Isolation of Paenibacillus illinoisensis that produces cyclodextrin glucanotransferase resistant to organic solvents.

Article abstract of DOI:10.1271/bbb.67.334

A bacterium that secreted cyclodextrin glucanotransferase (CGTase) in a medium overlaid with n-hexane was isolated and identified as Paenibacillus illinoisensis strain ST-12 K. The CGTase of the strain was purified from the culture supernatant. The molecular mass was 70 kDa. The enzyme was stable at pH 6 to 10 and active at pH 5.0 to 8.0. The optimum temperature at pH 7.0 was 65 degrees C in the presence of 5 mM CaCl2. The enzyme produced mainly beta-cyclodextrin. The total yield of alpha-, beta-, and gamma- cyclodextrins was increased 1.4-fold by the addition of ethanol. In particular, the yield of beta-cyclodextrins in the presence of 10% (vol/vol) ethanol was 1.6-fold that without ethanol. The CGTase was stable and active in the presence of large amounts of various organic solvents.

Full text of DOI:10.1271/bbb.67.334

Biosci. Biotechnol. Biochem., 67 (2), 334–340, 2003
Isolation of Paenibacillus illinoisensis That Produces Cyclodextrin
Glucanotransferase Resistant to Organic Solvents
Noriyuki DOUKYU,† Hirokazu KUWAHARA, and Rikizo AONO
Department of Biological Information, Graduate School of Bioscience and Biotechnology,
Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8501, Japan
Received August 13, 2002; Accepted September 26, 2002
A bacterium that secreted cyclodextrin glucanotran-
sferase (CGTase) in a medium overlaid with -hexane
was isolated and identiˆed as Paenibacillus illinoisensis
strain ST-12 K. The CGTase of the strain was puriˆed
from the culture supernatant. The molecular mass was
70 kDa. The enzyme was stable at pH 6 to 10 and active
at pH 5.0 to 8.0. The optimum temperature at pH 7.0
insoluble ‰avonoids in the presence of organic sol-
vent.5) However, organic solvents often aŠect enzyme
stability and activity.6,7) Some times, highly polar
organic solvents inactivate CGTases.4,5) Enzymes
with stability and activity in the presence of organic
solvents would be useful for technological applica-
tions in which such solvents are used. Organic-
solvent-resistant microorganisms are useful for the
screening for extracellular enzymes in the presence of
organic solvents. An organic solvent-stable lipase has
been found in a cyclohexane-tolerant microorgan-
ism.8) We have reported that cyclohexane-tolerant
Burkholderia cepacia strain ST-200 produces
cholesterol oxidase that resists organic solvents,
detergents, and heat; the enzyme is secreted into the
culture supernatant.9,10) In this study, we isolated a
microorganism that produced CGTase in a medium
n
was 65
produced mainly
-, and - cyclodextrins was increased 1.4-fold by the
addition of ethanol. In particular, the yield of
cyclodextrins in the presence of 10 (vol vol) ethanol
9
C in the presence of 5 m
M
CaCl2. The enzyme
b-cyclodextrin. The total yield of
a-,
b
g
b
-
%
W
was 1.6-fold that without ethanol. The CGTase was
stable and active in the presence of large amounts of
various organic solvents.
Key words: cyclodextrin glucanotransferase; cyclo-
dextrin; organic solvent; organic-solvent-
tolerant; Paenibacillus illinoisensis
overlaid with n-hexane, and puriˆed the CGTase to
examine its stability and activity in the presence of
various organic solvents.
Cyclodextrin glucanotransferase (EC 2.4.1.19;
CGTase) produces cyclodextrins from starch via
intramolecular transglycosylation. Cyclodextrins are
cyclic ring structures consisting of six, seven, or eight
Materials and Methods
Media. An organism that produced CGTase was
glucose residues joined by
a
-1-4 linkages (
a
-,
b
-, and
screened for with a screening medium consisting of
g
-cyclodextrin, respectively). Cyclodextrins have
1z potato starch (Wako Pure Chemical Industries,
been widely used in the ˆelds of food, pharmaceuti-
cals, agricultural chemistry, cosmetics, and perfumes
because of their ability to form inclusion complexes
with a wide variety of chemicals by partially encap-
sulating them into their cavities.1) Several attempts
have been made to improve the product selectivity of
CGTase by the addition of organic solvents.2,3)
CGTase from Bacillus circulans strain 251 produces
Osaka, Japan), 1
tories, Detroit, MI), 0.5
(Difco), 1 NaCl, and 10 m
z
Bacto Tryptone (Difco Labora-
z
Bacto Yeast Extract
MgSO4. When neces-
z
M
sary, the medium was solidiˆed with 1.5
agar. LBMg medium, consisting of 1 Bacto Tryp-
tone (Difco), 0.5 Bacto Yeast Extract, 1 NaCl,
MgSO4, was used at times to culture iso-
z
(wt vol)
W
z
z
z
and 10 m
lates.
M
a b-, and g-cyclodextrins in the ratio of 9:82:9 with
-,
the addition of
t
-butanol, although the product ratio
Isolation of an organism that produced CGTase on
a medium overlaid with -hexane. Soil samples were
is 15:65:20 without the solvent.3) The addition of
organic solvents can increase the overall yield of
cyclodextrin production.3,4) A CGTase from Bacillus
sp. strain BE101 produced twice as much cyclodex-
trin after the addition of ethanol.4) In addition,
CGTase is used for the transglycosylation of water-
n
gathered from several locations in the Kanto area in
Japan. A small amount of soil sample was suspended
in sterile 0.8
was spread on a screening-medium agar plate. The
surface of the agar was overlaid with -hexane to a
z
NaCl, and a portion of the suspension
n
†
To whom correspondence should be addressed. Fax: +81-45-924-5819; E-mail: ndoukyu
＠
bio.titech.ac.jp
Abbreviations: CGTase, cyclodextrin glucanotransferase; DNS, 3,5-dinitrosalicylic acid; LB medium, Luria-Bertani medium

