J. Biol. Macromol., 4 (2), 67-74 (2004)
67

ArticlePurification and enzymatic properties of a highly alkaline mannanasefrom alkaliphilic Bacillus sp. strain JAMB-750
Nobuhiro Takeda, 1 Kazumichi Hirasawa, 1 Kohsuke Uchimura, 2 Yuichi Nogi, 2
Yuji Hatada, 2 Ron Usami, 1 Yasuhiko Yoshida, 1 William D. Grant, 3Susumu Ito,2,* and Koki Horikoshi2
1

Faculty of Engineering, Toyo University (2100 Kuzirai, Kawagoe, Saitama 350-8585, Japan),
2
Japan Agency for Marine-Earth Science and Technology (JAMSTEC)(2–15, Natsushima, Yokosuka 237–0061, Japan), and3
Department of Microbiology and Immunology, University of Leicester (LE1 9HN, UK)

Received April 30, 2004; accepted May 21, 2004
An alkaliphilic Bacillus isolate (strain JAMB-750) was found to exoproduce a novel
alkaline mannanase (AmA). AmA was purified to homogeneity (about 98% pure) as judged
by SDS-polyacrylamide electrophoresis. The molecular mass was approximately 130 kDa.
The hydrolysis patterns of various substrates indicated that the enzyme was an endo-type
enzyme. The optimal temperature and pH for activity were 55oC and around 10,
respectively. The optimal pH is the highest among mannanases reported to date. Highly
alkaline mannanase is expected to have good wash performance in detergent solution andbe effective in biobleaching of kraft pulp. Therefore, AmA is highly applicable to the paperand detergent industries, as well as the food industry.
Keywords:
Bacillus, alkaliphile, alkaline enzyme, mannanase
Mannan and heteromannans, such as
locust bean gum, konjak mannan, guar gum,and ivory nut, are composed of a backbone of
β-1,4-linked mannose (and glucose) units,
which are often substituted with galactose andacetate residues depending on their origin [1].

* Corresponding author (Tel.:+81–46–
867–9644, Fax: +81–46–867–9645, E-mail:itos@jamstec.go.jp)

For complete hydrolysis of these mannans,many mannolytic microorganisms synthesize
the multiple mannolytic enzymes forcooperative actions. These enzymes include
endo-β-1, 4-mannanase (mannanase; EC
3.2.1.78), β-mannosidase (EC 3.2.1.25), andenzymes that cleave side chain sugars fromthe mannan backbone, such as
α-galactosidases (EC 3.2.1.22) and acetyl
esterases (EC 3.1.1.6) [2]. Mannanase is ahydrolase that catalyzes the endo-type68

Highly alkaline mannanase from Bacillus

hydrolysis of β-1,4-mannosidic linkages in
the main chain of naturally-occurring mannan
and heteromannans [3]. Many mannanases
have been found from various origins [4–8].
Some of the genes for the enzymes have beencloned and sequenced, and they are grouped
into glycoside hydorolase families 5 and 26(http://afmb.cnrs-mrs.fr/CAZY/).
Mannanases have many possible
applications, for instance, for use in pulp
bleaching, reduction of viscosity of instantcoffee, and clarification of fruit juices andwines [9]. An alkaline mannanase was foundby Horikoshi and his colleagues fromalkaliphilic Bacillus sp. strain AM-001 for the
first time [10]. And then, we found an alkaline
mannanase from an alkaliphilic Bacillus
isolate (strain JAMB-602) [11]. Recently,
alkaline mannanase was found to remove gum
stains of cosmetics and/or foods and thus beeffective additives for laundry detergents(WO 99/09128–WO 99/09133). Therefore,alkaline mannanase is a promising enzyme
and can be used in the detergent and paperindustries, as well as in the food industry.
In this report, we describe the purificationand properties of a highly alkaline mannanase(AmA) produced by Bacillus sp. strain
JAMB-750 isolated from a sample of soils.
EXPERIMENTAL
Materials
Unless otherwise stated, all chemicals
used were from Wako Pure Chemical.
N-Bromosuccinimide (NBS) was obtained
from Sigma. Iodoacetate, iodoacetamide, and1-ethyl-3-(3-dimethyl-aminopropyl)carbonate
were obtained from Nacalai Tesque.
Dithiothreitol was purchased from Amersham

