15.0; 20.0; 25.0 and 30.0% (w/v). Enzyme dilution used was 1:50 of the stock enzyme. The tests were carried out similarly as described under CGTase activity assay in Section 2.2. From the data on enzymatic activity as a function of substrate concentration up to 0.003 mol/L, the kinetic parameters Km and Vmax were obtained. The data was fitted to a standard Michaelis–Menten model using nonlinear least squares regression [21]. Keeping these values of Km and Vm constant, the whole set of data was used to determine the substrate inhibition constant, Ks, using the Haldane kinetic model [21]: V max S K m þ S þ ðS2 =K s Þ
Cassava starch
Hydrolyzed
2.3.3. Residual CGTase activity as function of pH and reaction time The pH stability of the enzyme was measured at pH 5.0 to 10.0 using disodium phosphate–citric acid buffer for pH 5–7 and boric acid–potassium chloride buffer for pH 8–10, 10% (w/v) maltodextrin and 50 8C. For each selected pH, the enzyme was prepared in the substrate solution and incubated for 240 min. Every 40 min, a sample of 1 mL was taken, diluted 1:50, and added to six test tubes containing 1 mL of a 10% substrate solution, and the residual activity was determined at 50 8C.
V¼
Potato Starch
(1)
where V is the initial substrate reaction rate (mol b-CD/(L h)), Vmax the maximum velocity, Km the Michaelis–Menten constant (mol/L), S the substrate concentration (mol/L) and Ks the substrate inhibition constant (mol/L).
2.5. Influence of products on CGTase activity The influence of the products was determined in tests in which a 1% (w/v) maltodextrin solution was prepared having in addition, one of the three different CDs (a-, b- and g-CD). The CGTase was diluted 1:50 and its activity was measured as described before. The exogenous CD concentrations at the beginning of these tests were 0.125, 0.25, or 0.5% (w/v).
3. Results and discussion 3.1. CGTase activity The enzyme eluted from the biospecific affinity column chromatography showed a 47-fold purification with a yield of 91.6%. The molecular weight of the enzyme was estimated to be 56,230 Da on the SDS-PAGE. The enzymatic activity of the CGTase from Bacillus firmus, strain 7B, was determined by colorimetric determination of the b-CD produced in the assay and by trichloroethylene precipitation (CD-TCE method) (Section 2.2). The activity observed in the cell-free supernatant was 0.1864 mmol of b-CD/(min mL) and up to a dilution of 29 it gave a precipitate by the CD-TCE method. The new
microorganism produced a more active cell-free supernatant medium than the CGTase from Bacillus firmus strain 37, previously studied by our group, which had an activity of 0.1155 mmol of b-CD/(min mL) and gave a precipitate up to a dilution of 28 by the CD-TCE method [14]. The action of this CGTase on different hydrolyzed and raw starches was investigated (Table 1). The enzyme could yield bCD from all tested hydrolyzed starches and cornstarch was the best substrate. However, when used raw, cornstarch could not be converted to b-CD, while cassava starch was the best substrate as raw starch. In contrast, for the CGTase from Klebsiella pneumoniae AS-22 [4], cornstarch was the worst substrate when used gelatinized and it produced very little or no b-CD when used raw. It is known that physical treatment opens up the structure of starch granules, which become susceptible to CGTase action, whereas raw starch as such should be relatively inaccessible to the enzyme [4]. This CGTase enzyme produced mainly b-CD from maltodextrin. The ratio of a-, b- and g-CD, in this study, was 20:59:21, respectively, after 30 min incubation in 50 mM Tris– HCl buffer and 5 mM CaCl2, pH 8.0, at 50 8C, 1% substrate. The different specific enzyme activities of the pure protein for cyclization, disproportionation and coupling were determined as described in Section 2.2. The disproportionation activity (341.3 U/mg) for this enzyme was the highest followed by the cyclization activity (79.8 U/mg) and coupling activity (46.1 U/ mg). The same order of activities was observed for the CGTase from Klebsiella pneumoniae AS-22 [4], but the activities were higher. Martins and Hatti-Kaul [22] have also reported different CGTase activities from Bacillus agaradhaerens LS-3C. For this enzyme the cyclization activity was higher, but all other activities were lower than the CGTase activities from this work. 3.2. Effect of pH and temperature on CGTase activity Fig. 1 shows the CGTase from Bacillus firmus, strain 7B, to be optimally active at pH 6.0, giving 0.7268 mmol of b-CD/ (min mL). However, the enzyme almost lost its activity below pH 5.0 and above pH 10.0. The value of the optimum pH obtained in this work is in accordance with the range of pH reported in literature. The optimum pH varies according to the microorganism species that produce the CGTase. In the literature, the range of optimum pH is quite wide, varying from 4.0, according the CGTase studied by Yu et al. [23], to 12, according the CGTase studied by Horikoshi [24]. The production of b-CD as a function of temperature was measured according to Section 2.3.1 and showed maximum activity at 50 8C. The enzyme was not very active at 70 8C or above (Fig. 2). For the industrial conversion of starch to CDs, it would be desirable an enzyme active at higher temperatures
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Fig. 1. Bacillus firmus, strain 7B, CGTase activity for the production of b-CD as a function of pH. Conditions: substrate is 1% (w/v) maltodextrin, in 50 mM buffer and 5 mM CaCl2, 50 8C. Buffers: disodium phosphate–citric acid buffer for pH 4.0–7.0 and boric acid–potassium chloride buffer for pH 8.0–10.0, Section 2.3.1.
