Production and characterization of recombinant Phanerochaete chrysosporium β-glucosidase in the methylotrophic yeast Pichia pastoris

Article abstract of DOI:10.1271/bbb.67.1

The extracellular β-glucosidase from the white-rot fungus Phanerochaete chrysosporium was expressed heterologously in the methylotrophic yeast Pichia pastoris. After 7 days' cultivation in an induction medium containing 1% (v/v) methanol, the expression level of the recombinant enzyme was 28.500 U/l, 38 times that of the wild-type enzyme. The specific activity of the crude recombinant enzyme for p-nitrophenyl-β-D-glucoside was 52 U/mg, 37 times that of the wild-type enzyme; this difference made the purification of the enzyme simple. On a SDS-PAGE, the molecular mass of the recombinant enzyme was 133 kDa, and that of the wild-type enzyme was 116 kDa, but the difference had no effect on the hydrolysis of cellobiose or p-nitrophenyl-β-D-glucoside. We concluded that the recombinant enzyme produced by Pichia pastoris retains the catalytic properties of the wild-type enzyme from Phanerochaete chrysosporium.

Full text of DOI:10.1271/bbb.67.1

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Production and Characterization of Recombinant
Phanerochaete chrysosporium β-Glucosidase in the
Methylotrophic Yeast Pichia pastoris
Rie KAWAIa, Makoto YOSHIDAa, Tomomi TANIa, Kiyohiko IGARASHIa, Tsuyoshi OHIRAa,
Hiromichi NAGASAWAa & Masahiro SAMEJIMAa
a Graduate School of Agricultural and Life Sciences, University of Tokyo Yayoi 1-1-1,
Bunkyo-ku, Tokyo 113-8657, Japan
Published online: 22 May 2014.
To cite this article: Rie KAWAI, Makoto YOSHIDA, Tomomi TANI, Kiyohiko IGARASHI, Tsuyoshi OHIRA, Hiromichi NAGASAWA
& Masahiro SAMEJIMA (2003) Production and Characterization of Recombinant Phanerochaete chrysosporium β-Glucosidase
in the Methylotrophic Yeast Pichia pastoris , Bioscience, Biotechnology, and Biochemistry, 67:1, 1-7, DOI: 10.1271/
bbb.67.1
To link to this article: http://dx.doi.org/10.1271/bbb.67.1
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Biosci. Biotechnol. Biochem., 67 (1), 1–7, 2003
Production and Characterization of Recombinant Phanerochaete
chrysosporium b-Glucosidase in the Methylotrophic Yeast Pichia pastoris
Rie KAWAI, Makoto YOSHIDA, Tomomi TANI, Kiyohiko IGARASHI, Tsuyoshi OHIRA
Hiromichi NAGASAWA, and Masahiro SAMEJIMA
,
†
Graduate School of Agricultural and Life Sciences, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku,
Tokyo 113-8657, Japan
Received March 14, 2002; Accepted August 16, 2002
The extracellular
b-glucosidase from the white-rot
acetyl-b-glucosaminidases and b-xylosidases from
fungus Phanerochaete chrysosporium was expressed
heterologously in the methylotrophic yeast Pichia
pastoris. After 7 days' cultivation in an induction medi-
various higher plants, fungi, and bacteria are includ-
ed.12) Although the structure of one family 3 enzyme,
the
b-glucan exohydrolase from barley, has been
um containing 1
%
(v v) methanol, the expression level
identiˆed by X-ray crystallography,13) the reaction
mechanism of P. chrysosporium BGL is still uncer-
tain because of the catalytic diversity of family 3
enzymes. In the cellulolytic culture of P. chrysospori-
um, production of BGL is lower than other cellulo-
lytic enzymes.14,15) Overexpression of recombinant
BGL would be useful to clarify the details of the cata-
lytic mechanism of P. chrysosporium BGL before
discussion of its physiological function.
