- Email: [email protected]
Immobilization of commercial cyclomaltodextrin glucanotransferase into controlled pore silica by the anchorage method and covalent bonding
Process Biochemistry 85 (2019) 68–77
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Immobilization of commercial cyclomaltodextrin glucanotransferase into controlled pore silica by the anchorage method and covalent bonding
T
Gabriela Gregolin Gimeneza, Richard Marllon Silvab, Carolina Pereira Franciscoc, Fabiana dos Santos Randod, João Henrique Dantasc, Hâmara Milaneze de Souzab, ⁎ Graciette Matiolia, a
Department of Pharmacy, State University of Maringa (UEM), Av. Colombo, 5790, Maringa, PR 87020-900, Brazil Food Science Department, State University of Maringa (UEM), Av. Colombo, 5790, Maringa, PR 87020-900, Brazil c Department of Chemical Engineering, State University of Maringa (UEM), Av. Colombo, 5790, Maringa, PR 87020-900, Brazil d Department of Cell Biology and Genetics, State University of Maringa (UEM), Av. Colombo, 5790, Maringa, PR 87020-900, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Immobilization CGTase Anchorage Covalent bond Cyclodextrins
Cyclomaltodextrin glucanotransferase (CGTase) is used mainly for the industrial production of cyclodextrins (CDs). Their potential use and availability have played a decisive role in increasing demand for CDs. The aim of the present study was to immobilize the commercial CGTase (Toruzyme® 3.0L) into controlled pore silica by surface anchoring (SCGT-A) and covalent bonding (SCGT-CB) and to apply the immobilized enzymes in the production of CDs. The SCGT-A method proved to be efficient due to its short immobilization time and, it was possible to immobilize 89.63% of CGTase, in terms of activity. Commercial CGTase immobilized by SCGT-CB provided an immobilization yield of 48.44%, also in terms of activity. There was an increase in total CD production of 60.97% and 34.90% for SCGT-A and SCGT-CB, respectively, in a reaction medium with 10% ethanol, compared to the same media without ethanol. Higher CD production occured at 24 and 48 h for SCGT-A and SCGT-CB methods, respectively. The use of SCGT-A was more efficient than SCGT-CB and free enzyme (CGT-F) after 24 h of reaction. The increase production of CDs obtained by the SCGT-A method and the demonstrated continuous production of CDs with the SCGT-CB method make the use of immobilized CGTase viable for industrial applications.
1. Introduction Cyclomaltodextrin glucanotransferase (CGTase) (EC 2.4.1.19) is a microbial amylolytic enzyme used mainly for the industrial production of cyclodextrins (CDs), by intramolecular the transglycosylation reaction (cyclization) from the degradation of starch and related sugars. The main CDs are composed of six (α-CD), seven (β-CD) or eight (γ-CD) glucose units linked by α-(1,4) bonds [1]. Cyclodextrins have a hydrophilic exterior, and so can be dissolved in water, a hydrophobic internal cavity, and can form inclusion complexes with a wide variety of nonpolar molecules. Due to this characteristic, they are used in food [1–5] pharmaceuticals and cosmetics [6–8], as well as the textile industry [9], paper industry, chemical technology, analytical chemistry, and other areas [10]. While their potential for use and availability have resulted in growing interest in CDs, their high cost of production is a limiting factor for their use in industry. In order to reduce costs, and owing to their
⁎
importance, a wide range of studies has emerged focusing on immobilization of the CGTase, and preparing it for industrial application [11–14]. The enzyme immobilization technique allows improving the stability of the biocatalyst and its continuous and repeated use, avoiding solubilization and enzymatic loss, and improving the stability of the biocatalyst [12,15,16]. Within what has been researched in the literature, until the moment it is not known the immobilization of the commercial CGTase (Toruzyme® 3.0L) on porous silica controlled by surface anchoring and covalent bonding employed in this work. Many enzymes have already been immobilized on a variety of silica matrices using different methods. Silica is by far the most studied mesoporous material regarding enzyme immobilization applications, and offer special properties such as an elevated surface area, thermal and mechanical stability, ease of handling, the ability to withstand high flow rates in continuous reactors and non-toxicity. They are not attacked by microorganisms and organic solvents [17,18].
Corresponding author at: State University of Maringa (UEM), Av. Colombo, 5790, Maringa, PR 87020-900, Brazil. E-mail address: [email protected] (G. Matioli).
https://doi.org/10.1016/j.procbio.2019.07.002 Received 24 August 2018; Received in revised form 22 December 2018; Accepted 1 July 2019 Available online 04 July 2019 1359-5113/ © 2019 Elsevier Ltd. All rights reserved.
Process Biochemistry 85 (2019) 68–77
G.G. Gimenez, et al.
controlled pore silica in a 250 mL Erlenmeyer condenser, followed by the addition of 60 mL of absolute ethanol. The reactor was maintained at 60 °C with 180 rpm orbital shaking. The reaction was started by the addition of 3 mL of OTMS (90% purity) and lasted 3 h. While still hot the material was vacuum filtered, washed twice with 120 mL of ethanol, followed by two washes with 120 mL of ultrapure water, dried at 105 °C for 24 h and stored in a desiccator until the enzymatic immobilization step [29,30]. The immobilization step was carried out by pouring 5 g of modified silica into a 125 ml Erlenmeyer flask containing 3 ml of the free enzyme (600 μl enzyme/g silica) suspended in 10 ml of hexane at room temperature. The suspension was shaken at 100 rpm for 5 min ensuring intimate contact of the solid phase with the liquid phase. The tube was then closed with aluminum foil with small perforations on its surface and placed in the desiccator, with the application of vacuum control until total evaporation of the solvent [31]. This technique was termed the "anchorage immobilization methodology", due to the attachment of the protein to the internal surface of the silica particles prepared by the evaporation of the solvent. After immobilization, the silica was washed twice with 10 mL of hexane and then twice with 10 mL of ultrapure water, and the solution obtained after washing was used to determine the enzymatic immobilization. The silica containing CGTase immobilized by anchorage (SCGT-A) was dried at room temperature and stored in the desiccator until the tests were carried out. The determination of the percentage of enzymatic immobilization was performed by enzyme activity measurements in the free enzyme solution (AEfree) and the silica wash solution (AEwash.sol) (Eq. 1):
The immobilization of biomolecules in mesoporous support can be effected by physical adsorption or chemical bonding. Silanol groups on the silica surface are involved in the immobilization of the enzyme by adsorption (mainly by means of Van der Waals forces, hydrogen bonding, electrostatic and dipolar interactions) or can be modified by silanization to activate these materials as a support for covalent immobilization [17,19–21] The technique of surface adsorption immobilization is easily applicable and tends to maintain enzymatic activity [22]. However, the immobilization of polar enzymes, such as the commercial CGTase Toruzyme, has some drawbacks such as low interaction with apolar surfaces, and a loss of enzymatic activity and functioning at low temperatures. The covalent immobilization technique is more complex, and agarose-based supports activated with glutaraldehyde, glyoxal or epoxy groups have been widely used because of their high compatibility with enzymes and ability to increase thermal stability as a consequence of enzymatic stiffening after covalent binding, without significantly affecting enzymatic activity. There are different pathways to performing multipoint covalent immobilization. In glyoxyl-activated agarose supports, multipoint covalent attachment, when promoted at alkaline pH, confers greater thermal stability to the enzyme than in other substrates [23]. This covalent immobilization occurs at pH 10.0 through the formation of Schiff bases between the aldehyde group of the support and the neutral amine groups of the enzyme and the subsequent stabilization of chemical bonding by reduction [24–26]. The enzyme commercially known as Toruzyme® is a CGTase solution from a Bacillus strain that received the CGTase gene from a Thermoanaerobacter sp strain sold by Novozymes A/S (Krogshoejvej 362,880 - Bagsvaerd – Denmark). It has a high starch hydrolysis capacity and is resistant to high temperatures, in addition to producing similar amounts of α- and β-CDs and very little γ-CD [27,28]. Considering the importance of CDs in various industrial areas, and taking into account their high cost of production, the objective of the present study was immobilize the commercial CGTase (Toruzyme® 3.0 L) into controlled pore silica controlled by surface anchoring and covalent bonding using octadecyltrimethoxysilane (OTMS) and 3-glycidoxypropyltrimethoxysilane (GPTMS), respectively, in order to try to improve the efficiency of CD production and, subsequently, compare the efficiency of the two methods of immobilization and the use of free CGTase in the production of CDs.
(1) The flowchart of the process of immobilization of the CGTase (Toruzyme®) by the surface anchoring method is shown in Flowchart 1.
2.2.1. Improvement of enzyme immobilization To obtain an ideal amount of immobilized and active enzyme in the controlled pore silica, enzymatic immobilization was optimized by means of three assay conditions (Flowchart 1). (1) evaluation of solvent used in silica washing after enzymatic immobilization: hexane (2 washes of 10 ml) followed by ultrapure water (2 washes of 10 ml); compared with washing with 10 mM sodium citrate buffer, pH 6.0 (4 washes of 10 ml); (2) evaluation of solvent used in enzymatic immobilization: pure hexane; hexane with isopropanol 90:10; and hexane with isopropanol 50:50; (3) evaluation of the concentration of CGTase added to the immobilization procedure: 50, 100, 200, 300 and 600 μL enzyme/g silica. The protein dosage of the enzymatic solution of CGTase Toruzyme® was determined by the Bradford method [32], its concentration was 3.64 mg/mL, so the use of 50, 100, 200, 300 and 600 μL enzyme/g silica represents the use of 0.182, 0.364, 0.728, 1.092, 2.185 mg enzyme/g silica, respectively. The determination of the best immobilization condition for each parameter was evaluated by the initial velocity method, in which the enzyme activity under production conditions is measured at the beginning of the reaction, so the enzyme activity is the parameter used to evaluate the condition of immobilization [33]. The reaction medium containing 15% (w/v) maltodextrin diluted in 10% (V/V) of 10 mM sodium citrate buffer pH 6.0 was prepared and placed in a jacketed glass reactor with a capacity of 50 ml, coupled to a bath thermostated at 65 °C under constant magnetic stirring. Aliquots of 1 mL were collected at 5 min intervals and inactivated by the addition of 1 mL of 0.02 N HCl and boiling at 100 °C for 10 min. The reaction lasted 30 min, and three consecutive operational cycles were performed for each analysis. The aliquots obtained were used for the spectrophotometric determination of β-CD. For commercial CGTase Toruzyme®, the optimum reaction conditions were previously evaluated and pH 6.0 was determined to be
2. Materials and methods 2.1. Reagents The reagents used were α-, β-, γ-CD (Sigma-Aldrich Brasil Ltda. Sao Paulo - Sao Paulo), the Toruzyme® 3.0 L enzyme (3 KNU-CP/MG, lot ACN 00220, donated by Novozyme, Bento Gonçalves, Rio Grande do Sul, Brazil), corn starch (Maizena®, Sao Paulo, Sao Paulo, Brazil), cassava starch (Zaeli Polvilho Doce/Sweet Starch® - Umuarama, Paraná Brazil) and maltodextrin (Lot 0000588861, Sygma Aldrich Brasil Ltda. Sao Paulo, Brazil). Controlled pore silica with a mean particle diameter of 0.42 mm, generously provided by Laboratory of Enzymatic Technology from the Chemical Engeneering Department of State University of Maringa, was supplied by Corning. The other reagents used were of analytical grade. 2.2. Immobilization of commercial CGTase in silica using anchoring method - (SCGT-A) For the immobilization of CGTase on silica by the anchoring method (SCGT-A) the support for the CGTase immobilization was prepared in two steps, the first of which was the chemical modification of the silica and the second of which was enzymatic immobilization. Chemical modification was performed by the silanization reaction of 10 g of 69
Process Biochemistry 85 (2019) 68–77
G.G. Gimenez, et al.
Flowchart 1. Flow diagram of CGTase immobilization process (Toruzyme®) by surface anchoring method. Optimized enzyme immobilization processstages: (1) solvent used to wash the silica after enzymatic immobilization; (2) solvent used in enzymatic immobilization; (3) concentration of CGTase added to the immobilization procedure.
