Download Isolation and Identification of Cellulase

Isolation from cattle manure and characterization of Bacillus
licheniformis NLRI-X33 Secreting Cellulase
Kim Tae-Ill, J. D. Han1, B. S. Jeon1,C.B. Yang1, K.N. Kim1and M.K. Kim2
1 National
Livestock Research Institute, Rural Development Administration, Suweon, 441-350, Korea
2 Division of applied Chemistry, school of Agricultural Biotechnology center for Plant Molecular Genetics and Breeding
Research, College of Agriculture and Life science, Seoul National University, Suwon, 441-744, Korea
ABSTRACT A bacterium producing extracellular cellulase was isolated from cow feces and has been identified
as Bacillus sp.. The isolate, NLRI-X33 was shown to be similar to Bacillus licheniformis on the basis of
morphological and biochemical properties as well as the composition of cellular fatty acids. When the isolate was
cultured in CMC media at 37C for 24hrs, CMCase, FPase and Avicelase activity was 1.65 U/ml, 0.13 U/ml and
0.18 U/ml whereas -glucosidase activity was not detected. The optimum pH and temperature for enzyme
induction was 7.5 and 50C. The maximum CMCase activity was observed at pH 7.5 and 75C. When crude
supernatant was used for zymogram analysis, four major bands were detected on CMC-SDS-PAGE.
Key Words : CMCase, Avicelase, -1,4-glucosidase, Bacillus licheniformis
INTRODUCTION
The function of cellulase is to hydrolyse cellulose by
breaking the -1,4-glycosidic linkage in the major
plant structural polysaccharide. There are three main
types of enzyme found in cellulase system that has
endoglucanase(CMCase, EC 3.2.1.4) which cleaves
internal -1,4-glycosidic bonds, exoglucanase
(Avicelase, EC 3.2.1.91) which releases cellobiose
from the non-reducing end of cellulose, and cellobiase
(-1,4-glycosidase, EC 3.2.1.2) which hydrolysis
cellobiose to glucose (Wood and Bhat, 1998.). These
enzymes work synergistically to hydrolyze cellulose
to single carbohydrate (Yu et al, 1998). Applications
of cellulase can be to improve the nutritional quality
and digestibility of consumption and digestibility of
ruminant feeds, to facilitate composting, to provide
sugar syrups for human or animal consumption feeds,
to provide sugar syrups for human or animal
consumption and to supply fine chemical through
fermentation (Beguin and Albert, 1985, Gilbert and
Hazlewood, 1993). Bacillus sp. (Fumiyasu et al., 1985,
Kim et al., 1997, Yoon and Jung, 1997), Pseudomonas
sp.(Berghem et al, 1976, Wood and Kim, 1968),
Clostridium sp. (Fagerstam and Pettersson, 1979) and
Cellulomonas sp.(Chey et al, 1990, 1992) were known
generally as cellulolytic bacteria. It has been revealed
that bacterial cellulases were inferior to those of fungi
for hydrolyzing cellulose but they show a tendency
to be more heat stable and are easier for genetic work.
In this paper, we describe the isolation and
identification of a cellulase-producing bacterium
from soil, compost and cow feces, and some properties
of the crude CMCase.
MATERIALS AND METHODS
Isolation and Identification of Bacteria
To isolate cellulolytic bacteria, the gathered soil,
compost and cow feces were suspended in 0.1%
Carboxymethyl cellulose(CMC medium viscosity,
Sigma Chem. Co.), 1.5% bacto-tryptone, 0.5%
bacto-soyton, 0.5% NaCl and 1.5% agar. After
incubation at 37C for 24hours, the colonies were
inoculated with needle onto CMC medium again.
After growth, the plates were stained with 0.1%
congo-red solution (Sigma Chem. Co.). When the
stained plates were destained with 0.1M NaCl, the
strains which formed a zone were selected as
cellulolytic bacteria.
General characteristics of the isolate were
determined according to the Bergeys Manual of
Systematic Bacteriology(Bergey’s 1986) and the
Manual for General Bacteriology (Gerhardt et al,
1981). Methyl esters of cellular fatty acids of the
isolate were analyzed by a gas liquid
chromatography(HP 6890 GC, Microbial ID, USA)
and microbial identification software.
