Appl Microbiol Biotechnol (2008) 77:1225–1232
DOI 10.1007/s00253-007-1263-7
BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Improvement in enzymatic desizing of starched cotton
cloth using yeast codisplaying glucoamylase
and cellulose-binding domain
Takeshi Fukuda & Michiko Kato-Murai &
Kouichi Kuroda & Mitsuyoshi Ueda & Shin-ichiro Suye
Received: 26 July 2007 / Revised: 24 October 2007 / Accepted: 25 October 2007 / Published online: 27 November 2007
# Springer-Verlag 2007
Abstract To utilize glucoamylase-displaying yeast cells for
enzymatic desizing of starched cotton cloth, we constructed
yeast strains that codisplayed Rhizopus oryzae glucoamy-
lase and two kinds of Trichoderma reesei cellulose-binding
domains (CBD1, CBD of cellobiohydrolase I (CBHI); and
CBD2, CBD of cellobiohydrolase II (CBHII)). In this study,
we aimed to obtain a high efficiency of enzymatic desizing
of starched cotton cloth. Yeast cells that codisplayed gluco-
amylase and CBD had higher activity on starched cotton
cloth than yeast cells that displayed only glucoamylase.
Glucoamylase and double CBDs (CBD1 and CBD2)
codisplaying yeast cells exhibited the highest activity ratio
(4.36-fold), and glucoamylase and single CBD (CBD1 or
CBD2) codisplaying yeast cells had higher relative activity
ratios (2.78- and 2.99-fold, respectively) than glucoamylase
single-displaying cells. These results indicate that the
glucoamylase activity of glucoamylase-displaying cells
would be affected by the binding ability of CBD codisplayed
on the cell surface to starched cotton cloth. These novel
strains might play useful roles in the enzymatic desizing of
starched cotton cloth in the textile industry.
T. Fukuda : S.-i. Suye (*)
Department of Applied Chemistry and Biotechnology,
Graduate School of Engineering,
University of Fukui,
3-9-1, Bunkyo,
Fukui 910-8507, Japan
e-mail: suye@acbio2.acbio.fukui-u.ac.jp
M. Kato-Murai : K. Kuroda : M. Ueda
Division of Applied Life Sciences,
Graduate School of Agriculture, Kyoto University,
Kitashirakawa, Sakyo-ku,
Kyoto 606-8502, Japan
Keywords Yeast cell surface engineering .
Rhizopus oryzae glucoamylase . Trichoderma reesei
cellulose-binding domain . Enzymatic desizing
Introduction
In the textile industry, large amounts of water, energy, and
auxiliary chemicals are consumed (Feitkenhauer and Meyer
2001). Especially, wastewater from the desizing process
causes environmental pollution. The sizing process is
needed to prevent abrasion, fluffiness, and cutting of the
warp during the weaving process. However, sizing reagents
such as starch inhibit the processes of bleaching, dyeing,
and treatment with surfactant. To remove the sizing
reagents from starched cotton cloth, large amounts of hot
water, surfactants, and other auxiliary chemicals are con-
sumed. To decrease the amount of wastewater produced
and energy consumed in the textile industry, enzymatic
treatment of wastewater has been attempted (Feitkenhauer
et al. 2003). Among the several methods of desizing starch-
ed cotton cloth, enzymatic desizing (e.g., using amylase) is
well known as an environmental-friendly method (Mori
et al. 1997).
Rhizopus oryzae glucoamylase (family 15 glycoside
hydrolase, Mertens and Skory 2007) is an exo-amylolytic
enzyme that cleaves α-1,4-linked and α-1,6-linked glucose
from starch (Ashikari et al. 1986; Murai et al. 1997). This
enzyme has been used to saccharify starchy materials for
glucose and ethanol production (Ashikari et al. 1989). This
enzyme could also be used for the enzymatic desizing of
starched cotton cloth.
The use of cell surface engineering to display R. oryzae
glucoamylase on the cell surface of yeast Saccharomyces
cerevisiae has been reported as a means of enabling yeast
1226
cells to utilize starch directly as the sole carbon source
(Murai et al. 1997). Cell surface engineering is a novel
genetic engineering method (Ueda et al. 1998; Ueda 2004)
that allows the display of proteins and peptides on the cell
surface of the yeast S. cerevisiae as fusion proteins linked
to the C-terminal half of α-agglutinin. Using cell surface
engineering techniques, enzymes or functional proteins
displayed on the yeast cell surface can be used as a protein
cluster, and the detection of activity of proteins displayed
on the cell surface can be carried out without tedious
purification and separation processes as whole-cell catalysts
(Fukuda et al. 2006).
In this study, to utilize glucoamylase-displaying yeast
cells for the enzymatic desizing of starched cotton cloth, we
constructed a yeast strain codisplaying R. oryzae gluco-
amylase and the cellulose-binding domains (CBD of CBHI
(CBD1) and CBD of CBHII (CBD2), family 1, Tomme et al.
1998) from two cellobiohydrolases of Trichoderma reesei
(Nam et al. 2002). The genes encoding CBD cellobiohydro-
lase I (CBHI) and cellobiohydrolase II (CBHII) were
expressed and their proteins were displayed. The binding
properties of each type of CBD-displaying yeast cells to
cellulose has been demonstrated (Ito et al. 2004). By
codisplaying glucoamylase and CBDs on the cell surface,
the yeast cell would acquire specific binding ability to cotton
cloth with glucoamylase activity (Fig. 1). Furthermore, the
codisplaying strain would have greater activity than a strain
displaying only glucoamylase. We compared the glucoamy-
lase activity of glucoamylase-displaying yeast cells with that
of glucoamylase and CBD codisplaying yeast cells toward
starched cotton cloth. The use of yeast cells codisplaying
glucoamylase and CBD was found to be a suitable method
for desizing starched cotton cloth.
Materials and methods
Strains and media
Escherichia