6.0 (1 wash of 30 ml), dried at room temperature and stored in a desiccator until the moment of use. The determination of the percentage of enzymatic immobilization was performed by the enzyme activity measurements in the enzyme-free solution (AEfree), and the residual solutions of the immobilization (AEresidual.sol) and washing of the silica (AEwashing.sol) (Eq.2):
optimal for this enzyme, and the optimum CD production temperature was 65 °C [27,34,35]. 2.2.2. Determination of best substrate for production of CDs The influence of the substrate concentration was evaluated in relation to β-CD production. The conditions used were corn starch, cassava starch, and maltodextrin at concentrations of 5, 10 and 15% (w/ V), diluted in 10% (V/V) of 10 mM sodium citrate buffer pH 6.0. The reaction medium was placed in a jacketed glass reactor coupled to a thermostated bath at 65 °C under constant magnetic stirring, and 500 mg of SCGT-A was added. At the start of the reaction, a 1 mL aliquot was collected from the medium and inactivated with 1 mL of 0.02 N HCl and boiling for 10 min. Then, 2 mL of distilled water was added and the sample was centrifuged at 8000 x g, at 40 °C for 20 min. The supernatant was separated for the spectrophotometric determination of β-CD by the phenolphthalein method. The other samples were collected at 5 min intervals for 30 min and the inactivation procedure described above followed. After the determination of the best concentration for all of the three substrates, the reaction was carried out with corn starch, cassava starch and maltodextrin at concentrations of 10% for 24 h in the same conditions as previously described, with samplings at 0, 20, 40, 60, 120, 180, 360, 720 and 1440 min.
(2) 2.4. Production of CDs in 10% cassava starch with and without ethanol and determination of length of CD production cycle This assay was conducted to evaluate the effect of ethanol on CD production. The reaction medium was placed in a jacketed glass reactor coupled to a thermostated bath at 65 °C under constant magnetic stirring, and the reactor was also coupled to a condenser to prevent loss of the medium and ethanol by evaporation. The reaction medium containing 10% (w/V) cassava starch as the substrate in the presence and absence of 10% (V/V) ethanol was used. The cycle was initiated by the addition of 500 mg of SCGT-A and, separately, 925.17 mg of SCGT-CB, with the weights of silica corresponding to the use of 89.63 μL of CGTase enzyme. The samples were collected at 0, 20, 40 min, 1, 6, 12, 24, 48, 72 and 120 h and immediately inactivated. The tubes were centrifuged at 8000 x g, 40 °C for 20 min. The supernatants were separated and analyzed for the chromatographic determination of CDs. The results were used to determine the cycle time and to evaluate the effect of ethanol on the production of CDs. In another experiment, 89.63 μL of commercial free CGTase (CGT-F) was used for the production of CDs in a reaction medium containing 10% (w/V) cassava starch substrate in the presence of 10% (V/V) ethanol, with collection and inactivation at the times mentioned above. The assays were performed in triplicate.
2.3. Immobilization of commercial CGTase by covalent bonding - (SCGTCB) In the same manner as SCGT-A, covalent bonding immobilization (SCGT-CB) was performed in two steps, the controlled pore silica (activated under vacuum at 200 °C) was silylated with 10% GPTMS solution, and the immobilization protocol of Bernal, Sierra and Mesa [23] was used. Silica containing CGTase immobilized by covalent bonding (SCGTCB) was prepared in 5 mL of enzyme-free solution (200 μL of Toruzyme® enzyme and 4,8 mL of 100 mM potassium bicarbonate buffer, pH 10) by adding 1 g of the modified silica. The resulting suspension was incubated at 4 °C for 24 h. Subsequently, NaBH4 solution (4 mg/mL) was added and incubated for 30 min at 4 °C [25]. When the reduction period was complete, the silica was filtered and washed with water (3 washes of 30 ml) followed by 10 mM sodium citrate buffer pH
2.5. CD production in five consecutive cycles In order to evaluate the operational stability of the two types of immobilization studied, five consecutive operating cycles were conducted with a CD production time of 24 h for SCGT-A and 48 h for SCGT-CB. A 10% (w/V) cassava starch substrate was used in the 70
Process Biochemistry 85 (2019) 68–77
G.G. Gimenez, et al.
immobilization method is the evaporation of the solvent in a low pressure environment. This causes the solvent to gradually pass to the vapor state and be removed from the immobilization environment. The solution temperature was lowered to an equilibrium value, determined experimentally as 13 °C, simultaneously with this process. The advantages of this approach in comparison with other methods of immobilization are that it is fast, occurs at low temperatures and causes constant removal of the solvent from the medium. These facts allow the concentration gradient in the liquid phase and the chemical potential of the surface to increase, forcing the enzyme to migrate from the free phase to the immobilized phase, resulting in a high enzyme density on the surface of the matrix. In addition to these advantages, the obtained biocatalyst is always dry and ready for use. Dantas [31] evaluated the hydrolytic activity of Burkholderia cepacia, Candida rugosa, Thermomyces lanuginosus and Porcine pancreatic immobilized on nonpolar and polar matrices by the anchoring method and observed that all the immobilized lipases exhibited hydrolyticactivity for para-nitrophenyl palmitate. In all the evaluated cases there was a reduction of the enzymatic activity in comparison with the free enzyme, but all the lipases were more active when immobilized in the apolar matrix, except for the Candida rugosa lipase, which exhibited greater activity when immobilized on polar silica. Dantas [31] also observed that the lipase obtained from Burkholderia cepacia exhibited the greatest enzymatic activity, while the enzyme immobilized on nonpolar surface was ten times more active than the same enzyme immobilized on polar surface.
presence of 10% ethanol (V/ V). After completion of each cycle, the silica was washed with buffer solution and reused in a new reaction medium. The supernatants of each operational cycle were collected, inactivated, centrifuged and analyzed by the chromatographic determination of CDs. 2.6. Spectrophotometric and chromatographic determination of CDs During the CD production assays, the evaluation of production was performed by spectrophotometric determination of β-CD concentration by the method described by Tardioli, Zanin and Moraes [36]. This method is based on the discoloration of a solution of phenolphthalein at 550 nm which occurs after the complexation of this dye with the β-CD present in the sample. As the relationship between absorbance and βCD concentration remains linear only at concentrations of up to 0.4 mmol/L, Complexation Theory was used, which provides the nonlinear relationship between the absorbance and concentration of β-CD [36]. The α-, β- and γ-CD concentration were determined by HPLC using a CG-480C liquid chromatograph (Varian), equipped with an IR-GC 410 refractive index detector and an aminopropylsilane (SGE) column (particle size 5 mm, length 25 cm, and internal diameter 4.6 mm). This was developed under isocratic conditions using a solution of acetonitrile and water (70:30) as the mobile phase and a flow of 0.7 ml/min at room temperature (21 °C). The standard solutions and the samples were filtered through a 0.45 μm membrane. The analytical curves were constructed for α-CD, β-CD and γ-CD in different concentration ranges. For α-CD, the values ranged from 1.0 to 7.0 mg/mL; for β-CD, the range was 1.0 to 11.0 mg/mL; and for γ-CD, the range was 1.0 to 3.0 mg/mL. The peak area results of each CD were adjusted by linear regression as a function of the concentration of the standard.
3.1.1. Improvement of enzyme immobilization The improvement of the immobilization process involved evaluating the solvent used in the washing of the silica following enzymatic immobilization. The results obtained indicated that washing with hexane solution followed by water was more suitable than washing with the buffer solution, as while there was no significant difference in the production of β-CD with the two washing methods in the first cycle, washing by hexane solution followed by water was more effective in the second and third cycles, and was therefore used in the continuation of the experiments (Fig. 1). The enzyme employed in this experiment has a polar character, by employing hexane in the wash, a nonpolar solvent, the enzyme was retained in the support, while the use of a buffer caused the enzyme to be carried along with the washing solution. The solvent used in enzymatic immobilization was evaluated with the pure hexane solvent, hexane with isopropanol 90:10 and hexane with isopropanol 50:50. Higher concentrations of isopropanol were not viable due to the difficulty of evaporating the same by the application of vacuum control. The greater efficiency of pure hexane in the first cycle, with greater β-CD production, made it more efficient than the other solvents, even with a reduction in yield in the second and third cycles. The higher production in the first cycle was more significant than the difference between the other cycles. Pure hexane was therefore used in the other immobilization experiments (Fig. 2). In this immobilization technique denominated anchoring, the use of hexane becomes advantageous because of the short time required for evaporation of the solvent under vacuum, and consequently speeds up the enzymatic immobilization. When solvent hexane was used with isopropanol, the time required for solvent evaporation under vacuum was higher, and the enzyme CGTase may have been carried out together with the solvent during the vacuum evaporation process, losing amount of enzyme and, consequently, decreasing the production of CDs. The increase in the concentration of the CGTase added during the immobilization procedure was evaluated using concentrations of 50, 100, 200, 300 and 600 μl enzyme/g silica, with hexane used as the immobilization solvent and washing after immobilization carried out with hexane followed by water. The choice of the best dilution took into account the coefficient of determination (R2), in the graph of the concentration of the β-CD produced versus time, as the model became more explanatory and was a better fit with the sample as the R² value
2.7. Statistical analysis The results of CD production were evaluated and compared by means of analysis of variance (ANOVA), and the data were compared by the Tukey test with a significance level of 5%. 3. Results and discussion 3.1. Immobilization of commercial CGTase into silica by the anchoring method - (SCGT-A) This technique was developed by Dantas [31] to overcome the drawbacks that are commonly found in classical immobilization methods, such as the diffusional limitation, the long equilibrium time, the low process temperature, and the biocatalyst drying. It is an innovative vacuum method for enzyme immobilization in a non-aqueous medium. During the anchorage immobilization process, after the silanization step, the polarity characteristics of the silica changed, due to the internal particle surface being covered with the OTMS, and becoming apolar. The immobilization step using hexane, an apolar solvent, allowed for greater interaction of the modified silica with the medium, reducing the diffusion restriction problems of the solvent/enzyme suspension through the silica pores [31,37]. In the immobilization stage of the Toruzyme® enzyme, the use of a vacuum proved to be very efficient. This was confirmed by an assay performed for β-CD production in 30 min of reaction using 15% maltodextrin as the substrate. In non-optimized immobilization conditions 6.85 mM β-CD was obtained. The greatest advantage of this method over the others was the shorter time required to complete the entire enzymatic immobilization process, which in this study occurred in 12.0 ± 0.5 min. At this time complete evaporation of the solvent from the suspension containing the immobilization matrix and the enzyme occurred. The principle of the 71
Process Biochemistry 85 (2019) 68–77
G.G. Gimenez, et al.
Fig. 1. Improvement of the silica wash solvent after enzymatic immobilization using hexane followed by water ( ) and buffer solution ( ) in the first (A), second (B) and third cycles (C).
Fig. 2. Improvement of enzymatic immobilization solvent using pure hexane ( ), hexane and isopropanol 90:10 ( ) and hexane and isopropanol 50:50 ( ) in the first (A), second (B) and third cycles (C).
increased (Fig.3). According to Fig. 3, concentrations of 50–300 μl of CGTase/g silica had a determination coefficient above 0.99 in the first cycle, and β-CD production was significantly higher at concentrations of 200 and 300 μl of CGTase/g of silica. A quantity of 200 μl CGTase/g silica was used, taking into account the high cost of the enzyme. Under optimized conditions it was possible to immobilize 89.63% of the offered CGTase in the controlled pore silica, corresponding to the use of 89.63 μl CGTase, in other words, 0.3264 mg CGTase, in a 50 ml reaction, due to the addition of 500 mg of SCGT-A. Thus, the enzymatic concentration in the solution was 0.18% (V/V), which resulted in the production of 4.31 mM β-CD in 30 min of reaction. When Calsavara et al. [27] used the same free enzyme in the concentrations 0.05; 0.1 and 0.2% (V/V), with corn starch at 15% (w/V) as substrate and ethanol 10% (V/V), the best results were obtained with a concentration of 0.1% (V/V) of enzyme.
