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Cyclodextrin glucanotransferase synthesis by semicontinuous cultivation of magnetic biocatalysts from cells of Bacillus circulans ATCC 21783
Process Biochemistry 42 (2007) 1454–1459 www.elsevier.com/locate/procbio
Short communication
Cyclodextrin glucanotransferase synthesis by semicontinuous cultivation of magnetic biocatalysts from cells of Bacillus circulans ATCC 21783 Mirka Safarikova a, Nikolina Atanasova b, Viara Ivanova c, Frantisek Weyda d, Alexandra Tonkova b,* a
Department of Biomagnetic Techniques, Institute of Systems Biology and Ecology AS CR, Na Sadkach 7, 370 05 Ceske Budejovice, Czech Republic b Department of Extremophilic Bacteria, Institute of Microbiology, Bulgarian Academy of Sciences, 26 Acad. G. Bonchev Street, 1113 Sofia, Bulgaria c Department of Organic Chemistry and Microbiology, University of Food Technologies, 26 Maritza Str., 4002 Plovdiv, Bulgaria d Institute of Entomology, Biological Centre AS CR, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic Received 2 March 2007; received in revised form 11 May 2007; accepted 14 June 2007
Abstract Cells of the alkalotolerant producer of cyclodextrin glucanotransferase (CGTase) Bacillus circulans ATCC 21783 were used as a model for preparing of magnetic biocatalysts applied for CGTase synthesis in batch and semicontinuous processes. The cell immobilization was carried out with four types of magnetic nano- and microparticles: magnetite microparticles (1–5 mm), entrapped in agar gel beads with bacterial cells (AMbiocatalyst); silanized magnetite (20–40 nm) covalently bound on the cell surface (SM-biocatalyst); and alkaline and citrate ferrofluids (10– 20 nm), attached on the cell wall by an ionic interaction (FF-alkaline and FF-citrate biocatalyst). The highest CGTase production was achieved after 96 h of semicontinuous process using SM-biocatalysts (particularly, these composed of 80 mg silanized magnetite and 140 mg bacterial cells) when the specific enzyme activity was 8.4-fold higher compared to that of free cells. Cells modified with magnetic alkaline and citrate ferrofluids exhibited 2.19- and 1.55-fold increase of the specific CGTase activities. Magnetic nanoparticles linked on the cell walls by ionic interactions were partially released during the cultivation, while the covalent bond between the activated magnetite and the cells was very stable. The data obtained demonstrate convincingly the effect of the magnetic technologies for an effective enzyme production. # 2007 Elsevier Ltd. All rights reserved. Keywords: Bacillus circulans ATCC 21783; Magnetic cell biocatalysts; Silanized magnetite; Ferrofluids; Cyclodextrin glucanotransferase; Cyclodextrins
1. Introduction In recent years, substantial progress has been made in developing magnetic techniques which have found applications in numerous fields of bioscience and biotechnology [1], biomedicine [2,3], protein isolation and purification [4] and cell and enzyme immobilization [5,6]. Magnetically responsive cells and enzymes offer several advantages, such as their easy manipulation in an external magnetic field. Moreover, cells bound on magnetic particles can be stably stored for a long period of time without loss of activity and repeatedly used in a
* Corresponding author. Tel.: +359 2 979 3163; fax: +359 2 870 0109. E-mail address: [email protected] (A. Tonkova). 1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2007.06.009
semicontinuous process [5]. Tyagi and Gupta [6] have reported that the magnetic immobilization of a purified xylanase resulted in a significant increase of enzyme thermal stability. Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) catalyses conversion of starch into cyclodextrins (CDs) which find numerous applications in medicine, agricultural, pharmaceutical and chemical industries because of their ability to encapsulate a wide range of organic and inorganic molecules and to change the stability, reactivity and solubility of the formed inclusion complexes with these molecules [7,8]. To our knowledge, there are no reports on magnetic cell immobilization of CGTase producers or of a purified CGTase by supports such as magnetic micro- and nanoparticles. Previous studies on CGTase production by free or agar- and membrane-immobilized cells of Bacillus circulans ATCC
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21783 in batch, semicontinuous and continuous processes demonstrated that the strain is a promising producer of CGTase on a large scale [9–12]. In the present work, cells of B. circulans ATCC 21783 were used as a model for preparing of magnetic biocatalysts (cells immobilized with magnetic carriers) and the effect of different magnetic matrices on the enzyme synthesis in batch and semicontinuous processes were studied.
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immobilized cells, named also ‘‘free’’ cells) was used as a control in all experiments and operated under the same growth conditions. For repeated batch experiments (semicontinuous cultivation), the magnetic biocatalysts were separated from the culture liquids by magnet and washed with sterile water for elimination of the released non-magnetic cells before reintroduction into fresh medium at every 48 h. Parallel experiments were also performed with free bacterial cells in order to establish the effect of the immobilization techniques on CGTase long-term production. The cells of the control were denoted ‘‘free cells’’ unlike the cells released from biocatalysts denoted ‘‘released cells’’.
2. Materials and methods 2.4. Analytical methods 2.1. Bacterial strain and media Bacillus circulans ATCC 21783 was supplied by the National Bank of Microorganisms and Cell Cultures (Sofia, Bulgaria). It was cultivated in a nutrient medium (initial pH 9.8–10.0) containing 2 g l 1 soluble starch [10,11].
