The molecular weight of the purified denatured CGTase was estimated to be 92 kDa (Figure 1A) by SDS-PAGE. The purified native and denatured enzymes showed single band with similar molecular weight on native PAGE and SDS-PAGE, respectively, suggesting that NPST-10 CGTase is a monomeric protein. Most of the reported CGTases are monomeric in nature with molecular weight between 60 and 110 kDa [23,27–29]. However, CGTases with lower molecular weight have been also reported, such as 33 kDa from Bacillus coagulans [30] and as 56 kDa from Bacillus sphaericus strain 41 [3].

The temperature profile of the enzyme was estimated by measurement of the enzyme activity at various temperatures (Figure 2). CGTase showed significant activity in a wide temperatures range, 45 °C–70 °C, showing maximal enzyme activity at 50 °C. The relative enzyme activities at 60, 65 and 70 °C were found to be 92%, 87.4% and 78.8%, respectively. However, increasing of the reaction temperature to 75 °C caused rapid decrease of the relative enzyme activity to 26.4%. Temperature optima in the range of 55–65 °C have been previously reported for CGTases from various alkaliphiles [20,23,27,29,31]. However, CGTase from thermophilic bacteria usually shows optimal activity at higher temperatures [28,32]. In addition, the thermal stability of the purified CGTase was investigated by pre-incubation of the enzyme at different temperatures at pH 8.0 for different time intervals before measurement of residual activities. The results illustrated in Figure 3, showed that the stability of the enzyme depended on the temperature and period of preheating. At 30 °C to 55 °C, the enzyme retained between 80% to 100% of its initial activity after enzyme treatment for 1 h, and retained 40–93% to 40% after 3 h of treatment. Moreover, CGTase retained 81.3%, 80.3% and 52.6% of the initial activity after treatment for 1 h at 60, 65 and 70 °C, respectively. More adverse effect was found by treating the enzyme for 3 h at 60–70 °C, at which the enzyme retained only 30% to 35% of its initial activity.

Earlier reports have shown that CGTase thermostability is enhanced in the presence of its substrate, products, and calcium ions [20,29,32]. Therefore, the influence of these additives on the thermostability of Amphibacillus sp. NPST-10 CGTase was studied. It was found that the thermal stability of CGTase was enhanced by about 1.3 fold in the presence of calcium ion, and slightly enhanced in the presence of maltodextrins or starch (Figure 4). Structural analysis of the CGTases have shown the presence of two calcium ions, one near the N-terminal end and the other near the active site region, provide stability to the conformational structure of flexible regions of the protein molecule including the active site [28].

The influence of pH on the CGTase activity was established by measurement of enzyme activity at varying pH values ranging from 4.0 to 11.0 at 50 °C under standard assay conditions. The results shown in Figure 5 indicated that the enzyme was active in a wide pH range from 6.0 to 9.0 with relative activities of 75% to 85%, showing maximal activity at pH 8. It has been reported that CGTases from alkaliphiles showed optimal cyclizing activities in pH range from 5.0 to 10.0 depending on the bacterial producer [3,20,27]. However, some CGTases exhibited two pH peaks at pH 6.0 and 9.0, such as CGTase from Bacillus sp. [7–12,23], Bacillus firmus [32], and Bacillus pseudalcaliphilus 20RF [29].

Investigation of the pH stability of Amphibacillus sp. NPST-10 CGTase was performed by pre-incubation of the enzyme in buffers of various pH values for 1 h at 25 °C prior to determination of the residual activities under the standard assay conditions. The enzyme retained about 93%–100% of its initial activity in a wide range of pH values, between pH 6.0 and 10.0 (Figure 6). Moreover, the enzyme retained more than 82% of its initial activity at pH 11.0, but was less stable in pH 5.0 and 12.0, showing residual activity of 55% and 49.2%, respectively. In comparison to other CGTases reported from alkaliphiles, Amphibacillus sp. NPST-10 enzyme showed high stability in wide pH range, slightly higher than alkaliphilic Bacillus pseudalcaliphilus 20RF CGTase [29]. Moreover, Amphibacillus sp. NPST-10 CGTase was much more stable than CGTase from alkaliphilic Bacillus sphaericus strain 41, where at pH 5.0, the activity retained was 34.2%, while in the range of pH 8.0–10.0 the average retention of activity was 16.8% [3], indicating that Amphibacillus sp. NPST-10 CGTase could be successfully applied for prodyction of cyclodextrins in range of pH from 5.0 to 11.0 for production of cyclodextrins.

