J. Biol. Macromol., 5(1), 11-16 (2005）

Production and characterization of β-glucosidase from Rhizopus oryzae MIBA348
Yukio Takii1), Kimiko Ikeda1), Chihiro Sato1), Megumu Yano1), Tsutomu Sato２）, and Hiroshi Konno２）
1)

Department of Food Science and Nutrition, School of Human Environmental sciences,
Mukogawa Women’s University.6-46 Ikebiraki-cho, Nishinomiya, Hyogo 663-8558
２)
Akita Konno Co. Ltd., Kariwano, Akita 019-2112
Received March 16, 2005; Accepted April 20, 2005

A β-glucosidase was purified from the cell extracts of Rhizopus oryzae MIBA348 to homogeneity by
successive anion exchange and size-exclusion chromatography and characterized. The enzyme has amolecular weight of 105,000. The optimum pH and the optimum temperature was 5.0 and 50℃,
respectively. The enzyme was active on p-nitrophenyl-β- D-glucopyranoside, cellobiose and salicine. Ithydrolyzed gentiobiose and amygdaline, but was inactive on Avicel, carboxymethylcellulose, maltose andp-nitrophenyl-α- D- glucopyranoside

Key words: Rhizopus oryzae, β-glucosidase, endo-β-D-1,4-glucanases

β-glucan in nature is a biological polymer
composed of β-1,3-, β-1,4-, and/or β-1,6-linkages
and includes laminarin, pachyman, carboxymethyl
cellulose, lichenan and barley beta-glucan (1-6).
Cellulose is the most abundant compound that
contains only β-1,4-linkages and is considered to be
degraded by the synergistic action of three types of
enzyme components: endo- glucanases (EC3.2.1.4,
endo-β-D-1,4-glucanases), cellobiohydrolases (EC
3.2.1.91), and β-glucosidases (EC3.2.1.21) (7-9).
β-Glucosidase is an important enzyme that was able
to produce reusable monosaccharide, glucose in the
final stage of degradation (10,11).
Zygomycetes group in mesophilic fungi arewidely distributed on stored grain, fruit andvegetables, in the air or in compost (12-15).
Therefore, they are well known sources of cellulases
and have been effectively used for industrial
purposes (13-15). For example, members ofMucor and Rhizopus are important for their use inproduction of organic acids, while some of themcause rot in ripe and harvested fruits and vegetables(16).
Recently, cellulases are found to becomposed of different glycosyl hydrolase families(17, 18). To elucidate the enzyme function in the

degradation system, the DNA sequences of therespective enzymes should provide important
information (19, 20).
In the present study, β-glucosidase was purified
from a mesophilic fungus, Rhizopus oryzae andcharacterized.

Materials and methods
Cultivation of fungi
Fungus cultivation was conducted at 30℃ for 2days, unless otherwise stated. Aliquots of colony
of Rhizopus species grown on potato dextrose agar
plate (21) were inoculated to Erlenmyer flask (200ml), containing solid medium (50 ml) which wascomposed of 20% wheat bran and carbon sources(1 %) and incubated. After adding 50 ml ofpotassium phosphate buffer (pH6.8), the cultures
were incubated for 24 hr at 4℃ and filtered througha glass filter (pore size, G1). The filtrate wascentrifuged at 10,000 rpm for 10 min and at 4℃ toremove insoluble materials and finally filled up to 55
ml with potassium phosphate buffer (pH 6.8).
In liquid culture, cultivation of R.oryzae 348
was performted by shaking 57 Erlenmeyer flasks

