Glucoamylase originating from Schwanniomyces occidentalis is a typical α-glucosidase

Article abstract of DOI:10.1271/bbb.69.1905

A starch-hydrolyzing enzyme from Schwanniomyces occidentalis has been reported to be a novel glucoamylase, but there is no conclusive proof that it is glucoamylase. An enzyme having the hydrolytic activity toward soluble starch was purified from a strain of S. occidentalis. The enzyme showed high catalytic efficiency (k_cat/K_m) for maltooligosaccharides, compared with that for soluble starch. The product anomer was α-glucose, differing from glucoamylase as a β-glucose producing enzyme. These findings are striking characteristics of α-glucosidase. The DNA encoding the enzyme was cloned and sequenced. The primary structure deduced from the nucleotide sequence was highly similar to mold, plant, and mammalian α-glucosidases of α-glucosidase family II and other glucoside hydrolase family 31 enzymes, and the two regions involved in the catalytic reaction of α-glucosidases were conserved. These were no similarities to the so-called glucoamylases. It was concluded that the enzyme and also S. occidentalis glucoamylase, had been already reported, were typical α-glucosidases, and not glucoamylase.

Full text of DOI:10.1271/bbb.69.1905

Biosci. Biotechnol. Biochem., 69 (10), 1905–1913, 2005
Glucoamylase Originating from Schwanniomyces occidentalis
Is a Typical ꢀ-Glucosidase
1
1
1
1
Fumiaki SATO, Masayuki OKUYAMA, Hiroyuki NAKAI, Haruhide MORI,
1
1;2;y
Atsuo KIMURA, and Seiya CHIBA
1
Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
Division of Food and Nutrition Sciences, Graduate School of Dairy Science, Rakuno Gakuen University,
2
Ebetsu 069-0851, Japan
Received May 6, 2005; Accepted July 15, 2005
A starch-hydrolyzing enzyme from Schwanniomyces
occidentalis has been reported to be a novel glucoamy-
lase, but there is no conclusive proof that it is
glucoamylase. An enzyme having the hydrolytic activity
toward soluble starch was purified from a strain of
S. occidentalis. The enzyme showed high catalytic
efficiency (kcat=Km) for maltooligosaccharides, com-
pared with that for soluble starch. The product anomer
was ꢀ-glucose, differing from glucoamylase as a ꢁ-
glucose producing enzyme. These findings are striking
characteristics of ꢀ-glucosidase. The DNA encoding the
enzyme was cloned and sequenced. The primary
structure deduced from the nucleotide sequence was
highly similar to mold, plant, and mammalian ꢀ-
glucosidases of ꢀ-glucosidase family II and other gluco-
side hydrolase family 31 enzymes, and the two regions
involved in the catalytic reaction of ꢀ-glucosidases were
conserved. These were no similarities to the so-called
glucoamylases. It was concluded that the enzyme and
also S. occidentalis glucoamylase, had been already
reported, were typical ꢀ-glucosidases, and not gluco-
amylase.
lase (GAM).7–9) The amino acid sequence deduced from
the GAM1 gene represented a high level of similarity to
those of human lysosomal ꢀ-glucosidase and rabbit
intestinal sucrose-isomaltase, and no similarity to those
of other fungal glucoamylases. Nevetheless, it has been
concluded that S. occidentalis glucoamylase is a novel
type of glucoamylase that might derive from the same
ancestral gene as those of mammalian ꢀ-glucosidase,
6
)
isomaltase, and sucrase. In addition, indispensable
confirmation, on the basis of the product anomer and the
substrate specificity, has hardly been made as to whether
S. occidentalis glucoamylase is glucoamylase or ꢀ-
1
0)
glucosidase. In the classification of glycosyl hydro-
1
1)
lases, S. occidentalis glucoamylase is grouped into the
family 31 and not into the family 15, implying the
possibility that the glucoamylase is ꢀ-glucosidase.
Therefore, we have looked upon the glucoamylase as
ꢀ-glucosidase, because it exhibits high similarity in the
amino acid sequences to many ꢀ-glucosidases from
1
2)
fungi, plants, and mammals. It seems significant to
ascertain whether the enzyme really is glucoamylase or
not.