Each CGTase and substrate concentration present their own characteristics of influence on the rate of cyclodextrin production. Li et al. [38] evaluated the influence of maltodextrin substrate solutions ranging from 1 mg/mL to 40 mg/mL on the production of CDs by α-CGTase from Paenibacillus macerans JFB05-01 and β-CGTase from Bacillus circulans STB01, and observed that the critical substrate concentrations to prevent substrate inhibition were 10 mg/mL for the α-cyclization reaction and 20 mg/mL for both the β- and γ-cyclization reactions. Mazzer et al. [39] evaluated the influence of variations in the concentration of maltodextrin (1.0–15%) on β-CD production using B. firmus strain 37 cells immobilized on inorganic matrices and observed that using maltodextrin concentration of 10% in the reaction medium was more economically advantageous. The analysis of the production of β-CD for 24 h (Fig. 5) showed that cassava starch, despite producing a small amount of β-CD in a short reaction time, surpassed the corn starch and maltodextrin production over longer periods, and was the most suitable for CD production under the test conditions used. Cassava is produced worldwide, with Africa and Asia being the largest producers, followed by Brazil, where it is found as a low-cost product. For the synthesis of CDs both amylopectin and amylose served as substrates for production, although amylopectin produced a better yield than amylose. The branched structure of amylopectin allows the enzyme reaction to start from a large number of points (non-reducing side chain ends) [40]. Alves et al. [41] reported that cassava starch has a
3.1.2. Determination of the best substrate for the production of CD The influence of the substrate concentration was analyzed, as it is known that inhibition of CGTase activity may occur when there is an excess of substrate in the medium. The production of β-CD by SCGT-A was evaluated using maltodextrin, corn starch and cassava starch for a 30 min reaction period. For all the substrates evaluated there was a higher production of β-CD when its concentration was 10% of the substrate (Fig. 4). A concentration of 10% for the three substrates was therefore used in the continuation of the experiments. 72
Process Biochemistry 85 (2019) 68–77
G.G. Gimenez, et al.
Fig. 5. Production of β-CD by SCGT-A using the substrates maltodextrin 10% ( ), corn starch 10% ( ) and cassava starch 10% ( ) for 24 h.
3.2. Immobilization of commercial CGTase by covalent bonding - (SCGTCB) For SCGT-CB it was possible to immobilize 48.44% of the enzyme, a significantly lower value than the amount of enzyme immobilized by the anchorage technique (89.63%). A quantity of 925.17 mg of SCGTCB was required to correspond to the same quantity of 500 mg of SCGTA. The difference in the immobilization efficiency of the two techniques employed may have occurred due to the use of apolar solvent in the anchoring technique, which increased the contact of the enzymatic suspension with the internal pores of the silica, resulting in an increase in the enzymatic immobilization surface. In the SCGT-CB technique, 100 mM potassium bicarbonate buffer solution was used in the enzymatic immobilization step, a polar solution which does not adequately interact with silylated silica. It is commonly reported that the use of silica may involve drawbacks such as resistance to mass transfer and difficult access to the enzyme within the pore due to differences in polarity or the presence of adsorbed gas molecules within the pores. Therefore, it is important to characterize the microenvironment inside the pores to improve the understanding of how material properties and solution composition can be used to control the enzymatic activity [18]. Enzymatic immobilization within the matrix pores may be an advantage when the process is not controlled by the effects of diffusion restriction. The reason for this is the formation of a microenvironment that is more suited to enzymatic activation within the pores, normally with polar characteristics, ionic strength and interactions which differ from those of the reaction medium away from the pores [43].
Fig. 3. Improvement of CGTase concentration used in enzymatic immobilization: 50 ( ), 100 ( ), 200 ( ), 300 ( ) and 600 ( ) μl of CGTase/g of silica in the first (A), second (B) and third cycles (C).
3.3. Production of CDs in 10% cassava starch with and without ethanol and determination of the length of the CD production cycle The analysis of CD production by SCGT-A and SCGT-CB was carried out for five consecutive days. It was found that the presence of ethanol did not modify CD production in 12 h, but that a significant difference began to appear from 24 h of reaction onwards (Fig. 6). For SCGT-A, the production of CDs in the reaction medium in the presence of ethanol was statistically greater with a 24 h than with a 12 h reaction time. With a 24 h reaction time 26.29 mmolxL−1 of α-CD and 23.10 mmolxL−1 of β-CD were obtained. With 48 h of reaction, the production of α-CD was 28.52 mmolxL−1 and the production of β-CD was 25.57 mmolxL−1. These results did not differ statistically, with shorter production times being more advantageous in industrial processes, reducing the production costs of CDs. It was chosen to use a 24 h CD production cycle for SCGT-A. For SCGT-CB, the production of CDs in the reaction medium in the presence of ethanol was statistically greater with a 48 h than with a 24 h reaction time. At 48 h of reaction it was possible to obtain 25.12
Fig. 4. Production of β-CD by SCGT-A using the substrates maltodextrin, corn starch and cassava starch at concentrations of 5% ( ), 10% ( ) and 15% ( ), for 30 min of reaction.
content of 79.9 ± 9% amylopectin and 20.8 ± 0.6% amylose. According to Weber, Collares-Queiroz and Chang [42], corn starch contains 27.8% of amylose, and consequently has less amylopectin than cassava starch, explaining its lower production on CDs.
73
Process Biochemistry 85 (2019) 68–77
G.G. Gimenez, et al.
Fig. 6. Production of α-CD ( ), β-CD ( ) and γ-CD ( ) by SCGT-A in the absence (A) and presence (B) of 10% ethanol (V/V) and by SCGT-CB in the absence (C) and presence (D) of 10% ethanol (V/V) using 10% cassava starch as a substrate, for 5 days.
mmolxL−1 of α-CD and 15.91 mmolxL−1 of β-CD. At 72 h of reaction, the production of α-CD was 25.62 mmolxL−1 and the production of βCD was 16.85 mmolxL−1. These results were not statistically different, and the use of shorter periods of CD production was once again chosen, with a 48 h reaction period selected for the CD production cycle for SCGT-CB. The production of γ-CDs was low for both SCGT-A and SCGT-CB, with no statistical difference between the reaction media in the presence or absence of ethanol. The highest production of α-CD and β-CD was obtained with the addition of ethanol for both SCGT-A and SCGT-CB. With 10% ethanol, there was a moderate increase in β-CD production and a significant increase in α-CD production. For SCGT-A, the increase in the production of α- and β-CD in 24 h of reaction in a reaction medium with 10% of ethanol was 143.88% and 26.30%, respectively, compared with the same reaction medium without ethanol, and there was a 60.97% increase in total CD production. As for SCGT-CB, the increase in α- and βCD production in 48 h of reaction in a reaction medium with 10% ethanol was 77.15 and 8.67%, respectively, compared to the same reaction medium without ethanol, and there was a 34.90% increase in total CD production. The increase in CD production in the presence of ethanol, which was much more significant for α-CD, agreed with the results obtained by Blackwood and Bucke [44]. The analysis of Fig. 6 indicates that the behavior of the enzyme changes from αβ-CGTase to α-CGTase with the addition of 10% ethanol (V/V) to the reaction medium. Blackwood and Bucke [42] suggested that these results could be explained by changes in the hydration and enzyme infrastructure, with the displacement of water molecules by the solvent on the surface of the protein or in the region of the active site. Mori et al. [45] assume that ethanol can change the aesthetic structures of starch and/or the enzyme in order to suppress β-CD formation. In the experiment performed by Calsavara et al. [27] for the production of CDs there was a 66% increase in the production of total CDs when 10% of ethanol (V/V) was used. The use of ethanol during the
production of the CDs prevents hydrolytic reactions, as it excludes water molecules from the active site of the enzyme, as well as reverse reactions, delaying the decomposition of the CDs formed. It is an additive that can be easily evaporated and reused and, in addition, reduces the possibility of microbial contamination during the process.
3.4. Comparison of the production of CDs using SCGT-A, SCGT-CB and commercial free CGTase (CGT-F) When the production of CDs by SCGT-A and CGT-F were compared, with both in a reaction medium containing 10% cassava starch in the presence of 10% ethanol, using equivalent amounts of commercial CGTase, the equivalent production of CDs occurred in up to 12 h of reaction. After this period, SCGT-A was more efficient in the production of α-CD, β-CD and γ-CD, producing 18.15% more total CDs at the end of 24 h of reaction, with the highest yield observed in β-CD, yielding 18.40 mmolxL−1 for CGT-F and 23.10 mmolxL−1 for SCGT-A. At the end of 120 h of reaction, a 56.99% increase was observed in the total CDs yield of SCGT-A in comparison with CGT-F (Fig. 7). In terms of the production of CDs by SCGT-CB in 48 h of reaction, the yield was very similar to that obtained with CGT-F, with an increase of total CD production of only 1.98%. There was no significant difference in α-CD and β-CD production, with a slight increase in γ-CD production. The production of total CDs using SCGT-CB continued to increase after 48 h of reaction, while the CDs produced with CGT-F began to suffer degradation and the enzyme began to lose its enzymatic activity. With 120 h of reaction, there was a significant statistical difference in the production of total CDs, with an increase of 49.01% in the total yield of CDs obtained from SCGT-CB in comparison with CGTF (Fig. 7). A continuous production of CDs by SCGT-CB was observed due to the covalent bond immobilization, which allows increases in thermal stability as a consequence of the enzymatic stiffening that occurs following covalent bonding, without significantly affecting the enzymatic activity. The production of total CDs by SCGT-A exhibited more promising 74
Process Biochemistry 85 (2019) 68–77
G.G. Gimenez, et al.
Fig. 8. Production of α-CD ( ), β-CD ( ) and γ-CD ( ) by SCGT-A (A) and SCGT-CB (B) using 10% cassava starch in the presence of 10% ethanol (V/V) as a substrate for five consecutive cycles.
immobilization yield and specific activity. However, covalent immobilization on activated silica conferred a thermal stability 120 times greater at 55 °C for β-galactosidase of B. circulans and Kluyveromyces lactis, with an immobilization yield of 60% in the presence of glycerin. 3.5. Production of CDs in five consecutive cycles When the production of CDs using SCGT-A and SCGT-CB for five consecutive cycles was performed it was observed that, although there was a high production of total CDs in the first reaction cycle, this declined significantly in the second cycle, with a decrease of 61.51% and 42.32% of total CD production for SCGT-A and SCGT-CB, respectively. In the fifth reaction cycle, total CD production was only 3.89% of the yield obtained in the first cycle for SCGT-A and 7.19% for SCGT-CB (Fig. 8). There was loss of enzymatic activity when SCGT-A and SCGT-CB was used for more than one reaction cycle, which may be related to the use of high reaction temperatures and the long reaction time required for CD production, which caused the enzyme to be released from its support, being lost in the operational medium. Anchorage immobilization consists of entrapment of the enzyme on the surface of the support by low-intensity forces such as Van der Walls forces, hydrophobic interactions or dispersion forces [31]. Thus, due to small adsorption forces involved, the enzyme can be released from the support, which results in a type of immobilization of low enzymatic binding stability. It is important to emphasize that this immobilization methodology is simple and supports high enzymatic activity on the first reaction cycle.
Fig. 7. Comparison of the production of α-CD (A), β-CD (B) and γ-CD (C) by CGT-F ( ), SCGT-A ( ) and SCGT-CB ( ), using 10% cassava starch in the presence of 10% ethanol (V/V) as a substrate, for 5 days.
results than production with SCGT-CB, as SCGT-A obtained a maximum yield in lesser reaction time. At 24 h of reaction, SCGT-A produced 70.49% more total CDs than SCGT-CB during 48 h of reaction, with this difference declining to 25.03% at the end of 120 h of reaction. The use of SCGT-A was therefore more efficient than SCGT-CB and CGT-F, as in addition to obtaining a higher production yield of total CDs than the others, a shorter period was required to obtain this yield. Loss of α-CD and γ-CD was observed over the 120 h of reaction, with loss of β-CD occurring after 48 h of reaction. Bernal, Sierra and Mesa [23] carried out a study comparing immobilization by covalent bonding on silica activated by glyoxal groups with the adsorption method, using the β-galactosidase enzyme. In immobilization by adsorption, the catalyst obtained exhibited lower 75
Process Biochemistry 85 (2019) 68–77
G.G. Gimenez, et al.
