World J Microbiol Biotechnol (2013) 29:865–873
DOI 10.1007/s11274-012-1241-9
ORIGINAL PAPER
b-cyclodextrin production by the cyclodextrin glucanotransferase
from Paenibacillus illinoisensis ZY-08: cloning, purification,
and properties
Yong-Suk Lee • Yi Zhou • Dong-Ju Park •
Jie Chang • Yong-Lark Choi
Received: 13 August 2012 / Accepted: 14 December 2012 / Published online: 23 December 2012
Ó Springer Science+Business Media Dordrecht 2012
Abstract The gene encoding the cyclodextrin glucano-
transferase (CGTase, EC2.4.1.19) of Paenibacillus illi-
noisensis was isolated, cloned, sequenced and expressed in
Escherichia coli. Sequence analysis showed that the mature
enzyme (684 amino acids) was preceded by a signal pep-
tide of 34-residues. The deduced amino acid sequence of
the CGTase from P. illinoisensis ZY-08 exhibited highest
identity (99 %) to the CGTase sequence from Bacillus li-
cheniformis (P14014). The four consensus regions of car-
bohydrate converting domain and Ca2? binding domain
could be identified in the sequence. The CGTase was
purified by using cold expression vector, pCold I, and His-
tag affinity chromatography. The molecular weight of the
purified enzyme was about 74 kDa. The optimum tem-
perature and pH of the enzyme were 40 °C and pH 7.4,
respectively. The enzyme activity was increased by the
addition of Ca2? and inhibited by Ba2?, Cu2?, and Hg2?.
The Km and Vmax values calculated were 0.48 mg/ml
and 51.38 mg of b-cyclodextrin/ml/min. The ZY-08
and recombinant readily converted soluble starch to
b-cyclodextrin but ZY-08 did not convert king oyster
mushroom powder and enoki mushroom powder. However
the recombinant CGTase converted king oyster mushroom
powder and enoki mushroom powder to b-cyclodextrin.
Y.-S. Lee  D.-J. Park  J. Chang  Y.-L. Choi (&)
Department of Biotechnology, College of Natural Resources
and Life Science, Dong-A University, Busan 604-714,
Republic of Korea
e-mail: ylchoi@dau.ac.kr
Y. Zhou
College of Agriculture, Yangtze University, Jingzhou,
Hubei Province, People’s Republic of China
Keywords Cyclodextrin glucanotransferase  CGTase 
Cyclodextrin  Paenibacillus illinoisensis  ZY-08 
King oyster mushroom  Enoki mushroom
Introduction
Cyclodextrin glucanotransferases (CGTases; E.C.2.4.1.19)
are starch degrading enzymes which produce cyclic oli-
gosaccharides termed cyclodextrins (CDs). This enzyme
belongs to the a-amylase family (glycosyl hydrolase
family 13). Members of this family contain conserved cat-
alytic residues and a conserved catalytic site architecture,
but their activities vary (Janecek 1997). CGTase usually
catalyzes four different reactions (cyclization, coupling,
disproportionation, and hydrolysis). The formation of CD
starts with binding of CGTase to a linear maltooligosac-
charide substrate, across multiple sugar binding subsites.
This is followed by cleavage of an a-1,4-glycosidic bond
resulting in an intermediate that is covalently bound to Asp
229 in subsite-1 (Uitdehaag et al. 2000). Subsequently, the
non-reducing end of the substrate moves into the site of the
covalent bond and is processed for the formation of a new
intramolecular a-1,4-glycosidic bond. As separation of a
specific form of CD from others is costly and time-con-
suming, the CGTase that predominantly produces one type
of CD is of interest (Martins and Hatii-Kaul 2002).
These oligosaccharides as three major types commonly
known as a, b, and c having 6, 7, and 8 glucose residues,
respectively (Rojtinnakorn et al. 2001; Martins and Hatii-
Kaul 2002). CDs have been mainly used as complexing
agents to increase the aqueous solubility of poorly water-
soluble drugs, to reduce or prevent gastrointestinal or
ocular irritation, to reduce or eliminate unpleasant
smells or tastes, to prevent drug–drug or drug-additive
123
866
interactions, or even to convert oils and liquid drugs
into microcrystalline or amorphous powders (Qi and
Zimmermann 2005). Hence, CDs offer a variety of indus-
trial applications in the areas of foods, cosmetics, and
pharmaceuticals as stabilizers, solubilizer, and control-
released substance (Schmid et al. 1998).
Paenibacillus is a widely distributed bacterial genus in
the environment. Some of the species belonging to this
genus have been isolated from soil (Axelrood et al. 2002;
Garbeva et al. 2003), water (Ross et al. 2001), and different
plant rhizospheres (von der Weid et al. 2000; Berge et al.
2002). Various bacteria use starch as a carbon and energy
source for growth and can produce CGTase, which
degrades starch into CDs. Evidence has been presented that