2.2. Magnetic particles Magnetite (iron(II, III) oxide) was from Aldrich. Water based magnetic fluids (ferrofluids, FF) stabilized with citrate and tetramethylammonium hydroxide were prepared using the precipitation procedure [13,14]. Silanized magnetite (SM) was obtained as follows: 400 mg magnetic nanoparticles (prepared by a standard precipitation of Fe(II) and Fe(III) in alkaline medium) and 3.2 ml 5% (3)-aminopropyltriethoxysilane (Sigma, pH 4.0) were mixed and heated in a water bath at 75 8C for 4 h under stirring. After washing with 0.1 M phosphate buffer (pH 7.0), 20 ml 5% glutaraldehyde (Fluka) in phosphate buffer was added to the particles followed by 1 h incubation at a room temperature under mixing. Finally, the activated magnetite was washed with phosphate buffer.
2.3. Preparation of magnetically immobilized cells and growth conditions Cells of alkalotolerant Bacillus circulans ATCC 21783 were immobilized by the following procedures: (1) entrapment in magnetite-agar gel beads using magnetite microparticles (1–5 mm); (2) covalent immobilization with silanized magnetite (20–40 nm) on the cell surfaces and (3) magnetic modification by alkaline- and citrate ferrofluids (10–20 nm), pH 13.5 and 7.9, respectively. The inoculum culture was grown in 500-ml Erlenmeyer flasks containing 100 ml nutrient medium (initial pH 9.8–10.0 adjusted by sterile sodium carbonate after autoclaving) at 40 8C. Sterile magnetic particles and wet bacterial cells were entrapped in agar beads according to Nilsson et al. [15] using sunflower oil as a hydrophobic phase. The corresponding quantities of a magnetic powder (1.6%, w/v) and wet cells (2%, w/v) were mixed with 25 ml of warm (50 8C) agar solution (3%, w/v) and 12 ml of this mixture was dropped into the chilled oil phase. After solidification (2 h at 4 8C) the biocatalysts (AM; agar beads with included magnetic powder and bacterial cells, diameter 3–5 mm) were washed abundantly with a sterile water and transferred into 100 ml flasks containing 20 ml of nutrient medium (pH 9.8–10.0). The covalent linking with a silanized magnetite was carried out in flasks containing 80 mg of activated magnetic particles, mixed with different amounts of cells (from 70 to 560 mg wet cells) and phosphate buffer (pH 7.1) to a final volume of 6 ml. After 20 h of stirring at a room temperature, SM-biocatalysts (cells bound to SM) were harvested by a magnet, washed with sterile water for elimination of non-immobilized cells and transferred into 20 ml of nutrient medium (pH 9.8–10.0). The magnetic modification of cells with ferrofluids (FFs) was carried out as follows: wet cells (0.6 g) were re-suspended in 1.8 ml 0.1 M glycine buffer (pH 2.2) in the experiments with citrate-FF or 0.2 M Tris–HCl buffer (pH 8.6) in the experiments with alkaline-FF. After 10 min, 0.3 ml of FF was added to 0.5 ml cell suspension under continuous stirring. The magnetic cells (FF-biocatalysts) were separated by a magnet after 30 min, washed two times with saline and transferred into 100 ml flasks containing 20 ml nutrient medium (pH 9.8–10.0). Batch cultivation of the biocatalysts obtained was performed at 40 8C on a shaker (New Brunswick, USA, 220 rpm). Free cell suspension (2%, v/v; non-
CGTase cyclization activity was assayed by the method of Kaneko et al. [16] based on the reduction in the colour intensity of phenolphthalein after complexation with b-CD. One unit of CGTase activity was defined as the amount of enzyme that formed 1 mg of b-CD min 1 under standard conditions (soluble starch, Fluka acc. to Zulkowsky as a substrate, phosphate buffer pH 6.0, 65 8C, 20 min reaction time). Total protein content was determined by the method of Bradford [17] using bovine serum albumin as a standard. Cell growth (the amount of released cells from the magnetic biocatalysts and free cells of the control) was measured by absorbance at 650 nm.
2.5. Electron microscopy For transmission electron microscopy (TEM), magnetic bacterial cells were fixed in 2% glutaraldehyde and 2.5% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), for 1 day at a room temperature. After washing with 0.1 M phosphate buffer and de-ionized water, the cells were dehydrated in 50%, 75% and absolute ethanol. Later, they were embedded into Spurr resin (standard mixture) and ultrathin sections were made with a Reichert Ultracut ultramicrotome. Jeol 1010 transmission electron microscope was used.
3. Results 3.1. CGTase production and operational stability of FFand AM-biocatalysts After 48 h of batch cultivation, the alkaline and citrate FF-modified cells showed 1.68- and 1.30-fold increase of the enzyme activities (542 and 420 U ml 1, respectively) and
Fig. 1. CGTase activity and operational stability of AM-entrapped and FFmodified cells of Bacillus circulans ATCC 21783. Data are mean values S.D. from 0.35 to 0.47, n = 3.
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Table 1 CGTase production by semicontinuous cultivation of AM- and FF-biocatalysts Run and biocatalyst
OD650 nm a
Protein (mg ml 1)b
Specific CGTase activity (U mg 1)
Increase (fold)
48 h (first run) AM FF-alkaline FF-citrate Free cells (control)
0.45 0.70 0.90 0.43
0.21 0.32 0.25 0.29
1320.0 1695.3 1680.0 1115.5
1.18 1.52 1.51 1.0
96 h (second run) AM FF-alkaline FF-citrate Free cells (control)
0.69 0.97 1.10 0.55
0.29 0.30 0.27 0.30
1524.0 1670.0 1183.0 763.1
1.99 2.19 1.55 1.0
144 h (third run) AM FF-alkaline FF-citrate Free cells (control)
0.80 0.60 1.15 0.60
0.27 0.28 0.20 0.27
1410.4 1153.6 1121.5 1073.0
1.30 1.07 1.04 1.0
192 h (fourth run) AM FF-alkaline FF-citrate Free cells
0.88 0.57 0.90 0.55
0.28 0.31 0.24 0.29
978.6 1061.3 1085.7 935.5
1.05 1.13 1.16 1.0
240 h (fifth run) AM FF-alkaline FF-citrate Free cells (control)
0.42 0.42 0.73 0.49
0.30 0.35 0.34 0.39
621.0 816.0 632.3 567.0
1.09 1.44 1.11 1.0
a b
Released cells; data are mean values S.D. 0.01, n = 3. Data are mean values S.D. 0.02, n = 3.
