Comparison of sucrose-hydrolyzing enzymes produced by Rhizopus oryzae and Amylomyces rouxii

Article abstract of DOI:10.1271/bbb.80346

Rhizopus oryzae produces lactic acid from glucose but not efficiently from sucrose, while Amylomyces rouxii, a species closely related to R. oryzae, ferments these sugars equally. The properties of two sucrose-hydrolyzing enzymes purified from culture filtrates of R. oryzae NBRC 4785 and A. rouxii CBS 438.76 were compared to assess lactic acid fermentation by the two fungi. The substrate specificity of the enzymes showed that the enzymes from strains NBRC 4785 and CBS 438.76 are to be classified as glucoamylase and invertase respectively. The entity of the enzyme from strain NBRC 4785 might be a glucoamylase, because eight residues of the N-terminal amino acid sequence coincided with those of the deduced protein from the amyB gene of R. oryzae. The enzyme from NBRC 4785 was more unstable than that from strain CBS 438.76 under conditions of lower pH and higher temperature. These observations mean that the culture conditions of R. oryzae for lactic acid production from sucrose should be strictly controlled to prevent inactivation of the glucoamylase hydrolyzing sucrose.

Full text of DOI:10.1271/bbb.80346

Biosci. Biotechnol. Biochem., 72 (12), 3167–3173, 2008
Comparison of Sucrose-Hydrolyzing Enzymes Produced
by Rhizopus oryzae and Amylomyces rouxii
y
Tsuyoshi WATANABE and Yuji ODA
Department of Food Science, Obihiro University of Agriculture and Veterinary Medicine,
Obihiro, Hokkaido 080-8555, Japan
Received May 20, 2008; Accepted September 6, 2008; Online Publication, December 7, 2008
[doi:10.1271/bbb.80346]
Rhizopus oryzae produces lactic acid from glucose but
include its use as a natural acidulant, preservative, and
flavor enhancer. One of the most important uses is as a
raw material for polylactide, which can partly replace
conventional plastics synthesized from fossil resources
as a biodegradable polyester.
not efficiently from sucrose, while Amylomyces rouxii, a
species closely related to R. oryzae, ferments these
sugars equally. The properties of two sucrose-hydro-
lyzing enzymes purified from culture filtrates of R.
oryzae NBRC 4785 and A. rouxii CBS 438.76 were
compared to assess lactic acid fermentation by the two
fungi. The substrate specificity of the enzymes showed
that the enzymes from strains NBRC 4785 and CBS
Microbial production of lactic acid is usually con-
ducted by anaerobic fermentation of lactic acid bacteria
in a medium containing mono- or disaccharides sup-
plemented with complex nitrogen sources. The addition
of natural components, such as yeast extract or
peptone, increases the culture costs and complicates
the purification procedures for lactic acid. Among
fungal strains aerobically forming organic acids, Rhi-
zopus oryzae is known as an alternative microorganism
4
38.76 are to be classified as glucoamylase and invertase
respectively. The entity of the enzyme from strain
NBRC 4785 might be a glucoamylase, because eight
residues of the N-terminal amino acid sequence coin-
cided with those of the deduced protein from the amyB
gene of R. oryzae. The enzyme from NBRC 4785 was
more unstable than that from strain CBS 438.76 under
conditions of lower pH and higher temperature. These
observations mean that the culture conditions of R.
oryzae for lactic acid production from sucrose should be
strictly controlled to prevent inactivation of the gluco-
amylase hydrolyzing sucrose.
2
,3)
for lactic acid fermentation.
The production yield
of lactic acid by R. oryzae is lower than that of lactic
acid bacteria, and this fungus primarily synthesizes
L-lactic acid in the presence of ammonium salts as
4
,5)
sole nitrogen source.
The costs of recovery and
purification of lactic acid produced by fungi are less
than those by lactic acid bacteria. R. oryzae con-
verts starchy materials to lactic acid by the action of
external amylases but does not degrade sucrose as
Key words: glucoamylase; ꢀ-fructofuranosidase; Rhizo-
pus oryzae; Amylomyces rouxii
6
)
efficiently.
