World Applied Sciences Journal 20 (6): 781-789, 2012

ISSN 1818-4952
IDOSI Publications, 2012
DOI: 10.5829/idosi.wasj.2012.20.06.6689

Bioconversion of Coffee Husk Cellulose and Statistical Optimization of

Process for Production of Exoglucanase by Rhizopus stolonifer
P.N. Navya, Roopali N. Bhoite and Pushpa S. Murthy
Department of Plantation Products, Spices and Flavour Technology,
Central Food Technological Research Institute,
Council of Scientific and Industrial Research, New Delhi, India
Abstract: Exoglucanase was produced using coffee husk, the by-product of coffee processing industries, as
the sole carbon source using economical fermentation strategies like solid-state fermentation (SSF). The effects
of process parameters such as pH, moisture and fermentation duration on exoglucanase synthesis was
investigated using response surface methodology (RSM) based on three factors and five level central
composite rotatable design (CCRD). Statistical analysis revealed moisture and fermentation duration has
significant effect on the exoglucanase production. The optimized conditions were pH 6.7, moisture 43.2% and
fermentation duration 99.8 hours. At these conditions, the predicted exoglucanase activity was 392.2 U/ml and
the actual experimental value was 390.3 U/ml. The experimental value (390.3 U/ml) related well with the predicted
one (392.2 U/ml) indicating the suitability of the model employed. The enzyme showed optimum activity at pH
6.0 and 40C. The enzyme activity was enhanced in presence of Mg2+ and Ca2+. The effect of solvents and
detergents on the enzyme activity was also studied. The results indicate excellent scope of utilizing coffee husk
as solid substrate for commercial production of exoglucanase employing Rhizopus stolonifer. This investigation
is the first report of utilizing coffee husk for the production of exoglucanase and is significantly higher than the
values reported for other potent fungi and bacteria and their utilization by biotechnological resources assume
social, economic and industrial importance.
Key words: Coffee Husk Solid-State Fermentation (SSF)
Surface Methodology (RSM)
INTRODUCTION

Rhizopus Stolonifer

Exoglucanase

Response

enzymes have attracted considerable attention in recent

years due to their great biotechnological and industrial
potential.
Enormous amounts of agricultural, industrial and
municipal cellulosic wastes have been accumulating or are
used inefficiently due to the high cost of their exploitation
processes [3]. Therefore, it has gained a considerable
economic interest to develop processes for the effective
treatment and utilization of cellulosic wastes as
inexpensive carbon sources.
Coffee is one of the most important agricultural
commodities in the world. The coffee industries discharge
huge quantity of by-products such as pulp, cherry husk,
silver skin and spent grounds during pulping, curing,
roasting and brewing of coffee and are available annually

Cellulases are a group of hydrolytic enzymes capable

of degrading cellulose to the smaller glucose units and
are chiefly endoglucanase (EC 3.2.1. 4), exoglucanase
(EC 3.2.1. 91) and -glucosidase (EC 3.2.1. 21) [1].
Cellulases account for approximately 20% of the world
enzyme market. The use of this enzyme has increased
incessantly, especially in food, brewery and wine
industries and has become the driving force for research
on cellulases. Amongst the cellulases, exoglucanase or
FPase are found to have potential applications in the
bioconversion of agricultural waste materials to valuable
products, such as single cell protein, fuels and chemical
feed stocks, food, animal feed, textile [2]. The cellulase

Corresponding Author: Pushpa S. Murthy, Department of Plantation Products Spices and Flavour Technology,
Central Food Technological Research Institute, Council of Scientific and Industrial Research,
New Delhi, India. Tel: + 91 821 2512352, Fax: +91 821 2517233.