Screening for Organic-solvent-tolerant Producer of CGTase
335
thickness of 3 to 5 mm. The plates were warmed at
30 C for 4 days in a tightly closed vessel. After incu-
bation, the -hexane was removed and 0.02 I2 in
0.2 KI solution (about 10 ml) was poured onto the
Puriˆcation of CGTase. Strain ST-12 K was grown
at 30 C for 14 h in LBMg medium containing 0.2
soluble starch, and the culture was centrifuged (6,000
9
9
z
n
z
×
z
g
, 15 min, 4
precipitated with (NH4)2SO4 (70
overnight. The precipitate was recovered by centrifu-
9
C). Proteins in the supernatant were
agar surface. Microorganisms that formed halos
around their colonies were isolated by repeated sin-
gle-colony isolation on the screening-medium agar.
z
saturation) at 4
9
C
×
g, 30 min, 49C) and dissolved in
gation (15,000
10 m Tris-HCl (pH 8.0). This solution was dialyzed
against the same buŠer at 4 C and put on a column
The microorganisms were grown at 30
the screening medium, and then the culture was
centrifuged (6,000 , 15 min, 4 C). The starch-
9
C for 48 h in
M
9
×
g
(2.5 by 10 cm) of DEAE-cellulose DE53 (Whatman,
Maidstone, England) equilibrated with the Tris-HCl
buŠer. The column was washed with 100 ml of the
9
hydrolyzing activity in the supernatants was meas-
ured by the two methods described below. Microor-
ganisms that hydrolyzed starch but did not generate
reducing sugars were selected as CGTase producers.
Cyclodextrin production by the culture supernatants
of the CGTase producers were examined by TLC as
described below. Microorganisms that produced
cyclodextrin from starch were selected. The enzyme
stability in the presence of an organic solvent was ex-
amined to select an organic-solvent-tolerant enzyme.
Seventy-ˆve microliters of chloroform or ethanol was
Tris buŠer at the ‰ow rate of 0.5 ml min and then
W
eluted with a linear gradient of NaCl concentrations
of 0 to 50 m
M
in 300 ml of the Tris buŠer at the ‰ow
W
rate of 0.5 ml min. The fractions with starch-
hydrolyzing activity were pooled. After the addition
of (NH4)2SO4 to 30
z
saturation, the solution was
put on a column (2.5 by 20 cm) of butyl-Toyopearl
650M (Tosoh, Tokyo, Japan) equilibrated with 30
saturated (NH4)2SO4-10 m Tris-HCl (pH 8.0). The
column was washed with 150 ml of the Tris buŠer
containing (NH4)2SO4 (30 saturation) and then
eluted with a decreasing linear gradient of (NH4)2SO4
concentrations of 30 saturation to 0 in 300 ml of
the Tris buŠer at the ‰ow rate of 0.5 ml min. The en-
z
M
added to 300
ture supernatant and 300
phate, pH 7.0). The mixture was shaken at 30
m
l of CGTase solution (30
l of 100 m sodium phos-
C for
ml of the cul-
m
M
z
9
12 h. The solution was diluted 4-fold with the sodium
phosphate buŠer and the activity was measured by
the iodine method described below. Thermal stability
