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Maltooligosaccharides production catalysed by cyclodextrin glycosyltransferase from Bacillus circulans DF 9R in batch and continuous operation
Process Biochemistry 47 (2012) 2562–2565
Contents lists available at SciVerse ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Short communication
Maltooligosaccharides production catalysed by cyclodextrin glycosyltransferase from Bacillus circulans DF 9R in batch and continuous operation Jorgelina Andrea Rodríguez Gastón, Hernán Costa, Ana Lía Rossi, Norberto Krymkiewicz, Susana Alicia Ferrarotti ∗ Universidad Nacional de Luján, Departamento de Ciencias Básicas, Luján (6700), Buenos Aires, Argentina
a r t i c l e
i n f o
Article history: Received 14 May 2012 Received in revised form 7 August 2012 Accepted 9 August 2012 Available online 15 August 2012 Keywords: Cyclodextrin glycosyltransferase Bacillus circulans Maltooligosaccharides Disproportionation Acceptor Starch conversion
a b s t r a c t Cyclodextrin glycosyltransferase catalyses the synthesis of cyclodextrins from starch by intramolecular transglycosylation. However, under certain conditions, the reaction can be directed towards the production of linear dextrins, suppressing the synthesis of cyclodextrins. Thus, the starch acts as donor molecule and low molecular weight oligosaccharides act as acceptors in intermolecular transglycosylation reactions. In this paper the optimal conditions to transform starch into maltooligosaccharides were determined employing glucose and maltose as acceptors and cyclodextrin glycosyltransferase from Bacillus circulans DF 9R as catalyst. Maltooligosaccharides production was optimised in a batch process reaching a yield of 60%. Besides, a continuous process was developed in a membrane reactor, increasing the yield to 72% and multiplying eight times the productivity. In this way, it was possible to obtain a high purity product with higher efficiency, using a cyclodextrin glycosyltransferase. This is even more advantageous when compared with the processes currently used in industry which uses three enzymes. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Cyclodextrin glycosyltransferase (CGTase, E.C. 2.4.1.19) is a member of the glycoside hydrolase family 13, also known as the ␣-amylase family [1]. This enzyme converts starch into mixtures of cyclic, linear and limit dextrins. The relative composition of these mixtures depends mainly on the enzyme source. Cyclodextrins (CD), the most important products of the CGTase, are non-reducing maltooligosaccharides with a hydrophilic surface and an apolar cavity. The most common types are ␣,  and ␥-CD which consist of 6, 7 or 8 glucose residues respectively, linked by ␣-1,4 bonds [2]. As these molecules can form inclusion complexes with many compounds, they are widely used in the food, pharmaceutical, cosmetic and chemical industries [3–5]. With regard to linear maltooligosaccharides (MOS), they are defined by the International Union of Pure and Applied Chemistry as polymers of monosaccharides with degree of polymerization (DP) between 3 and 10. However, DP up to 20–25 are often consider as MOS [6]. In this work, the disaccharide maltose is included as MOS. In general, food-grade oligosaccharides are mixtures of oligosaccharides with different DP including disaccharides and monosaccharides. These mixtures have different properties
∗ Corresponding author. Tel.: +54 2323 423171x366; fax: +54 2323 425795. E-mail addresses: [email protected], [email protected] (S.A. Ferrarotti). 1359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2012.08.008
depending on the profile of oligosaccharides which they are composed. Saccharides with high molecular weight affect the solubility and solution stability, while those with low molecular weight affect fermentability, viscosity, sweet power, humectancy and crystallisation. They are applied as coating agents, fat replacers, viscosity providers and flavour carriers; besides, they prevent sucrose crystallisation and have anti-staling effect on bread [7–9]. MOS are industrially produced from starch by the action of debranching enzymes such as pullulanase (EC 3.2.1.41) and isoamylase (EC 3.2.1.68), combined with different ␣-amylases. The length of the obtained chains depends on the ␣-amylase used [10]. Research has been conducted with the aim of enhancing the performance of CD production processes and to change the specificity of CGTases in order to increase the concentration of a particular CD [11]. However, there is little information regarding the use of CGTases for the production of linear dextrins. Martin et al. [12] described the conversion of soluble starch (Paselli SA2) into MOS catalysed by a CGTase from Thermoanaerobacter sp. In the presence of appropriate acceptors, CGTase is able to produce MOS rather than CD. The acceptor specificity of this enzyme is fairly broad and its transglycosylation capability seems to be dependent on the source of CGTase [13]. Generally, the enzymatic conversion of starch is performed in batch reactors, but it is interesting to develop systems with immobilized enzymes allowing the separation of the products as they are formed. Among these systems the enzymatic membrane reactor stands out, offering certain advantages such as the localization of
J.A. Rodríguez Gastón et al. / Process Biochemistry 47 (2012) 2562–2565