781

World Appl. Sci. J., 20 (6): 781-789, 2012

Preparation of the Inoculum: The Rhizopus stolonifer

was cultured in 100 mL Erlenmeyer flasks containing
potato dextrose broth. The flasks were incubated at 27C
for 72 h and the inoculum thus obtained was used for the
inoculation of the solid substrate medium.

worldwide. These could be excellent cost-effective

substrates for large-scale production of enzymes in
solid-state fermentation (SSF). These substrates are rich
in carbohydrates (57%), proteins (24%), celluloses and
hemicelluloses (43%) and minerals with potential for
utilization in bioprocesses [4]. These substrates are close
to natural habitats of microbes and may prove efficiency
in producing enzymes and metabolites [5].
SSF holds tremendous potential for the production of
enzymes and agro-industrial residues are generally
considered the best substrates [6, 7, 8]. It has been widely
accepted that Solid State Fermentation (SSF) is an
attractive means to produce cellulase economically
because of its low capital investment and operating cost.
Both bacteria and fungi are known to produce cellulases
using complex cellulosic substrates, however, fungal
enzymes are efficient to carry out cellulosic activities.
Cellulase production depends on a complex relationship
involving a variety of factors like pH, temperature,
fermentation period, cations, carbon and nitrogen sources
[9]. To establish a successful fermentation process, it is
necessary to choose best condition for high production
of the desired metabolite. RSM can be used to evaluate
the significance of several factors especially when
interactions exist among factors and they are complex to
determine. It is a collection of statistical techniques for
designing experiments, building models, evaluating the
effect of factors and obtaining optimum conditions for
desirable responses. Thus, the use of coffee husk as a
substrate to produce exoglucanase using Rhizopus
stolonifer by SSF could be useful and the study describes
the use of statistical techniques (response surface
methodology, RSM) to enhance the exoglucanase
production by optimizing the fermentation conditions and
to determine the pH and temperature optima and to study
the effect of metal ions and inhibitors on the exoglucanase
activity.

Substrate and Solid-State Fermentation: The coffee husk

was used as a substrate for SSF and was obtained from
the local coffee curing works, Mysore, India. The coffee
husk was pre-treated by steam at 121C for 15 min before
using as substrate in the culture media. The pH of the
substrate was adjusted using Mandels solution [10].
The contents were sterilized at 121C, 15 lbs for 30 min.
After sterilization the flasks were cooled and inoculated
with fresh culture of Rhizopus stolonifer and incubated at
30C for 96 hrs.
Enzyme Extraction and Assay: The enzyme was extracted
by suspending the fermented substrate in citrate buffer
(50 mM, pH 4.8) using orbital shaker (150 rpm) for
one hour. The suspended material and the fungal biomass
were separated by filtering through a Whatman No. 1 filter
paper and then by centrifugation (10000 rpm for 15 min).
The clarified supernatant was used as the crude enzyme.
Exoglucanase or Filter paper saccharifying activity in
the crude enzyme was determined as described by Ghose
[11]. The reaction mixture contained 50 mg of Whatman
No. 1 filter paper strips as a substrate, 1.4 mL of 50 mM
sodium citrate buffer (pH 4.8) and 0.1 mL of crude enzyme.
The control was prepared simultaneously by adding
buffer instead of enzyme. The samples were incubated at
50C for 60 minutes. The reducing sugar was estimated as
per Miller [12] and the absorbance was measured at
540 nm using spectrophotometer. One unit of enzyme
activity is defined as the amount of glucose (M) released
per mL enzyme solution per minute under assay
conditions.

MATERIALS AND METHODS

Experimental Design Using Response Surface

Methodology (RSM): A five-level and three variables
Central composite rotatable design (CCRD) (Statistica
software 9.0, StatSoft) was applied to determine the best
combination for exoglucanase production. The range and
the levels of the independent variables i.e., pH, moisture
and fermentation duration chosen for the current study
are presented in Table 1.
Each experiment was performed in duplicates
(it means that the same steps were repeated for two times
in all optimization experiments and the data in this study

Sources of Media and Analytical Chemicals: All

chemicals used were of analytical grade. Media and
chemicals used in this study were purchased from
HiMedia, India, Sigma-Aldrich, USA and Merck, Germany.
Micro-Organism: A strain of Rhizopus stolonifer, a
potential cellulose degrader was isolated and identified in
our laboratory was used in the study. It was maintained
on potato dextrose agar medium and was stored at 4C.
782

World Appl. Sci. J., 20 (6): 781-789, 2012

The precipitate was dissolved in 50 mM sodium citrate

buffer of pH 4.8.

