Physicochemical characterization of cellulase produced from Kurthia gibsonii isolated from cassava dumpsites in Ibadan, Nigeria

Full text

738
Ethiopian Journal of Environmental Studies & Management 8(Suppl. 1): 738 – 751, 2015.
ISSN:1998-0507 doi: http://dx.doi.org/10.4314/ejesm.v8i1.1S
Submitted: April 09, 2014 Accepted: September 14, 2015
PHYSICOCHEMICAL CHARACTERIZATION OF CELLULASE PRODUCED FROM
Kurthia gibsonii (CAC1) ISOLATED FROM CASSAVA DUMPSITES IN IBADAN,
NIGERIA
*ADU, K.T.,
1
KAYODE, R.M.O.
2
AND ADU, M.D.
3
1
Microbial Physiology and Biochemistry Research Unit, Department of Microbiology,
University of Ibadan, Ibadan, Nigeria
2
Division of Food Microbial Biotechnology and Toxicology, Department of Home Economics
and Food Science, University of Ilorin, P.M.B 1515, Ilorin, Nigeria
3
Department of Epidemiology and Medical Statistics, Faculty of Public Health, University of
Ibadan, Ibadan, Nigeria
Abstract
This experiment reports the physicochemical characterization of cellulase produced by
Kurthia gibsonii CAC1 isolated from cassava dumpsites in Ibadan, Nigeria. Lineweaver-Burk
plot of cellulase activities of K.gibsonii CAC1 was examined with K
max
and V
max
of the enzyme
assayed. At optimal pH of 5.0, 10% increase in enzymatic activities was observed. The
enzymatic activities of K. gibsonii CAC1 were optimal at 30
o
C and were still stable at 60
o
C
with 50% reduction in cellulase activities. Cellulase activities of K. Gibsonii CAC1 showed
significant difference (p≥0.05) with the different cations used. There was no significant
difference (p≤0.05) observed with increased concentrations (p≤0.05) using two-way ANOVA.
Ca
2+
highly enhanced the cellulase activities of K.gibsonii CAC1 by 60% at 10mM. Highest
inhibition by Hg
2+
was observed at 20mM with 50% inhibition. At increasing concentration of
the inhibitors, there was no significant difference (p≤0.05) in cellulase activities; although the
effect of each inhibitor on the enzymatic activity was significantly different (p≥0.05). Benzoic
acid gave the highest inhibition of the cellulase activities in K. gibsonii CAC1 by 30% at
20mM, while Ethylene diamine-tetraacetic acid (EDTA) boosted the enzymatic activities by
10% at 10mM. There was a significant difference (p≥0.05) in the effect of different
surfactants on the enzymatic activity but no significant difference (p≤0.05) with the
concentration of the surfactants on cellulase activity. Enzymatic activities of the bacterium
were enhanced by Polyoxyethylenesorbitan mono-oleate (Tween 80) with a boost of 50%.
Increasing the concentration of sodium dodecyl sulphate (SDS) and Polyethylene glycol p-
isooctylphenylether (Trition X-100) caused a 70% increase in cellulase activities. Cellulase of
K. gibsonii CAC1 had K
max
of 7.4 X 10
-2
mg/ml and V
max
of 6.7 X 10
-1
µg/sec.The cellulase
described in this work have many properties that are similar to those obtained from other
microbial sources and may be useful for various industrial applications.
Key Words:
Kurthia gibsonii, Cellulase activity, Substrate concentration, Cations, Inhibitors,
Surfactants
*Corresponding author: Adu, K.T.
Email: aduktus@gmail.com

