J. Biol. Macromol., 11(1), 23-30 (2011)

Note
Production of -Glucosidase by a Transformant of Aspergillus oryzae RIB40 in a
Liquid-Surface Immobilization (LSI) System
Kenji Ozeki1,*, Kana Kondo1, Hiroshi Nakagawa1, Kiyoko Ozeki2, Shinobu Oda1, andShinichi Ohashi1
1

Genome Biotechnology Laboratory, Kanazawa Institute of Technology, 3-1 Yatsukaho,

Hakusan, Ishikawa 924-0838, Japan and 2Graduate School of Humanities and Sciences, Nara
Women’s University, Kitauoyahigashi-machi, Nara 630-8506, Japan
Received January 15, 2011; Accepted April 8, 2011
The production of -glucosidase by Aspergillus oryzae RIB40 and a transformant,
RIB40-TF harboring the bglA gene was assessed in a liquid-surface immobilization(LSI) system that uses particles of a unique polymeric material, a ballooned microsphere.
In a submerged cultivation system, RIB40-TF produced significantly more -glucosidase
than RIB40, although the enzyme was not secreted into the medium. However, whenusing the LSI system, RIB40-TF secreted -glucosidase into the liquid medium to a levelof 1.36 U/ml. Furthermore, we showed that the LSI system was capable of supporting
repeated production of -glucosidase at least 3 cycles over a period of 39 days.
Key words: xylanase, Aspergillus, liquid-surface cultivation, immobilization, microsphere-Glucosidase(-D-glucosideendo-1,4--glucanase

(EC

and

3.2.1.91)

byglucohydrolase, EC 3.2.1.21) has recently

cellobiohydrolase

attracted considerable attention for use incellobiose. Thus, a multicomponent enzyme

bioethanol production from cellulose, asystemlinear polymer of D-glucose units linked byendo-1,4--glucanase, and cellobiohydrolase

1,4--D-glucosidic bonds. This enzyme

is necessary for the commercial productionhydrolyzes

of bioethanol from cellulose (1, 2).

counteract

cellobiose tothe

D-glucose

inhibition*Corresponding author: Tel.
+81-76-274-7944 Fax. +81-76-274-7511
E-mail: ozeki@neptune.kanazawa-it.ac.jp

to
of

(EC

3.2.1.4)

consistingof

-glucosidase,-Glucosidase is produced by various
fungi,

suchas

Trichoderma(3),

Paecilomyces(4),

Aureobasidium(5),PRODUCTION OF -GLUCOSIDASE IN LSI SYSTEM

24

Alternaria (6), and Aspergillus. Two species

enzymes, low levels of catabolite repressionthat have been particularly targeted for theand proteolytic digestion, and increased

production

stability of the secreted enzymes (14, 17).

andcharacterizationof

-glucosidase are Aspergillus niger (7-10)

However,andThe

disadvantages, such as poor heat dissipation

production of -glucosidase by these fungi

and slow diffusion of nutrients, products,

issubmerged

water, and oxygen in a packed bed (14, 18).

solid-state

Recently, we presented 3 types ofcultivation (SSC) has also been used (5, 12).

unique cultivation and application systems

It is well known that particularfor fungi: liquid-surface immobilization

cultivation methods influence the production(LSI) (19, 20), a liquid-liquid interface

and stability of enzymes. Compared to SmC,

bioreactor (L-L IBR) (19, 21, 22), and the

SSC generally enables high enzyme yield

extractive

and enhances the stability of produced(Ext-LSI) systems (23). Among these

enzymes (14). For example, the production

systems, LSI displayed enzyme productivityof tannase, pectinase, and polygalacturonasesuperior to SmC for lipase and xylanase. In

by A. niger in SSC is significantly higher

the LSI system (Fig. 1), which consists of athan that in SmC (15, 16). The higher

liquid medium overlaid by a mat ofproductivity with SSC results from various

fungus-microspherefactors:, higher titers and productivities ofdifferentiation, sufficient nutrient, water andAspergillus

oryzae(11-13).