of the enzyme also was examined. The residual activi-
ty at pH 7.0 was measured after incubation for 1 h at
z
z
W
zyme solution was dialyzed twice against the 10 m
Tris-HCl buŠer (pH 8.0) at 4
being used.
M
9 9
C and kept at 4 C until
40
9
C or 50
9
C by the iodine method.
Measurement of protein concentration. Protein
concentration was measured by the method of
Bradford12) with bovine serum albumin as the
standard.
Assays of starch-degrading activity. In one test,
the starch-hydrolyzing activity was assayed with
soluble starch as the substrate by measurement of
changes in iodine staining. A 20-
ml portion of enzyme
solution was added to 300 l of a reaction mixture
m
Zymograms. SDS-PAGE was done on a 10
z
consisting of 0.5
z
soluble starch (Wako Pure Chem-
(wt vol) polyacrylamide gel by the method of Laem-
W
icals) and 100 m
M
sodium phosphate buŠer (pH 7.0).
mli13) with slight modiˆcations. Soluble starch was
added to the polyacrylamide gel to a ˆnal concentra-
This mixture was incubated at 30
reaction was stopped by the addition of 0.5 ml of 5
acetic acid. Then, 0.5 ml of 0.02 I2 in 0.2 KI
9C for 10 min. The
M
tion of 1
gel was washed with the Tris-HCl buŠer containing
25 (vol vol) isopropanol. Then the gel was washed
with 100 m
in the same buŠer for 12 h at 30
starch was hydrolyzed was stained by soaking of the
gel in a solution of 0.2 I2-2 (wt vol) KI.
z
(wt vol). After the electrophoresis, the
W
z
z
solution was added to this mixture, and the absor-
bance of the ˆnal mixture was measured at 660 nm.
One unit of enzyme activity was deˆned as the
amount of enzyme that reduced the intensity of A660
z
W
M
Tris-HCl buŠer (pH 8.0) and incubated
C. A zone at which
9
of amylose-iodine complexes by 10
der the conditions described.
z
per minute un-
z
z
W
In the other assay of starch-hydrolyzing activity,
changes in the concentration of reducing sugars
during incubation with starch was measured by the
3,5-dinitrosalicylic acid (DNS) method.11) The reac-
tion mixture used was the same as above. This mix-
TLC. After the enzymatic reaction described
above with 0.5 soluble starch, 1 l of the reaction
z
m
mixture was spotted onto a 0.2-mm-thick silica gel 60
plate (Merck, Darmstadt, Germany) and the sample
was developed with a solvent mixture of acetic acid-
ethyl acetate-water (2:2:1, by vol). Glucose, maltose,
maltotriose, maltotetraose, maltopentaose, malto-
hexaose, and maltoheptaose were used as the stan-
dards for starch-hydrolyzed products. Cyclodextrins
also were standards. The oligosaccharides were de-
ture was incubated at 30
enzyme activity was deˆned as the amount of enzyme
forming reducing sugars corresponding to 1 mol of
9
C for 15 min. One unit of
m
glucose per minute in the 3,5-dinitrosalicylic acid
reaction.
tected by spraying of 1
z z
(wt vol) orcinol and 50
W

