J. Biol. Macromol., 14(1), 15-26 (2014)

Specific gene expression analysis in Aspergillus oryzae grown inwheat bran without starch.
Akihiro Kaneko*, Motoaki Sano, Shinichi Ohashi, Kenji Ozeki
G e n o m e B i o t e c h n o l o g y L a b o r a t o r y, K a n a z a w a I n s t i t u t e o f Te c h n o l o g y,
Ya t s u k a h o 3 - 1 , H a k u s a n , I s h i k a w a 9 2 4 - 0 8 3 8 , J a p a n

Received January 24, 2014; Accepted March 13, 2014

A starch removal method from a wheat bran medium wasestablished using citric acid. Secreted enzymes from cultivated
A. oryzae RIB40 were then investigated. The specific activity of
α-amylase decreased while that of xylanase and xy losidase
i n c r e a s e d 2 - f o l d a n d 7 - f o l d , r e s p e c t i v e l y. P r o t e o m i c s a n a l y s i sidentified three new α-glucosidases and two new α-glucanases.
DNA microarray identified high expression of three new endo glucanases

and

one

α-xylosidase.

The

expressionof

β-1,4-

xylanase was increased 27 -fold.

K e y w o r d s : A s p e rg i l l u s o r y z a e ; W h e a t b r a n ; D N A mi c r o a r r a y ; P r o t e o me

Introduction

by-product of flour milling and overWheat is one of the three major

500,000 tons per year are discharged as

cereals in the world. Wheat bran is abran. Although some wheat bran is usedas health food,1,2) most serves as feed or*To whom correspondence should be

food waste.3,4) Considering the global

addressed: E-mail:food problems, it is important that foodkatz_kaneko_ushizu@yahoo.co.jp, Tel:wastage is reduced and an effective use+81-76-274-7944;

for excess bran is designed. Wheat bran

16

Gene expression in A.oryzae grown in wheat bran without starch

is a cell-wall constituent of wheat,

Strains, media and culture conditions

containing hemicellulose, cellulose,

Aspergillus oryzae RIB40 was

starch and lignin.5-7)Approximately 8%

used throughout this study. A. oryzaeof wheat brans are starches. We report

RIB40 was isolated in 1950 from cereal

on an A. oryzae strain, cultivated on(raw material of shoyu) in Kyoto. All thesolid media, after starch removal from

cultivations were inoculated with 3.3×

wheat bran.

109 conidia of A. oryzae RIB40, andAspergillus oryzae is a safe

incubated at 30 °C for 72h. Wheat bran

filamentous fungus8,9), utilized insolid culture was cultivated using 2 g of

traditional Japanese fermentation food

dry wheat bran with 1.2 ml of water.

industries.10) A. oryzae produces andsecretes various enzymes into its growth

Preparation of wheat bran medium

medium.11-13) Analysis of the genome

without starchsequence of A. oryzae was completed in

During initial cultivation using

2005.14) DNA microarray andstarch-containing medium, amylase-likeproteomics analysis were used to detect

enzymes, such as α-amylase andgenes involved in starch breakdown inglucoamylase, were induced and

A. oryzae when grown in a wheat branhydrolytic enzyme expression was weak,medium.15-17) Previously, Aspergillus

making proteomics analysis difficult.

strains containing expression vectors

Therefore, starch removal from wheat

have been used, including a high-bran was performed. First, 2 g of wheat

producing xylanase strain.18,19) We reportbran was added to 20 ml of 50 mM

on A. oryzae RIB40 using cultivation

citrate or water before autoclaving atconditions which did not induce amylase

121 °C for 20 min. The media was called

as we aimed to isolate new“A”. Then, 100 ml boiling water was

hemicellulase and cellulose genes.

added, before the media was filteredusing a wire mesh filter (3 mm x 1 mm)

Materials and Methods

Residual substances were immersed inA. Kaneko et al.

100 ml of water for 1 minute. The media
was called “AC”.
After 1 minute, the AC media was

17

Sample collection method
Samples were collected from solid
culture using extraction buffer (40 ml ofwashed with 300 ml of water and

10 mM acetic acid buffer (pH 5.0), 1 g

residual substances were collected.

of 0.5% NaCl, and 160 ml sterile water).

