- Email: [email protected]
Production and single step purification of cyclodextrin glycosyltransferase from alkalophilic Bacillus firmus by ion exchange chromatography
Biochemical Engineering Journal 39 (2008) 510–515
Production and single step purification of cyclodextrin glycosyltransferase from alkalophilic Bacillus firmus by ion exchange chromatography Laxman S. Savergave, Santosh S. Dhule, Vitthal V. Jogdand ∗ , Sanjay N. Nene, Ramchandra V. Gadre Chemical Engineering and Process Development Division, National Chemical Laboratory, Pashan Road, Pune 411008, India Received 26 March 2007; received in revised form 29 September 2007; accepted 30 September 2007
Abstract Production and purification of starch digesting cyclodextrin glycosyl transferase (CGTase) from alkalophilic Bacillus firmus was investigated. Fermentation was carried out in 14 l bioreactor at 28 ◦ C using a medium containing dextrin, yeast extract, peptone, (NH4 )H2 PO4 and MgSO4 ·7H2 O. The extracellular enzyme was concentrated by tangential flow ultrafiltration. The concentrated enzyme was chromatographed using DEAEsepharose and phenyl sepharose. DEAE-sepharose could be used to purify CGTase in a single step with 23.1 fold purification and 80.6% recovery. The enzyme obtained had homogeneity and the molecular weight was 76 kDa confirmed by SDS-PAGE. © 2007 Elsevier B.V. All rights reserved. Keywords: CGTase; Bacillus firmus; Cyclodextrin; Alkalophilic; Purification
1. Introduction Cyclodextrin glycosyl transferase (CGTase), EC 2.4.1.19, is an extracelluar enzyme that converts starch into non-reducing, cyclic malto-oligosacchrarides called cyclodextrins (CDs). It is an important hydrolytic enzyme that carries out reversible intermolecular as well as intramolecular transglycosylation and performs cyclization, coupling and disproportionation of maltooligosaccharides [1]. Cyclodextrins have their systematic names of cyclic ␣-d-(1,4)-linked d-glucose oligosaccharides consisting of 6–8 glycosyl units, well known as ␣-, - and ␥-CDs [2]. CD molecules have the ability to form inclusion complexes with a variety of compounds therefore they are used in a wide range of application in food, pharmaceutical, cosmetic and agricultural industries [3,4]. CGTase is produced by species of Bacillus, Brevibacterium, Clostridium, Corynebacterium, Klebsiella, Micrococcus, Pseudomonas, Thermoanaerobacter and Thermoanaerobacterium [1]. Industrial production of CGTase became attractive only when alkalophilic Bacillus species were introduced as produc-
∗
Corresponding author. Tel.: +91 20 25902347; fax: +91 20 25903041. E-mail address: [email protected] (V.V. Jogdand).
1369-703X/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2007.09.020
tion organism. All the CGTase enzymes produce ␣-, - or ␥-CDs from starch in different ratios. Enzymes that can synthesize predominately one type of CD are preferred for industrial applications because separation of individual CDs from their mixture is expensive [5]. The characteristics of CGTase to transglycosylate various aglycone molecules like stevioside, rebaudioside, etc. are gaining attention of researchers in the field of biotransformation [6–9]. -Cyclodextrin forms colourless inclusion complex with phenolphthalein therefore, this property is used in the spectrophotometric method for the estimation of -CD concentration and thereby -CGTase activity. The main problem of this method is that the colour of the reagent is unstable and its optical density decreases linearly with time. Thus the decrease in colour intensity is both due to cyclodextrin concentration and time required for determination of optical density, which lead to erroneous results [10]. Various unit operations used in downstream processing for getting pure protein from fermentation broth constitutes the largest part of the production cost [11]. There are many reports on the purification of the CGTase where it required multiple steps of column chromatography after cell separation [12,13]. Purification strategies used mainly involve adsorption of CGTase on
L.S. Savergave et al. / Biochemical Engineering Journal 39 (2008) 510–515
starch, followed by gel filtration. However, drawback with the starch columns is that CGTase reacts with starch and produces cyclodextrins during elution and thus requires an additional step to exclude the CDs. Due to the additional step of removal of CDs very low recovery of the purified CGTases is reported. In the present work, attempt is made to purify CGTase produced by an alkalophilic Bacillus firmus, in a single step using column chromatography. DEAE-sepharose and phenyl sepharose were used as chromatographic matrices and evaluated for purification and recovery of purified CGTase. Based on these parameters, DEAEsepharose was found to be more efficient for the separation than other reported matrices. Homogeneity of the purified enzyme was confirmed by SDS-PAGE. 2. Materials and methods 2.1. Materials -Cyclodextrin and bovine serum albumin (fraction V) were purchased from Sigma. Soluble starch was purchased from E. Merck Mumbai, India, and dextrin was from Laxmi Starch, Coimbatore, India. All media components were procured from Hi-Media, Mumbai, India. Electrophoresis reagents were from SRL Pvt. Ltd., Mumbai, India. Molecular weight marker, DEAE-sepharose and phenyl sepharose were procured from Amersham Biosciences (GE Healthcare), Uppsala, Sweden. All other chemicals were of an analytical grade.