the CDs are transported into the cytoplasm via a specific
system and that they are hydrolyzed inside the cell by
CDase to form linear malto-oligosaccharides to be used as
energy source (Kaulpiboon and Pongsawasdi 2005).
Previously we reported that Paenibacillus illinoisensis
ZY-08 was isolated from the rhizosphere of Amorpho-
phallus konjac field and characterization of b-mannanase
(Lee et al. 2010). This paper describes the isolation and
cloning of the CGTase gene from P. illinoisensis ZY-08.
Purification and characterization of the enzyme is also
presented.
Materials and methods
Bacterial strains, plasmids, and media
Paenibacillus illinoisensis ZY-08 (KACC91465 P), a high
cyclodextrin glucanotransferase producer, was isolated
from soil in China and deposited with the Korean Agri-
cultural Culture Collection (KACC). Escherichia coli
JM109, BL21 (DE3), and EPI-100 were used as a trans-
formation and expression host. The plasmid pUC118/
BamHI/BAP (TaKaRa Bio, Otsu, Japan) and pEpiFOS-5
(Epicentre, Madison, WI, USA) were used to construct the
genomic library, and pCold I (TaKaRa) was used to purify
the fusion protein. E. coli strains were cultivated in Luria–
Bertani medium at 37 °C, and appropriate antibiotic was
added. King oyster mushroom (Pleurotus eryngii) powder
and enoki mushroom (Flammulina velvtipes) power were
got from mushroom farm in Korea.
Construction of the genomic library
from P. illinoisensis ZY-08
A genomic library of P. illinoisensis ZY-08 was con-
structed using a commercial fosmid vector, pEpiFOS-5
(Epicentre). A single colony of ZY-08 was inoculated into
10 ml of LB broth, incubated at 37 °C for overnight on
123
World J Microbiol Biotechnol (2013) 29:865–873
rotary shaker (180 rpm). Cells were harvested, and geno-
mic DNA was prepared by standard method, as described
by Sambrook et al. (1989). The extracted DNA (1 lg) was
efficiently sheared by hundreds of pipettings. It was treated
for end-repair to generate blunt ended and 50 -phosphory-
lated DNA. End-repaired DNA was electrophoresed in a
1 % low-melting point agarose at 50 V for 2 h, and DNA
fragments over 40 kb were isolated from the gel using
GELase (Epicentre). The prepared DNA was ligated into
the fosmid vector, and then ligation mixture was packaged
into lambda phages using MaxPlax Lambda Packaging
Extracts (Epicentre). The packaged library was transducted
into E. coli EPI-100.
Screening and subcloning of the CGTase
The genome library was cultivated on LB agar medium with
soluble starch (0.5 %) and chloramphenicol. The plates
were overlaid with phenolphthalein solution for evidence
activity after 2 days of incubation at 37 °C. The colonies
with halo zone on the pink background of the medium were
selected as CGTase producing recombinants. The plasmid
DNA of the selected clones was isolated, partially digested
with Sau3AI to have 1–4 kb fragment, and into a pUC118/
BamHI/BAP. The subclones were subjected to selection of
showing halo zones around the colonies on the pink back-
ground of the medium as b-CGTase producing recombi-
nants and sequenced. The analysis of sequence data and
sequence similarity searches were performed the BLAST
program of National Center for Biotechnology Information
(NCBI). Homology alignment was performed with CLUS-
TAL W program (Thomsom et al. 1994) using MacVector
6.5 software (Oxford Molecular Group). For the pro-
moter determination, the promoter prediction program
NNPP version 2.2 (http://www.fruitfly.org/seq_tools/promoter.
html) was used.
Construction of His-CGTase fusion plasmid
To construct the His-tag fusion vector, two oligonucleotide
primers, 50 -GCTTCCGCGATTGAGCTCGATGCGGAT
ACGGCT-30 and 50 -ATGAATTATAAGCTTTTATTGCC
AGTTTAC-30 , were synthesized by Bioneer Co. These
primers were modified to contain SacI and HindIII
restriction enzyme sites in order to facilitate cloning into
the His-tag fusion protein expression vector, pCold I, a
cold-shock expression vector (TaKaRa). The PCR mixture
included 100 pmol of primer, 200 ng of template DNA,
20 mM each deoxynucleoside triphosphates, and 1.0 U
Taq DNA polymerase in a 50 ll reaction volume. Thirty
rounds of amplification were done with the following
cycles: pre-denature step was 60 s at 95 °C, followed by 30
cycles of denaturation at 95 °C for 60 s, and annealing step
World J Microbiol Biotechnol (2013) 29:865–873
at 55 °C for 60 s and an extension step at 72 °C for 90 s,
followed final extension step at 72 °C for 10 min. The
amplified PCR fragments were double-digested by SacI/
HindIII and ligated into a pColdI/SacI/HindIII/CIAP vec-
