J. Biol. Macromol., 15 (1), 13-27 (2015)

ArticleTransglycosilation activity of Aspergillus oryzae-derived α-glucosidase
Emi Nagayoshia, Kenji Ozekib, Mai Hatac, Toshitaka Minetokid and Yukio Takiie*aDepartment of Bioscience, Faculty of Bio-environmental Science, Kyoto Gakuen University,
1-1 Nanjyo Otani, Sogabe-cho, Kameoka, Kyoto 621-8555, Japanb
Genome Biotechnology Laboratory, Kanazawa Institute of Technology, 3-1 Yatsukaho, Hakusan,
Ishikawa 924-0838, Japanc
Department of Food Science and Nutrition, Mukogawa Women’s University, 6-46 Ikebiraki-cho,Nishinomiya, Hyogo 663-8558, Japand
Ozeki General Research Laboratory, 4-9 Imadzu Dezaike-cho, Nishinomiya, Hyogo 663-8227
eLaboratory of Food Science and Health, 448-5 Creation Core Kyoto Mikuruma 215, Kajii-cho,
Kawaramachi Imadegawa, Kamigyou-ku, Kyoto 602-0841

Received May 12, 2015; Accepted June 19, 2015
An Aspergillus oryzae RIB40 (NBRC100959) α-glucosidase （designed as AgdB）gene (agdB)
was expressed at high levels in an A. oryzae host by self-cloning. The obtained transformant(MIBA1002) produced intracellular and extracellular α-glucosidase at levels 3- and 10-foldhigher, respectively, than the parent host strain. The base sequence of agdB consisted of a3036-kb structural gene containing three introns and encoding 963 amino acids, and the linear
sequence thus obtained from these amino acids was identical to A. oryzae RIB40 unknown
protein BAE64257.1. The amino acid sequence had 72% and 51% homology to α-glucosidase B
from Aspernillus nidulans and Acremonium implicatum, respectively, which exhibittransglycosylation activity. The sequence has conserved residues specific to glucosyl hydrase
family 31 (GH31) α-glucosidases. Tyr296 present in the β→α Loop1 in GH31 is important fortransglycosylation. The enzyme produced 2.2% isomaltose, 0.4% maltotriose, and 0.3%
kojibiose from 20% maltose substrate. This is the first report of the transglycosylation activityof α-glucosidase B cloned in A. oryzae (unknown protein BAE64257.1).

Key words: glycosyl hydrolases family 31, nitrate reductase gene-deficient strain, vitamin
glycoside, maltooligosaccharide,

Introduction

non-reducing

α-1,4-glucosideoligosaccharides
Alpha-glucosidase
3.2.1.20 )hydrolyzes(α-glucosidase,theECterminal,

suchmaltooligosaccharides

as(1–4).

bondofstarch

and

Water

is

typically an excellent glucosyl receptor for
carbohydrate hydrolases during catalysis.

To

whom

correspondence

shouldbe

addressed. Email: syokuken@gray.plala.or.jp

However, when the glucosyl receptor is asugar,

α-glucosidasecancatalyzeα-glucosidaseB of Aspergillus oryzae

14transglycosylation. Transglycosylation is thepharmaceutical industries demonstrates thetransferthesafety of enzymes produced by this organism.

α-glucoside,

The A. oryzae genome project was completed

maltooligosaccharide, or α-glucan to thein 2005 (14), Although A. oryzae likelyhydroxyl group at the 6- or 3-position of

produces

glucose or an oligosaccharide. α-Glucosidasetransglycosylation activity, to our knowledge

is widely distributed in animals, plants, andthere has been no report confirming this.

microbes (5–9).

