J.Biol.Macromol.,3(2), 57-60, 2003

Purification and properties of α-amylase from Aspergillus oryzae MIBA316
Makiko Kariya1), Yoko Shigemi1), Megumu Yano1), Hiroshi Konno2) and Yukio Takii1)
1)

Department of Food Science and Nutrition, School of Human Environmental Sciences, Mukogawa Women’s
University, Ikebiraki-cho, Nishinomiya, Hyogo 663-8558, Japan, 2)Akita Konno Co., Ltd., Kariwano, Akita 019-2112
Received February 14, 2003; Accepted July 1, 2003

An alpha-amylase was purified from a culture broth of Aspergillus oryzae MIBA316. The enzyme hydrolyzedeffectively amylopectin, amylose and starch, and cleaved maltopentaose to form a mixture of maltotriose andmaltose. Glucose was not produced from maltose. The N-terminal sequence of the first 10 residues and othermolecular properties were identical with those of Taka-amylase A.

Keywords: Aspergillus oryzae, α-amylase, glucoamylase, maltopentaose
Aspergillus species have attracted attention for their rolein fermentation of oriental food products or industrial
application of hydrolytic enzymes. Within the rangeof carbohydrolases produced by this species, themajority has been considered as α -amylase andglucoamylase1-3). Strain MIBA316 has been utilized forspecific fermentation of Japanese sake at Akita Konno
Co.
In the present study,α-amylase was purified fromthe culture broth of strain MIBA316 and its enzymaticand molecular properties, especially the cleavage
pattern of maltooligosaccharides, were characterized.

Materials and methods
Cultivation of A. oryzae MIBA316
MIBA316A was cultivated at 30℃ for 2 days on
medium I agar (3%) plate (pH 5.5), which contained
0.5% KH2PO4, 0.1% NaNO3, 0.5% soluble starch,
1.0% peptone, and 0.1% MgSO4.7H2O. Cells grown
were collected, suspended in 30 ml of 0.85% NaCl.
The 1-ml aliquots of cell suspensions obtained wereinoculated to 500 ml-flasks, containing 50 ml ofmedium I and shaken at 30℃ and at 100 rpm for 6days. The cultures were filtered through a glass filter(pore size, G1) and concentrated to 100 ml in vacuumat room temperature.
Purification of enzyme
Step 1. The culture concentrate was dialyzed against 10mM potassium phosphate buffer, pH 6.8 (named asPPB) and centrifuged at 10,000 rpm for 30 min at 4℃to remove insoluble materials.
Step 2. The clear supernatant solution was applied toDEAE-Sephacel column (1.5 x 36.2 cm), which wasequilibrated with PPB. After washing thoroughly withPPB, the column was eluted with a linear gradient (700ml) conducted between PPB in a mixing chamber andPPB/1.0 M NaCl in a reservoir (each 6 ml /tube)． The
active enzyme fractions were pooled, concentrated on
Amicon PM-10 membrane filter, and subjected tocentrifugation as described above.
Step 3. The concentrate (2.5ml) was applied to aSephacryl S-200 column (2.5 x 93.4 cm),which wasequilibrated with PPB/0.5 M NaCl/0.02% sodiumazide (each 3 ml/tube). The active fractions eluted
from the column were collected and concentrated.

Step 4. The concentrate was rechromatographedon Sephacryl S-200 column, as described in step 3.

58

Kariya et.al

The active fractions eluted from the column wereconcentrated, dialyzed against PPB and pooled as thefinal preparation.
Enzyme assay
α-Amylase activity was assayed as follows: Thereaction mixture (2.0 ml) contained 80 mM maleate-NaOH buffer (pH6.8), 10 mM amylopectin, and the
diluted enzyme solution. After incubation for 2-10
min at 37℃, the reaction was terminated by boiling for5min. Reducing power was determined by using
3,5-dinitrosalycilic acid4). When one of the oligosaccharides was employed as substrate, glucose formed
was determined by using glucose oxidase kit5). One
unit (U) of the enzyme activity was defined as theamount of enzyme hydrolyzing 1 μmol substrate for 1
min at 37℃ and at pH 6.8. Protein was assessed by
the method, as described before6).

Table 1. Purification of α-amylasefrom A .oryzae MIBA316
Step1234

TotalProtein

Total SpecificActivity activity

yield

purity(mg)(Unit)(U/mgProtein)(%)(fold)

817.2
250.0
73.3
17.4

4437
4878
2276
1124

5.1
19.5
31.1
64.5

100.0
110.0
51.3
25.3

1.0
3.6
5.8
12.6

Molecular weight determination
SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
was run according to the method, as previously
reported7,8). The molecular weights of proteins weredetermined by using standard molecular weightmarkers (29,900 – 112,000).