enzymes in a definite area while preserving its catalytic activity and enabling their reuse. On the other hand, when the reaction products are removed, their inhibitory effects on the enzyme decrease [14]. Another benefit of this system is that the final product obtained has a high purity since the enzyme and the unreacted substrate are retained within the reactor. In a previous paper we described the optimisation of CD production using a CGTase from Bacillus circulans DF 9R and cassava starch as substrate [15]. Furthermore, we studied the effect of glucose and oligosaccharides from maltose to maltoheptaose on the initial rate of -CD production. Maltose was the strongest inhibitor while the other maltodextrins had a lesser influence on the enzyme activity and glucose did not show an inhibitory effect at the assayed concentrations [16]. However, we observed that the addition of high concentrations of glucose inhibited CD production and favoured intermolecular transglycosylation, leading to the synthesis of MOS. The purpose of this work was to develop the optimal conditions to transform starch into MOS employing low molecular weight carbohydrates such as maltose and glucose as acceptors. Besides, MOS production was optimised in both batch and continuous processes, using in the latter case a reactor coupled to an ultrafiltration membrane that was able to retain molecules greater than 10 kDa. 2. Materials and methods 2.1. Reagents ␣,  and ␥-CD, soluble potato, corn, rice and wheat starches, glucose, maltose, maltotriose and Maltodextrins DE 16.5–19.5 were obtained from Sigma Chemical Co., Mo, USA. Glicemia, glucose oxidase enzymatic method for glucose determination was obtained from Wiener Lab., Rosario, Argentina. Food-grade cassava starch was obtained from a local supplier. Other chemicals used were AR grade from Merck, Darmstadt, Germany. 2.2. CGTase production The experiments were carried out using a CGTase obtained from Bacillus circulans DF 9R, isolated from rotten potatoes and purified by affinity chromatography on ␣-CD coupled to Sepharose-4B [17]. The microorganism was cultured in a minimum saline medium for enzyme production as described in a previous report [18]. 2.3. CGTase cyclizing activity The cyclizing activity of CGTase was determined according to the phenolphthalein method [19], measuring the -CD production spectrophotometrically at 550 nm on the basis of its ability to form a colourless inclusion complex with this dye. One unit of CGTase is defined as the amount of enzyme that catalyses the production of 1 mol of -CD per min under the reaction conditions. 2.4. Effect of acceptors on CGTase activity Enzyme working conditions used in these experiments were previously optimised [15]. In order to study the reaction products obtained by CGTase activity on starch in the presence of acceptors, flasks containing 10 mL of 5.0% cassava starch were incubated with 15 U of purified enzyme per gram of starch in a phosphate buffer 25 mM pH 6.4 at 56 ◦ C and 100 rpm at different time periods up to 24 h (4, 6, 8, 10, 12 and 24 h). Prior to incubation, starch suspensions were heated at 95 ◦ C until gelatinization was reached; then several concentrations of glucose or maltose from 1.25 to 5.0% were added. Control assays were carried out in the absence of acceptors. All the experiments were performed at least three times. The reaction products were analysed by colorimetric reactions and HPLC. 2.5. Colorimetric determination of CD and glucose concentration The ␣-CD concentration was assayed by the decrease in absorbance at 507 nm due to a methyl orange ␣-CD complex formation [20]. The -CD concentration was determined according to the method described in Section 2.3. The ␥-CD concentration was determined, measuring the absorbance at 630 nm due to the formation of an inclusion complex with bromocresol green [21]. The glucose concentration was determined using the glucose oxidase method mentioned in Section 2.1. 2.6. Analysis of products by HPLC The oligosaccharides obtained were analysed by HPLC using a Könik KNK-500 apparatus, with a column for carbohydrate analysis (LiChrospher® 100 NH2 – 5 m)
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at 30 ◦ C. Samples (20 L, 20–40 mg of carbohydrate/mL) were injected and oligosaccharides were eluted with acetonitrile:water (70:30), at a flow rate of 1.0 mL/min. Sugars were detected with a Shimadzu RID-10A differential refraction detector. ␣,  and ␥-CD, glucose, maltose, maltotriose and a commercial mixture of Maltodextrins DE 16.5–19.5, were employed as standards for HPLC analysis. 2.7. Batch production of MOS Once the most suitable acceptor and its optimal concentration were selected to produce MOS, the batch production was carried out adding the acceptor to flasks containing 50 mL of 5.0% gelatinized cassava starch, 15 U of purified enzyme per gram of starch in phosphate buffer 25 mM pH 6.4. Flasks were incubated at 56 ◦ C and 100 rpm during 10 h. Starches from other sources such as potato, corn, rice and wheat were also tested. All the experiments were performed at least three times. The reaction products were analysed by colorimetric reactions and HPLC. 2.8. Production of MOS in continuous operation The continuous production of MOS was carried out using as reactor a 3 L BioFlo 110 New Brunswick Scientific, USA, coupled to an Ultrafiltration cell, model 8200, Amicon, USA, with Amicon YM10 membrane (diameter 63.5 mm, NMWL 10 kDa). To 1 L of reaction medium consisting of 2.0% liquefied cassava starch solution plus 2.0% glucose in a phosphate buffer 25 mM pH 6.4, 15 U of purified enzyme per gram of starch were added. The incubation conditions were 56 ◦ C and 100 rpm. The flow rate was on average 2 mL/min using a vacuum pump. Fractions containing the products whose molecular weights below 10 kDa were collected at different times during 40 h. As the products were separated, the reactor was fed simultaneously, completing the volume to 1 L with fresh solution of starch and glucose in phosphate buffer through the action of a level sensor and a peristaltic pump. The reaction products were analysed by colorimetric reactions and HPLC. 2.9. Calculation of MOS yield of both processes To calculate the yield of the two processes, the initial mass of starch and the dry weight of products were considered. The mixture obtained in the batch process was previously ultrafiltered to separate limit dextrins and enzyme. Furthermore, 40 mL aliquots of reaction products were placed in plastic vials, frozen at −20 ◦ C and then lyophilised using a Labconco Free Zone 12 L, Freeze dry System, Model 77540. The mass of glucose determined by the enzymatic method was subtracted from the mass measured after lyophilisation. In parallel, the mass of total MOS and glucose was estimated by HPLC using as reference glucose and MOS. Glucose determined by colorimetric method was subtracted from the total mass.