In order to be establish the effect of various metal ions and reagents on CGTase activity, enzyme assays were carried out in presence of theses additives under the standard assay conditions. A significant lose of the enzyme activity was established in the presence of 10 mM Co²⁺, Zn²⁺, Cu²⁺, Cd²⁺ or Hg²⁺ with residual activities of 82.1%, 40.3%, 32.8% and 29.3% and 0.0%, respectively (Table 2). These results are similar to those reported by Atanasova et al. [29], significant decrease of CGTase activity of alkaliphilic Bacillus pseudalcaliphilus 20RF in the presence of 15 mM Zn²⁺ and Co²⁺ ions. However, the inhibitory effect of Co²⁺ on Amphibacillus sp. NPST-10 CGTase is in contrast to that reported by Martins and Hatti-Kaul [33], where cobalt had no effect on the activity of CGTase from Bacillus agaradhaerens LS-3C. In addition, Li et al. [21] has recently reported activation of CGTase from Paenibacillus macerans by Zn ion. CGTase of Amphibacillus sp. NPST-10 was slightly inhibited in the presence of manganese and magnesium ions. Ba²⁺ ion was found to significantly inhibit CGTase of Amphibacillus sp. NPST-10, which is similar to that reported for CGTase from B. agaradhaerens LS-3C [33], but in contrast to that reported by Li et al. [21] where Ba²⁺ has stimulatory effect on CGTase from Paenibacillus macerans. The effect of metal ions and reagents on the activity of CGTases depends on the bacterial producer. However, the inhibitory effect of ions is mostly attributed to the metal catalyzed oxidation of amino acid residues essential to the enzyme activity [29]. The most probable candidates would be tryptophan which is known to be present in the active site and specific tyrosine and histidine residues which are ascribed an important role in cyclization efficiency and in transition state stabilization [1]. The reducing agent, 2-mercaptoethanol, resulted in significant inhibition of Amphibacillus sp. NPST-10. EDTA, a metal chelating agent, had no effect on the enzyme activity, suggesting that this CGTase is not a metalloenzyme, which is similar to that reported for CGTase from alkaliphilic Bacillus pseudalcaliphilus 20RF [29]. Amphibacillus sp. NPST-10 CGTase activity was slightly stimulated in the presence of Ca²⁺, which appeared to be a characteristic feature of different bacterial CGTases [27,34].

Among the CDs, 1 mM of β- and γ-CD showed significant inhibitory effect on the enzyme activity with residual activity of 45.7% and 54.2%, respectively. Some authors have reported total inhibition of CGTase by the reaction products. For instance, CGTase from B. megaterium showed a total loss of activity in the presence of 12 mg/mL of β-CD [35], as did the CGTase from B. agaradhaerens LS-3C and Bacillus circulans DF 9R [33,36].

Kinetic studies of the purified CGTase were investigated by measuring initial rates of CGTase reaction at different concentrations of soluble starch in 50 mM Tris-HCl buffer (pH 8.0) at 50 °C. The kinetic parameters, K_m and V_max, were estimated using Michaelis-Menten equation and double reciprocal plot known as Lineweaver-Burk plot [33]. The K_m and V_max values using soluble starch as a substrate were estimated to be 1.7 ± 0.15 mg/mL and 100 ± 2.0 μmol/min, respectively (Figure 7). The low value of K_m indicates the high affinity of Amphibacillus sp. NPST-10 CGTase toward the substrate. K_m values ranging from 1.77–5.7 mg/mL and V_max from 43–1027 μmol/min have been previously reported for various CGTases [21–24,37].