Table 1: Process variables and their levels

Coded levels
------------------------------------------Independent variables
pH

Symbol
A

-1

+1

3.32

6.68

Moisture (%)

43.18

50

60

70

76.82

Fermentation duration (h)

79.64

96

120

144

160.36

Temperature and pH Optima and Stability: Optimal

temperature was determined by performing a standard
activity assay at different temperatures (20, 30, 40, 50, 60,
70 and 80C) in sodium citrate buffer (pH 4.8, 50 mM) and
thier thermal stability was carried out at (30, 40, 50, 60, 70
an 80C). Similarly, the optimization of pH and their
stability of the exoglucanase were determined at different
pH ranges from 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 for 30 min at
room temperature.

are the average values of the two experiments repeated)

and the average yield of exoglucanase was taken as the
response variable, Y. A second-order polynomial equation
was then fitted to the data by a multiple regression
procedure. This resulted in an empirical model that related
the responses measured to the independent variables of
the experiment.
For a three-factor system, the model equation is
given by eq. 1:

Effect of Metal Ions, Solvents and Detergents on

Exoglucanase Activity: The effect of various additives on
the partially purified exoglucanase activity was
determined by the presence of metal ions and other
reagents. The additives used in this study were CaCl 2,
KOH, MgSO4, ZnSO4, Fe2Cl3, NH4Cl, EDTA, Tween 20,
SDS, Triton X 100. Concentration of each additive was
1 mM. The solvents used were methanol, ethanol,
chloroform, isopropanol, acetone (20% v/v). The enzyme
with various additives were incubated for 60 min at room
temperature and the residual activity was assayed by DNS
method.

Y = 0 + 1A + 2B + 3C + 11A2 + 22B2 + 33C2

(1)
+ 12AB + 23BC + 13AC
where Y is the predicted response, 0 is the intercept, 1,
2, 3 are linear coefficients, 11, 22, 33 are squared
coefficients and 12, 23, 13 interaction coefficients and
A denoted pH, B was moisture (%) and C was the
fermentation duration (hours). The responses of the
CCRD design were fitted with a second-order
polynomial equation. The CCRD contained a total of
20 experimental trials that included 14 trials for non
central points and 6 trials for replications of the
central points. Statistical analysis of the data was
performed by Statistica 9.0, to evaluate the analysis of
variance (ANOVA), to determine the significance of
each term in the equations fitted and to estimate the
goodness of fit in each case. The fitted polynomial
equation was then expressed in the form of threedimensional response surface plots to illustrate the main
and interactive effects of the independent variables on the
dependent ones.

RESULTS AND DISCUSSION

Solid-State Fermentation of Coffee Husk by Rhizopus
Stolonifer: Exoglucanase was produced by solid-state
fermentation using Rhizopus stolonifer on pre-treated
coffee husk. The enzyme activity of 80.74 U/ml was
obtained at 96 hrs on fermentation at 30C. The enzyme
activity was further enhanced by optimizing the
fermentation conditions using statistical approach.
Optimization of Process Parameters by RSM: The level
of enzyme activity produced by organisms from the
natural environment is often low and needs to be elevated
for industrial production. Increase in enzyme levels is
often achieved by optimizing key growth conditions
(pH, moisture and fermentation duration) and the effect of
their interactions on exoglucanase production were
explored by the central composite rotatable design of
RSM. The experimental design and the results are outlined
in Table 2. Multiple regression analysis was performed on
the experimental data. The mathematical model that
represented exoglucanase activity within the region being
as the effects of independent variables was expressed in
the form of equation.

Partial Purification and Characterization of

Exoglucanase: The exoglucanase excreted by Rhizopus
stolonifer under solid-state fermentation was subjected to
purification. A more concentrated form of the crude
enzyme was obtained with limited amounts of sodium
citrate buffer (pH 4.8). The crude enzyme was purified
using 9% activated charcoal and centrifuged at 10000 rpm
for 15 min. The supernatant was transferred into another
flask and brought to 80% saturation with (NH4)2SO4.
The solution was left at 4C for 24 hrs and centrifuged.
783

World Appl. Sci. J., 20 (6): 781-789, 2012

Table 2: Treatment schedule for three-factor CCRD and response in terms of exoglucanase yield

Run

pH

Moisture (%)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

(-1) 4
(-1) 4
(-1) 4
(-1) 4
(+1) 6
(+1) 6
(+1) 6
(+1) 6
(- ) 3.32
(+ ) 6.68
(0) 5
(0) 5
(0) 5
(0) 5
(0) 5
(0) 5
(0) 5
(0) 5
(0) 5
(0) 5