Biosciences. Coomassie Brilliant Blue R-250(CBB), SDS, EDTA, and EGTA are theproducts of Bio-Rad. D-Mannose (M1;Sigma), locust bean gum (Sigma), ivory nut(Megazyme), konjak mannan (Kanto
Chemical), and guar gum (Sigma) wereobtained from the manufacturers inparentheses. Manno-oligosaccharides[mannobiose (M2) through mannohexaose(M6)] are the products of Megazyme.
Bacterial strains and culture conditions
Strain JAMB-750 had been isolated froma sample of soils as a mannanase-producer inthis laboratory. It was isolated at 30°C for
several days on 1.5% (w/v) agar containing(w/v) 0.2% locust bean gum, 0.5%
Polypepton S (Nihon Seiyaku), 0.5% yeast
extract (Difco), 0.1% K2HPO4, 0.02%
MgSO4.7H2O, and 0.5% Na2CO3 (separately
autoclaved). The isolate was grown at 30oC
for 2 days in an alkaline medium composed of
0.2% (w/v) locust bean gum, 5% (v/v) Mieki(Ajinomoto), 0.2% (w/v) yeast extract, 0.5%(w/v) bonito meat extract (Wako Pure
Chemical), 0.1% (w/v) K2HPO4, 0.1% (w/v)
methionine (Kanto Chemical), 0.02% (w/v)
MgSO4.7H2O, and 0.5% (w/v) Na2CO3(separately autoclaved). After removal of cellsby centrifugation (10,000 × g, 10 min) at 4oC,
the supernatant was used as the starting
material for purification of the enzyme.
Enzyme assay
A suitably diluted solution of enzyme
preparation was incubated routinely in 50 mM
glycine-NaCl-NaOH buffer (pH 9.0)
containing 0.4% (w/v) locust bean gum at
40°C for 30 min. Activity was expressed as
the initial rate of hydrolysis by measuring the

N. Takeda et al.
69

release of the reducing ends using the
3,5-dinitrosalicylic acid procedure [12] with
M1 as the standard. One unit (U) of
enzymatic activity was defined as the amountof protein that produced 1 µmol of reducing
sugar as M1 per min under the standard
conditions of enzyme assay. Protein wasdetermined using a DC-protein assay kit(Bio-Rad) with bovine serum albumin as thestandard protein.
Purification of the alkaline mannanase
Enzyme purification was done attemperatures below 4oC. The centrifugal
supernatant (3.58 liters) of the culture broth
was treated with ammonium sulfate, and thefraction that precipitated at 30–60% saturation
was collected. The precipitates formed weredissolved in a small volume of 10 mM sodium
phosphate buffer (pH 7.0), and the solution
was dialyzed against the same buffer
overnight. The retentate was then applied to acolumn of DEAE-Toyopearl 650M (2.5 × 20
cm, Tosoh) that had been equilibrated with 10
mM sodium phosphate buffer (pH 7.0). The
column was initially washed with 200 ml of
150 mM NaCl in the same buffer, andproteins were eluted with 1.0–liter linear
gradient of 150 to 300 mM NaCl in the same
buffer. The active fractions were combined
and loaded directly onto a column ofhydroxyapatite equilibrate with 10 mM
sodium phosphate buffer (pH 7.0). The
column was then washed with 200 ml of 50
mM sodium phosphate (pH 7.0), and proteins
were eluted with 1.0–liter linear gradient of
50 to 125 mM sodium phosphate (pH 7.0).
The active fractions were combined,
concentrated, and exchanged with 5 mM
Tris-HCl buffer (pH 7.5) by ultrafiltration on