Crude Enzyme Preparation
The isolated stains, grown in CMC agar, were
transferred to CMC broth and cultured at 37C. After
incubation for 24hours, cells were removed by
centrifugation at 14,000rpm for 20min at 4C and the
supernatant was precipitated by 80% ammonium
sulfate saturation for overnight at 4C. The precipitate
was dissolved with 0.1M Na-acetate buffer(pH 5.5).
The precipitate was used as crude enzyme for the
assay of enzyme activity.
Assay Cellulolytic Enzyme Activity
CMCase and Avicelase activity were assayed using
2% CMC and 2% Avicel as a substrate in 0.1M
Na-acetate buffer(pH5.5) at 50C for 15min. FPase
was assayed using Whatman No.1 Filter
paper(16cm) as a substrate in 0.1M Na-acetate
buffer(pH 5.5) at 50 C for 60min. The amount of
reducing sugar produced from CMC, Avicel and Filter
paper after reaction were determined by
Somogyi-Nelson method(Wood and Bhat, 1998). One
unit of enzyme activity was defined as the amount of
enzyme which catalyzed the production of 1mol of
glucose per min. -glucosidase assayed using 1mM
p-nitrophenyl--D-glucopyranoside(PNPG,
Sigma
Chem. Co.) as a substate in 0.1M Na-acetate
buffer(pH 5.5) at 50C. After reaction for 30min, 1M
Na2CO3 and distilled water were added to stop the
reaction, and liberated p-nitrophenol is measured at
430nm. One unit of enzyme activity is the amount of
enzyme required to release 1 mol p-nitrophenol per
min .
Determination of Enzyme Properties
To determine pH stability of CMCase, 100l of crude
enzyme solution was added to 400l of 0.1M
Na-acetate (pH45.5), 0.1M Na-phosphate (pH67),
0.1M
Tris-HCl
(pH7.59.0),
and
0.1M
Carbonate-bicarbonate buffers (pH9.511). After
standing at 4C for 24hour, the residual CMCase
activity was assayed using 200l of each solution in
the same way as described above.
The optimum temperature of CMCase activity was
determined by assaying at various temperatures. The
temperature effect on the stability of the enzyme was
tested through the residual activity after reaction for 1
hour.
Electrophoresis and Zymogram
Electrophoresis and Zymogram was performed
according to Laemmli(1970) and Park et al.(1997).
SDS-PAGE was carried out in 12.5% polyacrylamide
gel containing 0.1% CMC under constant 100 volts.
After running , proteins were stained with Coomassie
Brilliant blue R-250. Zymogram was carried out by
staining the gel with 0.5% congo-red solution after
removing SDS with 1% Triton X-100.
The molecular weight of activity bands were
estimated by plotting the log of the molecular weight
of standard markers, activity bands vs. the relative
mobility(Rf).
RESULTS AND DISCUSSION
Screening for Cellulase-producing bacterium
In the first step of screening, about 150 colonies from
soil, compost and cow feces were selected on CMC
agar by staining with 0.1% congo-red. And then, they
were tested if hemolysis occur on 5% sheep blood agar
or not.
Nine isolates without showing hemolysis were tested
for their ability to hydrolyse CM-cellulose at 50C. Of
them, X-33 strain is showing highest extracellular
CMCase activity (Table 1). There is no correlation
between diameter of clear zone and CMCase activity,
corresponding to Ha et al(1992.)
Table 1. Comparison of diameter of clear zone on
the medium and CMCase activity
Strain Source Clear
CMCase Hemolysisb
a
Zone (mm) activity(U/ml)
X-9 Cow feces 12
0.65
X-10 Cow feces 11
2.12
X-21 Cow feces 11
2.20
X-33 Cow feces 10
2.22
S-12 Soil
9
1.49
FT-16 Compost 11
0.69
T-6 Soil
10
0.41
T-20 Soil
9
0.99
T-24 Soil
9
1.29
aDiameter
of clear zone after cultivation at 37C on the
CMC agar medium
bThe stains were inoculated on he medium containing 5%
sheep blood, 5% beef heart, 1% Tryptone, 0.5% sodium
chloride and 1.5% agar and cultured at 37C for 24hrus
Identification of Cellulase-producing bacterium
Morphological and physiological characteristics of
isolate X-33 are shown in Table 2. X-33 is a
Gram-positive, spore-forming, and rod-shaped
bacterium. The cellular fatty acid composition of X-33
was similar to that of Bacillus licheniformis with a
similarity of 0.772. Major cellular fatty acids of X-33
were branched chain fattty acids such as 40.65% of 15:
0 iso, 33.04% of 15: 0 Anteiso and 8.33% of 17: 0
anteiso (Table 3). From the results, the isolate was
identified as Bacillus licheniformis and named
Bacillus licheniformis NLRI-X33.