tor. The recombinant fusion plasmid was transformed into
E. coli BL21 (DE3) for the fusion protein expression.
Expression and purification of fusion protein
The E. coli BL21 (DE3) cells containing the recombinant
plasmid were grown at 37 °C in LB medium with 100 lg/ml
ampicillin, by the addition of 0.1 mM isopropyl-b-D-thioga-
lactopyranoside (IPTG) when the optical density at 600 nm
reached 0.4–0.5. After induction for 24 h shaking at 15 °C, the
cells were harvested by centrifugation at 5,0009g for 15 min
at 4 °C, washed with binding buffer [20 mM sodium phos-
phate (pH 7.4), 0.5 M NaCl, and 5 mM imidazole], and then
resuspended in binding buffer. The cells were disrupted by
sonication (pulse on 30 s, pulse off 30 s, 5 times, on ice), and
the supernatant was collected by centrifugation at
15,0009g for 30 min at 4 °C. The clear supernatant was
loaded on to a HisTrap HP column (Amersharm Phamacia
Biotech.) equilibrated with binding buffer and eluted with
elution buffer [20 mM sodium phosphate (pH 7.4), 0.5 M
NaCl, and 0.5 M imidazole] at flow rate of 1 ml/min. The
eluted fractions were dialyzed overnight against 20 mM
sodium phosphate buffer and concentrated by using Amicon
Ultra-4 (Millipore, Bedford, MA). Sodium dodecyl sul-
fate–polyacrylamide gel electrophoresis (SDS-PAGE) was
carried out by the method of Laemmli (1970). The
concentrated protein was used to determine enzyme
characterizations.
Beta-cyclodextrin assay
Phenolphthalein (PHP) stock 4 mM in ethanol, 125 mM
Na2CO3 solution in distilled water, 20 mM sodium phos-
phate buffer (pH 7.4), b-CD in buffer (500 lg/ml) and
working PHP solution made by adding 1 ml of stock and
4 ml of ethanol in 100 ml Na2CO3 solution (Goel and Nene
1995). The reaction mixture containing 50 ll of 1 % sol-
uble starch, 50 mM sodium phosphate buffer (pH 7.4), and
the purified enzyme in a final volume of 250 ll was
incubated at 40 °C for 30 min. Then 0.75 ml of the
working PHP solution was added to the reaction mixture
and the tube was mixed. Enzyme activity was measured as
the decrease of extinction at 550 nm as indicated by the
formation of phenolphthalein-b-cyclodextrin inclusion
complexes. One unit of enzyme was defined as the amount
of enzyme that produced 1 lmol of b-CD per min under
standard conditions.
867
Effect of pH, temperature, and metal ions
on the CGTase
The purified CGTase was incubated with soluble starch at
different temperatures and pHs. For temperature effect,
reactions were performed at 20–80 °C for 30 min. The
temperature stability was pre-incubated at different tem-
perature, ranging from 20 to 80 °C for 30 min, without
substrate. The residual activity of the enzyme was assayed
with soluble starch at 40 °C for 30 min.
The 20 mM citrate buffer (pH 3–6), 20 mM sodium
phosphate buffer (pH 6–7.6), 20 mM Tris–HCl (pH 7.5–9),
and glycine-NaOH buffer (pH 8.6–10.6) were used as
reaction buffers. The pH stability of the enzyme was pre-
incubated with various buffers at 4 °C without substrate for
12 h. The residual activity of the enzyme was assayed with
soluble starch at 40 °C for 30 min. The reaction was car-
ried out using the CGTase assay procedure mentioned
earlier. The effects of different metal ions or some inhib-
itors on the CGTase activity were investigated by addition
of the test ions and reagents to reaction mixtures at final
concentration of 1 mM or 10 mM. The test ions and
reagents included BaCl2, CaCl2, CuCl2, HgCl2, NaCl,
NiCl2, MgCl2, and EDTA.
Substrate specificity of CGTase
Paenibacillus illinoisensis ZY-08 and E. coli JM109 har-
boring pUC-CGT were cultured in 500 ml of LB medium
containing 0.5 % (w/v) soluble starch, king oyster mush-
room powder, or enoki mushroom powder at 37 °C with
180 rpm. After 2 days, the supernatant of the medium were
obtained by centrifugation at 5,0009g for 15 min at 4 °C.
b-CD was checked in the supernatant of the medium.
Kinetic parameters of CGTase
The Km and Vmax values for the pure enzyme were deter-
mined by incubating purified CGTase in 50 mM sodium
phosphate buffer (pH 7.4) at various concentrations of
soluble starch solution, ranging from 0.4 to 6.0 mg/ml at
40 °C for 30 min. The values of Km and Vmax were then
determined using Michaelis–Menten equation and double
reciprocal plot known as Lineweaver-Burke plot (Martins
and Hatii-Kaul 2002).
Nucleotide sequence accession number
The nucleotide sequence of CGTase gene reported in this
article has been assigned GenBank accession numbers
FJ232954.
123
868 World J Microbiol Biotechnol (2013) 29:865–873