Although there was unsatisfactory operating stability for CD production, the use of SCGT-A produced significantly more CDs in one operating cycle than CGT-F. As it is a simple and inexpensive technique, the increase in the production of total CDs in one production cycle justifies the use of the enzyme immobilized by anchorage. SCGT-CB did not exhibit as effective performance as SCGT-A, but the CDs produced in the first during 120 h of reaction obtained a higher yield than the CDs produced by CGT-L, justifying its use when there is a need for long-term reactions. In addition, the silica used to immobilize CGTase can be reused for new immobilization processes due to its thermal, mechanical and microbiological stability.
review on the use of cyclodextrins in foods, Food Hydrocoll. 23 (2009) 1631–1634. [2] G. Astray, J.C. Mejuto, J. Morales, R. Rial-Otero, J. Simal-Gandara, Factors controlling flavors binding constants to cyclodextrins and their applications in foods, Food Res. Int. 43 (2010) 1212–1218. [3] Y. Li, Y. Chen, H. Li, Recovery and purification of cholesterol from cholesterolcyclodextrin inclusion complex using ultrasound-assisted extraction, Ultrason. Sonochem. 34 (2017) 281–288. [4] C. Yuan, L. Du, G. Zhang, Z. Jin, H. Liu, Influence of cyclodextrins on texture behavior and freeze-thaw stability of kappa-carrageenan gel, Food Chem. 210 (2016) 600–605. [5] X.-H. Zhao, C.-H. Tang, Spray-drying microencapsulation of CoQ10 in olive oil for enhanced water dispersion, stability and bioaccessibility: influence of type of emulsifiers and/or wall materials, Food Hydrocoll. 61 (2016) 20–30. [6] P.S.S. Lima, A.M. Lucchese, H.G. Araújo-Filho, P.P. Menezes, A.A.S. Araújo, L.J. Quintans-Júnior, J.S.S. Quintans, Inclusion of terpenes in cyclodextrins: preparation, characterization and pharmacological approaches, Carbohydr. Polym. 151 (2016) 965–987. [7] Z. Moussa, M. Hmadeh, M.G. Abiad, O.H. Dib, D. Patra, Encapsulation of curcumin in cyclodextrin-metal organic frameworks: dissociation of loaded CD-MOFs enhances stability of curcumin, Food Chem. 212 (2016) 485–494. [8] S. Pereva, T. Sarafska, S. Bogdanova, T. Spassov, Efficiency of cyclodextrin-ibuprofen inclusion complex formation, J. Drug Deliv. Sci. Technol. 35 (2016) 34–39. [9] M. Mihailiasa, F. Caldera, J. Li, R. Peila, A. Ferri, F. Trotta, Preparation of functionalized cotton fabrics by means of melatonin loaded β–cyclodextrin nanosponges, Carbohydr. Polym. 142 (2016) 24–30. [10] B.A. van der Veen, J.C. Uitdehaa, B.W. Dijkstra, L. Dijkhuizen, Engineering of cyclodextrin glycosyltransferase reaction and product specificity, Biochim. Biophys. Acta 1543 (2000) 336–360. [11] N.L. Cieh, S. Sulaiman, M.N. Mokhtar, M.N. Naim, Bleached kenaf microfiber as a support matrix for cyclodextrin glucanotransferase immobilization via covalent binding by different coupling agents, Process Biochem. 56 (2017) 81–89. [12] N. Graebin, J. Schöffer, D. Andrades, P. Hertz, M. Ayub, R. Rodrigues, Immobilization of glycoside hydrolase families GH1, GH13, and GH70: state of the art and perspectives, Molecules 21 (2016) 1074. [13] J.N. Schöffer, M.P. Klein, R.C. Rodrigues, P.F. Hertz, Continuous production of βcyclodextrin from starch by highly stable cyclodextrin glycosyltransferase immobilized on chitosan, Carbohydr. Polym. 98 (2013) 1311–1316. [14] J.N. Schöffer, C.R. Matte, D.S. Charqueiro, E.W. Menezes, T.M.H. Costa, E.V. Benvenutti, R.C. Rodrigues, P.F. Hertz, Directed immobilization of CGTase: the effect of the enzyme orientation on the enzyme activity and its use in packed-bed reactor for continuous production of cyclodextrins, Process Biochem. 58 (2017) 120–127. [15] C.R. Matte, M.R. Nunes, E.V. Benvenutti, J.N. Schöffer, M.A.Z. Ayub, P.F. Hertz, Characterization of cyclodextrin glycosyltransferase immobilized on sílica microspheres via aminopropyltrimethoxysilane as a “spacer arm”, J. Mol. Catal., B Enzym. 78 (2012) 51–56. [16] D.I. Bezbradica, C. Mateo, J.M. Guisan, Novel support for enzyme immobilization prepared by chemical activation with cysteine and glutaraldehyde, J. Mol. Catal., B Enzym. 102 (2014) 218–224. [17] S. Hudson, J. Cooney, E. Magner, Proteins in mesoporous silicates, Angew. Chem. 47 (2008) 8582–8594. [18] N. Carlsson, H. Gustafsson, C. Thörn, L. Olsson, K. Holmberg, B. Akerman, Enzymes immobilized in mesoporous silica: a physical–chemical perspective, Adv. Colloid Interface Sci. 205 (2014) 339–360. [19] C. Bernal, L. Sierra, M. Mesa, Application of Hierarchical porous silica with a stable large porosity for β-galactosidase immobilization, Chem Cat Chem 3 (2011) 1948–1954. [20] J. Kim, R.J. Desch, S.W. Thiel, V.V. Guliants, N.G. Pinto, Energetics of protein adsorption on amine-functionalized mesostructured cellular foam silica, J. Chromatogr. A 1218 (2011) 7796–7803. [21] D.N. Tran, K.J. Balkus, Perspective of recent progress in immobilization of enzymes, ACS Catal. 1 (2011) 956–968. [22] J.H. Dantas, L.D. de Paris, C.E. Barão, P.A.A. Arroyo, C.M.F. Soares, J.V. Visentainer, F. Faria, G.M. Zanin, Influence of alcohol: oil molar ratio on the production of ethyl esters by enzymatic transesterification of canola oil, Afr. J. Biotechnol. 12 (2013) 6968–6979. [23] C. Bernal, L. Sierra, M. Mesa, Design of β-galactosidase/silica biocatalysts: impact of the enzyme properties and immobilization pathways on their catalytic performance, Eng. Life Sci. 14 (2014) 85–94. [24] D. Brady, J. Jordaan, Advances in enzyme immobilisation, Biotechnol. Lett. 31 (2009) 1639–1650. [25] C. Mateo, J.M. Palomo, M.F. uentes, L. Betancor, V. Grazu, F. López-Gallego, B.C.C. Pessela, A. Hidalgo, G. Fernández-Lorente, R. Fernández-Lafuente, J.M. Guisán, Glyoxyl agarose, a fully inert and hydrophilic support for immobilization and high stabilization of proteins, Enzyme Microb. Technol. 39 (2006) 274–280. [26] C. Mateo, J.M. Palomo, G. Fernandez-Lorente, J.M. Guisan, R. Fernandez-Lafuente, Improvement of enzyme activity, stability and selectivity via immobilization techniques, Enzyme Microb. Technol. 40 (2007) 1451–1463. [27] L.P. Calsavara, A.R. Dias da Cunha, T.A. Balbino, G.M. Zanin, F.F. de Moraes, Production of cyclodextrins from cornstarch granules in a sequential batch mode and in the presence of etanol, Appl. Biochem. Biotechnol. 165 (2011) 1485–1493. [28] B.E. Norman, S.T. Jorgensen, Thermoanaerobacter sp. CGTase: its properties and applications, Denpun Kagaku 39 (1992) 101–108. [29] M. Kamori, T. Hori, Y. Yamashita, Y. Hirose, Y. Naoshima, Immobilization of lipase on a new inorganic ceramics support, toyonite, and the reactivity and
4. Conclusion This study described two methods of enzymatic immobilization based on the use of silica for the immobilization of commercial CGTase (Toruzyme®), that is, by internal surface anchorage of the enzyme and its covalent immobilization. The results showed that the anchorage immobilization method is a simple and effective manner for the fixation of the commercial CGTase on the internal surface of the immobilization matrix. A higher production yield of CDs was obtained with the improvement of the SCGT-A immobilization process, which evaluated the solvent used to wash the silica after enzymatic immobilization; the composition of the solvent used in enzymatic immobilization; and the concentration of CGTase added to the immobilization procedure. The method SCGT-CB gave a lower immobilization yield (48.44%) than SCGT-A (89.63%). There was an increase in the total production of CDs of 60.97% and 34.90% for SCGT-A and SCGT-CB, respectively, in a reaction medium containing 10% ethanol, in comparison with the same medium without ethanol, showing that the use of ethanol is very advantageous to obtain a higher production yield of CDs, mainly in relation to α-CD. The use of SCGT-A was more efficient than SCGT-CB and CGT-F, for employing a short reaction time, 24 h, SCGT-A produced 70.49% more total CDs than SCGT-CB, and 18.15% more than CGT-F. The production of CDs for five consecutive cycles showed that, although there was significant production of total CDs in the first reaction cycle for both SCGT-A and SCGT-CB, this progressively declined for the other cycles. Considering the simplicity of the two immobilization techniques evaluated and the immobilized enzyme performs well, it is suggested that the new biocatalysts described can be used in the production of CDs. The increase in CD production obtained by the SCGT-A method and the continuous production of CDs in long-term reactions conferred by the SCGT-CB method make the use of immobilized CGTase viable for industrial applications. Funding sources This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Author contribution All authors had materially participated in the research and article preparation was made by G.G.G. and G.M. Acknowledgements The authors are thankful to the Brazilian Agencies Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação Araucária for their financial support of this work. References [1] G. Astray, C. González-Barreiro, J.C. Mejuto, R. Rial-Otero, J. Simal-Gándara, A
76
Process Biochemistry 85 (2019) 68–77
G.G. Gimenez, et al.
[30]
[31] [32]
[33] [34] [35] [36]
[37] [38]
enantioselectivity of the immobilized lipase, J. Mol. Catal., B Enzym. 9 (2000) 269–274. J. Wang, G. Meng, K. Tao, M. Feng, X. Zhao, Z. Li, H. Xu, D. Xia, J.R. Lu, Immobilization of lipases on alkyl silane modified magnetic nanoparticles: effect of alkyl chain length on enzyme activity, PLoS One 7 (2012) e43478. J.H. Dantas, Modulação da hidrofobicidade da sílica para imobilização de lipase de Burkholderia cepacia, Doctoral thesis, State University of Maringa, 2016. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. K. Wilson, J. Walker, Principles and Techniques of Biochemistry and Molecular Biology, seventh ed., Cambridge University Press, New York, 2010. Z. Li, M. Wang, F. Wang, Z. Gu, G. Du, J. Wu, γ-Cyclodextrin: a review on enzymatic production and application, Appl. Microbiol. Biotechnol. 77 (2007) 245–255. T.-J. Kim, B.-C. Kim, H.-S. Lee, Production of cyclodextrin using raw corn starch without a pretreatment, Enzyme Microb. Technol. 20 (1997) 506–509. P.W. Tardioli, G.M. Zanin, F.F. Moraes, Characterization of Thermoanaerobacter cyclomaltodextrin glucanotransferase immobilized on glyoxilagarose, Enzyme Microb. Technol. 39 (2006) 1270–1278. R.M. Blanco, P. Terreros, N. Muñoz, E. Serra, Ethanol improves lipase immobilization on a hydrophobic support, J. Mol. Catal., B Enzym. 47 (2007) 13–20. C. Li, Q. Xu, Z. Gu, S. Chen, J. Wu, Y. Hong, L. Cheng, Z. Li, Cyclodextrin
[39]
[40]
[41] [42]
[43] [44]
[45]
77
glycosyltransferase variants experience different modes of product inhibition, J. Mol. Catal., B Enzym. 133 (2016) 203–210. C. Mazzer, L.R. Ferreira, J.R.T. Rodella, C. Moriwaki, G. Matioli, Cyclodextrin production by Bacillus firmus strain 37 immobilized on inorganic matrices and alginate gel, Biochem. Eng. J. 41 (2008) 79–86. B. Cheirsilp, S. Kitcha, S. Maneerat, Kinetic characteristics of β-cyclodextrin production by cyclodextrin glycosyltransferase from newly isolated Bacillus sp. C26, Electron. J. Biotechnol. 13 (2010) 4–5. V.D. Alves, S. Mali, A. Beléia, M.V.E. Grossmann, Effect of glycerol and amylose enrichment on cassava starch film properties, J. Food Eng. 78 (2007) 941–946. F.H. Weber, F.P. Collares-Queiroz, Y.K. Chang, Physicochemical, rheological, morphological, and thermal characterization of normal, waxy, and high amylose corn starches, Ciênc, Tecnol. Aliment. 29 (2009) 748–753. J.N. Talbert, J.M. Goddard, Enzymes on material surfaces, Colloids Surf. B Biointerfaces 93 (2012) 8–19. A.D. Blackwood, C. Bucke, Addition of polar organic solvents can improve the product selectivity of cyclodextrin glycosyltransferase: solvent effects on CGTase, Enzyme Microb. Technol. 27 (2000) 704–708. S. Mori, M. Goto, T. Mase, A. Matsuura, T. Ova, S. Kitahata, Reaction conditions for the production of γ-cyclodextrin by cyclodextrin glucanotransferase from Brevibacterium sp, Biosci. Biotechnol. Biochem. 59 (2000) 1012–1015.