1.5-fold increase of the specific enzyme activities compared to those of free cells (Fig. 1 and Table 1). The CGTase activity of cells entrapped in magnetic agar beads (AM) was lower, although the specific enzyme activity was 1.18-fold higher in comparison with free cells. Further, the long-term stability of the batch cultures was studied by a semicontinuous process. At every 48 h the biocatalysts were harvested by magnet, washed and transferred into fresh medium and the next run started. Free cells were also transferred into fresh medium by using 2% (v/v) cell suspension from their own previous batch. Table 1 contains the data from batch cultivation (until 48th h) and from semicontinuous cultivations (the next repeated batch runs, from 48th to 240th hour) in order to be avoided some repetition of data concerning the first 48 h of cultivation. The operational stability of AM- and FFs-biocatalysts was determined as a residual activity at the end of each batch run calculated towards the activities of the corresponding biocatalyst established in the first run. After 240 h semicontinuous cultivation these biocatalysts produced up to 51–67% of the respective initial enzyme yield (Fig. 1). The highest enzyme production was achieved at the end of the second run (96 h, Table 1). The specific CGTase activities were from 1.50- to 2.19fold higher, compared with free cells. An increase of the produced CGTase with 59% and 37% at 96th and 144th hour, respectively, compared with the first batch, was noted in the experiments with AM-immobilized cells (Fig. 1). Nevertheless, magnetite did not affect significantly CGTase production
because in experiments with agar-immobilized cells without magnetic particles, similar CGTase activities were reached [10]. AM-beads retained their integrity completely during 240 h semicontinuous process. In respect to the mechanical stability of the FF-biocatalysts, a certain release of the ferrofluids was observed at the fifth run (from 216th to 240th hour). The dark brown colour of the modified cells became lighter probably because of the release of magnetite from the cells or quite possibly because of the cell division. Magnetic nanoparticles attached on the cell walls by relatively weak ionic interactions remained on the cell wall surface, but during the cell division, the cell wall with FFs was also divided. Thus, after each cell generation FFs quantity on the cell wall diminished. Moreover, after cell lysis part of the FFs was eliminated or remained attached to empty cell walls. The quantity of released cells from FF-biocatalysts reached higher values as compared to the free cells because each sample was prepared from 150 mg wet cells, while in the control of free cells the respective inoculum cell concentration was 2% (v/v) (approximately 6 mg wet cells). Without immobilization these 150 mg wet cells could be lysed after 17–20 h demonstrating clearly one of the advantages of the magnetic modification. 3.2. CGTase production and operational stability of SM biocatalysts During 48 h of batch cultivation, the highest CGTase production was achieved by using of SM-biocatalysts in
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Table 2 CGTase production by semicontinuos cultivation of SM-biocatalysts Run and initial cell loading (mg)
OD650 nm a
Protein (mg ml 1)b
Specific CGTase activity (U mg 1)
Increase (fold)
48 h (first run) 70 140 210 280 560 Free cells (control)
0.04 0.03 0.02 0.05 0.80 0.34
0.066 0.066 0.067 0.072 0.38 0.37
5674.2 6575.7 5223.9 5395.8 950.5 802.2
7.07 8.20 6.51 6.73 1.18 1.0
96 h (second run) 70 140 210 280 560 Free cells (control)
0.09 0.18 0.20 0.14 0.97 0.51
0.052 0.078 0.11 0.09 0.35 0.38
5425.0 7179.5 5736.4 6813.3 2089.7 854.7
6.35 8.40 6.71 7.97 2.44 1.0
144 h (third run) 70 140 210 280 560 Free cells (control)
0.08 0.73 1.18 0.92 0.58 0.45
0.13 0.17 0.24 0.16 0.35 0.37
1943.8 2692.9 1435.0 1680.0 1116.0 650.8
2.98 4.14 2.20 2.58 1.71 1.0
192 h (fourth run) 70 140 210 280 560 Free cells (control)
0.43 0.66 0.67 0.68 0.58 0.48
0.14 0.19 0.22 0.21 0.33 0.32
2360.0 2188.9 1470.0 1596.2 1349.1 774.4
3.04 2.83 1.90 2.06 1.74 1.0
a b
Released cells; data are mean values S.D. 0.01, n = 3. Data are mean values S.D. 0.007, n = 3.
comparison to the other discussed above magnetically immobilized cells (Table 2 compared to Table 1). The covalently bound activated SM-magnetite on the bacterial cells did not allow a release of cells into the medium, except the variant with a large initial cell loading (560 mg). In the latter case, probably part of cells were not coupled with enough chemical bonds to the magnetic support. The very low quantities of released cells indicated that the established enzyme activities were due only to the immobilized cells (Table 2 and Fig. 2). The total protein content in the culture supernatants was also too low, which led to high specific CGTase activities, namely, seven- to eightfold higher compared to those of free cells using SM-biocatalyst, containing 70 and 140 mg wet cells, respectively. The repeated batch cultivation of SM-immobilized cells showed a significant increase of the enzyme yields and particularly the specific CGTase activities compared to FF- and AM-cells (Fig. 2 and Table 2). Similarly to the results obtained by AM- and FF-biocatalysts, the highest CGTase yields and specific enzyme activities were achieved after 96 h of semicontinuous process. Increased CGTase levels from 1.7to 2.2-fold and specific enzyme activities from 2.4- to 8.4-fold than those of free cells were established. In respect to the operational stability, SM-biocatalysts retained from 88% to 100% of their initial activity at the end of the fourth run (192 h) during the semicontinuous process (Fig. 2). The optimal
variant for cell immobilization with SM-matrix was 80 mg SM and 140 mg wet cells, providing the highest specific CGTase activities during 144 h repeated batch process (6575 U mg 1/48 h; 7179 U mg 1/96 h; 2693 U mg 1/144 h; Table 2). An increase of the released cells during the third and
Fig. 2. CGTase activity and operational stability of SM-immobilized cells of B. circulans ATCC 21783. Data are mean values S.D. from 0.51 to 0.67, n = 3.