coli strain DH5α (F − , endA1, hsdR17
þ −
r

K ; mK , supE44, thi-l, l rec A1, gyrA96, ΔlacU169
(φ80lacZΔM15) (Hanahan et al. 1983) was used as the host
cell for DNA manipulation. Yeast strain S. cerevisiae MT8-1
(MATa, ade, his3, leu2, trp1, ura3) (Tajima et al. 1985) was
used to display protein on its cell surface. E. coli was grown
in LB medium (1% [w/v] tryptone, 0.5% [w/v] yeast extract,
and 0.5% [w/v] sodium chloride). The yeast cells were grown
either in YPD medium (1% [w/v] yeast extract, 2% [w/v]
polypeptone, and 2% [w/v] glucose) and SD-W medium
(0.67% [w/v] yeast nitrogen base without amino acids
[Difco, MI, USA] and 2% [w/v] glucose with 0.002% [w/v]
adenine sulfate, 0.002% [w/v] uracil, 0.003% [w/v] L-leucine,
0.002% [w/v] L-histidine, and 2% [w/v] casamino acid).
Appl Microbiol Biotechnol (2008) 77:1225–1232
Plasmids
Plasmid pGA11 (Murai et al. 1997) was used to display
R. oryzae glucoamylase on the cell surface of S. cerevisiae
strain MT8-1. We used plasmids pMCBD1 (CBHI),
pMCBD2 (CBHII), and pMCBD3 (CBHI and CBHII)
(Nam et al. 2002) to display single and double T. reesei
CBDs on the cell surface of S. cerevisiae MT8-1 strain and
plasmid pCAS1 as a control (Shibasaki et al. 2001).
Yeast transformation
Plasmids pGA11, pMCBD1, pMCBD2, and pMCBD3
were introduced into S. cerevisiae strain MT8-1 by a lithium
acetate method using the Yeastmaker transformation system
(Takara Bio, Kyoto, Japan). The transformants used in this
study are summarized in Table 1.
Immunofluorescence microscopy
The localization of glucoamylase and CBD on the cell
surface of the yeast cell was confirmed by immunofluores-
cence labeling of the cells (Ito et al. 2004). After cultivation
in SD medium at 30°C for 48 h, yeast cells were harvested,
washed with phosphate-buffered saline (PBS, pH 7.4), and
resuspended in PBS containing 1% (w/v) bovine serum
albumin (BSA) for 30 min at 4°C. The yeast cells were then
incubated for 1.5 h at 4°C in PBS containing 1% (w/v) BSA
together with goat antiglucoamylase IgG antibody (Qiagen,
Valencia, CA, USA) used as the primary antibody at a
dilution rate of 1:500, after which they were incubated for
1 h at 4°C with the secondary antibody, a 1:300 dilution rate
of Texas Red™-conjugated rabbit antigoat IgG antibody
(Rockland Immunochemicals, PA, USA). To confirm the lo-
calization of CBD, mouse anti-His4 antibody (Qiagen) was
used as the primary antibody and Alexa Fluor 488-conjugated
rabbit antimouse IgG antibody (Molecular Probes, Eugene,
OR, USA) as the secondary antibody. After washing the cells
with PBS, they were subjected to fluorescence microscopy
(BH-BFL, Olympus Optical, Tokyo, Japan) where green fluo-
rescence indicated CBD (excitation at 490 nm and emission at
520 nm) and red fluorescence indicated glucoamylase
(excitation at 596 nm and emission at 620 nm).
Binding assay of transformants to phosphoric acid-swollen
Avicel as the free cellulose
After the cultivated yeast cells (6.7× 107 cells) were
harvested and washed twice with 3 ml of 50 mM potassium
phosphate buffer (pH 7.4) containing 50 mM NaCl, they
were reacted with 200 μl phosphoric acid-swollen Avicel
(Fukuda et al. 2006). After incubating at 25°C for 18 h, the
reaction mixture was filtered through an 11-μm nylon net
Appl Microbiol Biotechnol (2008) 77:1225–1232 1227

