Process Biochemistry 38 (2003) 1585 /1591 www.elsevier.

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Cyclodextrin glucanotransferase production by free and agar gel immobilized cells of Bacillus circulans ATCC 21783
Anna Vassileva a, Nigar Burhan b, Venko Beschkov b, Dimitrina Spasova c, Spasimira Radoevska c, Viara Ivanova d, Alexandra Tonkova a,*
a

Department of Extremophilic Bacteria, Institute of Microbiology, Bulgarian Academy of Sciences, 26, Acad. G. Bonchev str., 1113 Soa, Bulgaria b Institute of Chemical Engineering, Bulgarian Academy of Sciences, 103, Acad. G. Bonchev str., 1113 Soa, Bulgaria c Department of Morphology of Microorganisms and Electron Microscopy, Institute of Microbiology, Bulgarian Academy of Sciences, 26, Acad. G. Bonchev str., 1113 Soa, Bulgaria d Laboratory of Applied Microbiology, 26, Maritsa str., 4002 Plovdiv, Bulgaria Received 24 July 2002; accepted 29 January 2003

Abstract Cyclodextrins (CDs) are produced industrially from starch using bacterial cyclodextrin glucanotransferases (CGTase, EC 2.4.1.19). Batch cultivation of alkalophilic Bacillus circulans ATCC 21783 for CGTase synthesis was performed in a fluidized bed reactor. Under optimal growth conditions (40 8C, 150 ml working volume, 3.3 v/v/m, 1.0 /2.0 g l  1 initial starch concentration) enzyme activity was in a range 120 /150 U ml  1 at a specific growth rate 0.55 /0.60 h  1 (generation time 75 /80 min). CGTase was produced intensively after the end of the exponential phase (from 14th to 30th h) and thereafter the enzyme yield remained in the denoted range for 48 h. The use of agar-entrapped cells of B. circulans ATCC 21783 for CGTase production in a fluidized bed reactor led to enzyme activity in the range 180 /210 U ml  1 after 24 and 48 h cultivation, respectively. The operational stability of the biocatalysts was studied by repeated batch cultivation for 240 h (in a reactor). The residual activities represented 90 /95% of maximal. Scanning electron microscopy observations showed large groups of vegetative cells which continued to grow rapidly inside the agar beads indicating that high CGTase activity was due to the immobilization of the cells. # 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Bacillus circulans ; Cyclodextrins; Cyclodextrin glucanotransferase; Enzyme production; Cell immobilization; Scanning electron microscopy

1. Introduction Cyclodextrins (CDs) are cyclic, non-reducing malto oligosaccharides composed of 6 /60 glucose units linked with a-1,4-glucosidic bounds [1]. They possess a hydrophilic surface and a hydrophobic central cavity. The ability of CDs to encapsulate various chemical compounds and thus to change their physical and chemical properties, determines their wide application and importance for environmental protection, medicine, pharmaceutical and chemical industries [2,3]. For example, some of the main uses of b-CDs concern (a) detoxication

* Corresponding author. Tel.: /359-2-979-3163; fax: /359-2-700109. E-mail address: tonkova@microbio.bas.bg (A. Tonkova).

of wastewater from organic chemical and pharmaceutical industries by conversion of toxic substances to nontoxic b-CD-complexes resulting in their more rapid elimination from the activated sludge system; (b) acceleration of the hydrolysis of triglyceride; (c) improvement of the reaction rate for obtaining secondary glycosides (digoxin, acetyl-digoxin), important drugs; (d) serum substitute in mammalian cell structures by formation of CD-complexes with unsaturated fatty acids. Cyclodextrin glucanotransferase (CGTase EC 2.4.1.19) is a multifunctional enzyme which catalyzes four related reactions: cyclizing, coupling, disproportionation and hydrolysis [4]. By means of the cyclizing activity CGTases convert starch and related substrates into CDs. Most bacterial CGTases produce mainly a-CD, b-CD and g-CD consisting of six, seven, or eight glucose units,