These residual substances were washed

Filtration was performed using 90 mm

5 times with 40 ml of water. This “AC”

filter paper (Advantec).

wash media was called “AC+wash”(Fig.1). Furthermore, culture medium
using water instead of citric acid

Protein determination
The protein concentration in eachsolution was also prepared. Untreatedsample was measured using the Bradford

wheat bran medium was used as a

assay and bovine serum albumin as thecontrol.

standard (Bio-Rad).

18

Gene expression in A.oryzae grown in wheat bran without starch

SDS-PAGE
SDS-PAGE was performed

Quantitative analysis of xylose,obtained using two-step enzymatic

according to the method of Laemmli.20)

treatment, and arabinose was performed

Enzyme samples were denatured in 5%

using HPLC under the following(w/v) SDS by incubation for 5 min atconditions23): alliance dissolution test

96 °C.

system 2695 (Waters), RI detector 2414
(Waters) and a Shodex SPO 810 column.

Enzyme activity assays
α-Amylase activity was

The mobile phase was water, flow
velocity of 400 µL/min, temperature

determined using an α-amylase assay kit

65 °C, and analysis time of 35 minutes.

(Kikkoman Corp.).21) In order to

The retention time of xylose was 24-26

perform diastatic power determination,

minutes, and arabinose was 28-30

the diastatic-power-assay kit

minutes.

(Kikkoman) was used. One unit of αamylase activity was defined as theTwo-dimensional electrophoresis

amount of enzyme required to release 1

Protein concentration was

μmol of 2-chloro 4-nitrophenyl (CNP)

measured using the Bradford method.

from 2-chloro 4-nitrophenyl 6-azide-6-

Samples were adjusted to give 300 µg ofdeoxy-β-maltopentaside (N3-G5-β-CNP)

protein for two-dimensional

per minute at 37 °C.

electrophoresis analysis, performed as

Xylanase activity was measured

reported previously. 23)

using Xylanzyme AX tablets(Megazyme). Detection of β-xylosidase
activity used the substrate para-mRNA preparation
Total RNA was isolated from

nitrophenol-β-D- xylopyranosideliquid-grown mycelia using(Sigma) and the method of Ait et al.22)

ISOGEN(Wako, Osaka, Japan),according to the manufacturer's

HPLC analysis conditions

instructions. Briefly, mycelia wereA. Kaneko et al.

19

ground in liquid nitrogen to a finemeasure the fluorescence intensity of thepowder, and 1 g was mixed with 15 ml

two fluorophores using a GenePix

ISOGEN solution.

After addition of 3

4200A scanner (Axon Instruments at

ml chloroform, samples were vortexed

Molecular Devices, Sunnyvale, CA).

then centrifuged at 5000 g for 20 min.

Analysis was conducted using DOGAN

The supernatants were transferred to 7.5

at the National Institute of Technology

ml isopropanol in a fresh tube and theand Evaluation (NITE).

mixture was left at room temperature for(http://www.bio.nite.go.jp/dogan/project/

10 min before centrifugation at 5000 g

view/AO).

for 20 min. The pellet was rinsed in
100% ethanol, and centrifuged again at

Results

5000 g for 5 min. The pellet was

Starch removal from wheat bran by

dissolved in 1 ml of RNase-free watersolubilizationand stored at -80 °C until use.

A. oryzae RIB40 was incubated in

Total RNA was isolated from

A, AC and AC+Wash media, with or

mycelia cultured on solid medium

without citric acid, to remove starch.

according to the modified methods of

The starch removal was performed using

Cathala et al.24).

A, AC and AC+wash. When starch
removal was performed using water, a

DNA microarray experiments
DNA microarray experiments

50kDa protein, assumed to be αamylase, was detected. In comparison

were performed as describedwith the control, a small decrease in thepreviously.25) Two cDNA samples

50 kDa α-amylase band was observedlabeled with either Cy5 and Cy3 werewhen A and AC were used. A greater

mixed and applied onto 12 K A. oryzae

amount of α-amylase was removed whenoligonucleotide microarrays

AC+wash was used. Much larger(Fermlab/NovusGene, Tokyo, Japan) for

decreases were observed when citrichybridization. The slide was scanned toacid solution was used in the washes.