R.oryzae β-glucosidase

Enzyme assay
β-Glucosidase activity was determined photometrically at 37℃ in a reaction mixture (1.0 ml)
containing 30 mM potassium phosphate buffer (pH6.8), 2 mM p-nitrophenyl-β-D-glucopyranoside andenzyme. One unit (U) of enzyme activity wasdefined as the amount of enzyme hydrolyzing 1µmol of the nitrophenyl glucoside/min under theabove conditions (22). When various sugars were
employed as substrates, either reducing power or
glucose formed was assayed by using dinitrosalicylic
acid and glucose test kit, respectively (9). One unit(U) of enzyme activity was defined as the amount of
1 µmol sugars hydrolyzed/min. Protein was
assayed according to the method of Bradford using
bovine serum albumin as the standard (23).
Purification of enzyme
Step 1. The MIBA348 cells were thawed in 5ml
PPB and homogenized at 4℃ with pestle, and thencentrifuged as described above.
Step 2. The homogenate was dialyzed against
several changes of 1000 ml of 10 mM PPB (pH 6.8)
and centrifuged. The clear supernatant (350 ml)
was applied to DEAE-Sephacel column (1.5 x 40cm), which was equilibrated with PPB (pH 6.8).
After washing thoroughly with PPB, the column was
eluted with a linear gradient (500 ml) conducted
between PPB in a mixing chamber and PPB/1.0 M
NaCl in a reservoir .Fractions of 18 ml werecollected and assayed for glucosidase The active
enzyme fractions were pooled, dialyzed andcentrifuged as described above.
Step 3. The supernatant was applied to DEAESephacel column (1.6 x 8.0 cm), which was equilibrated and exhaustively washed with PPB. The
column was eluted with a linear gradient (200 ml)
conducted between PPB in a mixing chamber and
PPB/1.0 M NaCl in a reservoir. Fractions of 2.0 ml
were collectedand assayed for enzyme activity.
The active enzyme fractions were pooled, dialyzed

and concentrated to 2.5ml on Amicon PM-10
membrane.
Step 4.
The concentrate was applied to a
Sephacryl S-200 column (1.5 x 100 cm), which was
equilibrated with 10 mM PPB/0.3M NaCl andeluted with the same buffer. Fractions of 2.8 ml werecollected and assayed for enyme activity. As
shown in Fig. 1, the active enzyme fractions eluted
from the column were concentrated on Amicon
PM-10 membrane filter and subjected tocentrifugation and pooled as the final preparation.

18
16
14
12
10
8
6
4
2
0

0.10
0.08
0.06
0.04
0.02
0.00
0

10

20

30 40 50 60
FRACTION No.

70

80

90

Fig. 1.

Elution pattern of R. oryzae MIBA348
β-glucosidase from Sephacryl S-200 column.
---○---, absorbance at 280 nm, ------●-----: enzyme
activity. The activity is expressed in units (U) per
ml of eluate.

Results and Discussion
Twelve Rhizopus strains (MIBA341-352) fromcollections in this laboratory were tested for ability to
produce β-glucosidase.
R. oryzae MIBA348
produced the highest β-glucosidase (0.308 U/mg
protein), while other 11 strains gave activities lessthan 0.05 U/mg protein. The MIBA348 enzyme
production was maximum (1.52 U/mg protein)when CMC (0.2%) was used as a carbon source,
while the enzyme yield exhibited 80% and 48% of
the highest value, respectively when CMC was
substituted with 0.2% Avicel or 0.2% cellobiose.
The enzyme activity found outside the cells after
shaking culture for 2 days was11% of that foundinside the cells.
Table 1 shows a summary of the β-glucosidase

ABSORBANCE (280nm)(500 ml) containing 50 ml of the medium [0.2%carboxymethylcellulose (CMC), 0.1% KH2PO4,
0.3% K2HPO4, 0.3% peptone and 0.15% yeast
extract (pH 6.8)]. The cells collected by filtration
were kept frozen at -30℃ until use.

ACTIVITY（U/ml）

12

100

100

75

75

50

50

25

25

0

Total

Total

Yield SpecificPurifi-

Activity

cationProtein Activity
Step

(mg)

(U)

Crude extract200

231 100

1.2

1.0

1st DEAE-Sephacel

8.5

226 97.8

27

22

2nd DEAE-Sephacel

3.0

197 85.3

6554

Sephacryl S-200

1.0

103 44.5

103

86（％） （U/mg）

RELATIVE ACTIVITY （％）

Table 1. Purification of β–glucosidase from R.
oryzae MIBA348

RELATIVE ACTIVITY （％）

purification from strain R. oryzae MIBA348. The
final preparation (1.0 mg, 103 U/mg protein) was
purified 85.7-fold over the cell extracts, the yield
being 44.5 %. The protein was homogeneous interms of its behavior on SDS gel electrophoresis, as
shown in Fig. 2. The molecular weight wasestimated as105,000 on SDS gel electrophoresis andby gel filtration on Sephacryl S-200.

13

ACTIVITY RECOVERED（％）

Y. Takii et al.

0
15

35
55
75
TEMPERATURE（℃）

2

3

95

100
80
60
40
20
0
4

5

6

7

8

9

pH

Fig. 3. Effect of temperature on the activity andstability and pH profile of activity of R. oryzae
MIBA348 β-glucosidase.

