Study on the Substrate Specificity of -Amylases
That Contribute to Soil Removal in Detergents
Atsushi Tanaka and Eiichi Hoshino*
Household Products Research Laboratories, Kao Corporation, 1334 Minato, Wakayama, Wakayama 640-8580, Japan
ABSTRACT: The actions of -amylases in the degradation of
amylose and amylopectin were investigated, and the contribu-
tion of the enzymatic hydrolysis of high-viscosity gelatinized
starch to the removal of food stains that contain starch was char-
acterized. -Amylases from Bacillus amyloliquefaciens, B.
licheniformis, Aspergillus oryzae, and porcine pancreas were
used to test the removal of curry stains, which are hard to re-
move under low-temperature washing conditions and include
large amounts of starch. In a detergent, the enzyme from B.
amyloliquefaciens was the most effective in removing stains and
hydrolyzed amylopectin more efficiently than amylose as a re-
sult of the more efficient formation of an enzymesubstrate
complex in the former case. It is suggested that the cleaning
power of -amylases is due to their hydrolytic action, with
endo-degradation of amylopectin, which results in an efficient
reduction in the degree of polymerization. The liquefaction of
amylopectin then accelerates the removal of food stains.
Paper no. S1074 in JSD 2, 193199 (April 1999).
KEY WORDS: -Amylase, amylopectin, amylose, Bacillus
amyloliquefaciens, Bacillus licheniformis, curry stain, food
stain, laundry detergent, starch, substrate specificity.
-Amylase, [1,4--D-glucan-glucanohydrolase; EC 3.2.1.1],
which hydrolyzes -1,4-D-glucosidic linkages in amylose and
amylopectin with endo-degradation (1,2), has been included in
detergent products, in addition to its application to industrial
processes such as desizing and brewing (3,4). Resembling sebum
soil and skin grime, food stains containing starch are hard to re-
move during laundering under low-temperature washing condi-
tions that are common in Japan (5). In previous reports, we dis-
cussed the use and mechanisms of alkaline cellulase from Bacil-
lus sp. KSM-635 and a new protease from Bacillus sp.
KSM-K16 for removal of sebum soil trapped in the amorphous
region of cotton fibers and of skin grime, which contains human
epithelial cells that adhere to collars and sleeves, respectively
(68).
In general, the wheat powder present in curry paste and meat
sauce found in food stains on cloth consists of 60 to 80% (w/w)
starch and 20 to 40% (w/w) protein (9). Removal of such food
*To whom correspondence should be addressed at Sanitary Products
Research Laboratories, Kao Corp., 2606 Akabane, Ichikai, Haga,
Tochigi 321-3497, Japan.
E-mail: 157422@kastanet.kao.co.jp
Copyright 1999 by AOCS Press Journal of Surfactants and Detergents, Vol. 2, No. 2 (April 1999)
stains should be accelerated by the digestion of starch that forms
a network within the stain. Some -amylases have already been
incorporated into detergent products, but these are mainly used
for washing dishes. The mode of action of -amylases that con-
tribute to the cleaning power of detergents has not been fully elu-
cidated. In particular, the actions of -amylases that might im-
prove the cleaning power of laundry detergents have not been
fully investigated under low-temperature washing conditions.
In this study, we investigated the mode of action of typical
liquefying and saccharifying -amylases in the degradation of
amylose and amylopectin. We then studied the effects of enzy-
matic degradation of these substrates on the removal of food
stains from cotton cloth.
EXPERIMENTAL PROCEDURES
Sources of enzymes. Crude preparations of -amylases from B.
amyloliquefaciens (BAA) and B. licheniformis (BLA) were pur-
chased from Sigma Chemical Co. (St. Louis, MO) for washing
tests. Each enzyme was precipitated with 75% (wt/vol) ammo-
nium sulfate and then dissolved in a 1 mM solution of CaCl2.