20

Gene expression in A.oryzae grown in wheat bran without starch

Acidic hydrolysis of wheat bran using

measuring specific enzyme activities

50mM citrate, autoclaving (121 °C, 20(Table 1).

minutes) and hot water washes removed

The specific activity of α-amylasemost of the starch, probably due todecreased and the activities of xylanase

decomposition removal (Fig. 2).

and xylosidase increased in A. oryzae

Accordingly, α-amylase was not

RIB40 when cultivated in AC andconfirmed in the around 50kDa as a

AC+Wash with citric acid. AC+wash

result of proteome analysis (data notwith citric acid decreased α-amylase

shown). In addition, the effectiveness ofactivity and diastatic power whenstarch removal was also confirmed bycompared with the control (Table 1).

Fig. 2. SDS-PAGE of extracellular proteins produced in wheat bran extract medium.
M: marker, C: Control, AC: AC medium, AC+W: AC wash medium. Water: washed bywater. Citric acid: washed by citric acid solution.

A. Kaneko et al.

21

TABLE 1. Comparison of extracellular protein contents and enzyme activity.
First step
treatment
solution

Processing
step

Control

Beforeincubation
pH
6.0

After
incubation
pH
8.2

Proteinα-Amylase Diastatic

Xylanase Xylosidase

µg/ml
969

mU/µg
30.0

mU/µg
0.20

(mU/µg)1.2(mU/µg)
0.01

water

A

5.6

8.3

598

21.9

0.04

2.8

0.02

water

AC

6.7

7.8

410

24.1

0.1

4.6

0.02

water

AC+wash

6.9

7.7

345

13.5

0.0

3.4

0.02

citric acid

A

3.4

8.0

817

0.8

0.1

1.5

0.01

citric acid

AC

4.4

7.5

114

1.9

0.1

3.3

0.06

citric acidAC+wash

5.6

7.0

118

0.5

0.0

2.2

0.07

TABLE 2. Identification of secreted proteins after AC+Wash treatment (Fig. 3B).
Gene ID

AO90120000068
AO90103000423
AO90001000208
AO90009000356
AO90701000274
AO90012000135
DPP5_ASPOR
AO90003001036
AO90003000923
AO90005000639
AO90003000476

AO90003001062
AO90023000151
AO90012000445
AO90003001017
AO90020000289
AO90020000207
AO90003000990
AO90020000213
AO90026000003
AO90120000026

Gene namecatalasespot
15

beta-1,4-xylanasebeta-1,4-xylanasebeta-glucosidase-related glycosidases
beta-glucosidase-related glycosidases

21, 3544, 50, 51, 53, 54, 55
1, 2, 14,3, 4, 12beta-glucosidase-related glycosidases
13
dipeptidyl-peptidase 5 precursor
8
subtilisin-related protease/vacuolar protease B
47
predicted endo-1,3-beta-glucanase20
beta-N-acetylhexosaminidasemannosyl-oligosaccharide alpha-1,2mannosidase and related glycosylhydrolases
manganese superoxide dismutase
alpha-D-galactosidase (melibiase)beta-galactosidase
predicted protein
predicted protein
predicted protein
predicted protein
predicted protein
predicted protein
predicted protein

22

23
42
9
16

5, 6
11
24
31, 3234
36
45, 46

Analysis of two-dimensional

proteins secreted from A. oryzae RIB40

electrophoresis

grown in standard wheat bran medium inProteomics was conducted using two-

which starch had not been removed isdimensional electrophoresis.

shown in Fig. 3A and wheat bran

Comparative proteome analysis of

medium in which starch had been

22

Gene expression in A.oryzae grown in wheat bran without starch

removed by citric acid plus AC+Wash iswere found. Glucose is required for A.

shown in Fig. 3B. Spots surrounded by aoryzae to grow and if starch is removed,

circle are α-amylase, spots surrounded

the glucose must be produced from a

by a triangle are β-xylanase (Fig. 3A,

different source. Glucanase was highly

Table 2). Spots surrounded by a square

expressed and was considered to havewere only observed when starch was

generated glucose. In the wheat branremoved using citric acid plus

culture medium in which starch was

AC+Wash. α-Amylase spots were notremoved, α-amylase was not detected

observed in the citric acid plus

but many xylanases were. Two new β-

AC+Wash (Fig. 3). β-1,4-Xylanase was

glucanases were discovered and new

the most abundant protein. Three new β-

predicted proteins were observed (Table

glucosidases were discovered, in3). Exo-arabinanase, endo-1,4-β-addition, endo-1,3-β-glucanase and β-

xylanase A,

galactosidase of the cellulase family

A were discovered as a new enzyme,

glucan 1,3-β-glucosidase

Fig. 3. Comparative proteome analysis of secreted proteins using control wheatbran medium (without treatment) (A) and AC+Wash with citric acid wheat branmedium (B) . Sequential differential display of secreted proteins from A. oryzae .
○：α-amylase, △：beta-1,4-xylanase, □：The spot which is not seen as (A)A. Kaneko et al.