Fig. 2. SDS polyacryamide
gel electrophoresis of R.
oryzae MIBA 348 β-glucosidase.
The SDS gel contained 7.5% acryl
acryl-amide. Electrophoresis was
carried out for 90 min at 25 mA/geland at 25℃, using 10 μg protein.
After electrophoresis, the gel was
stained with Coomassie brilliant
blue. Lane1: standard molecular12weight markers (200,200,
116,250, 97,400, 66,200,
45,000), lane 2: purified
MIBA348 β-glucosidase(upper panel): The activity (●) was determined asgiven in the text, except that the incubation
temperature was changed. The enzyme activity
observed at 50℃ was expressed as 100%.
Thermostability（○） was examined after treating the
enzyme for 15 min at different temperatures and at
pH 6.8, as shown in the figure, and then the mixture
were assayed for the remaining activities. The activity
observed at 4℃ was defined as 100%.
(lower panel): The activity was determined as givenin the text, except that the enzyme was incubated in
10-fold volume each of 100 mM sodium acetate
buffer (pH 3.0 – 5.5), 100 mM Mcllvaine buffer(pH5.0 – 6.5) and 100 mM potassium phosphatebuffer (6.0-8.0), respectively prepared with different
pHs, as shown in the figure. The activity observedat pH 5.0 was taken as 100%.

14

R.oryzae β-glucosidase

The enzyme activity was maximal at 55℃ withhalf-optima at 48℃ and 62℃ (Fig. 3). The enzyme
was most active at pH 5.0 with half-optima at pH3.8
and 7.1 (Fig. 3). The β-1,4-linkages of cellobioseand salicin were cleaved by the enzyme､20% and8.4%, respectively, as compared with that of thenitrophenyl glucosaide. The following compounds
were not utilized as the substrates: p-nitrophenyl-αD-glucopyranoside, arbutin, maltose, and β-lactose(Table2). Neither Avicel nor CMC was effective

Table2 Hidrolysis of various sugars by R.oryzae
MIBA348 β-glucosidase

Sugar
β-pNPG
D-Cellobiose
Salicin
Arbutinβ-Lactose
AvicelCMC

Final concentration

Relative
activity (%)

Linkage

20mM
20mM
20mM
20mM
20mM
0.1%
0.1%

100.
20.3
8.38
0.0
0.0
0.0
0.0

β-1,4β-1,4β-1,4β-1,4β-1,4β-1,4β-1,4-

20mM
20mM
20mM
20mM

0.0
0.0
9.01
22.9

α-1,4α-1,4β-1,6β-1,6-p-nitrophenyl̶D-glucopyranoside

MaltoseAmygdalin
Gentiobiose

substrates for the enzyme. The enzyme lost no
activity at 4-50℃ for 15min at pH 6.8, and activity
recovered was 100% at 50℃, 50% at 62℃, and 20%
at 70℃, showing the enzyme was quite resistant toheat treatment. This stability of enzyme will beapplicable to hydrolyze β-glucan found in nature.
So far, fungal cellulases have been isolated
mainly from the members of Deuteromycotina (12,15). Trichoderma reesei produces eight cellulase
components belonging to seven different glycosylhydrolase families (CBHI [family 7], CBHII [family6], EGI [family 7], EGII [family 5], EGIII [family12], EGV [family 45], BGLI [family 3], and BGLII[family 1] (17, 19). These cellulase components havebeen shown to degrade crystalline cellulose

synergistically (1, 2). The variety of cellulase
components might arise mainly through multiple
gene transfer events rather than gene duplication
events, since it is thought that genes encoding
cellulase components belonging to the different
glycosyl hydrolase families evolved from different
ancestral genes (12).
Although many fungal cellulase genes havebeen isolated and characterized, the isolation of
glucosidase genes from food borne origins has notbeen reported (21, 22, 24, 26). To determine thecellulase system of Rhizopus species, we selected
β-glucosidase-producing fungi.
As a result,
β-glucosidase of Rhizopus oryzae MIBA348 was
purified to electrophoretically homogeneous state.
The final preparation was found to be active towards
gentiobiose and amygdaline which contained β- 1, 6linkages, as shown in Table 2. This glucosidase
was able to hydrolyze β- 1, 6- linkages as well as β1,4- linkages, since it exhibited the similar activity on
gentiobiose as that on D-cellobiose.
Aspergillus oryzae α-amylase production is
stimulated by soluble starch, glycogen and malto-oligosaccharides (26). The molecular mechanism
study of the α-amylase induction in this fungus
indicated a gene, designated amyR was atranscriptional activator gene involved in starch
induced efficient expression of the amylolytic genes(30). On the other hand, cellulose and its oligo‐
saccharides were reported to be inducers in
Tricoderma species (27, 28). Recently, genes
encoding FI-carboxymethylcellulase (CMCase) and
β-glucosidase from the fungus Aspergillus aculeatus
were individually fused with the gene encoding the
C-terminal half (320 amino acid residues from the C
terminus) of yeast alpha-agglutinin and introduced
into S. cerevisiae. (29). The genes were expressedby the glyceraldehyde-3-phosphate dehydrogenase
promoter from S. cerevisiae. The cells displaying
these cellulases could grow on cellobiose orwater-soluble cellooligosaccharides as the solecarbon source. The degradation and assimilation ofcellooligosaccharides were confirmed by thin-layer
chromatography. This result showed that the cellsurface-engineered yeast with these enzymes could
be endowed with the ability to assimilate
cellooligosaccharides. This is the first step in the