HereThe α-glucosidases are classified into threecharacteristics of α-glucosidase

types according to their substrate specificity.

from A. oryzae exhibiting transglycosylation

Type 1 α-glucosidases are members of theactivity and obtained high-producing strains

GH13

through self-cloning of the gene in A. oryzae.

ofanon-reducing

andcontaining

glucosyl
terminushydrolyze
aryl

group
of

at

hetero-substrates

groups,such

p-nitrophenyl-α-D-glucopyranoside,

α-glucosidases

weinvestigated

the

with

enzymatic(AgdB)

asfasterMaterials and Methods

than maltooligosaccharides. However, GH13
enzymes have almost no effect on α-glucan.

Chromosomal DNA library construction

Type 2 α-glucosidases have higher activity

Chromosomal DNA was isolated from A.

towards maltooligosaccharides than towards

oryzaehetero-substrates and can hydrolyze α-glucan.

procedure (15). The DNA was digested withType 3 α-glucosidases are similar to those

restriction enzyme BamHI, separated byclassified as Type 2, and can hydrolyze

agaroseoligosaccharides and starch at similar rates.

subjected to Southern hybridization using

Type 2 and 3 enzymes belong to the GH31.

AlkPhos Direct (Amersham Biosciences, Inc.

In addition to hydrolysis, α -glucosidases

Piscataway, NJ, USA). The probes used were

catalyzes transglucosylation to form new

generatedglucosidic linkages, and thus it is used for thereaction (PCR) as described below. A.

production ofα-glucosides(10–13).

nidulans belongs to the same family as A.

The fungus A. oryzae is essential in the

oryzae.

traditional Japanese fermentation industry

α-glucosidase B (5) is conserved between A.

and is used in the production of sake,oryzae and A. nidulans. The following

soybean paste, and soy sauce. The longprimers were designed: SP1 degenerate

history of A. oryzae in the food andprimer (5′-TGGATHGAYATGAAYGARCC-

strain

gel

by

The

RIB40

using

electrophoresis,

nested

amino

a

and

polymerase

acidroutine

then

chain

sequenceofE. Nagayoshi et al.

153′), ASP2 primer (5′-TGRTCNCKRAARAA

Japan). The construct was then transfected

NGTRTA-3′), and ASP3 primer (5′-CCNCC

into Escherichia coli strain LE392 to gener-

RAANCCRCANACRTC-3′),

ate a chromosomal DNA library.

where

H

represents A, C, or T, Y represents C or T, R
represents A or G,, and N represents A, C, G,

Cloning into phage

or T. Nested PCR was performed first using

The chromosomal DNA was cloned into

the chromosomal DNA, SP1 primer, andphage and plaques were formed. Clones

ASP3 primer, and then with the obtained

containing the agdB were selected by plaque

PCR product, SP1 primer, and ASP2 primer,

hybridization using ECL Direct (Amersham

to provide a PCR fragment approximately

Biosciences, Tokyo, Japan). The probes used

900 bp long (15) which was used as a probe

were the same as those used to construct thefor Southern blot analysis.

chromosomal DNA library. Several positive

A band approximately 5.5 kb in size was de-plaques were obtained.

tected using Southern blot analysis. The
chromosomal DNA was digested again with

Sequencing of agdB gene

BamHI and separated by agarose electropho-

A DNA fragment from A. oryzae was ampli-

resis as follows. The 5.5 kb band was cut out

fied from the positive plaques. PCR was per-of the gel using a razor, crushed in a mi-formed using the T7 and T3 promoter se-

cro-centrifuge tube using a plastic pipet tip,quences adjacent to the vector-cloning site

and incubated with constant shaking in 50

and the 3′- and 5′-terminal sequences of themM Tris-HCl /80 mM NaCl (pH 7.5) at 37°C

probe. The nucleotide sequence of the vector

for 1 h. The agarose pieces were then re-insert was determined by sequencing the am-

moved by centrifugation at 3,000 x g at room

plified PCR fragment using a BigDye Ter-

temperature for 1 min. The fragment recov-

minator v1.1 Cycle Sequencing Kit (Applied

ered from the supernatant by ethanol (final

Biosystems, Life Technologies, Tokyo, Ja-

concentration: 75%) precipitation was dis-pan).