3. Results and discussion 3.1. Effects of acceptors on CGTase activity CGTase activity on cassava starch under the conditions described in Section 2.4 led mainly to the CD production. However, low concentrations of MOS were also obtained. Fig. 1 shows the presence of glucose, maltose and maltotriose as well as CD. MOS higher than maltotriose may be present in very low concentrations but their retention times match those of the CD. In the presence of small sugars such as glucose and maltose, which act as acceptors, the intermolecular transglycosylation was favoured. Thus, disproportionation reactions predominated over cyclization reactions and linear oligosaccharides were the main reaction products [13]. In order to enhance MOS production, glucose or maltose were added into the cassava starch solutions. The addition of these small saccharides to reaction mixtures led to an increase in MOS production and a decrease in CD production. Fig. 2 shows that glucose has stronger suppressive effect than maltose on CD synthesis. Furthermore, when the glucose concentration was equal to the starch concentration (5.0%), the three kinds of CD became undetectable. In these assays, each type of CD was quantified by colorimetric reactions as described in Section 2.5, because the presence of high concentration of maltodextrins in the reaction medium interferes with the CD determination by HPLC. Once it was found that the samples had very low CD concentration they were analysed by HPLC. The chromatographic profiles of reaction products showed glucose and a mixture of MOS. Higher glucose concentrations gave rise to molecules of DP up to M10 (Fig. 3). These results were observed
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Fig. 1. Reaction products obtained by the action of CGTase on starch HPLC elution pattern (LiChrospher® 100 NH2 – 5 m) of the reaction products obtained by the action of CGTase on cassava starch after 4 h reaction. The elution solvent was acetonitrile:water (70:30) and the flow rate was 1.0 mL/min. Abbreviations indicate Glu, glucose; M2 , maltose; M3 , maltotriose; CD, cyclodextrin.
after an incubation time of 24 h. To optimise the reaction time, aliquots were taken from 4 to 24 h incubation. Reaction mixtures analysis showed that an incubation time of 10 h was enough to achieve the same results as 24 h; because of that, the reaction was concluded after 10 h. There were no variations in the length of the MOS obtained as a result of different incubation times and the chromatographic profiles obtained were the same as in Fig. 3. Using maltose as acceptor, MOS until M12 were obtained with all concentrations tested but it should be considered that the CD were also present in these mixtures (data not shown). 3.2. MOS production in batch The highest yield of MOS production with absence of CD was obtained with the addition of 5.0% glucose to 5.0% starch solution with 15 U of purified enzyme per gram of starch. The reaction mixture was incubated in buffer 25 mM pH 6.4 at 56 ◦ C and 100 rpm for 10 h. Process yield, calculated according to Section 2.9, was 60.0 ± 4.2% and productivity was 80.0 ± 5.2 mg MOS per unit of enzyme. Under these conditions, several tests were carried out using starches from different sources such as potato, corn, rice and wheat. HPLC chromatograms showed similar elution profiles to those obtained using cassava starch as substrate. Based on those
Fig. 2. Acceptors effect on CGTase cyclization activity. Effect of the addition of glucose (left) or maltose (right) at concentrations from 1.25 to 5.00% in the reaction mixture containing cassava starch and CGTase on the CD production. Black bars indicate ␣-CD; grey bars, -CD and white bars, ␥-CD.
Fig. 3. HPLC elution diagrams of the products obtained by the action of CGTase on the starch in the presence of glucose HPLC elution pattern (LiChrospher® 100 NH2 – 5 m) of the reaction products obtained by the action of CGTase on 5.0% cassava starch plus 5.0% glucose after 10 h incubation. The elution solvent was acetonitrile:water (70:30) and the flow rate 1.0 mL/min. Abbreviations indicate M2 , maltose; M3 , maltotriose; M4 , maltotetraose; M5 , maltopentaose; M6 , maltohexaose; M7 , maltoheptaose; M8 , maltooctaose; M9 , maltononaose; M10 , maltodecaose. The area corresponding to each oligosaccharide taking as 100% the sum of all of them were: M2 , 36.3%; M3 , 24.1%; M4 , 15.6%; M5 , 9.2%; M6 , 5.5%; M7 , 3.8%; M8 , 1.3%; M9 , 1.8%; M10 , 2.4%.