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fourth runs (typically for immobilization procedures with living bacterial cells) and a reduction of the specific activities were observed. The integrity of the SM-biocatalysts was retained completely during the whole process. 3.3. TEM micrographs of FF- and SM-biocatalysts The attachment of magnetic particles on the cell wall was confirmed by transmission electron microscopy (TEM). Activated silanized magnetite (SM) was bound in a form of agglomerates to the cell surface only in a few points (Fig. 3A). Nevertheless, this amount of magnetite was sufficient to form
stable magnetically responsive cells. Similarly, magnetic particles originated from ferrofluid stabilized with tetramethylammonium hydroxide (FF-alkaline) were attached to the cell wall, but the density of magnetic particles was slightly higher (Fig. 3B). On the contrary, magnetic nanoparticles from citratestabilized ferrofluid (FF-citrate) did not form bigger complexes and were bound more regularly on the cell surface (Fig. 3C). Free magnetite aggregates not attached to cells were also present in the samples. 4. Discussion The studies concerning the use of magnetic carriers for immobilization of bacterial cells or enzymes are very scanty. Thus, the report of Guobin et al. [5] have shown the successful application of entrapped in magnetic polyvinyl alcohol beads cells from Pseudomonas delafieldii used for biodesulfurization. In relation to the magnetic immobilization of enzymes, the studies with purified xylanase immobilized on magnetic latex beads are known [6]. The immobilized enzyme preparation has been shown 80% of the total activity and a significant thermal stability. The data obtained in the present work demonstrate convincingly the effect of the used magnetic carriers for a significant enhancing of the CGTase production by immobilized Bacillus circulans ATCC 21783 cells. The possible explanation for these results is based on the good linking of a large amount of bacterial cells on the used magnetic carriers. Generally, in all immobilization procedures with living bacterial cells (irrespectively of the used support), cells are released from the biocatalysts during the cultivation process which continue to grow in the culture medium. In the case with SM-biocatalysts, probably some of these released cells were taken hold again of the free magnetite aggregates present in the culture liquid (Fig. 3). Thus, the measured cell growth as well as the total protein content in the culture liquid remained low, but the enzyme synthesis continued. In fact, a production of almost pure enzyme in clear culture liquids was achieved using of SMbiocatalyst (particularly, these composite from 80 mg SM and 140 bacterial cells). The released non-magnetic cells were few (observed under the microscope). Therefore, SM-biocatalysts offer the opportunity for an effective CGTase synthesis and a possible application for a direct cyclodextrin production. These biocatalysts could decrease the cost of the cyclodextrin production. The present results were not compared with other CGTase producing cells immobilized on magnetic supports because to our knowledge such studies are not available. Acknowledgements
Fig. 3. TEM micrographs of SM-biocatalyst (A, bar 0.5 mm), FF-alkaline biocatalyst (B, bar 0.5 mm) and FF-citrate biocatalyst (C, bar 0.5 mm) using B. circulans ATCC 21783 cells.
This work was supported by the Bulgarian Ministry of Science and Education (grant B-1521 NSF), bilateral grant P44/04 between the Bulgarian and Czech Academies of Sciences, the Grant Agency of the Czech Academy of Sciences (project no. IBS6087204), ISBE Research Intention no. AV0Z60870520.