Results and discussions

Molecular cloning of the CGTase gene

from P. illinoisensis ZY-08

To clone the CGTase gene from ZY-08, the genome library

was constructed by Fosmid Library Production Kit. A
number of the transformants (approximately 2,000) were
screened and only one colony showed hydrolytic activity
on LB-chloramphenicol-soluble starch (0.5 %) agar plate.
The selected clone was partially digested with Sau3AI and
ligated into a pUC118/BamHI/BAP. The secondary library
was subjected to selection of subclones, pUC-CGT,
showing clear zones around the colonies on the LB-ampi-
cillin soluble starch agar plate.
The nucleotide sequence analysis of the CGTase gene
from ZY-08 revealed the presence of a single open reading
frame (ORF) with two potential initiation ATG codons
(Fig. 1). A typical sequence for the ribosome-binding site
(50 -GAAGGGTGA-30 ) exhibited a good rationally com-
plementary with the 30 end of the ZY-08 16S rRNA gene
was located on 7 bases upstream from the first ATG start
codon. Consequently, the latter is most likely the true ini-
tiator codon (Jemli et al. 2008). Thus, the ORF of CGTase
gene from ZY-08 was composed of 2,157 bp, encoded a
protein having 718 amino acids. By using the promoter
prediction program, a potential -10 region (50 -TATTA
TT-30 ) and -35 region (50 -AAGTCT-30 ) were found at 60
and 115 bp upstream of the initiator codon, respectively.
The N-terminal sequence analysis was predicted according
to the SignalP site (http://www.cbs.dtu.dk/services/SignalP
). It is presumable that the most likely cleavage sites are
between Ala-34 and Asp-35 (Nielsen et al. 1997; Bendtsen
et al. 2004). So, mature protein was consisted of 684 amino
acids with an estimated molecular weight about 74,352 Da.

Amino acid sequence analysis

The deduced amino acid sequence of the CGTase from
P. illinoisensis ZY-08 was compared with other CGTase
sequences using the BLAST program (Table 1). The
enzyme showed identities and positives with other bacterial
CGTases such as Bacillus licheniformis (BACLI, P14014,
99 %, 99 %), B. circulans 18 (BACCI2, P30920, 91 %,
96 %), Bacillus sp. 6.6.3 (BACSS, P31747, 91 %, 95 %),
alkalophilic Bacillus sp. 17.1 (BAC11, P30921, 73 %,
85 %), B. circulans 251 (BACCI1, P43379, 73 %, 83 %),
Bacillus sp. 1018 (BACS8, P17692, 73 %, 83 %), alkalophilic
Bacillus sp. 1.011 (BACS0, P05618, 72 %, 84 %) alkalophilic
Bacillus sp. 38.2 (BACS3, P09121, 71 %, 83 %), Thermoan-
aerobacterium thermosulfurines EM1 (THETU, P26827,
66 %, 80 %), Paenibacillus marcerans (PAEMA1, P31835,
65 %, 80 %), P. marcerans (PAEMA2, P04830, 64 %, 76 %),
Fig. 1 Nucleotide sequences and deduced amino acid sequences of
the cyclodextrin glucanotransferase (CGTase) gene from Paenibacil-
lus illinoisensis ZY-08. The possible -35 and -10 sequence in the
promoter region and the possible ribosome-binding site, SD, are
double underlined. The possible signal peptide cleavage site is
indicated by arrow. The four highly conserved regions and Ca2?
binding domain in different CGTases are underlined. The start and
end of the A to E domains of CGTases are marked. The important
amino acids are shaded
B. stearothermophylus NO2 (BACST, P31797, 60 %,
75 %), Brevibacillus brevis CD162 (BREBE, O30565, 59 %,
74 %), alkalophilic Bacillus sp. 1.1 (BACS2, P31746,
58 %, 75 %), B. obhensis (BACOH, P27036, 58 %, 74 %),
and Klebsiella pneumoniae (KLEOX, P08704, 35 %, 53 %)
(Fig. 2a). From the multiple alignment, the five structural