solved in 10 mM Tris-HCl (pH7.5). The
DNA in an aliquot of this sample was ligatedConstruction of vectors for use in A. oryzae

into the BamHI site of λDASH II phage using

PCR was performed on the obtained plaques

T4 ligase. The obtained phage DNA wasusing a primer containing a start codon andpackaged using an in vitro packaging kit

NotI sequence and another primer containing(Gigapack-Gold: TOYOBO Co. Ltd, Osaka,

a stop codon and XbaI sequence to amplify

α-glucosidaseB of Aspergillus oryzae

16

the -glucosidase B structural gene. The ob-

dextrin, 1% polypeptone, 0.1% KH2PO4,

tained PCR fragment was ligated into the

0.05% MgSO4・7H2O, pH 5.5) was inoculated

pNEN8142 vector, which can replicate in A.

with 2.5 × 106 spores, then cultured at 30°C

In brief, 10 μg of the PCR

for 1–9 d with shaking. The cultures were

fragment was double-digested with NotI andpassed through a 3GI glass filter and the

XbaI and subjected to 0.8% agarose gel elec-

filtrate was used as the extracellular crude

trophoresis to separate a 3-kb fragment. This

enzyme preparation. Cells were washed withfragment was cut from the gel and recovered

ultrapure water and homogenized in bufferin TE solution (10 mM Tris-HCl, 1 mM

(50 mM NaH2PO4 (pH 7.0), 10 mM EDTA,

EDTA, pH 8.0) using a Gene Clean Kit-Bio

0.1% Triton X-100, 0.1% N-lauroylsarcosine

101 (MP Biomedicals, Japan, Tokyo, Japan).

sodium salt, 10 mM β-mercaptoethanol) and

The pNEN8142 vector was double-digested

centrifuged. The supernatant was used as thewith NotI and XbaI and then ligated with theintracellular crude enzyme preparation.

oryzae (16).

3-kb. DNA fragment, obtained as describedabove, to generate pNEN142-agdB(17).

-Glucosidase activity (hydrolytic assay)
-Glucosidase activity was measured with an

Preparation of A. oryzae producing highα-glucosidase assay kit (Kikkoman Co., To-levels of agdB

kyo, Japan) (18). using

A.oryzae was transformed with thep-nitrophenyl-α-D-glucopyranoside as thepNEN142-agdB (17). The cells were

substrate and measuring the amount of

cultured at 30°C in dextrin Czapek-Dox (DC;p-nitrophenol released after incubating at

2% dextrin, 0.3% NaNO3, 0.2% KCl, 0.1%

37°C for 10 min. One unit of α-glucosidase

KH2PO4, 0.05% MgSO4 ・ 7H2O, 0.002%

was defined as the amount of enzyme releas-

FeSO4, pH 5.5) containing 0.8 M NaCl. Since

ing 1 μmol of product per minute. McIlvaine

pNEN142-agdB

broad buffer (0.1 M citric acid /0.2 M

contains

thenitratereductase gene (niaD), transformants were

di-sodium phosphate buffer: pH 3.0–8.0) was

selected by their ability to grow on DC

used to determine the optimal pH for enzyme

medium.

activity and its pH stability. The pH of thesolution was adjusted to 5.0 with 10 vol. each

Conditions for cell culture and enzyme

ofextraction

then glucose was assayed using a Glucose C

Dextrin peptone medium (100 mL; 2%

0.1 M citric acid or 0.2 M Na2HPO4,

E. Nagayoshi et al.

17

test kit (Wako Pure Chemical Co., Osaka,present it seems to be more suitable for us toJapan).

transform the respective strain by using
BLAST

searchA.

oryzae(geneBankTransglycosylation activity and conditions

BAE64257.1: 82% identity).

for HPLC analysis

Sequencing the insert of the recombinant

To measure the transglycosylation activity ofphage showed that the agdB gene was a

α-glucosidase, 20% maltose, 10mM acetate

3036-bp structural gene containing threebuffer (pH 5.5), and 0.023 U of enzyme (50introns and encoding 963 amino acid residues.