Glucoamylase (EC 3.2.1.3, 1,4-ꢀ-D-glucan glucohy-
drolase) and ꢀ-glucosidase (EC 3.2.1.20, ꢀ-D-glucoside
glucohydrolase) are a group of typical exo-type hydro-
lases, which catalyze the splitting of ꢀ-glucosidic
linkage at the non-reducing terminal side of the
substrates, such as ꢀ-glucosides, maltooligosaccharides,
and ꢀ-glucans, to release D-glucose. Various types of ꢀ-
Key words: Schwanniomyces occidentalis; glucoamy-
lase; ꢀ-glucosidase; product anomer analy-
sis
It has been reported that yeast genus Schwanniomyces
spp. secretes ꢀ-amylase and glucoamylase to hydrolyze
soluble starch to glucose using the extracellular en-
zymes. The two ꢀ-amylase genes (AMY1 and AMY2:
1
2)
glucosidases
are widespread in microorganisms,
plants, mammals, and insects. On the other hand, the
distribution of glucoamylase is limited only to micro-
organisms. The two enzymes are not, apparently,
distinguished in view of releasing only D-glucose, but
can be essentially differentiated by the anomer type of
the product D-glucose. ꢀ-Glucosidase produces ꢀ-D-
glucose by retaining the anomeric configuration of
substrate, and glucoamylase produces ꢁ-D-glucose by
inverting. The two enzymes are quite different from
1
–4)
SWA1 and SWA2, respectively)
S. occidentalis and S. castellii.
were cloned from
As for glucoamylase, the S. occidentalis GAM1
gene,5,6) encoding a starch-hydrolyzing enzyme, was
cloned and expressed in Saccharomyces cerevisiae. The
enzymes of Schwanniomyces spp., showing hydrolytic
activities not only on soluble starch but also on maltose
and isomaltose, have long been designated glucoamy-
y
To whom correspondence should be addressed. Fax: +81-11-706-2808; E-mail: schiba@abs.agr.hokudai.ac.jp

1906
F. SATO et al.
each other in the reaction mechanism.
Protein determination and amino acid sequencing.
The enzyme concentration was spectrophotometrically
determined on the basis of the value that E1 at 280 nm
The present paper describes the purification, charac-
terization, substrate specificity, and product anomer of an
original enzyme purified from S. occidentalis. Addition-
ally, the similarity in the primary structures between the
original enzyme and S. occidentalis glucoamylase al-
ready reported, and small discrepancies in enzymatic
properties between the original enzyme and the recombi-
nant enzyme expressed in Pichia pastris, are discussed
on the basis of the structure of Escherichia coli ꢀ-
xylosidase, which is the only enzyme in the family 31
glycoside hydrolases whose three-dimensional structure
1
%
cm
was 13.1, which was calculated from the amino acid
contents of protein hydrolyzate (6 M HCl, 24 h, and
ꢀ
110 C) determined using JEOL JLC/500V equipped
with the ninhydrin detection system. In the purification
steps, however, the value, based on the assumption that
1
1
%
E
at 280 nm was 10, was also used. The analysis of
cm
amino acid sequence in the N-terminal of the purified
enzyme was carried out on an Applied Biosystems
model 477A protein sequencer with an on-line phenyl-
thiohydantoin analyzer (model 120A; Applied Biosys-
tems Inc., Foster City, CA, U.S.A.).
1
3,14)
has recently been solved.
The data on the product
anomer and the substrate specificity provide conclusive
evidence that S. occidentalis glucoamylase is a typical
ꢀ
-glucosidase, and not a so-called glucoamylase.
Estimation of molecular weight. To estimate the
molecular weight (M ) of the purified enzyme, sodium
r
Materials and Methods
dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS–PAGE) was carried out according to the method
of Laemmli, using 7.5% gel and the standard proteins
(Invitrogen Corp., Carlsbad, CA, U.S.A.): myosin H-
chain (Mr, 200,000), phosphorylase b (Mr, 97,400),
bovine serum albumin (Mr, 68,000), ovalbmin (Mr,
1
7)
Yeast strain. The yeast strain used in this study was
Schwanniomyces occidentalis ATCC26074. The use of
S. occidentalis ATCC26076 was desirable, but unfortu-
nately we could not obtain that strain.
43,000), and carbonic anhydrase (Mr, 29,000). Proteins
Materials. Arabitol, maltose (SP-grade), p-nitrophen-
yl ꢀ-glucoside, p-nitrophenyl ꢀ-maltoside, trehalose,
and soluble starch (SP-grade) were purchased from
Nacalai Tesque Chemical Inc. (Kyoto, Japan). To
remove possible impurities, maltose was further purified
by repeated recrystallization, and soluble starch was
washed with ice-cold water. ꢀ-Glucose was purchased
from Wako Pure Chemical Ind., Ltd. (Osaka, Japan);
were stained with Rapid CBB KANTO (Kanto Kagaku
Co., Tokyo, Japan).
Gas-liquid chramatography (GLC). GLC of anomeric
forms of the enzyme reaction products was conducted by
the novel quantitative method described in our previous
1
8,19)
papers.