0032-9592/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0032-9592(03)00060-8

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respectively. CGTases are produced mainly by members of the genus Bacillus . Alkalophilic bacilli have received the major attention for industrial applications because of their high activity over a wide range of pH and temperatures [5]. These bacilli have been regarded as the most promising strains for producing b-type CGTase, which creates mainly b-CD without accumulation of aCD, and thus the purification of b-CD from the reaction mixture is facilitated using the solubility difference between b-CD and g-CD [5 /9]. Bacillus circulans ATCC 21783 is capable of growing in high alkaline pH media containing 1% of Na2CO3 and produces mainly b-CGTase [10,11]. The lack of reports about the optimization of CGTase production by B. circulans ATCC 21783, as well as the significant demand of b-CDs on the world market determined the aim of the work presented: to develop b-CGTase yield by free and agar gel immobilized cells of B. circulans ATCC 21783 comparable or higher than that of other Bacillus strains used for an industrial enzyme production.

2.2. CGTase synthesis by immobilized cells 2.2.1. Batch cultivation Agar entrapment of bacterial cells was performed according to Nilsson et al. [12] using sunflower oil as hydrophobic phase. The optimization of the immobilization conditions were carried out in flasks (100 ml). Different agar gel concentrations (2, 3 and 4%, w/v) and an initial cell loading (ICL, 0.5 /2.0%, w/v) in the agar beads were tested. The corresponding quantity of wet cells harvested by centrifugation from an exponential growth phase were resuspended in 0.4 ml sterile water and mixed with 25 ml of warm (50 8C) agar solution. Seven ml of this mixture was dropped into the cooled oil phase. After solidification (at least 1 h, 4 8C) the biocatalysts (agar beads with entrapped bacterial cells) were washed thoroughly with sterile water and transferred into 20 ml production medium. Batch cultivation was performed at 40 8C on a shaker (220 rpm). Parallel experiments with free cells were carried out as a control (inoculum cell concentration 1 and 2%, v/v). 2.2.2. Repeated batch cultivation Repeated batch experiments with agar entrapped cells of B. circulans ATCC 21783 were carried out in a fluidized bed reactor under optimal growth and immobilization conditions (air flow rate 6.1 v/v/min). At the end of each batch circle (time interval 48 h) the biocatalysts were washed abundantly with sterile water and then introduced into fresh production medium. 2.3. Analytical methods CGTase cyclization activity was assayed by the method of Kaneko et al. [13] 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 bCD min 1 under standard conditions (phosphate buffer pH 6.0, 60 8C, 20 min reaction time). Total protein content was determined by the method of Bradford using bovine serum albumin as a standard. Cell growth was measured by absorbance at 650 nm. Thin-layer chromatography (TLC) of saccharides obtained by starch conversion was performed on silica gel 60 pre-coated aluminium sheets (Merck, Darmstadt, Germany, 20 /20 cm). Reaction mixtures containing 40 mg soluble starch (Fluka) in 1.0 ml 0.1 M sodium phosphate buffer (pH 6.0) and 0.1 ml enzyme solution (cultural supernatants after 30 h cell growth) were incubated at 60 8C. Samples were taken after 2 h, mixed with 2 volumes of methanol, centrifuged and 15 ml portions of the starch hydrolyzates obtained applied to a thin-layer chromatography sheet. The saccharide contents of the supernatants were determined on the same sheet. A mixture of n-propyl alcohol/ethyl acetate/water

