Production of CGTase by a Bacillus alkalophilic CGII strain isolated from wastewater of a manioc flour industry

Produção de CGTase por Bacillus alkalophilic CGII isolado de água residuária de uma fecularia de mandioca

BIOTECHNOLOGY

Production of CGTase by a Bacillus alkalophilic CGII strain isolated from wastewater of a manioc flour industry

Produção de CGTase por Bacillus alkalophilic CGII isolado de água residuária de uma fecularia de mandioca

Telma Luisa de Freitas^I; Rubens Monti^II; Jonas Contiero^III

^IDepartamento de Bioquímica e Tecnologia, Universidade Estadual Paulista Júlio Mesquita Filho, Araraquara, SP, Brasil

^IIDepartamento de Alimentos e Nutrição, Faculdade de/ FCF/ Universidade Estadual Paulista Júlio Mesquita Filho, Araraquara, SP, Brasil

^IIIDepartamento de Bioquímica e Microbiologia, Instituto de Biociências, Universidade Estadual Paulista Júlio Mesquita Filho, Rio Claro, SP, Brasil

^{Correspondence}

ABSTRACT

GCTase production by a new strain of Bacillus alkalophilic CGII isolated from Brazilian wastewater of manioc flour industry was examined. The growth medium used was composed by 1.5% starch, 1.5% nitrogen and 1% Na₂CO₃. Higher activity was obtained with starch, maltodextrin and galactose. When glucose was added to the medium, no enzyme production was observed. High enzyme activity and growth were reached when aeration was increased (88.6 U/mL). The enzyme characterization showed an optimum pH and temperature 8.0 and 55ºC for starch hydrolyses, respectively. Mg⁺ and Ca⁺⁺ showed small activation; however, Hg⁺ and Cu⁺ showed a strong enzyme inhibition.

Key words:Bacillus alkalophilic CGII; CGTase; b-cyclodextrin; starch hydrolysis; enzyme activity

RESUMO

Estudou-se a produção de CGTase por uma nova cepa de Bacillus alkalophilic CGII, isolada de água residuária de uma fecularia de mandioca, durante cultivo em meio composto de 1,5% de amido, 1,5% de fonte de nitrogênio e 1% Na₂CO₃.A atividade enzimática foi alta quando se utilizou amido, maltodextrina e galactose como fontes de carbono. Quando se utilizou glicose no meio de cultivo não se observou produção da enzima. Atividade enzimática alta (88,6 U/mL) e melhor crescimento foram obtidos quandose aumentou a aeração. A caracterização da enzima mostrou um pH ótimo de 8,0 e temperatura ótima de 55ºC sendo que a enzima sofreu uma pequena ativação por Mg⁺ e Ca⁺⁺. A enzima foi fortemente inibida por Hg⁺ e Cu⁺.

Palavras-chave:Bacillus alkalophilic CGII, CGTase, b-ciclodextrina, hidrólise do amido, atividade enzimática

INTRODUCTION

The enzyme CGTase (cyclodextrin glycosyltransferase) converts starch into cyclic maltooligosaccharide known as cyclodextrin (1,20,21,27).Cyclodextrin (CD) may contain six (a-CD, cyclohexamilose cG₆), seven (b-CD cycloheptamilose cG₇), or eight (g-CD, cyclooctamilose cG₈) units linked by a-1,4 glycosidic bonds (3,31). The main difference among them is the apolar cavity size and solubility in water (2). The CDs have the capability to form inclusion complexes with a variety of molecules, modifying their physical chemical properties. In general, this is the reason why CDs are being widely used in food, pharmacy and cosmetics industries (2,23,26,27). Several microorganisms are CGTase producers, but the genus Bacillus is the most frequently cited in the literature (1,3,5,9,11,12,13, 14,15,17,18,19,20,21,24,25,30). In this work an alkalophilic CGTase producer microorganism, characterized as Bacillus alkalophilic CGII was isolated from wastewater of manioc flour industry. The main objective of the work was to improve the production of CGTase and to characterize the enzyme partially.

MATERIALS AND METHODS

Isolation of the CGTase producer microorganism

To isolate the CGTase producer microorganism, the method described by Park et al. (22) was used. Soil samples of 1g were suspended in 50 mL of sterilized water and 0.1 mL of the suspensions was inoculated in plates containing culture medium prepared with 2.0% soluble starch (Ecibra); 0.5% polypeptone (Oxoid); 0.5% yeast extract (Oxoid); 0.1% K₂HPO₄ (Synth); 0.02% MgSO₄ x 7H₂O (Synth) 1.0% Na₂CO₃ (Mallinckrodt); 2.0% Agar (Difco); 0.03% phenolphthalein (Synth) and 0.01% methyl orange (Merck), pH 10.5, and incubated at 37ºC. Light zones were observed around the CGTase producer colonies due to phenolphthalein inclusion in the medium by the cyclodextrin produced. The CGTase producer colonies were transferred into tubes containing the same culture medium but without phenolphthalein and methyl orange.