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Immobilization of commercial cyclomaltodextrin glucanotransferase into controlled pore silica by the anchorage method and covalent bonding
T
Gabriela Gregolin Gimeneza, Richard Marllon Silvab, Carolina Pereira Franciscoc, Fabiana dos Santos Randod, João Henrique Dantasc, Hâmara Milaneze de Souzab, ⁎ Graciette Matiolia, a
Department of Pharmacy, State University of Maringa (UEM), Av. Colombo, 5790, Maringa, PR 87020-900, Brazil Food Science Department, State University of Maringa (UEM), Av. Colombo, 5790, Maringa, PR 87020-900, Brazil c Department of Chemical Engineering, State University of Maringa (UEM), Av. Colombo, 5790, Maringa, PR 87020-900, Brazil d Department of Cell Biology and Genetics, State University of Maringa (UEM), Av. Colombo, 5790, Maringa, PR 87020-900, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Immobilization CGTase Anchorage Covalent bond Cyclodextrins
Cyclomaltodextrin glucanotransferase (CGTase) is used mainly for the industrial production of cyclodextrins (CDs). Their potential use and availability have played a decisive role in increasing demand for CDs. The aim of the present study was to immobilize the commercial CGTase (Toruzyme® 3.0L) into controlled pore silica by surface anchoring (SCGT-A) and covalent bonding (SCGT-CB) and to apply the immobilized enzymes in the production of CDs. The SCGT-A method proved to be efficient due to its short immobilization time and, it was possible to immobilize 89.63% of CGTase, in terms of activity. Commercial CGTase immobilized by SCGT-CB provided an immobilization yield of 48.44%, also in terms of activity. There was an increase in total CD production of 60.97% and 34.90% for SCGT-A and SCGT-CB, respectively, in a reaction medium with 10% ethanol, compared to the same media without ethanol. Higher CD production occured at 24 and 48 h for SCGT-A and SCGT-CB methods, respectively. The use of SCGT-A was more efficient than SCGT-CB and free enzyme (CGT-F) after 24 h of reaction. The increase production of CDs obtained by the SCGT-A method and the demonstrated continuous production of CDs with the SCGT-CB method make the use of immobilized CGTase viable for industrial applications.
1. Introduction Cyclomaltodextrin glucanotransferase (CGTase) (EC 2.4.1.19) is a microbial amylolytic enzyme used mainly for the industrial production of cyclodextrins (CDs), by intramolecular the transglycosylation reaction (cyclization) from the degradation of starch and related sugars. The main CDs are composed of six (α-CD), seven (β-CD) or eight (γ-CD) glucose units linked by α-(1,4) bonds [1]. Cyclodextrins have a hydrophilic exterior, and so can be dissolved in water, a hydrophobic internal cavity, and can form inclusion complexes with a wide variety of nonpolar molecules. Due to this characteristic, they are used in food [1–5] pharmaceuticals and cosmetics [6–8], as well as the textile industry [9], paper industry, chemical technology, analytical chemistry, and other areas [10]. While their potential for use and availability have resulted in growing interest in CDs, their high cost of production is a limiting factor for their use in industry. In order to reduce costs, and owing to their
⁎
importance, a wide range of studies has emerged focusing on immobilization of the CGTase, and preparing it for industrial application [11–14]. The enzyme immobilization technique allows improving the stability of the biocatalyst and its continuous and repeated use, avoiding solubilization and enzymatic loss, and improving the stability of the biocatalyst [12,15,16]. Within what has been researched in the literature, until the moment it is not known the immobilization of the commercial CGTase (Toruzyme® 3.0L) on porous silica controlled by surface anchoring and covalent bonding employed in this work. Many enzymes have already been immobilized on a variety of silica matrices using different methods. Silica is by far the most studied mesoporous material regarding enzyme immobilization applications, and offer special properties such as an elevated surface area, thermal and mechanical stability, ease of handling, the ability to withstand high flow rates in continuous reactors and non-toxicity. They are not attacked by microorganisms and organic solvents [17,18].
Corresponding author at: State University of Maringa (UEM), Av. Colombo, 5790, Maringa, PR 87020-900, Brazil. E-mail address: [email protected] (G. Matioli).
https://doi.org/10.1016/j.procbio.2019.07.002 Received 24 August 2018; Received in revised form 22 December 2018; Accepted 1 July 2019 Available online 04 July 2019 1359-5113/ © 2019 Elsevier Ltd. All rights reserved.
Process Biochemistry 85 (2019) 68–77
G.G. Gimenez, et al.
controlled pore silica in a 250 mL Erlenmeyer condenser, followed by the addition of 60 mL of absolute ethanol. The reactor was maintained at 60 °C with 180 rpm orbital shaking. The reaction was started by the addition of 3 mL of OTMS (90% purity) and lasted 3 h. While still hot the material was vacuum filtered, washed twice with 120 mL of ethanol, followed by two washes with 120 mL of ultrapure water, dried at 105 °C for 24 h and stored in a desiccator until the enzymatic immobilization step [29,30]. The immobilization step was carried out by pouring 5 g of modified silica into a 125 ml Erlenmeyer flask containing 3 ml of the free enzyme (600 μl enzyme/g silica) suspended in 10 ml of hexane at room temperature. The suspension was shaken at 100 rpm for 5 min ensuring intimate contact of the solid phase with the liquid phase. The tube was then closed with aluminum foil with small perforations on its surface and placed in the desiccator, with the application of vacuum control until total evaporation of the solvent [31]. This technique was termed the "anchorage immobilization methodology", due to the attachment of the protein to the internal surface of the silica particles prepared by the evaporation of the solvent. After immobilization, the silica was washed twice with 10 mL of hexane and then twice with 10 mL of ultrapure water, and the solution obtained after washing was used to determine the enzymatic immobilization. The silica containing CGTase immobilized by anchorage (SCGT-A) was dried at room temperature and stored in the desiccator until the tests were carried out. The determination of the percentage of enzymatic immobilization was performed by enzyme activity measurements in the free enzyme solution (AEfree) and the silica wash solution (AEwash.sol) (Eq. 1):
The immobilization of biomolecules in mesoporous support can be effected by physical adsorption or chemical bonding. Silanol groups on the silica surface are involved in the immobilization of the enzyme by adsorption (mainly by means of Van der Waals forces, hydrogen bonding, electrostatic and dipolar interactions) or can be modified by silanization to activate these materials as a support for covalent immobilization [17,19–21] The technique of surface adsorption immobilization is easily applicable and tends to maintain enzymatic activity [22]. However, the immobilization of polar enzymes, such as the commercial CGTase Toruzyme, has some drawbacks such as low interaction with apolar surfaces, and a loss of enzymatic activity and functioning at low temperatures. The covalent immobilization technique is more complex, and agarose-based supports activated with glutaraldehyde, glyoxal or epoxy groups have been widely used because of their high compatibility with enzymes and ability to increase thermal stability as a consequence of enzymatic stiffening after covalent binding, without significantly affecting enzymatic activity. There are different pathways to performing multipoint covalent immobilization. In glyoxyl-activated agarose supports, multipoint covalent attachment, when promoted at alkaline pH, confers greater thermal stability to the enzyme than in other substrates [23]. This covalent immobilization occurs at pH 10.0 through the formation of Schiff bases between the aldehyde group of the support and the neutral amine groups of the enzyme and the subsequent stabilization of chemical bonding by reduction [24–26]. The enzyme commercially known as Toruzyme® is a CGTase solution from a Bacillus strain that received the CGTase gene from a Thermoanaerobacter sp strain sold by Novozymes A/S (Krogshoejvej 362,880 - Bagsvaerd – Denmark). It has a high starch hydrolysis capacity and is resistant to high temperatures, in addition to producing similar amounts of α- and β-CDs and very little γ-CD [27,28]. Considering the importance of CDs in various industrial areas, and taking into account their high cost of production, the objective of the present study was immobilize the commercial CGTase (Toruzyme® 3.0 L) into controlled pore silica controlled by surface anchoring and covalent bonding using octadecyltrimethoxysilane (OTMS) and 3-glycidoxypropyltrimethoxysilane (GPTMS), respectively, in order to try to improve the efficiency of CD production and, subsequently, compare the efficiency of the two methods of immobilization and the use of free CGTase in the production of CDs.
(1) The flowchart of the process of immobilization of the CGTase (Toruzyme®) by the surface anchoring method is shown in Flowchart 1.
2.2.1. Improvement of enzyme immobilization To obtain an ideal amount of immobilized and active enzyme in the controlled pore silica, enzymatic immobilization was optimized by means of three assay conditions (Flowchart 1). (1) evaluation of solvent used in silica washing after enzymatic immobilization: hexane (2 washes of 10 ml) followed by ultrapure water (2 washes of 10 ml); compared with washing with 10 mM sodium citrate buffer, pH 6.0 (4 washes of 10 ml); (2) evaluation of solvent used in enzymatic immobilization: pure hexane; hexane with isopropanol 90:10; and hexane with isopropanol 50:50; (3) evaluation of the concentration of CGTase added to the immobilization procedure: 50, 100, 200, 300 and 600 μL enzyme/g silica. The protein dosage of the enzymatic solution of CGTase Toruzyme® was determined by the Bradford method [32], its concentration was 3.64 mg/mL, so the use of 50, 100, 200, 300 and 600 μL enzyme/g silica represents the use of 0.182, 0.364, 0.728, 1.092, 2.185 mg enzyme/g silica, respectively. The determination of the best immobilization condition for each parameter was evaluated by the initial velocity method, in which the enzyme activity under production conditions is measured at the beginning of the reaction, so the enzyme activity is the parameter used to evaluate the condition of immobilization [33]. The reaction medium containing 15% (w/v) maltodextrin diluted in 10% (V/V) of 10 mM sodium citrate buffer pH 6.0 was prepared and placed in a jacketed glass reactor with a capacity of 50 ml, coupled to a bath thermostated at 65 °C under constant magnetic stirring. Aliquots of 1 mL were collected at 5 min intervals and inactivated by the addition of 1 mL of 0.02 N HCl and boiling at 100 °C for 10 min. The reaction lasted 30 min, and three consecutive operational cycles were performed for each analysis. The aliquots obtained were used for the spectrophotometric determination of β-CD. For commercial CGTase Toruzyme®, the optimum reaction conditions were previously evaluated and pH 6.0 was determined to be
2. Materials and methods 2.1. Reagents The reagents used were α-, β-, γ-CD (Sigma-Aldrich Brasil Ltda. Sao Paulo - Sao Paulo), the Toruzyme® 3.0 L enzyme (3 KNU-CP/MG, lot ACN 00220, donated by Novozyme, Bento Gonçalves, Rio Grande do Sul, Brazil), corn starch (Maizena®, Sao Paulo, Sao Paulo, Brazil), cassava starch (Zaeli Polvilho Doce/Sweet Starch® - Umuarama, Paraná Brazil) and maltodextrin (Lot 0000588861, Sygma Aldrich Brasil Ltda. Sao Paulo, Brazil). Controlled pore silica with a mean particle diameter of 0.42 mm, generously provided by Laboratory of Enzymatic Technology from the Chemical Engeneering Department of State University of Maringa, was supplied by Corning. The other reagents used were of analytical grade. 2.2. Immobilization of commercial CGTase in silica using anchoring method - (SCGT-A) For the immobilization of CGTase on silica by the anchoring method (SCGT-A) the support for the CGTase immobilization was prepared in two steps, the first of which was the chemical modification of the silica and the second of which was enzymatic immobilization. Chemical modification was performed by the silanization reaction of 10 g of 69
Process Biochemistry 85 (2019) 68–77
G.G. Gimenez, et al.
Flowchart 1. Flow diagram of CGTase immobilization process (Toruzyme®) by surface anchoring method. Optimized enzyme immobilization processstages: (1) solvent used to wash the silica after enzymatic immobilization; (2) solvent used in enzymatic immobilization; (3) concentration of CGTase added to the immobilization procedure.