Amylomyces is a monotypic genus containing the
single variable species A. rouxii, which is closely
Sugar beet is a principal rotation crop in Hokkaido,
the northernmost island of Japan. It is used in the
manufacture of crystallized sucrose, but circumstances
surrounding sugar beet become more and more severe
on the domestic sugar industry. Annual consumption
of sugar is decreasing in Japan, having reached to 3
million tons in 1976 because of expanded use of high
fructose corn syrup, increasing import of sugar-contain-
ing foods, and dietary changes of consumers to less
sweet tastes. To find a way out of these difficulties,
valuable products synthesized from sucrose are re-
quired. Hence, we have paid attention to lactic acid as a
source of such products.
7
)
related to R. oryzae, as shown by the formation of
8
)
rhizoids, stolons, and black-pigmented sporangia.
distinct morphological characteristic of A. rouxii is the
A
enormous number of chlamydospores produced in the
8
)
aerial and substrate mycelium. Ellis et al. reported
that five strains of A. rouxii grew on glucose and
sucrose equally, although A. rouxii was inferior to
8
)
R. oryzae in growth and the production rate of lactic
9
)
acid. We have surveyed the strains of R. oryzae, and
we found that some strains ferment sucrose vigorously.
In the present experiments, sucrose-hydrolyzing en-
zymes of selected strains of R. oryzae and A. rouxii
were purified to compare their properties for the
efficient and stable production of lactic acid from
sucrose by R. oryzae.
Lactic acid is widely used in the food, cosmetics,
1
)
chemical, and pharmaceutical industries. The func-
tional applications of lactic acid as a food ingredient
y
To whom correspondence should be addressed. Fax: +81-155-49-5554; E-mail: yujioda@obihiro.ac.jp

3168
T. WATANABE and Y. ODA
Materials and Methods
Reagents. Fructooligosaccharides, which contain 1-
F
15)
kestose, nystose, and 1 -ꢀ-fructofuranosyl nystose,
Organisms. Twenty-six strains of R. oryzae accumu-
lating lactic acid predominantly were obtained from the
NITE Biological Research Center (Chiba, Japan). The
NBRC numbers of these strains are as follows: 4705,
and a purified preparation of glucoamylase from
Rhizopus species with an approximate molecular weight
of 70,000 were obtained from Wako Pure Chemical
Industries (Osaka, Japan).
4
4
5
706, 4707, 4716, 4744, 4758, 4780, 4783, 4785, 4798,
804, 4809, 5318, 5319, 5378, 5379, 5380, 5384, 5413,
418, 5438, 5440, 5780, 6155, 9364, and 31005.
Analysis of DNA sequences. The primers, designed
based on the amyB gene sequence (DQ219822) of
0
A. rouxii CBS 438.76 was used as the reference strain.
R. oryzae NRRL 395, were BF10 (5 -CACTGAAA-
0
0
CCGGGAAATATGAC-3 ) and BR14 (5 -TAGAAGA-
0
Culture. Cells were grown on potato dextrose agar
prepared horizontally in a standing test tube. The
mycelium was taken from the surface of the agar and
CATCAAGCCACTCAC-3 ). These were used in the
polymerase chain reaction. Fragments of about 2.1 kb
amplified from the genomic DNA of strains NBRC 4785
and NBRC 5384 (= NRRL395) were sequenced on both
strands with an automated DNA sequencer.
inoculated in 100 ml of a medium containing 10% sugar,
.
0
7H O, and 2.5% CaCO in a 300-ml Erlenmeyer flask.
.2% (NH4)2SO4, 0.065% KH2PO4, 0.025% MgSO4
2
3
Sugar and other components of the medium were
autoclaved separately and mixed just before inoculation.
After cultivation of R. oryzae for 6 d and A. rouxii for
Results
Production of lactic acid from sucrose and other
sugars
ꢀ
1
4 d at 30 C with shaking (150 rpm), unless otherwise
stated, the filtrate of the medium was dialyzed against
distilled water and used as the crude enzyme.
