α-, β- and γ-Cyclodextrin were purchased from Pharmacia Biotech. Soluble starch, Fractogel EMD BioSec(s), yeast extract and peptone was purchased from Sigma. Cocoyam starch was of industrial grade. Phenolphthalein was purchased from Merck. All other chemicals were of analytical grade.
Screening and Isolation of bacteria
Bacteria were isolated from Colocacia esculenta rizospheric soil. Samples were suspended in sterile water, serial diluted, and then plated on Horikoshi II agar plate containing (w/v): 1.0 % soluble starch, 0.5 % yeast extract, 0.5 % peptone, 0.1 % KH2PO4, 0.02 % MgSO4.7H2O, 0.02 % phenolphthalein and 1.0 % Na2CO3 (separately autoclaved). Plates were incubated at 37°C for 24 h. Bacterial colonies which produced the largest clear halo zones were selected for further studies (Illias et al. 2002).
CGTase production and purification
The selected strain was cultivated in flasks containing 200 mL of Horikoshi II broth culture medium and incubated at 37°C during 48 hours at 200 rpm. Cells and insoluble material were removed by centrifugation at 2012 g for 15 min at 4°C, and the cell-free supernatant was used as the source of the enzyme. The crude extract (25 ml) was concentrated to a final volume of 5 ml by rotoevaporation to 30°C. Later, the sample was purified to 4°C in chromatographic column of gel filtration(1.6X 60cm) using Fractogel EMD BioSec(s) as matrix with mobile phase buffer Tris/HCl pH 8.0 20 mM. The column was washed with the same buffer at flow rate 60 mL/h at fractions of 4ml was collected. The protein concentration was estimated using the Bradford method (Bradford 1976).
Cyclizing activity of CGTase
The cyclizing activity of CGTase was determined applying the phenolphthalein method, measuring the production of β-CD spectrophotometrically at 550 nm, on the basis of its ability to form a colourness inclusion complex with this dye. Phenolphtalein (4 mM) in ethanol was diluted in 125 mM Na2CO3 pH 11 just before starting the assay. 2% Starch in 0.05 M citrate-phosphate buffer pH 5.0 were used as substrate. One unit of CGTase is defined as the amount of enzyme catalyzing the production of 1 μmol of β-CD per minute under the reaction conditions (Goel and Nene 1995).
The effect of pH on CGTase activity was measured in the range from 3.0 to 10.5, at 55°C for 10 minutes using 50mM citrate-phosphate (pH 3.0-7.5), 50mM Tris–HCl (pH 8.0-9.0) and 50mM sodium carbonate (pH 9.0–11) buffers.
Stability against pH
Enzyme preparations (0.57 U) were incubated at 4°C in 100mM sodium acetate/acetic acid (pH 3.0-5.5), 100mM K2HPO4/ KH2PO4 (pH 6.0-7.5), 100mM Tris/HCl (pH 8.0-9.0) and Na2CO3 /NaHCO3 (pH 9.5-11.5) buffers. Aliquots were removed after 60 minutes of incubation, diluted in 0.1 M K2HPO4/ KH2PO4 buffer (pH 7.0) and assayed for cyclizing activity of CGTase.
The effect of temperature on CGTase activity was evaluated in the range of 35-75°C in 50mM citrate phosphate (pH 5.0) buffer. After incubation for 10 minutes, the cyclizing activity was measured.
Enzyme preparations (0.57 U) were incubated at different temperatures between 25°C and 85°C in 50mM sodium citrate phosphate buffer, pH 5.0. Aliquots were removed after 30 minutes incubation, chilled quickly, and assayed for cyclizing activity.
Determination of CDs concentration
The enzyme (0.27mg/mL) was incubated at 55°C for 60 minutes with 1.5% starch in 50mM citrate phosphate buffer (pH=5.0). The concentration of the various CDs produced by the action of the purified CGTase on soluble and cocoyam starch was colorimetrically determined. The concentration of α-CD was assayed by the decrease in absorbance at 507 nm due to the formation of inclusion complexes with methyl orange (Higuti et al. 2004). The concentration of β-CD was determined according to the method described above (Goel and Nene 1995). The concentration of γ-CD was quantified by measuring the absorbance at 630 nm due to the formation of inclusion complexes with bromocresol green (Kato and Horikoshi 1984).
All experiments were carried out in triplicate under identical conditions.