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References [1] Safarik I, Safarikova M. Magnetic nanoparticles and biosciences. Monats Chem 2002;133:737–59. [2] Pankhurst QA, Connolly J, Jones SK, Dobson J. Applications of magnetic nanoparticles in biomedicine. J Phys D: Appl Phys 2003;36:R167–81. [3] Saiyed ZM, Telang SD, Ramchand CN. Application of magnetic techniques in the field of drug discovery and biomedicine. Biomagn Res Technol 2003;1:2–15. [4] Safarik I, Safarikova M. Magnetic techniques for the isolation and purification of proteins and peptides. Biomagn Res Technol 2004;2:7–40. [5] Guobin S, Jianmin X, Chen G, Huizhou L, Jiayong C. Biodesulfurization using Pseudomonas delafieldii in magnetic polyvinyl alcohol beads. Lett Appl Microbiol 2005;40:30–7. [6] Tyagi R, Gupta MN. Immobilization of Aspergillus niger xylanase on magnetic latex beads. Biotechnol Appl Biochem 1995;21:217–22. [7] Biwer A, Antranikian G, Heinzle E. Enzymatic production of cyclodextrins. Appl Microbiol Biotechnol 2002;59:609–17. [8] Singh M, Sharma R, Banerjee U. Biotechnological applications of cyclodextrins. Biotechnol Adv 2002;20:341–59. [9] Tonkova A. Microbial starch converting enzymes of the a-amylase family. In: Ray RC, Ward OP, editors. Microbial Biotechnology in Horticulture, New Hampshire, USA: Science Publishers; 2006. vol. 1 p. 421–72. [10] Vassileva A, Burhan N, Beschkov V, Spasova D, Radoevska S, Ivanova V, et al. Cyclodextrin glucanotransferase production by free and agar gel
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immobilized cells of Bacillus circulans ATCC 21783. Process Biochem 2003;38:1585–91. Vassileva A, Burhan N, Beschkov V, Ivanova V, Tonkova A. Immobilization of Bacillus circulans ATCC 21783 cells for cyclodextrin glucanotransferase production. In: Proceedings of the XIth International Workshop on Bioencapsulation, 25–27 May; 2003. p. 201–4. Vassileva A, Beschkov V, Ivanova V, Tonkova A. Continuous cyclodextrin glucanotransferase production by free and immobilized cells of Bacillus circulans ATCC 21783 in bioreactor. Process Biochem 2005; 40:3290–5. Massart R. Preparation of aqueous magnetic liquids in alkaline and acidic media. IEEE Trans Magn 1981;17:1247–8. Domingo JC, Mercadal M, Petriz J, De Madariaga MA. Preparation of PEG-grafted immunomagnetoliposomes entrapping citrate stabilized magnetite particles and their application in CD34+ cell sorting. J Microencapsul 2001;18:41–54. Nilsson K, Birnbaum S, Flygare S, Linse L, Schro¨der U, Jeppsson U, et al. A general method for the immobilization of cells with preserved viability. Eur J Appl Microbiol Biotechnol 1983;17:319–26. Kaneko T, Kato T, Nakamura N, Horikoshi K. Spectrophotometric determination of cyclization activity of b-cyclodextrin-forming cyclomaltodextrin glucanotransferase. J Jpn Soc Starch Sci 1987;34:45–8. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 1976;72:248–54.
Short communication
Cyclodextrin glucanotransferase synthesis by semicontinuous cultivation of magnetic biocatalysts from cells of Bacillus circulans ATCC 21783 Mirka Safarikova a, Nikolina Atanasova b, Viara Ivanova c, Frantisek Weyda d, Alexandra Tonkova b,* a
Department of Biomagnetic Techniques, Institute of Systems Biology and Ecology AS CR, Na Sadkach 7, 370 05 Ceske Budejovice, Czech Republic b Department of Extremophilic Bacteria, Institute of Microbiology, Bulgarian Academy of Sciences, 26 Acad. G. Bonchev Street, 1113 Sofia, Bulgaria c Department of Organic Chemistry and Microbiology, University of Food Technologies, 26 Maritza Str., 4002 Plovdiv, Bulgaria d Institute of Entomology, Biological Centre AS CR, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic Received 2 March 2007; received in revised form 11 May 2007; accepted 14 June 2007
Abstract Cells of the alkalotolerant producer of cyclodextrin glucanotransferase (CGTase) Bacillus circulans ATCC 21783 were used as a model for preparing of magnetic biocatalysts applied for CGTase synthesis in batch and semicontinuous processes. The cell immobilization was carried out with four types of magnetic nano- and microparticles: magnetite microparticles (1–5 mm), entrapped in agar gel beads with bacterial cells (AMbiocatalyst); silanized magnetite (20–40 nm) covalently bound on the cell surface (SM-biocatalyst); and alkaline and citrate ferrofluids (10– 20 nm), attached on the cell wall by an ionic interaction (FF-alkaline and FF-citrate biocatalyst). The highest CGTase production was achieved after 96 h of semicontinuous process using SM-biocatalysts (particularly, these composed of 80 mg silanized magnetite and 140 mg bacterial cells) when the specific enzyme activity was 8.4-fold higher compared to that of free cells. Cells modified with magnetic alkaline and citrate ferrofluids exhibited 2.19- and 1.55-fold increase of the specific CGTase activities. Magnetic nanoparticles linked on the cell walls by ionic interactions were partially released during the cultivation, while the covalent bond between the activated magnetite and the cells was very stable. The data obtained demonstrate convincingly the effect of the magnetic technologies for an effective enzyme production. # 2007 Elsevier Ltd. All rights reserved. Keywords: Bacillus circulans ATCC 21783; Magnetic cell biocatalysts; Silanized magnetite; Ferrofluids; Cyclodextrin glucanotransferase; Cyclodextrins
1. Introduction In recent years, substantial progress has been made in developing magnetic techniques which have found applications in numerous fields of bioscience and biotechnology [1], biomedicine [2,3], protein isolation and purification [4] and cell and enzyme immobilization [5,6]. Magnetically responsive cells and enzymes offer several advantages, such as their easy manipulation in an external magnetic field. Moreover, cells bound on magnetic particles can be stably stored for a long period of time without loss of activity and repeatedly used in a
* Corresponding author. Tel.: +359 2 979 3163; fax: +359 2 870 0109. E-mail address: [email protected] (A. Tonkova). 1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2007.06.009
semicontinuous process [5]. Tyagi and Gupta [6] have reported that the magnetic immobilization of a purified xylanase resulted in a significant increase of enzyme thermal stability. Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) catalyses conversion of starch into cyclodextrins (CDs) which find numerous applications in medicine, agricultural, pharmaceutical and chemical industries because of their ability to encapsulate a wide range of organic and inorganic molecules and to change the stability, reactivity and solubility of the formed inclusion complexes with these molecules [7,8]. To our knowledge, there are no reports on magnetic cell immobilization of CGTase producers or of a purified CGTase by supports such as magnetic micro- and nanoparticles. Previous studies on CGTase production by free or agar- and membrane-immobilized cells of Bacillus circulans ATCC
M. Safarikova et al. / Process Biochemistry 42 (2007) 1454–1459
21783 in batch, semicontinuous and continuous processes demonstrated that the strain is a promising producer of CGTase on a large scale [9–12]. In the present work, cells of B. circulans ATCC 21783 were used as a model for preparing of magnetic biocatalysts (cells immobilized with magnetic carriers) and the effect of different magnetic matrices on the enzyme synthesis in batch and semicontinuous processes were studied.
