J. Biol. Macromol., 4(1) 3-11 (2004)

Review
Promising α-Amylases for Modern Detergents
Susumu Ito and Koki Horikoshi
Japan Marine Science and Technology Center (JAMSTEC)
2-15 Natsushima, Yokosuka 237-0061, Japan
Received February 29, 2004; Accepted March 1, 2004
Keywords：
α-amylase, Bacillus, detergent, site-directed mutagenesis
Introduction
Amylose is a linear polysaccharide that isformed by α-1,4-linked α-D-glucose units.
Amylopectin is also a polysaccharide of
α-D-glucose monomers, but it contains
α-1,6-branching points. α-Amylases(1,4-α-D-glucan glucanohydrolase; EC 3.2.1.1)
catalyze the hydrolysis of the α-1,4-glucosidic
linkages of starch. It randomly cleaves theinternal bonds in amylose and amylopectin.
Various starch-hydrolyzing enzymes catalyze asimilar reaction (1). Cyclodextrin
glucanotransferase (2), maltogenic α-amylases(3,4), and maltotetraose-forming amylases (5).
Pullulanase (6,7) and isoamylase (8,9) hydrolyze
the α-1,6-glucosidic linkages. Neopullualanase(10,11) and amylopullulanase (12) hydrolyze
both the α-1,4-glucosidic linkages and the
α-1,6-glucosidic linkages of starch. Suchstarch-hydrolyzing enzymes should be named
the α-amylase family, because they are similar to
α-amylases (13). According to the sequence andactive site topology, α-amylase is grouped intofamily 13 (14). Well-known bacterial α-amylases
are liquefying ones from Bacillus species, suchas Bacillus amyloliquefaciens (BAA),

Geobacillus stearothermophilus (BSA), andBacillus licheniformis (BLA) (15-19). Their
molecular masses and optimal pH for activity arearound 55 kDa and. between 5 and 7.5,
respectively. BLA is thermostable and its optimal
temperature is around 80°C, depending upon thepresence of Ca2+ ions (20).
The tertiary structures of α-amylases havebeen reported for TAKA-amylase A (21), porcine
pancreas (22), Aspergillus nigar (23), bareley(24), and Pseudomonas stutzeri (25). Recently,
Matius et al. (26,27) reported on the crystal
structure of the industrially important BLA.
Similar to the other α-amylases, BLA has athree-domain structure with the domains A, B,
and C. The N-terminal domain A forms a
so-called (β/α)8-barrel (TIM-barrel) domain.
Domain B is located in a looped out part of thepolypeptide chain between the third β-strand andthe third helix of TIM-barrel. It forms a largepart of the substrate-binding cleft and ispresumed to be important for the substratespecificity differences observed between
α-amylases. The C-terminal domain C forms anantiparallel β-sheet. Three Ca2+ and one Na+ ionswere found in BLA, a linear triad CaI-Na-CaII4

α―Amylases for Detergents

arrangement, is located at the interface between
domain A and B. Another Ca2+ (CaIII) bridges
between domains A and C.
α-Amylases are important, especially in the
starch and food industries. The demand forliquefying α-amylase for use in laundry andautomatic dishwashing detergents has been
growing for several years (28). Detergent
enzymes have to function in washing machinesor dishwashers under conditions that are very
unfavorable for the stability of the enzyme. The
pH is high alkaline in washing conditions. The
high temperature (55~60°C) in a dishwasher
requires thermostable enzymes. Enzymes preferto be resistant to various detergent ingredients,
such as surfactants, chelators, and oxidants(bleach). However, most of bacterial liquefying
α-amylases, such as BAA and BSA, including
BLA, has optimal pH of between 5 and 7.5.
Therefore, they are not practically suitable foruse in laundry and dishwashing detergents withhigh alkalinity. We have found an alkaline,
liquefying α-amylase, designated AmyK, fromalkaliphilic Bacillus sp. strain KSM-1378 (29).
Here, we describe the enzymatic properties,
improvement in thermostability and oxidative
stabilization of AmyK.
Enzymatic properties of AmyKThe homogeneous preparation of AmyK had
the specific activity of 5,000 units (U)/mg whenmeasured at 50°C and at pH 8.5 in 50 mM
Tris-HCl buffer. The molecular mass of thepurified enzyme was approximately 53 kDa by
SDS-PAGE. The isoelectric point was around pH
9 (29). When AmyK was preincubated with 10
mM EDTA or EGTA, the activity decreased to
10% or 9% of the initial activity, respectively.
AmyK was resistant to incubation at 40°C for 1
h with various surfactants, such as SDS,
polyoxyethylene alkyl ether, sodium