W
of the recombinant enzyme was 28,500 U l, 38 times
that of the wild-type enzyme. The speciˆc activity of the
crude recombinant enzyme for
W
p
-nitrophenyl-
b- -gluco-
D
side was 52 U mg, 37 times that of the wild-type en-
W
zyme; this diŠerence made the puriˆcation of the en-
zyme simple. On a SDS-PAGE, the molecular mass
of the recombinant enzyme was 133 kDa, and that of
the wild-type enzyme was 116 kDa, but the diŠerence
had no eŠect on the hydrolysis of cellobiose or
p-
Up to now, several groups have produced fungal
and bacterial family 3 BGLs with active forms in het-
nitrophenyl- -glucoside. We concluded that the
b-
D
recombinant enzyme produced by Pichia pastoris
retains the catalytic properties of the wild-type enzyme
from Phanerochaete chrysosporium
erologous expression systems using Saccharomyces
16–18)
cerevisiae
.
In such systems, however, the recom-
.
binant enzymes often cause overglycosylation,19–21)
and have activity diŠerent than that of wild-type en-
zymes.19,20) Extracellular enzymes of S. cerevisiae
hinder the puriˆcation of recombinant proteins.22) In
contrast, in an expression system with the methylo-
trophic yeast Pichia pastoris, recombinant protein is
Key words: -glucosidase; glycoside hydrolase; Pichia
b
pastoris Phanerochaete chrysosporium
heterologous expression
;
;
b
-Glucosidase (BGL, EC 3.2.1.21) is glycoside
hydrolase that catalyzes the hydrolysis of various
-glucosides with the release of -glucose from
glycosylated less extensive than with S. cerevisiae; P.
pastoris typically adds only 8 to 14 mannose residues
per side chain, but S. cerevisiae adds 50 to 150 such
residues.23,24) Moreover, P. pastoris secretes little na-
tive protein into the culture solution, and production
of recombinant proteins is generally greater.22,25,26)
We recently reported high production of a recom-
binant P. chrysosporium cellobiose dehydrogenase
b
b
-
D
the nonreducing end. In the cellulose-degrading
culture of the wood-rotting fungus Phanerochaete
chrysosporium, BGL is produced extracellularly with
other cellulolytic enzymes.1) Therefore, the extracel-
lular BGL has been investigated as a terminal enzyme
of the cellulose hydrolyzing system that cleaves cel-
lobiose into two glucose units.2–5)
(CDH) by the methylotrophic yeast Pichia pastoris
;
the enzyme has the same catalytic properties as wild-
type CDH and the molecular mass of the recom-
binant enzyme is the almost same as that of wild-type
CDH.27) In this report, we describe the e‹cient
production of recombinant P. chrysosporium BGL
in P. pastoris and its steady-state kinetic and cel-
lulose-binding properties.
Currently, glycoside hydrolases are classiˆed into
more than 80 families on the basis of their amino acid
sequences and hydrophobic cluster analysis.6–10) Ac-
cording to the classiˆcation, most BGLs from vari-
ous sources belong to family 1 or 3, and the extracel-
lular BGL from P. chrysosporium has been classiˆed
into family 3,11) in which not only BGLs but also
N-
†
To whom correspondence should be addressed. Tel: +81-3-5841-5255; Fax: +81-3-5841-5273; E-mail: amsam
-glucosidase; CDH, cellobiose dehydrogenase; MeU G, methylumbelliferyl- -glucoside;
, recombinant; wt, wild-type; YPG, yeast extract peptone glycerol; YPM, yeast extract peptone methanol
＠
mail.ecc.u-tokyo.ac.jp
Abbreviations: BGL,
-glucoside;
b
b
b
-
D
p
NPG, -nitrophenyl-
p
b
-
D
r

2
R. KAWAI et al.
Materials and Methods
Cultivation of Phanerochaete chrysosporium. P.