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immobilized cells, named also ‘‘free’’ cells) was used as a control in all experiments and operated under the same growth conditions. For repeated batch experiments (semicontinuous cultivation), the magnetic biocatalysts were separated from the culture liquids by magnet and washed with sterile water for elimination of the released non-magnetic cells before reintroduction into fresh medium at every 48 h. Parallel experiments were also performed with free bacterial cells in order to establish the effect of the immobilization techniques on CGTase long-term production. The cells of the control were denoted ‘‘free cells’’ unlike the cells released from biocatalysts denoted ‘‘released cells’’.
2. Materials and methods 2.4. Analytical methods 2.1. Bacterial strain and media Bacillus circulans ATCC 21783 was supplied by the National Bank of Microorganisms and Cell Cultures (Sofia, Bulgaria). It was cultivated in a nutrient medium (initial pH 9.8–10.0) containing 2 g l 1 soluble starch [10,11].
2.2. Magnetic particles Magnetite (iron(II, III) oxide) was from Aldrich. Water based magnetic fluids (ferrofluids, FF) stabilized with citrate and tetramethylammonium hydroxide were prepared using the precipitation procedure [13,14]. Silanized magnetite (SM) was obtained as follows: 400 mg magnetic nanoparticles (prepared by a standard precipitation of Fe(II) and Fe(III) in alkaline medium) and 3.2 ml 5% (3)-aminopropyltriethoxysilane (Sigma, pH 4.0) were mixed and heated in a water bath at 75 8C for 4 h under stirring. After washing with 0.1 M phosphate buffer (pH 7.0), 20 ml 5% glutaraldehyde (Fluka) in phosphate buffer was added to the particles followed by 1 h incubation at a room temperature under mixing. Finally, the activated magnetite was washed with phosphate buffer.
2.3. Preparation of magnetically immobilized cells and growth conditions Cells of alkalotolerant Bacillus circulans ATCC 21783 were immobilized by the following procedures: (1) entrapment in magnetite-agar gel beads using magnetite microparticles (1–5 mm); (2) covalent immobilization with silanized magnetite (20–40 nm) on the cell surfaces and (3) magnetic modification by alkaline- and citrate ferrofluids (10–20 nm), pH 13.5 and 7.9, respectively. The inoculum culture was grown in 500-ml Erlenmeyer flasks containing 100 ml nutrient medium (initial pH 9.8–10.0 adjusted by sterile sodium carbonate after autoclaving) at 40 8C. Sterile magnetic particles and wet bacterial cells were entrapped in agar beads according to Nilsson et al. [15] using sunflower oil as a hydrophobic phase. The corresponding quantities of a magnetic powder (1.6%, w/v) and wet cells (2%, w/v) were mixed with 25 ml of warm (50 8C) agar solution (3%, w/v) and 12 ml of this mixture was dropped into the chilled oil phase. After solidification (2 h at 4 8C) the biocatalysts (AM; agar beads with included magnetic powder and bacterial cells, diameter 3–5 mm) were washed abundantly with a sterile water and transferred into 100 ml flasks containing 20 ml of nutrient medium (pH 9.8–10.0). The covalent linking with a silanized magnetite was carried out in flasks containing 80 mg of activated magnetic particles, mixed with different amounts of cells (from 70 to 560 mg wet cells) and phosphate buffer (pH 7.1) to a final volume of 6 ml. After 20 h of stirring at a room temperature, SM-biocatalysts (cells bound to SM) were harvested by a magnet, washed with sterile water for elimination of non-immobilized cells and transferred into 20 ml of nutrient medium (pH 9.8–10.0). The magnetic modification of cells with ferrofluids (FFs) was carried out as follows: wet cells (0.6 g) were re-suspended in 1.8 ml 0.1 M glycine buffer (pH 2.2) in the experiments with citrate-FF or 0.2 M Tris–HCl buffer (pH 8.6) in the experiments with alkaline-FF. After 10 min, 0.3 ml of FF was added to 0.5 ml cell suspension under continuous stirring. The magnetic cells (FF-biocatalysts) were separated by a magnet after 30 min, washed two times with saline and transferred into 100 ml flasks containing 20 ml nutrient medium (pH 9.8–10.0). Batch cultivation of the biocatalysts obtained was performed at 40 8C on a shaker (New Brunswick, USA, 220 rpm). Free cell suspension (2%, v/v; non-
CGTase cyclization activity was assayed by the method of Kaneko et al. [16] based on the reduction in the colour intensity of phenolphthalein after complexation with b-CD. One unit of CGTase activity was defined as the amount of enzyme that formed 1 mg of b-CD min 1 under standard conditions (soluble starch, Fluka acc. to Zulkowsky as a substrate, phosphate buffer pH 6.0, 65 8C, 20 min reaction time). Total protein content was determined by the method of Bradford [17] using bovine serum albumin as a standard. Cell growth (the amount of released cells from the magnetic biocatalysts and free cells of the control) was measured by absorbance at 650 nm.
2.5. Electron microscopy For transmission electron microscopy (TEM), magnetic bacterial cells were fixed in 2% glutaraldehyde and 2.5% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), for 1 day at a room temperature. After washing with 0.1 M phosphate buffer and de-ionized water, the cells were dehydrated in 50%, 75% and absolute ethanol. Later, they were embedded into Spurr resin (standard mixture) and ultrathin sections were made with a Reichert Ultracut ultramicrotome. Jeol 1010 transmission electron microscope was used.