511
in absorbance was calculated with respect to blank containing Tris–HCl buffer instead of -CD solution. 2.4. Modified β-cyclodextrin glycosyltransferase assay One ml of appropriately diluted enzyme sample was incubated at 60 ◦ C for 15 min with 5 ml of 1% (w/v) gelatinized soluble starch in 50 mM, 7.0-pH Tris–HCl buffer. Reaction was terminated by boiling the reaction mixture for 3 min and reaction volume was made to 10 ml with distilled water. Two ml of above reaction mixture was withdrawn and mixed with 3 ml of Tris–HCl buffer, 5 ml of 125 mM Na2 CO3 , and 0.5 ml of phenolphthalein (25 mg phenolphthalein/100 ml absolute alcohol). Absorbance was measured at 550 nm. The percent decrease of sample was calculated with respect to control containing 5 ml of buffer, 5 ml of sodium carbonate and 0.5 ml of phenolphthalein. % decrease in absorbance =
Acontrol − Atest × 100 Acontrol
where Acontrol = absorbance of control and Atest = absorbance of sample. The amount of -cyclodextrin (-CD) produced was estimated from the standard graph of 0–500 g/ml -CD concentration against % decrease in absorbance. One unit of CGTase was defined as the amount of enzyme required to produce 1 m of -CD/min. 2.5. Production of CGTase
2.2. Strain Bacterial strain was B. firmus 5119 from NCIM (National Collection of Industrial Microorganism) Pune, India [14]. 2.3. Phenolphthalein reagent stability study Phenolphthalein assay reagent was prepared according to Goel and Nene [15]. The reagents prepared were phenolphthalein stock solution (4 mM) in ethanol, 125 mM sodium carbonate, 0.05 M Tris–HCl buffer (pH 7.0) and 300 g/ml CD in Tris–HCl buffer. Advantage of making phenolphthalein stock solution in ethanol is that it could be diluted with sodium carbonate buffer just before CD analysis where 1 ml of this solution was added to 4 ml ethanol and its volume was made to 100 ml with Na2 CO3 . To 1 ml of appropriately diluted -CD solution (to get 50–300 g/ml -CD) 4 ml of phenolphthalein (corresponding to 60 g phenolphthalein per assay) reagent was added and its optical density was measured at 550 nm. Percentage decrease in optical density was calculated with respect to a control that contained 1 ml Tris–HCl buffer instead of -CD solution. In the modified method, standard graph was prepared by diluting various volumes of -CD stock solution (500 g/ml -CD) to 5 ml with 50 mM Tris–HCl buffer (pH 7.0). To this, 5 ml of 125 mM sodium carbonate and 0.5 ml of phenolphthalein stock (25 mg phenolphthalein/100 ml absolute ethanol) were mixed and used for -CD estimation as above and percent decrease
Gawande et al. [16] optimized media for Klebsiella pneumoniae pneunoniae AS-22. They found soluble carbon in the form of dextrin was better than starch for cyclodextrin glucanosyltransferase enzyme production. Use of dextrin as carbon source is also advantageous as it can be used in higher concentration and its solution can be easily pumped for operating the fermenter in fed batch mode. Therefore we used dextrin in the medium. During inoculum build up, a tube containing 5 ml basal medium containing dextrin 40 g/l, yeast extract 10 g/l, peptone 10 g/l, (NH4 )H2 PO4 4 g/l, MgSO4 ·7H2 O 0.5 g/l was autoclaved and separately autoclaved Na2 CO3 10 g/l was added to it and inoculated with a loop full of freshly prepared slant of B. firmus. The tube was incubated at 28 ◦ C on rotary shaker at 210 rpm for 48 h and 0.5 ml from culture broth was transferred to 10 tubes each containing 4.5 ml of basal medium and incubated for 12 h. These 10 tubes were transferred to ten 250 ml flasks containing 45 ml basal medium and incubated for 12 h. All the flasks were pooled together and used as seed culture for 14 l New Brunswick Scientific Bioflow-2000 fermenter. Fermentation medium was also as in the inoculation medium and separately autoclaved Na2 CO3 10 g/l was added to it before inoculation. Fermentation was carried out at 28 ◦ C with 10 l working volume. Airflow rate of 0.5-vvm (volume of air per unit volume of medium per minute) was used throughout the run. The pH of the medium was initially maintained at 9 ± 0.5 with 1% sodium carbonate. The dissolved oxygen (DO) was
512
L.S. Savergave et al. / Biochemical Engineering Journal 39 (2008) 510–515
maintained above 20% of oxygen saturation using an automatic agitation control and silicon oil was used as antifoam. Fermentation was carried out in batch mode till the stationary phase was reached. Samples were removed at regular interval to check the growth and CGTase activity. Cell mass was measured as optical density at 600 nm. An aliquot was centrifuged at 10,000 rpm for 10 min and CGTase activity in the supernatant was determined. Protein was estimated according to Lowry et al. [17]. 2.6. Cell separation and CGTase concentration Fermentation broth was cooled to 10 ◦ C and centrifuged at 18,000 rpm using a Sharples centrifuge having provision of continuous feeding. Supernatant was subjected to microfiltration with hollow fiber membrane module with membrane area of 3.75 ft2 (AgTech USA). The enzyme in the permeate was concentrated by ultrafiltration using a 10-kDa MWCO (molecular weight cut-off) hollow fiber membrane module (Nitto Denko Corporation, surface area of membrane 0.375 m2 ).
Fig. 1. Decrease in phenolphthalein absorbance with time. Profile of blank readings observed by () reproducing the method of Goel and Nene and () modified method.
2.7. Phenyl sepharose chromatography AKTA purifier, a protein purification system from Amersham Biosciences, was used for purification studies. Binding study was carried out at different ammonium sulphate concentrations from 0.8 M to 1.2 M. Tricorn 10/200 column with phenyl sepharose was equilibrated with 25 mM, pH 7.0 Tris–HCl buffer containing 0.8 M to 1.2 M (NH4 )2 SO4 , respectively. One millilitre of concentrated enzyme sample was supplemented with 1 M (NH4 )2 SO4 and loaded at linear flow rate of 38.2 cm/h. Elution was carried out by stepwise decrease in the ionic strength of (NH4 )2 SO4 ranging from 0.25 M to 0 M, at linear flow rate of 76.4 cm/h. Fractions were analyzed for CGTase activity and protein concentration. 2.8. Purification of CGTase using DEAE-sepharose DEAE-sepharose was packed in Tricorn 10/200 column and equilibrated with 25 mM, pH 7.0 Tris–HCl buffer. One ml of concentrated enzyme sample with 70 units and 7.87 mg of protein was loaded at linear velocity of 76.4 cm/h. The column was washed with the equilibrating buffer to remove unbound proteins. Five millilitre fractions were collected. For elution, ionic strength of NaCl was increased and its linear velocity was 76.4 cm/h. The collected fractions were analyzed for protein and CGTase activity.
Fig. 2. Growth profile and CGTase production by Bacillus firmus.
containing myosin (220 kDa), ␣2 -macroglobulin (170 kDa), -galactosidase (116 kDa), transferrin (76 kDa) and glutamic dehydrogenase (53 kDa) was used. Protein bands were envisaged by silver staining protocol.
2.9. SDS-PAGE Homogeneity of protein in eluted fractions was checked by SDS-PAGE on a vertical slab gel electrophoresis using 7.5% acrylamide gel at constant current of 30 mA for 2 h. Gel (8 cm × 12 cm) was run according to the method of Laemmli [18]. Twenty-five microlitres of appropriately diluted marker and samples were applied into the wells. Molecular weight marker from Amersham Biosciences
Fig. 3. Binding and elution profile of CGTase using DEAE-sepharose on AKTA purifier.