chrysosporium strain K-328) was grown on Kremer
and Wood medium29) modiˆed by the exclusion of
MnO2 and containing 2
z cellulose powder (What-
man CF-11) as the source of carbon. The initial pH
of the culture was adjusted to 5.0 by the addition of
8
×
phosphoric acid. Some 4.0 10 spores were used to
inoculate 400 ml of the medium in a 1-liter Erlenmey-
er ‰ask and the culture was cultivated at 37
9C with
shaking at 150 rpm (q 20 mm). After 3 days of culti-
vation, mycelia were collected by ˆltration, frozen
with liquid nitrogen, and ground to a ˆne powder
with a pestle.
Fig. 1. Construction of BGL-pPIC9K.
-Factor, a secretion-signal sequence attached to the BGL se-
quence; 5 AOX1, promoter fragment; kanamycin, kanamycin
a
?
Construction of the expression vector. Total RNA
was extracted from the mycelia with an E.Z.N.A.
Fungal RNA Kit (Omega Bio-tek, Inc., Doraville,
GA). The ˆrst-strand cDNA was synthesized from
the total RNA with a Ready To Go T-Primed First-
Strand Kit (Amersham Biosciences, Piscataway, NJ).
On the basis of the nucleotide sequence of the cDNA
encoding BGL from P. chrysosporium OGC101
(GenBank accession no. AF036872),11) the following
oligonucleotide primers were designed for prepara-
gene that confers resistance to geneticin disulfate; HIS4, Pichia
pastoris wild-type gene encoding histidinol dehydrogenase.
Activity screening of transformants was done with
4-methylumbelliferyl-
b- -glucoside (MeU bG) as the
D
substrate.31) Geneticin-disulfate-resistant colonies
were transferred to a YPM plate (10 g l yeast extract,
W
20 g l peptone, 1
z
methanol by vol., and 20 g l
W
W
agar). After 1 day of cultivation at 26.5
0.5 sodium acetate buŠer (pH 4.5) containing 5 m
MeU G was sprayed on the plate. After 2.5 h of in-
cubation at 26.5 C, the release of methylumbellifer-
9
C, 1 ml of
tion of the cDNA insert. The 5
?
-primer (5
?-TTTT-
M
M
TTGAATTCCAGTCCGGCCTGTATCAG-3
?
) con-
b
tained six thymine residues, an EcoRI site (shown in
italics), and 17 nucleotide residues encoding the N-
terminal six amino acid residues of the mature BGL.
9
one by extracellular
light.
rBGL was detected with UV
The
3
?
-primer (5
?
-TTTTTTGAATTCTAGTTCG-
TGATTGAGCCCTT-3
?
)
contained six thymine
Production of rBGL. The colonies selected by
MeU G screening were used to inoculate 500 ml of
YPG medium (10 g l yeast extract, 20 g l peptone
residues, an EcoRI site (shown in italics), the stop
codon (bold characters), and 18 nucleotide residues
encoding the C-terminal six amino acid residues of
the mature BGL. PCR was done with the two primers
and the ˆrst-strand cDNA as a template. The ampliˆ-
cation protocol was as follows; denaturation was
b
W
W
and 10 ml l glycerol) in a 1-liter Erlenmeyer ‰ask and
W
incubated at 30
9C with shaking at 150 rpm (q
20 mm) for 2 days. The cells were harvested by cen-
trifugation (30 min, 2,000
of YPM medium (YPM plate formula without agar)
and cultivated for another 7 days at 26.5 C. One mil-
liliter of methanol was added every day for the induc-
tion of BGL. Cycle cultivation was also done with
×
g), suspended in 100 ml
done at 94
9
C for 5 min, followed by 28 cycles at
94 C for 30 s, 55
9
9
C for 30 s, and 72 C for 3 min
9
9
(10 min for the last cycle only). The ampliˆed cDNA
was sequenced and inserted into the expression vector
pPIC9K (Invitrogen, Carlsbad, CA), which contains
a histidinol dehydrogenase gene, HIS4, and a
kanamycin resistance gene for screening for
geneticin-disulfate-resistant transformants as de-
scribed previously.27,30) The vector obtained was
designated BGL-pPIC9K (Fig. 1).