336
N. DOUKYU et al.
(vol vol) sulfuric acid on the TLC plate, which was
The 1480 bp sequences, corresponding to 96
complete sequence, was similar to the 16S rDNA
sequences of Paenibacillus species as follows: 99
P. illinoisensis (DDBJ EMBL GenBank accession
z of the
W
then heated at 120
9C for 10 min. Cyclodextrins were
detected by being sprayed with 1
z
I2 in methanol.
z
,
W
W
EŠects of pH on enzyme activity and stability. The
enzyme activity was assayed at 30 C at various pHs
with 100 m citric acid-NaH2PO4 (pH 3.0–5.5),
no. D85397); 96
z
, P. amylolyticus (no. D85396);
9
and 94z P. borealis (no. AJ011322). The microbio-
M
logical and biochemical characteristics of strain
CH3COOH–CH3COONa (pH 4.0–5.5), KH2PO4–
Na2HPO4 (pH 5.5–7.5), Tris-HCl (pH 7.5–9.0),
Na2CO3–NaHCO3 (pH 9.0–11.0), and NaCl–NaOH
(pH 11.0–13.0). Enzyme stability was measured at
ST-12 K were examined by methods reported for the
15)
classiˆcation of species of Paenibacillus
.
Cells of
strain ST-12 K were rods measuring 0.8 to 1.0 by 3.0
to 4.0 m, they were Gram positive, motile, and
m
30
9
listed.
C after 1 h of incubation at the various pHs just
spore-forming. The colonies were ‰at, smooth, circu-
lar, entire, and yellowish gray. No soluble pigment
was produced on a nutrient agar. Strain ST-12 K had
positive catalase test results but negative oxidase test
results. Gelatin and starch were hydrolyzed and
casein was weakly hydrolyzed. Acetate was used, but
Measurement of cyclodextrin. A sample was ana-
lyzed with an NH2-1251-N (4.6 by 250 mm) column
(Senshu Science Co., Tokyo) attached to an HPLC
apparatus. The column was eluted with acetonitrile-
citrate, lactate, succinate,
were not. Strain ST-12 K grew at 40
the presence of 2 NaCl. Growth was inhibited at
C or in the presence of 5 NaCl. Acid was
produced from -arabinose, glycerol, -mannose,
and ra‹nose, but not from lactose or -rhamnose.
These microbiological characteristics were identical
to those of P. illinoisensis
Strain ST-12 K grew on LBMg medium overlaid
with a 10 volume of dodecane (log ow 7.0), decane
(log Pow 6.0), nonane (log Pow 5.5), -octane (log Pow
4.9), or diphenyl ether (log Pow 4.2), but not in that
overlaid with cyclohexane (log Pow 3.4), or -xylene
L
-malate, and
L
-glutamate
water (6:4, vol vol) at the ‰ow rate of 0.5 ml min.
9
C, pH 5.7, or in
W
W
The elution was monitored by an refractive index de-
tector (RID-300, Japan Spectroscopic Co., Ltd.).
z
4
9
z
L
D
Commercial CGTase preparations. CGTase from
Bacillus macerans was the product of Amano En-
zyme, Inc. (Nagoya, Japan).
L
.
z
P
n
Results
Isolation of
a microorganism that produced
CGTase tolerant of organic solvents
p
First, we screened for microorganisms that pro-
duced starch-hydrolyzing enzymes on the screening-
medium agar overlaid with cyclohexane. Although
we examined about 500 soil samples, CGTase-
producing microorganisms were not found. Next, we
screened for the microorganisms on a screening-
(log Pow 3.1). The growth was weak on the LBMg
medium in the presence of n-hexane (log Pow 3.9).
Puriˆcation of CGTase
The activity in the presence of dodecane, decane,
and nonane was similar to that without any solvent.
medium plate overlaid with
n
-hexane, which is less
The activity in the presence of
n-octane, diphenyl
toxic than cyclohexane. From about 400 soil samples,
452 strains formed halos around their colonies. Of
the strains, 38 strains had strong starch-hydrolyzing
activity in the culture supernatant by iodine method.
Of the 38 strains, the culture supernatants of 12
strains did not contain a detectable starch-hydrolytic
activity generating reducing sugars by the DNS
method. These 12 strains were selected as CGTase
producers. Cyclodextrins were detected as the major
products yielded by the culture supernatants of the 12
strains. Of the 12 selected strains, strain ST-12 K pro-
duced the CGTase with the highest thermal stability.
This enzyme was more stable in the presence of chlo-
roform and ethanol than the enzymes of the other 11
strains. Consequently, strain ST-12 K was selected as
a producer of a CGTase tolerant of organic solvents.
ether, or -hexane was one tenth that without a sol-
n
vent. Therefore, the CGTase was puriˆed from the
culture supernatant of ST-12 K grown on the medium
without any solvent. Table 1 summarizes the puriˆca-
tion steps used. The starch-hydrolyzing activity
detected by the iodine method was eluted from the
DE53 column with NaCl at 15 m
column of butyl-Toyopearl 650M with (NH4)2SO4 at
10 saturation. In the end, the CGTase was puriˆed
M
and from the
z
4.5-fold from the culture supernatant. The puriˆed
CGTase had a speciˆc activity of 1200 U mg of pro-
W
tein. The puriˆed preparation gave a single band by
SDS-PAGE (Fig. 1). The molecular mass was esti-
mated to be 70 kDa. A 70-kDa protein from the cul-
ture supernatant was seen also on the zymogram
when ST-12 K grew on a medium with dodecane, de-
cane, and nonane (not shown).
Characterization and identiˆcation of strain
ST-12 K
EŠects of pH and temperature
The CGTase was active at pH 5.0 to 9.0 and most
active at pH 5.0 to 8.0. The enzyme was stable at
We analyzed a partial sequence of the 16S rDNA
gene sequence of ST-12 K as described by Lane.14)