L.S. Savergave et al. / Biochemical Engineering Journal 39 (2008) 510–515
513
Table 1 Summary of CGTase purification from alkalophilic Bacillus firmus Step
Total volume (ml)
Total protein (mg)
Total units
Specific activity (U/mg)
Purification fold
Percent yield
Crude enzyme (microfiltered) Ultrafiltration DEAE-sepharose
7650 670 12.1
50,949 5,274 2.6
51,255 46,900 61.7
1.0 8.8 23.3
– 8.8 23.1
100 91.5 80.6
3. Results and discussion
specific activity of CGTase from 1.0 U/mg to 8.8 U/mg of protein.
3.1. Modification in the phenolphthalein method The phenolphthalein method is based on decrease in absorbance due to formation of inclusion complex of phenolphthalein with -cyclodextrin. The percent reduction in absorbance can be directly correlated with the concentration of CD. However, phenolphthalein reagent is unstable and its colour intensity decreases with time [19]. This is due to isomerization of the indicator [10] at higher pH, which was about 11 in the method reported by Goel. In the modified method we were able to control the pH of the reagent from 10 to 10.2, which increased the stability of the reagent Fig. 1. Decrease in pH was achieved either by increasing the volume of Tris–HCl buffer in the assay mixture or by increasing Tris–HCl buffer concentration to 250 mM or decreasing sodium carbonate concentration to 50 mM. However, it was observed that sodium carbonate concentration also affected the absorbance of the reagent. Makela et al. observed 3.5-fold increase in the colour intensity by increasing buffer concentration from 0.004 M to 0.1 M [19]. Similarly increase in the Tris–HCl buffer concentration also showed decrease in the colour intensity. Therefore, we used the modification where volume of the buffer was increased so that blank value was more stable.
3.3. Purification of CGTase During the hydrophobic interaction chromatography, optimum concentration of ammonium sulphate for hydrophobic interaction and purity of CGTase was found to be eluted at 0.095 M (NH4 )2 SO4 from phenyl sepharose column with 8.9-fold purification and percent recovery of 64.7, but single band on SDS-PAGE was not observe (data not presented). Tachibana et al. purified the enzyme by applying it consecutively to anion-exchange chromatography (Resource Q), hydrophobic interaction chromatography (phenyl superose), and affinity chromatography (␣-CD-(epoxy)-sepharose 6B). After these three steps, CGTase was purified 1750-fold with a yield of only 10% [22]. Volkova et al. used butyl-toyopearl column followed by DEAE-sephacel and purified the CGTase with 13-fold concentration [23]. In our study DEAE-sepharose was found to be more efficient for the purification of CGTase of B. firmus. CGTase eluted
3.2. Production and concentration of CGTase In most of the microorganisms, CGTases are extracellular enzymes and differ in their amount and type of CDs produced. Conventionally, CGTase is produced by submerged fermentation and maximal production is observed in the stationary phase [20]. It is reported that CGTase is produced along with cellular growth [21]. During our experiments, the culture growth took place rapidly and dissolved oxygen decreased correspondingly. During initial period exponential growth of the culture was observed but there was hardly any enzyme produced up to 9 h of fermentation. PH was automatically controlled in the range of 8.5–9.5. After 12 h, an increase in enzyme activity was observed which reached its maximum in late log phase as depicted in Fig. 2. Maximum CGTase activity was 6.8 U/ml at 33 h and biomass OD reached 45. The fermentation broth was centrifuged to remove the biomass and CGTase. The clear supernatant enzyme was filtered using AgTech (U.S.A.) microfiltration hollow fiber module having pore size of 0.1 m and area of 3.75 ft2 . Then the enzyme was concentrated by ultrafiltration. The ultrafiltration removed undesired low molecular weight proteins from the broth, which resulted in increased
Fig. 4. SDS-PAGE. Lane 1: crude CGTase, lane 2: molecular weight marker, lane 3: purified CGTase from DEAE-sepharose, lane 4: purified CGTase from phenyl sepharose.
514
L.S. Savergave et al. / Biochemical Engineering Journal 39 (2008) 510–515
with gradient elution from 0.1 M to 0.2 M NaCl and the corresponding chromatogram is shown in Fig. 3. Other tightly bound proteins were eluted at 1 M NaCl. Table 1 indicates the purification of CGTase by DEAE-sepharose with 80% recovery and purification fold of 23. Yim et al. purified the CGTase using DEAE-sephadex A-50 followed by DEAE-sepharose CL-6B and reported recovery of 26.6% [24]. Cao et al. have reported increase in specific activity from an average of 603 U/mg of protein in crude broth to 5753 U/mg of protein after DEAE-cellulose and sepharose CL-6B gel filtration step [25]. 3.4. Homogeneity and molecular weight of the enzyme Purified enzyme from DEAE-sepharose showed a single band (Fig. 4.) by SDS-PAGE indicating one-step purification for CGTase whereas complete purification could not be achieved using phenyl sepharose. Molecular weight of CGTase was estimated as 76 kDa. Molecular weight of the previously purified CGTases from B. firmus was reported to be 80 kDa [26]. Kobayashi et al. reported that Bacillus macerans cyclodextrin glucanotranferase could be dissociated into two subunits by SDS-PAGE [27]. However, present study verified that the enzyme from B. firmus was found to be monomeric in nature. 4. Conclusion We have modified the CGTase assay, which excludes the variations due to phenolphthalein colour instability and provides a quick and reliable estimation of CGTase activity. Starch adsorption chromatography is one of the popular methods for the initial capture of the CGTase, but it demands gel filtration for the separation of CDs formed during elution of enzyme from the column. We have successfully purified CGTase in a single chromatographic step using DEAE-sepharose. Between two chromatographic techniques used, namely HIC and IEC, the later gave highly purified enzyme with better recovery. Phenyl sepharose gave partially purified enzyme with two more contaminating proteins and low recovery. Enzyme was found to elute at 0.2 M NaCl and 0.095 M (NH4 )2 SO4 during ion exchange and hydrophobic interaction chromatography, respectively. Purification protocol resulted in increased specific activity from 1 U/mg to 23.3 U/mg with purification fold of 23.1 and 80% recovery. CGTase reported in this study was monomeric with molecular weight of 76 kDa. Single band in SDS-PAGE showed homogeneity of the purified CGTase. Acknowledgement The Department of Biotechnology (DBT), India, supported this research. References [1] G. Biwer, E. Antranikian, Heinzle, Enzymatic production of cyclodextrins, Appl. Microbiol. Biotechnol. 59 (2002) 609–617.