Each enzyme solution was dialyzed against 1 mM CaCl2 for 24
h. Enzymes were further purified, as reported previously (10),
for studies on their modes of action in the hydrolysis of amylose
and amylopectin. After dialysis, crude BAA was loaded on a gel
filtration column (2.6 80 cm) of Toyopearl HW-55 (Tosoh,
Tokyo, Japan) that had been equilibrated with 10 mM Tris-HCl
(pH 9.0). The fraction containing -amylase was dialyzed
against 1 mM CaCl2 for 24 h. Crude BLA was similarly loaded
on a gel filtration column. The fraction containing -amylase
was loaded on an ion-exchange column (5.2 50 cm) of a
DEAE-Toyopearl 650S (Tosoh) that had been equilibrated with
10 mM Tris-HCl (pH 9.0), and then it was eluted with a linear
gradient of NaCl (0 to 0.1 M) in the same buffer and dialyzed
against 1 mM CaCl2. Preparations of -amylase from As-
pergillus oryzae (TAA), porcine pancreas (PPA), and -amylase
from barley were purchased from Sigma Chemical Co.
Substrates. Amylopectin from potato was purchased from
Sigma Chemical Co. and dissolved in a reaction mixture by heat-
ing at 100C. Water-soluble amylose, with an average degree of
polymerization (DP) of 18, was purchased from Hayashibara
Biochemical Laboratories (Okayama, Japan). A large excess of
the reducing end of the soluble amylose was decomposed by pre-
193
194 A. TANAKA AND E. HOSHINO
treatment with sodium borohydride before measurements of the
hydrolytic activity of enzymes (11). D-Glucose (G1), maltose
(G2), maltotriose (G3), maltotetraose (G4), maltopentaose (G5),
maltohexaose (G6), and maltoheptaose (G7) were purchased
from Seikagaku Kogyo (Tokyo, Japan).
Washing tests. The heavy-duty detergent consisted of 20%
(w/w) sodium ether sulfate (mean length of alkyl chain, 12), 20%
(w/w) polyoxyethyleneglycol monododecyl ether (mean length
of ethylene oxide, 7), 2% (w/w) fatty acid, 2% (w/w) citric acid,
1% (w/w) polyethyleneglycol (molecular weight, 6,000), 3%
(w/w) monoethanol amine, and 5% (w/w) ethanol, with the re-
mainder being water. Artificially soiled test cloth with food stains
was prepared by dipping a cotton cloth (cotton count, 20; shirt-
ing, standard cotton plain woven cloth from the Laundry Re-
search Association, Tokyo, Japan; basis weight, 142.9 g/m2; den-
sity, 0.32 g/cm3) into an aqueous suspension of curry paste (Ot-
suka Shokuhin, Osaka, Japan). The average weight of curry paste
that adhered to the cotton cloth was 6.6 mg/cm2; as determined
after air-drying. Swatches (measuring 5 5 cm2) were cut from
the prepared test cloth after air-drying and then washed with de-
tergent that included -amylase in a Terg-O-Tometer (Ueshima
Co., Tokyo, Japan). Five test swatches were first soaked in 1 L
of a 1.33% (wt/vol) solution of detergent, which had been ad-
justed to pH 7.0 with dilute HCl, at 30C for 1 h, and then they
were agitated (100 rpm) at 30C for 10 min in the Terg-O-Tome-
ter. Ordinary Japanese tap water, containing about 55 ppm of
CaCl2, was used. The reflectance of test swatches was measured
at 460 nm before and after washing, and detergency was calcu-
lated as follows:
detergency (%) = [(RW RS) / (R0 RS)] 100 [1]
where RW, RS, and R0 are the reflectance of washed (after washing),
soiled (before washing), and unsoiled test swatches, respectively.
Electron microscopic examination of stains. The surface con-
dition of test cloth stained with curry paste was examined with a
scanning electron microscope (SEM; S-4000; Hitachi, Tokyo,
Japan) at 15 kV after critical-point drying. Cross-sections of cot-
ton fibers in the test cloth were also examined with a transmis-
sion electron microscope (TEM; JEM-2000FX; JEOL, Tokyo,
Japan) as described previously (12).