739
Introduction
Cellulase has attracted many scientific
and industrial attention for its applications
in many fields such as animal feed, textile,
pulp and paper industries, ethanol
production, laundry (Voragen, 1992;
Godfrey and West, 1996; Tolan and
Foody, 1999); de-inking of recycled paper
(Smook, 1992; Moekerbak and
Zimmermann, 1998). The importance of
cellulase and allied enzymes is increasing
in the global market. In the year 2000, the
estimated world sale of industrial enzymes
was put at 1.6 billion US dollars with
cellulase occupying significant part
(Demain, 2000). Microorganisms
especially bacteria and fungi have played
major roles in the production of enzymes
such as cellulase and hemicellulase mainly
due to low cost of production and the
process is less expensive and not laborious
(Karmakar and Ray, 2010).
Cellulase consists of three major
components which are the endoglucanase,
exoglucanase and β-glucosidase.
Endoglucanase acts on carboxy methyl
cellulose (CMC) and breaks the cellulose
chains with resultant formation of glucose
and cello-oligosaccharides; exoglucanase
acts on microcrystalline cellulose (avicel),
converting it to cellobiose as the primary
product and beta-glucosidase causes the
hydrolysis of cellobiose to glucose
(Karmakar and Ray, 2011). The end
product from this enzyme is glucose
(Wood and McCrae, 1979).
Kurthia gibsonii CAC1 is a motile,
aerobic, rod-shaped, non-pigmented and
Gram positive bacterium which grow best
at incubation temperature of 30
o
C and pH
5.5 with lactose and ammonium chloride
supplemented medium as best carbon and
nitrogen sources respectively (Adu et al.,
2014). It has been investigated that Kurthia
gibsonii CAC1 isolated from cassava
dumpsites are capable of producing
cellulase necessary to degrade cellulolytic
components of cassava (Adu et al., 2014).
Hence, researching into factors that may
influence enzymatic activity of the
cellulase produced by Kurthia gibsonii
CAC1 is the focus of this work.
Materials and Methods
Sample Collection
The bacterial isolate (Kurthia gibsonii
CAC1) used for this study was collected
from the culture collection centre of the
Department of Microbiology, University of
Ibadan and transported aseptically to the
postgraduate laboratory of the department
of Microbiology for further microbial
analysis.
Cellulase Enzyme Production
This was done according to the
modified method of Todar (2008). The
composition of the optimized cellulase
production medium was as follow: Lactose
(10g/L), K
2
HP0
4
(2g/L), KH
2
P0
4
(2.5g/L),
urea (1.0g/L), MgS0
4
.7H
2
0 (0.2g),
FeS0
4
.7H
2
0 (0.01g/L) and MnS0
4
.7H
2
0
(0.007g/L). Production medium was
prepared by dissolving the above chemical
constituents into conical flasks and then
autoclaved at 121°C for 15min. The
medium was later inoculated with 2ml of a
24hr old bacterial culture. A control
experiment without bacterial inoculum was
set up. They flasks were incubated for 24
to 72hr before harvesting the crude
enzyme. The supernatant containing the
crude enzyme was harvested by cold
centrifuging at 5000rpm for 20min using a
Himac CR21GII high speed refrigerated
centrifuge. The harvested supernatant was
stored at 4°C for further analysis.
Characterization of the Cellulase
Enzymes
The activities of the cellulase enzyme
from the bacteria were characterized
considering the effects of temperature, pH,
substrates, metal ions, inhibitors, and
surfactants.
Physicochemical Characterization of Cellulase Produced from
Kurthia
gibsonii
................
ADU et al.