[2] H. Bender, Production, characterization and application of cyclodextrins, Adv. Biotechnol. Process. 6 (1986) 31–71. [3] J. Szejtli, Industrial applications of cyclodextrins, Carbohyd. Polym. 12 (1992) 375–392. [4] A.R. Hedges, Cyclodextr: production, properties and applications, in: F. Schenk, R.E. Hebeda (Eds.), Starch Hydrolysis Products, VCH Publishers, New York, 1992, pp. 319–333. [5] B.N. Gawande, A. Goel, A.Y. Patkar, S.N. Nene, Purification and properties of a novel raw starch degrading cyclomaltodextrin glucanotransferase from Bacillus firmus, Appl. Microbiol. Biotechnol. 51 (1999) 504–509. [6] V.T. Kochikyan, A.A. Markosyan, L.A. Abelyan, A.M. Balayan, V.M. Abelyan, Combined enzymatic modification of stevioside and rebaudioside A, Appl. Biochem. Microbiol. 42 (2006) 31–37. [7] D.C. Park, T.A. Kim, Y.H. Lee, Characteristics of transglycosylation reaction of cyclodextrin glucanotransferase in the heterogeneous enzyme reaction system using extrusion starch as glucosyl donor, Enzyme Microb. Technol. 22 (1998) 217–222. [8] M. Sato, K. Nakamura, Y. Hiroto Nagano, K. Yagi, Koizumi, Synthesis of glucosyl-inositol using a CGTase, isolation and characterization of the positional isomers and assimilation profiles for intestinal bacteria, Biotechnol. Lett. 14 (1992) 654–659. [9] S. Riva, Enzymatic modification of the sugar moieties of natural glycosides, J. Mol. Catal. B: Enzyme 19 (2002) 43–45. [10] N.G. Usanov, E.A. Gil’vanova, P.A. Elizar’ev, E.A. Prutsakova, I.A. Melent’ev, An improved method of photometric determination of cyclodextrin glucanotransferase activity, Appl. Biochem. Microbiol. 43 (2007) 105–110. [11] G. Leaver, J.R. Conder, J.A. Howell, A method development study of the production of albumin from animal blood by ion-exchange chromatography, Separ. Sci. Technol. 22 (1987) 2037–2059. [12] B.N. Gawande, A. Goel, A.Y. Patkar, Purification and properties of novel starch degrading ␣-cyclodextrin glycosyltransferease from Klebsiella pneumoniae pneunoniae AS-22, Enzyme Microb. Technol. 28 (2001) 735–743. [13] R.F. Martins, R. Hatti-Kaul, A new cyclodextrin glycosyltransferase from an alkaliphilic Bacillus agaradhaerens isolate; purification and characterization, Enzyme Microb. Technol. 30 (2002) 116–124. [14] A. Goel, S. Nene, A novel raw starch degrading cyclomaltodextrin glucanotransferase from Bacillus firmus, Biotechnol. Lett. 17 (1995) 411– 416. [15] A. Goel, S.N. Nene, Modifications in phenolphthalein method for spectrophotometric estimation of beta cyclodextrin, Starch/Starke 47 (1995) 399–400. [16] B.N. Gawande, A. Goel, A.Y. Patkar, Application of factorial design for optimization of cyclodextrin glycosyltransferease production from Klebsiella pneumoniae pneunoniae AS-22, Biotechnol. Bioeng. 64 (1999) 168–173. [17] O.H. Lowry, N.J. Rosenbrough, R.J. Randdall, Protein measurement with folin phenol reagent, J. Biol. Chem. 193 (1951) 265–274. [18] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [19] M. Makela, T. Korpela, S. Laakso, Colorimetric determination of cyclodextrin: two assay modifications based on molecular complexation of phenolphthalein, J. Biochem. Biophys. Methods 14 (1987) 85– 92. [20] Tonkova, Bacterial cyclodextrin glucanotransferase, Enzyme Microb. Technol. 22 (1998) 678–686. [21] J.H. Lee, K.H. Choi, J.Y. Choi, Y.S. Lee, I.B. Kwon, I.H. Yu, Enzymatic production of alpha cyclodextrin with the cyclodextrin glucanotransferase of Klebsiella oxytoca 19-1, Enzyme Microb. Technol. 14 (1992) 1020–1217. [22] Y. Tachibana, A. Kuramura, N. Shirasaka, Y. Suzuki, T. Yamamoto, S. Fujiwara, M. Takagi, T. Imanaka, Purification and characterization of an extremely thermostable cyclomaltodextrin glucanotransferase from a newly isolated hyperthermophilic Archaeon, a Thermococcus sp., Appl. Environ. Microbiol. 65 (1999) 1991–1997. [23] D.A. Volkova, S.A. Lopatin, I.M. Gracheva, V.P. Varlamov, Preparation of high-purity cyclodextrin glucanotransferase from Bacillus sp. 1070, Appl. Biochem. Microbiol. 37 (2001) 138–141.
L.S. Savergave et al. / Biochemical Engineering Journal 39 (2008) 510–515 [24] D.G. Yim, H.H. Sato, Y.H. Park, Y.K. Park, Production of cyclodextrin from starch by cyclodextrin glycosyltransferase from Bacillus firmus and characterization of purified enzyme, J. Ind. Microbiol. Biotechnol. 18 (1997) 402–405. [25] X. Cao, Z. Jin, F. Chen, X. Wang, Purification and properties of cyclodextrin glucanotransferase from an alkalophilic Bacillus sp. 7-12, J. Food Biochem. 28 (2004) 463–475.
515
[26] C.B. Sohn, S.A. Kim, Y.A. Park, M.H. Kim, S.K. Moon, S.A. Jang, Characterization of cyclodextrin glycosyltransferase from Bacillus firmus, J. Korean Soc. Food Sci. Nutr. 26 (1997) 351–357. [27] S. Kobayashi, K. Kainuma, S. Suzuki, Purification and some properties of Bacillus macerans cycloamylose (cyclodextrin) glucanotransferase, Carbohyd. Res. 61 (1978) 229–238.