123
World J Microbiol Biotechnol (2013) 29:865–873 869

Table 1 List of CGTases used

Homologous protein Origin Identity (%) Positive (%) GenBank
in sequence alignment and
phylogenetic tree construction BACLI Bacillus licheniforms 99 99 P14014
BACCI2 Bacillus circulans 8 91 96 P30920
BACSS Bacillus sp. 6.6.3 91 95 P31747
BAC11 Alkalophilic Bacillus sp. 17.1 73 85 P30921
BACCI1 Bacillus circulans 251 73 83 P43379
BACS8 Bacillus sp. 1018 73 83 P17692
BACS0 Alkalophilic Bacillus sp. 1011 72 84 P05618
BACS3 Alkalophilic Bacillus sp. 38.2 71 83 P09121
THETU T. thermosulfirines EM1 66 80 P26827
PAEMA1 Paenibacillus maserans 65 80 P31835
PAEMA2 Paenibacillus maserans 64 76 P04830
BACST Bacillus stearothermophylus NO2 60 75 P31797
BREBE Brevibacillus brevis CD162 59 74 O30565
BACS2 Alkalophilic Bacillus sp. 1.1 58 75 P31746
BACOH Bacillus obhensis 58 74 P27036
KLEOX Klebsiella pneumonia M5a1 35 53 P08704

domains (A, B, C, D, and E) and four highly consensus regions
of carbohydrate-converting enzymes labelled I–IV and Ca2?
binding domain could be identified in the CGTase of ZY-08.
The conserved amino acids in the acceptor binding site, Lys-
93, Tyr-123, Asn-128, Phe-217, Asn-227, Leu-228, Tyr-229,
Asp-230, Phe-317, and Asp-405, reported as the main deter-
minants for cyclization reaction, were also identified (Ui-
tdehaag et al. 1999; van der Veen et al. 2001) (Figs. 1, 2b;
Table 2).
Purification of the CGTase
The recombinant CGTase was expressed in E. coli BL21
(DE3) using pCold I as the expression vector. The purified
enzyme showed a single band by SDS-PAGE (Fig. 3). The
molecular mass was estimated to be 74 kDa. Most of the
reported CGTases are monomeric, ranging in molecular
weight between 60 and 90 kDa (Saha and Zeikus 1990;
Galvin et al. 1994; Feederle et al. 1996; Kim et al. 1998;
Hashimoto et al. 2001). Only the CGTases produced by
alkalophilic B. sphaericus E-244 and Bacillus sp. 199 were
found to be homodimeric (Oguma et al. 1990; Yoshida
et al. 1991).
Enzymatic properties of the purified CGTase
The temperature and pH profile of the enzyme were esti-
mated by measurement of the enzyme activity at various
temperature and pH conditions. The CGTase showed sig-
nificant activity in a wide temperatures range 30–50 °C,
showing optimal temperature at 40 (Fig. 4a). Several
CGTases had optimum temperature in the range of
35–65 °C (Galvin et al. 1994; Kim et al. 1998;
Matzke et al. 2000). The effect of pH on the CGTase
activity and stability were also determined (Fig. 4B). The
enzyme exhibited an optimum activity at pH 7.4 and was
stable from pH 6.0 to 9.0 with a gradual loss of activity at
higher and lower pH values in the range of 6–9. Similar pH
stability (pH 6.0–9.0) was observed in Paenibacillus pabuli
US132 (Jemli et al. 2008). The enzyme from Paenibacillus
sp. A11 was stable in a wide pH range of 6.0–10.0
(Yoshida et al. 1991). Most of the reported CGTases
exhibited optimum pH ranging from 5.0 to 8.0 (Sohn et al.
1997). In order to reveal the effect of various metal ions
and reagents on CGTase activity, enzyme assays were
carried out in presence of the Ba2?, Ca2?, Cu2?, Hg2?,
Na?, Ni2?, Mg2?, and EDTA (Table 3). A significant lose
of the enzyme activity was established in the presence of
the 10 mM Mg2?, Ba2?, Cu2?, and Hg2? with residual
activities of 89 ± 2, 68, 25 ± 2, and 0 %, respectively.
Ni2? and Na2? ion had weakly effect on the enzyme
activity. EDTA, a metal chelating angent, had no effect on
the enzyme activity, suggesting that this CGTase is not a
metalloenzyme, which is similar to that reported for
CGTase from alkaliphilic Bacillus pseudalcaliphilus 20RF
(Atanasova et al. 2011). The enzyme activity was slightly
stimulated in the presence of Ca2? ion, which is similar to
that reported for CGTase from alkaliphilic Amphibacillus
sp. NPST-10 (Ibrahim et al. 2012).
The initial reaction rate of the CGTase was measured at
various concentrations of soluble starch and estimated
using Michaelis–Menten equation and double reciprocal
plot known as Lineweaver–Burk plot (Martins and Hatii-
Kaul 2002). The Km and Vmax values were estimated to be
0.48 mg/ml and 51.38 mg of b-cyclodextrin/ml/min,
respectively. Km values for several CGTases have been