an Amicon Ultra PL-10 (Millipore). The
resulting concentrate was used exclusively forfurther experiments as the final preparation of
purified enzyme.
Electrophoresis
SDS-polyacrylamide gel electrophoresis(SDS-PAGE) was performed essentially as
described by Laemmli [13] on slab gels [10%(w/v) acrylamide, 80 × 100 mm, 1.0-mm
thick], and the gels were stained for proteinwith Coomassie Brilliant Blue R-250 (CBB,
Bio-Rad). The molecular mass was estimatedby SDS-PAGE with Precision Plus ProteinStandards (Bio-Rad)
Activity staining
After SDS-PAGE, the gel was washedtwice with 50 mM glycine-NaCl-NaOH buffer(pH 9.0) with gentle shaking to remove SDS.
Onto the gel, the agar sheet containing 0.7%(w/v) agar, 0.25% (w/v) konjak mannan, and
50 mM glycine-NaCl-NaOH (pH 9.0) was
overplayed, followed by incubation for 1 h at
40oC. The agar sheet was then soaked in a
0.1% (w/v) solution of Congo red for 10 min
at room temperature and washed several times
with 1% (w/v) NaCl.
Sequencing of amino-terminal regions
The enzyme sample was blotted on a
polyvinylidene difluoride membrane (Applied
Biosystems) that had been wetted withmethanol. The N-terminal amino acidsequence of the protein was determined
directly using a protein sequencer (model
476A, Applied Biosystems).
Analysis of products of hydrolysis ofmannans and manno-oligosaccharides

70Highly alkaline mannanase from Bacillus

Table 1.
Purification
step
Culture supernatantAmmonium sulfate
DEAE-Toyopearl
Hydorxyapatite

Summary of typical purification of AmA

Total
protein(mg)

Total
activity(U)

Specific
activity(U/mg)

Yield

Purification(%)(-fold)

10600
540
16.2

924
486
220

0.087
0.90
13.6

100
52.6
23.8

1.0
10.3
156

83.1

39.5

9.0

454

2.1

Thin-layer chromatography (TLC) wasused to identify products. Enzymatichydrolysis of ivory nut, locust bean gum, guar
gum, konjak mannan, andmanno-oligosacchrides [0.4% (w/v) each] wascarried out at 40oC in 50 mM
glycine-NaCl-NaOH buffer (pH 9.0). At
appropriate times, aliquots (5 µl each) were
removed and developed by TLC (silica gel 60
plates, 10 × 20 cm, Merck) with
1-butanol-acetic acid-H20 (2:1:1, v/v) as asolvent system. The sugar spots were detectedby spraying with 10% (v/v) H2SO4, andheating. MI and manno-oligosaccharides fromM2 through M6 were used as authentic
standards.
RESULTS AND DISCUSSION

Bacillus. The isolate is positive for catalase,oxidase, gelatinase, protease, DNase, andproduction of H2S, and negative for amylase,
reduction of nitrite and nitrate, and the
producton of indole. It utilizes D-xylose,
L-arabinose, cellobiose, D-fructose,
D-galactose, D-glucose, glycerol, D-lactose,
D-mannitol, D-mannose, maltose, andD-trehalose, but does not D-raffinose,
L-rhamnose, myo-inositol and D-sorbitol. The
major isoprenoid quinone type is MK-7, and

a

b

kDa
250
150
100
75
50
37

Taxonomic analysis of the isolate
Strain JAMB-750 occurs as
Gram-positive rods 3.0–3.8 µm in length and
0.4–0.5 µm in width and is motile by meansof peritrichous flagella and contains
subterminal endospore. It is absolutely
aerobic. The temperature range for growth is
13–40°C with an optimum of around 34°C.
The pH range for growth is 7–11 with an
optimum of pH 9. These results indicate that
this isolate is a facultatively alkaliphilic

25
20
M P
Fig. 1. Electrophoresis of purified AmA.
(a) SDS-PAGE of the purified AmA (1 µgof protein) on a 10% (w/v) polyacrylamide
gel is shown. The protein was stained with
CBB (lane P). Protein mass markers (inkDa) are indicated on the left (lane M). (b)Activity staining of the enzyme after
SDS-PAGE.