Fig. 3. Arrhenius plot of the activity for the production of b-CD as a function of the inverse of the absolute temperature for the CGTase from Bacillus firmus, strain 7B. Conditions: substrate is 1% (w/v) maltodextrin, in 50 mM Tris–HCl buffer and 5 mM CaCl2, pH 8.0, Section 2.3.1.
than observed in this work, because this would reduce the risk of microbial contamination. In the literature, few enzymes are reported with optimum activity at temperatures above 60 8C, as the CGTases from Bacillus amyloquefaciens [23], Bacillus stearothermophilus [25] and Thermoanaerobacter sp. [17], with maximal activity at 70, 80 and 85 8C, respectively. The effect of the temperature on enzymatic activity was further analyzed, using the Arrhenius equation by plotting activity (Ae expressed in mmol of b-CD/(min mL)) against the inverse of temperature (1/T expressed in K1), which were further adjusted to the equation Ae ¼ QeðEa =RTÞ (r = 0.998 for T 50 8C). The activation energy (Ea) was determined as 9.4 kcal/mol (Fig. 3). The reported Ea of CGTases from Bacillus sp. [26], Bacillus agaradhaerens [12] and Bacillus macerans [27] are 8.1 kcal/mol, 17.0 kcal/mol and 27.4 kcal/mol, respectively. Therefore, the Ea of the CGTases depends on their source.
The data presented in Fig. 4 show that the enzyme from Bacillus firmus, strain 7B, retained almost 100% of its initial
activity in a wide range of temperatures, between 35 and 60 8C for a reaction period of 4 h. Comparison with other alkalophylic bacilli CGTases revealed that this range is practically the same [12]. At 65 8C and above, the enzyme presented increasingly higher thermal deactivation, particularly for 75 8C, where it did not show any residual activity after 240 min. The deactivation energy of the enzyme, in the temperature range between 65 and 85 8C, was determined with the values of thermal inactivation coefficient (Kd) obtained by the method described by Matioli et al. [9]. Fig. 5 shows the slope of the adjusted straight line that correlates the natural logarithm of Kd with the inverse of the absolute temperature (T) and the resulting deactivation energy is 37.8 kcal/mol. The half-life of the CGTase enzyme was calculated by the exponential decay model also described by Matioli et al. [9]. For the enzyme incubated in a solution of maltodextrin 10% (w/v), pH 8.0, the half-life was higher than 20 h for temperatures lower than 60 8C, and dropped to just 30 min at 80 8C. These results are better than that obtained for the Bacillus firmus strain 37 CGTase, which showed a half-life lower than 12 h for temperatures lower than 60 8C [9]. In addition, it can be
Fig. 2. Bacillus firmus, strain 7B, CGTase activity for the production of b-CD as a function of temperature. Conditions: substrate is 1% (w/v) maltodextrin, in 50 mM Tris–HCl buffer and 5 mM CaCl2, pH 8.0, Section 2.3.1.
Fig. 4. Bacillus firmus, strain 7B, CGTase activity for the production of b-CD as a function of time. Conditions: substrate is 10% (w/v) maltodextrin, in 50 mM Tris–HCl buffer and 5 mM CaCl2, pH 8.0, Section 2.3.2.
3.3. Effect of temperature and pH on CGTase stability
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Fig. 5. Arrhenius plot of the residual activity for the production of b-CD as a function of the inverse of the absolute temperature for the CGTase from Bacillus firmus, strain 7B. Conditions: substrate is 1% (w/v) maltodextrin, in 50 mM Tris–HCl buffer and 5 mM CaCl2, pH 8.0, Section 2.3.2.
calculated that at 60 8C and 24 h of reaction time, the CGTase from bacillus strain 7B had a deactivation of 56%, while for the enzyme from Bacillus firmus strain 37 it was 73%. Nonetheless, for long reaction periods, the results from this work do not warrant the use of the new enzyme for temperatures above 60 8C, in 1% substrate solution, because the enzyme deactivates faster in low concentration substrate solutions. The maximum stability of the enzyme occurred at pH 8.0 (Fig. 6). This pH was selected for all the enzyme characterization tests.
Fig. 7. Initial velocity of b-CD production (V) by the CGTase of Bacillus firmus, strain 7B as a function of substrate concentration (S), and model comparison. Conditions were: pH 8.0, 50 8C, 50 mM Tris–HCl buffer, 5 mM CaCl2, and the substrate was maltodextrin (DE 10).
3.5. Kinetic parameters
Fig. 7 shows data at the initial rate of reaction (V) as a function of the substrate concentration. The substrate inhibition is clearly seen because the initial rate of CD production at the reaction beginning increased until a maltodextrin concentration of 0.0029 mol/L was reached, and then, decreased with further increase in substrate concentration. The enzyme produced by Bacillus firmus strain 37 showed similar results [8].
The Vmax and Km values obtained by maltodextrin as substrate were 0.0706 mol of (-CD/(L h) and 0.0011 mol/L, respectively. Fig. 8 shows the Hanes plots used with points of low substrate concentration to determine Vmax and Km. The Km value (0.0011 mol/L) of this work is smaller than the Km from Bacillus firmus strain 37 CGTase (0.0033 mol/L) [8]. Considering that the Km parameter is correlated to the affinity of the enzyme for its substrate [18], the low value observed in this work indicates that the enzyme has comparatively higher affinity for the substrate. In the literature, some Km values measured for CGTase are lower than the value obtained in this work (e.g., Bacillus sp., 0.00026 mol/L [26]; Bacillus sphaericus, 0.00038 mol/L [28]), but many values are higher (e.g., Bacillus circulans, 0.00502 mol/L [29]; Bacillus agaradhaerens, 0.01076 mol/L [12]; Bacillus macerans, 0.00265 mol/L [27]; Bacillus coagulans, 0.0028 mol/L [28]). The substrate inhibition constant, Ks, calculated as described in Section 2.4 gave 0.0294 mol/L showing that the new CGTase enzyme is more inhibited by high concentrations of the
Fig. 6. Bacillus firmus, strain 7B, CGTase activity for the production of b-CD as a function of pH and reaction time. Conditions: substrate is 1% (w/v) maltodextrin, in 50 mM buffer and 5 mM CaCl2, 50 8C. Buffers: disodium phosphate–citric acid buffer for pH 5.0–7.0 and boric acid–potassium chloride buffer for pH 8.0–10.0, Section 2.3.3.