Twenty-six strains of R. oryzae were cultured in a
medium containing 10% sucrose as the principal carbon
source for 10 d. All strains except for two, NBRC 4798
and NBRC 5418, produced lactic acid to some extent,
but no sucrose-hydrolyzing activity was detected in the
culture filtrate (data not shown).
Enzyme assays. The reaction mixture for the assay of
the sucrose-hydrolyzing enzyme contained a 100 mM
acetate buffer (pH 5.0), 150 mM sucrose, and the crude
enzyme in a total volume of 0.25 ml. The sucrose was
replaced with 10 mg/ml of soluble starch or 20 mg/ml
Strain NBRC 4785, which reproducibly produced
lactic acid, was selected as a representative strain of
R. oryzae and compared with A. rouxii CBS 438.76
ꢀ
of inulin when necessary. After incubation at 30 C for
3
ꢀ
0 min, the reaction was stopped by the addition of a
,5-dinitrosalicylic acid reagent, and the reducing sugars
at 30 C for lactic acid fermentation of some sugars
(Table 1). Appreciable amounts of ethanol accompa-
nied the formation of lactic acid in the two strains,
3
produced were determined.
1
0)
The activity hydrolyz-
1
2)
ing p-nitrophenyl ꢁ-D-glucopyranoside was measured
by the formation of p-nitrophenol as ꢁ-glucosidase
activity. One unit was defined as the amount of enzyme
that released 1 mmol of reducing sugar equivalent to
glucose or p-nitrophenol per min under the above
conditions.
as usually observed. Lactic acid was produced from
glucose, sucrose, maltose, fructooligosaccharides, and
soluble starch by R. oryzae NBRC 4785, and from
glucose, sucrose, raffinose, fructooligosaccharides, and
soluble starch by A. rouxii CBS 438.76. The latter
strain produced a detectable amount of lactic acid from
inulin. These fermentation profiles suggested differ-
ences in the hydrolysis of fructosyl-linkage by the two
strains.
Molecular weight. SDS-polyacrylamide gel electro-
phoresis (SDS–PAGE) was carried out in 7.5% gel to
estimate the molecular weight of the enzyme.11) The
protein bands were visualized by silver staining. The
standards for molecular weight determination were
obtained from Bio-Rad Laboratories (Hercules, CA).
Table 1. Production of Lactic Acid and Ethanol from Sugars by the
Two Fungal Strains
Analytical procedures. Lactic acid and ethanol were
determined by high-performance liquid chromatography
(
R. oryzae
NBRC 4785
A. rouxii
CBS 438.76
Component
HPLC).1 Soluble sugars were analyzed by thin-layer
2)
Lactic acid Ethanol Lactic acid Ethanol
mg/ml) (mg/ml) (mg/ml) (mg/ml)
chromatography (TLC) using a solvent system of 1-
butanol/2-propanol/acetic acid/water (7:5:2:4, v/v).
Spots were visualized by spraying the plate with an
(
Glucose
Sucrose
77.8
77.3
74.1
<0:1
24.6
<0:1
70.1
6.5
5.7
71.2
73.1
<0:1
38.5
70.8
1.5
6.0
4.9
1
3)
anisaldehyde-sulfuric acid reagent. Protein was meas-
1
Maltose
Raffinose
8.2
<0:1
1.8
4)
<0:1
<0:1
<0:1
3.8
ured by the method of Bradford.
The N-terminal
Fructooligosaccharides
Inulin
Soluble starch
5.1
0.5
amino acid sequence of the enzyme was analyzed by
automated Edman degradation in a protein sequencer
66.2
5.5
(
Procise 494, Applied Biosystems, Foster City, CA).

Sucrose-Hydrolyzing Enzymes
3169
100
a
a
Monosaccharides
Sucrose
b
50
0
2
4
6
8
10 12 14
Culture period (d)
0
Fig. 2. Residual Sugars in the Media Containing Sucrose during
Growth of R. oryzae NBRC 4785 (a) and A. rouxii CBS 438.76 (b).