Fig. 1. Effect of pH on the activity and stabilityof AmyK. A. The pH-activity curves ofpurified AmyK and BLA (each at 0.2 U/ml) are
shown by solid and dotted lines, respectively.
The buffers used (50 mM each) were as
follows: glycine-HCl, pH 3.0-3.5 (▲); acetate,
pH 4.0-6.0 (■); Tris-HCl, pH 6.5-8.5 (●);
glycine-NaOH, pH 9.0-10.5 (□);
glycine-NaCl-NaOH, pH 8.0-10.5 (○);
carbonate, pH 10.0-11.0 (△). The values areshown as percentages of the maximum specificactivity of AmyK observed at pH 8.0-8.5,
which is taken as 100%. B. To assess the pH
stability of AmyK, the enzyme (2.0 U/ml) was
preincubated at the indicated pH in 10 mM
Britton-Robinson buffer and at 40°C for 30
min, and then samples (0.1 ml) were used forthe measurements of the residual activity under
the standard conditions of enzymatic assay. The
values are shown as percentages of the originalactivity, which is taken as 100%.

S. Ito and K. Horikoshi

α-sulfonated fatty acid ester, linear alkylbenzene
sulfonate, and alkyl glucoside (each added at
0.1%, w/v). Moreover, the enzyme is unique inthat at the early stage of hydrolysis of soluble
starch, the enzyme produced G5 to G9 inquantities much larger than G2 and G3. G1 andG4 formed at this stage were trace. On further
incubation, G3 and G5 increased while G6 to G8
decreased. These results indicate that AmyK isclassified as liquefying α-amylase (20, 30).
As shown in Fig. 1A, the maximum activity
was observed around pH 8.0-8.5 when measured
in various buffers at 50 mM with soluble starchas substrate. Between pH 5 and 10, the specificactivity was 2- to 5-fold greater than that of BLA.
To assess the pH stability of AmyK, the enzyme
was preincubated at 40°C for 30 min in 10 mM
Britton-Robinson buffer and assayed at 50°C in
50 mM Tris-HCl buffer at pH 8.5. The enzyme
was stable over a range between pH 6 and pH 10(Fig. 1B).
The optimal temperature for activity wasaround at 55°C at pH 8.5 in 50 mM Tris-HCl
buffer (Fig. 2A), while that of BLA was aroundat 80°C. At 80°C, the specific activities of BLA
and AmyK were almost equal, and they wereapproximately 25% of the maximum activity of
AmyK observed at 55°C. Time course of thethermal inactivation of AmyK was followed atvarious temperatures and at pH 8.5 in 20 mM
Tris-HCl buffer (Fig. 2B). The enzyme retained
its full activity after 60 min of incubation at
45°C, but only 32% and 3% of original activity
remained after 60 min of incubation at 50°C and
60°C, respectively. However, in the presence of
0.1 mM CaCl2, nearly 100% and 65% of theoriginal activity remained at 50°C and 60°C,
respectively. The enzymatic activity wasabolished after heating at 70°C for 60 min evenin the presence of 0.1 mM Ca2+ ions. In contrast,

5

BLA was quite stable to incubation at 70°C at
least up to 60 min under our test conditions.
Deduced amino acid sequence of AmyKThe entire gene for AmyK was cloned andsequenced (29). It contained an open reading
frame of 1545-bp encoding 515 amino acids

Fig. 2. Effect of temperature on the activity andstability of AmyK. A. The temperature-activity
curves of purified AmyK (●) and BLA (○) (each at
0.18 U/ml) are shown. The reactions were done at
the indicated temperatures for 10 min and at pH 8.5
in 50 mM Tris-HCl buffer. The values are shown aspercentages of the maximum activity of AmyK
observed at 55°C, which is taken as 100%. B. To
asses the thermostability of AmyK, the enzyme (1.8
U/ml) was heated at the indicated temperatures in thepresence at 50°C (□), 60°C (△), and 70°C (○)), or in
the absence at 50°C (■) and 60°C (▲) of 0.1 mM
Ca2+ ions in 50 mM Tris-HCl buffer (pH 8.5).
Samples (0.1 ml) were used for the determination of
the residual activity under the standard conditions ofenzyme assay.