r
the protocol described above, except for transferal of
the cells to new YPM medium after 4 days of cultiva-
tion. One milliliter of culture was collected daily and
×
g). The BGL activity in
centrifuged (5 min, 15,000
the supernatant was measured in terms of the release
of -nitrophenol from NPG as described in Enzyme
assay below.
p
p
Screening for transformants. The methylotrophic
yeast Pichia pastoris strain KM71, which is a
histidine-deˆcient strain (his„), was transformed
with the linearized BGL-pPIC9K with a multicopy
Pichia expression kit (Invitrogen). Transformants in-
dependent of histidine (his+) and resistant to geneti-
cin disulfate (Sigma, St. Louis, MO) were screened
for as described before.22)
Puriˆcation of wtBGL and rBGL. wtBGL was
puriˆed from the culture supernatant of P.
chrysosporium grown on cellulose as described
elsewhere (in preparation).
rBGL expressed by P.
pastoris was puriˆed as follows. The culture was
×
spun down (30 min, 1,500
g
) and the culture super-
(w v) bentonite
natant was incubated with 5
z
W

Recombinant Phanerochaete chrysosporium
b-Glucosidase
3
(Wako Pure Chemicals, Osaka, Japan) for 30 min at
pH 4.0–6.0. The amount of
p
-nitrophenol was meas-
room temperature. After the bentonite was removed
ured by monitoring of changes in absorbance at
×
＝
by centrifugation (30 min, 1,500
g
), the solution
405 nm (e405 17.0 m
M
„1 cm„1 ). One unit of
p
NPG
was concentrated 20-fold with a PM-30 ultraˆltration
membrane (Millipore Corporation, Bedford, MA)
and was put through a fast desalting column PD-10
hydrolyzing activity was deˆned as the amount of en-
zyme necessary to release 1 mol of -nitrophenol
per minute. Various concentrations of NPG (0.1–
5 m ) and gluconolactone (40–200 ) were used to
estimate the steady-state kinetic parameters Km kcat
and i. These parameters were calculated from the e-
mt, where
quation catS S+(1+I Ki is the
speciˆc activity, kcat is the catalytic constant, S is the
substrate concentration, I is the concentration of
gluconolactone, and Ki is the competitive inhibition
constant. All kinetic parameters were calculated
from nonlinear ˆtting to the data with DeltaGraph
4.0.5 (SPSS Inc., Chicago, IL) and KaleidaGraph
3.08 (Synergy Software, Reading, PA).
m
p
p
(Amersham Biosciences) for dialysis against 20 m
Tris-HCl buŠer (pH 8.0) containing 0.1 m
M
M
m
M
M
,
phenylmethylsulfonyl ‰uoride and 0.02
z
sodium
K
azide. The crude BGL was puriˆed by HPLC with a
r
k
＝
k
s
W
)
K
k
W
×
Super Q-5PW column (7.5 mm 75 mm; Tosoh,
Tokyo, Japan) equilibrated with the same buŠer. The
enzyme was eluted from the column with a linear
gradient of 0–500 m
containing NPG hydrolyzing activity was collected,
M
NaCl in 35 ml. The fraction
p
concentrated by ultraˆltration with an Ultrafree-0.5
ˆlter unit (Millipore Corporation) and put through a
×
Super SW 3000 column (4.6 mm 300 mm; Tosoh)
equilibrated with 0.1 potassium phosphate (pH
7.0), 0.5 sodium sulfate, and 0.05 sodium azide.