One ml of the culture filtrate after 10-fold dilution was applied to
TLC.
8
6
4
b
and left a detectable amount of sucrose that was not
hydrolyzed by the loss of sucrose-hydrolyzing activity.
Incomplete hydrolysis of sucrose might cause defects in
the industrial production of lactic acid from sucrose by
R. oryzae. From these reasons, the sucrose-hydrolyzing
enzymes of the two fungi were purified in order to
compare their properties.
2
0
0
0
0
.4
c
.3
.2
.1
0
Purification of sucrose-hydrolyzing enzymes
The culture filtrate (1,560 ml) of R. oryzae NBRC
785, which included maximal sucrose-hydrolyzing
4
activity, was dialyzed against distilled water and con-
centrated 80-fold by ultrafiltration. The concentrated
filtrate was mixed with a 1.0 M acetate buffer (pH 5.0)
to adjust it to 20 mM, and was passed through CM-
Toyopearl 650 M in a column (1:5 ꢁ 13 cm). The
column was washed with the buffer and eluted with a
linear gradient of an NaCl concentration from 0 to
500 mM in the buffer. The active fractions eluted with
0
5
10
15
Culture period (d)
Fig. 1. Lactic Acid Production (a), pH (b), and Sucrose-Hydrolyzing
Activity (c) in the Medium Containing Sucrose during Growth of the
Two Fungal Strains.
Symbols: , R. oryzae NBRC 4785; , A. rouxii CBS 438.76.
130 mM NaCl were collected and saturated with solid
ammonium sulfate to 40%. The enzyme solution was
applied to a Butyl-Toyopearl 650 M column (1:5 ꢁ 13
cm) equilibrated with a buffer containing 40% saturated
ammonium sulfate. The column was washed with the
same buffer and eluted with a decreasing gradient of
40% to 0% saturated ammonium sulfate. The enzyme
eluted with 18% saturated ammonium sulfate was
recovered.
The culture filtrate (1,610 ml) of A. rouxii CBS
438.76 grown for 14 d was purified according to the
methods described above. The dialyzed and concen-
trated filtrate was mixed with 1.0 M Tris–HCl (pH 8.5) to
adjust it to 20 mM and was passed through a DEAE-
Toyopearl 650 M column. The active fractions eluted
with 160 mM NaCl were collected and applied to a
Butyl-Toyopearl 650 M column. The active fractions
eluted with 22% saturated ammonium sulfate were
put on a column of Toyopearl HW-55 (1:5 ꢁ 90 cm)
equilibrated with a 20 mM phosphate buffer (pH 6.8)
containing 50 mM NaCl.
Synthesis of sucrose-hydrolyzing enzymes
Lactic acid production and sucrose-hydrolyzing
activity were followed during the growth of R. oryzae
NBRC 4785 and A. rouxii CBS 438.76 in the medium
containing 10% sucrose (Fig. 1). Strain NBRC 4785
formed lactic acid more rapidly than strain CBS 438.76,
with a pH reduction. There was a remarkable difference
in the change in sucrose-hydrolyzing activity between
the two strains. The activity of strain NBRC 4785
increased with a slight lag period after inoculation and
disappeared for 8 d. On the other hand, strain CBS
4
38.76 continued to synthesize the sucrose-hydrolyzing
enzyme throughout the culture periods. A decrease in
sucrose in the media of both strains was accompanied
by the appearance of monosaccharides, which were
consumed, but not completely (Fig. 2). An external pH
below 4.0 might inhibit further fermentation of mono-
