Partial purification and properties of cyclodextrin glycosiltransferase (CGTase) from alkalophilic Bacillus species
© Mora et al.; licensee Springer. 2012
Received: 10 October 2012
Accepted: 30 November 2012
Published: 12 December 2012
Cyclodextrin glucanotransferase (CGTase, EC 126.96.36.199) is an unique enzyme capable of converting starch and related substrates into cyclodextrins (CDs). In this paper, we report an one step gel purification method of CGTase from Bacillus sp. and later enzyme characterization. The Bacillus sp. strain was isolated from a Colocacia esculenta rizospheric soil sample and the CGTase production was carried out in alkaline medium (pH=10). The CGTase purification from the culture supernatant was performed by gel filtration. The enzyme was purified in one step with a recovery of 87.3% activity and 40-fold purification for specific enzymatic activity of 2.24 U/mg. Optimal activity was observed at pH 5.0 in citrate-phosphate buffer, and the enzyme retained almost 100 % of its activity between pH 5.5 and 10 after incubation for 1 h at 4°C. The enzyme exhibited maximum activity at 55°C and showed a T50% of 70°C. The ratio of α:β:γ CD formed by the enzyme was 0.74:1:0.61 for soluble starch and 0.29:1:0.85 for cocoyam starch.
KeywordsCyclodextrins glucanotransferase Alkalophilic Bacillus sp. Enzyme purification Enzyme characterization Cyclodextrin production
The alkaliphilic bacilli are the best producers of the enzyme cyclodextrin glucanotransferase (CGTase, EC 188.8.131.52). Cyclodextrin glucanotransferase is a multifunctional enzyme which catalyzes four related reactions: cyclizing, coupling, disproportionation, and hydrolysis. By means of the cyclizing activity, CGTase is an unique enzyme capable of converting starch and related substrates into cyclodextrins (CDs) (Jemli et al. 2007).
Cyclodextrins (CDs) are non-reducing cyclic oligosaccharides with the spatial structure of a doughnut. The interior of CDs is hydrophobic and its external surface is hydrophilic. Due to this feature, CDs are able to form inclusion complexes with either organic or inorganic molecules (Abdel et al. 2011). As a result, CDs can change physical and chemical properties of encapsulated guest compounds. Therefore, CDs are becoming increasingly popular and are extensively used in industries such as pharmaceutical, textile, agriculture, cosmetic, chemical and food, where CDs help to increase the solubility and stability, reduce volatility, and improve the control of the release of drugs, and mask odours and tastes (Atanosova et al. 2008, Hamoudi et al. 2011, Marcon et al. 2009, Sian et al. 2005, Wang et al. 2011).
CGTase is an extracellular, inducible enzyme produced only by microbial cells. The roles of CGTase production in microorganisms are still unclear; however, some researchers believe that starch is converted by CGTase into CDs that cannot be used by other competing organisms. In this way, the CDs can be used as substrate by the CGTase producer.
Most bacterial CGTases mainly produce α-CD, β-CD and γ-CD consisting of six, seven, or eight glucose units, respectively. Thus, CGTase is sometimes classified into three different types (α-, β- and γ-CGTase), depending on the major CD produced. CGTases that synthesize predominantly one type of CD have great commercial importance, because separation of one type of CDs from a mixture of products is time-consuming, costly and tedious (Jemli et al. 2007). The yields and ratios of α-, β- and γ-CD produced from starch differ depending on the origin of the CGTase and the environmental conditions (Schmid 1989). For this reason, purification and characterization of new CGTases attract a great attention in the CDs production field. Alkalophilic bacilli have received the major attention for industrial applications because of their high activity over a wide range of pH and temperatures.
The main objective of this study was to purify CGTase from alkalophilic Bacillus species isolated from soil and to characterize some relevant properties of the enzyme.
Result and Discussion
Summary of CGTase purification results
Total volumen (mL)
Protein concentration (mg/mL)
Enzimatic activity (U/mL)
Especific activity. (U/mg)
Gel filtration (Fractogel)
Cyclodextrin glucanotransferase (CGTase) from alkalophilic Bacillus has been successfully purified and effectively used for conversion of starch into cyclodextrin under a wide range of pH conditions. The enzyme exhibits a good thermostability and shows optimum temperature at 55°C. Furthermore, it is a good candidate to obtain increased production of γ-CD production when used cocoyam starch as substrate.
Material and Methods
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.
These authors: MMMM, RVS, JJT-L have made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data. These authors: KHS, APR, HLR have been involved in drafting the manuscript or revising it critically for important intellectual content. All authors read and approved the final manuscript.
This research was supported by the International Foundation for Science, Stockholm, Sweden, and the Organization for the Prohibition of Chemical Weapons, The Hague, The Netherlands, through a Grant to H. Ramirez (Grant F/3004-67).
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