Screening for Organic-solvent-tolerant Producer of CGTase
337
Table 1. Puriˆcation of CGTase from Strain ST-12 K
Volume
(ml)
Total protein
(mg)
Total activity
(U)
Speciˆc activity
Puriˆcation
(fold)
Yield
Step
(U mg)
(z)
W
Culture supernatant
(NH4)2SO4 precipitation
DEAE-cellulose chromatography
Butyl-Toyopearl chromatography
3,520
64
79.5
54.1
9.15
4.75
21,000
16,900
9,760
264
312
1,070
1,200
1
100
80
46
1.2
4.1
4.5
95
72
5,720
27
from pH 6.0 to 10.0 after incubation for 1 h at 30
9
C.
The optimum temperature at pH 7.0 was at 60
9
C
(Fig. 2). The enzyme was stable at temperatures from
4 to 45 C. In the presence of 5 m CaCl2, the enzyme
had optimal activity at pH 7.0 at 65 C and still had
85 and 40 of its activity after incubation for
30 min at 60 C and 70 C, respectively. However, the
enzyme had lost almost all activity after 30 min at
80 C.
9
M
9
z
z
9
9
9
Formation of cyclodextrins from starch
Mostly, -cyclodextrin was produced during the
ˆrst hour (Fig. 3). Some - and -cyclodextrins
formed up to 3.26 mg ml (33 of the starting
amount of starch) was produced in 24 h, and the
ratio of -, -, and -cyclodextrins was 24:69:7. By
the addition of 10 (vol vol) ethanol, up to
4.52 mg ml cyclodextrins were produced (45
b
a
g
z
W
a
b
g
z
W
z
of the
starting amount of starch) in 24 h, and the ratio of
-, - and -cyclodextrins was 12:82:6. The eŠects of
Fig. 1. SDS-PAGE of Fractions Obtained during Puriˆcation of
the CGTase.
W
Samples containing 1 U of CGTase were electrophoresed on
a
b
g
an SDS-10
The gel was stained with Coomassie brilliant blue R-250 (CBB)
(A) or 0.2 I2-2 (wt vol) KI solution (B). Lane 1, proteins
precipitated with 70
z (wt vol) polyacrylamide-1z soluble starch gel.
W
ethanol concentration on the yield and product selec-
tivity of cyclodextrins are shown in Table 2. The
z
z
W
highest conversion yield was 46
ethanol was added to 20 . This yield was close to
that with 10 ethanol. The proportion of -cyclo-
z, reached when
z
saturated (NH4)2SO4 from the culture su-
pernatant; lane 2, proteins in a solution gathered from fractions
with activity from the DEAE-cellulose DE53 column; lane 3,
proteins in a solution gathered from fractions with activity from
the butyl-Toyopearl 650M column. M, Molecular size markers
(kilodaltons).
z
z
b
dextrin produced was highest in the presence of 10
z
ethanol.
Fig. 2. EŠects of Temperature on CGTase Activity and Stability.
(A) Enzyme activity was assayed by the iodine method at pH 7.0 at the temperatures indicated in the ˆgure in the absence of CaCl2
) or in the presence of 5 m CaCl2 ( ). (B) Enzyme dissolved in 100 m phosphate buŠer (pH 7.0) was incubated for 30 min at the
temperatures indicated in the absence of CaCl2 ( ) or in the presence of 5 m CaCl2 ( ), and the relative activity was assayed by the
iodine method at 30 C.
(