The reaction mixture consisting of 120 ml of
6.25 mM p-nitrophenyl ꢀ-maltoside or maltotriose and
ꢁ
-glucose, from Sigma Chemical Co. (St. Louis, MO,
30 ml of the original enzyme (0.95 mg) in 25 mM sodium
acetate buffer (pH 5.0) was incubated at 25 C for 4 min.
ꢀ
U.S.A.). Malto-triose, -tetraose, -pentaose, -hexaose,
and -heptaose, and isomaltose were kindly supplied by
Nihon Shokuhin Kako Co., Ltd. (Fuji, Japan). Nigerose
and kojibiose were enzymatically synthesized through
The reaction mixture was immediately frozen with
ꢀ
liquid nitrogen and then lyophilized at ꢁ50 C. The
freeze-dried sample was converted to the trimethylsilyl
(TMS) compound by incubating with 0.05 ml of a
1
5)
transglucosylation by buckwheat ꢀ-glucosidase. High
ꢀ
ꢀ
maltotetraose syrup (TETRUP ) was kindly supplied by
TMSI-H reagent at 80 C for 10 min, and then subjected
Hayashibara Biochemical Lab., Inc. (Okayama, Japan).
Polypentone and yeast extract were purchased from
Seikagaku Co. (Tokyo, Japan); endoglycosidase F (N-
glycosidase F-freetype), from Boeringer Mannheim
Biochemia (Mannheim, Germany); and trimethylsilyla-
tion reagent (TMSI-H) containing hexamethyldisilazane
and trimethylchlorosilane in pyridine, from GL Sciences
Inc. (Tokyo, Japan).
to GLC. The TMS derivative of arabitol was used as the
internal standard. The TMS derivatives of ꢀ- and ꢁ-
glucose and the equilibrated maltose were also used as
the standard sugars. The column temperature was
ꢀ
ꢀ
elevated at a rate of 10 C per min from 150 to 270 C.
ꢀ
Culture media. Yeast cells were grown at 21 C for
21 h with shaking in synthetic media, a modified PYN
7
,20)
medium
extract, 0.4% KH PO , 0.2% MgSO 7H O, 0.2%
containing 0.7% polypeptone, 0.6% yeast
.
(NH ) SO , 2% CaCO , and 4% TETRUP . Three
Enzyme assay. Maltase activity was determined by the
glucose oxidase method16) with our modification, using
Glucose AR-II (Wako Pure Chemical Ind., Ltd.). The
standard reaction mixture, containing 0.2 ml of 0.5%
maltose, 0.2 ml of 0.1 M sodium acetate buffer (pH 5.0),
and 0.1 ml of the enzyme solution in a total 0.5 ml
2
4
4
2
ꢀ
4
2
4
3
liters of the culture medium was centrifuged at
22;000 ꢂ g for 60 min, and the supernatant (2,200 ml)
was recovered as the crude enzyme solution.
ꢀ
volume, was incubated at 37 C. One unit of the enzyme
Enzyme. An enzyme was purified from the culture
supernatant (2,200 ml). To the supernatant (the crude
enzyme solution), solid ammonium sulfate was slowly
added with stirring up to 100% saturation (about 70 g of
activity was defined as the amount of enzyme that
hydrolyzed 1 mmol of maltose per min under the
conditions just described.

Schwanniomyces occidentalis Glucoamylase Is ꢀ-Glucosidase
1907
ammonium sulfate per 100 ml of the solution), and the
turbid solution was allowed to stand overnight in a cold
room. The resulting precipitate was collected by
centrifugation at 25;000 ꢂ g for 60 min, and then
dissolved in 350 ml of 0.01 M sodium acetate buffer
three times using KOD DNA polymerase (TOYOBO
Biochem., Tokyo, Japan) with high fidelity. The ampli-
fied DNA fragment was ligated into pBluescript II SK
(Stratagene, La Jolla, CA, U.S.A.) at the EcoRV site,
and the generating plasmid pBS-SOG was sequenced by
using 310 Genetic analyzer (Applied Biosystems, Foster
City, CA, U.S.A.).
(pH 4.3). The solution was thoroughly dialyzed against
the same buffer.