chromatograms the yield of MOS production for each starch was estimated: cassava starch (60.0 ± 3.7%), potato starch (59.6 ± 3.5%), corn starch (63.3 ± 2.2%), rice starch (64.4 ± 1.6%) and wheat starch (62.6 ± 1.5%). The analysis of variance, one-way ANOVA test, was used to compare differences between yield with different starches tested. No significant differences (p < 0.001) were observed. Therefore, starches from different source as those tested could be used as substrate. 3.3. Continuous process for MOS production In order to increase conversion yield from cassava starch and allow the reuse of CGTase, an ultrafiltration membrane reactor system was employed. Operating conditions for the continuous process were based on those previously used for batch production (Section 3.2). With regard to the substrate, it was observed that high starch concentrations significantly decreased the filtration rate due to the high viscosity of these solutions. Because of this, it was necessary to reduce the substrate concentration although this was disadvantageous for system performance. Anyway, when the starch concentration was fixed at 2.0%, an adequate flow rate was maintained while a quite good performance was obtained. For this reason, working conditions were fixed: 2.0% cassava starch, 2.0% glucose, 15 U of purified enzyme per gram of starch in buffer 25 mM pH 6.4 at 56 ◦ C and 100 rpm. In this way, the flow rate was maintained on average at 2 mL/min. The assay was started with 1 L medium volume in the reactor. CGTase and unconverted starch were retained within the reactor system by the membrane with a cut-off of 10 kDa. The cut-off size was selected based on reports of other authors who found that working with CD whose molecular weights are similar to those of MOS, lower membrane cut-off leads to a reduction in the permeate flux [22]. It should be noted that the enzyme is retained by the membrane because their molecular weight is 74.47 kDa. As the reaction progressed, synthesized MOS were removed from the reactor by ultrafiltration
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Acknowledgements We thank Dr. Natalia Szerman for critically reading the manuscript and Prof. Susan Levin for language supervision. The work was supported by grants from the Universidad Nacional de Luján, Luján, Buenos Aires, Argentina. References
Fig. 4. Maltooligosaccharides concentrations obtained in the continuous process. Maltooligosaccharides concentration determined in the fractions collected over 40 h in a continuous production process employing a reactor coupled to ultrafiltration membrane.
through the membrane. On the fractions collected, volume, CD and MOS concentration were measured. CD concentration was determined by colorimetric techniques. The three kinds of CD remained undetectable in all fractions. MOS concentration and relative composition of the mixtures were determined by HPLC. Furthermore, the process yield, calculated as was described in Section 2.9, was 72.4 ± 3.6% for the continuous process, 12% higher than the one obtained in the classical batch operation described in Section 3.2. Also the efficiency of enzyme use was increased by employing the membrane reactor, yielding 641.5 ± 18.6 mg of MOS per unit of CGTase whereas in the batch process, 80.0 mg per unit of CGTase were obtained. After 40 h of continuous operation, residual enzyme activity was 81% of initial activity. This would indicate that the enzyme could still be used for longer periods. Fig. 4 shows MOS concentration measured in all fractions collected. Moreover, all fractions had equal relative composition as observed in the HPLC profiles (data not shown). The product obtained by the process in the membrane reactor contains only glucose and MOS. No further purification is necessary. This type of mixtures of oligosaccharides has application in milk and food for children, soft drinks, sports drinks and energy supplements [8]. 4. Conclusion CGTase from B. circulans DF 9R allowed the production of short chain MOS, up to M10, with a yield of 60% using starch from several sources as donor substrate and glucose as acceptor in a batch process. Processing performance was improved by employing a membrane reactor which allowed retaining the enzyme and substrate; while reaction products were removed as they are being produced, obtaining a yield of 72%. The productivity per unit of enzyme achieved was eight times higher than that obtained in the batch process. Thus, it was possible to obtain MOS with high purity using a single enzyme.
[1] Stam MR, Danchin EGJ, Rancurel C, Coutinho PM, Henrissat B. Dividing the large glycoside hydrolase family 13 into subfamilies: towards improved functional annotation of ␣-amylase-related proteins. Protein Eng Des Sel 2006;1(12):555–62. [2] Singh R, Bharti N, Madan J, Hiremath SN. Characterization of cyclodextrin inclusion complexes – a review. J Pharm Sci Technol 2010;2:171–83. [3] Loftsson T, Duchêne D. Cyclodextrins and their pharmaceutical applications. Int J Pharm 2007;329:1–11. [4] Parrot-Lopez H, Perret F, Bertino-Ghera B. Amphiphilic cyclodextrins and their applications. Preparation of nanoparticles based on amphiphilic cyclodextrins for biomedical applications. Ann Pharm Fr 2010;68:12–26. [5] Szente L, Szejtli J. Cyclodextrins as food ingredients. Trends Food Sci Technol 2004;5:137–42. [6] Barreteau H, Delattre C, Michaud P. Production of oligosaccharides as promising new food additive generation. Food Technol Biotechnol 2006;44(3): 323–33. [7] Nagarajan DR, Rajagopalan G, Krihnan C. Purification and characterization of maltooligosaccharide-forming ␣-amylase from a new Bacillus subtilis KCC103. Appl Microbiol Biotechnol 2006;73:591–7. [8] Marchal LM, Beeftink HH, Tramper J. Towards a rational design of commercial maltodextrins. Trends Food Sci Technol 1999;10:345–55. [9] Plácido Moore GR, Rodríguez do Canto L, Amante ER. Cassava and corn starch in maltodextrin production. Quim Nova 2005;28:596–600. [10] Doukyu N, Yamagishi W, Kuwahara H, Ogino H, Furuki N. Purification and characterization of a maltooligosaccharide-forming