Partial purification and properties of cyclodextrin glycosiltransferase (CGTase) from alkalophilic Bacillus species.pdf
RESEARCH Open Access Partial purification and properties of cyclodextrin glycosiltransferase (CGTase) from alkalophilic Bacillus species Marlene M Martínez Mora1, Karel Hernández Sánchez1, Reynaldo Villalonga Santana2, Arley Pérez Rojas3, Héctor L Ramírez1 and Juan José Torres-Labandeira4* Abstract Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.9) 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. Keywords: Cyclodextrins glucanotransferase, Alkalophilic Bacillus sp., Enzyme purification, Enzyme characterization, Cyclodextrin production Background The alkaliphilic bacilli are the best producers of the enzyme cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19). Cyclodextrin glucanotransferase is a multifunc- tional 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 sub- strates into cyclodextrins (CDs) (Jemli et al. 2007). Cyclodextrins (CDs) are non-reducing cyclic oligosac- charides 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 in- clusion 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 increas- ingly popular and are extensively used in industries such as pharmaceutical, textile, agriculture, cosmetic, chem- ical 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 (Ata- nosova 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 organ- isms. 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 * Correspondence: calvarezlorenzo@gmail.com 4Department of Pharmacy and Pharmaceutical Technology, University of Santiago de Compostela, Faculty of Pharmacy, Santiago de Compostela 15782, Spain Full list of author information is available at the end of the article a SpringerOpen Journal © 2012 Mora et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mora et al. SpringerPlus 2012, 1:61 http://www.springerplus.com/content/1/1/61 importance, because separation of one type of CDs from a mixture of products is time-consuming, costly and te- dious (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 atten- tion in the CDs production field. Alkalophilic bacilli have received the major attention for industrial applica- tions 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 The CGTase producing strain was identified as Bacillus sp. in previous work Martinez et al. (2012).The CGTase purification from the culture supernatant was performed by gel filtration using Fractogel EMD BioSec(s) as matrix (Figure 1). The active fraction used for the biochemical characterization of the enzyme was located between tube 10 and tube 17, where the enzymatic fraction with the highest purification level was found. In the tube interval from 20 to 30, the concentration in protein (because of the culture medium composition), the cyclodextrin pro- duction and the enzymatic activity were maxima, and the enzyme could not be isolated. The enzyme could be sufficiently purified in one step with a recovery of 87.3% activity and 40-fold purification for specific enzymatic activity of 2.24 U/mg (one unit of enzyme activity refers to the amount of enzyme that cat- alyzes the production of 1 μmol of β-CD per minute under the reaction condition) (Table 1). A similar result has been reported for B. agaradhaerens LS-3C CGTase when sufficiently purified using starch adsorption as the sole purification step with a recovery of 50% activity and 43-fold purification (Martin and Hatti 2002). Enzyme activity was measured at 55°C using the stand- ard assay method by varying the pH values from 3.0 to 10.5. The optimum pH of the purified CGTase was 5.0 (Figure 2), which is in agreement with the values reported for purified CGTases from Bacillus sp. 7–12 (Cao et al. 2005) and B. megaterium (Pishtiyski et al. 2008). Most CGTase exhibit optimum pH ranging from 5.0 to 8.0 (Sian et al. 2005), although the enzyme from Brevibacterium sp. no. 9605 exhibits the highest activity at pH 10.0 (Mori et al. 1994). The enzyme was then incubated for 60 minutes at 4°C under various pH conditions, prior to the determination of residual activity under standard assay conditions. The enzyme retained 85% of its initial activity at pHs be- tween 6 and 10.0. At pH 5.0 the activity retained was 70%; below this pH value a drastic reduction in enzyme activity was observed (Figure 3). When compared to other Bacillus CGTases, a similar behavior was observed Figure 2 0 0,5 1 1,5 2 2,5 3 1 6 11 16 21 26 31 Fraction Numbers / 4ml A 280 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 Enzimatic Activity AE (U/ml) A280 AE(U/ml) conc.CD Figure 1 Elution profile of the CGTase from gel filtration chromatography column using Fractogel EMD BioSEC (S). Fraction 10–17 was collected and used for subsequent characterization steps. Mora et al. SpringerPlus 2012, 1:61 Page 2 of 6 http://www.springerplus.com/content/1/1/61 Material and Methods Materials α-, β- and γ-Cyclodextrin were purchased from Pharma- cia 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 incu- bated at 37°C during 48 hours at 200 rpm. Cells and in- soluble 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 apply- ing the phenolphthalein method, measuring the produc- tion of β-CD spectrophotometrically at 550 nm, on the basis of its ability to form a colourness inclusion com- plex with this dye. Phenolphtalein (4 mM) in ethanol was diluted in 125 mM Na2CO3 pH 11 just before start- ing 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). Optimum pH 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. 