The dialyzed solution was applied to a DEAE-
Sepharose CL-6B (2:6 ꢂ 31 cm) equilibrated with the
same buffer (pH 4.3). Linear gradient elution was
carried out by increasing the concentration of sodium
chloride (0 to 0.5 M) in the same buffer; the flow rate was
Production of the recombinant enzyme. The pBS-
SOG was amplified by PCR to construct an expression
0
vector in Pichia pastoris, using the forward primer, 5 -
0
ATT ATA GAA TTC ACC ATG ATT TTT C-3
(containing a EcoRI site, bold characters), and the
2
4
4 ml/h, and each fraction, 10 ml (fractions 1 to 66) or
ml (fractions 67 to 178). The active fractions (nos. 143
0
reverse primer, 5 -ATG TTA GGG CCC CCA AGT
0
to 149) were combined and concentrated up to 3 ml. The
concentrate (3 ml) was subjected to gel-chromatography
in a Bio-Gel P-200 column (2:1 ꢂ 40 cm) equilibrated
with 0.01 M sodium acetate buffer containing 0.2 M
sodium chloride (Fig. 1). The active fractions (nos. 33
to 45) were recovered and used as a purified enzyme
preparation. The enzyme gave a broad and smeary band
on the usual polyacrylamide gel electrophoresis. Such a
band is often observed in the electrophoresis of
glycoprotein. Therefore, the elution pattern is shown in
Fig. 1, so as to confirm the homogeneity of the enzyme.
AAT GGT-3 (containing an ApaI site, bold characters).
The resulting fragment was digested by corresponding
restriction enzymes and ligated into pPICZA (Invitro-
gen) at corresponding sites, developing the plasmid
pPICZA-SOG encoding the S. occidentalis enzyme,
with the His ꢂ 6 sequence added to its C-terminal
end. The pPICZA-SOG was propagated in E. coli DH
5ꢀ, purified by a method with alkaline-lysis of the
E. coli cell, and digested with BglII to generate a linear-
form plasmid. The linear pPICZA-SOG (10 mg) was
electroporated into P. pastoris GS115 cells (Invitogen)
according to the supplier’s protocol, and positive
colonies were selected. The colonies selected were
cultured to check for the enzyme secretion by 3-ml
Cloning of a glycoside hydrolase family 31 enzyme
gene. The gene encoding the enzyme was amplified
from genomic DNA of S. occidentalis ATCC26074 by
ꢀ
minicultures grown overnight at 30 C in BMGY
0
PCR, using the forward primer 5 -GAA ATT TTA AAA
0
(13.4 g/l yeast nitrogen base without amino acids, 20
g/l peptone, 10 g/l yeast extract, 0.5% glycerol, 0.4 mg/
l biotin, and 0.1 M potassium phosphate, pH 6.0) in 50-
ml tubes. Cells were harvested, resuspended in 12 ml
BMMY (13.4 g/l yeast nitrogen base without amino
acids, 20 g/l peptone, 10 g/l yeast extract, 0.5% meth-
anol, 0.4 mg/l biotin, and 0.1 M potassium phosphate,
0
0
TGA ACT CAT GAC TG-3 and the reverse primer 5 -
AAA ACT AAA ATG ATA TCA TTC AAA TAA T-3 ,
which were designed from the sequence of S. occiden-
talis ATCC26076 glucoamylase (GAM1) gene (acces-
sion no. M60207). The PCRs were done independently
ꢀ
pH 6.0), and incubated for 96 h at 30 C under vigorous
shaking. After centrifugation, culture supernatants were
tested for maltase activity, as described above.
Purification of the recombinant enzyme. A yeast
transformant was grown in BMGY (200 ml in a 500-ml
flask) up to 2.5 in OD600, and the cells were harvested by
centrifugation, resuspended in BMMY (200 ml in a 500-
ml flask), and the culture was continued for 70 h at
ꢀ
30 C. One ml of methanol was added every 24 h to
maintain induction. To the culture supernatant (190 ml),
ammonium sulfate was added up to 90% saturation and
ꢀ
the suspension was allowed to stand overnight at 5 C.
The resulting precipitant was collected by centrifuge
ꢀ
(
20;000 ꢂ g, 15 min, 4 C) and dissolved in 20 mM
sodium phosphate buffer (pH 7.5) containing 500 mM
sodium chloride, and loaded onto a Ni-Sepharose Fast
Flow (Amersham Bioscience AB) column prepared
according to the method in the supplier’s manual, and
equilibrated with the same buffer. The column was
washed with 20 mM sodium phosphate buffer (pH 7.5),
followed by washing with the same buffer (pH 6.0),
Fig. 1. Gel Chromatography of the Enzyme from Schwanniomyces
occidentalis on a Bio-Gel P-200 Column.
Enzyme solution, 3 ml; column, 2:1 ꢂ 40 cm; equlibrium, 0.01 M
sodium acetate buffer (pH 5.0) containing 0.2 M NaCl; elution, the
same buffer; flow rate, 6 ml/h.