(-1) 50
(-1) 50
(+1) 70
(+1) 70
(-1) 50
(-1) 50
(+1) 70
(+1) 70
(0) 60
(0) 60
(- ) 43.18
(+ ) 76.82
(0) 60
(0) 60
(0) 60
(0) 60
(0) 60
(0) 60
(0) 60
(0) 60

Fermentation duration (h)

(-1) 96
(+1) 144
(-1) 96
(+1) 144
(-1) 96
(+1) 144
(-1) 96
(+1) 144
(0) 120
(0) 120
(0) 120
(0) 120
(- ) 79.64
(+ ) 160.36
(0) 120
(0) 120
(0) 120
(0) 120
(0) 120
(0) 120

Exoglucanase activty (U/ml)

-------------------------------------------------------Experimental
Predicted
234.78
217.83
162.26
143.53
377.01
175.92
208.57
80.74
192.36
192.65
243.58
104.85
324.98
150.63
201.52
224.06
204.11
225.05
213.97
224.46

247.13
206.23
208.99
159.02
361.98
174.47
220.64
68.86
181.85
202.60
253.16
94.58
318.47
156.47
215.41
215.41
215.41
215.41
215.41
215.41

CCRD, Central composite rotatable design.

Table 3: Analysis of variance and lack of fit for exoglucanase production
Source
Model
A
B
C
AB
AC
BC
A2
B2
C2
Residual
Lack of fit
Pure error
Cor total

Sum of squares

Df

Mean square

F-value

p value Prob > F

80861.52
520.64
30345.25
31685.77
1705.28
10748.71
638.67
983.61
3126.23
866.46
2100.47
1528.21
572.27
82961.99

9
1
1
1
1
1
1
1
1
1
10
5
5
19

8984.61
520.64
30345.25
31685.77
1705.28
10748.71
638.67
983.61
3126.23
866.46
210.05
305.64
114.4

42.77
2.48
144.47
150.85
8.12
51.17
3.04
4.68
14.88
4.13

< 0.0001
0.1465
< 0.0001
< 0.0001
0.0173
< 0.0001
0.1118
0.0557
0.0032
0.0697

2.67

0.1524

Df, Degrees of freedom.

R2; 0.9747, Adj R2; 0.9519, Pred R2; 0.8368, Adeq precission; 28.602, C.V %; 7.06.

Y = +215.55 47.14B 48.17C 14.60AB 36.65AC

14.73B2
(2)

The subsequent analysis of variance addressed the

aptness of the model for exoglucanase production.
The computed F-value of 42.77 implies significance of the
model. The model was found to be highly significant and
sufficient to represent the actual relationship between
the response and the significant variables as indicated
by the model p-value (<0.0001), large lack-of-fit p-value
(0.1524), suitable coefficient of determination (R2 = 0.9747)
and adjusted coefficient of determination (R2 adjusted
= 0.9519) from ANOVA (Table 3). The adequate precision
value of the model is 28.602. The precision value is an

where Y is exoglucanase activity and A, B and C were the

coded variables for the pH, moisture (%) and fermentation
duration (h) respectively. This equation was used to
predict the exoglucanase activity presented in Table 2.
Apart from explaining the linear effects of pH, moisture
(%) and fermentation duration (h) on exoglucanase
production, the CCRD approach described the quadratic
and interaction effects of the parameters too.
784