740
Assessment of the Optimum pH for Crude
Cellulase Production
One millitre each of 0.1M citrate-
phosphate buffer adjusted to various pH
values (4.0-9.0) was inoculated with 1ml
of the enzyme and incubated at 30°C for
1hr. Volume of 30µL of each cellulase
enzyme was inoculated aseptically into a
5mm well on CMC agar medium. The agar
plate was then incubated at 30°C for 24hr.
This was done according to the modified
method of Bertrand et al. (2004). After the
incubation period; Gram’s iodine solution
was used to flood the plates and allowed to
stand for 30min. The diameter of the
hydrolytic zone formed around the site of
inoculation was measured and taken to
represent the activity of the enzyme.
Determination of the Optimum
Temperature for Crude Cellulase
Production
One millilitre of the crude enzyme
extract was introduced into four (4) test
tubes. One test tube each was incubated at
25, 30, 37, and 45°C respectively for
15min. At the end of incubation, the crude
enzyme extract was aseptically introduced
into a 5mm well on CMC agar medium.
The agar plates were then incubated at
30°C for 24hr according to the modified
method of Bertrand et al. (2004). After the
incubation period, Gram’s iodine solution
was used to flood the plates and was
allowed to stand for 30min. The diameter
of the hydrolytic zone formed around
inoculation site, after the addition of
iodine, was measured and taken to
represent the activity of the enzyme. Each
treatment was carried in three replicates.
Assessment of Temperature Stability of
the Crude Enzyme
This was done according to the
modified method of Bertrand et al. (2004).
The enzyme sample was divided into four
groups in glass test tubes and incubated at
various temperatures of 25, 30, 37, and
45°C respectively before incubation for
24hr. Volume of 30µL of each crude
cellulase enzyme in the test tube was
aseptically inoculated into a 5mm well on
CMC agar plates. The agar plates were
then incubated at 30°C for 24hr. Gram’s
iodine solution was used to flood the plates
and allowed to stand for 30min. The
diameter of the hydrolytic zone formed
around inoculation site was measured and
taken to represent the residual activity of
the enzymes.
Investigation on the Effects of Cations,
Inhibitors and Surfactants on Cellulase
Activity
One millliitre of enzyme samples was
introduced into varying concentration of
the cations (5, 10, 15 and 20mM),
inhibitors and surfactants (0.25, 0.5, 0.75,
1.0 and 1.25%) and incubated at 30°C for
1hr. Then 30µ L of each cellulase enzyme
was aseptically inoculated into a 5mm well
on CMC agar medium. Control sample
was inoculated with equal quantity of
enzyme and buffer mixture. The agar
plates were incubated at 30°C for 24hr
according to the modified method of
Bertrand et al. (2004). Gram’s iodine
solution was used to flood the plates and
allowed to stand for 30min. The diameter
of the hydrolytic zone formed around
inoculation site was measured and taken to
represent the residual activity of the
enzymes. Chemical used include: NaCl,
CaCl
2
, HgCl
2
, MgCl
2
, NH
4
Cl, Urea,
Benzoic acid, EDTA
(Ethylenediaminetetraacetic acid), SDS
(sodium dodecyl sulphate), Triton X-100
(Polyethylene glycol p-
isooctylphenylether), and Tween 80
(Polyoxyethylenesorbitan mono-oleate).
Assessment on the Effect of Substrate
Concentrations on Crude Cellulase
Activity
This was done according to the
modified method of Bertrand et al. (2004).
Increasing concentrations (0.5, 1, 1.5, 2.0
and 2.5% w/v) of carboxy-methyl cellulose
was dissolved in 0.1M citrate phosphate
buffer at pH 6.0 and used for enzyme assay
Ethiopian Journal of Environmental Studies and Management Vol. 8 (Suppl. 1) 2015