123
870 World J Microbiol Biotechnol (2013) 29:865–873

Fig. 2 Phylogenetic tree

(a) and the sequence alignment
(b) of closers published CGTase
sequences. The tree is
implemented in the ClustalX
program. Asterisk indicate the
four conserved residues.
Identity is dark background and
similarity is light background

Table 2 Consensus regions I to IV of amylase in CGTases

reported. The CGTase from Bacillus sp. TS1-1 exhibited
Km values of 0.52 mg/ml (Rahman et al. 2006), Bacillus
circulans E 192 exhibited Km values of 5.7 mg/ml (Bovetto
et al. 1992), and Bacillus firmus exhibited Km values of
1.21 mg/ml (Gawande et al. 1999).
The CGTase from the wild type or recombinant con-
verted soluble starch to b-CD without difficulty. The wild
type easily converted soluble starch to b-CD, but did not
use king oyster mushroom and enoki mushroom. However
the recombinant converted soluble starch, king oyster
mushroom, and enoki mushroom to b-CD. The soluble
starch was easier used than king oyster mushroom and
enoki mushroom (Fig. 5). Currently in Korea, the king
oyster mushroom and enoki mushroom are cultivated in
bottle mushroom cultivating farm houses. The value of
those as an exportable item is very high due to their lesser
moisture content and expected to become a potential source
of agricultural revenue (Cho et al. 2001). Mushroom cul-
tivation has long been a bioconversion process and
simultaneously a useful approach to treating solid agri-
cultural and industrial wastes in both developed and
developing countries (Chang and Chiu 1992). The recom-
binant CGTase was more useful for the mushroom bio-
conversion and industrial application than the wild type.

123
World J Microbiol Biotechnol (2013) 29:865–873 871

Table 3 Effect of various metal ions on activity of CGTase

1 mM 10 mM

Ca2? 105 ± 1 113 ± 3

EDTA 103 102 ± 1
Ni2? 97 ± 2 105 ± 2
?
Na 98 ± 1 95 ± 2
Mg2? 95 ± 1 89 ± 2
Ba2? 87 ± 2 68
Cu2? 76 ± 2 25 ± 2
Hg2? ND ND

Fig. 3 Purification of CGTase from P. illinoisensis ZY-08. Lane 1

protein marker (EBM-1014, Elpis Biotech, Korea), lane 2 soluble
fraction, lane 3 purified CGTase

Fig. 5 Comparison of CD production of ZY-08 by using various

polysaccharides. a wild type b recombinant clone. Line control, dash
starch, dash-dot King oyster mushroom, dash-dot-dot Enoki
mushroom

Further we will work on revealing why the wild type

CGTase does not convert oyster mushroom powder or
enoki mushroom.

Fig. 4 Effect of temperature (a) and pH (b) on activity of CGTase. Acknowledgments This work was supported by Dong-A University
Optimum activity (filled circle), enzyme stability (open circle) Research Fund.