Fig. 8. Determination of Km and Vmax by the Hanes method for the CGTase from Bacillus firmus, strain 7B. Only the points shown in Fig. 6 with low substrate concentration were used. Experimental conditions were as in Fig. 6.
3.4. Influence of substrate concentration on CGTase activity
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deactivation of the enzyme was smaller at 60 8C and 24 h of reaction time; enzyme affinity for the substrate is comparatively higher.
Fig. 9. Influence of products a-, b- and g-CD, on the activity of Bacillus firmus strain 7B CGTase for production of (-CD. Conditions were: pH 8.0, 50 8C, 50 mM Tris–HCl buffer, 5 mM CaCl2, and the substrate was maltodextrin 1% (w/v) (DE 10). The exogenous cyclodextrin concentration is given over the bars.
We have studied the CGTase from Bacillus firmus strain 37 since 1994, but now we have got a new strain with improved characteristics for production of the enzyme. Thus, the method that we have been using to select strains that are good producers of CGTase [14] is valid and can lead to improved results. After obtaining good strains for producing CGTase, the next steps for improving CGTase production are, according to Martins and Kaul [12], and we agree with them: ‘‘cloning and expression of the CGTase gene are important for making changes and improving the critical features of the enzyme’’. Acknowledgment
substrate than the CGTase from Bacillus firmus strain 37 (Ks = 0.0928 mol/L) [8]. Other known CGTases have the following Ks values: Bacillus macerans, 5.89 mg/mL [30]; Bacillus macerans, 0.01 mg/mL [31]. In these works the substrate molar mass was not given. 3.6. Influence of products on CGTase activity The influence of the reaction products a-, b- and g-CD on the cyclization activity is shown at Fig. 9. The enzyme showed loss of activity in the presence of increasing a-, b- and g-CD concentrations but its activity was higher in the presence of low a-CD concentration. The higher activity observed with added a-CD may not be enzyme activation, but indicate that this CD is serving as substrate for producing b-CD. The CGTase activity from Bacillus agaradhaerens [12] had a slight inhibitory effect from a- and g-CD and a slight activation effect by b-CD. These results indicate that the influence of reaction products on CGTase activity varies according to the source of enzyme, even though the inhibitory effect is more common than the activation effect. Inhibition by b- and g-CD has been reported for the CGTases from Bacillus firmus strain 37 [8] and Bacillus autolyticus [32]. 4. Conclusions The new CGTase from Bacillus firmus, strain 7B, has been reported and the results obtained in this work were compared with those from Bacillus firmus strain 37 CGTase, previously studied by our group [5,8,9,14]. Some characteristics of these enzymes showed to be close (enzymatic activity as a function of temperature and pH, thermal stability, activation and deactivation energy, influence of products), but for others characteristics, the new CGTase was shown to be better, for example: observed enzyme activity in the cell-free supernatant was higher as determined by colorimetric and CD-TCE methods; enzyme half-life showed to be higher for temperatures lower than 60 8C;
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[16] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–5. [17] Tardioli EW, Zanin GM, Moraes FF. Characterization of Thermoanaerobacter cyclomaltodextrin glucanotransferase immobilized on glyoxilagarose. Enzyme Microbial Technol 2006;39:1270–8. [18] Dixon M, Webb EC. Enzymes. London: Longman Group Limited; 1979. p.1116. [19] Lima HOS, Moraes FF, Zanin GM. Beta-cyclodextrin production by simultaneous fermentation and cyclization. Appl Biochem Biotechnol 1998;70–72:789–804. [20] Nomoto M, Shew DC, Chen SJ, Yen CWL, Yang CP. Cyclodextrin glucanotransferase from alkalophilic bacteria of Twain. Agric Biol Chem 1984;48:1337–8. [21] Segel IH. Enzyme kinetics. New York: John Wiley & Sons; 1975. p. 957. [22] Martins RF, Hatti-Kaul R. Bacillus agaradhaerens LS-3C cyclodextrin glycosyltransferase: activity and stability features. Enzyme Microbial Technol 2003;33:819–27. [23] Yu EKC, Aoki H, Misawa M. Specific alpha-cyclodextrin production by a novel thermostable cyclodextrin glycosyltransferase. Appl Microbiol Biotechnol 1988;28:377–9. [24] Horikoshi K. Production of alkaline enzymes by alkalophilic microorganism. Part I. Alkaline protease produced by Bacillus n8 221. Agric Biol Chem 1971;35:1407–14. [25] Chung H-J, Yoon S-H, Kim M-J, Kweon K-S, Lee I-W, Kim J-W, Oh B-H, Lee H-S, Spiridonova VA, Park KH. Characterization of a thermostable
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cyclodextrin glucanotransferase isolated from Bacillus stearothermophilus ET1. J Agric Food Chem 1998;46:952–9. Hamon V, Moraes FF. Etude Preliminaire a L’immobilsation de L’enzyme CGTase WACKER. In Research Report, Laboratoire de Tecnologie Enzymatique. Compie`gne, France: Universite´ de Tecnologie de Compie`gne; 1990. De Pinto JA, Campbell LL. Purification and properties of the cyclodextrinase of Bacillus macerans. Biochemistry 1968;7:114–20. Oguma T, Kikuchi M, Mizusawa K. Purification and some properties of cyclodextrin-hydrolyzing enzyme from Bacillus sphaericus. Biochim Biophys Acta 1990;1036:1–5. Villette J, Bouquelet S, Leleu JB, Sicard PJ. Isolation and mechanism of action of the cycloglucosyl transferase from Bacillus circulans. In: Ducheˆne D, editor. Minutes of the 5th International Symposium on Cyclodextrins, Paris: Editions Sante´. 1990. p. 32–8. Lee KCP, Tao BY. A kinetic-study of cyclodextrin glycosyltransferase – substrate and product inhibitions. Biotechnol Appl Biochem 1995;21: 111–21. Arya SK, Srivastava SK. Kinetics of immobilized cyclodextrin gluconotransferase produced by Bacillus macerans ATCC 8244. Enzyme Microbial Technol 2006;39:507–10. Tomita K, Kaneda M, Kawamura K, Nakanishi K. Purification and properties of a cyclodextrin glucanotransferase from Bacillus autolyticus 11149 and selective formation of b-cyclodextrin. J Ferm Bioeng 1993;75: 89–92.