6.0 (1 wash of 30 ml), dried at room temperature and stored in a desiccator until the moment of use. The determination of the percentage of enzymatic immobilization was performed by the enzyme activity measurements in the enzyme-free solution (AEfree), and the residual solutions of the immobilization (AEresidual.sol) and washing of the silica (AEwashing.sol) (Eq.2):
optimal for this enzyme, and the optimum CD production temperature was 65 °C [27,34,35]. 2.2.2. Determination of best substrate for production of CDs The influence of the substrate concentration was evaluated in relation to β-CD production. The conditions used were corn starch, cassava starch, and maltodextrin at concentrations of 5, 10 and 15% (w/ V), diluted in 10% (V/V) of 10 mM sodium citrate buffer pH 6.0. The reaction medium was placed in a jacketed glass reactor coupled to a thermostated bath at 65 °C under constant magnetic stirring, and 500 mg of SCGT-A was added. At the start of the reaction, a 1 mL aliquot was collected from the medium and inactivated with 1 mL of 0.02 N HCl and boiling for 10 min. Then, 2 mL of distilled water was added and the sample was centrifuged at 8000 x g, at 40 °C for 20 min. The supernatant was separated for the spectrophotometric determination of β-CD by the phenolphthalein method. The other samples were collected at 5 min intervals for 30 min and the inactivation procedure described above followed. After the determination of the best concentration for all of the three substrates, the reaction was carried out with corn starch, cassava starch and maltodextrin at concentrations of 10% for 24 h in the same conditions as previously described, with samplings at 0, 20, 40, 60, 120, 180, 360, 720 and 1440 min.
(2) 2.4. Production of CDs in 10% cassava starch with and without ethanol and determination of length of CD production cycle This assay was conducted to evaluate the effect of ethanol on CD production. The reaction medium was placed in a jacketed glass reactor coupled to a thermostated bath at 65 °C under constant magnetic stirring, and the reactor was also coupled to a condenser to prevent loss of the medium and ethanol by evaporation. The reaction medium containing 10% (w/V) cassava starch as the substrate in the presence and absence of 10% (V/V) ethanol was used. The cycle was initiated by the addition of 500 mg of SCGT-A and, separately, 925.17 mg of SCGT-CB, with the weights of silica corresponding to the use of 89.63 μL of CGTase enzyme. The samples were collected at 0, 20, 40 min, 1, 6, 12, 24, 48, 72 and 120 h and immediately inactivated. The tubes were centrifuged at 8000 x g, 40 °C for 20 min. The supernatants were separated and analyzed for the chromatographic determination of CDs. The results were used to determine the cycle time and to evaluate the effect of ethanol on the production of CDs. In another experiment, 89.63 μL of commercial free CGTase (CGT-F) was used for the production of CDs in a reaction medium containing 10% (w/V) cassava starch substrate in the presence of 10% (V/V) ethanol, with collection and inactivation at the times mentioned above. The assays were performed in triplicate.
2.3. Immobilization of commercial CGTase by covalent bonding - (SCGTCB) In the same manner as SCGT-A, covalent bonding immobilization (SCGT-CB) was performed in two steps, the controlled pore silica (activated under vacuum at 200 °C) was silylated with 10% GPTMS solution, and the immobilization protocol of Bernal, Sierra and Mesa [23] was used. Silica containing CGTase immobilized by covalent bonding (SCGTCB) was prepared in 5 mL of enzyme-free solution (200 μL of Toruzyme® enzyme and 4,8 mL of 100 mM potassium bicarbonate buffer, pH 10) by adding 1 g of the modified silica. The resulting suspension was incubated at 4 °C for 24 h. Subsequently, NaBH4 solution (4 mg/mL) was added and incubated for 30 min at 4 °C [25]. When the reduction period was complete, the silica was filtered and washed with water (3 washes of 30 ml) followed by 10 mM sodium citrate buffer pH
2.5. CD production in five consecutive cycles In order to evaluate the operational stability of the two types of immobilization studied, five consecutive operating cycles were conducted with a CD production time of 24 h for SCGT-A and 48 h for SCGT-CB. A 10% (w/V) cassava starch substrate was used in the 70
Process Biochemistry 85 (2019) 68–77
G.G. Gimenez, et al.
immobilization method is the evaporation of the solvent in a low pressure environment. This causes the solvent to gradually pass to the vapor state and be removed from the immobilization environment. The solution temperature was lowered to an equilibrium value, determined experimentally as 13 °C, simultaneously with this process. The advantages of this approach in comparison with other methods of immobilization are that it is fast, occurs at low temperatures and causes constant removal of the solvent from the medium. These facts allow the concentration gradient in the liquid phase and the chemical potential of the surface to increase, forcing the enzyme to migrate from the free phase to the immobilized phase, resulting in a high enzyme density on the surface of the matrix. In addition to these advantages, the obtained biocatalyst is always dry and ready for use. Dantas [31] evaluated the hydrolytic activity of Burkholderia cepacia, Candida rugosa, Thermomyces lanuginosus and Porcine pancreatic immobilized on nonpolar and polar matrices by the anchoring method and observed that all the immobilized lipases exhibited hydrolyticactivity for para-nitrophenyl palmitate. In all the evaluated cases there was a reduction of the enzymatic activity in comparison with the free enzyme, but all the lipases were more active when immobilized in the apolar matrix, except for the Candida rugosa lipase, which exhibited greater activity when immobilized on polar silica. Dantas [31] also observed that the lipase obtained from Burkholderia cepacia exhibited the greatest enzymatic activity, while the enzyme immobilized on nonpolar surface was ten times more active than the same enzyme immobilized on polar surface.
presence of 10% ethanol (V/ V). After completion of each cycle, the silica was washed with buffer solution and reused in a new reaction medium. The supernatants of each operational cycle were collected, inactivated, centrifuged and analyzed by the chromatographic determination of CDs. 2.6. Spectrophotometric and chromatographic determination of CDs During the CD production assays, the evaluation of production was performed by spectrophotometric determination of β-CD concentration by the method described by Tardioli, Zanin and Moraes [36]. This method is based on the discoloration of a solution of phenolphthalein at 550 nm which occurs after the complexation of this dye with the β-CD present in the sample. As the relationship between absorbance and βCD concentration remains linear only at concentrations of up to 0.4 mmol/L, Complexation Theory was used, which provides the nonlinear relationship between the absorbance and concentration of β-CD [36]. The α-, β- and γ-CD concentration were determined by HPLC using a CG-480C liquid chromatograph (Varian), equipped with an IR-GC 410 refractive index detector and an aminopropylsilane (SGE) column (particle size 5 mm, length 25 cm, and internal diameter 4.6 mm). This was developed under isocratic conditions using a solution of acetonitrile and water (70:30) as the mobile phase and a flow of 0.7 ml/min at room temperature (21 °C). The standard solutions and the samples were filtered through a 0.45 μm membrane. The analytical curves were constructed for α-CD, β-CD and γ-CD in different concentration ranges. For α-CD, the values ranged from 1.0 to 7.0 mg/mL; for β-CD, the range was 1.0 to 11.0 mg/mL; and for γ-CD, the range was 1.0 to 3.0 mg/mL. The peak area results of each CD were adjusted by linear regression as a function of the concentration of the standard.
3.1.1. Improvement of enzyme immobilization The improvement of the immobilization process involved evaluating the solvent used in the washing of the silica following enzymatic immobilization. The results obtained indicated that washing with hexane solution followed by water was more suitable than washing with the buffer solution, as while there was no significant difference in the production of β-CD with the two washing methods in the first cycle, washing by hexane solution followed by water was more effective in the second and third cycles, and was therefore used in the continuation of the experiments (Fig. 1). The enzyme employed in this experiment has a polar character, by employing hexane in the wash, a nonpolar solvent, the enzyme was retained in the support, while the use of a buffer caused the enzyme to be carried along with the washing solution. The solvent used in enzymatic immobilization was evaluated with the pure hexane solvent, hexane with isopropanol 90:10 and hexane with isopropanol 50:50. Higher concentrations of isopropanol were not viable due to the difficulty of evaporating the same by the application of vacuum control. The greater efficiency of pure hexane in the first cycle, with greater β-CD production, made it more efficient than the other solvents, even with a reduction in yield in the second and third cycles. The higher production in the first cycle was more significant than the difference between the other cycles. Pure hexane was therefore used in the other immobilization experiments (Fig. 2). In this immobilization technique denominated anchoring, the use of hexane becomes advantageous because of the short time required for evaporation of the solvent under vacuum, and consequently speeds up the enzymatic immobilization. When solvent hexane was used with isopropanol, the time required for solvent evaporation under vacuum was higher, and the enzyme CGTase may have been carried out together with the solvent during the vacuum evaporation process, losing amount of enzyme and, consequently, decreasing the production of CDs. The increase in the concentration of the CGTase added during the immobilization procedure was evaluated using concentrations of 50, 100, 200, 300 and 600 μl enzyme/g silica, with hexane used as the immobilization solvent and washing after immobilization carried out with hexane followed by water. The choice of the best dilution took into account the coefficient of determination (R2), in the graph of the concentration of the β-CD produced versus time, as the model became more explanatory and was a better fit with the sample as the R² value
2.7. Statistical analysis The results of CD production were evaluated and compared by means of analysis of variance (ANOVA), and the data were compared by the Tukey test with a significance level of 5%. 3. Results and discussion 3.1. Immobilization of commercial CGTase into silica by the anchoring method - (SCGT-A) This technique was developed by Dantas [31] to overcome the drawbacks that are commonly found in classical immobilization methods, such as the diffusional limitation, the long equilibrium time, the low process temperature, and the biocatalyst drying. It is an innovative vacuum method for enzyme immobilization in a non-aqueous medium. During the anchorage immobilization process, after the silanization step, the polarity characteristics of the silica changed, due to the internal particle surface being covered with the OTMS, and becoming apolar. The immobilization step using hexane, an apolar solvent, allowed for greater interaction of the modified silica with the medium, reducing the diffusion restriction problems of the solvent/enzyme suspension through the silica pores [31,37]. In the immobilization stage of the Toruzyme® enzyme, the use of a vacuum proved to be very efficient. This was confirmed by an assay performed for β-CD production in 30 min of reaction using 15% maltodextrin as the substrate. In non-optimized immobilization conditions 6.85 mM β-CD was obtained. The greatest advantage of this method over the others was the shorter time required to complete the entire enzymatic immobilization process, which in this study occurred in 12.0 ± 0.5 min. At this time complete evaporation of the solvent from the suspension containing the immobilization matrix and the enzyme occurred. The principle of the 71
Process Biochemistry 85 (2019) 68–77
G.G. Gimenez, et al.
Fig. 1. Improvement of the silica wash solvent after enzymatic immobilization using hexane followed by water ( ) and buffer solution ( ) in the first (A), second (B) and third cycles (C).
Fig. 2. Improvement of enzymatic immobilization solvent using pure hexane ( ), hexane and isopropanol 90:10 ( ) and hexane and isopropanol 50:50 ( ) in the first (A), second (B) and third cycles (C).
increased (Fig.3). According to Fig. 3, concentrations of 50–300 μl of CGTase/g silica had a determination coefficient above 0.99 in the first cycle, and β-CD production was significantly higher at concentrations of 200 and 300 μl of CGTase/g of silica. A quantity of 200 μl CGTase/g silica was used, taking into account the high cost of the enzyme. Under optimized conditions it was possible to immobilize 89.63% of the offered CGTase in the controlled pore silica, corresponding to the use of 89.63 μl CGTase, in other words, 0.3264 mg CGTase, in a 50 ml reaction, due to the addition of 500 mg of SCGT-A. Thus, the enzymatic concentration in the solution was 0.18% (V/V), which resulted in the production of 4.31 mM β-CD in 30 min of reaction. When Calsavara et al. [27] used the same free enzyme in the concentrations 0.05; 0.1 and 0.2% (V/V), with corn starch at 15% (w/V) as substrate and ethanol 10% (V/V), the best results were obtained with a concentration of 0.1% (V/V) of enzyme.