2. Materials and methods

2.1. CGTase synthesis by free cells

2.1.1. Bacterial strain and growth conditions B. circulans ATCC 21783 was supplied by the National Bank of Microorganisms and Cell Cultures (Sofia, Bulgaria). The seed medium comprised (g l1): soluble starch (Poland), 2; peptone (Oxoid, Basingstoke, UK), 5; yeast extract (Oxoid), 5; MgSO4, 0.2 and K2HPO4, 1. Sterile sodium carbonate was used to adjust the medium to pH 9.8 /10.0 after autoclaving. A production medium with the same composition and various initial starch concentrations (0.4, 0.8, 1.0, 2.0 and 10 g l1) was used for studying CGTase synthesis in batch cultivation in a fluidized bed reactor. The inoculum culture was grown in 100-ml Erlenmeyer flasks containing 20 ml nutrient medium (initial pH 9.8 /10.0) at 40 8C. After overnight incubation in a water bath shaker (Julabo SW1) at 220 rpm, the cell suspension (2%, v/v; OD650nm 1.4 /1.5) was transferred into a bubble column reactor of 175 mm height, 50 mm inner diameter and total volume 300 ml. Sterile air was introduced via a rotameter and a sterile air filter into the nozzle at the bottom of the reactor. Experiments were carried out under the following conditions: temperature 40 8C (maintained by thermostat), working volume 150 ml and air flow rate 3.3 v/v/min.

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(7:1:2, v/v/v) was used as a developing solvent. Saccharides were detected by spraying the air-dried plates with staining reagent containing ethanol/acetic acid/sulphuric acid/anisaldehyde (9:0.1:0.5:0.5, v/v/v/v). Carbohydrates were revealed after heating for 10 min at 120 8C and were visualized as dark green spots. 2.4. Scanning electron microscopy (SEM) SEM observations was performed as described by Tonkova et al. [14] with a scanning device attached to a Zeiss electron microscope (model 10 C) at 20 kV accelerating voltage with a 5 /6 nm electron beam.

3. Results 3.1. Growth and CGTase production by free cells of Bacillus circulans ATCC 21783 A set of preliminary runs were performed to determine the optimal working volume (150 ml), temperature (40 8C) and aeration rate (3.3 v/v/min) during study of the influence of substrate concentration on CGTase synthesis. Cell growth, CGTase activity and pH at initial starch concentrations of 1 and 10 g l 1 are presented in Fig. 1A,B. The exponential growth phase lasted 9 h in all experiments irrespective of the initial substrate content. The specific growth rate was in the range 0.55 /0.60 h1 (generation time 75 /80 min). CGTase synthesis began in the early exponential phase and intensive enzyme production was established after the end of exponential growth (9 h). Maximal CGTase yield was determined from the 14th to the 30th h of the processes (Fig. 1A,B). The highest CGTase activity (120 /150 U ml1) was achieved in the presence of 1.0 /2.0 g l 1 starch in the medium (Fig. 1A). From the beginning of the process to the end of exponential growth pH-values decreased to 8.0 /9.0 at 0.4 /2.0 g l 1starch (Fig. 1A) and to 6.0 at 10 g l 1 starch (Fig. 1B). After that, at low substrate concentrations the pH in the culture medium was gradually increased to 9.5 /10.0, whereas at high starch content (10 g l1) pH-values were retained at 6.0. The different courses of pH and CGTase yield-curves depending on the initial starch content in the medium correlated with the regulation of enzyme synthesis. TLC of the supernatants from 30 h culture liquids demonstrated that the high starch concentration (Fig. 2, lane 10) led to an accumulation of glucose, maltose, maltotriose, and traces of maltotetraose in the cultural medium, whereas at low starch concentration (Fig. 2, lanes 2, 4, 6 and 8) saccharides were not detected (substrate was completely consumed). Glucose and maltose exerted a repressive effect on CGTase synthesis (general phenomenon for the synthesis of extracellular

Fig. 1. Growth and CGTase production during batch cultivation of Bacillus circulans ATCC 21783 in a uidized bed reactor. (A) 1.0 g l  1 initial starch concentration; (B) 10.0 g l  1 initial starch concentration.