1908
F. SATO et al.
Table 1. Summary for Purification of Schwanniomyces occidentalis Enzyme
Protein
mg)
Activity
(unit)
Specific Activity
(unit/mg)
Purification
(-fold)
Procedure
(
Culture medium
Ammonium sulfate
2,200a
3,000
3,300
1.3
2.1
1
1
,600a
1.6
(100% saturation)
DEAE-Sepharose CL-6B
Bio-Gel P-200
290a
92b
3,000
2,500
10
27
7.7
16
aCalculated under the assumption that E
1%
1
at 280 nm was 10.
cm
bCalculated according to the fact that E 11 % at 280 nm was 13.1.
cm
containing 500 mM sodium chloride. The absorbed
components were eluted with 20 mM sodium phosphate
buffer (pH 4.5) containing 500 mM sodium chloride. The
active fractions containing the enzyme were pooled and
dialyzed against 20 mM sodium acetate buffer (pH 4.5).
The dialyzed solution (3.2 ml) was used as the recombi-
ꢀ
nant enzyme. All purification steps were done at 4 C.
Biochemical assay. Optimum pH was examined with
0.2% malose and 4.6 nM original enzyme or 1.9 nM
recombinant enzyme in McIlvaine buffer (pH 2.5 to
ꢀ
8
(
.0), at 37 C for 5 min. For pH stability, 13.8 nM
original) or 19 nM (recombinant) of the enzyme was
ꢀ
incubated at 4 C for 24 h in 25 mM Britton–Robinson
buffer at various pHs from 2.5 to 10.7. The pH-treated
sample was added to a solution of 0.2% maltose and
Fig. 2. SDS–PAGE of the Purified Original (A) and the Recombinant
(
B) Enzymes.
Lane 1 in A and B, molecular marker proteins; lane 2 in A,
ꢀ
incubated at 37 C in 200 mM sodium acetate buffer
purified original enzyme; lane 3 in A, endogylcosidase F-treated
enzyme; N-terminal amino acid sequences of a, b and c, APAXXIG,
(
pH 4.5). For thermal stability, 6.8 nM (original) or 8 nM
recombinant) of the enzyme was incubated for 15 min
APAXXIG, and DGIWA, respectively; lane 2 and
3 in B,
(
in 75 mM sodium acetate buffer (pH 4.5) at various
temperatures from 25 to 60 C, followed by measure-
recombinant enzyme and endglycosidase F-treated recombinant
enzyme.
ꢀ
ment of the activity at pH 4.5. The kinetic constants
were calculated by Lineweaver–Burk plot, Hanes–
Woolf plot, and fitting to the Michaelis–Menten equa-
tion by nonlinear regression using the computer program
Curve Expert 1.3, in which the initial velocities were
purified enzyme preparation, which appeared to be
chromatographically homogeneous (Fig. 1), was sub-
jected to SDS–PAGE. As shown in Fig. 2, however, the
enzyme preparation was separated into two components
measured under various concentrations from 0:5 ꢃ Km to
having the relative molecular masses (M s) of 130,000
r
ꢀ
2
ꢃ Km in 40 mM sodium acetate buffer (pH 4.5) at 37 C.
and 69,000. Separation of the two components as active
enzymes was attempted, but was unsuccessful by all
chromatographic methods.
The molecular weights of the two components were
diminished by treatment with endoglycosidase F, im-
plying that the components were glycoproteins; the
The enzyme concentrations used were made to vary
from 3.8 to 38 nM. Liberated glucose was measured by
Glucose AR-II.
Results and Discussion
major component gave an M of 126,000, and the minor
r
Purification of the enzyme from S. occidentalis cul-
ture medium
one, an M of about 66,000. Especially, the minor
r
component indicated two sharp bands. The proteins
corresponding to the three bands were transferred on
The purification procedure for the original enzyme is
summarized as maltase in Table 1, in which the total
activity in the procedure of salting-out with ammonium
sulfate is higher than that in the crude enzyme solution.
The reason why the activity increased by the procedure
is obscure, but it seems reasonable to assume that an
unknown inhibitor against maltase might be removed
from the culture media by salting-out with ammonium
sulfate, although the possibility cannot be ruled out of an
inhibitor against glucose oxidase in Glucose AR-II. The
2
1)
PVDF membrane
from SDS–PAGE gel, and then
subjected to amino acid sequence analysis. The N-
terminal amino acid sequence for the band of the major
component (band a in lane 3 of Fig. 2A, M , 126,000)
r
was identified to be APAXXIG, and those for the upper
and lower bands of the minor component (bands b and c
r
in lane 3 of Fig. 2A, M , 66,000), were identified to be
APAXXIG and DGIWA, respectively. The sequence of
APAXXIG agreed with an internal sequence, 21-

Schwanniomyces occidentalis Glucoamylase Is ꢀ-Glucosidase
1909
QAAPASSIGSSAS-, in the primary structures deduced
from the DNA sequence and also from the ATCC26076
in the upper stream of Trp325 up to N-terminal Met1
was entirely identical with that of the strain ATCC26076
glucoamylase (accession no. M60207), but in the lower
stream a few substitutions were found in the two strains.