Fig. 1 Interaction between

yeasts codisplaying glucoamy-
lase and CBDs and starched
cotton cloth. Binding toward
starched cotton cloth by single
glucoamylase-displaying yeast.
Yeasts codisplaying glucoamy-
lase and CBDs were able to
bind starched cotton cloth
more strongly than single
glucoamylase-displaying yeast

Table 1 List of strains used in

this study Strain Relevant feature(s) Reference or source

MT8-1/pCAS1 Control strain Nam et al. (2002)

CBD1: cellulose-binding
domain of T. reesei cellobio-
hydrolase I, CBD2: cellulose-
binding domain of T. reesei
cellobiohydrolase II
MT8-1/pMCBD1 Display of CBD1 Mori et al. (1997)
MT8-1/pMCBD2 Display of CBD2 Mori et al. (1997)
MT8-1/pMCBD3 Display of CBD1 and CBD2 Mori et al. 1997
MT8-1/pGA11 Display of glucoamylase Feitkenhauer and Meyer (2001)
MT8-1/pGA11/pMCBD1 Display of glucoamylase and CBD1 Present study
MT8-1/pGA11/pMCBD2 Display of glucoamylase and CBD2 Present study
MT8-1/pGA11/pMCBD3 Display of glucoamylase, CBD1, and CBD2 Present study
1228
filter (pore size, 11 μm) (NY1102500, Millipore, MA,
USA) for removing the bound cells against Avicel, and the
filtrate was refiltered through the same filter. The absor-
bance of the filtrate at 600 nm was measured to estimate the
number of unbound cells. The binding ability of yeast cells
to phosphoric acid-swollen Avicel was determined by
measuring the optical density at 600 nm of the unbound
cells. The binding ratio (%) was calculated as follows (Nam
et al. 2002):
Binding ratioð%Þ
¼ ððO:D:600 ðt ¼ 0Þ
O:D:600 ðt ¼ 24ÞÞ=O:D:600 ðt ¼ 0ÞÞ  100
Binding assay of transformants to cotton cloth
The cultivated yeast cells (6.7×107 cells) were harvested by
centrifugation (1,500×g, 5 min) and washed twice with
1 ml of 50 mM potassium phosphate buffer (pH 7.4)
containing 50 mM NaCl before reacting with square pieces
of cotton cloth (1 cm2). After incubating at 25°C for 18 h,
the cotton cloth was removed from the reaction solution.
The absorbance of the reaction solution without cotton
cloth at 600 nm was measured to estimate the number of
unbound cells. The calculation is described in the previous
section.
Measurement of glucoamylase activity
Glucoamylase activity for soluble starch
The glucoamylase activity of transformants was measured
using the hexokinase from Bacillus sp. (EC 2.7.1.1)/
glucose-6-phosphate dehydrogenase (G-6-PDH) from Ba-
cillus sp. (EC 1.1.1.49) method with soluble starch (Bondar
and Mead 1974). Yeast cells were cultivated in 100 ml of
SD-W medium and collected by centrifugation at 1,500×g,
4°C for 5 min. The cells (A600=10) had been washed with
PBS (pH 7.4). The reaction solution contained 1.1 mM ATP
(Wako Pure Chemical Industries, Japan), 1.3 mM NAD+
(Oriental Yeast, Japan), 0.18 mM MgCl2, and 0.5% (w/v)
soluble starch and was prepared by dissolving each com-
ponent in 50 mM α-glycerophosphate buffer (pH 6.0).
Hexokinase (0.01 ml; 1,200 U/ml; Oriental Yeast) and G-6-
PDH (1,200 U/ml) (Oriental Yeast) were added to the
reaction solution (3 ml), which was then placed in a water
bath at 37°C for 10 min. Yeast cells (0.1 ml, A600=10)
were added to the reaction solution and incubated 37°C for