23

TABLE 3. BLAST analysis of the protein spots(Fig. 2B).
Spot No.
31,32
5,6

45,46
11

34
36

FungusGene name

Aspergillus oryzae

glucan 1,3-beta-glucosidase A

Aspergillus flavus

probable glucan 1,3-beta-glucosidase A

Aspergillus oryzae

predicted protein

Aspergillus flavus

BNR/Asp-box repeat domain protein

Penicillium chrysogenum

exo-arabinanase

Aspergillus oryzae

endo-1,4-beta-xylanase A

Aspergillus flavus

probable endo-1,4-beta-xylanase A

Aspergillus oryzae

predicted protein

Aspergillus oryzae

glutaminase A

Aspergillus flavus

glutaminase GtaA

Aspergillus oryzae

predicted protein

Aspergillus fumigatus

putative uncharacterized protein

Aspergillus oryzae

predicted protein

Aspergillus flavus

oxidocyclase

Aspergillus flavus

FAD dependent oxidoredutase

when A. oryzae RIB40 was cultivatedremoved medium, although the cellulase

wheat bran without starch. It is

activity were high. The expression level

suggested that newly observed proteinsof β-1,4-xylanase was increased 27-fold

are induced when α-amylase is

and the expression levels of three newly

decreased, in particular, enzymes fromidentified endo-glucanases were 73, 70,

the cellulase or hemicellulase families.

and 10-fold higher than their expression
in starch-containing medium. The

DNA microarray
RNA was extracted from A. oryzae

expression level of β-xylosidase
increased 3 fold. The genes in which the

RIB40 after treatment with AC+Wash

expression levels increased wereplus citric acid and the control sample inconsidered to participate inwhich starch was not removed. DNA

solubilization of hemicellulose rather

microarray was then performed.

than the starch of wheat bran, cellulose,

The number of secreted celluloserelated genes decreased in the starchand lignin.

24

Gene expression in A.oryzae grown in wheat bran without starch

TABLE 5. Hemicellulase and cellulose genes in which significant
variation in expression was observed between treated and untreated
wheat bran medium.
Fold difference
in expression

Gene Namebeta-1,4-xylanase27

beta-xylosidase3

endo-glucanase73

endo-glucanase70

endo-glucanase10

beta-glucanase related glycosidase

3alpha-glucosidase family31of glycosil hydrolase

38

beta-glucosidsse related glycosidase

5

glucsosidaseⅠ

2

Discussion
Removal of starch from wheat

The specific activities of xylanaseand xylosidase were increased after

bran medium was investigated. We

starch removal. It was considered thatsuggest citric acid is an effective method

the specific activities increased becauseof starch removal from wheat bran:

the nutrient source was altered;

Autoclave treatment with citric acid andhemicellulose, cellulose, and lignin,wash with hot water sufficiently

basic components of wheat bran, were

removed starch such that α-amylase ofutilized instead of starch and we suggest

A. oryzae RIB40 was not induced.

the activities of xylanase and xylosidase

A. Kaneko et al.

25

were increased because A. oryzae

Med., 106, 24-27 (1999).

produced xylan as a nutrient source.

3) Angulo J, Mahecha L, Yepes S,

Induction of xylanase and xylosidase,

Yepes A, Bustamante G, Jaramillo H,

without induction of enzymes in the

Valencia E, Villamil T, and Gallo J, J

amylase family, can be achieved in A.

Environ Manage., 95, 203-209

oryzae RIB40 grown in treated wheat( 2012).

bran culture medium.
In addition, we identified β-glucosidase
and endo-1,3-β-glucanase using twodimensional electrophoresis andproteomics.
DNA microarray analysis of A.
oryzae RIB40 grown in culture medium

4) Watanabe H, J. Jpn. Inst. Energy, 87,
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Nobuaki I, Takashi S, and Hajime T,

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Biosci. Biotech. Biochemi., 58, 288-

and one β-xylosidase.

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Communicated by Ishijima Sumio