saccharides for both strains. In strain CBS 438.76, the
rate of sucrose hydrolysis appeared to be higher than
that of sugar consumption, resulting in an accumulation
of monosaccharides during 2 to 6 d of cultivation. Strain
NBRC 4785 consumed the monosaccharides formed
from sucrose more rapidly than strain CBS 438.76
A single peak of the activity was found in each
chromatograph of the two enzymes. By these proce-
dures, the enzymes were purified, 3.5-fold for strain
NBRC 4785 and 7.4-fold for strain CBS 438.76

3170
T. WATANABE and Y. ODA
Table 2. Purification of the Sucrose-Hydrolyzing Enzymes Produced by the Two Fungal Strains
Total activity
U)
Total protein
(mg)
Specific activity
(U/mg)
Purification
(fold)
Yield
(%)
Strain
Step
(
R. oryzae NBRC 4785
Culture filtrate
CM-Toyopearl
Butyl-Toyopearl
215
135
76
19.3
7.77
1.96
11.1
17.4
38.8
1.0
1.6
3.5
100
62.8
35.3
A. rouxii CBS 438.76
Culture filtrate
652
157
81
3.91
167
350
1.0
2.1
4.2
7.4
100
24.1
12.5
7.5
DEAE-Toyopearl
Butyl-Toyopearl
Toyopearl HW55
0.448
0.117
0.0398
698
1,231
49
a
b c d
a
b
d
1
00
100
80
60
40
20
0
8
6
4
2
0
0
0
0
0
Molecular weight
16,250
1
9
7,400
c
100
100
80
60
40
20
8
6
4
2
0
0
0
0
0
6
4
6,200
5,000
0
2
3
4
5
6
7
20
40
60
80
Temperature (°C )
pH
Fig. 4. Effects of the pH (a) and Temperature (b) on the Activity of
the Enzymes and Those of pH (c) and Temperature (d) on the
Stability of the Enzymes.
Fig. 3. SDS-Polyacrylamide Gel Electrophoresis of the Purified
Enzymes from the Two Fungal Strains.
The purified enzymes of R. oryzae NBRC 4785 (0.083 mg) and
A. rouxii CBS 438.76 (0.144 mg) before (a, c) and after (b, d)
treatment with endoglycosidase H, respectively, were electrophor-
esed on 7.5% gel.
Buffers used: citrate, pH 2.5 to 3.8; acetate, pH 4.0 to 5.5;
,
phosphate, 6.0 to 7.2. Symbols: , R. oryzae NBRC 4785;
A. rouxii CBS 438.76.
ꢀ
(
Table 2). The enzymes were dialyzed against a 20 mM
assay, the enzymes were incubated at 40 C for 30 min at
various pHs and temperatures for 30 min in a 20 mM
phosphate buffer (pH 6.8) (Fig. 4c, d). The enzyme from
strain NBRC 4785 was more unstable than that from
CBS 438.76.
phosphate buffer (pH 6.8) and used in subsequent
experiments.
Properties of the purified enzymes
Each purified enzyme gave a single band on SDS–
PAGE with molecular weights showing homogeny
through electrophoresis (Fig. 3). The molecular weights
of the enzymes from strains NBRC 4785 and CBS
The effects of various concentrations of sucrose on
the enzyme activity were examined. The values of Km
(mM) and Vmax (U/mg protein) were calculated to be
200 and 103 for the enzyme from strain NBRC 4785,
and 52.4 and 1,710 for that from strain CBS 438.76,
respectively.
The products of sugars incubated with the purified
enzymes were analyzed by TLC (Fig. 5). The enzyme
from strain NBRC 4785 split both sucrose and maltose
into monosaccharides almost completely and produced
monosaccharides from fructooligosaccharides and solu-
ble starch. The terminal moiety of glucose appeared
to be hydrolyzed from fructooligosaccharides by this
4
38.76 were estimated to be 59,000 and 69,000, and fell
to 47,000 and 60,000 after treatment with endoglycosi-
dase H, respectively.