The sources of starch could affect the CGTase activity, which is probably due to the differences in the starch granules structure and properties among various starch sources [37]. Hence, the action of the purified CGTase on hydrolyzed and raw substrates was studied. The results presented in Figure 8 indicated that the enzyme possesses activity on all of the tested substrates, whether partially hydrolyzed (soluble starch) or raw starch. Interestingly, Amphibacillus sp. NPST-10 CGTase showed higher activity using raw cornstarch than soluble starch (partially hydrolyzed starch). However, the cyclization activity using maltodextrin, short oligosaccharides (17 glucose residues) was the highest. The raw starch has a compact crystalline structure that is not easily degraded, and therefore CGTases with high activity toward raw starch is highly recommended for industrial production of CDs [31].

The production of CDs was analyzed by HPLC using pure CGTase and 1% (w/v) of soluble starch as a substrate, at 50 °C and pH 8.0. A maximum yield of CDs with about 67.2% conversion of soluble starch into CDs could be obtained after 4 h and the products ratio at that point was 10.2% α-CD, 86.4% β-CD and 3.4% γ-CD. The production yield and ratio of the different CDs formed by CGTases are dependent not only on the microbial source producing the enzyme, but also on the nature of the substrate and reaction conditions, such as temperature, pH and reaction time [5]. Depending on the most abundant form of cyclodextrin that CGTase produces, the enzyme is sometime classified as α-, β- and γ-CGTase [1,25,37]. By this classification, Amphibacillus sp. NPST-10 CGTase could be considered as β-CGTase. Of the three kinds of CDs, β-CD is of most practical use because its inclusion complexes are easily prepared and more stable. The size of the β-CD polar cavity is optimum for many molecules such as drugs and preservatives. Furthermore, β-CD is easily separated from the reaction mixtures due to its low solubility in water [38].

Cyclization activity of CGTase was measured as β-CD forming activity according to a method previously described with some modification [33]. Seven hundred and fifty micro liter of 1% (w/v) starch solution prepared in 50 mM Tris-HCl buffer, pH 8 was pre-incubated at 50 °C for 5 min. One hundred micro liter of enzyme sample was added to the reaction mixture and after incubating for 20 min at 50 °C, the reaction was quenched by adding 375 μL of 0.15 M NaOH. Subsequently, 100 μL of 0.02% (w/v) phenolphthalein prepared in 5 mM Na₂CO₃ was added, and after standing at room temperature for 15 min, the color intensity was measured at 550 nm. One unit of CGTase activity was defined as the amount of enzyme releasing 1 μmol of β-CD per min under the defined assay conditions. A calibration curve was made using 0.001–0.5 μmol of β-CD in 50 mM Tris-HCl (pH 8). Protein concentration was determined according to the method described by Bradford [41] with bovine serum albumin as the standard protein.

Hydrolytic activity of CGTase was performed by incubation of 750 μL 1% starch solution, prepared in 50 mM Tris-HCl buffer (pH 8), for 30 min at 50 °C. To terminate the enzymatic reactions, the mixtures were boiled for 5 min. The release of reducing sugars was followed by dinitrosalicylic acid method [42]. 0.1 mL of the reaction mixture was added to 1 mL of 3,5-dinitrosalycilic acid reagent and incubated in boiling water bath for 10 min, cooled and the absorbance was measured at 546 nm. One unit of hydrolytic activity of CGTase was defined as the amount of enzyme releasing 1 μmol of reducing sugars per minute under the defined assay conditions. A calibration curve was made using 0.1–1 mg/mL d-glucose.