Measurement of the hydrolytic activity of enzymes. The reac-
tion mixture consisted of 0.5% (wt/vol) soluble amylose (DP, 18)
or amylopectin, 20 mM phosphate buffer (pH 7.0), and 0.5 to 15
g/mL enzyme. After incubation at 30C for an appropriate pe-
riod, the reducing power that had been generated in the super-
natant was measured by the dinitrosalicylic acid method (13).
One unit of amylase activity was defined as the amount of en-
zyme that catalyzed the liberation of reducing sugar equivalent
to 1.0 mol of D-glucose per min from a 0.5% (wt/vol) solution
of soluble amylose at 30C and pH 7.0. The activity and rate pa-
rameters were determined by least-squares analysis of initial ve-
locity data using the general equation of linear regression as a
function of incubation time and enzyme concentration. Kinetic
studies were performed with a reaction mixture consisting of
0.05 to 0.5% (wt/vol) of soluble amylose or amylopectin, 25 mM
Journal of Surfactants and Detergents, Vol. 2, No. 2 (April 1999)
Tris-acetate buffer (pH 7.0), 1 mM CaCl2 , and 0.5 to 15 g/mL
of enzyme. The maximal velocity of enzymatic hydrolysis (Vmax)
and the dissociation constant of the enzymesubstrate (ES)
complex (Km) were estimated from Lineweaver-Burk plots (14).
The values of Km were calculated with units of anhydrous glu-
cose instead of mole of substrate because the DP of the substrate
changed with hydrolysis time. Changes in enthalpy (Ho) and in
entropy (So) for formation of the ES complex were calculated
from the Km value at a given temperature (15 to 35C) according
to the following equation:
Km1 = exp ( Ho/RT + So/R ) [2]
where R and T are the gas constant and the absolute temperature,
respectively. The activation enthalpy (H) and entropy (S)
for enzymatic hydrolysis were calculated from the Vmax value at
a given temperature (15 to 35C) according to the following
equations:
Vmax = k0[E0] [3]
k0 = kT/h exp(H/RT + S/R) [4]
where k0,k,h, and [E0] are rate constant, Boltzmanns constant,
Plancks constant, and the initial concentration of enzyme, re-
spectively.
The initial velocity for enzymatic hydrolysis in the detergent
liquor (pH 7.0) was measured in a 1.33% (wt/vol) solution of de-
tergent containing 0.5% (wt/vol) soluble amylose as substrate.
Liberated reducing sugars were quantitated by the dinitrosali-
cylic acid method, and then the relative velocity of hydrolysis in
the detergent liquor was calculated by reference to the velocity
of hydrolysis in 20 mM phosphate buffer (pH 7.0):
relative velocity of hydrolysis (%) = Vdet/Vbuff 100 [5]
where Vdet and Vbuff are the initial velocities of hydrolysis of soluble
amylose in detergent liquor and in phosphate buffer, respectively.
Characterization of hydrolysis products. The reaction prod-
ucts released from soluble amylose were identified by high-per-
formance liquid chromatography (HPLC) (15). The reaction
mixture consisted of 0.5% (wt/vol) soluble amylose, 20 mM
phosphate buffer (pH 7.0), and 0.5 units/mL enzyme. After incu-
bation at 30C for an appropriate period, aliquots (10 to 25 L)
of the reaction mixture were subjected to HPLC on a LiChros-
pher 100NH2 column (E. Merck, Darmstadt, Germany) equipped
with a refractometer (L-6200; Hitachi). A mixture of acetonitrile
and water (60:40, v/v) was used as eluent with a flow rate of 0.8
mL/min at 35C. The products of enzymatic hydrolysis of amy-
lopectin were also identified by HPLC (16). The reaction mix-
ture consisted of 0.5% (wt/vol) amylopectin, 20 mM phosphate
buffer (pH 7.0), and enzyme. The amount of enzyme used in 1.0
mL of the reaction mixture was equivalent to the amount that cat-
alyzed the liberation of 2.0 mol of reducing sugars as D-glu-
cose per min from a 0.5% (wt/vol) solution in 20 mM phosphate
buffer of amylopectin at 30C and pH 7.0. After incubation for
an appropriate period at 30C, aliquots (10 to 25 L) of the reac-