Sucrose-hydrolyzing activity was assayed in a pH
ꢀ
ꢀ
range of 3.1 to 7.5 at 30 C and in the range of 30 C to
ꢀ
7
0 C at pH 5.0 (Fig. 4a, b). The two enzymes gave
ꢀ
maximal activities at pH 4.5 at 30 C, while the optimal
temperature for the enzyme from strain CBS 438.76 was
higher than that from strain NBRC 4785. Before the

Sucrose-Hydrolyzing Enzymes
3171
identical except at position 90. As for the enzyme from
strain CBS 438.76, determination of the N-terminal
amino acid sequence was unsuccessful.
a
Discussion
There are several types of microbial enzymes that can
1
7)
be involved in the degradation of sucrose. The first
enzyme is invertase (ꢀ-fructofuranoside fructohydro-
lase: EC 3.2.1.26), hydrolyzing sucrose and other ꢀ-
fructofuranosides, and the second enzyme is inulinase
b
(2,1-ꢀ-D-fructan fructanohydrolase, EC 3.2.1.7), hydro-
lyzing inulin. These enzymes commonly attack the
substrates from the fructose moiety, but show distin-
guishable properties. The ratio of the activity on inulin
versus sucrose was found to be less than 0.02 for
invertase and higher than 0.1 for inulinase. Fungal
invertases have been purified and characterized from
C
T
C
T
C
T
C
T
C
T
C
T
C
T
1
8)
1
8)
19)
Aspergillus ficuum, Aspergillus nidulans, Aspergil-
2
0–23)
Aspergillus oryzae,24) and Aspergillus
lus niger,
2
5)
ochraceus. However, invertase has not been found in
the genus Rhizopus, and inulinase has been reported
to participate in the hydrolysis of sucrose in spite of
2
6)
its lower affinity. The third enzyme is ꢁ-glucosidase
ꢁ-D-glucoside glucohydrolase, EC 3.2.1.20), which
Fig. 5. Hydrolysis Products of Sugars Due to the Purified Enzymes
from R. oryzae NBRC 4785 (a) and A. rouxii CBS 438.76 (b).
The reaction mixture contained a 100 mM acetate buffer (pH 5.0),
(
hydrolyzes maltose and its oligosaccharides from the
glucose moiety. ꢁ-Glucosidases from different origins
vary in substrate specificity, and the enzyme that
preferentially hydrolyzes sucrose has been alternatively
5
mg/ml of each sugar, and the purified enzyme (15 U) in a total
ꢀ
volume of 0.10 ml. After incubation of the mixture at 30 C for 20 h,
the reaction was stopped by placing the mixture in boiling water for
5 min. One ml of each mixture before (C) and after (T) the reaction
was applied to TLC. The arrow indicates the position of the
17)
called glucoinvertase or glucosidosucrase. Exogenous
maltose has been incorporated into the cells of Saccha-
monosaccharides.
1
5)
27)
romyces cerevisiae
and Candida albicans
and
hydrolyzed by ꢁ-glucosidase. The enzyme from the
yeast Torulaspora pretoriensis showed much higher
affinity to sucrose than to maltose.
enzyme, leaving residual oligosaccharides. When com-
pared with the commercial enzyme preparation, the
specific activities (U/mg protein) for sucrose, soluble
starch, and p-nitrophenyl ꢁ-D-glucopyranoside were
2
8)
In the present experiments, sucrose-hydrolyzing en-
zymes produced by R. oryzae NBRC 4785 and A. rouxii
CBS 438.76 were assumed to be distinct types based on
the results of lactic acid production from some sugars.
The profiles of sugar hydrolysis showed that the
enzymes from strains NBRC 4785 and CBS 438.76
are to be classified as glucoamylase and invertase
respectively.
3
4
5.9, 1.26, and 0.01 in the enzyme from strain NBRC
785, and 0.04, 3.09, and 0.04 in the purified enzyme of
glucoamylase from the Rhizopus species, respectively.
The enzyme from strain NBRC 4785 can be classified
as glucoamylase irrespective of its high activity for
sucrose.