commonly examined incultivation(SmC),

although

SSC

has

some

liquid-surface

practical

immobilization(MS),

Fig. 1. Production of -glucosidase in the liquid-surface immobilization (LSI)system. Spores of A. oryzae RIB40 (A) or its transformant RIB40-TF (B) were
floated on the surface of YPD medium with ballooned microsphere (MS)particles. Cultivation in the thick fungus-MS mat used nutrients and water in theliquid medium, and oxygen in the atmosphere. The fungus-MS mat exhibitedhigh yield of -glucosidase.

fungalOzeki, K., et. al.

oxygensupply,andhigher25

enzymeTACTGGGCCTTAGGCAG-3’/-2565-2610

production were achieved, despite thein bglA, 48-mer) which includes a NotI sitestationary conditions (19, 20). We expected(underlined)that the LSI system would also be suitabletermination

for the production of other enzymes, such as

carried out by using LA Taq polymerase

other hydrolytic enzymes and peroxidases.

(Takara-bio Co., Kyoto) in a thermal cycler

Here, we report the application of the LSI(30 s at 95 °C; 40 s at 55 °C; 60 s at 72 °C;

system to the production of -glucosidase an

25 cycles). The 2627-bp PCR product was

important enzyme in the production of

double digested with SalI and NotI. Then,bioethanol from cellulolytic materials with athebglA transformant of A. oryzae RIB40. The

corresponding sites between P-enoA142 andenzyme productivity was compared with

T-agdA, a fungal high-level expressionthat of SmC with A. oryzae RIB40; thevector pNEN142 (9.3 kb) (24). P-enoA142

transformant was shown to have a higher

is the promoter from the A. oryzae enolase

-glucosidase production capability.

gene, improved by introducing 12 copies ofdownstreamcodon.

fragment

from

the

Amplification

was

was

clonedintothe

By analyzing an A. oryzae DNA“region III” that had previously been

microarray, the-glucosidase gene (bglA:

discovered to contain cis-acting sequence

AO090009000356) was seen to be more

involved in the positive regulation of A.

highly expressed in the liquid culture

oryzae amylases, P-enoA, the promoter A.

medium of the rice bran (data not shown).

oryzae enolase gene.

An expression vector containing bglA,

The resulting plasmid, pNEN-BGLA,pNEN-BGLA (12.2 kb), was constructed.

was introduced into A. oryzae niaD300

Plasmid pBGLA, which has a genomic(niaD) according to a previously methodfragment containing bglA, was used as a(25). Transformants were selected based on

template for PCR with the following

the ability to grow on nitrate resulting fromprimers:(5’-

the introduction of the entire niaD gene.

ACGCGTCGACGCGTATGAAGCTTGGT

Recombinant -glucosidase activity was

TGGATCGAGGTG-3’/-14-24

bglA,

measured in 50 mM sodium acetate buffer

38-mer) which introduces a SalI site

(pH 5.0) at 40 °C for 10 min with 1 mM

(underlined)

p-nitrophenyl

primerjust

1

upstream

in
from

the

-D-glucopyranoside.

of -glucosidasetranslational initiation codon of A. oryzae

unit

celA,

2

supernatant, isolated by filtration (pore size,(5’-ATAGTTTAGCGGCCGCATTCTTATT

0.45 m), was defined as the amount ofandprimeractivity in

One
the

PRODUCTION OF -GLUCOSIDASE IN LSI SYSTEM

26

enzymethat

released

1

mol

of

of -glucosidase. Spore suspension (final
concentration, 1 × 106 spores/ml) and 10 ml

p-nitrophenol per min.
We then examined the -glucosidaseof ballooned polyacrylnitrile microspheres

yield of the transformants. The transformant(MMS-DE-1 [former MFL-80SDE]: mean

and the untransformed host were grown for

diameter, 40 μm; density, 0.06; Matsumoto

3 days in YPD medium (1% yeast extract,

Yushi-Seiyaku, Co., Ltd., Osaka) were2% peptone, 2% dextrose, pH 6.5), after

mixed with 50 ml of YPD medium andwhich they were analyzed for -glucosidase

poured into a polypropylene vessel (55 mm

activity. The specific -glucosidase activity,

i.d. × 70 mm). After precultivation (30 °C,

prepared from a cell extract of transformant

without shaking, 3 days), -glucosidase

RIB40-TF, was 130-fold higher than that ofaccumulation in the YPD medium was

RIB40, and reached 1.03 U/ml in SmC.