123
872
References
Atanasova N, Kitayska D, Bojadjieva I, Yankov D, Tonkova A (2011)
A novel cyclodextrin glucanotransferase from alkaliphilic
Bacillus pseudalcaliphilus 20RF: purification and properties.
Process Biochem 46:116–122
Axelrood PE, Chow ML, Arnold CS, Lu K, McDermott JM, Davies J
(2002) Cultivation-dependent characterization of bacterial diver-
sity from British Columbia forest soils subjected to disturbance.
Can J Microbiol 48:643–654
Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved
prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783–795
Berge O, Guinebretière MH, Achouak W, Normand P, Heulin T (2002)
Paenibacillus graminis sp. nov. and Paenibacillus odorifer sp.
nov., isolated from plant roots, soil and food. Int J Syst Evol
Microbiol 52:607–616
Bovetto LJ, Backer DP, Villette JR, Sicard PJ, Bouquelet SJL (1992)
Cyclomaltodextrin glucanotransferase from Bacillus circulans E
192. Biotechnol Appl Biochem 5:48–58
Chang ST, Chiu SW (1992) Mushroom production—an economic
measure in maintenance of food security. In: DaSilva EJ, Ratledge
C, Sasson A (eds) Microbial technology: economic and social
aspect. Cambridge University Press, Cambridge, pp 110–141
Cho SH, Lee SD, Ryu JS, Kim NG, Lee DS (2001) Changes in quality
of king oyster mushroom (Saesongi) during modified atmosphere
strage. Korean J Postharvest Sci Technol 8:367–373
Feederle R, Pajatsch M, Kremmer E, Bock A (1996) Metabolism of
cyclodextrins by Klebsiella oxytoca M5a1: purification and
characterization of a cytoplasmically located cyclodextrinase.
Arch Microbiol 165:206–212
Galvin NM, Kelly CT, Fogarty WM (1994) Purification and
properties of the cyclodextrinase of Bacillus sphaericus ATCC
7055. Appl Microbiol Biotechnol 42:46–50
Garbeva P, van Veen JA, van Elsas JD (2003) Predominant Bacillus
spp. in agricultural soil under different management regimes
detected via PCR-DGGE. Microbial Ecol 45:302–316
Gawande BN, Goel A, Patkar AY, Nene SN (1999) Purification and
properties of a novel raw starch degrading cyclomaltodextrin
glucanotransferase from Bacillus firmus. Appl Microbiol Bio-
technol 51:504–509
Goel A, Nene S (1995) Modifications in the phenolphthalein method
for spectrophotometric estimation of beta cyclodextrin. Starch
47:399–400
Hashimoto Y, Yamamoto T, Fujiwara S, Takagi M, Imanaka T (2001)
Extracellular synthesis, specific recognition, and intracellular
degradation of cyclomaltodextrins by the hyperthermophilic
archaeon Thermococcus sp. strain B1001. J Bacteriol 183:
5050–5057
Ibrahim AS, Al-Salamah AA, El-Tayeb MA, El-Badawi YB,
Antranikian G (2012) A novel cyclodextrin glycosyltransferase
from alkaliphilic Amphibacillus sp. NPST-1: purification and
properties. Int J Mol Sci 13:10505–10522
Janecek S (1997) a-Amylase family: molecular biology and evolu-
tion. Prog Biophys Mol Biol 67:67–97
Jemli S, Messaoud EB, Mabrouk SB, Bejar S (2008) The cyclodextrin
glycosyltransferase of Paenibacillus pabuli US132 strain:
molecular characterization and overproduction of the recombi-
nant enzyme. J Biomed Biotech 2008:1–9
Kaulpiboon J, Pongsawasdi P (2005) Purification and characterization
of cyclodextrinase from Paenibacillus sp. A11. Enzyme Microb
Technol 36:168–175
Kim MH, Sohn CB, Oh TK (1998) Cloning and sequencing of a
cyclodextrin glycosyltransferase gene from Brevibacillus brevis
CD162 and its expression in Escherichia coli. FEMS Microbiol
Lett 164:411–418
123
World J Microbiol Biotechnol (2013) 29:865–873
Laemmli UK (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227:680–685
Lee YS, Zhou Y, Park IH, Subosh Chandra M, Ahn SC, Choi YL
(2010) Isolation and purification of thermostable b-mannanase
from Paenibacillus illinoisensis ZY-08. J Korean Soc Appl Biol
Chem 53:1–7
Martins FR, Hatii-Kaul R (2002) A new cyclodextrin glycosyltransferase