Table
2.
Biochemical
and
physiolagical
characteri-stics of isolates NLRI-X33
Characteristics
Result
Gram stainig / Shape
+, Rod
Catalase
+
Oxidase

Spore
+
Motility at 22C
+
Acid production from:
Glucose
+
Mannitol
+
Lactose

Maltose

Saccharose + Esculin
+
Arabinose
+ Arginine
+
Cellobiose
+ Galactose 
Raffinose
 Salicin
+
Sorbitol
+
Sucrose
+
Trehalose
+
Table 3. Composition of the cellar fatty acids of the
isolate X-33
Fatty acid contents(%) Fatty acid contents(%)
13:0 iso
0.21
14: 0 iso
0.78
14:0
0.41
15: 0 iso
40.65
15:0Anteriso 33.04
16:1w7calcohol 0.31
16:0iso
2.35
16:0wllc
0.38
16:0
2.23
16:02oh
0.18
iso 17: 1 w10c 1.15
17: 0 iso
8.63
17: 0 anteriso 0.29
18:0
0.26
Activity of Cellulolytic Enzymes on Various
Substrates
B. licheniformis NLRI-X33 was grown at 37C for
24hour in substate-containing medium, which is
either Filter Paper No. 1, CM-celllose or Avicel, to
test for cellulolytic activity with a substrate as the
carbon source(Table 4). Maximum enzyme activity
appeared in CMCase with 0.1% CM-cellulose.
Activity of FPase and avicelase was detected whereas
-glucosidase was not done at all. This result indicated
that CM-cellulose was revealed as effective substate
for celluloytic production. However, this result was
not corresponding to the suggestion of Chey et
al(1990) that avicelase was the most inducible enzyme
in cellulolytic bacteria .
Table 4. Comparison of activities of FPase,
Avicelase and -glucosidase on three substrates
Substrate
Activity(U/ml)
FPase CMCase avicease -glucosidase
Filter Paper 0.18
0.88
0
0
CMC
0.13
1.65
0.18
0
Avicel
0.14
1.03
0
0
a
Effect of initial pH and Temperature on Enzyme
Production
Initial pH and incubation temperature of CMC
medium examined to improve enzyme production of B.
licheniformis NLRI-X33. To determine effect of initial
pH on enzyme production, pH of CMC medium was
adjusted from 4.5 to 8 by adding 0.1N HCl and 0.1N
NaOH to medium. Fig. 1 shows that the optimal initial
pH for enzyme production was at pH 7.5. To
determine the effect of incubation temperature on
enzyme production , the medium was incubated at
3060C for 24hrs. Fig. 2 shows that the optimal
temperature was at 50C. The temperature below
40C and above 60C did not support high enzyme
production.
To determine the optimal time for harvesting the
culture to obtain maximum enzyme yield and the
growth curve at the optimal condition for enzyme
production, initial pH 7.5 and 50C, culture was
harvested at intervals of 12 hour during incubation
(Fig. 3.). The growth and the enzyme production
increased rapidly during 0 to 36hrs but the growth kept
in stationary phase at 48hrs whereas the enzyme
production increased continuously. When cultured for
48hrs, the maximum quantity of CMCase and the
growth were 1.5U/ml and 7.2 Log CFU/ml
Relative activity(%)
100
80
60
40
20
0
4
5
6
7
8
9
Initial pH
Fig. 1 Effect of initial pH on the CMCase
production by B. licheniformis NLRI-X33.
Relative activity (%)
100
80
60
40
20
0
30
Activity (units/mL)
0.1% Avicel, 0.1% CM-cellulose or Filter Paper No.1
(212cm)
50
60
6
0.5
5
12
24
36
48
60
4
84
72
Incubation time (hrs)
Fig. 3 Time course of growth and CMCase
production in a culture of B. licheniformis
NLRI-X33.