741
without increasing the enzyme volume.
One millilitre of each solution was added
to one millilitre enzyme sample and
incubated at 50
o
C for 30 min. Quantities of
reducing sugar released was measured
according to Miller (1959). A reciprocal of
quantity of reducing sugar recorded was
plotted against a reciprocal of the substrate
concentration. The V
max
, K
max
and the
regression equation were determined from
the line equation obtained.
Statistical Analysis
The analysis of variance (ANOVA)
was carried out with 95% confidence level
on the effect of physicochemical properties
on the growth and cellulase activities of
isolates CAC1 and CAC2.
Results
Effect of pH on cellulase activities
Figure 1 shows the effect of increasing
pH on cellulase activity by K.gibsonii
CAC1. As pH was increased from 5.0 to
8.0, there was a gradual decrease in the
cellulase activity produced by K.gibsonii
CAC1 from optimal pH of 5.0 until
minimum pH was observed at 8.0. Increase
in pH from 5.0 to 8.0 did not result in an
increase in cellulase activity, rather a 40%
reduction in activity was observed.
Effect of Temperature on Cellulase
Activities
The effect of varying incubation
temperature on cellulase activity by
K.gibsonii CAC1 is illustrated in Figure 2.
At 25
0
C, there was an increase in cellulase
activity (100%). Highest activity (400%)
was recorded at 30
0
C and this represented
300% increase in the cellulase activity of
K.gibsonii CAC1enzyme. Further increase
in temperature to 37
0
C led to 250%
increase in enzyme activity.
Effect of Temperature Stability on
Cellulase Activities
Figure 3 illustrates the response of K.
gibsonii CAC1 cellulase to incubation
temperature relative to time. It was
observed that the enzyme was stable at
high temperature of 60
o
C. When incubated
for 30min at 30
o
C, there was a sharp
increase in residual activity (100%) and
further incubation resulted in the reduction
of the activity. Incubation at 37
o
C initially
resulted in increase in the residual activity
(50%) but a slight reduction in activity
(20%) was noted after 30 min of
incubation before a gradual rise in activity
was recorded.
Effect of Cations on Cellulase Activities
Figure 4 shows the effect of cations on
cellulase activity of K.gibsonii CAC1.
Addition of Ca
2+
at increasing
concentration (5,10,15 and 20mM) highly
enhanced the cellulase activity with
optimal enhancement at 10mM giving 60%
increase in cellulase activity after which
there was a decline in the boost. NH
4+
also
enhanced enzymatic activity of the
cellulase, at lesser degree compared to
Ca
2+
. At 5Mm NH
4+
, enzymatic activity
was increased by 30% but only 10%
increase was observed when concentration
was increased to 10Mm. An increase of
20% was recorded as the concentration
was further increased to 15Mm. At
reduced concentration of 5mM, the
enzymatic activities were boosted (10%)
by the addition of Mg
2+
after which further
increase in concentration of the cations did
inhibit the cellulase activities. The higher
the concentration added, the higher the
inhibition recorded. The highest inhibition
(50%) was recorded with Hg
2+
at 20mM.
The peak enhancement concentration of
Na
+
(10%) was observed at 15mM after
which there was a decline, with lower
concentrations giving a mild inhibition of
cellulase activity.
Effect of Enhancers or Inhibitors on
Cellulase Activities
Figure 5 shows the effect of
enhancers/inhibitors on cellulase activity
of K.gibsoniiCAC1 at increasing
concentration (5, 10, 15 and 20mM). Urea
did not show any observable effect on the
cellulase activities as the concentration
Physicochemical Characterization of Cellulase Produced from
Kurthia
gibsonii
................
ADU et al.