Y. Takii et al.

assimilation of cellulosic materials by S. cerevisiae
expressing heterologous cellulase genes.
To investigate the substrate specificity observed
towards compounds such as amygdaline andgentiobiose which were composed of β-1,6-linkages,
detailed molecular analysis of the enzyme is required.
This approach is underway to clone genes coding for
MIBA348 enzyme and expression in our strong
expression system using Aspergillus oryzae as ahost.

References
1) Morimoto, K., Karita, S., Kimura, T., Sakka, K.
and Ohmiya, K. Characterization of Clostridium
paraputrificum Chitinase A from a Recombinant
Escherichia coli. J. Biosci. Bioeng , 92(5):466-468 (2001).
2) Kimura, T., Suzuki, H., Fukumura, H., Aburatani,T., Morimoto, K., Karita, S., Sakka, K. andOhmiya, K. Molecular cloning, overexpression,and purification of major xylanase from
Aspergillus oryzae. Biosci. Biotechnol. Biochem.,
64: 2734-2738 (2000).
3) Ohara, H., Karita, S., Kimura, T., Sakka, K. andOhmiya, K. Characterization of the cellulolytic
complex (cellulosome) from Ruminococcus albus.
Biosci. Biotechnol. Biochem., 64: 254-260 (2000).
4) Yamaoka, H., Hayashi, H., Karita, S., Kimura, T.,
Sakka, K. and Ohmiya, K. Purification and someproperties of a chitinase from Xanthomonas sp.
strainAK. J. Ferment. Bineng., 88 , 328-330(1999).
5) Kawazu, T., Sun, T., Shibata, M., Kimura, T.,
Sakka, K. and Ohmiya, K. Expression of abacterial endoglucanase gene in tobacco increases
digestibility of its cell wall fibers. J. Biosci. Bioeng.
88: 421-425 (1999).
6) Kimura, T., Kitamoto, N., Kito, Y., Karita, S.,
Sakka, K. and Ohmiya, K. Molecular cloning ｏf
xylanase gene xynG1 from Aspergillus orzae
KVN 616, a shoyu koji mold, and analysis of its
expression. J. Ferment. Bioeng., 85 10-16 (1998).
7) Henrissat, B., H. Driguez, C. Viet, and M.
Schulein.. Synergism of cellulases from cellulases
from Trichoderma reesei in the degradation ofcellulose. Bio/Technology 3:722-726 (1985).