World Appl. Sci. J., 20 (6): 781-789, 2012

index of the signal-to-noise ratio and values greater than

4.0 are pre-requisites for a model to be a good fit. The
lower value of the coefficient of variation (C.V% = 7.06)
showed that the experiments were precise and reliable.
According to the p-value (The value, in case of below
0.05, indicated significance level) B, C, AB, AC, B2 were
significant, but A, BC, A2 and C2 were not significant
(Table 3).
Response-surface curves for the variation in
exoglucanase activity as a function of two variables,
with the other one kept at the central value were
constructed. From the response for the interaction of pH
and moisture (Fig. 1a), exoglucanase activity increased
with increase in pH. It is exemplified that the pH change
observed during the growth of microbes also effects
product stability in the medium [13]. Decrease in the
enzyme activity when moisture was increased proved that
moisture is an important factor for the production of
enzyme by SSF. These results indicated a positive
relationship between cellulase production and moisture
content. High and low water content adversely affects the
primary metabolic activities of microbes leading to feeble
enzyme synthesis. Increase in moisture level is alleged to
decrease enzyme production due to substrate porosity,
alteration in substrate particle structure, lowering oxygen
transfer, higher soluble protein and enhance aerial
mycelial growth [14]. Fig. 1(b) relates the interaction
between pH and fermentation duration. The activity
increased significantly when pH was increased, whereas
non significant increase in enzyme activity was observed
with increase in fermentation duration. The decrease in
enzyme activity with fermentation duration may be due to
the presence of impurities beside cellulose which could
result in hindering the increase of cellulase activity [15].
Fig. 1(c) revealed the interaction effect of moisture and
fermentation duration on exoglucanase production.
The moisture in a range of 50%-60% on fermentation
for 72-96 hrs produced maximum enzyme activity with
377 U/mL and 324 U/mL respectively. However the
exoglucanase decreased with increase in moisture and
fermentation period. The exoglucanase activity was
maximum at 96 hrs and rapidly decreased. The decrease in
the enzyme activity when the moisture and fermentation
duration was increased may be due to protein solubility
and as well as effect on swelling at low moisture of the
substrate [16] and probably due to the depletion of
nutrients in the medium which stressed the fungal
physiology resulting in the inactivation of enzyme
secretion.

600
500
400
300
200
7
6

160
5

130
4

100

(a)
450
350
250
150
50
80
70
60
50

(b)

400
300
200
100
80
70
60
50

95

120

145

170

(c)
Fig. 1: Response surfaces showing the effects of (a) pH
and moisture, (b) pH and fermentation duration
moisture and (c) fermentation duration
Compared to the reported literature on production of
exoglucanase on various fungal sources and substrates
such as T. reesei and Rhizopus oryzae PR 7 [17, 18]
R. stolonifer grown on coffee husk is found to be
potential producer with maximum production of 390.28
U/ml. Maximum activity was observed at pH 6.68, with
43% moisture at 99 h of fermentation. It has been reported
that maximum exogluacanase activity of 48 U/mL FPase
was achieved from SSF of water hyacinth from
Rhizopus oryzae and the highest production was
achieved at 48 hours of cultivation [18].
785

World Appl. Sci. J., 20 (6): 781-789, 2012

1.0000

3.3182

6.6818
6.6818

43.182
43.182

76.818

79.637
99.818

160.36

Fig. 2: Desirability profiles showing the optimum conditions for exoglucanase production
Observed vs. Predicted Values

3 factors, 1 Blocks, 20 Runs; MS Residual=210.3466

DV: EXOGLUCA
400

Predicted Values (U/mL)

350
300
250
200
150
100
50
0

50

100

150

200

250

300

350

400

450

Observed Values (U/mL)

Fig. 3: Plot showing the distribution of experimental and predicted values of exoglucanase activity
Statistica plot illustrates the interaction between pH,
moisture and fermentation duration corresponding to the
response. The optimum conditions to obtain maximal
exoglucanase production were pH 6.68, moisture 43.18%
and fermentation duration of 99.8 h. Fig. 2 illustrates the
desirability profiles of exoglucanase showing the optimum
conditions. The desirability profiles indicate that the
maximum exoglucanase activity can be achieved at the
suggested points.

at 99 hrs of cultivation. The crude extract from the culture

medium was taken through the two-step purification
process. The enzyme was purified with activated charcoal
and ammonium sulphate precipitation. After precipitation
total protein content was reduced by about 91%
indicating 3.1 fold purification with a yield of 31.4%.
Optimum Temperature and pH on Exoglucanase Activity
and Stability: The optimum temperature for exoglucanase
was found to be 40C (Fig. 4). The relative activity
decreased to 65%, 51% and 42% at 50, 60 and 70C
respectively. The activity decreased significantly above
50C and below 30C. The activity of the partially purified
exoglucanase decreased with the increase in temperature.
The residual activity decreased to 70% and 50% at 40 and
50C, respectively. The stability of exoglucanase
decreased rapidly above 40C.
As shown in Fig. 5, the optimum pH for the coffee
husk exoglucanase was around 6.0. The relative activity
of exoglucanase decreased to 85.3% and 50% at pH 6 and
7, respectively. The exoglucanase activity decreased
rapidly below pH 5.0 and above pH 7.0. The pH stability
of exoglucanase indicated that the enzyme was
comparatively stable in pH range of 5-7. In contrast, the
activity decreased markedly below pH 4 and above pH
8 and only 39.2% and 20% was retained at pH 3 and 12
respectively.