6

α―Amylases for Detergentsincluding a putative signal peptide of 31 aminoacids. The molecular mass of the mature enzyme
was 55,391 Da, a value close to the 53 kDa
determined by SDS-PAGE of the purified AmyKfrom the culture broth of Bacillus sp. strain
KSM-1378. When suitably aligned, the deduced
amino sequence of AmyK exhibited only 66.7,
68.6, and 68.9% identity to those of BAA (31),
BSA (32), and BLA (33), respectively, but almost
no homology to those of saccharifying
α-amylases reported to date. Essentially, AmyKhas the (β/α)8 barrel structure. Four conserved
regions, which are necessary for the catalytic
activity of α-amylases (35) were conserved in
AmyK as Asp102 to His107 (region I), Gly232
to His240 (region II), Glu266 to Lys269 (regionIII), and Phe328 to Asp333 (regions IV). They
form the active center, the substrate binding sites,
and the Ca2+ binding sites.
For the evaluation of properties of AmyK, weused an excretion vector pHSP64 to
hype-rproduce it with Bacillus subtilis as thehost (36,37). The gene for AmyK was amplified
by PCR and cloned into SalI-SmaI site of
pHSP64. The transformed B. subtilis
hyper-produced the AmyK activity,
corresponding to approximately 1.0 g protein/liter of an optimized liquid culture (38).
Improvement of thermostability of AmyK
Compared with BAA and BSA, BLA is more
thermostable. Tomazic and Klibanov (39,40) and
Declerck et al. (41) reported that temperature
optima of native or engineered thermophilic
enzymes are generally higher than those of
mesophilic enzymes, possibly due to the stiff
structures which stay folded at higher
temperatures than mesophilic enzymes. Declerck
et al. (41,42) and Joyet et al. (43) independently
reported hyperthermostable mutants of BLA;
two substitutions in the amino acid sequence,

His133Ile (or His133Tyr) and Ala 209Val (or Ala
209Ile), can together increase in the half life of
BLA at 90°C up to 10 folds. It is very interestingthat the original amino acid sequence of AmyK
conserves the corresponding amino acid residues
at amino acids 135 (Tyr) and 214 (Ile),
respectively. Van der Laan (44) and Aehle (45)showed that two substitutions,
Val128Glu/His133Tyr or His133Tyr/Asn188Asp,
improved the thermostability of BLA in thepresence of excess CaCl2 (1.5 mM). Again, weemphasize the unique amino acid sequence of
AmyK in that the substituted residues in BLA are
conserved as Tyr135 and Glu130 in the enzyme(although the substituted Asp188 corresponds toIle193). Therefore, there may be anotherunidentified structural element(s) responsible forthermostabilization of AmyK, if its
thermostability could be further improved ashigh as that of BLA or the engineered BAA.
Suzuki et al. (46) demonstrated that
thermostability of BAA was drastically improvedby deletion of Arg176-Gly177 and substitutionof Lys269 for Ala, using site-directedmutagenesis. They proposed that an increase inhydrophobicity by changes in charged residues
enhanced the thermostability of this enzyme.
Machius et al. (27) also suggested that the loop
containing the Arg-Gly residues in BAA has twomore amino acid residues than BLA and that this
could cause increased mobility of this region anddecreased thermostability of the whole protein.
In fact, the amino acid sequences of AmyK,BAA and BSA, all conserved the corresponding
Arg-Gly residues at the respective amino acidpositions. However, substituted Ala269 in BAA
corresponds to Ala274 for AmyK and Ala269 for
BLA in their original amino acid sequences. In
support of this scenario, we deleted
Arg181-Gly182 residues from the AmyK
molecule by site-directed mutagenesis (47).