bacterial enzymes). Catabolite repression stopped enzyme synthesis and CGTase activity remained in a low level of 50 /53 U ml1 (Fig. 1B). Substrate was completely depleted at low initial concentrations (Fig. 2, lane 2, 4, 6 and 8), catabolite repression did not appear and CGTase activities achieved 120 to 150 U ml 1. The enzyme solutions catalyzed the formation of bCDs at pH 6.0 and 8.5 (Table 1) and the established high specific cyclizing activities showed the possibility of using the enzyme in a wide pH range. This is of great importance for industrial applications. CD-production was analyzed by TLC. Saccharides formed from 4% (w/v) soluble starch (Fluka) after 2 h enzyme reaction under the assay conditions (pH 6.0, 60 8C) were clearly visible (Fig. 2, lanes 3, 5, 7, 9 and 11). The starch hydrolyzates obtained from all batch experiments showed a predominant formation of b-CDs and linear saccharides, such as glucose, maltose, maltotriose and traces of maltopentaose.

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Fig. 2. Thin-layer chromatography of saccharides. A mixture of standard linear saccharides (G1 glucose to G7 maltoheptaose, 0.5% [w/v] each) was applied to lane 1; standard a-, b- and g- CDs (0.5% [w/v] each) were applied to lanes 12, 13 and 14; supernatants from batches at: 0.4 g l  1 starch-lane 2, at 0.8 g l  1 starch-lane 4, at 1.0 g l  1 starch-lane 6, at 2.0 g l  1 starch-lane 8 and at 10 g l  1 starch-lane 10; starch hydrolyzates after 2 h reaction time (lanes 3, 5, 7, 9 and 11; samples are arranged in the same order as above).

Table 1 Specic cyclizing CGTase activities of cultural supernatants obtained after 30 h batch growth of Bacillus circulans ATCC 21783 Batch cultivation at: Specific activity (U mg 1) pH 6.0 0.4 g l starch 0.8 g l  1 starch 1.0 g l  1 starch 2.0 g l  1 starch 10.0 g l  1 starch
1

pH 8.5 115.71 414.78 513.33 445.90 270.95

184.86 583.17 623.12 444.40 248.90

3.2. CGTase production by immobilized cells of Bacillus circulans ATCC 21783 3.2.1. Batch cultivation It is known that gel concentration and initial cell loading (ICL) in the agar beads are significant factors affecting enzyme production by the biocatalysts. The mechanical stability of the beads and diffusion rate of nutrients and oxygen through the gel are dependent on the gel concentration. The results obtained showed that during the cultivation process (72 h) the integrity of the beads was completely retained in all experiments but

some cells were released from the agar beads into the medium. It was established that the higher ICL led to a smaller quantity of released cells in the medium (data not shown) and higher enzyme activity. The course of CGTase activity curves by immobilized and free cells was different (Fig. 3A /C). During a 72 h process the CGTase yield by free cells was maintained in a range of 80 /100 U ml1. Using immobilized cells enzyme activities were significantly increased from 24 to 48 h and achieved 180 /200 U ml 1 (2-fold higher compared to the control) at 0.7 /2.0% ICL. Over 72 h CGTase activities decreased to 150 /170 U ml1 (1.7 /1.8-fold higher than control) probably because of the depletion of the nutrients (all experiments were performed in flasks at 2.0 g l1 initial starch concentration). The optimal immobilization conditions including 3% (w/v) agar concentration and 2% (w/v) ICL provided 170 /200 U ml1 enzyme yield during 24 /48 h batch cultivation at an optical density (650 nm) of the released cells of 0.80 /0.87. 3.2.2. SEM observations From the results above it was clear that the gel concentration did not exert a significant influence on CGTase production (Fig. 3A /C). This fact could be

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under the optimal immobilization conditions during five cycles (48 h each) in a fluidized bed reactor (Fig. 5). The results obtained at the end of the first cycle (48 h) showed the same range of CGTase activity (185 U ml 1) compared to those of the immobilized cells in flasks (at 48 h). During the next four cycles enzyme activity was maintained in the high level of 190 /210 U ml 1, i.e. 90 /95% of the maximal activity was retained during 240 h of cultivation. The optical density (650 nm) of the released cells was maintained between 0.60 and 0.75 and pH-values of cultural medium 9.8 /10.0. In respect to mechanical stability of the biocatalysts no fragments of the gel beads were found and their integrity was completely retained.