CGTase production

The selected strain was cultivated in flasks containing 200 mL of culture medium and incubated at 37ºC during 18 hours at 200 rpm. This culture was used to inoculate (10% V/V) 2L of culture medium, in a fermentator (5 L capacity) containing 2 mL of antifoaming agent. The incubation was done at 37ºC, 200 rpm and aeration 1.5 vvm. Samples were with drawn periodically to analyze the production of the enzyme, the pH and the enzymatic activity. To improve the enzyme production, many attempts were made, such as substitution of starch by different carbohydrates, and variation in nitrogen and Na₂CO₃ concentration. After enzyme production, the culture was centrifuged, and 3% of starch was added to the supernatant containing the enzyme for adsorption and desorption in phosphate buffer 10mM (pH 7.0) containing NaCl 2M, acetone 3V and ammonium sulphate 70%. The enzyme was dialyzed in distilled water during 12 hours and stored under refrigerated conditions.

Enzymatic activity

Cell-free enzymatic activity was measured according to Mäkela et al. (16) and Goe and Nene (8). Five mL of the supernatant and 5.0 mL of a 1% starch solution [0.1 g of soluble starch; 1.0 mL CaCL₂ (0.05 M) and pure water for a total volume of 10 mL] were mixed in a thermostatic reactor at 50ºC. Samples were taken periodically from the reactors, and inactivated in water at 100ºC for 5 minutes. The concentration of cyclodextrin was measured by addition of 2.5 mL of a 3mM phenolphthalein solution (5.0 mL of 0.6 M Na₂CO₃ buffer, pH 10.5, and the volume completed with 2.5 mL of distilled water in a volumetric flask) to 0.5 mL of the inactivated samples. The absorbance of the final solution was analyzed in spectrophotometer at 550 nm. A unit of the enzymatic activity was defined as the quantity of enzyme that produces 1 mmol of b-CD per minute under standard conditions.

RESULTS AND DISCUSSION

Isolation of the microorganism

During the selection of CGTase producer microorganisms, four strains of alkalophilic Bacillus were isolated. The strain that showed highest activity b-CD production was characterized as Bacillus alkalophilic CGII.

CGTase production

Table 1 shows the effect of the carbon source on the enzyme synthesis after 48 hours of fermentation. According to the results, the microorganism grew very well in all studied carbon sources, but the growth was slower in mannitol and glucose. Higher specific activities were obtained with starch, maltodextrin and galactose. When glucose was added to the medium, enzyme production was not observed. Fig.1 shows the effect of starch concentration, showing that the highest activity was reached at 1.5%. Fig. 2 shows the effect of nitrogen concentration (yeast extract and peptone) on the cellular growing and enzyme production. It is important to note that the initial pH is lower for higher nitrogen concentration present in the medium. However; the DpH was not altered at any concentration. It was verified that, the maximum growth occurred at about 24-30 hours in all concentrations. A diauxie phase was observed at lower concentrations (Fig. 3). At the same time as the nitrogenous concentration increases, the exponential phase becomes longer. The 1.5% nitrogenous concentration was chosen for the next assays because in this concentration it was possible to achieve a better enzymatic activity in a short period of time, that is, at around 36 hours of fermentation, as shown in Fig. 4.

The effect of Na₂CO₃ on cellular growth and enzyme production was studied varying the concentration from 0.25% to 1.5%. As shown in Fig. 5, the difference between the starting and final pH in all concentrations tested, did not change, that is, the DpH was kept almost constant. Regarding to cellular growth, it was observed a larger exponential phase at higher concentrations of Na₂CO₃ and an enhanced diauxie at lower concentrations (Fig. 6). At higher concentrations of Na₂CO₃ the fermentation time to obtain a good enzymatic activity was the same as for lower concentrations (Fig. 7). Based in this fact, the 1.0% Na₂CO₃ concentration was chosen for the other assays, because the adition to the culture medium is only to increase its pH, and to buffer the medium for the growth of a alkalophilic microorganism.

It was observed that the use of antifoaming at 0.05% (v/v) did not influence the enzymatic production, and that the aeration of 1.5 vvm allowed a production of 88.6 U/mL.