Each CGTase and substrate concentration present their own characteristics of influence on the rate of cyclodextrin production. Li et al. [38] evaluated the influence of maltodextrin substrate solutions ranging from 1 mg/mL to 40 mg/mL on the production of CDs by α-CGTase from Paenibacillus macerans JFB05-01 and β-CGTase from Bacillus circulans STB01, and observed that the critical substrate concentrations to prevent substrate inhibition were 10 mg/mL for the α-cyclization reaction and 20 mg/mL for both the β- and γ-cyclization reactions. Mazzer et al. [39] evaluated the influence of variations in the concentration of maltodextrin (1.0–15%) on β-CD production using B. firmus strain 37 cells immobilized on inorganic matrices and observed that using maltodextrin concentration of 10% in the reaction medium was more economically advantageous. The analysis of the production of β-CD for 24 h (Fig. 5) showed that cassava starch, despite producing a small amount of β-CD in a short reaction time, surpassed the corn starch and maltodextrin production over longer periods, and was the most suitable for CD production under the test conditions used. Cassava is produced worldwide, with Africa and Asia being the largest producers, followed by Brazil, where it is found as a low-cost product. For the synthesis of CDs both amylopectin and amylose served as substrates for production, although amylopectin produced a better yield than amylose. The branched structure of amylopectin allows the enzyme reaction to start from a large number of points (non-reducing side chain ends) [40]. Alves et al. [41] reported that cassava starch has a
3.1.2. Determination of the best substrate for the production of CD The influence of the substrate concentration was analyzed, as it is known that inhibition of CGTase activity may occur when there is an excess of substrate in the medium. The production of β-CD by SCGT-A was evaluated using maltodextrin, corn starch and cassava starch for a 30 min reaction period. For all the substrates evaluated there was a higher production of β-CD when its concentration was 10% of the substrate (Fig. 4). A concentration of 10% for the three substrates was therefore used in the continuation of the experiments. 72
Process Biochemistry 85 (2019) 68–77
G.G. Gimenez, et al.
Fig. 5. Production of β-CD by SCGT-A using the substrates maltodextrin 10% ( ), corn starch 10% ( ) and cassava starch 10% ( ) for 24 h.
3.2. Immobilization of commercial CGTase by covalent bonding - (SCGTCB) For SCGT-CB it was possible to immobilize 48.44% of the enzyme, a significantly lower value than the amount of enzyme immobilized by the anchorage technique (89.63%). A quantity of 925.17 mg of SCGTCB was required to correspond to the same quantity of 500 mg of SCGTA. The difference in the immobilization efficiency of the two techniques employed may have occurred due to the use of apolar solvent in the anchoring technique, which increased the contact of the enzymatic suspension with the internal pores of the silica, resulting in an increase in the enzymatic immobilization surface. In the SCGT-CB technique, 100 mM potassium bicarbonate buffer solution was used in the enzymatic immobilization step, a polar solution which does not adequately interact with silylated silica. It is commonly reported that the use of silica may involve drawbacks such as resistance to mass transfer and difficult access to the enzyme within the pore due to differences in polarity or the presence of adsorbed gas molecules within the pores. Therefore, it is important to characterize the microenvironment inside the pores to improve the understanding of how material properties and solution composition can be used to control the enzymatic activity [18]. Enzymatic immobilization within the matrix pores may be an advantage when the process is not controlled by the effects of diffusion restriction. The reason for this is the formation of a microenvironment that is more suited to enzymatic activation within the pores, normally with polar characteristics, ionic strength and interactions which differ from those of the reaction medium away from the pores [43].
Fig. 3. Improvement of CGTase concentration used in enzymatic immobilization: 50 ( ), 100 ( ), 200 ( ), 300 ( ) and 600 ( ) μl of CGTase/g of silica in the first (A), second (B) and third cycles (C).
3.3. Production of CDs in 10% cassava starch with and without ethanol and determination of the length of the CD production cycle The analysis of CD production by SCGT-A and SCGT-CB was carried out for five consecutive days. It was found that the presence of ethanol did not modify CD production in 12 h, but that a significant difference began to appear from 24 h of reaction onwards (Fig. 6). For SCGT-A, the production of CDs in the reaction medium in the presence of ethanol was statistically greater with a 24 h than with a 12 h reaction time. With a 24 h reaction time 26.29 mmolxL−1 of α-CD and 23.10 mmolxL−1 of β-CD were obtained. With 48 h of reaction, the production of α-CD was 28.52 mmolxL−1 and the production of β-CD was 25.57 mmolxL−1. These results did not differ statistically, with shorter production times being more advantageous in industrial processes, reducing the production costs of CDs. It was chosen to use a 24 h CD production cycle for SCGT-A. For SCGT-CB, the production of CDs in the reaction medium in the presence of ethanol was statistically greater with a 48 h than with a 24 h reaction time. At 48 h of reaction it was possible to obtain 25.12
Fig. 4. Production of β-CD by SCGT-A using the substrates maltodextrin, corn starch and cassava starch at concentrations of 5% ( ), 10% ( ) and 15% ( ), for 30 min of reaction.
content of 79.9 ± 9% amylopectin and 20.8 ± 0.6% amylose. According to Weber, Collares-Queiroz and Chang [42], corn starch contains 27.8% of amylose, and consequently has less amylopectin than cassava starch, explaining its lower production on CDs.
73
Process Biochemistry 85 (2019) 68–77
G.G. Gimenez, et al.
Fig. 6. Production of α-CD ( ), β-CD ( ) and γ-CD ( ) by SCGT-A in the absence (A) and presence (B) of 10% ethanol (V/V) and by SCGT-CB in the absence (C) and presence (D) of 10% ethanol (V/V) using 10% cassava starch as a substrate, for 5 days.
mmolxL−1 of α-CD and 15.91 mmolxL−1 of β-CD. At 72 h of reaction, the production of α-CD was 25.62 mmolxL−1 and the production of βCD was 16.85 mmolxL−1. These results were not statistically different, and the use of shorter periods of CD production was once again chosen, with a 48 h reaction period selected for the CD production cycle for SCGT-CB. The production of γ-CDs was low for both SCGT-A and SCGT-CB, with no statistical difference between the reaction media in the presence or absence of ethanol. The highest production of α-CD and β-CD was obtained with the addition of ethanol for both SCGT-A and SCGT-CB. With 10% ethanol, there was a moderate increase in β-CD production and a significant increase in α-CD production. For SCGT-A, the increase in the production of α- and β-CD in 24 h of reaction in a reaction medium with 10% of ethanol was 143.88% and 26.30%, respectively, compared with the same reaction medium without ethanol, and there was a 60.97% increase in total CD production. As for SCGT-CB, the increase in α- and βCD production in 48 h of reaction in a reaction medium with 10% ethanol was 77.15 and 8.67%, respectively, compared to the same reaction medium without ethanol, and there was a 34.90% increase in total CD production. The increase in CD production in the presence of ethanol, which was much more significant for α-CD, agreed with the results obtained by Blackwood and Bucke [44]. The analysis of Fig. 6 indicates that the behavior of the enzyme changes from αβ-CGTase to α-CGTase with the addition of 10% ethanol (V/V) to the reaction medium. Blackwood and Bucke [42] suggested that these results could be explained by changes in the hydration and enzyme infrastructure, with the displacement of water molecules by the solvent on the surface of the protein or in the region of the active site. Mori et al. [45] assume that ethanol can change the aesthetic structures of starch and/or the enzyme in order to suppress β-CD formation. In the experiment performed by Calsavara et al. [27] for the production of CDs there was a 66% increase in the production of total CDs when 10% of ethanol (V/V) was used. The use of ethanol during the
production of the CDs prevents hydrolytic reactions, as it excludes water molecules from the active site of the enzyme, as well as reverse reactions, delaying the decomposition of the CDs formed. It is an additive that can be easily evaporated and reused and, in addition, reduces the possibility of microbial contamination during the process.
3.4. Comparison of the production of CDs using SCGT-A, SCGT-CB and commercial free CGTase (CGT-F) When the production of CDs by SCGT-A and CGT-F were compared, with both in a reaction medium containing 10% cassava starch in the presence of 10% ethanol, using equivalent amounts of commercial CGTase, the equivalent production of CDs occurred in up to 12 h of reaction. After this period, SCGT-A was more efficient in the production of α-CD, β-CD and γ-CD, producing 18.15% more total CDs at the end of 24 h of reaction, with the highest yield observed in β-CD, yielding 18.40 mmolxL−1 for CGT-F and 23.10 mmolxL−1 for SCGT-A. At the end of 120 h of reaction, a 56.99% increase was observed in the total CDs yield of SCGT-A in comparison with CGT-F (Fig. 7). In terms of the production of CDs by SCGT-CB in 48 h of reaction, the yield was very similar to that obtained with CGT-F, with an increase of total CD production of only 1.98%. There was no significant difference in α-CD and β-CD production, with a slight increase in γ-CD production. The production of total CDs using SCGT-CB continued to increase after 48 h of reaction, while the CDs produced with CGT-F began to suffer degradation and the enzyme began to lose its enzymatic activity. With 120 h of reaction, there was a significant statistical difference in the production of total CDs, with an increase of 49.01% in the total yield of CDs obtained from SCGT-CB in comparison with CGTF (Fig. 7). A continuous production of CDs by SCGT-CB was observed due to the covalent bond immobilization, which allows increases in thermal stability as a consequence of the enzymatic stiffening that occurs following covalent bonding, without significantly affecting the enzymatic activity. The production of total CDs by SCGT-A exhibited more promising 74
Process Biochemistry 85 (2019) 68–77
G.G. Gimenez, et al.
Fig. 8. Production of α-CD ( ), β-CD ( ) and γ-CD ( ) by SCGT-A (A) and SCGT-CB (B) using 10% cassava starch in the presence of 10% ethanol (V/V) as a substrate for five consecutive cycles.
immobilization yield and specific activity. However, covalent immobilization on activated silica conferred a thermal stability 120 times greater at 55 °C for β-galactosidase of B. circulans and Kluyveromyces lactis, with an immobilization yield of 60% in the presence of glycerin. 3.5. Production of CDs in five consecutive cycles When the production of CDs using SCGT-A and SCGT-CB for five consecutive cycles was performed it was observed that, although there was a high production of total CDs in the first reaction cycle, this declined significantly in the second cycle, with a decrease of 61.51% and 42.32% of total CD production for SCGT-A and SCGT-CB, respectively. In the fifth reaction cycle, total CD production was only 3.89% of the yield obtained in the first cycle for SCGT-A and 7.19% for SCGT-CB (Fig. 8). There was loss of enzymatic activity when SCGT-A and SCGT-CB was used for more than one reaction cycle, which may be related to the use of high reaction temperatures and the long reaction time required for CD production, which caused the enzyme to be released from its support, being lost in the operational medium. Anchorage immobilization consists of entrapment of the enzyme on the surface of the support by low-intensity forces such as Van der Walls forces, hydrophobic interactions or dispersion forces [31]. Thus, due to small adsorption forces involved, the enzyme can be released from the support, which results in a type of immobilization of low enzymatic binding stability. It is important to emphasize that this immobilization methodology is simple and supports high enzymatic activity on the first reaction cycle.
Fig. 7. Comparison of the production of α-CD (A), β-CD (B) and γ-CD (C) by CGT-F ( ), SCGT-A ( ) and SCGT-CB ( ), using 10% cassava starch in the presence of 10% ethanol (V/V) as a substrate, for 5 days.
results than production with SCGT-CB, as SCGT-A obtained a maximum yield in lesser reaction time. At 24 h of reaction, SCGT-A produced 70.49% more total CDs than SCGT-CB during 48 h of reaction, with this difference declining to 25.03% at the end of 120 h of reaction. The use of SCGT-A was therefore more efficient than SCGT-CB and CGT-F, as in addition to obtaining a higher production yield of total CDs than the others, a shorter period was required to obtain this yield. Loss of α-CD and γ-CD was observed over the 120 h of reaction, with loss of β-CD occurring after 48 h of reaction. Bernal, Sierra and Mesa [23] carried out a study comparing immobilization by covalent bonding on silica activated by glyoxal groups with the adsorption method, using the β-galactosidase enzyme. In immobilization by adsorption, the catalyst obtained exhibited lower 75
Process Biochemistry 85 (2019) 68–77
G.G. Gimenez, et al.