10 min. The activity of glucoamylase was determined by
measuring the formation of NADH at 340 nm. One unit of
enzyme activity was defined as the amount of enzyme that
resulted in the formation of 1 μmol of NADH per min. In
Appl Microbiol Biotechnol (2008) 77:1225–1232
calculating the amount of NADH produced, a molar
absorption coefficient for NADH of 6.3×103 M cm−1 was
useds.
Glucoamylase activity for starched cotton cloth
Glucoamylase activity toward starched cotton cloth was
measured in the same way as it was measured for soluble
starch. The substrate, starched cotton cloth, was prepared
by boiling the cotton cloth in soluble starch solution boiled
for 5 min and drying-up at 60°C for 24 h.
Yeast cells were cultivated in 100 ml of SD-W medium
and collected by centrifuging at 1,500×g, 4°C for 5 min.
The cells (A600=10) were washed with PBS (pH 7.4). The
reaction solution, containing 1.1 mM ATP, 1.3 mM NAD+,
and 0.18 mM MgCl2, was prepared by dissolving each
component in 50 mM α-glycerophosphate buffer (pH 6.0).
Measurement of activity was initiated by adding yeast cells
(0.1 ml) and starched cotton cloth to the reaction solution
and incubating for 37°C for 10 min, and the assay was
performed using hexokinase and G-6-PDH with the same
procedure as described above (see the “Glucoamylase
activity for soluble starch” section).
Results
Confirmation of singly displayed protein and codisplay
of dual proteins on the cell surface of transformants
The expression plasmids constructed in this study—pGA11
for display of the R. oryzae glucoamylase, pMCBD1 and
pMCBD2 for display of the CBD1 (T. reesei CBHI) and
CBD2 (T. reesei CBHII), and pMCBD3 for display of the
double CBDs (CBD1 and CBD2)—were transformed or
cotransformed into the S. cerevisiae strain MT8-1 strain si-
multaneously. The resulting transformants were designated
strains MT8-1/pGA11, MT8-1/pMCBD1, MT8-1/pMCBD2,
MT8-1/pMCBD3, MT8-1/pGA11/pMCBD1, MT8-1/
pGA11/pMCBD2, and MT8-1/pGA11/pMCBD3 (Table 1).
To confirm the codisplay of glucoamylase and CBDs on
the yeast cell surface, immunofluorescence labeling of trans-
formants was performed. No fluorescence signal of Alexa
Fluor 488 for CBD (Fig. 2, center column) or of Texas
Red™ for glucoamylase (Fig. 2, right column) was observed
on the cell surface of the MT8-1/pCAS1 strain, the negative
control (Fig. 2a, center and right columns). The green
fluorescence of Alexa Fluor 488, indicating the localization
of CBD, was observed for strains MT8-1/pMCBD1
(Fig. 2b), MT8-1/pMCBD2 (Fig. 2c), MT8-1/pMCBD3
(Fig. 2d), MT8-1/pGA11/pMCBD1 (Fig. 2f), MT8-1/
pGA11/pMCBD2 (Fig. 2g), and MT8-1/pGA11/pMCBD3
(Fig. 2h). The red fluorescence of Texas Red™ to detect the
Appl Microbiol Biotechnol (2008) 77:1225–1232 1229

Fig. 2 Immunofluorescence
labeling of transformants.
Phase-contrast micrographs (left
column) and fluorescence
micrographs (center and right
columns). Cells were labeled
with mouse anti-His4 antibody
and Alexa Fluor 488-conjugated
rabbit antimouse IgG antibody
(center column) and with goat
antiglucoamylase IgG antibody
and Texas Red™-conjugated
rabbit antigoat IgG antibody
(right column)