Enzyme characterization

For partial enzyme characterization in the supernatant three concentration methods were used: a) precipitation with ammonium sulphate (70%); b) precipitation with 3V acetone; c) adsorption with starch and elution with NaCl 2N. The specific activity was determined. Precipitation with ammonium sulphate and acetone showed the best yields. The enzyme obtained by these two methods was submitted to chromatography in DEAE-Trysacryl, achieving a purification of 11.7 times. SDS-PAGE of the active fraction showed three proteic bands, and one of them was CGTase. Other studies of characterization were made with the partially purified enzyme. Figs. 8 and 9 show the effect of pH and temperature on the enzymatic activity, respectively. The optima pH and temperature for starch hydrolysis were 8.0 and 55ºC respectively. Thatai et al. (29) determined a pH of 6.6 and temperature of 65ºC and Gawande et al. (7) found the optimal temperature of 65ºC for CGTase from Bacillus firmus. Table 2 shows the results obtained with various metallic ions and EDTA. Mg⁺ and Ca⁺ showed little activation. However, Hg⁺ and Cu⁺ showed a strong inhibition, while the remaining ions showed weak inhibition. These results contrast with the ones obtained by Fujita et al. (6) who verified a strong inhibition for Zn²⁺, Fe²⁺and Cu²⁺ and a stability in the presence of Hg²⁺ for the enzyme of Bacillus sp. AL-6.

The results indicate that B. alkalophilic CGII is a good CGTase producer when compared to the other microorganisms. Further studies are needed in order to optimize the enzyme production and the quantification of b-cyclodextrin production by HPLC analysis and to explore the biotechnological potential of this enzyme.

Submitted: September 19, 2003; Returned to authors for corrections: April 28, 2004; Approved: July 27, 2004