The fractions containing NPG-hydrolyzing activity
M
Cellulose-binding ability. The cellulose-binding
ability of rBGL with and without papain treatment
M
z
p
was compared as previously reported.14,27)
(2.9 ) was incubated with 0.83 papain (Elastin
Products Co., Inc., Owensville, MO) at 30 C in
100 m potassium phosphate buŠer (pH 7.0) con-
taining 2 m EDTA and dithiothreitol for 18 h. The
solution was mixed with 5 cellulose (Funacel SF;
Funakoshi Co., Ltd., Tokyo, Japan) in 100 m sodi-
um acetate buŠer, pH 4.5, and incubated at 30 C for
1 h. The amount of BGL that adsorbed on the cellu-
NPG
rBGL
were concentrated and desalted by ultraˆltration as
described above. SDS-PAGE was done according to
m
M
m
M
9
the procedure of Laemmli32) with 12
z
poly-
M
acrylamide gel and a Mini-PROTEAN II cell (Bio-
Rad Laboratories, Hercules, CA). The protein con-
centration in the culture solution was measured with
a Protein Assay kit II (Bio-Rad Laboratories) based
on Bradford's method33) with bovine serum albumin
as the standard protein. The concentrations of puri-
M
z
M
9
r
lose was estimated from the decrease of
p
ˆed
r
BGL and wtBGL were calculated from the ab-
hydrolyzing activity after removal of the cellulose
particles by centrifugation (10 min, 15,000
sorbance at 280 nm (e280 119 m „1 cm„1).11)
M
g). For a
＝
×
control, the same experiment was done without incu-
bation with cellulose.
Identiˆcation of N-terminal amino acid sequence.
The
rBGL puriˆed by SDS-PAGE as described
above was transferred to a poly vinylidene di‰uoride
membrane (Millipore Corporation) with a Trans-Blot
SD cell (Bio-Rad Laboratories). After staining of the
manuscript with Coomassie brilliant blue R-250, part
Results and Discussion
Construction of expression vector and screening
for transformants
of the membrane, including the
r
BGL, was cut out
The cDNA fragment encoding mature BGL of P.
chrysosporium strain K-3 was cloned on the basis of
the nucleotide sequence of the cDNA encoding BGL
from P. chrysosporium strain OGC101 reported by
Li and Renganathan.11) The nucleotide sequence was
and put on a protein sequencer (Applied Biosystems
model 491 cLC) in the pulsed-liquid mode.
Enzyme assay. All enzyme activities were assayed
in 100 m
M
sodium acetate buŠer, pH 4.5, at 30
9
C.
deposited in the DDBJ EMBL GenBank with the
W
W
Cellobiose hydrolyzing activity was assayed by
monitoring of the release of glucose from cellobiose.
accession no. AB081121. Both mature BGLs of
strain OGC101 and K-3 consisted of 802 amino acid
residues. However, eight amino acid residues were
diŠerent in the BGL of strain K-3 compared with that
of strain OGC101. The nucleotide sequence was iden-
tiˆed by analysis of clones derived from diŠerent
rounds of PCR, so these replacements were not PCR
errors but arose because of the diŠerence in strains.
The expression vector pPIC9K, which contained
the P. chrysosporium BGL cDNA downstream from
The enzyme solution (5
m
l) was incubated with
l for
1–20 m cellobiose in the ˆnal volume of 50
M
m
15 min and the concentration of glucose was esti-
mated by a Glucose CII-Test Wako (Wako Pure
Chemicals).
p
-Nitrophenyl-
activity was assayed by monitoring of the release of
-nitrophenol from
NPG as described previously.15)
The pH-dependence of wtBGL and BGL was exam-
ined by the same protocol, except that the incubation
was carried out in 100 m sodium acetate buŠer at
b
-
D
-glucoside ( pNPG) hydrolyzing
p
p
r
an alcohol oxidase I (AOXI) promoter and a-factor
secretion-signal sequence, was constructed and desig-
M
nated BGL-pPIC9K (Fig. 1). P. pastoris KM71 was

4
R. KAWAI et al.
Fig. 2. Activity Screening of Clones of Pichia pastoris Secreting
r
BGL.