Production and characterization of a new cyclodextrin glycosyltransferase from Bacillus firmus isolated from Brazilian soil Cristiane Moriwaki a, Glauciane L. Costa a, Rubia Pazzetto a, Gisella M. Zanin b, Fla´vio F. Moraes b, Ma´rcia Portilho a, Graciette Matioli a,* a
Pharmacy and Pharmacology Department, State University of Maringa´, Av. Colombo, 5790 - 87020-900 Maringa´, PR, Brazil b Chemical Engineering Department, State University of Maringa´, Av. Colombo, 5790 - 87020-900 Maringa´, PR, Brazil Received 30 June 2006; received in revised form 3 May 2007; accepted 11 July 2007
Abstract A new CGTase was obtained from Bacillus firmus, strain 7B, isolated from oat soil culture, using a high alkaline pH medium containing 1% Na2CO3. The enzyme was characterized in soluble form, for pH 5–11, temperature from 30 to 85 8C, using a 1% maltodextrin substrate solution and appropriate buffers. It produced mainly b-CD and the cell-free supernatant had a precipitating activity measured by the trichloroethylene method that is a 100-fold greater than that of the enzyme of Bacillus firmus, strain 37, previously studied by our group. The molecular weight of the pure protein was measured as 56,230 Da with SDS-PAGE. The optimum temperature for the enzyme activity was 50 8C and it was most active at pH 6.0. Thermal deactivation was noticeable above 65 8C and the enzyme was highly stable below 60 8C. The influence of substrate or product concentration on the initial rate of CD production was studied and the kinetic parameters were determined. The enzyme showed cyclization activity on different raw and hydrolyzed starches and hydrolyzed cornstarch gave the highest activity. # 2007 Elsevier Ltd. All rights reserved. Keywords: Cyclodextrin glycosyltransferase; CGTase; Cyclodextrin; Alkalophylic bacillus; Activation energy; Deactivation energy
1. Introduction One of the areas of importance in biotechnology and bioengineering is the phenomenon of molecular complexation, which is useful in selecting, separation and solubilization of various bio-molecules. Cyclodextrins (CDs) are advantageous molecular complexation agents [1]. CDs are cyclic oligosaccharides consisting of six (a-CD), seven (b-CD), eight (g-CD) or more glucopyranosyl units linked by a-(1,4) bonds. They are also known as cycloamylose, cyclomaltose and Schardinger dextrins [2]. They are produced by reacting liquefied starch with the enzyme cyclodextrin glycosyltransferase (CGTase). Usually, a mixture of CDs is formed and their concentration ratios depend on the enzyme source. Under normal conditions, b-CD is produced in a greater amount, in some cases a-CD, but g-CD is seldom produced in high yields. Depending on which CD a-, b- or g-CD is the main product, the enzyme is called an a-, b- or g-CGTase,
* Corresponding author. Tel.: +55 44 3261 4301; fax: +55 44 3261 4119. E-mail address: [email protected] (G. Matioli). 1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2007.07.007
respectively [3–5]. The CGTases from most microorganisms are extracellular enzymes [6]. The CD molecules have a unique structure with a hydrophobic cavity and a hydrophilic surface. Due to this feature, they can form inclusion complexes with a wide variety of solid, liquid and gaseous compounds [2,7]. In the complexes, a guest molecule is held within the cavity of the CD host molecule. Complex formation is a dimensional, geometrically limited fit, between host cavity and guest molecule [1]. Generally, hydrophobic molecules or even hydrophilic ones have greater affinity for the CD cavity when they are in a water solution [8]. Moreover, the molecular encapsulation changes the physical and chemical properties of the included molecules. Accordingly, the use of CDs is largely increasing in the pharmaceutical, agricultural, chemical, cosmetic and food industries [4–6]. In addition to the common use of CDs, they are used in separation science because they allow discriminating between positional isomers, functional groups, homologues and enantiomers. This property makes them a useful agent for a wide variety of separations [1]. The CGTase is usually a monomeric enzyme, with a molecular weight of the order of 74,500 Da that presents a
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sequence of amino acids which reveals a structural similarity to the enzyme a-amylase. Besides the cyclization reaction, which forms the CDs, CGTase catalyzes the coupling reaction and the disproportionation of linear maltodextrins [9,10], which opens the ring structure and exchanges segments of maltodextrin linear chains, respectively. Since the discovery of CGTase from B. macerans in 1903, the production of the CGTase enzyme has been studied in several lineages of bacteria, such as B. elaterium, B macerans, Klebsiella pneumoniae, and B. stearothermophillus [3,11]. Bacillus species constitute the major producers of industrial enzymes. Various applications (e.g., in detergents, pulp and paper industry) have prompted the isolation of strains from a variety of alkaline environments, as a source of enzymes with suitable activities. More than 10 alkalophylic Bacillus species have been identified so far [12]. The enzymes obtained from these microorganisms present different properties, such as thermal stability, optimal pH, molecular weight, and capacity for the formation of CDs [11]. Enzymes having the capability of producing predominantly a particular type of CD can decrease downstream purification costs and hence are commercially valuable [4]. We have isolated a microorganism, which produces a new CGTase that makes predominantly b-CD from starch. Its free cell culture supernatant dextrinizing activity was greater than that of the enzyme of Bacillus firmus, strain 37, previously studied by our group. In this paper, we report the production and properties of this new enzyme from an alkalophylic bacillus. 