μL total volume) were incubated at 37°C, the

Homology searches of this base sequence

enzyme was heat denatured at 100°C for 5

using

min to terminate the reaction, then the

nih.gov/Blast.cgi) (19) returned an unnamed

mixture was analyzed by high performanceprotein product from A. oryzae (GenBank:liquidchromatography

BAE64257.1; 100% sequence identity), a

column(6 x 150 mm) of Shodex SUGAR(HPLC).

HPLC

putative

BLAST

lysosomal
flavus(http://blast.ncbi.nlm.

α-glucosidase

from

NRRL3357(XP

Series® 6 µm SZ5532 100 Å was used andAspergillus

eluted at 1 mL/min with an acetonitrile to002380576.1; 99% identity), α/β-glucosidasewater ratio ranging from 80:20 (0–10 min) to

AgdC from A. oryzae (XP 001825390.2; 95%

70：30 (10–30 min). A RID-10A differential

identity), and Pc21g13670 from Penicillium

refractometer (Shimadzu, Kyoto, Japan) was

chrysogenum

used as a detector.

002568384.1; 88% identity). These proteins

Wisconsin

54-1255(XP

with high sequence identity had been
Results and Discussion

speculated based on the base sequence of thecorresponding isolated DNA (GH31) andBase sequence of the agdB gene

their functions likewise were speculative.

Initially, the present study was conducted

The results for α-glucosidases that reportedly

throughout under lacking the references

have

about the corresponding genome structure

α-glucosidase B from A. nidulans FGSC A4

since total genome sequences of A. oryzae(XP 682222.1, 82% homology) (5)were kept unknown at that time. We obtained

α-glucosidase from Acremonium implicatum

those sequences by applying the information(BAD08418.1, 51% homology) (6). Theseof A. nidulans agdB genes to those that

results indicate that the isolated enzyme mayreportedly had transglycosylation activity. At

be involved in the synthesis of noveltransglycosylation

activity

were

and

α-glucosidaseB of Aspergillus oryzae

18

glycosides.

A search for this gene in DOGAN, a website

CLUSTLW (20) analysis results obtainedthatusing these amino acid sequences are shown(http://www.bio.nite.go.jp/dogan/Top),in Fig. 1. Tyrosine (Tyr)296 was present inyielded maltase-glucoamylase and related

the β→α Loop 1, which contains bothhydrolases

conserved and catalytic residues specific to(AO090038000471). Although several other

GH31

is

enzymes belonging to the GH31 family are

theirannotated for A. oryzae, they showed low

α-glucosidases

considered(21–23),

important

for

and

transglycosylation activity (23).

annotatesallA.

oryzaebelongingtoproteins

GH31

sequence homology (Table 1).

A.oryzae (This study) ･･････YGAHPIYFDHRQTGTHGVFLLNSNGMDIFID--NNSTQFLEYNIIGGVLDFYFIAGPTPRDVAIQYAEITQTPLMTPYWGLGYHQCKYGY
A.nidulans･･････YGAHPIYFDHRQDGTHGVFLLNSNGMDIYID--NEGGQFLEYNIIGGVFDFYFIAGPSPQDVARQYAEIVQPPLMVPYWGLGFHQCRYGY
A.implicatum･･････YGAQPVYLEHRETGTHGVLFLNSNGMDVIIDRDNNGNQYLEYNTLGGVLDLYFFAAPTPIEAAQKYSEIAGLPALSPYWGLGFQQCKYGY***:*:*::**: *****::*******: ** *:. *:**** :***:*:**:*.*:* :.* :*:**. * : ******::**:***