amylase that improves product selectivity in water-miscible organic solvents, from dimethylsulfoxide-tolerant Brachybacterium sp. strain LB25. Extremophiles 2007;11:781–8. [11] Leemhuis H, Kelly RM, Dijkhuizen L. Engineering of cyclodextrin glucanotransferases and the impact for biotechnological applications. Appl Microbiol Biotechnol 2010;85:823–35. [12] Martin MT, Alcalde M, Plou FJ, Dijkhuizen L, Ballesteros A. Sintesis of malto-oligosaccharides via the aceptor reaction catalyzed by cyclodextrin glycosyltransferases. Biocatal Biotransform 2001;19:21–35. [13] Plou FJ, Martin MT, Gómez de Segura S, Alcalde M, Ballesteros A. Glucosyltransferases acting on starch or sucrose for the synthesis of oligosaccharides. Can J Chem 2002;80:743–52. [14] Ma J, Zhang L, Liang Z, Zhang W, Zhang Y. Recent advances in immobilized enzymatic reactors and their applications in proteome analysis. Anal Chim Acta 2009;632:1–8. [15] Szerman N, Schroh I, Rossi AL, Rosso AM, Krymkiewicz N, Ferrarotti S. Cyclodextrins production by cyclodextrin glycosyltransferase from Bacillus circulans DF 9R. Bioresour Technol 2007;98(15):2886–91. [16] Rodríguez Gastón JA, Szerman N, Costa H, Krymkiewicz N, Ferrarotti SA. Cyclodextrin glycosyltransferase from Bacillus circulans DF 9R: activity and kinetic studies. Enzyme Microb Technol 2009;45:36–41. [17] Ferrarotti SA, Rosso AM, Maréchal MA, Krymkiewicz N, Maréchal LR. Isolation of two strains (S-R type) of Bacillus circulans and purification of a cyclomaltodextrin-glucanotransferase. Cell Mol Biol 1996;42:653–7. [18] Rosso AM, Ferrarotti SA, Krymkiewicz N, Nudel BC. Optimisation of batch culture conditions for cyclodextrin glucanotransferase production from Bacillus circulans DF 9R. Microb Cell Factories 2002:1–10. [19] Goel A, Nene S. Modifications in the phenolphthalein method for spectrophotometric estimation of beta cyclodextrin. Starch 1995;47:399–400. [20] Higuti IH, da Silva PA, Papp J, Mayumi de Eiróz Okiyama V, Alves de Andrade E, Abreu Marcondes A, et al. Colorimetric determination of ␣ and -cyclodextrins and studies on optimization of CGTase production from B. firmus using factorial designs. Braz Arch Biol Technol 2004;47(6):837–41. [21] Kato T, Horikoshi K. Colorimetric determination of ␥-cyclodextrin. Anal Chem 1984;56:1738–40. [22] Slominska L, Szostek A, Grzeskowiak A. Studies on enzymatic continuous production of cyclodextrins in an ultrafiltration membrane reactor. Carbohydr Polym 2002;50:423–8.
Contents lists available at SciVerse ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Short communication
Maltooligosaccharides production catalysed by cyclodextrin glycosyltransferase from Bacillus circulans DF 9R in batch and continuous operation Jorgelina Andrea Rodríguez Gastón, Hernán Costa, Ana Lía Rossi, Norberto Krymkiewicz, Susana Alicia Ferrarotti ∗ Universidad Nacional de Luján, Departamento de Ciencias Básicas, Luján (6700), Buenos Aires, Argentina
a r t i c l e
i n f o
Article history: Received 14 May 2012 Received in revised form 7 August 2012 Accepted 9 August 2012 Available online 15 August 2012 Keywords: Cyclodextrin glycosyltransferase Bacillus circulans Maltooligosaccharides Disproportionation Acceptor Starch conversion
a b s t r a c t Cyclodextrin glycosyltransferase catalyses the synthesis of cyclodextrins from starch by intramolecular transglycosylation. However, under certain conditions, the reaction can be directed towards the production of linear dextrins, suppressing the synthesis of cyclodextrins. Thus, the starch acts as donor molecule and low molecular weight oligosaccharides act as acceptors in intermolecular transglycosylation reactions. In this paper the optimal conditions to transform starch into maltooligosaccharides were determined employing glucose and maltose as acceptors and cyclodextrin glycosyltransferase from Bacillus circulans DF 9R as catalyst. Maltooligosaccharides production was optimised in a batch process reaching a yield of 60%. Besides, a continuous process was developed in a membrane reactor, increasing the yield to 72% and multiplying eight times the productivity. In this way, it was possible to obtain a high purity product with higher efficiency, using a cyclodextrin glycosyltransferase. This is even more advantageous when compared with the processes currently used in industry which uses three enzymes. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Cyclodextrin glycosyltransferase (CGTase, E.C. 2.4.1.19) is a member of the glycoside hydrolase family 13, also known as the ␣-amylase family [1]. This enzyme converts starch into mixtures of cyclic, linear and limit dextrins. The relative composition of these mixtures depends mainly on the enzyme source. Cyclodextrins (CD), the most important products of the CGTase, are non-reducing maltooligosaccharides with a hydrophilic surface and an apolar cavity. The most common types are ␣,  and ␥-CD which consist of 6, 7 or 8 glucose residues respectively, linked by ␣-1,4 bonds [2]. As these molecules can form inclusion complexes with many compounds, they are widely used in the food, pharmaceutical, cosmetic and chemical industries [3–5]. With regard to linear maltooligosaccharides (MOS), they are defined by the International Union of Pure and Applied Chemistry as polymers of monosaccharides with degree of polymerization (DP) between 3 and 10. However, DP up to 20–25 are often consider as MOS [6]. In this work, the disaccharide maltose is included as MOS. In general, food-grade oligosaccharides are mixtures of oligosaccharides with different DP including disaccharides and monosaccharides. These mixtures have different properties
∗ Corresponding author. Tel.: +54 2323 423171x366; fax: +54 2323 425795. E-mail addresses: [email protected], [email protected] (S.A. Ferrarotti). 1359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2012.08.008