S. Ito and K. Horikoshi

AmyK and the dipeptide-deleted mutant protein(dRG) were very similar with respect to specificactivity, pH-activity curve, temperature-active
curve, susceptibility to inhibitors, and pattern ofhydrolysis products from soluble starch andmalto-oligosaccharides. As expected from thesequence alignment of AmyK and related
amylolytic enzymes, dRG was found to acquire
increased thermostability, regardless of whether
CaCl2 was present or not. It was stable to
incubation up to 50°C and 60°C in the absence
and presence of CaCl2, respectively. In contrast,
AmyK was stable up to 35-40°C and 45°C,
respectively (Fig. 3).
To evaluate the possible role of calcium
against thermoinactivation of AmyK and dRG,
we examined their resistance to EDTA and
EGTA by incubating for 30 min at varying
concentrations of the individual chelating
reagents at 40°C and pH 8.5. Wild-type AmyK
lost its activity completely at very low
concentrations of EDTA and EGTA (0.05 mM),
whereas dRG remained approximately 60-70%
active, independent of calcium concentrations up
to 2 mM examined. The residual activities ofboth enzymes after incubation for various times
at 45°C and at pH 8.5 in the presence of 0.25
mM EDTA or 0.05% SDS. Fifty % inactivation
by 0.25 mM EDTA occurred after 3 min for
AmyK and after 35 min for dRG, and that by
0.05% SDS occurred after 1.5 h for the former.
However, the activity of dRG remained constantat 45°C for over a 20-h period even in thepresence of SDS.
Proline residues in proteins are known to
restrict backbone bond rotation because of theirpyrrolidine rings. Suzuki et. al. (48) proposed a“proline rule” which states that many prolines atsecond position of β-turn make proteins more
thermostable. Matthews et al. (49) improved thethermostability of T4 lysozyme by replacing

7

Fig. 3. Comparison of thermostability of
AmyK and dRG. To assess thethermostability of AmyK and dRG, theenzymes (2.0 U/ml) were individually heatedfor 2 h at the indicated temperatures in theabsence or presence of 2.0 mM CaCl2 in 50
mM Tris-HCl buffer (pH 8.5). Samples weretransferred after 2 h to wet ice (0°C) and theresidual activity in them (0.1-ml aliquot) was
assayed by measuring the rate of soluble
starch hydrolysis under the standard
conditions of the assay. The residual
activities of AmyK (○) and dRG (●) in theabsence (solid lines) or presence of CaCl2(dotted lines) are expressed as percentages of
the respective original activities, which areeach taken as 100%.

Ala87 at one of its β-turns with a proline residue,
so as to decreased the backbone entropy of
unfolding.
We showed that AmyK could also bethermostabilized by replacing proline for Arg124(50). According to the homology alignment of
AmyK with BLA, the amino acid residues of
BAA, BSA, and BLA corresponding to Arg124
in AmyK were all proline. According to thestructure of BLA, the position of Arg124 of
AmyK is presumed to be located on a loop
connecting between a two-stranded antiparallel
β-sheet involving residues at amino acids from

8

α―Amylases for Detergents

information was given about the positions of
β-turn in the BLA structure, thethermostabilization of the Arg124Pro mutant iscaused as a result of the “proline rule”.

Fig. 4. Effects of temperature on the stability of
wild-type AmyK (●), R124P (○), dRG (■), anddRG -R124P (□). Each enzyme (0.04 µg/ml) was
heated for 10 min at different temperatures in 10
mM Tris-HCl buffer (pH 8.5) without (A) andwith (B) 0.1 mM CaCl2, and assayed for theactivity remaining. The activity of each enzyme
after treatment at 30°C is taken as 100%.

113 to 121 and from 135 to 142 in domain B. We
chose the Arg124 residue in AmyK to examine
the effects of proline substitution on
thermostability and catalytic activity. Half lives
at 50°C of the wild-type enzyme and an
Arg124Pro mutant enzyme were 4.4 min and11.3 min, respectively. Furthermore, thethermostabilization due to double mutation with
Arg124Pro and dRG is cumulative andremarkable even in the absence of any
exogenous additives. The temperature at which
the enzyme is inactivated in 10 min is 47.4°C for
the wild-type enzyme and 62.5°C for the double
mutant enzyme (Fig. 4). Although no

Oxidative stabilization of AmyKWhen AmyK was incubated with 1%, 2%, and
3% each of H2O2 in 10 mM Tris-HCl buffer (pH8.5) plus 2 mM CaCl2 at 30ºC for 30 min, theactivity decreased to 27%, 4%, and 2% of thecontrol, respectively. Such oxidative inactivation
has also been reported for an alkaline serine
protease (subtilisin) (51,52). Oxidative stabilitiesof detergent proteases have been improved byreplacing a Met residue for non-oxidizableamino acids by site-directed mutagenesis.
In the AmyK molecule, there are 10 Met
residues at positions 9, 10, 105, 116, 202, 208,
261, 309, 382, and 430, as deduced from its
nucleotide sequence. When the amino acid