Viable cell count() and CMCase activity() in the culture
medium (initial pH 7.5)
Effects of Temperature and pH on CMCase
Activity in Crude Extract
The enzyme activity was assayed at temperature
ranging 40-80C for 15min. Figure 4 shows that the
optimum temperature for hydrolyzing CMcellulose
was 65C. The crude enzyme stability was tested by
various temperatures for 1 hour and then measured
the remaining activity. This enzyme was stable at
temperatures below 50C for 1 hour (about 10% loss).
However, residual activity was 50% above 70C for 1
hour. The reported optimum temperature of the
bacterial CMCase is 65C (Kim et al, 1997), and that
of CMCase from Trichoderma sp. C-4 is 50C (Son et
al,1997).
To study the effect of pH on CMCase activity,
released reducing sugar was measured after the crude
enzyme was incubated with 2% CMcellulose in
various buffers raging pH 4.011.0 at 50C for 15min.
The crude enzyme was found to be most active in the
range of pH 6.5-7.5 at 50C with above 90% of its
activities(Fig.5). The crude enzyme effectively
hydrolyzed 2% CMcellulose in the 0.1M Tris-HCl
(pH7.5). To investigate the effect of pH on enzyme
stability, samples of the crude enzyme incubated was
assayed. Fig. 5 shows that the activities for hydrolysis
of CMcellulose were stable between pH 7.5 and 8.0
The residual activities(%) are the values exchanged
pH for the enzyme activity in 0.1M Tris-HCl (pH7.5)
is settled to 100%. The optimal pH for the enzyme
activity shows the pH range for enzyme stability of the
CMCase-producing bacteria in the range of the pH
7.0-8.0. The reported pH stability of the bacterial
CMCase is between pH 4.0 and 7.0 (Son et al, 1997),
that of Bacillus stearothemophilus No.236 is between
pH 6.0 and 7.0 (Kim et al ,1997).
lati
Incubation Temperature (¡É)
Fig. 2 Effect of incubation temperature on
CMCase Production of CMCase from B.
licheniformis NLRI-X33.
1.0
0.0
0
Re
40
7
Log CFU/mL
a
8
1.5
ve
100
80
60
40
ac
20
tivi
0
ty
(%
30
40
50
60
Temperature (? )
70
80
Fig. 4 Effect of temperature on activity and
stability of the CMCase from B. licheniformis
NLRI-X33.
The enzyme activity was assayed at various temperature for
15min()and the residual activity was measured activity
was measured after incubation of enzyme various
temperature for 1hr()
Relative activity(%)
100
80
60
40
20
0
4
5
6
7
8
9
10
11
pH
Fig. 5 Effect of pH on activity and stability of the
CMCase from B. licheniformis NLRI-33.
The enzyme activity was assayed at various pH() and
residual activity was measured activity was measured after
incubation of the enzyme at various pH for 24hr().
Patterns of extracellular proteins and Zymogram
Crude enzymes, collected at intervals of 12 hrs, were
electrophoresed on 12.5% SDS-PAGE containing
0.1% CM-cellulose to study patterns of extracellular
proteins and CMCase activity of Bacillus
licheniformis NLRI-X33 on a gel. It appeared that
patterns of proteins come to increase between 45 and
31kDa by passing incubation time (not shown). 4
major active bands, Cel 1, Cel 2, Cel 3 and 4, were
detected by staining with 0.5% congo-red
solution(Fig. 6). The molecular weight of the bacterial
CMCase is 95kDa and 92kDa(Fumiyasu et al,1985,
Kim et al,1997), and that of CMCase from Erwina sp.
is 42,39,35,31 and 27kDa (Park et al,1997, Saarilahti
et al,1990).
5.0
Cel 1
Log MW
4.8
Cel 2
Cel 3
4.6
Cel 4
4.4
4.2
4.0
0.0
0.2
0.4
0.6
0.8
1.0
Rf
Fig 6. Molecular weights of Cel 1, Cel 2, Cel 3, and
Cel 4
The molecular weight of Cel 1, Cel 2, Cel 3 and Cel 4 was
estimated by platting the log of the molecular weights of
standard markers, Cel 1, Cel 2, Cel 3 and 4 vs. the relative
mobility(Rf)
ACKNOWLEDGEMENT
This work was supported by the 97 projects of
Agricultural R&D promotion center, Ministry of
Agriculture and Forestry, Korea
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