depending on the profile of oligosaccharides which they are composed. Saccharides with high molecular weight affect the solubility and solution stability, while those with low molecular weight affect fermentability, viscosity, sweet power, humectancy and crystallisation. They are applied as coating agents, fat replacers, viscosity providers and flavour carriers; besides, they prevent sucrose crystallisation and have anti-staling effect on bread [7–9]. MOS are industrially produced from starch by the action of debranching enzymes such as pullulanase (EC 3.2.1.41) and isoamylase (EC 3.2.1.68), combined with different ␣-amylases. The length of the obtained chains depends on the ␣-amylase used [10]. Research has been conducted with the aim of enhancing the performance of CD production processes and to change the specificity of CGTases in order to increase the concentration of a particular CD [11]. However, there is little information regarding the use of CGTases for the production of linear dextrins. Martin et al. [12] described the conversion of soluble starch (Paselli SA2) into MOS catalysed by a CGTase from Thermoanaerobacter sp. In the presence of appropriate acceptors, CGTase is able to produce MOS rather than CD. The acceptor specificity of this enzyme is fairly broad and its transglycosylation capability seems to be dependent on the source of CGTase [13]. Generally, the enzymatic conversion of starch is performed in batch reactors, but it is interesting to develop systems with immobilized enzymes allowing the separation of the products as they are formed. Among these systems the enzymatic membrane reactor stands out, offering certain advantages such as the localization of
J.A. Rodríguez Gastón et al. / Process Biochemistry 47 (2012) 2562–2565
enzymes in a definite area while preserving its catalytic activity and enabling their reuse. On the other hand, when the reaction products are removed, their inhibitory effects on the enzyme decrease [14]. Another benefit of this system is that the final product obtained has a high purity since the enzyme and the unreacted substrate are retained within the reactor. In a previous paper we described the optimisation of CD production using a CGTase from Bacillus circulans DF 9R and cassava starch as substrate [15]. Furthermore, we studied the effect of glucose and oligosaccharides from maltose to maltoheptaose on the initial rate of -CD production. Maltose was the strongest inhibitor while the other maltodextrins had a lesser influence on the enzyme activity and glucose did not show an inhibitory effect at the assayed concentrations [16]. However, we observed that the addition of high concentrations of glucose inhibited CD production and favoured intermolecular transglycosylation, leading to the synthesis of MOS. The purpose of this work was to develop the optimal conditions to transform starch into MOS employing low molecular weight carbohydrates such as maltose and glucose as acceptors. Besides, MOS production was optimised in both batch and continuous processes, using in the latter case a reactor coupled to an ultrafiltration membrane that was able to retain molecules greater than 10 kDa. 2. Materials and methods 2.1. Reagents ␣,  and ␥-CD, soluble potato, corn, rice and wheat starches, glucose, maltose, maltotriose and Maltodextrins DE 16.5–19.5 were obtained from Sigma Chemical Co., Mo, USA. Glicemia, glucose oxidase enzymatic method for glucose determination was obtained from Wiener Lab., Rosario, Argentina. Food-grade cassava starch was obtained from a local supplier. Other chemicals used were AR grade from Merck, Darmstadt, Germany. 2.2. CGTase production The experiments were carried out using a CGTase obtained from Bacillus circulans DF 9R, isolated from rotten potatoes and purified by affinity chromatography on ␣-CD coupled to Sepharose-4B [17]. The microorganism was cultured in a minimum saline medium for enzyme production as described in a previous report [18]. 2.3. CGTase cyclizing activity The cyclizing activity of CGTase was determined according to the phenolphthalein method [19], measuring the -CD production spectrophotometrically at 550 nm on the basis of its ability to form a colourless inclusion complex with this dye. One unit of CGTase is defined as the amount of enzyme that catalyses the production of 1 mol of -CD per min under the reaction conditions. 2.4. Effect of acceptors on CGTase activity Enzyme working conditions used in these experiments were previously optimised [15]. In order to study the reaction products obtained by CGTase activity on starch in the presence of acceptors, flasks containing 10 mL of 5.0% cassava starch were incubated with 15 U of purified enzyme per gram of starch in a phosphate buffer 25 mM pH 6.4 at 56 ◦ C and 100 rpm at different time periods up to 24 h (4, 6, 8, 10, 12 and 24 h). Prior to incubation, starch suspensions were heated at 95 ◦ C until gelatinization was reached; then several concentrations of glucose or maltose from 1.25 to 5.0% were added. Control assays were carried out in the absence of acceptors. All the experiments were performed at least three times. The reaction products were analysed by colorimetric reactions and HPLC. 2.5. Colorimetric determination of CD and glucose concentration The ␣-CD concentration was assayed by the decrease in absorbance at 507 nm due to a methyl orange ␣-CD complex formation [20]. The -CD concentration was determined according to the method described in Section 2.3. The ␥-CD concentration was determined, measuring the absorbance at 630 nm due to the formation of an inclusion complex with bromocresol green [21]. The glucose concentration was determined using the glucose oxidase method mentioned in Section 2.1. 2.6. Analysis of products by HPLC The oligosaccharides obtained were analysed by HPLC using a Könik KNK-500 apparatus, with a column for carbohydrate analysis (LiChrospher® 100 NH2 – 5 m)