M

M

M

9

338
N. DOUKYU et al.
Fig. 3. Formation of a-, b-, and g-Cyclodextrins from Starch.
Thirty microliters of the enzyme solution (20 U ml) was added to the reaction mixture (300 ml) consisting of 1
z
soluble starch and
W
100 m
at 30
M
sodium phosphate buŠer (pH 7.0) (A) or to a reaction mixture containing 10
z (vol vol) ethanol (B). The mixture was incubated
W


b
9
C. The amount of cyclodextrins is shown as the percentage of soluble starch converted. ( ), a-Cyclodextrin; ( ), -cyclodextrin;
$

), g-cyclodextrin; ( ), total cyclodextrin.
(
Table 2. EŠects of Ethanol on Yield and Product Selectivity of
Table 4. EŠects of Organic Solvents on Activity of CGTase
Cyclodextrinsa)
Relative activitya) of
CGTase from:
Product ratio (
z)
Solvent
log Pow
b)
Ethanol (
z
)
Yield (z)
ST-12 K
100b)
B. macerans
a
b
g
None
Methanol
Ethanol
2-Propanol
Chloroform
Benzene
Toluene
—
„0.76
„0.24
0.28
1.9
2.1
2.6
3.1
3.4
3.9
100b)
0
5
10
20
50
33
30
45
46
26
24
16
12
12
5
69
77
82
77
72
7
7
6
11
23
78
104
80
91
106
104
±
±
±
±
±
±
4
9
8
9
6
6
18
20
15
65
88
88
88
98
94
±
±
±
±
±
±
±
±
±
3
4
5
3
5
8
6
2
4
a
Thirty microliters of the enzyme solution (20 U ml) was added to the
W
reaction mixture (300
sodium phosphate buŠer (pH 7.0) in 0 to 50
Yields of cyclodextrins are shown as the percentage of converted soluble
starch after 24 h of incubation at 30 C.
m
l) containing of 1
z
soluble starch and 100 m
(vol vol) ethanol.
M
±
93 11
p
-Xylene
Cyclohexane
-Hexane
z
W
±
5
9
102
94
b
±
n
9
a)
An organic solvent (75
(0.2 U ml in 100 m sodium phosphate buŠer, pH 7.0) containing
soluble starch. The mixture was shaken at 30 C for 2 to 10 min.
The activity was measured by the iodine method.
The activity is relative to that in a control CGTase solution not exposed
to any organic solvent.
ml) was added to 300 ml of CGTase solution
M
W
0.5
z
9
Table 3. EŠects of Organic Solvents on Stability of CGTase
b)
Relative residual activitya)
of CGTase from:
Solvent
log Pow
Means and SD for three independent experiments are shown.
ST-12 K
100b)
B. macerans
None
Methanol
Ethanol
2-Propanol
Chloroform
Benzene
Toluene
—
„0.76
„0.24
0.28
1.9
2.1
2.6
3.1
3.4
3.9
100b)
EŠects of organic solvents on enzyme activity and
stability
102
84
82
47
88
102
92
88
±
±
±
±
±
±
±
±
±
9
2
6
2
11
6
2
8
5
98
99
96
75
92
100
100
91
±
±
±
±
±
±
±
±
±
1
4
6
2
2
3
6
3
1
Table 3 shows the stability of the puriˆed CGTase
of ST-12 K in the presence of various solvents and
also the stability of commercially available CGTase
from Bacillus macerans. The enzyme from B. mace-
rans has been extensively studied.16) Highly polar
p
-Xylene
Cyclohexane
-Hexane
hydrophobic solvents with log Pow values (less than 2)
n
84
90
inactivate most enzymes through structural denatura-
tion.17) Both enzymes were moderately stable in
presence of methanol, ethanol, 2-propanol, benzene,
a)
An organic solvent (75
(0.2 U ml in 100 m sodium phosphate buŠer, pH 7.0). The mixture
C for 12 h. The solution was diluted 4-fold with the
sodium phosphate buŠer and the activity was measured by the iodine
method.
The activity is relative to that in a control CGTase solution not exposed
to any organic solvent.
ml) was added to 300 ml of CGTase solution
M
W
was shaken at 30
9
toluene,
enzymes were partially inactivated by the addition of
chloroform after incubation for 12 h at 30 C.
p-xylene, cyclohexane, and n-hexane. The
b)
9
Means and SD for three independent experiments are shown.
Table 4 shows activity in the presence of organic
solvents. The enzyme from B. macerans was
inactivated by methanol, ethanol, 2-propanol, chlo-
roform. The enzyme of ST-12 K was active in the