N. Takeda et al.

Relative activity (%)

major fatty acids are iso-C15:0, andanteiso-C15:0. The G + C content of strain
JAMB-750 genomic DNA was determined to
be 36.2–38.4 mol% by the method of
Tamaoka and Komagata [14]. To determine
the phylogenetic position of strain JAMB-750,
100

a

80
60
40
20

Residual activity (%)

0

100

5

7
9
pH

11

b

80
60
40
20
0

5

7
9
pH

11

Fig. 2. Effects of pH on activity andstability. (a) The pH–activity curve of
purified AmA (0.2 U/ml) is shown. The
buffers (50 mM each) used were as
follows: acetate, pH 5.6–6.1, closed
circles; sodium phosphate, pH 6.3–8.5,
closed squares; glycine-NaCl-NaOH, pH
7.7–10.9, closed triangles. The reactions
were done at 40°C for 5 min. The values
are shown in percentages of the maximal
activity observed at pH 10, which is taken
as 100%. (b) To assess the pH stability of
AmA, the enzyme (0.3 U/ml) was
preincubated at the indicated pH in 10 mM
Britton-Robinson universal buffers at
40°C for 30 min. And then, samples (0.1ml) were used for the measurements of theresidual activity under the standard
conditions of enzyme assay. The values
are shown as percentages of the originalactivity, which is taken as 100%.

71

its 16S rDNA sequence was determined andanalyzed using comparative sequence analysis
against known 16S rDNA sequences. The16S
rDNA sequence of the isolate had a closest
match of 98.9% homology with that from
Bacillus alcalophilus DSM485 T. The next
highest similarity was with Bacillus
pseudoalcaliphilus DSM8725T (97.9%
homology). Based on these taxonomic
characteristics, strain JAMB-750 is closely
related to B. alcalophilus. The
GenBank/EMBL/DDBJ accession number forthe 16S rDNA of the strain is AB128830.
Purification of alkaline mannanase
When Bacillus sp. strain JAMB-750 wasgrown in an alkaline mannan medium, several
extracellular mannanases were detected in theculture supernatant. Activity staining after
SDS-PAGE showed that the major mannanaseband had a molecular mass of 130 kDa(AmA). AmA was purified 454-fold, with a
specific activity of 39.5 U/mg and a final
yield of 9.0% (Table 1). The SDS-PAGE andactivity staining of the purified enzyme gave asingle band (approximately 98% pure) with
an apparent molecular mass of 130 kDa, as
shown in Fig. 1. The N-terminal amino acidsequence of AmA was
Glu-Ser-Lys-Ile-Pro-Lys-Asp-X-Glu-Gly.
Effects of pH on activity and stability
The activity of AmA at various pH
values was measured with locust bean gum (agalactomannan) as substrate. The enzyme
showed high activity at alkaline side, with an
optimum at pH 10, in 50 mM
glycine-NaCl-NaOH buffer, as shown in Fig.
2a. This optimal pH value is the highest
among those of other mannanase reported toHighly alkaline mannanase from Bacillus
72

Effects of chemicals and cations
AmA (0.2 U/ml) was preincubated withchemicals or cations (1.0 mM each) at 40 oC
for 30 min in 5 mM Tris-HCl buffer (pH 7.5).
An aliquot (0.1 ml) was used to determine theresidual activity under the standard conditionsof enzyme assay. Iodoacetate, iodoacetamide,N-ethylmaleimide, dithiothreitol,
1-ethyl-3-(3-dimethyl-aminopropyl)carbonate,
diethyl pyrocarbonate, EDTA, and EGTA did
not essentially affect the AmA activity. NBS

Relative activity (%)

Effects of temperature on activity andstability
The optimal temperature of AmA wasdetermined at various temperatures at pH 9.0
in 50 mM glycine-NaCl-NaOH buffer, as
shown in Fig. 3a. The enzyme had an optimal
temperature of 55oC. The thermal stability of
AmA was examined after incubation at 40, 50,
and 60oC in 5 mM Tris-HCl buffer (pH 7.5)
for various periods. AmA was stable to
incubation up to 40oC for 60 min. The
first-order constants of irreversible
thermoinactivation, k, at 50 and 60oC, wereobtained by linear regression insemi-logarithmic coordinates. Each enzyme
half-life was calculated using the equation
t1/2=ln2/k. The half-lives of AmA were 117
min at 50oC and 65 min at 60oC (Fig. 3b).

abolished the activity of AmA completely.
The inhibition of some enzymes by NBS is
caused by oxidation of Trp residues inproteins [15]. Therefore, tryptophan residue(s)may participate in the catalysis or maintainthe structure of AmA. Cations (Cl forms) such

a

100
80
60
40
20
0
10

Residual activity (%)

date [10,11]. Moreover, this is the first reportof alkaline mannanase from B. alcalophilus.
The pH stability of AmA was assessed byincubating at 40°C for 30 min at different pHvalues in 10 mM Britton-Robinson universal
buffers, and the residual activity wasmeasured by the standard conditions ofenzyme assay. The enzyme was stablebetween pH 6.5 and pH 10 (Fig. 2b).

