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Cyclodextrin glucanotransferase production by free and agar gelimmobilized cells ofBacillus 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. Bonchevstr., 1113 Sofia, Bulgariab Institute of Chemical Engineering, Bulgarian Academy of Sciences, 103, Acad. G. Bonchevstr., 1113 Sofia, Bulgaria

c Department of Morphology of Microorganisms and Electron Microscopy, Institute of Microbiology, Bulgarian Academy of Sciences, 26, Acad. G.

Bonchev str., 1113 Sofia, Bulgariad 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 l1 initial starch concentration)

enzyme activity was in a range 120/150 U ml1 at a specific growth rate 0.55 /0.60 h1 (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 ml1 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% ofmaximal. Scanning electron microscopy observations showed large groups ofvegetative 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 hydro-philic surface and a hydrophobic central cavity. The

ability of CDs to encapsulate various chemical com-

pounds and thus to change their physical and chemical

properties, determines their wide application and im-

portance for environmental protection, medicine, phar-

maceutical and chemical industries [2,3]. For example,

some of the main uses ofb-CDs concern (a) detoxication

of wastewater from organic chemical and pharmaceu-

tical industries by conversion of toxic substances to

nontoxic b-CD-complexes resulting in their more rapid

elimination from the activated sludge system; (b) accel-

eration of the hydrolysis of triglyceride; (c) improvementof 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, disproportio-

nation 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

andg-CD consisting of six, seven, or eight glucose units,

* Corresponding author. Tel.: /359-2-979-3163; fax: /359-2-700-

109.

E-mail address: tonkova@microbio.bas.bg(A. Tonkova).

Process Biochemistry 38 (2003) 1585/1591

www.elsevier.com/locate/procbio

0032-9592/03/$ - see front matter# 2003 Elsevier Science Ltd. All rights reserved.

doi:10.1016/S0032-9592(03)00060-8

mailto:tonkova@microbio.bas.bgmailto:tonkova@microbio.bas.bgmailto:tonkova@microbio.bas.bgmailto:tonkova@microbio.bas.bg

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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 ofa-CD, and thus the purification ofb-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 Na2CO3and 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 thanthat of other Bacillus strains used for an industrial

enzyme production.

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 con-

centrations (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 Erlen-

meyer 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.

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 immobili-

zation 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 trans-

ferred into 20 ml production medium. Batch cultivation

was performed at 40 8

C on a shaker (220 rpm). Parallelexperiments 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 im-

mobilization 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 com-

plexation withb-CD. One unit of CGTase activity was

defined as the amount of enzyme that formed 1 mg ofb-

CD min1 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 saccharidesobtained 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 con-

tents of the supernatants were determined on the same

sheet. A mixture of n-propyl alcohol/ethyl acetate/water

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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 themedium. 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 ml1 (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

Fig. 2. Thin-layer chromatography of saccharides. A mixture of standard linear saccharides (G1glucose to G7maltoheptaose, 0.5% [w/v] each) was

applied to lane 1; standarda-,b- andg- CDs (0.5% [w/v] each) were applied to lanes 12, 13 and 14; supernatants from batches at: 0.4 g l1 starch-lane

2, at 0.8 g l1 starch-lane 4, at 1.0 g l1 starch-lane 6, at 2.0 g l1 starch-lane 8 and at 10 g l1 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

Specific cyclizing CGTase activities of cultural supernatants obtained

after 30 h batch growth ofBacillus circulans ATCC 21783

Batch cultivation at: Specific activity (U mg1)

pH 6.0 pH 8.5

0.4 g l1 starch 184.86 115.71

0.8 g l1 starch 583.17 414.78

1.0 g l1 starch 623.12 513.33

2.0 g l1 starch 444.40 445.90

10.0 g l1 starch 248.90 270.95

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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

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

ml1) 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 Uml1, 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 optimizationof CGTase synthesis by B. circulans ATCC 21783, a

potential industrial producer. Concerning the maximi-

zation 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 ml1 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-immo-

bilized cells (190/210 U ml1). Moreover, a maximal

enzyme activity was attained in the relatively short

cultivation time of 24/30 h in contrast to other reportedstudies with B. circulans ATCC 21783 where a max-

imum 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 ml1).

Alginate-immobilized cells of B. cereus RJ 30 [18]produced enzyme activity of 30/40 Uml1 during 240 h

semicontinuous cultivation. Under optimal immobiliza-

tion conditions agar-entrapped cells of B. circulans

ATCC 21783 produced 190/210 U ml1 enzyme

activity during 240 h repeated batch cultivation.

An alkalophilicB. firmus var. alkalophilus (excreting

b-CGTase) has been selected for achieving overproduc-

tion 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.

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