Screening for Organic-solvent-tolerant Producer of CGTase
339
presence of all organic solvents tested in this assay
with a 10-min reaction.
removal of cyclodextrins by precipitation from the
reaction system shifts the equilibrium in favor of
cyclodextrin production. However, precipitation of
cyclodextrins by the addition of ethanol was not
observed in our study. The eŠects of solvent addi-
tion, such as increased yield and improved product
selectivity, were related to reduced competition from
the intermolecular transglycosylation reaction that
causes degradation of cyclodextrin products.3) Final
concentrations of cyclodextrin products were higher
when degradation of cyclodextrin by CGTase was in-
hibited by the addition of organic solvents. Inhibi-
tion of cyclodextrin degradation by CGTase was
aŠected by the solvent added and the kind of
cyclodextrin. Therefore, product selectivity seemed
to depend on the solvent and the cyclodextrin.
ST-12 K CGTase was stable and active in the
presence of various organic solvents. Although
CGTase from Bacillus macerans was stable in the
presence of methanol, ethanol, 2-propanol, or chlo-
roform, the enzyme was not active in these solvents.
The inactivation of Bacillus macerans CGTase by the
addition of the organic solvents seemed to be reversi-
ble. The stability of CGTase from Bacillus sp. strain
BE101 decreases in the presence of ethanol.4) The
residual activity of CGTase from Bacillus sp. strain
Discussion
Depending on the most abundant kind of
cyclodextrin that CGTase produces, the enzyme is
sometimes classiˆed as
a-, b-, or g-CGTase. By this
classiˆcation, the enzyme from strain ST-12 K is a
b
b
-CGTase. Of the three kinds of cyclodextrins,
-cyclodextrin is of the most practical use because its
inclusion complexes are easily prepared and stable;
the size of the apolar cavity is optimum for many
molecules such as drugs and preservatives.1) In addi-
tion, b-cyclodextrin is easily separated from reaction
mixtures because of its low solubility in water. In
view of these points, we wanted to isolate microor-
ganisms producing enzymes that synthesize
cyclodextrin. The CGTases from Bacillus mega-
terium Bacillus circulans, a Bacillus sp., Bacillus
harophilus Thermoanaerobacterium thermosulfu-
b-
,
,
rigenes, and Bacillus ohbensis produce mainly
b-
cyclodextrin.16) On the basis of its 16S rDNA se-
quence, and its morphological and physiological
characteristics, strain ST-12 K was classiˆed as P.
illinoisensis. No CGTase from P. illinoisensis was
reported.
BE101 is about 40
presence of 10 (vol vol) ethanol. Under the same
conditions, ST-12 K CGTase maintained 99
z at 459C after 12 h in the
There have been several studies on improvement
of product selectivity or increasing the yield of
cyclodextrin production by CGTase in the presence
of organic solvents.2–4) However, there is no report
about screening for a microbe that produces organic-
solvent-tolerant CGTase. Cyclodextrins are mostly
used in food, cosmetics, and pharmaceuticals,
ethanol is a possible solvent to use in the production
of cyclodextrins. Addition of ethanol also can im-
prove the overall process economics by reducing bac-
terial contamination in enzymatic processes. CGTase
z
W
z
of its
activity in the presence of ethanol. Our CGTase was
stable and active in the presence of various organic
solvents. The CGTase should be useful for techno-
logical applications with organic solvents. In particu-
lar, this enzyme can be used to improve the overall
process economics of the enzymatic production of
cyclodextrins in the presence of ethanol.
b-
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