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at 30 ◦ C. Samples (20 L, 20–40 mg of carbohydrate/mL) were injected and oligosaccharides were eluted with acetonitrile:water (70:30), at a flow rate of 1.0 mL/min. Sugars were detected with a Shimadzu RID-10A differential refraction detector. ␣,  and ␥-CD, glucose, maltose, maltotriose and a commercial mixture of Maltodextrins DE 16.5–19.5, were employed as standards for HPLC analysis. 2.7. Batch production of MOS Once the most suitable acceptor and its optimal concentration were selected to produce MOS, the batch production was carried out adding the acceptor to flasks containing 50 mL of 5.0% gelatinized cassava starch, 15 U of purified enzyme per gram of starch in phosphate buffer 25 mM pH 6.4. Flasks were incubated at 56 ◦ C and 100 rpm during 10 h. Starches from other sources such as potato, corn, rice and wheat were also tested. All the experiments were performed at least three times. The reaction products were analysed by colorimetric reactions and HPLC. 2.8. Production of MOS in continuous operation The continuous production of MOS was carried out using as reactor a 3 L BioFlo 110 New Brunswick Scientific, USA, coupled to an Ultrafiltration cell, model 8200, Amicon, USA, with Amicon YM10 membrane (diameter 63.5 mm, NMWL 10 kDa). To 1 L of reaction medium consisting of 2.0% liquefied cassava starch solution plus 2.0% glucose in a phosphate buffer 25 mM pH 6.4, 15 U of purified enzyme per gram of starch were added. The incubation conditions were 56 ◦ C and 100 rpm. The flow rate was on average 2 mL/min using a vacuum pump. Fractions containing the products whose molecular weights below 10 kDa were collected at different times during 40 h. As the products were separated, the reactor was fed simultaneously, completing the volume to 1 L with fresh solution of starch and glucose in phosphate buffer through the action of a level sensor and a peristaltic pump. The reaction products were analysed by colorimetric reactions and HPLC. 2.9. Calculation of MOS yield of both processes To calculate the yield of the two processes, the initial mass of starch and the dry weight of products were considered. The mixture obtained in the batch process was previously ultrafiltered to separate limit dextrins and enzyme. Furthermore, 40 mL aliquots of reaction products were placed in plastic vials, frozen at −20 ◦ C and then lyophilised using a Labconco Free Zone 12 L, Freeze dry System, Model 77540. The mass of glucose determined by the enzymatic method was subtracted from the mass measured after lyophilisation. In parallel, the mass of total MOS and glucose was estimated by HPLC using as reference glucose and MOS. Glucose determined by colorimetric method was subtracted from the total mass.
3. Results and discussion 3.1. Effects of acceptors on CGTase activity CGTase activity on cassava starch under the conditions described in Section 2.4 led mainly to the CD production. However, low concentrations of MOS were also obtained. Fig. 1 shows the presence of glucose, maltose and maltotriose as well as CD. MOS higher than maltotriose may be present in very low concentrations but their retention times match those of the CD. In the presence of small sugars such as glucose and maltose, which act as acceptors, the intermolecular transglycosylation was favoured. Thus, disproportionation reactions predominated over cyclization reactions and linear oligosaccharides were the main reaction products [13]. In order to enhance MOS production, glucose or maltose were added into the cassava starch solutions. The addition of these small saccharides to reaction mixtures led to an increase in MOS production and a decrease in CD production. Fig. 2 shows that glucose has stronger suppressive effect than maltose on CD synthesis. Furthermore, when the glucose concentration was equal to the starch concentration (5.0%), the three kinds of CD became undetectable. In these assays, each type of CD was quantified by colorimetric reactions as described in Section 2.5, because the presence of high concentration of maltodextrins in the reaction medium interferes with the CD determination by HPLC. Once it was found that the samples had very low CD concentration they were analysed by HPLC. The chromatographic profiles of reaction products showed glucose and a mixture of MOS. Higher glucose concentrations gave rise to molecules of DP up to M10 (Fig. 3). These results were observed
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Fig. 1. Reaction products obtained by the action of CGTase on starch HPLC elution pattern (LiChrospher® 100 NH2 – 5 m) of the reaction products obtained by the action of CGTase on cassava starch after 4 h reaction. The elution solvent was acetonitrile:water (70:30) and the flow rate was 1.0 mL/min. Abbreviations indicate Glu, glucose; M2 , maltose; M3 , maltotriose; CD, cyclodextrin.
after an incubation time of 24 h. To optimise the reaction time, aliquots were taken from 4 to 24 h incubation. Reaction mixtures analysis showed that an incubation time of 10 h was enough to achieve the same results as 24 h; because of that, the reaction was concluded after 10 h. There were no variations in the length of the MOS obtained as a result of different incubation times and the chromatographic profiles obtained were the same as in Fig. 3. Using maltose as acceptor, MOS until M12 were obtained with all concentrations tested but it should be considered that the CD were also present in these mixtures (data not shown). 3.2. MOS production in batch The highest yield of MOS production with absence of CD was obtained with the addition of 5.0% glucose to 5.0% starch solution with 15 U of purified enzyme per gram of starch. The reaction mixture was incubated in buffer 25 mM pH 6.4 at 56 ◦ C and 100 rpm for 10 h. Process yield, calculated according to Section 2.9, was 60.0 ± 4.2% and productivity was 80.0 ± 5.2 mg MOS per unit of enzyme. Under these conditions, several tests were carried out using starches from different sources such as potato, corn, rice and wheat. HPLC chromatograms showed similar elution profiles to those obtained using cassava starch as substrate. Based on those
Fig. 2. Acceptors effect on CGTase cyclization activity. Effect of the addition of glucose (left) or maltose (right) at concentrations from 1.25 to 5.00% in the reaction mixture containing cassava starch and CGTase on the CD production. Black bars indicate ␣-CD; grey bars, -CD and white bars, ␥-CD.