Methylumbelliferone released was observed under UV light as
described in Materials and Methods. 1, Negative clone trans-
formed with pPIC9K; 2, positive clone transformed with BGL-
pPIC9K.
transformed with the vector and His+ transformants
were screened for. The transformants with multico-
pies of BGL cDNA were then selected as geneticin-
disulfate-resistant colonies. When the concentration
of geneticin disulfate in the medium was 0.25 mg ml,
W
hundreds of colonies appeared on the plates. For that
reason, 10 large colonies were selected from plates
with higher concentration of geneticin disulfate
Fig. 3. Time Course Changes of BGL Activity in the Culture Su-
pernatant of a Single-batch (A) and Cycle System (B).
BGL activity was monitored in terms of pNPG as described in
Materials and Methods. The arrow indicates the time when the
medium was changed. Values are means of four replicates and
(0.5–3.0 mg ml). The selected clones were trans-
W
ferred to a YPM plate and the secretion of
monitored in terms of the release of methylumbel-
liferone from MeU G. Bright ‰uorescence due to
rBGL was
error bars show the S.D., and have been multiplied by 10„3
.
b
methylumbelliferone was clearly observed around the
selected clone, but the clone with the pPIC9K vector
without BGL cDNA showed no ‰uorescence (Fig. 2).
This result suggested that the extracellular BGL
were 14,300 and 17,400 U l, respectively, and the
W
speciˆc activity was identical 52 U mg both times.
W
This result suggested that cycle cultivation main-
tained the productivity and speciˆc activity of
r
BGL.
activity was
r
BGL under these conditions. In the
The speciˆc activity of rBGL in the cycle culture was
screening, one of geneticin-disulfate-resistant clones
was not ‰uorescent, suggesting that activity screening
37 times that of wtBGL in the culture supernatant of
P. chrysosporium (1.4 U mg).
W
is excluded clones unable to secrete
medium.
rBGL in the
Physical properties of rBGL
The cellulolytic culture of P. chrysosporium con-
tains many proteins, so at least ˆve steps of column
chromatography were needed to purify wtBGL.14) In
contrast, as the culture supernatant of P. pastoris
Production of rBGL by Pichia pastoris
BGL activity in the induction medium was detected
at day 1 and it reached 28,500 U l at day 7
W
(Fig. 3(A)). In our previous study, BGL activity was
contained mainly rBGL, anion exchange and gel-
about 750 U l after 16 days of cultivation in the cel-
ˆltration puriˆed the enzyme to give a single band on
SDS-PAGE (Fig. 4). Although the molecular mass of
wtBGL was 116 kDa, almost identical to the molecu-
lar mass (114 kDa) reported previously,14) that of
W
15)
lulolytic culture of P. chrysosporium
,
so that the
production yield of BGL in the P. pastoris system
r
was 38 times that of wtBGL. However, in single-
batch cultivation, the speciˆc activity (activity total
r
BGL was estimated to be 133 kDa. The amino acid
sequence of BGL includes multiple N- and O-
glycosylation sites. Incubation of BGL with -man-
W
protein) in the culture supernatant at day 5,
53 U mg, decreased to 48 U mg by day 6. The
r
a
W
W
decrease seemed to be due to proteolytic activity or
production of other proteins, so a cycle system with 4
days of cultivation was done twice (Fig. 3(B)). The
nosidase and endoglycosidase H resulted in an 18
kDa decrease in molecular mass. Such treatment
removes only N-glycan from protein, so molecular
production of
r
BGL in the ˆrst and second cultures
mass between wtBGL and rBGL might diŠer because

Recombinant Phanerochaete chrysosporium
b-Glucosidase
5
Fig. 4. SDS-PAGE of Puriˆed
rBGL and wtBGL.