4. Discussion Few studies have been reported on the optimization of CGTase synthesis by B. circulans ATCC 21783, a potential industrial producer. Concerning the maximization of CGTase production by the denoted strain (in fluidized bed reactor) only the investigation of Jamuna et al. [16] was known. According to these authors the CGTase yield of 60 U ml 1 achieved in culture liquid with calcium alginate immobilized cells was 2.25-fold lower in comparison to the presented enzyme yield of 120 /150 U ml1 in batch cultivation of free cells and 3.3-fold lower than CGTase production of agar-immobilized cells (190 /210 U ml 1). Moreover, a maximal enzyme activity was attained in the relatively short cultivation time of 24 /30 h in contrast to other reported studies with B. circulans ATCC 21783 where a maximum cyclizing activity appeared after 48 /55 h [16 /18] or 72 /96 h [19] of growth. Bacillus cereus RJ 30 [18] and Bacillus firmus var. alkalophilus [5] were reported as other organisms useful for CGTase production. B. cereus RJ 30 exhibited maximal CGTase activity of 106 U ml1. Further investigations with the same producer [20] evaluating solid, slurry and submerged fermentations for CGTase production have shown the same range of enzyme yield (110 U ml 1). Alginate-immobilized cells of B. cereus RJ 30 [18] produced enzyme activity of 30 /40 Uml 1 during 240 h semicontinuous cultivation. Under optimal immobilization conditions agar-entrapped cells of B. circulans ATCC 21783 produced 190 /210 U ml1 enzyme activity during 240 h repeated batch cultivation. An alkalophilic B. firmus var. alkalophilus (excreting b-CGTase) has been selected for achieving overproduction of CGTase [5]. The b-CGTase gene from this strain has been cloned and expressed in Escherichia coli . The b-CGTase of transformant E. coli has been mostly produced after the stationary phase of growth. The highest enzyme yield of about 20 U ml1 (calculated

Fig. 3. CGTase production by agar-entrapped Bacillus circulans ATCC 21783 cells as a function of gel concentration and ICL. (A) 2% agar; (B) 3% agar; (C) 4% agar; dotted lines, free cells; compact lines, immobilized cells.

explained by the ability of the strain to grow also under anaerobic conditions [15]. For that reason the reduced diffusion of oxygen at a larger gel concentration did not affect cell growth in/on the inner and outer layers of the agar beads (Fig. 4A /D). Samples taken after 48 h cultivation under optimal immobilization conditions showed spores, some single vegetative and lyzed cells in layers from the outer surface of the beads (Fig. 4A,B). SE-micrographs of the sliced agar beads (inner layer) had a similar appearance but vegetative cells continued to grow rapidly and large clumps of cells were visible (Fig. 4C,D). At the same time only lyzed cell material and spores were found in culture medium indicating that high CGTase activity was due to the immobilized cells. 3.2.3. Operational stability of biocatalysts A main advantage of the biocatalysts, besides the enhanced enzyme activity, is their multiple use by a repeated batch cultivation. The long-term stability of the biocatalysts was studied by semicontinuous cultivation

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Fig. 4. Scanning electron micrographs of agar-entrapped cells of Bacillus circulans ATCC 21783 after 48 h of cultivation in asks (bars /0.5 mm). (A) Spores and a few vegetative cells on the bead surface. (B) Spores and single lyzed cells in an outer layer of the agar bead. (C) Heaps of vegetative cells and single spores in the inner layer of an agar bead. (D) Group of growing vegetative cells inside the agar bead.

could be applied in continuous enzyme and CD production.

Acknowledgements This work was supported by a research grant No X1112/01 of the National Scientic Foundation (Bulgarian Ministry of Science and Education).

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