158) Schwarz, W. H.. The cellulosome and cellulose
degradation by anaerobic bacteria.
Appl.
Microbiol. Biotechnol. 56:634-649 (2001).
9) Takii,Y., Kuriyama, N. Purification andcharacterization of two extracellular cellulases
from Thermomonospora curvata KP1246. J. Biol.
Macromol., 0, 17-20 (2000).
10) Odoux E, Chauwin A, Brillouet JM. Purificationand characterization of vanilla bean (Vanilla
planifolia Andrews) beta-D-glucosidase J Agric
Food Chem. 7;51(10):3168-73. (2003 ).
11) de Palma-Fernandez ER, Gomes E, da Silva R.
Purification and characterization of two
beta-glucosidases from the thermophilic fungus
Thermoascus aurantiacus. Folia Microbiol(Praha).; 47:685-90 (2002)
12) Teeri, T. T. Crystalline cellulose degradation:
new insight into the function of cellobiohydrolases. Trends Biotechnol. 15:160-167(1997).
13) Takii, Y. Food-borne fungi kingdom. J. Biol.
Macromol., 1, 3-7 (2001).
14) Tomme, P., R. A. Warren, and N. R. Gilkes.
Cellulose hydrolysis by bacteria and fungi. Adv.
Microb. Physiol. 37:1-81(1995.)
15) Himmel, M. E., M. F. Ruth, and C. E. Wyman..
Cellulase for commodity products from cellulosic
biomass. Curr. Opin. Biotechnol. 10:358-364(1999).
16) Murashima, K., T. Nishimura, Y. Nakamura, J.
Koga, T. Moriya, N. Sumida, T. Yaguchi, and T.
Kono. Purification and characterization of newendo-1,4-β-D-glucanases from Rhizopus oryzae.
Enzyme Microb. Technol. 30:319-326 (2002).
17). Sheppard, P. O., F. J. Grant, P. J. Oort, C. A.
Sprecher, D. C. Foster, F. S. Hagen, A. Upshall, G.
L. McKnight, and P. J. O’Hara. The use ofconserved cellulase family-specific sequences toclone cellulase homologue cDNAs from
Fusarium oxysporum. Gene 150:163-167 (1994).
18) Takashima, S., H. Iikura, A. Nakamura, M.
Hidaka, H. Masaki, and T. Uozumi. Comparisonof gene structures and enzymatic properties
between two endoglucanases from Humicola
grisea. J. Biotechnol. 67:85-97 (1999).
19) Teeri, T. T., P. Lehtovaara, S. Kauppinen, I.
Salovuori, and J. Knowles. Homologous domains

16

R.oryzae β-glucosidasein Trichoderma reesei cellulolytic enzymes: genesequence and expression of cellobiohydrolase II.
Gene 51:43-52 (1987.).
20) Van Arsdell, J. N. Cloning, characterization,and expression in Saccharomyces cerevisiae ofendoglucanase I from Trichoderma reesei.
Bio/Technology 5:60-64 (1987.).
21) Handbook of microbiological media. Atlas,
R.M., Parks, L.C. (ed.) CRC Press, Boca Raton,
Ann Arbor, London, Tokyo (1993)22) Takii, Y., Daimon, K, and Suzuki, Y. Cloning
and expression of a thermostable α-1,4-gllucosidase gene from Bacillus stearothermophilus
ATCC12016 in Escherichia coli. Appl. Microbiol. Biotechnol., 38:243-247 (1992)
23) Bradford,M.M. A rapid and sensitive method
for the quantitation of microgram quantities ofprotein utilizing theprinciple of protein dye
binding. Anal. Biochem. 72, 248-254, (1976).
24) Laemmli, U.K., Cleavage of structural proteinsduring the assembly of the head of bacteriophage
T4. Nature 227, 680-685 (1970).
25) Murai T, Ueda M, Kawaguchi T, Arai M,
Tanaka A. Assimilation of cellooligo- saccharides
by a cell surface-engineered yeast expressing
beta-glucosidase and carboxy- methylcellulase
from Aspergillus aculeatus. Plant Cell. 16:
2192-203. (2004).

26) Tonomura K., Suzuki H., Nakamura N., Kuraya
K. and Tanabe O. On the inducers of formation in
Aspergillus oryzae. Agric. Biol. Chem., 25 1-6,
(1961).
27) Nagoya M., Goto, M., Okada, H. and Morikawa,
Y. L-Sophorose induces cellulase gene transcription in the celluolytic fungus Tricoderem
reesei. Curr. Genet., 38, 329-334 (2001).
28) Aro, N., Ilmen, M., Saloheimo, A. and Penttla,
M. ACEI of Tricoderem reesei is a repression ofcellulose and xylanase expression. .Appl.Environ.
Microbiol., 69, 56-65, (2003).
29) Murai T, Ueda M, Kawaguchi T, Arai M,
Tanaka A. Assimilation of cellooligosaccharides
by a cell surface-engineered yeast expressing
beta-glucosidase and carboxymethylcellulase
from Aspergillus aculeatus.
Appl Environ
Microbiol. 64 4857-4861 (1998).
30) Gomi, K., Akeno, T., Minetoki, T., Ozeki, K.,
Kumagai, C., Okazaki, N., and Imura, Y.
Molecular cloning and characterization of atranscriptional activator gene, amyR, involved in
the amylolytic gene expression in Aspergillus
oryzae. Biosci. Biotechnol. Biochem., 64: 816-827(2000).

Communicated by Ishijima Sumio