Fig. 5. Rates of oxidative inactivation by H2O2 of
the wild-type and mutant enzymes.
Each enzyme was incubated in the presence of 0.5
M H2O2 at 30ºC and at pH 8.9 in 50 mM Tris-HCl
buffer. Timed samples were withdrawn and mixed
with 140 µg/ml catalase to quench excess H2O2, andthe residual activities were measured under the
standard conditions of enzyme assay. The originalactivity of each enzyme is taken as 100%. The
wild-type and mutant enzymes retained full
activities during the incubation without H2O2 up to
2 h. Symbols used are: ○, wild-type AmyK; ♦,Met202Ala; ▲, Met202Ser; ■, Met202Val, □,Met202Leu; ●, Met202Ile; △, Met202Thr.

S. Ito and K. Horikoshi

sequence was suitably aligned with those of BSA,
BLA, and BAA, Met10, 202 and 309 (in AmyK
numbering) are conserved. This suggests thatoxidation by H2O2 of some of the three residues
would cause the reduced enzymatic activity. The
oxidative stabilization of AmyK was achieved
significantly by replacing Met202 with
non-oxidizable amino acid residues, such as Thr,
Leu, Ile, Ser, and Ala. These mutant enzymes
showed strong oxidative stability whenincubated with 2% H2O2 in 10 mM Tris-HCl
buffer (pH 8.5) plus 2 mM CaCl2 at 30°C for up
to 60 min (Fig. 5). A computer-aided modelingof AmyK which was constructed using the 3D
structure of BLA (27) as a template suggested
that Met202 is located on the enzyme surface
and may correspond to the substrate binding
residue in AmyK. Substitutions with smaller
amino acid residues such as Ser and Ala caused
drastic increase in the Km values toward aborohydride-reduced amylose, whilesubstitutions with bulky Thr and Ile had
essentially little effect. These results suggest thatoxidation of Met202 to the sulfoxide derivative
resulted in interference with substrate binding,thereby abolishing the enzyme activity (53).
Recently, we found an alkaline, Ca2+-free
α-amylase, designated AmyK38 (54,55), froman alkaliphilic Bacillus isolate that is highly
resistant to chelating reagents and chemical
oxidants and requires Na+ for activity. We havealready clarified why AmyK38 is resistant to
EDTA and chemical oxidants by analyzing the
crystal structure of the enzyme (56). Thus,
detergent enzyme technologies will further
develop with microbial screening for novel
enzymes and protein engineering based on
enzyme structures.
In this review, protein engineering of adetergent α-amylase AmyK was summarized.

9

We showed that the deletion of Arg181-Gly182
or Thr183-Gly184 on a loop of domain B in
AmyK caused enhanced thermostability andchelator resistance, and that the enzyme can also
be thermostabilized by replacing proline forArg124 on a loop region in domain B. The
double mutant enzyme is suitable for practicaluse at high temperatures and high chelator
concentrations under high alkalinity. α-Amylase
is very promising for use not only in the starchand food industries but also in the detergent
industry.
References(1) Yamamoto, T. and Shinke, R. in Enzyme
chemistry and molecular biology ofamylase and related enzymes. (TheAmylase Research Society of Japan ed.),pp. 3-5, CRC Press, Inc., Japan (1995).
(2) Kitahata, S. in Handbook of amylases
and related enzymes. (The Amylase
Research Society of Japan ed.), pp.
154-164, Pergamon Press, Oxford, UK(1988).
(3) Outtrup, H. and Norman, B.E. Starch/
Stärke 36, 405-410 (1984).
(4) Wako, K., Takahashi, C., Hashimoto, S.
and Kanaeda, J. J. Jpn. Soc. Starch Sci.
25, 155-161 (1978).
(5) Sakano, Y., Kashiwagi, Y. and Kobayashi,
T. Agric. Biol. Chem. 46, 639-646 (1982).
(6) Bender, H. and Wallenfels, K. Biochem. Z.
234, 79-95 (1961).
(7) Ara, K., Igarashi, K., Saeki, K., Kawai, S.
and Ito, S. Biosci. Biotech. Biochem. 56,
62-65 (1992).
(8) Yokobayashi, K., Misaki, A. and Harada
T. Biochim. Biophys. Acta 212, 458-469(1970).
(9) Ara, K., Saeki, K. and Ito, S. J. Gen.
Microbiol. 139, 781-786 (1993).