2. Materials and methods 2.1. Enzyme production and purification CGTase enzyme was obtained from Bacillus firmus, strain 7B, which was isolated from a Brazilian soil of oat culture. An alkaline medium, pH 10.3, with the following composition was used for CGTase production: 2.0% (w/v) soluble starch; 0.5% (w/v) polypeptone; 0.5% (w/v) yeast extract; 0.1% (w/v) K2HPO4; 0.02% (w/v) MgSO47H2O; 1.0% (w/v) Na2CO3. The microorganism was inoculated in 5 L of a liquid medium and the cultivation proceeded at 37 8C for 5 days, with stirring at 150 rpm. Thus, the cells and insoluble material were removed by centrifugation at 8800 g for 15 min at 4 8C. The cell-free supernatant was mixed with ammonium sulfate (80% saturation) and left in a refrigerator overnight. A second centrifugation at 8800 g for 30 min was conducted and the precipitate was dissolved in 10 mM Tris–HCl buffer, pH 8.0. The enzyme was further purified by biospecific affinity column chromatography using Sepharose 6B gel and b-CD as ligand [13,14]. Protein concentration was determined by the method of Bradford [15], using bovine serum albumin as standard. The molecular mass of CGTase was estimated under denaturing conditions by electrophoresis. SDS-PAGE was performed on 12.5% polyacrilamide gel [16]. The following standards protein molecular weight markers (SDS7B2 kit-Sigma) were used: triosephosphate isomerase (26,600 Da), fumarase (48,500 Da), pyruvate kinase (58,000 Da), lactic dehydrogenase (84,000 Da), lactoferrin (90,000 Da), b-galactosidase (116,000 Da) and a2-macroglobulin (180,000 Da). Protein bands were visualized by Coomassie blue dye.
2.2. CGTase activity assay CGTase activity (or cyclization activity) was measured as a function of the b-CD production rate. The b-CD produced in the assay was determined by the method of dye extinction, i.e. color fading at 550 nm that happens
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after complexation of phenolphthalein with b-CD [17]. One unit of activity (U) corresponds to the amount of CGTase that liberates 1 mmol of b-CD/min at the reaction conditions. The activity assay conditions consisted of a substrate solution containing 1% (w/v) maltodextrin (D.E. 10) in 50 mM Tris–HCl buffer and 5 mM CaCl2, pH 8.0, at 50 8C. For several different tests in this work, pH 8.0 was chosen, because the enzyme was more stable at this pH. The diluted buffer solutions of enzyme and substrate were separately heated at 50 8C. In six test tubes, 1 mL of substrate solution was placed and soon after 1 mL of enzyme solution was added. The tube was agitated and it was incubated for 30 min at 50 8C, and one tube was removed every 5 min. CGTase was inactivated by heating the tube in boiling water for 10 min [13,14]. The reaction time and enzyme dilution were selected by allowing a linear relationship between the concentration of the formed CD and time, seeking to reduce the effect of product inhibition and reagent depletion, according to the criteria established by the method of the initial velocities [18]. The control was a reaction time of 0 min, and in this case, the CGTase was deactivated first by boiling and then, the substrate solution was added. In the same conditions as above, the CGTase activity was also measured replacing maltodextrin by cornstarch, cassava starch and potato starch raw and partially hydrolyzed up to D.E. 10. The preparation of hydrolyzed starch solutions followed the methodology of Lima et al. [19] using a-amylase. CGTase activity in the culture medium was also assayed by the method of successive dilutions of the supernatant after centrifugation and then, reaction of the diluted samples with starch. The amount of CD produced in this case was assayed through precipitation with TCE (trichloroethylene), i.e., the CD-TCE method [20]. For the assay of the CGTase activity for producing different CDs, a 1:50 dilution of the stock enzyme was used. The concentration of different CDs produced was determined by HPLC under the following conditions: aminopropylsilane column, acetonitrile and filtered distilled water (65:35) as the mobile phase, flow rate of 0.7 mL/min and refractive index detector. The coupling activity was determined by measuring the disappearance of bCD in the presence of glucose. Appropriately diluted enzyme was incubated with a mixture of 1% (w/v) b-CD and 1% (w/v) glucose in 50 mM Tris–HCl buffer and 5 mM CaCl2, pH 8.0, at 50 8C for 30 min. The concentration of b-CD was determined by the phenolphthalein colorimetric method [17]. One unit of coupling activity is defined as the amount of enzyme that can convert 1 mmol of b-CD/min. The disproportionation activity was determined by measuring conversion of maltose to larger maltooligosacharides oligomers. Appropriately diluted enzyme was incubated with a mixture of 1% (w/v) maltose in 50 mM Tris– HCl buffer and 5 mM CaCl2, pH 8.0, at 50 8C for 10 min. The conversion of maltose to others malto-oligosaccharides was estimated by the HPLC method above described. One unit of disproportionation activity is defined as the amount of enzyme that can convert 1 mmol of maltose/min.