Although there was unsatisfactory operating stability for CD production, the use of SCGT-A produced significantly more CDs in one operating cycle than CGT-F. As it is a simple and inexpensive technique, the increase in the production of total CDs in one production cycle justifies the use of the enzyme immobilized by anchorage. SCGT-CB did not exhibit as effective performance as SCGT-A, but the CDs produced in the first during 120 h of reaction obtained a higher yield than the CDs produced by CGT-L, justifying its use when there is a need for long-term reactions. In addition, the silica used to immobilize CGTase can be reused for new immobilization processes due to its thermal, mechanical and microbiological stability.
review on the use of cyclodextrins in foods, Food Hydrocoll. 23 (2009) 1631–1634. [2] G. Astray, J.C. Mejuto, J. Morales, R. Rial-Otero, J. Simal-Gandara, Factors controlling flavors binding constants to cyclodextrins and their applications in foods, Food Res. Int. 43 (2010) 1212–1218. [3] Y. Li, Y. Chen, H. Li, Recovery and purification of cholesterol from cholesterolcyclodextrin inclusion complex using ultrasound-assisted extraction, Ultrason. Sonochem. 34 (2017) 281–288. [4] C. Yuan, L. Du, G. Zhang, Z. Jin, H. Liu, Influence of cyclodextrins on texture behavior and freeze-thaw stability of kappa-carrageenan gel, Food Chem. 210 (2016) 600–605. [5] X.-H. Zhao, C.-H. Tang, Spray-drying microencapsulation of CoQ10 in olive oil for enhanced water dispersion, stability and bioaccessibility: influence of type of emulsifiers and/or wall materials, Food Hydrocoll. 61 (2016) 20–30. [6] P.S.S. Lima, A.M. Lucchese, H.G. Araújo-Filho, P.P. Menezes, A.A.S. Araújo, L.J. Quintans-Júnior, J.S.S. Quintans, Inclusion of terpenes in cyclodextrins: preparation, characterization and pharmacological approaches, Carbohydr. Polym. 151 (2016) 965–987. [7] Z. Moussa, M. Hmadeh, M.G. Abiad, O.H. Dib, D. Patra, Encapsulation of curcumin in cyclodextrin-metal organic frameworks: dissociation of loaded CD-MOFs enhances stability of curcumin, Food Chem. 212 (2016) 485–494. [8] S. Pereva, T. Sarafska, S. Bogdanova, T. Spassov, Efficiency of cyclodextrin-ibuprofen inclusion complex formation, J. Drug Deliv. Sci. Technol. 35 (2016) 34–39. [9] M. Mihailiasa, F. Caldera, J. Li, R. Peila, A. Ferri, F. Trotta, Preparation of functionalized cotton fabrics by means of melatonin loaded β–cyclodextrin nanosponges, Carbohydr. Polym. 142 (2016) 24–30. [10] B.A. van der Veen, J.C. Uitdehaa, B.W. Dijkstra, L. Dijkhuizen, Engineering of cyclodextrin glycosyltransferase reaction and product specificity, Biochim. Biophys. Acta 1543 (2000) 336–360. [11] N.L. Cieh, S. Sulaiman, M.N. Mokhtar, M.N. Naim, Bleached kenaf microfiber as a support matrix for cyclodextrin glucanotransferase immobilization via covalent binding by different coupling agents, Process Biochem. 56 (2017) 81–89. [12] N. Graebin, J. Schöffer, D. Andrades, P. Hertz, M. Ayub, R. Rodrigues, Immobilization of glycoside hydrolase families GH1, GH13, and GH70: state of the art and perspectives, Molecules 21 (2016) 1074. [13] J.N. Schöffer, M.P. Klein, R.C. Rodrigues, P.F. Hertz, Continuous production of βcyclodextrin from starch by highly stable cyclodextrin glycosyltransferase immobilized on chitosan, Carbohydr. Polym. 98 (2013) 1311–1316. [14] J.N. Schöffer, C.R. Matte, D.S. Charqueiro, E.W. Menezes, T.M.H. Costa, E.V. Benvenutti, R.C. Rodrigues, P.F. Hertz, Directed immobilization of CGTase: the effect of the enzyme orientation on the enzyme activity and its use in packed-bed reactor for continuous production of cyclodextrins, Process Biochem. 58 (2017) 120–127. [15] C.R. Matte, M.R. Nunes, E.V. Benvenutti, J.N. Schöffer, M.A.Z. Ayub, P.F. Hertz, Characterization of cyclodextrin glycosyltransferase immobilized on sílica microspheres via aminopropyltrimethoxysilane as a “spacer arm”, J. Mol. Catal., B Enzym. 78 (2012) 51–56. [16] D.I. Bezbradica, C. Mateo, J.M. Guisan, Novel support for enzyme immobilization prepared by chemical activation with cysteine and glutaraldehyde, J. Mol. Catal., B Enzym. 102 (2014) 218–224. [17] S. Hudson, J. Cooney, E. Magner, Proteins in mesoporous silicates, Angew. Chem. 47 (2008) 8582–8594. [18] N. Carlsson, H. Gustafsson, C. Thörn, L. Olsson, K. Holmberg, B. Akerman, Enzymes immobilized in mesoporous silica: a physical–chemical perspective, Adv. Colloid Interface Sci. 205 (2014) 339–360. [19] C. Bernal, L. Sierra, M. Mesa, Application of Hierarchical porous silica with a stable large porosity for β-galactosidase immobilization, Chem Cat Chem 3 (2011) 1948–1954. [20] J. Kim, R.J. Desch, S.W. Thiel, V.V. Guliants, N.G. Pinto, Energetics of protein adsorption on amine-functionalized mesostructured cellular foam silica, J. Chromatogr. A 1218 (2011) 7796–7803. [21] D.N. Tran, K.J. Balkus, Perspective of recent progress in immobilization of enzymes, ACS Catal. 1 (2011) 956–968. [22] J.H. Dantas, L.D. de Paris, C.E. Barão, P.A.A. Arroyo, C.M.F. Soares, J.V. Visentainer, F. Faria, G.M. Zanin, Influence of alcohol: oil molar ratio on the production of ethyl esters by enzymatic transesterification of canola oil, Afr. J. Biotechnol. 12 (2013) 6968–6979. [23] C. Bernal, L. Sierra, M. Mesa, Design of β-galactosidase/silica biocatalysts: impact of the enzyme properties and immobilization pathways on their catalytic performance, Eng. Life Sci. 14 (2014) 85–94. [24] D. Brady, J. Jordaan, Advances in enzyme immobilisation, Biotechnol. Lett. 31 (2009) 1639–1650. [25] C. Mateo, J.M. Palomo, M.F. uentes, L. Betancor, V. Grazu, F. López-Gallego, B.C.C. Pessela, A. Hidalgo, G. Fernández-Lorente, R. Fernández-Lafuente, J.M. Guisán, Glyoxyl agarose, a fully inert and hydrophilic support for immobilization and high stabilization of proteins, Enzyme Microb. Technol. 39 (2006) 274–280. [26] C. Mateo, J.M. Palomo, G. Fernandez-Lorente, J.M. Guisan, R. Fernandez-Lafuente, Improvement of enzyme activity, stability and selectivity via immobilization techniques, Enzyme Microb. Technol. 40 (2007) 1451–1463. [27] L.P. Calsavara, A.R. Dias da Cunha, T.A. Balbino, G.M. Zanin, F.F. de Moraes, Production of cyclodextrins from cornstarch granules in a sequential batch mode and in the presence of etanol, Appl. Biochem. Biotechnol. 165 (2011) 1485–1493. [28] B.E. Norman, S.T. Jorgensen, Thermoanaerobacter sp. CGTase: its properties and applications, Denpun Kagaku 39 (1992) 101–108. [29] M. Kamori, T. Hori, Y. Yamashita, Y. Hirose, Y. Naoshima, Immobilization of lipase on a new inorganic ceramics support, toyonite, and the reactivity and
4. Conclusion This study described two methods of enzymatic immobilization based on the use of silica for the immobilization of commercial CGTase (Toruzyme®), that is, by internal surface anchorage of the enzyme and its covalent immobilization. The results showed that the anchorage immobilization method is a simple and effective manner for the fixation of the commercial CGTase on the internal surface of the immobilization matrix. A higher production yield of CDs was obtained with the improvement of the SCGT-A immobilization process, which evaluated the solvent used to wash the silica after enzymatic immobilization; the composition of the solvent used in enzymatic immobilization; and the concentration of CGTase added to the immobilization procedure. The method SCGT-CB gave a lower immobilization yield (48.44%) than SCGT-A (89.63%). There was an increase in the total production of CDs of 60.97% and 34.90% for SCGT-A and SCGT-CB, respectively, in a reaction medium containing 10% ethanol, in comparison with the same medium without ethanol, showing that the use of ethanol is very advantageous to obtain a higher production yield of CDs, mainly in relation to α-CD. The use of SCGT-A was more efficient than SCGT-CB and CGT-F, for employing a short reaction time, 24 h, SCGT-A produced 70.49% more total CDs than SCGT-CB, and 18.15% more than CGT-F. The production of CDs for five consecutive cycles showed that, although there was significant production of total CDs in the first reaction cycle for both SCGT-A and SCGT-CB, this progressively declined for the other cycles. Considering the simplicity of the two immobilization techniques evaluated and the immobilized enzyme performs well, it is suggested that the new biocatalysts described can be used in the production of CDs. The increase in CD production obtained by the SCGT-A method and the continuous production of CDs in long-term reactions conferred by the SCGT-CB method make the use of immobilized CGTase viable for industrial applications. Funding sources This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Author contribution All authors had materially participated in the research and article preparation was made by G.G.G. and G.M. Acknowledgements The authors are thankful to the Brazilian Agencies Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação Araucária for their financial support of this work. References [1] G. Astray, C. González-Barreiro, J.C. Mejuto, R. Rial-Otero, J. Simal-Gándara, A
76
Process Biochemistry 85 (2019) 68–77
G.G. Gimenez, et al.
[30]
[31] [32]
[33] [34] [35] [36]
[37] [38]
enantioselectivity of the immobilized lipase, J. Mol. Catal., B Enzym. 9 (2000) 269–274. J. Wang, G. Meng, K. Tao, M. Feng, X. Zhao, Z. Li, H. Xu, D. Xia, J.R. Lu, Immobilization of lipases on alkyl silane modified magnetic nanoparticles: effect of alkyl chain length on enzyme activity, PLoS One 7 (2012) e43478. J.H. Dantas, Modulação da hidrofobicidade da sílica para imobilização de lipase de Burkholderia cepacia, Doctoral thesis, State University of Maringa, 2016. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. K. Wilson, J. Walker, Principles and Techniques of Biochemistry and Molecular Biology, seventh ed., Cambridge University Press, New York, 2010. Z. Li, M. Wang, F. Wang, Z. Gu, G. Du, J. Wu, γ-Cyclodextrin: a review on enzymatic production and application, Appl. Microbiol. Biotechnol. 77 (2007) 245–255. T.-J. Kim, B.-C. Kim, H.-S. Lee, Production of cyclodextrin using raw corn starch without a pretreatment, Enzyme Microb. Technol. 20 (1997) 506–509. P.W. Tardioli, G.M. Zanin, F.F. Moraes, Characterization of Thermoanaerobacter cyclomaltodextrin glucanotransferase immobilized on glyoxilagarose, Enzyme Microb. Technol. 39 (2006) 1270–1278. R.M. Blanco, P. Terreros, N. Muñoz, E. Serra, Ethanol improves lipase immobilization on a hydrophobic support, J. Mol. Catal., B Enzym. 47 (2007) 13–20. C. Li, Q. Xu, Z. Gu, S. Chen, J. Wu, Y. Hong, L. Cheng, Z. Li, Cyclodextrin
[39]
[40]
[41] [42]
[43] [44]
[45]
77
glycosyltransferase variants experience different modes of product inhibition, J. Mol. Catal., B Enzym. 133 (2016) 203–210. C. Mazzer, L.R. Ferreira, J.R.T. Rodella, C. Moriwaki, G. Matioli, Cyclodextrin production by Bacillus firmus strain 37 immobilized on inorganic matrices and alginate gel, Biochem. Eng. J. 41 (2008) 79–86. B. Cheirsilp, S. Kitcha, S. Maneerat, Kinetic characteristics of β-cyclodextrin production by cyclodextrin glycosyltransferase from newly isolated Bacillus sp. C26, Electron. J. Biotechnol. 13 (2010) 4–5. V.D. Alves, S. Mali, A. Beléia, M.V.E. Grossmann, Effect of glycerol and amylose enrichment on cassava starch film properties, J. Food Eng. 78 (2007) 941–946. F.H. Weber, F.P. Collares-Queiroz, Y.K. Chang, Physicochemical, rheological, morphological, and thermal characterization of normal, waxy, and high amylose corn starches, Ciênc, Tecnol. Aliment. 29 (2009) 748–753. J.N. Talbert, J.M. Goddard, Enzymes on material surfaces, Colloids Surf. B Biointerfaces 93 (2012) 8–19. A.D. Blackwood, C. Bucke, Addition of polar organic solvents can improve the product selectivity of cyclodextrin glycosyltransferase: solvent effects on CGTase, Enzyme Microb. Technol. 27 (2000) 704–708. S. Mori, M. Goto, T. Mase, A. Matsuura, T. Ova, S. Kitahata, Reaction conditions for the production of γ-cyclodextrin by cyclodextrin glucanotransferase from Brevibacterium sp, Biosci. Biotechnol. Biochem. 59 (2000) 1012–1015.