determined over an 18-day period. As shown

However, neither strains secreted significant

in Fig. 2A, although RIB40

-glucosidase into the medium, similar tolittletheVolvariella

RIB40-TF secreted significant amounts ofvolvacea (26) and Trichoderma reesei (27).

-glucosidase (maximum 1.36 U/ml) in the

Next, RIB40 and RIB40-TF were

medium up to day 11. -Glucosidase

applied to the LSI system for the production

specific activity of RIB40-TF was 667-foldsituation

reportedfor-glucosidaseinaccumulatedthemedium,Fig. 2. Time course of -glucosidase and total protein production by A. oryzae
RIB40 and its transformant, RIB40-TF, in the LSI system. (A) Production of
-glucosidase; (B) production of total protein. Open circles/triangles: RIB40; closed
circles/triangles: RIB40-TF.

Ozeki, K., et. al.

27

higher than that of RIB40. However,-glucosidase activity gradually reduced
after this time. It is assumed that prolonged
cultivation

leads

to

digestionof

-glucosidase by endogenous proteases. The
levels of other proteins in the medium werehigher with RIB40 than with RIB40-TF (Fig.
2B). This might have resulted from titrationof common regulatory proteins that interact
with Region III (pNEN-BGLA) (28).
Finally, production of -glucosidasewith RIB40-TF was performed in an LSI
system that used a 600 ml polypropylene
vessel with inlet and outlet ports. A mixtureof spore suspension (final concentration, 106
spores/ml), 40 ml of MMS-DE-1, and 300

Fig. 3. Repeated batch production of
-glucosidase by A. oryzae RIB40-TF
in the LSI system. At days 15 and 27,
300 ml of YPD medium was
exchanged with fresh medium.

ml of YPD medium was poured to the vessel.

-glucosidase produced by RIB40-TF was

A magnet was put to a bottle in the vessel,

1.36

and a polypropylene net was included tocomparison,stabilize

activities

thefungus-MS

mat.

After

U/ml-medium
thereported

(Fig.

2A).

-glucosidaseforBy

specific

Aureobasidium

precultivation (30 °C, without shaking, 3

pullulans (5), Alternaria alternata (6),days), -glucosidase production in the

Aspergillus terreus (29), Aspergillus niger

medium was periodically determined. The(30), and Schizophyllum commune (31) are

medium was exchanged with fresh YPD

0.46, 2.5, 2.18, 8.5, and 22.2 U/ml-medium,medium (300 ml) at days 15 and 27. As

respectively. Therefore, our -glucosidaseshown in Fig. 3, repeated batch production

titer is currently insufficient for practicalof -glucosidase was stably achieved 3

production purposes. It will be necessary to

times

was

modify the media composition, culture

demonstrated that A. oryzae RIB40-TF was

condition, and strain to achieve competitive

stably maintained in the LSI system, andproduction rates.

over

39

days.

Thus,

it

that it secreted active enzyme over thisperiod.
The maximum specific activity of

We express our sincere thanks to the
Ozeki Co., for providing the pNEN142

PRODUCTION OF -GLUCOSIDASE IN LSI SYSTEM

28

vector, and Director Nobuo Ichimaru and Mr.

-glucosidase from a color variant strain

Tadashiof Aureobasidium pullulans.

Hiraideof

MatsumotoYushi-Seiyaku Co., for the kind gift ofEnviron.

microspheres.

(1994).
6.

Microbiol.,

3774-3780

B.

J.:

Production

andcharacterizationofcellulase

and

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Communicated by Tanida Seiichi