3. Results 3.1. CGTase production and operational stability of FFand AM-biocatalysts After 48 h of batch cultivation, the alkaline and citrate FF-modified cells showed 1.68- and 1.30-fold increase of the enzyme activities (542 and 420 U ml 1, respectively) and
Fig. 1. CGTase activity and operational stability of AM-entrapped and FFmodified cells of Bacillus circulans ATCC 21783. Data are mean values S.D. from 0.35 to 0.47, n = 3.
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Table 1 CGTase production by semicontinuous cultivation of AM- and FF-biocatalysts Run and biocatalyst
OD650 nm a
Protein (mg ml 1)b
Specific CGTase activity (U mg 1)
Increase (fold)
48 h (first run) AM FF-alkaline FF-citrate Free cells (control)
0.45 0.70 0.90 0.43
0.21 0.32 0.25 0.29
1320.0 1695.3 1680.0 1115.5
1.18 1.52 1.51 1.0
96 h (second run) AM FF-alkaline FF-citrate Free cells (control)
0.69 0.97 1.10 0.55
0.29 0.30 0.27 0.30
1524.0 1670.0 1183.0 763.1
1.99 2.19 1.55 1.0
144 h (third run) AM FF-alkaline FF-citrate Free cells (control)
0.80 0.60 1.15 0.60
0.27 0.28 0.20 0.27
1410.4 1153.6 1121.5 1073.0
1.30 1.07 1.04 1.0
192 h (fourth run) AM FF-alkaline FF-citrate Free cells
0.88 0.57 0.90 0.55
0.28 0.31 0.24 0.29
978.6 1061.3 1085.7 935.5
1.05 1.13 1.16 1.0
240 h (fifth run) AM FF-alkaline FF-citrate Free cells (control)
0.42 0.42 0.73 0.49
0.30 0.35 0.34 0.39
621.0 816.0 632.3 567.0
1.09 1.44 1.11 1.0
a b
Released cells; data are mean values S.D. 0.01, n = 3. Data are mean values S.D. 0.02, n = 3.
1.5-fold increase of the specific enzyme activities compared to those of free cells (Fig. 1 and Table 1). The CGTase activity of cells entrapped in magnetic agar beads (AM) was lower, although the specific enzyme activity was 1.18-fold higher in comparison with free cells. Further, the long-term stability of the batch cultures was studied by a semicontinuous process. At every 48 h the biocatalysts were harvested by magnet, washed and transferred into fresh medium and the next run started. Free cells were also transferred into fresh medium by using 2% (v/v) cell suspension from their own previous batch. Table 1 contains the data from batch cultivation (until 48th h) and from semicontinuous cultivations (the next repeated batch runs, from 48th to 240th hour) in order to be avoided some repetition of data concerning the first 48 h of cultivation. The operational stability of AM- and FFs-biocatalysts was determined as a residual activity at the end of each batch run calculated towards the activities of the corresponding biocatalyst established in the first run. After 240 h semicontinuous cultivation these biocatalysts produced up to 51–67% of the respective initial enzyme yield (Fig. 1). The highest enzyme production was achieved at the end of the second run (96 h, Table 1). The specific CGTase activities were from 1.50- to 2.19fold higher, compared with free cells. An increase of the produced CGTase with 59% and 37% at 96th and 144th hour, respectively, compared with the first batch, was noted in the experiments with AM-immobilized cells (Fig. 1). Nevertheless, magnetite did not affect significantly CGTase production
because in experiments with agar-immobilized cells without magnetic particles, similar CGTase activities were reached [10]. AM-beads retained their integrity completely during 240 h semicontinuous process. In respect to the mechanical stability of the FF-biocatalysts, a certain release of the ferrofluids was observed at the fifth run (from 216th to 240th hour). The dark brown colour of the modified cells became lighter probably because of the release of magnetite from the cells or quite possibly because of the cell division. Magnetic nanoparticles attached on the cell walls by relatively weak ionic interactions remained on the cell wall surface, but during the cell division, the cell wall with FFs was also divided. Thus, after each cell generation FFs quantity on the cell wall diminished. Moreover, after cell lysis part of the FFs was eliminated or remained attached to empty cell walls. The quantity of released cells from FF-biocatalysts reached higher values as compared to the free cells because each sample was prepared from 150 mg wet cells, while in the control of free cells the respective inoculum cell concentration was 2% (v/v) (approximately 6 mg wet cells). Without immobilization these 150 mg wet cells could be lysed after 17–20 h demonstrating clearly one of the advantages of the magnetic modification. 3.2. CGTase production and operational stability of SM biocatalysts During 48 h of batch cultivation, the highest CGTase production was achieved by using of SM-biocatalysts in
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Table 2 CGTase production by semicontinuos cultivation of SM-biocatalysts Run and initial cell loading (mg)
OD650 nm a
Protein (mg ml 1)b
Specific CGTase activity (U mg 1)
Increase (fold)
48 h (first run) 70 140 210 280 560 Free cells (control)
0.04 0.03 0.02 0.05 0.80 0.34
0.066 0.066 0.067 0.072 0.38 0.37
5674.2 6575.7 5223.9 5395.8 950.5 802.2
7.07 8.20 6.51 6.73 1.18 1.0
96 h (second run) 70 140 210 280 560 Free cells (control)
0.09 0.18 0.20 0.14 0.97 0.51
0.052 0.078 0.11 0.09 0.35 0.38
5425.0 7179.5 5736.4 6813.3 2089.7 854.7
6.35 8.40 6.71 7.97 2.44 1.0
144 h (third run) 70 140 210 280 560 Free cells (control)
0.08 0.73 1.18 0.92 0.58 0.45
0.13 0.17 0.24 0.16 0.35 0.37
1943.8 2692.9 1435.0 1680.0 1116.0 650.8
2.98 4.14 2.20 2.58 1.71 1.0
192 h (fourth run) 70 140 210 280 560 Free cells (control)
0.43 0.66 0.67 0.68 0.58 0.48
0.14 0.19 0.22 0.21 0.33 0.32
2360.0 2188.9 1470.0 1596.2 1349.1 774.4
3.04 2.83 1.90 2.06 1.74 1.0
a b
Released cells; data are mean values S.D. 0.01, n = 3. Data are mean values S.D. 0.007, n = 3.