Production and single step purification of cyclodextrin glycosyltransferase from alkalophilic Bacillus firmus by ion exchange chromatography Laxman S. Savergave, Santosh S. Dhule, Vitthal V. Jogdand ∗ , Sanjay N. Nene, Ramchandra V. Gadre Chemical Engineering and Process Development Division, National Chemical Laboratory, Pashan Road, Pune 411008, India Received 26 March 2007; received in revised form 29 September 2007; accepted 30 September 2007
Abstract Production and purification of starch digesting cyclodextrin glycosyl transferase (CGTase) from alkalophilic Bacillus firmus was investigated. Fermentation was carried out in 14 l bioreactor at 28 ◦ C using a medium containing dextrin, yeast extract, peptone, (NH4 )H2 PO4 and MgSO4 ·7H2 O. The extracellular enzyme was concentrated by tangential flow ultrafiltration. The concentrated enzyme was chromatographed using DEAEsepharose and phenyl sepharose. DEAE-sepharose could be used to purify CGTase in a single step with 23.1 fold purification and 80.6% recovery. The enzyme obtained had homogeneity and the molecular weight was 76 kDa confirmed by SDS-PAGE. © 2007 Elsevier B.V. All rights reserved. Keywords: CGTase; Bacillus firmus; Cyclodextrin; Alkalophilic; Purification
1. Introduction Cyclodextrin glycosyl transferase (CGTase), EC 2.4.1.19, is an extracelluar enzyme that converts starch into non-reducing, cyclic malto-oligosacchrarides called cyclodextrins (CDs). It is an important hydrolytic enzyme that carries out reversible intermolecular as well as intramolecular transglycosylation and performs cyclization, coupling and disproportionation of maltooligosaccharides [1]. Cyclodextrins have their systematic names of cyclic ␣-d-(1,4)-linked d-glucose oligosaccharides consisting of 6–8 glycosyl units, well known as ␣-, - and ␥-CDs [2]. CD molecules have the ability to form inclusion complexes with a variety of compounds therefore they are used in a wide range of application in food, pharmaceutical, cosmetic and agricultural industries [3,4]. CGTase is produced by species of Bacillus, Brevibacterium, Clostridium, Corynebacterium, Klebsiella, Micrococcus, Pseudomonas, Thermoanaerobacter and Thermoanaerobacterium [1]. Industrial production of CGTase became attractive only when alkalophilic Bacillus species were introduced as produc-
∗
Corresponding author. Tel.: +91 20 25902347; fax: +91 20 25903041. E-mail address: [email protected] (V.V. Jogdand).
1369-703X/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2007.09.020
tion organism. All the CGTase enzymes produce ␣-, - or ␥-CDs from starch in different ratios. Enzymes that can synthesize predominately one type of CD are preferred for industrial applications because separation of individual CDs from their mixture is expensive [5]. The characteristics of CGTase to transglycosylate various aglycone molecules like stevioside, rebaudioside, etc. are gaining attention of researchers in the field of biotransformation [6–9]. -Cyclodextrin forms colourless inclusion complex with phenolphthalein therefore, this property is used in the spectrophotometric method for the estimation of -CD concentration and thereby -CGTase activity. The main problem of this method is that the colour of the reagent is unstable and its optical density decreases linearly with time. Thus the decrease in colour intensity is both due to cyclodextrin concentration and time required for determination of optical density, which lead to erroneous results [10]. Various unit operations used in downstream processing for getting pure protein from fermentation broth constitutes the largest part of the production cost [11]. There are many reports on the purification of the CGTase where it required multiple steps of column chromatography after cell separation [12,13]. Purification strategies used mainly involve adsorption of CGTase on
L.S. Savergave et al. / Biochemical Engineering Journal 39 (2008) 510–515
starch, followed by gel filtration. However, drawback with the starch columns is that CGTase reacts with starch and produces cyclodextrins during elution and thus requires an additional step to exclude the CDs. Due to the additional step of removal of CDs very low recovery of the purified CGTases is reported. In the present work, attempt is made to purify CGTase produced by an alkalophilic Bacillus firmus, in a single step using column chromatography. DEAE-sepharose and phenyl sepharose were used as chromatographic matrices and evaluated for purification and recovery of purified CGTase. Based on these parameters, DEAEsepharose was found to be more efficient for the separation than other reported matrices. Homogeneity of the purified enzyme was confirmed by SDS-PAGE. 2. Materials and methods 2.1. Materials -Cyclodextrin and bovine serum albumin (fraction V) were purchased from Sigma. Soluble starch was purchased from E. Merck Mumbai, India, and dextrin was from Laxmi Starch, Coimbatore, India. All media components were procured from Hi-Media, Mumbai, India. Electrophoresis reagents were from SRL Pvt. Ltd., Mumbai, India. Molecular weight marker, DEAE-sepharose and phenyl sepharose were procured from Amersham Biosciences (GE Healthcare), Uppsala, Sweden. All other chemicals were of an analytical grade.