Almost all mutations are silent, but variants of A1682C,
C1927G, and G2575T lead to amino acid substitutions,
Tyr547 to Ser, Arg629 to Gly, and Ala845 to Ser.
Moreover, a six-nucleotide insertion causes the generat-
ing of two amino acid gaps and some amino acid
5
,6,10)
glucoamylase (GAM1) gene.
The -XX- amino acid
residues, corresponding to -SS- in the deduced amino
acid sequence, could not be identified by protein
sequencer, probably because the serine residues were
considered to be modified by the sugar chain of the O-
linked type. The sequence of DGIWA also agreed with
an internal sequence, 465-PFDGIWADMN-, in the
primary structure of S. occidentalis enzyme, as describ-
ed below. These findings imply that the minor compo-
nent consisting of two polypeptides may be generated
from the major component by limited proteolysis. It is
thought that the two polypeptides maintain a three-
dimensional structure similar to the main component as
the active enzyme even after the limited proteolysis.
Consequently, the major and minor components could
not be separated into each other without inactivation.
The cleavage site corresponds to the loop region
substitution,
436-PGYTVFPDFLAENIQEYMN-454
(ATCC26074) and 436-QVTLFSRFLSRKHSDMD-452
(ATCC26076).
The alignment with E. coli ꢀ-xylosidase as an enzyme
in the family 31 glycoside hydrolases shows that the
different residues must be located far from an active
site pocket, as stated below (Fig. 3). Gly629 (26074)
corresponds to Gly471 (yicI) in E. coli ꢀ-xylosidase
residue, located in the back face of ðꢁ=ꢀÞ barrel of the
8
three-dimensional structure. Ser547 (26074) is situated
in an extra region which has been found only in fungi ꢀ-
glucosidases, and Ser845 (26074), in the C-terminal
domain of the ꢀ-xylosidase. The region of Pro436 to
Asn454 (26074) is comparable to that of Gln381 to
Asp400 (yicI) in E. coli ꢀ-xylosidase, which is located
in a portion of the protruded domain between ꢁ3 and ꢀ3
of the catalytic domain, and is deviated from the active
site. Therefore, these substitutions would not affect the
catalytic activity, especially, the reaction mechanism,
such as retaining or inverting of product anomer.
between ꢀ3 and ꢁ4, the backside of ðꢁ=ꢀÞ barrel
8
(Met408-Asp411 of E. coli ꢀ-xylosidase), in the three-
dimensional structure of E. coli ꢀ-xylosidase, which is
the only available structure in glycoside hydrolase
family 31.1
3,14)
The region might be susceptible to
digestion by protease, since it is exposed to the
surrounding solution. Such limited proteolytic modifi-
2
2–24)
cations have been often found in ꢀ-glucosidases,
although their physiological and functional significance
has not been ascertained. Therefore, the enzyme
preparation containing a small amount of the enzyme
modified by limited proteolysis was used itself in the
experiment described below.
Production and purification of the recombinant
enzyme
Minicultures of P. pastoris transformations of resist-
ance to zeocin were analyzed for ꢀ-glucosidase secre-
tion by measuring maltase activity. Supernatants from
transformants contained 0:213 ꢄ 0:031 U/ml of ꢀ-glu-
cosidase. In large-scale cultures using 200 ml of in-
duction medium, the level of ꢀ-glucosidase was about
14.2 mg/l, estimated from the final specific activity of
ꢀ-glucosidase, 29.5 U/mg. The purified recombinant
enzyme was subjected to SDS–PAGE, and no limited
proteolysis was observed in the recombinant enzyme
(Fig. 2). This implies that protease involved in digestion
of the original enzyme was absent in P. pastoris culture
supernatant. The N-terminus of the recombinant enzyme
was APAXXIGXX, the same as that of the original
enzyme. That is, the signal peptidases derived from
S. occidentalis and P. pastoris would recognize the
substrate in the same manner.
Molecular cloning of S. occidentalis enzyme DNA
The gene encoding the enzyme was amplified by
PCR using primers designed from the S. occidentalis
ATCC26076 glucoamylase gene (accession no.
M60207), and sequenced. The gene from start to stop
codon had 2,883 base, encoding a protein of 960 amino
acids with a calculated molecular mass of 10,6584.7 Da,
and no intron was found. Figure 3 shows the alignment
of the only amino acid sequence from the catalytic
domain to the C-terminal domain of the S. occidentalis
ATCC26074 enzyme, together with S. occidentalis
ATCC26076 glucoamylase and E. coli ꢀ-xylosidase.