2.3. Effect of temperature and pH on CGTase stability 2.3.1. CGTase activity as function of temperature and pH The enzyme activity at different temperatures and pH was determined as given in Section 2.2. The only difference being that, instead of the usual value of 50 8C and pH 8.0, the temperature and pH were set to different desired values (pH 5–10 and temperature 35–70 8C). 2.3.2. Residual CGTase activity as function of temperature and reaction time CGTase residual activity was determined by the method of initial velocities [18], at the temperatures from 35 to 85 8C with an interval of 5 8C. A solution of 10% (w/v) maltodextrin, pH 8.0, in 50 mM Tris–HCl buffer and 5 mM CaCl2, was prepared to dilute the enzyme. For each selected temperature, the diluted enzyme solution was incubated for 240 min. Every 40 min, a sample of 1 mL was taken, diluted 1:50, and added to six test tubes containing 1 mL of a 10% substrate solution, and the residual activity was determined at 50 8C. The substrate solution 10% (w/v) was prepared in 50 mM Tris–HCl buffer and 5 mM CaCl2, pH 8.0. The CDs produced from the residual activity test were determined by the colorimetric methods, according to the section on CGTase activity assay (Section 2.2).
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Table 1 Specific enzyme activity of the new CGTase for different substrates Substrate
Activity (U/mg, 1% substrate, pH 8.0, 50 8C)
Maltodextrin
79.8
Cornstarch Raw
Hydrolyzed
Raw
Hydrolyzed
Raw
97.1
0
90.9
6.4
90.3
12.3
2.4. Influence of substrate concentration on CGTase activity The substrate solutions were made with maltodextrin (D.E. 10) (average mol. wt. of 1672 kD; Fluka Chemie AG, Buchs, Switzerland). The influence of substrate concentration was measured in relation to the production of b-CD, and this allowed determining the occurrence of substrate inhibition. The test was carried out at 50 8C and pH 8.0. The substrate solutions were prepared in the following concentrations: 0.05; 0.1; 0.25; 0.5; 1.0; 1.5; 2.5; 5.0; 7.5; 10.0; 12.5; 15.0; 20.0; 25.0 and 30.0% (w/v). Enzyme dilution used was 1:50 of the stock enzyme. The tests were carried out similarly as described under CGTase activity assay in Section 2.2. From the data on enzymatic activity as a function of substrate concentration up to 0.003 mol/L, the kinetic parameters Km and Vmax were obtained. The data was fitted to a standard Michaelis–Menten model using nonlinear least squares regression [21]. Keeping these values of Km and Vm constant, the whole set of data was used to determine the substrate inhibition constant, Ks, using the Haldane kinetic model [21]: V max S K m þ S þ ðS2 =K s Þ
Cassava starch
Hydrolyzed
2.3.3. Residual CGTase activity as function of pH and reaction time The pH stability of the enzyme was measured at pH 5.0 to 10.0 using disodium phosphate–citric acid buffer for pH 5–7 and boric acid–potassium chloride buffer for pH 8–10, 10% (w/v) maltodextrin and 50 8C. For each selected pH, the enzyme was prepared in the substrate solution and incubated for 240 min. Every 40 min, a sample of 1 mL was taken, diluted 1:50, and added to six test tubes containing 1 mL of a 10% substrate solution, and the residual activity was determined at 50 8C.
V¼
Potato Starch
(1)
where V is the initial substrate reaction rate (mol b-CD/(L h)), Vmax the maximum velocity, Km the Michaelis–Menten constant (mol/L), S the substrate concentration (mol/L) and Ks the substrate inhibition constant (mol/L).
2.5. Influence of products on CGTase activity The influence of the products was determined in tests in which a 1% (w/v) maltodextrin solution was prepared having in addition, one of the three different CDs (a-, b- and g-CD). The CGTase was diluted 1:50 and its activity was measured as described before. The exogenous CD concentrations at the beginning of these tests were 0.125, 0.25, or 0.5% (w/v).