511
in absorbance was calculated with respect to blank containing Tris–HCl buffer instead of -CD solution. 2.4. Modified β-cyclodextrin glycosyltransferase assay One ml of appropriately diluted enzyme sample was incubated at 60 ◦ C for 15 min with 5 ml of 1% (w/v) gelatinized soluble starch in 50 mM, 7.0-pH Tris–HCl buffer. Reaction was terminated by boiling the reaction mixture for 3 min and reaction volume was made to 10 ml with distilled water. Two ml of above reaction mixture was withdrawn and mixed with 3 ml of Tris–HCl buffer, 5 ml of 125 mM Na2 CO3 , and 0.5 ml of phenolphthalein (25 mg phenolphthalein/100 ml absolute alcohol). Absorbance was measured at 550 nm. The percent decrease of sample was calculated with respect to control containing 5 ml of buffer, 5 ml of sodium carbonate and 0.5 ml of phenolphthalein. % decrease in absorbance =
Acontrol − Atest × 100 Acontrol
where Acontrol = absorbance of control and Atest = absorbance of sample. The amount of -cyclodextrin (-CD) produced was estimated from the standard graph of 0–500 g/ml -CD concentration against % decrease in absorbance. One unit of CGTase was defined as the amount of enzyme required to produce 1 m of -CD/min. 2.5. Production of CGTase
2.2. Strain Bacterial strain was B. firmus 5119 from NCIM (National Collection of Industrial Microorganism) Pune, India [14]. 2.3. Phenolphthalein reagent stability study Phenolphthalein assay reagent was prepared according to Goel and Nene [15]. The reagents prepared were phenolphthalein stock solution (4 mM) in ethanol, 125 mM sodium carbonate, 0.05 M Tris–HCl buffer (pH 7.0) and 300 g/ml CD in Tris–HCl buffer. Advantage of making phenolphthalein stock solution in ethanol is that it could be diluted with sodium carbonate buffer just before CD analysis where 1 ml of this solution was added to 4 ml ethanol and its volume was made to 100 ml with Na2 CO3 . To 1 ml of appropriately diluted -CD solution (to get 50–300 g/ml -CD) 4 ml of phenolphthalein (corresponding to 60 g phenolphthalein per assay) reagent was added and its optical density was measured at 550 nm. Percentage decrease in optical density was calculated with respect to a control that contained 1 ml Tris–HCl buffer instead of -CD solution. In the modified method, standard graph was prepared by diluting various volumes of -CD stock solution (500 g/ml -CD) to 5 ml with 50 mM Tris–HCl buffer (pH 7.0). To this, 5 ml of 125 mM sodium carbonate and 0.5 ml of phenolphthalein stock (25 mg phenolphthalein/100 ml absolute ethanol) were mixed and used for -CD estimation as above and percent decrease
Gawande et al. [16] optimized media for Klebsiella pneumoniae pneunoniae AS-22. They found soluble carbon in the form of dextrin was better than starch for cyclodextrin glucanosyltransferase enzyme production. Use of dextrin as carbon source is also advantageous as it can be used in higher concentration and its solution can be easily pumped for operating the fermenter in fed batch mode. Therefore we used dextrin in the medium. During inoculum build up, a tube containing 5 ml basal medium containing dextrin 40 g/l, yeast extract 10 g/l, peptone 10 g/l, (NH4 )H2 PO4 4 g/l, MgSO4 ·7H2 O 0.5 g/l was autoclaved and separately autoclaved Na2 CO3 10 g/l was added to it and inoculated with a loop full of freshly prepared slant of B. firmus. The tube was incubated at 28 ◦ C on rotary shaker at 210 rpm for 48 h and 0.5 ml from culture broth was transferred to 10 tubes each containing 4.5 ml of basal medium and incubated for 12 h. These 10 tubes were transferred to ten 250 ml flasks containing 45 ml basal medium and incubated for 12 h. All the flasks were pooled together and used as seed culture for 14 l New Brunswick Scientific Bioflow-2000 fermenter. Fermentation medium was also as in the inoculation medium and separately autoclaved Na2 CO3 10 g/l was added to it before inoculation. Fermentation was carried out at 28 ◦ C with 10 l working volume. Airflow rate of 0.5-vvm (volume of air per unit volume of medium per minute) was used throughout the run. The pH of the medium was initially maintained at 9 ± 0.5 with 1% sodium carbonate. The dissolved oxygen (DO) was
512
L.S. Savergave et al. / Biochemical Engineering Journal 39 (2008) 510–515
maintained above 20% of oxygen saturation using an automatic agitation control and silicon oil was used as antifoam. Fermentation was carried out in batch mode till the stationary phase was reached. Samples were removed at regular interval to check the growth and CGTase activity. Cell mass was measured as optical density at 600 nm. An aliquot was centrifuged at 10,000 rpm for 10 min and CGTase activity in the supernatant was determined. Protein was estimated according to Lowry et al. [17]. 2.6. Cell separation and CGTase concentration Fermentation broth was cooled to 10 ◦ C and centrifuged at 18,000 rpm using a Sharples centrifuge having provision of continuous feeding. Supernatant was subjected to microfiltration with hollow fiber membrane module with membrane area of 3.75 ft2 (AgTech USA). The enzyme in the permeate was concentrated by ultrafiltration using a 10-kDa MWCO (molecular weight cut-off) hollow fiber membrane module (Nitto Denko Corporation, surface area of membrane 0.375 m2 ).
Fig. 1. Decrease in phenolphthalein absorbance with time. Profile of blank readings observed by () reproducing the method of Goel and Nene and () modified method.
2.7. Phenyl sepharose chromatography AKTA purifier, a protein purification system from Amersham Biosciences, was used for purification studies. Binding study was carried out at different ammonium sulphate concentrations from 0.8 M to 1.2 M. Tricorn 10/200 column with phenyl sepharose was equilibrated with 25 mM, pH 7.0 Tris–HCl buffer containing 0.8 M to 1.2 M (NH4 )2 SO4 , respectively. One millilitre of concentrated enzyme sample was supplemented with 1 M (NH4 )2 SO4 and loaded at linear flow rate of 38.2 cm/h. Elution was carried out by stepwise decrease in the ionic strength of (NH4 )2 SO4 ranging from 0.25 M to 0 M, at linear flow rate of 76.4 cm/h. Fractions were analyzed for CGTase activity and protein concentration. 2.8. Purification of CGTase using DEAE-sepharose DEAE-sepharose was packed in Tricorn 10/200 column and equilibrated with 25 mM, pH 7.0 Tris–HCl buffer. One ml of concentrated enzyme sample with 70 units and 7.87 mg of protein was loaded at linear velocity of 76.4 cm/h. The column was washed with the equilibrating buffer to remove unbound proteins. Five millilitre fractions were collected. For elution, ionic strength of NaCl was increased and its linear velocity was 76.4 cm/h. The collected fractions were analyzed for protein and CGTase activity.
Fig. 2. Growth profile and CGTase production by Bacillus firmus.
containing myosin (220 kDa), ␣2 -macroglobulin (170 kDa), -galactosidase (116 kDa), transferrin (76 kDa) and glutamic dehydrogenase (53 kDa) was used. Protein bands were envisaged by silver staining protocol.
2.9. SDS-PAGE Homogeneity of protein in eluted fractions was checked by SDS-PAGE on a vertical slab gel electrophoresis using 7.5% acrylamide gel at constant current of 30 mA for 2 h. Gel (8 cm × 12 cm) was run according to the method of Laemmli [18]. Twenty-five microlitres of appropriately diluted marker and samples were applied into the wells. Molecular weight marker from Amersham Biosciences
Fig. 3. Binding and elution profile of CGTase using DEAE-sepharose on AKTA purifier.