10

α―Amylases for Detergents(10) Kuriki, T., Okada, S. and Imanaka, T. J.
Bacteriol. 170, 1554-1559 (1988).
(11) Igarashi, K., Ara, K., Saeki, K., Ozaki,
K., Kawai, S. and Ito, S. Biosci. Biotech.
Biochem. 56, 514-516 (1992).
(12) Ara, K., Saeki, K., Igarashi, K., Takaiwa,
M., Uemura, T., Hagihara, H., Kawai, S.
and Ito S. Biochim. Biophys. Acta 1243,
315-324 (1995).
(13) Kuriki, T and Imanaka, T. J. Biosci.
Bioeng. 87, 557-565 (1999).
(14) Henrissat , B. Biochem. J. 280, 309-316(1991).
(15) UpaDek H. and Kottwitz, B. in Enzymes
in Detergency (Van Ee, J.H., Misset, O.
and Baas, E.J. eds) pp.203-212, Marcel
Dekker, Inc., New York, USA (1997).
(16) Bisgaard-Frantzen, H., Svendsen, A.,
Norman, B., Pedersen, S., Kjærulff, S.,
Outtrup, H. and Borchert, T.V. J. Appl.
Glycosci. 46, 199-206 (1999).
(17) Saito, N. Arch. Biochem. Biophys. 155,290-298 (1973).
(18) Welker, N.E. and Campbell, L.L. J.
Bacteriol. 94, 1124-1130 (1967).
(19) Manning, G.B. and Campbell, L.L. J.
Biol. Chem. 236, 2952-2957 (1961).
(20) Yamamoto, T. in Handbook of amylases
and related enzymes. (The Amylase
Research Society of Japan ed.) pp. 40-45,
Pergamon Press, Oxford, UK (1988).
(21) Matsuura, Y., Kusunoki, M., Harada, W.
and Kakudo, M. J. Biochem. 95, 697-702(1984).
(22) Buisson, G., Duee, E., Haser, R. andPayan. F. EMBO J. 6, 3909-3916 (1987).
(23) Boel, E., Brady, L., Brzozowski, A. M.,
Derewanda, Z., Dodson, G. G., Jensen, V.
J., Petersen, S. B., Swift, H., Thim, L.
and Woldile, H. F. Biochemistry 29,6244-6249 (1990).

(24) Kadziola, A., Abe, J., Svensson, B. andHaser, R. J. Mol. Biol. 239, 104-121(1994).
(25) Yoshioka, Y., Hasegawa, K., Matsuura,Y., Katsube, Y. and Kubota, M. J. Mol.
Biol. 271, 619-628 (1997).
(26) Machius, M., Wiegand, G. and Huber, R.
J. Mol. Biol. 246, 545-559 (1995).
(27) Machius, M., Declerck, N., Huber, R.
and Wiegand, G. Structure 6, 281-292(1998).
(28) Ito, S., Kobayashi, T. and Ozaki, K. J.
Appl. Glycosci., 47, 243-251 (2000).
(29) Igarashi, K., Hatada, Y., Hagihara, H.,
Saeki, K., Takaiwa, M., Uemura, K., Ara,
K., Ozaki, K., Kawai, S., Kobayashi, T.
and Ito, S. Appl. Environ. Microbiol. 64,
3282-3289 (1998).
(30) Iwasa, S., Aoshima, H., Hiromi, K. and
H. Hatano J. Biochem. 75, 969-978(1974).
(31) Takkinen, K., Pettersson, R.F.,
Kalkkinen, N., Palva, I., Soderlund, H.
and Kaariainen,. L. J. Biol. Chem. 258,
1077-1013 (1983).
(32) Nakajima, R., Imanaka, T. and Aiba, S.
J. Bacteriol. 163, 401-406 (1985).
(33) Yuuki, T., Nomura, T., Tezuka, H.,
Tsuboi, A., Yamagata, H., Tsukagoshi, N.
and Udaka, S. J. Biochem. 98, 1147-1156(1985).
(34) Tsukamoto, A., Kimura, K., Ishii, Y.,
Takano, T. and Yamane, K. Biochem.
Biophys. Res. Commun. 151, 25-31(1988).
(35) Nakajima, R., Imanaka, T. and Aiba, S.
Appl. Microbiol. Biotechnol. 23, 355-360(1986).
(36) Sumitomo, N., Ozaki, K., Kawai, S. andIto, S. Biosci. Biotechnol. Biochem. 56,
872-877 (1992).