742
increased. Inhibition was recorded with
benzoic acid as the concentration increased
with highest inhibition (30%) observed at
20mM. Increase in the concentration of
EDTA resulted in enhanced activities
(10%) of the enzyme at 10mM.
Effect of Surfactants on Cellulase
Activities
Figure 6 shows the effect of surfactants
concentrations on cellulase activities of
K.gibsonii CAC1. Increase in
concentration of Tween 80 initially
increased enzyme activities of K. gibsonii
CAC1until highest activity increase (40%)
was observed at 0.5% after which further
increase in concentration of Tween 80
caused a gradual decrease in cellulase
activity. Increase in concentration of Triton
X-100 resulted in gradual increase in
cellulase activity of K.gibsonii CAC1 with
peak increase (80%) recorded at 1.25%.
There was an initial increase in cellulase
activity (20%) of K. gibsonii CAC1 at
0.25% SDS after which further increase in
concentration resulted in gradual decrease
in enzymatic activity (10%) until least
activity was recorded at 1.25%.
K
max
and V
max
Determination and Effect
of Increasing Substrate Concentration
Effect of different concentrations of
carboxymethyl cellulose on cellulase
activity of K.gibsonii CAC1 was studied
and the Michaelis-Menten kinetic
constants K
max
and V
max
for the cellulase
were determined. Cellulase of K.gibsonii
CAC 1 showed maximum activity at 0.8%
carboxymethyl cellulose concentration
with a linear increase up to this
concentration. Above this concentration, a
decrease in the enzyme activity was
observed. Line weaver-Burk plot for the
enzyme activity is shown in Figure 7.
Cellulase of K.gibsonii CAC1 had a
Michaelis constant (K
max
) of 7.4 X 10
-2
mg/ml and a peak enzyme activity (V
max
)
of 6.7 X 10
-1
µg/sec.
Discussion
At increasing pH values, cellulase
activities of Kurthia gibsonii CAC1 were
not significantly different (p ≤0.05)
considering their two-way analysis of
variance. Cellulase activity of K. gibsonii
CAC1 however was optimum at pH 5.0
while, further increase in pH did not favour
the enzymatic activities. This is similar to
earlier report by Immanuel et al. (2006),
who reported that Micrococcus sp, Bacillus
sp, and Cellulomas sp had maximum
activities at pH 7.0. Odeniyi et al. (2009)
reported the cellulase activity of a Bacillus
coagulans strain isolated from a
fermenting palm-fruit residue to be pH-
tolerant at 4.0 to 9.0. A similar report by
Gautam et al. (2010) on a cellulase enzyme
from Pseudomonas sp showed a broad
range activity at pH optimum of 7.5.
There gradual increase in cellulase
activities of K.gibsonii CAC1 at 25
0
C with
optimal temperature observed at 30
0
C
which lead to sharp decrease in enzyme
activities is similar to the report given by
Itoandon et al. (2011) which showed that
Aspergillus niger had optimum
temperature for cellulase activities at 30
0
C.
Report by Otajevwo and Aluyi, (2010),
showed that Psuedomonas aeruginosa had
peak cellulase activities at 40
0
C and
similarly, optimal temperature of 40
0
C was
reported for Aspergillus niger Z10 strain
by Gokhancoral et al. (2002). Cellulase
activities from Trichoderma sp and other
mesophilic cellulolytic fungi are at their
optimum when assayed at about 50
0
C
(Mandels et al., 1974; Tangnu et al., 1981
and Kawamori et al., 1987). The least
growth of K. gibsonii CAC1 was observed
at pH 3.5. The cellulase activities of K.
gibsonii CAC1 at various temperatures
used were not significantly different
(p≤0.05) when incubated at different
incubation period.
Cellulase activities of K.gibsonii CAC1
showed a significant difference (p≥0.05)
with different cations used but no
Ethiopian Journal of Environmental Studies and Management Vol. 8 (Suppl. 1) 2015