L.S. Savergave et al. / Biochemical Engineering Journal 39 (2008) 510–515
513
Table 1 Summary of CGTase purification from alkalophilic Bacillus firmus Step
Total volume (ml)
Total protein (mg)
Total units
Specific activity (U/mg)
Purification fold
Percent yield
Crude enzyme (microfiltered) Ultrafiltration DEAE-sepharose
7650 670 12.1
50,949 5,274 2.6
51,255 46,900 61.7
1.0 8.8 23.3
– 8.8 23.1
100 91.5 80.6
3. Results and discussion
specific activity of CGTase from 1.0 U/mg to 8.8 U/mg of protein.
3.1. Modification in the phenolphthalein method The phenolphthalein method is based on decrease in absorbance due to formation of inclusion complex of phenolphthalein with -cyclodextrin. The percent reduction in absorbance can be directly correlated with the concentration of CD. However, phenolphthalein reagent is unstable and its colour intensity decreases with time [19]. This is due to isomerization of the indicator [10] at higher pH, which was about 11 in the method reported by Goel. In the modified method we were able to control the pH of the reagent from 10 to 10.2, which increased the stability of the reagent Fig. 1. Decrease in pH was achieved either by increasing the volume of Tris–HCl buffer in the assay mixture or by increasing Tris–HCl buffer concentration to 250 mM or decreasing sodium carbonate concentration to 50 mM. However, it was observed that sodium carbonate concentration also affected the absorbance of the reagent. Makela et al. observed 3.5-fold increase in the colour intensity by increasing buffer concentration from 0.004 M to 0.1 M [19]. Similarly increase in the Tris–HCl buffer concentration also showed decrease in the colour intensity. Therefore, we used the modification where volume of the buffer was increased so that blank value was more stable.
3.3. Purification of CGTase During the hydrophobic interaction chromatography, optimum concentration of ammonium sulphate for hydrophobic interaction and purity of CGTase was found to be eluted at 0.095 M (NH4 )2 SO4 from phenyl sepharose column with 8.9-fold purification and percent recovery of 64.7, but single band on SDS-PAGE was not observe (data not presented). Tachibana et al. purified the enzyme by applying it consecutively to anion-exchange chromatography (Resource Q), hydrophobic interaction chromatography (phenyl superose), and affinity chromatography (␣-CD-(epoxy)-sepharose 6B). After these three steps, CGTase was purified 1750-fold with a yield of only 10% [22]. Volkova et al. used butyl-toyopearl column followed by DEAE-sephacel and purified the CGTase with 13-fold concentration [23]. In our study DEAE-sepharose was found to be more efficient for the purification of CGTase of B. firmus. CGTase eluted
3.2. Production and concentration of CGTase In most of the microorganisms, CGTases are extracellular enzymes and differ in their amount and type of CDs produced. Conventionally, CGTase is produced by submerged fermentation and maximal production is observed in the stationary phase [20]. It is reported that CGTase is produced along with cellular growth [21]. During our experiments, the culture growth took place rapidly and dissolved oxygen decreased correspondingly. During initial period exponential growth of the culture was observed but there was hardly any enzyme produced up to 9 h of fermentation. PH was automatically controlled in the range of 8.5–9.5. After 12 h, an increase in enzyme activity was observed which reached its maximum in late log phase as depicted in Fig. 2. Maximum CGTase activity was 6.8 U/ml at 33 h and biomass OD reached 45. The fermentation broth was centrifuged to remove the biomass and CGTase. The clear supernatant enzyme was filtered using AgTech (U.S.A.) microfiltration hollow fiber module having pore size of 0.1 m and area of 3.75 ft2 . Then the enzyme was concentrated by ultrafiltration. The ultrafiltration removed undesired low molecular weight proteins from the broth, which resulted in increased
Fig. 4. SDS-PAGE. Lane 1: crude CGTase, lane 2: molecular weight marker, lane 3: purified CGTase from DEAE-sepharose, lane 4: purified CGTase from phenyl sepharose.
514
L.S. Savergave et al. / Biochemical Engineering Journal 39 (2008) 510–515
with gradient elution from 0.1 M to 0.2 M NaCl and the corresponding chromatogram is shown in Fig. 3. Other tightly bound proteins were eluted at 1 M NaCl. Table 1 indicates the purification of CGTase by DEAE-sepharose with 80% recovery and purification fold of 23. Yim et al. purified the CGTase using DEAE-sephadex A-50 followed by DEAE-sepharose CL-6B and reported recovery of 26.6% [24]. Cao et al. have reported increase in specific activity from an average of 603 U/mg of protein in crude broth to 5753 U/mg of protein after DEAE-cellulose and sepharose CL-6B gel filtration step [25]. 3.4. Homogeneity and molecular weight of the enzyme Purified enzyme from DEAE-sepharose showed a single band (Fig. 4.) by SDS-PAGE indicating one-step purification for CGTase whereas complete purification could not be achieved using phenyl sepharose. Molecular weight of CGTase was estimated as 76 kDa. Molecular weight of the previously purified CGTases from B. firmus was reported to be 80 kDa [26]. Kobayashi et al. reported that Bacillus macerans cyclodextrin glucanotranferase could be dissociated into two subunits by SDS-PAGE [27]. However, present study verified that the enzyme from B. firmus was found to be monomeric in nature. 4. Conclusion We have modified the CGTase assay, which excludes the variations due to phenolphthalein colour instability and provides a quick and reliable estimation of CGTase activity. Starch adsorption chromatography is one of the popular methods for the initial capture of the CGTase, but it demands gel filtration for the separation of CDs formed during elution of enzyme from the column. We have successfully purified CGTase in a single chromatographic step using DEAE-sepharose. Between two chromatographic techniques used, namely HIC and IEC, the later gave highly purified enzyme with better recovery. Phenyl sepharose gave partially purified enzyme with two more contaminating proteins and low recovery. Enzyme was found to elute at 0.2 M NaCl and 0.095 M (NH4 )2 SO4 during ion exchange and hydrophobic interaction chromatography, respectively. Purification protocol resulted in increased specific activity from 1 U/mg to 23.3 U/mg with purification fold of 23.1 and 80% recovery. CGTase reported in this study was monomeric with molecular weight of 76 kDa. Single band in SDS-PAGE showed homogeneity of the purified CGTase. Acknowledgement The Department of Biotechnology (DBT), India, supported this research. References [1] G. Biwer, E. Antranikian, Heinzle, Enzymatic production of cyclodextrins, Appl. Microbiol. Biotechnol. 59 (2002) 609–617.