S. Ito and K. Horikoshi(37) Sumitomo, N., Ozaki, K., Hitomi, J.,
Kawaminami, S., Kobayashi, T., Kawai,
S. and Ito, S. Biosci. Biotechnol. Biochem.
59, 2172-2175 (1998).
(38) Ikawa, K., Araki, H., Tsujino, Y.,
Hayashi, Y., Igarashi, K., Hatada, Y.,
Hagihara, H., Ozawa, T., Ozaki, K.,
Kobayashi, T. and Ito, S. Biosci.
Biotechnol. Biochem. 62, 1720-1725(1998).
(39) Tomazic, S. J. and Klibanov A. M. J.
Biol Chem. 263, 3086-3091 (1988).
(40) Tomazic, S. J. and Klibanov A. M. J.
Biol. Chem. 263, 3092-3096 (1988).
(41) Declerck, N., Machius, M., Chambert,R., Wiegand, G., Huber, R. and Gaillardin,
C. Protein Eng. 8, 1029-1037 (1995).
(42) Declerck, N., Machius, M., Chambert,R., Wiegand, G., Huber, R., andGaillardin, C. Protein Eng. 10, 541-549(1997).
(43) Joyet, P., Declerck, N., and Gaillardin,
C. Biotechnology, 10, 1579-1583 (1992).
(44) Van der Laan, J. and May, M.
International patent WO 95/35382(1995).
(45) Aehle, W. in Enzymes in Detergency(Van Ee, J.H., Misset .O. and Baas, E.J.
eds.) pp.213-229, Marcel Dekker, Inc.,New York, USA (1997).
(46) Suzuki, Y., Ito, N., Yuuki,. T., Yamagata,
H. and Udaka, S. J. Biol. Chem. 264,
18933-18938 (1989).
(47) Igarashi, K., Hatada, Y., Ikawa, K.,
Araki, H., Ozawa, T., Kobayashi, T.,
Ozaki, K. and Ito, S. Biochem. Biophys.

11Res. Commun. 248, 372-377 (1998).
(48) Suzuki, Y. Proc. Jpn. Acad. Ser. B Phys.
Biol. Sci. 65, 146-148 (1989).
(49) Matthews, B. W., Nicholson, H., andBecktel, W. J. Proc. Natl. Acad. Sci. USA
84, 6663-6667 (1987).
(50) Igarashi, K., Ozawa, T.,
Ikawa-Kitayama, K., Hayashi, Y., Araki,
H., Endo, K., Hagihara, H., Ozaki, K.,
Kawai, S. and Ito, S. Biosci. Biotechnol.
Biochem. 63, 1535-1540 (1999).
(51) Estell, D.A., Graycar, T.P. and Wells,
J.A. J. Biol. Chem. 260, 6518-6521(1985).
(52) Bott, R., Ultsch, M., Kossiakoff, A.,
Graycar, T., Katz, B. and Power, S. J.
Biol. Chem. 263, 7895-7906 (1988).
(53) Hagihara, H., Hatada, Y., Ozawa T.,
Igarashi, K., Araki, H., Ozaki, K.,
Kobayashi, T., Kawai, S. and Ito, S. J.
Appl. Glycosci. 50, 67-372 (2003).
(54) Hagihara, H., Igarashi, K., Hayashi, Y.,
Endo, K. Ikawa-Kitayama, K. Ozaki, K.
Kawai, S. and Ito, S Appl. Environ.
Microbiol. 67, 1744-1750 (2001).
(55) Hagihara, H., Hayashi, Y., Endo, K.,
Igarashi, K., Ozawa, T., Kawai, S., Ozaki,
K. and Ito, S. Eur. J. Biochem. 268,
3974-3982 (2001).
(56) Nonaka, T., Fujihashi, M., Kita, A.,
Hagihara, H., Ozaki, K., Ito, S. and Miki,
K. J. Biol. Chem. 278, 24818-24824(2003).

Communicated by Takahisa Kanda