Fig. 3. HPLC elution diagrams of the products obtained by the action of CGTase on the starch in the presence of glucose HPLC elution pattern (LiChrospher® 100 NH2 – 5 m) of the reaction products obtained by the action of CGTase on 5.0% cassava starch plus 5.0% glucose after 10 h incubation. The elution solvent was acetonitrile:water (70:30) and the flow rate 1.0 mL/min. Abbreviations indicate M2 , maltose; M3 , maltotriose; M4 , maltotetraose; M5 , maltopentaose; M6 , maltohexaose; M7 , maltoheptaose; M8 , maltooctaose; M9 , maltononaose; M10 , maltodecaose. The area corresponding to each oligosaccharide taking as 100% the sum of all of them were: M2 , 36.3%; M3 , 24.1%; M4 , 15.6%; M5 , 9.2%; M6 , 5.5%; M7 , 3.8%; M8 , 1.3%; M9 , 1.8%; M10 , 2.4%.
chromatograms the yield of MOS production for each starch was estimated: cassava starch (60.0 ± 3.7%), potato starch (59.6 ± 3.5%), corn starch (63.3 ± 2.2%), rice starch (64.4 ± 1.6%) and wheat starch (62.6 ± 1.5%). The analysis of variance, one-way ANOVA test, was used to compare differences between yield with different starches tested. No significant differences (p < 0.001) were observed. Therefore, starches from different source as those tested could be used as substrate. 3.3. Continuous process for MOS production In order to increase conversion yield from cassava starch and allow the reuse of CGTase, an ultrafiltration membrane reactor system was employed. Operating conditions for the continuous process were based on those previously used for batch production (Section 3.2). With regard to the substrate, it was observed that high starch concentrations significantly decreased the filtration rate due to the high viscosity of these solutions. Because of this, it was necessary to reduce the substrate concentration although this was disadvantageous for system performance. Anyway, when the starch concentration was fixed at 2.0%, an adequate flow rate was maintained while a quite good performance was obtained. For this reason, working conditions were fixed: 2.0% cassava starch, 2.0% glucose, 15 U of purified enzyme per gram of starch in buffer 25 mM pH 6.4 at 56 ◦ C and 100 rpm. In this way, the flow rate was maintained on average at 2 mL/min. The assay was started with 1 L medium volume in the reactor. CGTase and unconverted starch were retained within the reactor system by the membrane with a cut-off of 10 kDa. The cut-off size was selected based on reports of other authors who found that working with CD whose molecular weights are similar to those of MOS, lower membrane cut-off leads to a reduction in the permeate flux [22]. It should be noted that the enzyme is retained by the membrane because their molecular weight is 74.47 kDa. As the reaction progressed, synthesized MOS were removed from the reactor by ultrafiltration
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Acknowledgements We thank Dr. Natalia Szerman for critically reading the manuscript and Prof. Susan Levin for language supervision. The work was supported by grants from the Universidad Nacional de Luján, Luján, Buenos Aires, Argentina. References
Fig. 4. Maltooligosaccharides concentrations obtained in the continuous process. Maltooligosaccharides concentration determined in the fractions collected over 40 h in a continuous production process employing a reactor coupled to ultrafiltration membrane.
through the membrane. On the fractions collected, volume, CD and MOS concentration were measured. CD concentration was determined by colorimetric techniques. The three kinds of CD remained undetectable in all fractions. MOS concentration and relative composition of the mixtures were determined by HPLC. Furthermore, the process yield, calculated as was described in Section 2.9, was 72.4 ± 3.6% for the continuous process, 12% higher than the one obtained in the classical batch operation described in Section 3.2. Also the efficiency of enzyme use was increased by employing the membrane reactor, yielding 641.5 ± 18.6 mg of MOS per unit of CGTase whereas in the batch process, 80.0 mg per unit of CGTase were obtained. After 40 h of continuous operation, residual enzyme activity was 81% of initial activity. This would indicate that the enzyme could still be used for longer periods. Fig. 4 shows MOS concentration measured in all fractions collected. Moreover, all fractions had equal relative composition as observed in the HPLC profiles (data not shown). The product obtained by the process in the membrane reactor contains only glucose and MOS. No further purification is necessary. This type of mixtures of oligosaccharides has application in milk and food for children, soft drinks, sports drinks and energy supplements [8]. 4. Conclusion CGTase from B. circulans DF 9R allowed the production of short chain MOS, up to M10, with a yield of 60% using starch from several sources as donor substrate and glucose as acceptor in a batch process. Processing performance was improved by employing a membrane reactor which allowed retaining the enzyme and substrate; while reaction products were removed as they are being produced, obtaining a yield of 72%. The productivity per unit of enzyme achieved was eight times higher than that obtained in the batch process. Thus, it was possible to obtain MOS with high purity using a single enzyme.
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