It has been reported that, in glucoamylases, two Glu
residues function as the acid and base groups in the
1
2)
catalytic reaction.
However, the common regions
conserving such catalytic Glu residues could not be
found in the primary structures of either S. occidentalis
enzyme. The amino acid sequence showed high sim-
ilarity with those of ꢀ-glucosidases in glycoside hydro-
lase family 31 enzymes, and two invariant amino acid
residues, Asp472 and Asp640, which function as the
catalytic nucleophile and acid/base, respectively, are
conserved.2 Therefore, the enzyme must hydrolyze or
generate O-glucosidic bond through a retaining mech-
anism. The sequence of the strain ATCC26074 enzyme
Effects of pH and temperature
The effects of pH and temperature on enzyme activity
were examined using maltose as substrate. The pH-
optimum of the original enzyme was observed in a broad
pH range from 3.5 to 5.0, and that of the recombinant
enzyme was also observed in a broad pH range from 3.7
to 4.7. The original enzyme was stable in a range from
pH 3.5 to 8.5, but appeared to be labile below pH 3.0.
5)

1910
F. SATO et al.
Fig. 3. Multiple Alignment for Catalytic and C-Terminal Domains of Glycoside Hydrolase Family 31 ꢀ-Glycosidases, from E. coli (yicI),
S. occidentalis ATCC 26074 (26074), and S. occidentalis ATCC 26076 (26076).
Structural features are represented by squiggles (ꢀ-helices), arrows (ꢁ-strands), and T (ꢁ-turns) with element of the ðꢁ=ꢀÞ barrel core
8
numbered. Residues conserved among the three enzymes are expressed by reversal letters, and similar amino acids are boxed. The catalytic
acidic-residues and Thr642 (26074) focused in context are indicated by closed and open triangles, respectively. The polymorphisms between
ATCC 26074 and 26076 are displayed by double underlines. The N-terminus of the minor component of the original enzyme is indicated by
dashed underline.

Schwanniomyces occidentalis Glucoamylase Is ꢀ-Glucosidase
1911
Table 2. Kinetic Parameters of Original and Recombinant Enzymes for Hydrolysis of Various Substrates
Original
Recombinant
kcat
Km
kcat=Km
kcat
ꢁ1
Km
kcat=Km
Substrate
Maltose
Maltotriose
ꢁ
1)
(sꢁ1ꢃmM
ꢁ1
(sꢁ1ꢃmM
ꢁ1
(
s
(mM)
)
(s
)
(mM)
)
190
230
180
200
140
140
140
0.096
0.064
0.051
0.087
0.078
0.11
0.66
0.48
0.12
0.61
3.0
2,000
3,590
3,500
2,300
1,800
1,270
210
210
330
250
180
190
190
220
0.13
1,600
4,500
6,000
4,200
2,600
2,300
1,100
0.074
0.042
0.043
0.073
0.083
0.20
Maltotetraose
Maltopentaose
Maltohexaose
Maltoheptaose
Soluble starch
pNP ꢀ-glucoside
pNP ꢀ-maltoside
Kojibiose
8.0
16.7
2,170
54.1
10.2
ꢅ
0.62
ꢅ
16.5
ꢅ
260
33
—
97
—
—
0.76
3.3
128
54.5
117
Nigerose
Isomaltose
59
130
19.7
81.2
180
140
1.6
1.2
ꢅ
Not measured.
The recombinant enzyme was stable in a range from
pH 3.1 to 8.1.
Both of the original and the recombinant enzymes
þ3. It would be significant also to check the substrate
specificity to learn whether the enzyme is glucoamylase
or ꢀ-glucosidase. The distinction between glucoamylase
and ꢀ-glucosidase has been explained by the substrate
specificities for maltooligosaccharides and soluble
ꢀ
were stable up to 45 C, but lost the activity completely
by incubation at 60 C for 15 min.
ꢀ
1
2)
These results show that there are a little or no
differences in the effects of pH and temperature between
the original and recombinant enzymes.