298

A.oryzae
A.nidulans
A.implicatu

QDVYEVAAVVANYSTNNIPLETIWTDIDYMDRRRIFTIDPERFPADLYKDLVDTIHARDQHYIVMVDPAVYYKESNPALDEGLKYDIFMKENNGSEYQGV

398

QDVYEVAAVTANYSVHDIPLETIWTDIDYMDRRRIFTLDPERFPPELVKDLVDTLHARDQHYIVMVDPAVYYSEPNPALDAGLKYDAFMKELNGTHYQGV

398

RDVFEVAEVVYNYSQANIPLEVMWTDIDYMDRRRVFSLDPERYPIEKVRALVDHLHENDQHYIVMVDPAVAYVESD-TLSRGIEDDIWLLHSNGSVWLGV:**:*** *. *** :****.:***********:*::****:* : : *** :* .************ * *.: :*. *:: * :: . **: : **385

A.oryzae
A.nidulans
A.implicatum

VWAGPSHFPDWFHPDSQQYWSEQFLAFFDGTNGPDIDALWIDMNEPANFYNHPYPGNNTTPENFAEVDGDPPAAPAVRDGPDAPIPGFPASLQPNWVQGN

498

VWAGPSYFPDWFHPNAQEYWTEQFLNFFDGVNGPDIDALWIDMNEPANFYNRPYPGNNTTPEEFAEANDNPPEPPAVRDGPDAPIPGFPDSLQPNFASGQ498

VWPGVTVFPDWFAKNIGKYWNNEFALFFDKDEGVDIDGLWIDMNEPS-----SFPCFFPCDDPYGSAKGYPPEPPPVREFP-RELPGFPCALQP---EGT

476**.* : ***** : :**.::* *** :* ***.********:.:*. : :..... ** .*.**: *:**** :***

298
286

.*

A.oryzae
A.nidulans
A.implicatum

ATEKRSTAAVVKRQRSQSRRNLGAGHWKSPKGKF--DARAGWQHGKQTGSGCGPNECKGLPNRHLIRPPYMIQN---------GAGPTLADSTADTDLVQ

587

TNEKRAVVTVERRARSQSHRQLGAGRWRSAVRHWPRDPKAGWQHGRKSGSGCGPHECRGLPNRELIRPPYMIQN---------GAGPTLADNTADTDIVQ

589

ECEDGSVAGSSKRDTS----LLHTAEYIARDTKFN-NLRAPALPRQSDG------DQKGLPDRDLLYPEYAIHNKAAFKDSWNAAEGGISNKTVLTDVIH*. :.. :* ** :..: : :: : :*:. *: :***:*.*: * * *:*.* :::.*. **:::

565

A.oryzae
A.nidulans
A.implicatum

SGGYVQYDTHNLYGAMMSSHSHNAMRARRPDDRALVITRSTFAGSGKDVSHWLGDNVSGWLWYQLSISQILQFASLYQIPVVGPDVCGFGGNVTETLCARSGGYVQYDTHSLYGAMMSTHSHNAMRARRPDDRALVITRSTFAGSGKDVSHWLGDNISDWLSYRLSISQILQFASLYQIPVVGPDVCGFGGNVTETLCAR

684
686

QNGLAEYDVHNLYGAMMSTASYDAMLARRPGLRPYVITRSTFPGAGHKVGHWLGDNLSNWDQYRQSIRTMLAFTSIFQFGMVGSDVCGFGGNTTEELCAR..* .:**.*.*******: *::** ****. *. *******.*:*:.*.******:*.* *: ** :* *:*::*: :** ********.** ****

662

A.oryzae
A.nidulans

WATLGSFYTFFRNHAEIYANSQEFYRWPTVAQAARNGISIRYQLLDYIYTAIYKQNQTGTPALNPLFFNYPNDPNTYPIDLQFFYGDGIL･･････････
WATLGSFYTFFRNHAEIFANPQEFYRWPIVAEAARNGIAIRYQLLDYIYTAIYKQTQTGTPSLNPLFFNYPFDQNTYGIDLQFFYGPGIL･･････････