comparison to the other discussed above magnetically immobilized cells (Table 2 compared to Table 1). The covalently bound activated SM-magnetite on the bacterial cells did not allow a release of cells into the medium, except the variant with a large initial cell loading (560 mg). In the latter case, probably part of cells were not coupled with enough chemical bonds to the magnetic support. The very low quantities of released cells indicated that the established enzyme activities were due only to the immobilized cells (Table 2 and Fig. 2). The total protein content in the culture supernatants was also too low, which led to high specific CGTase activities, namely, seven- to eightfold higher compared to those of free cells using SM-biocatalyst, containing 70 and 140 mg wet cells, respectively. The repeated batch cultivation of SM-immobilized cells showed a significant increase of the enzyme yields and particularly the specific CGTase activities compared to FF- and AM-cells (Fig. 2 and Table 2). Similarly to the results obtained by AM- and FF-biocatalysts, the highest CGTase yields and specific enzyme activities were achieved after 96 h of semicontinuous process. Increased CGTase levels from 1.7to 2.2-fold and specific enzyme activities from 2.4- to 8.4-fold than those of free cells were established. In respect to the operational stability, SM-biocatalysts retained from 88% to 100% of their initial activity at the end of the fourth run (192 h) during the semicontinuous process (Fig. 2). The optimal
variant for cell immobilization with SM-matrix was 80 mg SM and 140 mg wet cells, providing the highest specific CGTase activities during 144 h repeated batch process (6575 U mg 1/48 h; 7179 U mg 1/96 h; 2693 U mg 1/144 h; Table 2). An increase of the released cells during the third and
Fig. 2. CGTase activity and operational stability of SM-immobilized cells of B. circulans ATCC 21783. Data are mean values S.D. from 0.51 to 0.67, n = 3.
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fourth runs (typically for immobilization procedures with living bacterial cells) and a reduction of the specific activities were observed. The integrity of the SM-biocatalysts was retained completely during the whole process. 3.3. TEM micrographs of FF- and SM-biocatalysts The attachment of magnetic particles on the cell wall was confirmed by transmission electron microscopy (TEM). Activated silanized magnetite (SM) was bound in a form of agglomerates to the cell surface only in a few points (Fig. 3A). Nevertheless, this amount of magnetite was sufficient to form
stable magnetically responsive cells. Similarly, magnetic particles originated from ferrofluid stabilized with tetramethylammonium hydroxide (FF-alkaline) were attached to the cell wall, but the density of magnetic particles was slightly higher (Fig. 3B). On the contrary, magnetic nanoparticles from citratestabilized ferrofluid (FF-citrate) did not form bigger complexes and were bound more regularly on the cell surface (Fig. 3C). Free magnetite aggregates not attached to cells were also present in the samples. 4. Discussion The studies concerning the use of magnetic carriers for immobilization of bacterial cells or enzymes are very scanty. Thus, the report of Guobin et al. [5] have shown the successful application of entrapped in magnetic polyvinyl alcohol beads cells from Pseudomonas delafieldii used for biodesulfurization. In relation to the magnetic immobilization of enzymes, the studies with purified xylanase immobilized on magnetic latex beads are known [6]. The immobilized enzyme preparation has been shown 80% of the total activity and a significant thermal stability. The data obtained in the present work demonstrate convincingly the effect of the used magnetic carriers for a significant enhancing of the CGTase production by immobilized Bacillus circulans ATCC 21783 cells. The possible explanation for these results is based on the good linking of a large amount of bacterial cells on the used magnetic carriers. Generally, in all immobilization procedures with living bacterial cells (irrespectively of the used support), cells are released from the biocatalysts during the cultivation process which continue to grow in the culture medium. In the case with SM-biocatalysts, probably some of these released cells were taken hold again of the free magnetite aggregates present in the culture liquid (Fig. 3). Thus, the measured cell growth as well as the total protein content in the culture liquid remained low, but the enzyme synthesis continued. In fact, a production of almost pure enzyme in clear culture liquids was achieved using of SMbiocatalyst (particularly, these composite from 80 mg SM and 140 bacterial cells). The released non-magnetic cells were few (observed under the microscope). Therefore, SM-biocatalysts offer the opportunity for an effective CGTase synthesis and a possible application for a direct cyclodextrin production. These biocatalysts could decrease the cost of the cyclodextrin production. The present results were not compared with other CGTase producing cells immobilized on magnetic supports because to our knowledge such studies are not available. Acknowledgements
Fig. 3. TEM micrographs of SM-biocatalyst (A, bar 0.5 mm), FF-alkaline biocatalyst (B, bar 0.5 mm) and FF-citrate biocatalyst (C, bar 0.5 mm) using B. circulans ATCC 21783 cells.
This work was supported by the Bulgarian Ministry of Science and Education (grant B-1521 NSF), bilateral grant P44/04 between the Bulgarian and Czech Academies of Sciences, the Grant Agency of the Czech Academy of Sciences (project no. IBS6087204), ISBE Research Intention no. AV0Z60870520.
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