Some 1.5 g of puriˆed BGL or wtBGL was put on a 12z
m
r
polyacrylamide gel as described in Materials and Methods. Lane
S, Prestained SDS-PAGE Standards, broad range (Bio-Rad
Laboratories; units, kDa); lane 1, rBGL; lane 2, wtBGL.
of O-glycosylation as well as N-glycosylation. The N-
terminal amino acid sequence of the puriˆed enzyme,
Tyr-Val-Glu-Phe-Gln-Ser-Gly-Leu-Tyr-Gln, corres-
ponds to a secretion-signal sequence (ˆrst four
residues) and N-terminal sequence of mature BGL
from P. chrysosporium (last six residues) deduced
from the nucleotide sequence of the cDNA.11) This
ˆnding conˆrms that the puriˆed enzyme is the
Fig. 5. Plot of Substrate Concentration Versus Velocity of Cel-
lobiose (A) and NPG (B) Hydrolysis by BGL ( ) and wtBGL
).
Cellobiose and
from the release of glucose and
p
r

(

p
NPG hydrolyzing activities were estimated
-nitrophenol, respectively, as
p
described in Materials and Methods.
product of the BGL cDNA from P. chrysosporium
.
Table 1. Steady-state Kinetic Parameters of
r
BGL and wtBGLa
wtBGL
Kinetic properties of rBGL
Steady-state kinetic properties of rBGL were com-
r
BGL
Substrate
Km
(m
kcat
Km
(m
kcat
M
)
(s„1
)
M
)
(s„1
)
pared with those of wtBGL in terms of cellobiose
and NPG hydrolyzing activities (Fig. 5). The plots
p
±
± ± ±
29.8 1.4 4.06 0.85 27.5 1.8
Cellobiose 5.05 0.67
of substrate concentration versus velocity were both
showed a simple hyperbola, obeying the Michaelis-
Menten equation. The kinetic parameters obtained
from Fig. 5 are summarized in Table 1. The Km and
± ± ± ±
0.198 0.019 103.1 2.6 0.117 0.006 96.2 0.9
p
NPG
a
The experimental results shown in Fig. 5 were ˆtted to the Michaelis-
Menten equation as described in Materials and Methods. Values are
means and S.D.s.
kcat for
pNPG hydrolysis of wtBGL were similar to
the values reported previously.4,14) Those values for
± ±
wtBGL were estimated as 2.20 0.30 and 2.22
cellobiose and
p
NPG hydrolysis by
r
BGL were iden-
M
, respectively (mean S.D.). Inhibition by
±
tical to those by wtBGL, so the diŠerence in molecu-
0.35
gluconolactone during hydrolysis of
wtBGL is competitive, according to previous reports,
and the is of gluconolactone estimated in this study
were similar to the values reported previously
).4,14) All of these results suggested that
BGL
expressed in P. pastoris functioned as well as did
wtBGL produced by P. chrysosporium
m
lar mass had little if any eŠect on the kinetic activity.
p
NPG by
Both wtBGL and rBGL activity showed the same pH
dependence, with the optimum pH of about 4.5 (not
shown).
K
The eŠects of gluconolactone on
ing activity of BGL and wtBGL are shown in Fig. 6.
The NPG hydrolyzing activities of both enzymes
p
NPG hydrolyz-
(¿
4
m
M
r
r
p
.
were similarly inhibited in proportion to the
gluconolactone concentration and the inhibition
pattern was of the competitive type. Ki values of
P. chrysosporium BGL has N-terminal cellulose
binding domain in addition to the catalytic domain,11)
and these domains can be separated by papain treat-
ment.14) Therefore, the cellulose binding ability of
gluconolactone for
pNPG hydrolysis by rBGL and

6
R. KAWAI et al.
Acknowledgments
We thank Dr. Emi Murayama for technical sup-
port in protein sequencing and for discussions of this
work. This work was supported by a Grant-in-Aid
for Scientiˆc Research to M.S. (No. 14360094) from
the Japanese Ministry of Education, Science, and
Culture.
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