787
789

A.implicatum

WASLGAFQTFYRNHGQFEFSYQEFFLWDTVAESARKAIDIRYRLLDYMYTALWRQSKDGTPAILPMFYIYPEDKNTWDLELQYFYGQGVI･･････････**:**:* **:***.:: . ***: * **::**:.* ***:****:***:::*.: ***:: *:*: ** * **: ::**:*** *::765

Fig.1. CLASTALW alignment of glycoside GH31 members that have reported
transglycosylation activity.
ｗ is catalytic residues, ｗ is conserved residues,
ｗ is important aromatic resi
dues in GH31 α-glucosidase. The β→α Loop1 is involved in the transglycosylation

specificity of the GH31 α-glucosidase. → is the β→α Loop1 of GH31 α-glucosidase.

E. Nagayoshi et al.

19



Table 1. BLAST search results for A.oryzae GH31 members (performed using

DOGAN).



Product

ident

DOGAN ID

maltase glucoamylase and related hydrolases, glycosylhydrolase family 31

53.75

AO090005001084

maltase glucoamylase and related hydrolases, glycosylhydrolase family 31

34.92

AO090003001209

glucosidase II catalytic (alpha) subunit and related
enzymes, glycosyl hydrolase family 31

31.48

AO090102000559

alpha-glucosidases, family 31 of glycosyl hydrolases

24.75

AO090001000649

alpha-glucosidases, family 31 of glycosyl hydrolases

27.83

AO090023000288

alpha-glucosidases, family 31 of glycosyl hydrolases

26.67

AO090005000767

alpha-glucosidases, family 31 of glycosyl hydrolases

28.47

AO090005000768

alpha-glucosidases, family 31 of glycosyl hydrolases

22.78

AO090026000111

alpha-glucosidases, family 31 of glycosyl hydrolases

26.34

AO090701000639

alpha-glucosidases, family 31 of glycosyl hydrolases

24.39

AO090701000558

serine/threonine protein kinase of the CDC7 subfamily
involved in DNA synthesis, repair and recombination

23.57

AO090020000026



predicted protein

35.38

AO090003001426



Na+/K+ ATPase, alpha subunit

31.82

AO090005000141












Glucosidase production by transformed

relative of A. oryzae, is reportedly localized

MIBA1002

in the cell wall. -Glucosidase synthesized

Self-cloning of agdB in A. oryzae yielded 65

by MIBA1002 appears to have a signaltransformant strains. The MIBA1002 strain

sequence, as demonstrated in A. nidulans (5).

showed the highest α-glucosidase production

The sequence information obtained here

and was cultured with shaking in CD medium

indicates that MIBA1002 strain releases

for 1–9 d to assess its enzyme production

α-glucosidase into the extracellular spaceability.

following recombination.

MIBA1002

had

approximately

10-fold higher intracellular -glucosidase
activity on day 3 of culture (0.12 U/mL) and

SDS-PAGE electrophoresis

an approximately 3-fold higher extracellular

The extracellular crude enzyme preparation

enzyme activity on day 8 (0.021 U/mL) (Fig.

was subjected to sodium dodecyl sulfate2) compared with the wild-type strain.

polyacrylamide

α-Glucosidase B from A. nidulans, a close

Electrophoresis was performed using angelelectrophoresis.

20

α-glucosidaseB of Aspergillus oryzae

Fig. 2. Comparison of agdB production by MIBA1002 and wild-type strain.
Left panel, intracellular enzyme production by MIBA1002 and wild-type strain cells.
Right panel, extracellular enzyme production by MIBA1002 and wild-type strain.