Results and discussion
The final preparation of enzyme was purified
12.6-fold over culture broth, the yield being 25.3 %(Table 1). This preparation gave a single protein bandin both native PAGE and in SDS-PAGE (Fig. 1).
The molecular weight of the enzyme was estimated tobe 50,000. The N-terminal amino acid sequence ofthe first 10 was determined to be Ala-Thr-Pro-Ala-AspTrp-Arg-Ser-Gln-Ser-. The activity was maximal at55℃, and the enzyme was most active at pH4.5.
These properties were identical to those for
Taka-amylase A9).
The enzyme effectively hydrolyzed amylopectin andamylose (Table 2). On the other hand, glycogen,pullulan and panose were not effective substrates.
Neither glucose liberation nor an increase in reducing
power was confirmed when one of the following
saccharides was employed as the substrate; isomaltose,isomaltotriose, and isomaltotetraose.

1234Fig.1 Native and SDS-PAGE of A. oryzae MIBA316
α-amylase
Lanes1 and 2, molecular weight markers (112,000, 81,000,49,900, 36,200, 21,000) and (68,000, 45,000, 29,900 and14,100): lanes 3 and 4, purifiedα-amylase (2.5 μg each) inSDS-gel and in native gel,respectively.

Hydrolysis of amylose and amylopectin, respectively
by this enzyme was followed by incubating 20-μl each
at 0.5, 1, 2, 4 and 24 h intervals. Maltose increased
throughout the reaction, whereas maltotriose decreased
continuously, and glucose appeared after 1 h incubation.
Such hydrolysis pattern indicates the linkage to be split
in maltosaccharides is the non-reducing terminal one.

A.oryzae α-amylase

59Ｇ1Ｇ1Ｇ2Ｇ2Ｇ3Ｇ3Ｇ4Ｇ4Ｇ5Ｇ5S 0 10 20 30 40 50 60(min)S 0 10 20 30 40 50 60 S(min)Fig. 2. A paperchromatogram of the product from maltopentaose (in left panel) and maltose (in right panel) attime intervals indicated of A.oryzae MIBA316 α-amylase. S,.standard maltooligosaccharides. G1, glucose; G2，maltose; G3, maltotriose; G4, maltotetraose; G5, malttopentaose. Twenty μl aliquots of the respective reaction mixtures wereapplied on Advantec Toyo NO.51A filter paper. N-butanol/ pyridine/deionized water (6:4:3, v/v) as solvent, and spots detected using
silver nitrate, as described by Walker and Campbell (J. Bacteriol. 86:681-688, 1963).

Table 2 Hydrolysis of various Substrates
by A. oryzae MIBA316 α-amylase
Ｒｅｌａｔｉｖｅ activity(％)
ＳｕｇａｒｓIII

100

0.25% amylose

99

0.25% soluble starch

37.5‐‐‐

0.25% glycogen2.10‐

0.25% pullulan

010mM panose

0.00

0.5% amylopectine

10mM maltose
10mM maltotriose
10mM maltotetraose
10mM isomaltose
10mM isomaltotrise
10mM isomaltotetrase

‐‐‐‐‐
‐

I, assayed by using dinitrosalycilic acidII, determined by using glucose oxidase.

‐‐
1.07
100
68.80
0.00
0.00
0.19

Maltopentaose was degraded to maltotriose andmaltose. However, maltose could not be degraded toglucose (Fig.2). It was also observed maltotetraosee
was converted to a mixture of maltotriose and maltose,
and that maltotriose was slowly decomposed to maltose
and glucose. Maltose was not effective substrate of thisenzyme to yield glucose (Table2, Fig.2).
Amylose (0.5 %) was incubated for 30 and 60 min
with 0.2 U enzymes, and subjected to the polarimetricanalysis. Comparable experiments were conductedwith same units of glucoamylase from Rhizopus sp. andα-amylase from Bacillus subtilis, respectively. After
60 min treatment, the optical rotation was 0.4 degreewith glucoamylase. But the degree was 1.5 and 1.8 for
MIBA enzyme and α -amylase, respectively. The
anomeric configuration of products revealed that thisenzyme was anα-amylase.
MIBA enzyme had the same N-terminal amino acidsequence (10 residues) as Taka-amylase, and the othermolecular properties were not discriminated. Fromthese findings along with the results reported in thereferences, MIBA enzyme is assigned toα- amylase.
Today the strain has been extensively employed as
useful host for cloning of carbohydrolase derived fromthe Aspergillus species and the gene encoding forαglucosidase which might be classified into the same60

Kariya et.alamylase family was isolated from A. oryzae10).
The genes for A. oryzae MIBA316 glucoamylase
were cloned in specific strain of A. oryzae that has been
employed as safety host-vector system12). A similar
type of A. oryzae enzyme was successfully constructedin A.nidulans strain and well characterized11).
It should be noted that strain MIBA316 could
produce α -amylase and glucoamylase. Recently,
these enzymes secreted by A.niger are reported to be
products formed by proteolytically processing of aprecursor12). These activities have been effectively
used fermentation of rice and other starches.
More detailed study on the enzyme structure ofMIBA316 strain deduced from its nucleotide sequences
is under progress13).

Acknowledgment
The present study was supported in part by the grantfrom The San-Ei Gen Foundation for Food ChemicalResearch.

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Communicated by Masatake Ohnishi