743
significant difference (p≤0.05) was
observed with increasing concentration
using two-way ANOVA. Of all the cations
studied, it was discovered that the addition
of Ca
2+
resulted in highest cellulase
activities of K.gibsonii CAC1 with optimal
activity recorded at 10mM. Addition of
Ca
2+
at increasing concentration (5, 10, 15
and 20mM) highly enhanced the cellulase
activities with optimal boost giving 60%
increase. At reduced concentration of
5mM, the enzymatic activities were
boosted (10%) by the addition of Hg
2+
and
Mg
2+
. Increasing concentration of Hg
2+
and
Mg
2+
resulted in inhibition of the activities
of the cellulase but enhanced activities at
low concentrations. The higher the
concentration of Hg
2+
and Mg
2+
added; the
higher the inhibition recorded. The highest
inhibition (50%) was recorded with Hg
2+
at
20mM. This was in contrast to the findings
of Odeniyi et al. (2009) who recorded total
inhibition of cellulase activities in Bacillus
coagulans after the addition of 1mM
HgCl
2
solution. Igbal et al. (2011) reported
that Hg
2+
had partial inhibition on purified
cellulase activities from Trichoderma
viride isolated from wheat straw.
At increasing concentration of the
inhibitors, there was no significant
difference (p ≤0.05) in the cellulase
activities of K.gibsonii CAC1 but the
effect of each inhibitor on the enzymatic
activity was significantly different
(p≥0.05). Urea showed similar effect
shown by the control on the cellulase
activities of K.gibsonii CAC1 as the
concentration increased. Benzoic acid
showed pronounced inhibitory effect on
the enzyme. Inhibition was recorded with
benzoic acid as the concentration increased
with highest inhibition (30%) observed at
20mM. Increase in EDTA concentration
resulted in enhanced cellulase activities
with highest boost recorded at 10mM after
which there was decrease in activities of
the cellulase enzyme.
At lower concentrations of Tween 80
up till 0.5%, cellulase activities of
K.gibsonii CAC1 were enhanced, although
this was followed by decrease in the
enzymatic activities as the concentration
was increased. Enzymatic activities of the
bacteria were enhanced by Tween 80 with
highest boost of 50% observed in K.
gibsonii CAC1. There was 70% increase in
cellulase activities when Trition X-100
was added. Igbal et al., 2011, reported that
SDS, and EDTA showed inhibitory effect
on purified cellulase activity from
Trichoderma viride isolated from wheat
straw.
Using carboxy methyl cellulose as
substrate, the enzyme showed maximum
activity (V
max
) of 0.67µg/sec with its
corresponding K
max
value of 0.074 mg/ml
for K.gibsonii CAC1. In literature,
different ranges of K
max
and V
max
for
different microorganisms, especially
fungal species have been reported.
According to Ekperigin (2007), K
max
values for A. anitratus and Branhamella
sp. were 0.32 and 2.54mM respectively for
cellobiose as substrate, while for CMC
substrate the values recorded were 4.97
and 7.90 mg/mL for the same species
respectively. Similarly, K
m
value of 3.6
mg/mL for Pseudomonas fluorescens and
1.1 mg/mL for Trichoderma reesei were
reported by Bakare et al. (2005) and
Cascalheira and Queiroz, (1999)
respectively were reported. Odeniyi et al.
(2009) reported the K
max
and V
max
of a
carboxy-methyl-cellulase from Bacillus
coagulans strain to be 0.65 mg/ml and
1.36µg/sec respectively.
Conclusion
Kurthia gibsonii CAC1 produced
cellulase which is a thermostable enzyme
whose activities are enhanced by some
cations such as Ca
2+
and Mg
2+
and partially
inhibited by Hg
2+
. They were able to grow
at pH range of 5-7 and optimum
temperature of 30
o
C. The novel
Physicochemical Characterization of Cellulase Produced from
Kurt
hia
gibsonii
................
ADU et al.

744
microorganism can be used for the
production of cellulase with various
industrial applications and to aid in
degradation of cellulolytic wastes.
Acknowledgements
The authors would wish to appreciate
support received from the postgraduate
laboratory technologists, Department of
Microbiology, University of Ibadan in
course of the research work.
Figure 1: Effect of pH on the cellulase activities of K. gibsonii CAC1
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745
Figure 2: Effect of temperature on cellulase activities of K. gibsonii CAC1
Physicochemical Characterization of Cellulase Produced from
Kurthia
gibsonii
................
ADU et al.

746
Figure 3: Response of K. gibsonii CAC1cellulase to incubation temperature relative to time
Ethiopian Journal of Environmental Studies and Management Vol. 8 (Suppl. 1) 2015

747
Figure 4: Effect of cations on cellulase activity of K. gibsonii CAC1
Physicochemical Characterization of Cellulase Produced from
Kurthia
gibsonii
................
ADU et al.

748
Figure 5: Effect of inhibitors/enhancers on cellulase activity of K. gibsonii CAC1
Ethiopian Journal of Environmental Studies and Management Vol. 8 (Suppl. 1) 2015

749
Figure 6: Effect of surfactants on cellulase activity of K. gibsonii CAC1
Physicoc
hemical Characterization of Cellulase Produced from
Kurthia
gibsonii
................
ADU et al.

750
Figure 7: Lineweaver-Burk plot of CMCase activity of K. gibsonii CAC1
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