30
50
70
Temperature ( )

b

100

10

1

0

20
40
Time (min)60

Fig. 3. Effects of temperature on activity
and stability. (a) The temperature–activity
curve of AmA (0.2 U/ml) at pH 9.0 in 50
mM glycine-NaCl-NaOH buffer is shown.
The reactions were done at the indicatedtemperatures for 5 min. The values areshown in percentages of the maximal
activity observed at 55°C, which is taken as
100%. (b) To assess the thermostability of
AmA, the enzyme (0.3 U/ml) was heated at
40°C, 50°C or 60°C in 5 mM Tris-HCl
buffer (pH 7.5). Aliquots (0.1 ml) were
removed at different times and used for themeasurements of the residual activity under
the standard conditions of enzyme assay.
The residual activities at 40°C (closed
circles), 50°C (closed squares), and at 60°C
(closed triangles) are shown. The values areshown in percentages of the originalactivity, which is taken as 100%.

N. Takeda et al.

as Fe3+, Fe2+, Pb2+, Hg2+, and Cd2+ inactivatedthe enzyme activity (60–100% inhibition).
Under the same conditions Mn2+, Ca2+, Ni2+,
Mg2+, and Co2+ were essentially without
effect.

73

gum, konjak mannann, ivory nut, and guar
gum is approximately 100:100:60:30. The
time courses of hydrolysis products fromthese natural mannans were examined at pH 9
and at 40°C for up to 17 h, as shown in Fig. 4a.
AmA hydrolyzed these substrates to yield
various oligosaccharides during the reactions.
It could not hydrolyze M2 and M3, whereas it
acted slightly on M4 and efficiently on M5

Analysis of hydrolysis products and substratespecificity
The relative activity toward locust bean

a
Ivory

LocustKonjak

Guar

M1
M2
M3
M4
M5
M6

0 1/6 1 17 0 1/6 1 17 0 1/6 1 170 1/6 1 17

StTime (h)

b
M2

M3

M4

M5

M6

M1
M2
M3
M4
M5
M6

0 1/6 17 0 1/6 17 0 1/6 17 0 1/6 17 0 1/6 17 St

Time (h)Fig. 4. TLC of the products of reaction by AmA. (a) The reactions were carried out
at 40°C and at pH 9.0 in 50 mM glycine-NaCl-NaOH buffer with 0.05 U/ml of
enzyme and 0.4% (w/v) ivory nut (Ivory), locust been gum (Locust), guar gun(Guar), and konjak mannan (Konjak). (b) The reactions were carried out at 40°C
and at pH 9.0 in 50 mM glycine-NaCl-NaOH buffer with M2 through M6 as
substrates [0.4% (w/v) each] and 0.05 U/ml of enzyme. The incubation times were
0 min, 10 min, and 17 h for all the reactions. At intervals, aliquots (5 µl) werewithdrawn from the reaction mixtures and developed by TLC, as described in
EXPERIMENTAL. Standard markers (St) are shown on the right.

Highly alkaline mannanase from Bacillus
74

and M6 to yield M3 as the major product andM2 as the minor product, as shown in Fig. 4b.
These results show that AmA hydrolyzes
mannan, manno-oligosacchrides larger thanM4 and that it is an endo-acting enzyme. The
hydrolysis pattern is essentially similar to thatof alkaliphilic Bacillus sp. strain JAMB-602[11], although the other enzymatic andphysicochemical properties are quite different
between the two enzymes.
Overall, AmA from Bacillus sp. strain
JAMB-750 is very promising for use in thepaper and detergent industries, because it isvery active at high pH and stable at hightemperature. Many nucleotide sequences ofneutral mannanases from various
microorganisms have been reported [16–19];
however, those of alkaline enzymes are only
two to date [11,20]. We are now cloning andsequencing the gene for the highly alkaline
AmA.
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Communicated by Hiroshi Ueno