Fig. 3. Electrophoresis profiles of
agdB.
Electrophoresis was performed at 25°C
for 1.5h at 25 mA/gel, using 3 µg and 5µg protein in the presence (A) or absence (B) of SDS. The numerals in theleft panel denote molecular weightstandards (from top to bottom): myosin(200,000), β-galactosidase (116,000),
phosphorylase-b (97,000), bovine serum albumin (66,000), and ovalbumin(45,000).

polyacrylamide gel and proteins were stainedProtein

bands

were

observed

atusing Coomassie Brilliant Blue R250 (24)approximately 78 kDa and 63 kDa and had(Fig. 3). Myosin (200 kDa), β-galactosidase

high purity. The N-terminal amino acid(116 kDa), phosphorylase b (97 kDa), serum

sequences of the 73- and 63-kDa subunits

albumin (66 kDa), and ovalbumin (45 kDa)

were identified at residues 21 to 28 and 515

were used as molecular weight markers.

to 522 of the derived amino acid sequence,

E. Nagayoshi et al.

21

respectively. The first 20 amino acids at theTransglycosylation activity

N terminus of AgdB shows a typical featureTheof signal peptides.

transglycosylation activity of MIBA1002

Native PAGE of the crude enzyme on a 10%

enzyme was analyzed with an HPLC. Malt-

polyacrylamide gel yielded a single band,ose at an initial concentration of 20% (w/v)

indicating that the protein was composed of

was decreased to 1.1% by incubationg with

two subunits. Although Aspergillus species

the enzyme for 120 min and completely de-generally produce enzymes in solid-state

graded after reacting for 180 min. The reac-

culture, Aspergillus was here induced totion products generated after 240 min were

produce enzymes in liquid culture, allowing

identified as follows: 2.2 % isomaltose, 0.4%

us to obtain high-purity crude enzymes.

maltotriose, and 0.3% kojibiose (Fig. 5, 6).

hydrolyzing

ability

togetherwithOn the other hand, when, instead of the exEnzymatic characteristics

tracellular recombinant protein, we used sim-

The optimal temperature, heat resistance,

ilar amounts of enzyme obtained from thethermal stability, optimal pH, and pH

wild type strain, 7.9% of the maltose sub-stability of this enzyme were determined. The

strate was left unreacted, and no significant

optimalamount of oligosaccharides was detected af-

temperatureforactivitywasdetermined to be 60°C after incubating for 10

ter 300 min incubation.

min (Fig. 4A). The enzyme was stable up to

These results confirmed that agdB encoded

45°C (Fig. 4B). When incubated at 4°C, 20°C

a protein exhibiting transglycosylation activ-(room temperature), and 30°C for 24 h, theity and indicated that 20% maltose as a sub-enzyme maintained ≥ 90% activity. Even

strate likely favored transglycosylation overafter

thawing,hydrolysis. Liquid culture offers the ad-

enzymeactivity remained relatively stablevantage of easy control of culture conditions(data not shown). The optimal pH was 5.0

compared to solid-state culture. Liquid cul-when incubated for 10 min at pH values fromture of MIBA1002 enabled the release of

3.0 to 8.0 (Fig. 4C). The enzyme maintained

highly pure AgdB product into the extracel-≥ 60% a incubated at each pH value for 24 hlular space. MIBA1002 can facilitate amy-

and was most stable at pH 5.0 (Fig. 4D).

lolysis, producing glucose in the presence ofProcedures for these assays are provided inamylase, and has many applications in thethe figure legends.