Coupling activity of the purified CGTase was measured according to the disappearance of β-CD in the presence of glucose using modification a protocol described previously [3]. Enzyme solution (100 μL) was added to 750 μL of 50 mM Tris-HCl buffer containing 0.5 mM β-CD and 1% glucose starch solution prepared in 50 mM Tris-HCl buffer (pH 8) and incubated for 30 min at 50 °C. The reaction was stopped by boiling for 5 min and the amount of residual β-CD was determined calorimetrically as described in Section 3.5.1. One unit of coupling activity was defined as the amount of enzyme that can convert 1 μmol of β-CD/min.

The molecular weight of the purified enzyme was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), [43]. SDS-PAGE was performed on 7.5% polyacrylamide gel, using standard protein markers with molecular weights ranging from 10.5 to 175 kDa (Pink plus prestained protein ladder, GeneDirex). The non-specific staining of proteins with silver reagent in the polyacrylamide gel was carried out in order to detect minute concentration of proteins in the gel [44].

For CGTase activity staining, the enzyme samples were applied to 10% native PAGE. After gel electrophoresis, the gel was washed twice with distilled water, and twice with 50 mM glycine-NaOH buffer, pH 8. Then, the gel was immersed in 50 mM glycine-NaOH buffer (pH 10) containing 1% (w/v) soluble starch, 0.02% (w/v) phenolphthalein and 1% (w/v) agar. After agar solidification, it was incubated for 1 h at 50 °C, and the CGTase activity was seen as a colorless band on a red background due to formation of stable colorless CD-phenolphthalein inclusion complex [14]. For detection of starch degrading activity, the native gel was immersed in solution of 1% (w/v) soluble starch in 50 mM Tris-HCl buffer (pH 8), and incubated for 1 h at 50 °C. Then, the starch solution was decanted and iodine solution was poured on the gel. The starch-degrading activity was seen as a clear band on dark blue background.

The effect of temperature on the cyclization activity of CGTase was examined at various temperatures ranging from 35 °C to 70 °C under the standard assay conditions described above. The thermostability of the purified CGTse was determined by incubating the enzymes in 50 mM Trise-HCl (pH 8) at different temperatures (35 °C to 70 °C) and at various times interval (10–180 min), aliquots of enzyme were withdrawn and assayed for residual cyclization activity under the standard assay conditions. The thermal stability of the enzyme was further investigated in the presence of 1 mM calcium salt, 1% (w/v) of starch or maltodextrin, respectively, at varying temperatures from 50 °C to 70 °C for 1 h prior of determination of the residual enzyme activities under the standard assay conditions.

The effect of pH on the activity of CGTase was examined at various pH values ranging from pH 4.0 to 12.0, using suitable buffers including 50 mM sodium acetate (pH 5.0 and 6.0), 50 mM Trise-HCl (pH 7.0 and 8.0), 50 mM glycine-NaOH buffer (9.0 and 10.0) and 50 mM carbonate-bicarbonate buffer (11.0 and 12.0), respectively. In addition, the pH stability of purified enzyme was investigated by incubating the purified enzymes in buffers with different pH values for 1 h at room temperature, and the residual cyclization activities were assayed under the standard assay conditions. All experiments and enzyme assays were performed in triplicate and the mean values were reported.

The effects of different metal ions and some inhibitors on the CGTase activity were investigated by addition of the test ions and reagents to reaction mixtures at final concentration of 1 mM or 10 mM. The test ions and reagents included Cu²⁺, Ca²⁺, Zn²⁺, Mn²⁺, Mg²⁺, Cd²⁺, Na⁺, Ba²⁺, Hg²⁺, Ni²⁺, Co²⁺, EDTA, 2-mercaptoethanol, α-, β-, and γ-CD.

Kinetic studies were performed by measuring the cyclization activity of CGTase at various concentrations of soluble starch ranging from 0% to 1.5 mg/mL. The kinetic constants, K_m and V_max, were estimated using Michaelis-Menten equation and double reciprocal plot known as Lineweaver-Burk plot [33].