30 40 50 60 70 80 40 50 60 70 80 90 100 Residual Activity (%) Temperature  0C Figure 4 Optimun temperature of CGTase. 20 30 40 50 60 70 80 90 40 60 80 100 Residual Activity  (%) Temperature  0C Figure 5 Thermal stability of CGTase. Figure 6 Production of cyclodextrins on soluble (black) and cocoyam starch (gray). Mora et al. SpringerPlus 2012, 1:61 Page 4 of 6 http://www.springerplus.com/content/1/1/61 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. Optimum temperature The effect of temperature on CGTase activity was evalu- ated 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. Thermostability Enzyme preparations (0.57 U) were incubated at differ- ent temperatures between 25°C and 85°C in 50mM so- dium 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 sol- uble and cocoyam starch was colorimetrically deter- mined. 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 for- mation of inclusion complexes with bromocresol green (Kato and Horikoshi 1984). All experiments were carried out in triplicate under identical conditions. Abbreviations CGTase: Cyclodextrin glucanotransferase; CDs: Cyclodextrins. Competing interests The authors declare that they have no competing interests. Author's contributions 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. Acknowledgements 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). Author details 1Center for Enzyme Technology, University of Matanzas, Matanzas, C.P. 44740, Cuba. 2Department of Analytical Chemistry, Faculty of Chemistry, Complutense University of Madrid, Madrid, Spain. 3Center for Environmental Studies, University of Matanzas, Matanzas, C.P. 44740, Cuba. 4Department of Pharmacy and Pharmaceutical Technology, University of Santiago de Compostela, Faculty of Pharmacy, Santiago de Compostela 15782, Spain. Received: 10 October 2012 Accepted: 30 November 2012 Published: 12 December 2012 References Jemli S, Messaoud E, Ayadi-Zouari D, Naili B, Khemakhem B, Bejar S (2007) A β- cyclodextrin glycosyltransferase from a newly isolated Paenibacillus pabuli US132 strain: Purification, properties and potential use in bread-making. Biochem Eng J 34:44–50 Abdel M, El H, Abdel A (2011) Biosynthesis of cyclodextrin glucosyltransferase by the free and immobilized cells of Bacillus cereus NRC7 in batch and continuous cultures. J Appl Microbiol 111:1129–1137 Atanosova N, Petrova P, Ivanova V (2008) Isolation of novel alkaliphilic bacillus strains for cyclodextrin glucanotransferase production. Appl Biochem Biotechnol 149:155–167 Hamoudi M, Fattal E, Gueutina C, Nicolas V, Bochota A (2011) Beads made of cyclodextrin and oil for the oral delivery of lipophilic drugs: In vitro studies in simulated gastro-intestinal fluids. 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Starch 47:399–400 Mora et al. SpringerPlus 2012, 1:61 Page 5 of 6 http://www.springerplus.com/content/1/1/61 Higuti I, Silva P, Papp V, Okiyama M, Alves de Andrade E, Marcondes A, Nascimento A (2004) Colorimetric determination of α and β-cyclodextrins and studies on optimization of CGTase production from B. firmus using factorial designs. Braz Arch Biol Technol 47:837–841 Kato T, Horikoshi K (1984) Colorimetric determination of γ-cyclodextrin. Anal Chem 56:1738–1740 doi:10.1186/2193-1801-1-61 Cite this article as: Mora et al.: Partial purification and properties of cyclodextrin glycosiltransferase (CGTase) from alkalophilic Bacillus species. SpringerPlus 2012 1:61. 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Title: Partial purification and properties of cyclodextrin glycosiltransferase (CGTase) from alkalophilic Bacillus species ​
Authors: Marlene M Martínez Mora, Karel Hernández Sánchez, Reynaldo Villalonga Santana, Arley Pérez Rojas, Héctor L Ramírez, Juan José Torres-Labandeira ​
Publisher: SpringerPlus
Date: 12 December 2012 ​
Abstract: Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.9) is a unique enzyme capable of converting starch and related substrates into cyclodextrins (CDs). ​ In this paper, we report a one-step gel purification method of CGTase from Bacillus sp. ​ and later enzyme characterization. ​ The Bacillus sp. ​ strain was isolated from a Colocacia esculenta rhizospheric soil sample, and the CGTase production was carried out in an 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 hour 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. ​