fermentation, food, and pharmaceutical inrepeatedfreezingand22

α-glucosidaseB of Aspergillus oryzae

Fig. 4. Effects of temperature and pH on the activity and stability of agdB.
The activity of agdB was determined as described in the Materials and Methods,
except that the temperature was changed. The maximum activity is taken as 100%.
A, agdB (0.12 U/mL) was incubated for 10 min at different temperatures in acetate
buffer (pH 5.0). B, agdB was divided into aliquots (0.18 U/mL each) and one aliquot
was heated for 15 min at the indicated temperature (30 – 70°C). Each mixture wasthen assayed for residual activity. The activity observed after incubation at 4°C
was considered to be 100%. C, The activity of agdB at different pH values was assessed as described in the Materials and Methods. The maximum activity wasconsidered to be 100%. D, agdB was divided into aliquots (0.18 U/mL each) and one
aliquot was incubated at 20°C for 24 h at the indicated (pH3.0 – 8.0). The pH ofeach mixture was then adjusted to 5.0 and the activity was assayed. The activity
measured after incubation for 24 h at pH 6.8 and 4°C was considered to be 100%.

E. Nagayoshi et al.

23

Fig.5. Structures of the glucooligosaccharides used in this study.

Fig.6.

HPLC

analysisofchromatographytransglycosylation

by

MIBA1002 enzyme.
Using 20% maltose as a substrate,transglycosylation

reactions

wereperformed with 0.023 U enzyme in
10mM acetate buffer (pH 5.5) in a
total volume of 50 μL at 37°C for 30,
60, 120, 180, 240, and 300 min.
Maltose(○),

isomaltose

(□),

and

glucose(●),

maltotriose

(■),

and

kojibiose (▲) were used as standard
markers.

dustries. Although isomaltose and kojibi-

maltosesubstrate.

Currently,

ose can be produced from sucrose using su-

α-glucosaccharides are too expensive for thecrose phosphorylase from Leuconostoc mes-

mass production of these oligosaccharides.

entroides, the process is complicated and the

Very recently, A.niger α-glucosidase wasyields are extremely low (10). MIBA1002

expressed in yeast and optimized (25) and theshows great promise for the development ofsimilar enzyme was purified from thermo-

foods

on

philic fungus to achieve more effective pro-non-conventional carbohydrate processing.

duction (26). Our aim to synthesize useful

MIBA1002 enzyme may be useful as aoligosaccharides from maltose will be ac-transglycosylation catalyst for these applica-

complished in future by using AgdB

tions since it formed 2.2% isomaltose, 0.4%transglycosylation activity. The present study

maltotriose, and 0.3% kojibiose from 20%

is the first report of the enzymatic character-andcosmetics

basedα-glucosidaseB of Aspergillus oryzae

24

Glucoamylase. Biosci. Biotechnol.

istics and transglycosylation ability of an un-

Biochem. 61:1233–1239.

named protein product from A. oryzae.
3.

Duan K, Sheu D, Lin C. 1995.

theTransglucosylation of a fungal

Fermentation Research Institution (deposit

alpha-glucosidase. The enzyme

number: NITE P-214) (Tokyo, Japan).

properties and correlation of

MIBA1002

wasdepositedwithisomaltooligosaccharide production.
Ann. N. Y. Acad. Sci. 31:325–328.

Disclosure statement
4.

Frandsen T, Svensson B. 1998.

A patent relating to this work has been

Plant alpha-glucosidases of the

publishedglycoside hydrolase family 31.

(Patent

Publication

No.

Molecular properties, substrate

2008-72956) (Tokyo, Japan).

specificity, reaction mechanism,and comparison with family

Acknowledgements

members of different origin. Plant
Mol. Biol. 37:1–13.

This study was supported by the Ministry of
Education, Culture, Sports, Science and5.

Kato N, Suyama S, Shirokane M,
Kato M. 2002. Novel α -Glucosidase

Technology Grant-in-Aid for Scientific Re-from Aspergillus nidulans withsearch, number B-22300265. We express

Strong Transglycosylation Activity.

our sincere thanks to the Ozeki Co. forAppl. Environ. Microbiol.

providing the pNAN8142 vector and68:1250–1256.

host (A.oryzae)

6.

Yamamoto T, Unno T, Watanabe Y,
Yamamoto M, Okuyama M, Mori H,
Chiba S, Kimura A. 2004.

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Communicated by Ishijima Sumio