3. Results and discussion 3.1. CGTase activity The enzyme eluted from the biospecific affinity column chromatography showed a 47-fold purification with a yield of 91.6%. The molecular weight of the enzyme was estimated to be 56,230 Da on the SDS-PAGE. The enzymatic activity of the CGTase from Bacillus firmus, strain 7B, was determined by colorimetric determination of the b-CD produced in the assay and by trichloroethylene precipitation (CD-TCE method) (Section 2.2). The activity observed in the cell-free supernatant was 0.1864 mmol of b-CD/(min mL) and up to a dilution of 29 it gave a precipitate by the CD-TCE method. The new
microorganism produced a more active cell-free supernatant medium than the CGTase from Bacillus firmus strain 37, previously studied by our group, which had an activity of 0.1155 mmol of b-CD/(min mL) and gave a precipitate up to a dilution of 28 by the CD-TCE method [14]. The action of this CGTase on different hydrolyzed and raw starches was investigated (Table 1). The enzyme could yield bCD from all tested hydrolyzed starches and cornstarch was the best substrate. However, when used raw, cornstarch could not be converted to b-CD, while cassava starch was the best substrate as raw starch. In contrast, for the CGTase from Klebsiella pneumoniae AS-22 [4], cornstarch was the worst substrate when used gelatinized and it produced very little or no b-CD when used raw. It is known that physical treatment opens up the structure of starch granules, which become susceptible to CGTase action, whereas raw starch as such should be relatively inaccessible to the enzyme [4]. This CGTase enzyme produced mainly b-CD from maltodextrin. The ratio of a-, b- and g-CD, in this study, was 20:59:21, respectively, after 30 min incubation in 50 mM Tris– HCl buffer and 5 mM CaCl2, pH 8.0, at 50 8C, 1% substrate. The different specific enzyme activities of the pure protein for cyclization, disproportionation and coupling were determined as described in Section 2.2. The disproportionation activity (341.3 U/mg) for this enzyme was the highest followed by the cyclization activity (79.8 U/mg) and coupling activity (46.1 U/ mg). The same order of activities was observed for the CGTase from Klebsiella pneumoniae AS-22 [4], but the activities were higher. Martins and Hatti-Kaul [22] have also reported different CGTase activities from Bacillus agaradhaerens LS-3C. For this enzyme the cyclization activity was higher, but all other activities were lower than the CGTase activities from this work. 3.2. Effect of pH and temperature on CGTase activity Fig. 1 shows the CGTase from Bacillus firmus, strain 7B, to be optimally active at pH 6.0, giving 0.7268 mmol of b-CD/ (min mL). However, the enzyme almost lost its activity below pH 5.0 and above pH 10.0. The value of the optimum pH obtained in this work is in accordance with the range of pH reported in literature. The optimum pH varies according to the microorganism species that produce the CGTase. In the literature, the range of optimum pH is quite wide, varying from 4.0, according the CGTase studied by Yu et al. [23], to 12, according the CGTase studied by Horikoshi [24]. The production of b-CD as a function of temperature was measured according to Section 2.3.1 and showed maximum activity at 50 8C. The enzyme was not very active at 70 8C or above (Fig. 2). For the industrial conversion of starch to CDs, it would be desirable an enzyme active at higher temperatures
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Fig. 1. Bacillus firmus, strain 7B, CGTase activity for the production of b-CD as a function of pH. Conditions: substrate is 1% (w/v) maltodextrin, in 50 mM buffer and 5 mM CaCl2, 50 8C. Buffers: disodium phosphate–citric acid buffer for pH 4.0–7.0 and boric acid–potassium chloride buffer for pH 8.0–10.0, Section 2.3.1.
Fig. 3. Arrhenius plot of the activity for the production of b-CD as a function of the inverse of the absolute temperature for the CGTase from Bacillus firmus, strain 7B. Conditions: substrate is 1% (w/v) maltodextrin, in 50 mM Tris–HCl buffer and 5 mM CaCl2, pH 8.0, Section 2.3.1.
than observed in this work, because this would reduce the risk of microbial contamination. In the literature, few enzymes are reported with optimum activity at temperatures above 60 8C, as the CGTases from Bacillus amyloquefaciens [23], Bacillus stearothermophilus [25] and Thermoanaerobacter sp. [17], with maximal activity at 70, 80 and 85 8C, respectively. The effect of the temperature on enzymatic activity was further analyzed, using the Arrhenius equation by plotting activity (Ae expressed in mmol of b-CD/(min mL)) against the inverse of temperature (1/T expressed in K1), which were further adjusted to the equation Ae ¼ QeðEa =RTÞ (r = 0.998 for T 50 8C). The activation energy (Ea) was determined as 9.4 kcal/mol (Fig. 3). The reported Ea of CGTases from Bacillus sp. [26], Bacillus agaradhaerens [12] and Bacillus macerans [27] are 8.1 kcal/mol, 17.0 kcal/mol and 27.4 kcal/mol, respectively. Therefore, the Ea of the CGTases depends on their source.
The data presented in Fig. 4 show that the enzyme from Bacillus firmus, strain 7B, retained almost 100% of its initial
activity in a wide range of temperatures, between 35 and 60 8C for a reaction period of 4 h. Comparison with other alkalophylic bacilli CGTases revealed that this range is practically the same [12]. At 65 8C and above, the enzyme presented increasingly higher thermal deactivation, particularly for 75 8C, where it did not show any residual activity after 240 min. The deactivation energy of the enzyme, in the temperature range between 65 and 85 8C, was determined with the values of thermal inactivation coefficient (Kd) obtained by the method described by Matioli et al. [9]. Fig. 5 shows the slope of the adjusted straight line that correlates the natural logarithm of Kd with the inverse of the absolute temperature (T) and the resulting deactivation energy is 37.8 kcal/mol. The half-life of the CGTase enzyme was calculated by the exponential decay model also described by Matioli et al. [9]. For the enzyme incubated in a solution of maltodextrin 10% (w/v), pH 8.0, the half-life was higher than 20 h for temperatures lower than 60 8C, and dropped to just 30 min at 80 8C. These results are better than that obtained for the Bacillus firmus strain 37 CGTase, which showed a half-life lower than 12 h for temperatures lower than 60 8C [9]. In addition, it can be
Fig. 2. Bacillus firmus, strain 7B, CGTase activity for the production of b-CD as a function of temperature. Conditions: substrate is 1% (w/v) maltodextrin, in 50 mM Tris–HCl buffer and 5 mM CaCl2, pH 8.0, Section 2.3.1.
Fig. 4. Bacillus firmus, strain 7B, CGTase activity for the production of b-CD as a function of time. Conditions: substrate is 10% (w/v) maltodextrin, in 50 mM Tris–HCl buffer and 5 mM CaCl2, pH 8.0, Section 2.3.2.
3.3. Effect of temperature and pH on CGTase stability
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Fig. 5. Arrhenius plot of the residual activity for the production of b-CD as a function of the inverse of the absolute temperature for the CGTase from Bacillus firmus, strain 7B. Conditions: substrate is 1% (w/v) maltodextrin, in 50 mM Tris–HCl buffer and 5 mM CaCl2, pH 8.0, Section 2.3.2.
calculated that at 60 8C and 24 h of reaction time, the CGTase from bacillus strain 7B had a deactivation of 56%, while for the enzyme from Bacillus firmus strain 37 it was 73%. Nonetheless, for long reaction periods, the results from this work do not warrant the use of the new enzyme for temperatures above 60 8C, in 1% substrate solution, because the enzyme deactivates faster in low concentration substrate solutions. The maximum stability of the enzyme occurred at pH 8.0 (Fig. 6). This pH was selected for all the enzyme characterization tests.