[2] H. Bender, Production, characterization and application of cyclodextrins, Adv. Biotechnol. Process. 6 (1986) 31–71. [3] J. Szejtli, Industrial applications of cyclodextrins, Carbohyd. Polym. 12 (1992) 375–392. [4] A.R. Hedges, Cyclodextr: production, properties and applications, in: F. Schenk, R.E. Hebeda (Eds.), Starch Hydrolysis Products, VCH Publishers, New York, 1992, pp. 319–333. [5] B.N. Gawande, A. Goel, A.Y. Patkar, S.N. Nene, Purification and properties of a novel raw starch degrading cyclomaltodextrin glucanotransferase from Bacillus firmus, Appl. Microbiol. Biotechnol. 51 (1999) 504–509. [6] V.T. Kochikyan, A.A. Markosyan, L.A. Abelyan, A.M. Balayan, V.M. Abelyan, Combined enzymatic modification of stevioside and rebaudioside A, Appl. Biochem. Microbiol. 42 (2006) 31–37. [7] D.C. Park, T.A. Kim, Y.H. Lee, Characteristics of transglycosylation reaction of cyclodextrin glucanotransferase in the heterogeneous enzyme reaction system using extrusion starch as glucosyl donor, Enzyme Microb. Technol. 22 (1998) 217–222. [8] M. Sato, K. Nakamura, Y. Hiroto Nagano, K. Yagi, Koizumi, Synthesis of glucosyl-inositol using a CGTase, isolation and characterization of the positional isomers and assimilation profiles for intestinal bacteria, Biotechnol. Lett. 14 (1992) 654–659. [9] S. Riva, Enzymatic modification of the sugar moieties of natural glycosides, J. Mol. Catal. B: Enzyme 19 (2002) 43–45. [10] N.G. Usanov, E.A. Gil’vanova, P.A. Elizar’ev, E.A. Prutsakova, I.A. Melent’ev, An improved method of photometric determination of cyclodextrin glucanotransferase activity, Appl. Biochem. Microbiol. 43 (2007) 105–110. [11] G. Leaver, J.R. Conder, J.A. Howell, A method development study of the production of albumin from animal blood by ion-exchange chromatography, Separ. Sci. Technol. 22 (1987) 2037–2059. [12] B.N. Gawande, A. Goel, A.Y. Patkar, Purification and properties of novel starch degrading ␣-cyclodextrin glycosyltransferease from Klebsiella pneumoniae pneunoniae AS-22, Enzyme Microb. Technol. 28 (2001) 735–743. [13] R.F. Martins, R. Hatti-Kaul, A new cyclodextrin glycosyltransferase from an alkaliphilic Bacillus agaradhaerens isolate; purification and characterization, Enzyme Microb. Technol. 30 (2002) 116–124. [14] A. Goel, S. Nene, A novel raw starch degrading cyclomaltodextrin glucanotransferase from Bacillus firmus, Biotechnol. Lett. 17 (1995) 411– 416. [15] A. Goel, S.N. Nene, Modifications in phenolphthalein method for spectrophotometric estimation of beta cyclodextrin, Starch/Starke 47 (1995) 399–400. [16] B.N. Gawande, A. Goel, A.Y. Patkar, Application of factorial design for optimization of cyclodextrin glycosyltransferease production from Klebsiella pneumoniae pneunoniae AS-22, Biotechnol. Bioeng. 64 (1999) 168–173. [17] O.H. Lowry, N.J. Rosenbrough, R.J. Randdall, Protein measurement with folin phenol reagent, J. Biol. Chem. 193 (1951) 265–274. [18] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [19] M. Makela, T. Korpela, S. Laakso, Colorimetric determination of cyclodextrin: two assay modifications based on molecular complexation of phenolphthalein, J. Biochem. Biophys. Methods 14 (1987) 85– 92. [20] Tonkova, Bacterial cyclodextrin glucanotransferase, Enzyme Microb. Technol. 22 (1998) 678–686. [21] J.H. Lee, K.H. Choi, J.Y. Choi, Y.S. Lee, I.B. Kwon, I.H. Yu, Enzymatic production of alpha cyclodextrin with the cyclodextrin glucanotransferase of Klebsiella oxytoca 19-1, Enzyme Microb. Technol. 14 (1992) 1020–1217. [22] Y. Tachibana, A. Kuramura, N. Shirasaka, Y. Suzuki, T. Yamamoto, S. Fujiwara, M. Takagi, T. Imanaka, Purification and characterization of an extremely thermostable cyclomaltodextrin glucanotransferase from a newly isolated hyperthermophilic Archaeon, a Thermococcus sp., Appl. Environ. Microbiol. 65 (1999) 1991–1997. [23] D.A. Volkova, S.A. Lopatin, I.M. Gracheva, V.P. Varlamov, Preparation of high-purity cyclodextrin glucanotransferase from Bacillus sp. 1070, Appl. Biochem. Microbiol. 37 (2001) 138–141.
L.S. Savergave et al. / Biochemical Engineering Journal 39 (2008) 510–515 [24] D.G. Yim, H.H. Sato, Y.H. Park, Y.K. Park, Production of cyclodextrin from starch by cyclodextrin glycosyltransferase from Bacillus firmus and characterization of purified enzyme, J. Ind. Microbiol. Biotechnol. 18 (1997) 402–405. [25] X. Cao, Z. Jin, F. Chen, X. Wang, Purification and properties of cyclodextrin glucanotransferase from an alkalophilic Bacillus sp. 7-12, J. Food Biochem. 28 (2004) 463–475.
515
[26] C.B. Sohn, S.A. Kim, Y.A. Park, M.H. Kim, S.K. Moon, S.A. Jang, Characterization of cyclodextrin glycosyltransferase from Bacillus firmus, J. Korean Soc. Food Sci. Nutr. 26 (1997) 351–357. [27] S. Kobayashi, K. Kainuma, S. Suzuki, Purification and some properties of Bacillus macerans cycloamylose (cyclodextrin) glucanotransferase, Carbohyd. Res. 61 (1978) 229–238.