The enzyme from strain CBS 438.76 bore mono-
saccharides from sucrose, raffinose, fructooligosaccha-
rides, inulin, and levan, but not maltose or soluble starch
The N-terminal amino acid sequence and molecular
weight confirmed that the entity of the enzyme from
strain NBRC 4785 might correspond with one of the
glucoamylases encoded by the amyB gene in addition to
(
Fig. 5). The complete hydrolysis of fructooligosaccha-
1
6)
rides indicates that this enzyme hydrolyzed the terminal
moiety of fructose exogenously. The ratio of the activity
on inulin versus sucrose was 0.013, indicating a higher
preference for sucrose.
the amyA gene in R. oryzae NRRL 395. The AmyA
and AmyB proteins share homologous amino acid
sequences, but the AmyB protein lacks a starch-binding
domain, differently from the AmyA protein. The
commercial glucoamylase from the Rhizopus species
used in the present experiments might be the AmyA
The N-terminal amino acid sequence of the enzyme
from strain NBRC 4785 was SKPATFPT, which agreed
with that of the protein from the amyB gene (DQ219822)
1
6)
protein based on its molecular weight of 70,000. Loss
of a starch-binding domain appears to cause drastic
changes in the substrate specificity of the AmyA protein.
The physiological role of the AmyB protein is not
understood, because the amyB gene has not been highly
1
6)
of R. oryzae NRRL 395 (= NBRC 5384).
sequence analysis of amyB genes from strains NBRC
785 (AB444724) and NBRC 5384 (AB444726) re-
vealed that the deduced amino acid sequences were
DNA
4

3172
T. WATANABE and Y. ODA
expressed in shake-flask cultures with various carbon
sources, and no glucoamylase activity has been detected
when recombinant expression of the amyB gene was
genus Amylomyces. Mycologia, 68, 131–143 (1976).
Saito, K., Abe, A., Sujaya, I. N., Sone, T., and Oda, Y.,
Comparison of Amylomyces rouxii and Rhizopus oryzae
in lactic acid fermentation of potato pulp. Food Sci.
Technol. Res., 10, 224–226 (2004).
9
)
1
6)
conducted in Pichia pastoris.
The enzyme from R. oryzae NBRC 4785 was more
sensitive to external pH and temperature than that from
A. rouxii CBS 438.76. Acidification of the medium for
1
0) Oda, Y., and Tonomura, K., Purification and character-
ization of invertase from Torulaspora pretoriensis YK-1.
Biosci. Biotechnol. Biochem., 58, 1155–1157 (1994).
1) Laemmli, U. K., Cleavage of structural proteins during
the assembly of the head of bacteriophage T4. Nature,
227, 680–685 (1970).
12) Oda, Y., Yajima, Y., Kinoshita, M., and Ohnishi, M.,
Differences of Rhizopus oryzae strains in organic acid
synthesis and fatty acid composition. Food Microbiol.,
8
d after inoculation was severe in the enzyme secreted
1
from strain NBRC 4785, and might have limited the
activity. The other R. oryzae strains used in the present
experiments also appeared to possess an AmyB protein
carrying sucrose-hydrolyzing activity, because no activ-
ity was detected after 10 d of cultivation. The presence
of invertase in strain CBS 438.76 does not delimit
the species A. rouxii, because some strains accepted in
2
0, 371–375 (2003).
1
3) Saito, K., Kondo, K., Kojima, I., Yokota, A., and
Tomita, F., Purification and characterization of 2,6-ꢀ-D-
fructan 6-levanbiohydrolase from Streptomyces exfolia-
tus F3-2. Appl. Environ. Microbiol., 66, 252–256 (2000).
4) Bradford, M. M., A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing
the principle of protein-dye binding. Anal. Biochem., 72,
248–254 (1976).
8
)
A. rouxii did not grow on sucrose or raffinose. At least,
it can be stated that AmyB protein plays a crucial role in
the lactic acid fermentation of sucrose in R. oryzae. The
lower expression of the amyB gene and instability of
the AmyB protein can explain why the fermentation
of sucrose by R. oryzae lacks efficiency, as reported
previously.
1
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This study was supported in part by a grant from
the Ministry of Agriculture, Forestry, and Fisheries
of Japan (MAFF) Rural Bio-Mass Research Project
(
BUM-Cm1200).
1
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