localization of glucoamylase was observed for strains MT8-1/
pGA11, MT8-1/pGA11/pMCBD1, MT8-1/pGA11/
pMCBD2, and MT8-1/pGA11/pMCBD3 (Fig. 2e–h, right
column). Single display of glucoamylase and CBD and
codisplay of glucoamylase and CBDs on the yeast cell
surface were confirmed by immunofluorescence labeling.
Binding assay of CBD-displaying transformants
toward cellulose
To examine the binding ability of glucoamylase and CBD
codisplaying cells toward cellulose, we first investigated the
binding ability toward phosphoric acid-swollen Avicel as
the cellulose in the reaction solution. The binding ratio of
transformants is shown in Fig. 3a. The MT8-1/pCAS1 cells
with nonharboring glucoamylase and CBD as a control
exhibited a very low binding ratio (Fig. 3a–A). The binding
ratio of glucoamylase and double CBDs codisplaying strain
(MT8-1/pGA11/pMCBD3) had a higher binding ratio than
other codisplaying strains (MT8-1/pGA11/pMCBD1 and
MT8-1/pGA11/pMCBD2; Fig. 3a–B to D).
Then, the binding ability toward cotton cloth as the
industrial cellulose in the reaction solution was investigated
for each codisplaying transformant. The binding ratios of
1230 Appl Microbiol Biotechnol (2008) 77:1225–1232

Table 2 Glucoamylase activity of transformants toward soluble starch

Strain Glucoamylase activitya

(U/l of culture broth)

MT8-1/pGA11 32.4
MT8-1/pGA11/pMCBD1 23.6
MT8-1/pGA11/pMCBD2 20.2
MT8-1/pGA11/pMCBD3 30.9
MT8-1/pMCBD1 ND
MT8-1/pMCBD2 ND
MT8-1/pMCBD3 ND
MT8-1/pCAS1 ND

Data are the average of three independent measurements. Deviations

are all less than 10%.
ND: not detected
a
Activity was measured by the hexokinase/G-6-PDH method.

Measurement of glucoamylase activity of transformants

toward starched cotton cloth

The glucoamylase activity of the transformants toward

Fig. 3 a Binding ratio of transformants toward phosphoric acid-
swollen Avicel. A MT8-1/pCAS1 as a control, B MT8-1/pGA11/
pMCBD1, C MT8-1/pGA11/pMCBD2, and D MT8-1/pGA11/
pMCBD3. Data are the average of three independent measurements.
Deviations were all less than 10%. b Binding ratio of transformants
toward cotton cloth. A MT8-1/pCAS1 as a control, B MT8-1/pGA11/
pMCBD1, C MT8-1/pGA11/pMCBD2, and D MT8-1/pGA11/
pMCBD3. The binding ability of yeast cells was determined by
measuring the optimal density at 600 nm of the unbound cells. Data
are the average of three independent measurements. Deviations were
all less than 10%
starched cotton cloth was measured by the hexokinase/G-6-
PDH method. The results are shown in Table 3. In strains
MT8-1/pGA11/pMCBD1, MT8-1/pGA11/pMCBD2, and
MT8-1/pGA11/pMCBD3, activity was higher than the
MT8-1/pGA11 strain. Strains codisplaying glucoamylase
and CBD showed higher glucoamylase activity toward
starched cotton cloth than that of the yeast strain displaying
only glucoamylase. Glucoamylase activity was not detected
in strains MT8-1/pMCBD1, MT8-1/pMCBD2, MT8-1/
pMCBD3, and MT8-1/pCAS1 (negative control).
Discussion