• ¹
  Abelyan, V.A.; Yamamoto, T.; Afrykyan, E.G. On the mecanism of action of cyclomaltodextrin glucotransferase of alkalophilic, termophilic and mesophilic microorganisms. Biochem. 59(8):839-844, 1994.
• ²
  Allegre, M.; Deratani, A. Cyclodextrin uses: from concept to industrial reality. Agro-Food Industry Hi-Tech, 1994, p. 9-17.
• ³
  Bender, H. An improve method for the preparation of cyclodextrin octaamylose, using starches and the cyclodextrin glicosyltransferase of Klebsiella pneumoniae M 5 al. Carbohydr. Res.124:225-233, 1986.
• ⁴
  Bender, H. Production, characterization and application of cyclodextrins. Adv. Biotechnol. Process. 6:37-71,1986.
• ⁵
  Freitas T L. Produção e caracterização da CGTase produzida por Bacillus sp. Araraquara, 2001, 77 p. (M. Sc. Dissertação, Instituto de Química de Araraquara, Unesp).
• ⁶
  Fugita, Y; Tsubouchi, H.; Inagi, Y.; Tomita, K.; Ozaki, A.; Nakanishi, K. Purification and properties of Cyclodextrin Glycosiltransferase from Bacillus sp AL-6. J. Ferm. Bioeng. 70(3):150-154,1990.
• ⁷
  Gawande, B.N.; Goel, A.; Patkar, A.Y.; Nene, S.N. Purification and properties of a novel raw starch degrading cyclodextrin glucanosiltransferase from Bacillus firmus Appl. Microbiol. Biotechnol. 51:504-509,1999.
• ⁸
  GoeL, A.; Nene, S.N. Modifications in the phenolphthalein method for spectrophotometric estimation of beta-cyclodextrin. Starch/Stärke 47(10):399-400, 1995.
• ⁹
  HorikoshI, K. Production of alkaline enzymes by alkalophilic microorganisms. Part II. Alkaline amilase produced by Bacillus Nº. A-40-2. Agr. Biol. Chem. 35(11):1783-1791, 1971.
• ¹⁰
  Horikoshi, K. Production and industrial applications of b-cyclodextrin. Process. Biochem. 14(5):26-30,1979.
• ¹¹
  Jamuna, R.; Saswathi, N.; Sheela, R.; Ramakrishna, S.V. Syntesis of cyclodextrin-Glucosiltransferase by Bacillus cereus for the production of cyclodextrins. Appl. Biochem. Biotechnol. 43:163-176,1993.
• ¹²
  Kitahata, S.; Tsuyama, N.; Okada, S. Purification and some properties of Cyclodextrin Glycosyltransferase from a strain of Bacillus sp. Agr. Biol. Chem. 38(2):387-393, 1974.
• ¹³
  Kitahata, S.; Okada, S. Purification and some properties of cyclodextrin glycosyltransferase from Bacillus Stearothermophilus TC-60. J. Jap. Soc. Starch Sci. 29(1):7-12,1982.
• ¹⁴
  Kobayashi, S.; Kainuma, K.; SuzukI, S. Purification and some properties of Bacillus macerans cycloamylose (cyclodextrin) glucanotransferase. Carbohydr. Res. 61:229-238, 1977.
• ¹⁵
  KometanI, T.; Terada, Y.; Nishimura, T.; Takii, H.; Okada, S. Purification and characterization of cyclodextrin glucanotransferase from an alkalophilic Bacillus species and transglycosylation at alkaline pHs. Biosci. Biotechnol. Biochem. 58(3):517-520, 1994.
• ¹⁶
  Mäkelä, M.J.; Korpela, T.; Laakso, S. Colorimetric determination of b-cyclodextrin: two assay modifications based on molecular complexation of phenolphtalein. J. Biochem. Biophys. Meth. 14: 85-92, 1987.
• ¹⁷
  Mäkelä, M.J.; Paavilainen, S.K.; Korpela, T.K. Growth dynamics of cyclomaltodextrin glucanotransferase producing Bacillus circulans var. alkalophilus Can. J. Microbiol. 36:176-182,1990.
• ¹⁸
  Maréchal, L.R.; Rosso, A.M,; Maréchal, M.A.; Krymkiewicz, N.; Ferrarotti, S.A. Some properties of a cyclomaltodextrin-glucanotransferase from Bacillus circulans DF 9R type. Cell. Mol. Biol. 42(5):659-664, 1996.
• ¹⁹
  Matioli, G.; Zanin, G.M,; Guimaraes, M.F.; De Moraes, F.F. Production and purification of CGTase of alkalophylic Bacillus isolated from Brazilian soil. Appl. Biochem. Biotechnol. 70(2):267-275, 1998.
• ²⁰
  Nakamura, N.; Horikoshi, K. Characterization and some cultural conditions of a cyclodextrin-glycosyltransferase-producing alkalophilic Bacillus sp. Agr. Biol. Chem. 40(4):753-757, 1976a.
• ²¹
  Nakamura, N.; Horikoshi, K. Purification and properties of cyclodextrin glycosyltransferase of alkalophilic Bacillus sp. Agric. Biol. Chem. 40(9):935-941, 1976b.
• ²²
  Park, C.S.; Park, K.H.; Kim, S.H. A rapid screening method for alkaline b-cyclodextrin glucanotransferase using phenolphthalein-methylorange containing solid medium. Agric. Biol. Chem. 53(4):1167-1169,1989.
• ²³
  Pczczola, D.E. Production and potencial food application of cyclodextrins. Food Technol. 42: 96-100,1988.
• ²⁴
  Sabioni, J.G.; Park, Y.K. Production and characterization of cyclodextrin glycosyltransferase from Bacillus lentus Starch/Stärke. 44(6):225-229, 1992.
• ²⁵
  Sin, K.; Nakamura, A.; Masaki, H.; Uozumi, T. Extracellular production of Bacillus ohbensis cyclodextrin glucosiltransferase by Bacillus subtilis Biosci. Biotechnol. Biochem. 57(2):346-347,1993.
• ²⁶
  Starnes, R.L. Industrial potencial of cyclodextrin glycosyltransferases. Cereal Food World. 35:1094-1099,1990.
• ²⁷
  Szejtli, J. Utilization of cyclodextrins in industrial products and process. J. Mater. Chem. 7(4): 575-587,1997.
• ²⁸
  Terada, Y.; Yanase, M,; Takata, T.; Okada, S. Cyclodextrins are not the major cyclic a-1,4-glucans produced by the initial action of cyclodextrin glucanotransferase on amylase. J. Biol. Chem. 272(25):15729-15733,1997.
• ²⁹
  Thatai, A.; Kumari, M.; Mukherjee, K.J. A single step purification process for cyclodextrin glucanosiltransferase from a Bacillus sp. Isolated from soil. Preparative Biochem. Biotechnol. 29(1):35-47,1999.
• ³⁰
  Tomita, K.; Kaneda, M.; Kawamura, K.; Nakanishi, K. Purification and properties of a cyclodextrin glucanotransferase from Bacillus autolyticus 1149 and selective formation of b-cyclodextrin. J. Ferm. Bioeng. 75(2):89-92, 1993.
• ³¹
  Yim, D.G.; Sato, H.H.; Park, Y.H.; Park, Y.K. Production of cyclodextrin from starch by cyclodextrin glycosyltransferase from Bacillus firmus and characterization of purified enzyme. J. Ind. Microb. Biotechnol. 18:402-405, 1997.

Publication Dates

• Publication in this collection
  25 Nov 2005
• Date of issue
  Sept 2004

History

• Received
  19 Sept 2003
• Reviewed
  28 Apr 2004
• Accepted
  27 July 2004