Fig. 7. Initial velocity of b-CD production (V) by the CGTase of Bacillus firmus, strain 7B as a function of substrate concentration (S), and model comparison. Conditions were: pH 8.0, 50 8C, 50 mM Tris–HCl buffer, 5 mM CaCl2, and the substrate was maltodextrin (DE 10).
3.5. Kinetic parameters
Fig. 7 shows data at the initial rate of reaction (V) as a function of the substrate concentration. The substrate inhibition is clearly seen because the initial rate of CD production at the reaction beginning increased until a maltodextrin concentration of 0.0029 mol/L was reached, and then, decreased with further increase in substrate concentration. The enzyme produced by Bacillus firmus strain 37 showed similar results [8].
The Vmax and Km values obtained by maltodextrin as substrate were 0.0706 mol of (-CD/(L h) and 0.0011 mol/L, respectively. Fig. 8 shows the Hanes plots used with points of low substrate concentration to determine Vmax and Km. The Km value (0.0011 mol/L) of this work is smaller than the Km from Bacillus firmus strain 37 CGTase (0.0033 mol/L) [8]. Considering that the Km parameter is correlated to the affinity of the enzyme for its substrate [18], the low value observed in this work indicates that the enzyme has comparatively higher affinity for the substrate. In the literature, some Km values measured for CGTase are lower than the value obtained in this work (e.g., Bacillus sp., 0.00026 mol/L [26]; Bacillus sphaericus, 0.00038 mol/L [28]), but many values are higher (e.g., Bacillus circulans, 0.00502 mol/L [29]; Bacillus agaradhaerens, 0.01076 mol/L [12]; Bacillus macerans, 0.00265 mol/L [27]; Bacillus coagulans, 0.0028 mol/L [28]). The substrate inhibition constant, Ks, calculated as described in Section 2.4 gave 0.0294 mol/L showing that the new CGTase enzyme is more inhibited by high concentrations of the
Fig. 6. Bacillus firmus, strain 7B, CGTase activity for the production of b-CD as a function of pH and reaction time. Conditions: substrate is 1% (w/v) maltodextrin, in 50 mM buffer and 5 mM CaCl2, 50 8C. Buffers: disodium phosphate–citric acid buffer for pH 5.0–7.0 and boric acid–potassium chloride buffer for pH 8.0–10.0, Section 2.3.3.
Fig. 8. Determination of Km and Vmax by the Hanes method for the CGTase from Bacillus firmus, strain 7B. Only the points shown in Fig. 6 with low substrate concentration were used. Experimental conditions were as in Fig. 6.
3.4. Influence of substrate concentration on CGTase activity
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deactivation of the enzyme was smaller at 60 8C and 24 h of reaction time; enzyme affinity for the substrate is comparatively higher.
Fig. 9. Influence of products a-, b- and g-CD, on the activity of Bacillus firmus strain 7B CGTase for production of (-CD. Conditions were: pH 8.0, 50 8C, 50 mM Tris–HCl buffer, 5 mM CaCl2, and the substrate was maltodextrin 1% (w/v) (DE 10). The exogenous cyclodextrin concentration is given over the bars.
We have studied the CGTase from Bacillus firmus strain 37 since 1994, but now we have got a new strain with improved characteristics for production of the enzyme. Thus, the method that we have been using to select strains that are good producers of CGTase [14] is valid and can lead to improved results. After obtaining good strains for producing CGTase, the next steps for improving CGTase production are, according to Martins and Kaul [12], and we agree with them: ‘‘cloning and expression of the CGTase gene are important for making changes and improving the critical features of the enzyme’’. Acknowledgment
substrate than the CGTase from Bacillus firmus strain 37 (Ks = 0.0928 mol/L) [8]. Other known CGTases have the following Ks values: Bacillus macerans, 5.89 mg/mL [30]; Bacillus macerans, 0.01 mg/mL [31]. In these works the substrate molar mass was not given. 3.6. Influence of products on CGTase activity The influence of the reaction products a-, b- and g-CD on the cyclization activity is shown at Fig. 9. The enzyme showed loss of activity in the presence of increasing a-, b- and g-CD concentrations but its activity was higher in the presence of low a-CD concentration. The higher activity observed with added a-CD may not be enzyme activation, but indicate that this CD is serving as substrate for producing b-CD. The CGTase activity from Bacillus agaradhaerens [12] had a slight inhibitory effect from a- and g-CD and a slight activation effect by b-CD. These results indicate that the influence of reaction products on CGTase activity varies according to the source of enzyme, even though the inhibitory effect is more common than the activation effect. Inhibition by b- and g-CD has been reported for the CGTases from Bacillus firmus strain 37 [8] and Bacillus autolyticus [32]. 4. Conclusions The new CGTase from Bacillus firmus, strain 7B, has been reported and the results obtained in this work were compared with those from Bacillus firmus strain 37 CGTase, previously studied by our group [5,8,9,14]. Some characteristics of these enzymes showed to be close (enzymatic activity as a function of temperature and pH, thermal stability, activation and deactivation energy, influence of products), but for others characteristics, the new CGTase was shown to be better, for example: observed enzyme activity in the cell-free supernatant was higher as determined by colorimetric and CD-TCE methods; enzyme half-life showed to be higher for temperatures lower than 60 8C;
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