from an alkaliphilic Bacillus agaradhaerens isolate: purification
and characterization. Enzyme Microb Tehcnol 30:116–124
Matzke J, Herrmann A, Schneider E, Bakker EP (2000) Gene cloning,
nucleotide sequence and biochemical properties of a cytoplasm
cyclomaltodextrinase (neopullulanase) from Alicyclobacillus
acidocaldarius, reclassification of a group of enzymes. FEMS
Microbiol Lett 183:55–61
Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) Identifi-
cation of prokaryotic and eukaryotic signal peptides and
prediction of their cleavage sites. Protein Eng 10:1–6
Oguma T, Kikuchi M, Mizusawa K (1990) Purification and some
properties of cyclodextrin hydrolysing enzyme from Bacillus
sphaericus E-244. Biochem Biophys Acta 1036:1–5
Qi Q, Zimmermann W (2005) Cyclodextrin glucanotransferase: from
gene to applications. Appl Microbiol Biotechnol 66:475–485
Rahman K, Illias RM, Hassan O, Mahmood NAN, Rashid NAA
(2006) Molecular cloning of a cyclodextrin glucanotransferase
gene from alkalophilic Bacillus sp. TS1-1 and characterization of
the recombinant enzyme. Emxyme Microbial Technol 39:74–84
Rojtinnakorn J, Kim P, Laloknam S, Tongsima A, Kamolsiripichaip-
orn S, Limpaseni T, Pongsawasdi P (2001) Immunoaffinity
purification and characterization of cyclodextrin glycosyltrans-
ferase from Bacillus circulans A11. Sci Asia 27:105–112
Ross N, Villemur R, Marcandella E, Deschênes L (2001) Assessment
of changes in biodiversity when a community of ultramicrobac-
teria isolated from groundwater is stimulated to form a biofilm.
Microbial Ecol 42:56–68
Saha BC, Zeikus JG (1990) Characterization of thermostable
cyclodextrinase from Clostridium thermohydrosulficum 39E.
Appl Environ Microbiol 56:2941–2943
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a
laboratory manual, 2nd edn. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor
Schmid G, Englbrecht A, Schmid D (1998) Cloning and nucleotide
sequence of a cyclodextrin glycosyltransferase gene from the
alkalophilic Bacillus sp. 1-1. In: Huber O, Szejtli J (eds)
Proceedings of the fourth international symposium on cyclo-
dextrins. Kluwer, Dordrecht, pp 77–81
Sohn CB, Kim SA, Park A, Kim MH, Moon SK, Jang SA, Lee MS
(1997) Purification and characterization of cyclodextrin glyco-
syltransferase from Bacillus firmus. J Korean Soc Food Sci Nutr
26:351–357
Thomsom JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving
the sensitivity of progressive multiple sequence alignment through
sequence weighting, positions-specific gap penalties and weight
matrix choice. Nucleic Acids Res 22:4673–4680
Uitdehaag JCM, Kalk KH, van der Veen BA, Dijkhuizen L, Dijkstra
BW (1999) The cyclization mechanism of cyclodextrin glyco-
syltransferase (CGTase) as revealed by a c-cyclodextrin-CGTase
complex at 1.8 Å resolution. J Biol Chem 274:34868–34876
Uitdehaag JCM, van Alebeek GWM, van der Veen BA, Dijkhuizen L,
Dijkstra BW (2000) Structures of maltohexaose and maltohep-
taose bound at the donor sites of cyclodextrin glycosyltransferase
give insight into the mechanisms of transglycosylation activity
and cyclodextrin size specificity. Biochemistry 39:7772–7780
van der Veen BA, Leemhuis H, Kralj S, Uitdehaag JCM, Dijkstra
BW, Kijkhuizen L (2001) Hydrophobic amino acid residues in
the acceptor binding site are main determinants for reaction
World J Microbiol Biotechnol (2013) 29:865–873 873

mechanism and specificity of cyclodextrin- glycosyltransferase. Yoshida A, Iwasaki Y, Akiba T, Horikoshi K (1991) Purification and
J Biol Chem 276:44557–44562 properties of cyclomaltodextrinase from alkalophilic Bacillus sp.
von der Weid I, Nobrega A, Paiva E, van Elsas JD, Seldin L (2000) J Ferment Bioeng 71:226–229
Diversity of Paenibacillus polymyxa strains isolated from the
rhizosphere of maize planted in cerrado soil. Res Microbiol
151:369–381

123