To utilize glucoamylase-displaying yeast cells for the enzy-

transformants are shown in Fig. 3b. The MT8-1/pCAS1 matic desizing of starched cotton cloth, we constructed yeast
cells with nonharboring glucoamylase and CBD as a con-
trol showed no binding (Fig. 3b–A). On the other hand, Table 3 Glucoamylase activity of transformants toward starched
binding of strains codisplaying glucoamylase and CBDs cotton cloth
(MT8-1/pGA11/pMCBD1, MT8-1/pGA11/pMCBD2MT8-1/ Strain Glucoamylase activitya
pGA11/pMCBD3) was detected at a higher level than that (U/l of culture broth)
toward phosphoric acid-swollen cellulose (Fig. 3b).
MT8-1/pGA11 8.0
Glucoamylase activity toward soluble starch MT8-1/pGA11/pMCBD1 22.2
MT8-1/pGA11/pMCBD2 23.9
MT8-1/pGA11/pMCBD3 34.9
The glucoamylase activity of the transformants toward
MT8-1/pMCBD1 ND
soluble starch was measured by the hexokinase/G-6-PDH MT8-1/pMCBD2 ND
method. Glucoamylase activity was confirmed in strains MT8-1/pMCBD3 ND
MT8-1/pGA11, MT8-1/pGA11/pMCBD1, MT8-1/pGA11/ MT8-1/pCAS1 ND
pMCBD2, and MT8-1/pGA11/pMCBD3 (Table 2). On the
Data are the average of three independent measurements. Deviations
other hand, glucoamylase activity was not detected in
are all less than 10%.
strains MT8-1/pMCBD1, MT8-1/pMCBD2, MT8-1/ a
Glucoamylase activity toward starched cotton cloth was measured by
pMCBD3, and MT8-1/pCAS1 (negative control). the hexokinase/G-6-PDH method.
Appl Microbiol Biotechnol (2008) 77:1225–1232
strains codisplaying glucoamylase and cotton (cellulose)-
binding protein. These strains showed much higher activity
toward starched cotton cloth than that of a yeast strain dis-
playing only glucoamylase (Table 3). Previously, by using
yeast cell surface engineering, we produced yeast cells dis-
playing R. oryzae glucoamylase (Murai et al. 1997, 1999),
and yeast cells displaying T. reesei CBHI CBD1, CBHII
CBD2, and double CBDs (CBD1 and CBD2) have also
been reported (Nam et al. 2002). However, to utilize the
enzymatic desizing of starched cotton cloth on an industrial
scale, there had been no attempt to create a yeast strain
codisplaying glucoamylase and CBD, which would provide
glucoamylase activity and cellulose-binding ability. This is
the first report on yeasts displaying both CBD and gluco-
amylase on the cell surface, and its practical and effective
application.
We constructed seven transformant yeast strains (Table 1):
a glucoamylase-displaying strain; strains displaying CBDs;
and strains displaying both glucoamylase and CBDs. First,
we compared the binding ability toward phosphoric acid-
swollen Avicel as free cellulose in the reaction solution
between cells codisplaying glucoamylase and CBD and
control cells (Fig. 3). The strains codisplaying glucoamy-
lase and CBD (MT8-1/pGA11/pMCBD1, MT8-1/pGA11/
pMCBD2, and MT8-1/pGA11/pMCBD3) had higher bind-
ing ability toward phosphoric acid-swollen Avicel than that
of the control strain (MT8-1/pCAS1) (Fig. 3a). In the bind-
ing assay toward cotton cloth, similar results were obtained
(Fig. 3b). These results indicate that the glucoamylase-
displaying and CBD codisplaying cells have binding ability
toward cellulose. Among the transformants, the yeast strain
displaying only glucoamylase (MT8-1/pGA11) showed
higher activity than that of the strain codisplaying glucoa-
mylase and CBDs (MT8-1/pGA11/pMCBD1, MT8-1/
pGA11/pMCBD2, and MT8-1/pGA11/pMCBD3) toward
soluble starch (Table 2).
It is interesting to note that when we studied the gluco-
amylase activities of transformants toward starched cotton
cloth, the glucoamylase and CBD codisplaying cells had
much higher activity than the cells displaying only
glucoamylase. This indicates that the glucoamylase activity
of glucoamylase-displaying cells would be significantly
affected by the binding ability of codisplayed CBD toward
starched cotton cloth. The experiments using starched
cotton cloth in the enzymatic desizing process demonstrated
a useful function of glucoamylase and CBD codisplaying
yeast cells by showing the effect of the binding ability of
CBD on codisplayed glucoamylase.
The N-terminal region in CBH II contributes in the
binding onto both amorphous and crystalline cellulose
types; the homologous C-terminal peptide in CBH I,
however, only affects the interaction with microcrystalline
cellulose (Tomme et al. 1988). CBD3 (CBD1–CBD2) on
1231
the surface of yeast cell might be corroborated and bind
cotton synergistically.
Large amounts of water and energy are consumed in the
textile industry. By using yeasts that codisplay glucoamy-
lase and CBD, the enzymatic desizing process might be
carried out with a smaller amount of enzyme than the
quantities of commercial amylases used at present. Further-
more, in our system using glucoamylase-displaying and
CBD codisplaying yeasts, the reaction temperature was
37°C compared with 60°C in the present system using
commercial amylases; thus, the amount of energy neces-
sary for the reaction using codisplaying yeasts is much
lower than that needed when commercial amylase is used.
Therefore, these newly constructed strains could have a
useful role in decreasing the amount of wastewater
produced and decreasing energy consumption in the textile
industry.
Acknowledgement The authors thank Professor Yasushi Morikawa,
Department of Bioengineering, Nagaoka University of Technology for
providing the cDNA of T. reesei CBDI and CBDII. The authors also
thank Mr. Toshiyuki Baba, Technical Center, Sakai Ovex for kindly
providing the cotton cloth.
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