starch. In the case of ꢀ-glucosidase, the molecular
activity kcat value for the smallest substrate such as
maltose in a series of maltooligosaccharides is not much
different from the values for other maltooligosacchar-
ides. In the case of glucoamylase, however, the kcat
values are highly dependent on the polymerization
degree for substrate, resulting in remarkable differences
in the values between maltose and other maltooligosac-
Substrate specificity of the enzyme
The kinetic parameters for the hydrolysis of various
substrates by the original and the recombinant enzymes
are listed in Table 2. The tendencies of substrate
specificity toward maltooligosaccharides, glucobioses,
and soluble starch were similar in the original and the
recombinant enzymes. Neither enzyme favored soluble
starch, as compared with maltooligosaccharides. The
enzymes showed relatively low Km values for gluco-
bioses such as kojibiose, nigerose, and isomaltose,
having ꢀ-1,2-, ꢀ-1,3-, and ꢀ-1,6-glucosidic linkages,
respectively. The noteworthy difference between the
original and recombinant enzymes was observed in
hydrolysis of maltotetraose and maltopentaose, for
1
2)
charides,
and the catalytic efficiency (kcat=Km) for
soluble starch is overwhelmingly large in comparison
with any other substrates. The result of substrate
specificity analysis strongly suggests that the enzyme
is not glucoamylase, but is ꢀ-glucosidase.
Anomeric form of glucose liberated from substrates
by the enzyme
Determination of the anomeric configuration of the
product is significant for understanding the reaction
mechanism of glycosidases. The anomeric form of
glucose produced from p-nitrophenyl ꢀ-maltoside and
maltotriose by the original enzyme was examined by
which the k =Km values of the ꢀ-glucosidase from
cat
P. pastoris were 1.7- and 1.8-fold higher, respectively,
than the original enzyme from S. occidentalis (Table 2).
Additionally, the kcat=Km values of recombinant enzyme
for maltotetraose were higher than that for maltotriose,
meaning that, unlike the original enzyme, the recombi-
nant enzyme possess positive subsite affinity at subsite
þ3. This variation appears to be caused by a difference
in post-translational modification with a sugar chain.
According to the three-dimensional structure of E. coli
1
8,19)
GLC.
The chromatograms are shown in Figs. 4A
and B. ꢀ-Glucose, p-nitrophenyl ꢀ-glucoside, and a
trace amount of ꢁ-glucose, which appeared to occur
through sponteneous mutarotation of the product ꢀ-
glucose, were detected as the products from p-nitro-
phenyl ꢀ-maltoside (Fig. 4A). From maltotriose, ꢀ-
glucose, maltose equilibrated between ꢀ- and ꢁ-anom-
ers, and a small amount of ꢁ-glucose, which appears to
be somewhat greater in Fig. 4B than in Fig. 4A, were
detected as products. In this case, the maltose released
from the non-reducing terminal side of maltotriose, in
which ꢁ-anomer is usually greater than ꢀ-anomer, may
be additionally cleaved into ꢀ- and ꢁ-glucose. There-
fore, the amount of ꢁ-glucose detected in GLC appears
ꢀ
-xylosidase and the alignment between the primary
structures of the S. occidetalis enzyme and E. coli
-xylosidase, Asp233-Pro234 and Asn641-Thr642
ꢀ
(
ATCC26074) may be situated near the subsite þ3
and on the protein surface. Among them, Thr642 could
be modified by O-glycosylation, and thus the difference
in O-glycosylation might affect the affinity at subsite

1912
F. SATO et al.
Fig. 4. Gas-Liquid Chromatograms of TMS Derivatives of Products from p-Nitrophenyl ꢀ-Maltoside (A) and Maltotriose (B) by Shwanniomyces
occidentalis Enzymes.
C, D, E, and F are chromatograms of standard sugars. TMS derivative of arabitol was used as the internal standard. Column temperature was
ꢀ
ꢀ
ꢀ
elevated at a rate of 10 C/min from 150 to 270 C, and kept constant at 280 C. Symbols a, b, c, d, and e corresponds to ꢀ-glucose, p-nitrophenyl
-glucoside, ꢀ-maltose, and ꢁ-maltose, respectively.
ꢀ
to be over that of ꢁ-glucose occurring through sponte-
neous mutarotation from ꢀ-glucose. These results imply
that the anomeric configuration of the glucose produced
from p-nitrophenyl ꢀ-maltoside and maltotriose is ꢀ-
glucose, and that the enzyme is a typical ꢀ-glucosidase.
Jim e´ nez, A., Molecular structure of the SWA2 gene
encoding an AMY1-related ꢀ-amylase from Schwannio-
myces occidentalis. Curr. Genet., 24, 75–83 (1993).
Dohmen, R. J., Strasser, A. W. M., Dahlems, U. M., and
Hollenberg, C. P., Cloning of the Schwanniomyces
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(1990).
Naim, H. Y., Niermann, T., Kleinhans, U., Hollenberg,
C. P., and Strasser, A. W. M., Striking structural and
functional similarities suggest that intestinal sucrase-
isomaltase, human lysosomal ꢀ-glucosidase and
Schwanniomyces occidentalis glucoamylase are derived
from a common ancestral gene. FEBS Lett., 294, 109–
112 (1991).
5
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