Validation of the Model: In order to confirm the model

adequacy, experiments were conducted in duplicates in
flasks at the optimum conditions to obtain the maximum
secretion of the enzyme. Results showed excellent
correlation between predicted and experimental values
which justifies the validity of the response model and
the existence of an optimum point. The validity of the
model can be cross checked against the actual data
set by using an actual versus predicted plot (Fig. 3).
The relationship between the experimental values and
predicted values showed that the plotted points cluster
around the diagonal line, indicating good fitness of the
model.
Partial Purification, Characterization and Kinetics of
Exoglucanase: The SSF of coffee husk for exoglucanase
production by Rhizopus stolonifer produced 390.28 U/ml
786

World Appl. Sci. J., 20 (6): 781-789, 2012

100

80

Residual relative activity (%)

Relative activity (%)

100

80

60
60
40
40

20
Residual relative activity (%)

20

Relative activity (%)

0
20

30

40

50

60

70

80

Temperatureo(C)

110

110

100

100
90

90

80

80

70

70

60

60

50

50

40

40

Residual relative activity (%)

30

Relative acctivity (%)

30
2

Residual relative activity (%)

Relative activity (%)

Fig. 4: Effect of temperature on exoglucanase activity and thermal stability

20
8

10

12

pH

Fig. 5: Effect of pH on coffee husk exoglucanase activity and pH stability

Table 4: Effect of additives on Exoglucanase activity from Rhizopus
stolonifer
Chemicals (1 mM)
None
Mg2+
NH4+
Ca2+
K
Zn2+
Fe3+
Fe2+
EDTA

Relative activity (%)

100.001.00
126.380.94
99.130.17
158.981.30
94.822.50
74.710.23
100.000.58
83.610.42
130.250.91

Thus, maximum activity of the enzyme was observed

at pH 6.0 and 40C, which is similar to exoglucanase from
maize stover [19]. Few reports on exoglucanase from

Fig. 6: Effect of organic solvents and detergents on

exoglucanase activity
787

World Appl. Sci. J., 20 (6): 781-789, 2012

Aspergillus niger NRRL 567 was found to be 3.5 and 30C

[20]. The temperatures higher than 40C suppressed
production of exoglucanase and supported the finding of
Rajoka et al. [21]. Cellulases in general show optimum
temperature between 30 to 55C [22].

brewery industries. This also addresses valorisation of

agro wastes which otherwise cause environmental
pollution. Further increase in production by microbial
engineering techniques is in progress.
ACKNOWLEDGMENTS

Effect of Additives on Exoglucanase Activity: Metal ions

were assayed for their effects on partially purified
exoglucanase activity. The results are presented in
Table 4. The results indicated that the metal ions such as
Mg2+ and Ca2+ increased 1.26 and 1.58 fold of enzyme
activity. The results are in corroboration with cellulase
produced from T. harzianum utilizing Mahua cake [23].
The positive effect of EDTA on exoglucanase activity
might be attributed to the reduction of metal ions in the
solution through chelation. An increase in the
exoglucanase activity in presence of Ca2+, EDTA and
Mg2+ indicated the absence of thiol groups at the catalytic
site. It seems that heavy metals attack some certain
groups at the active site of enzyme, for example the thiol
groups, leading to the inactivation, whereas few metal
ions could enhance the substrate binding affinity of the
enzyme and stabilize the confirmation of the catalytic site
[24] and require Ca2+ and Mg2+ for maximal activity [25].
On the other hand, Zn2+ and Fe2+ reduced the enzyme
activity. Zn2+ and Fe2+ were strongly inhibitory, while the
other cations tested exhibited either no effect or moderate
levels of inhibition.
The addition of organic solvents (acetone, ethanol,
isopropanol, methanol, chloroform) and detergents
(Tween 20, Triton X 100 and SDS) tested lead to reduction
in exoglucanase activity (Fig. 6). Around 40% loss in the
exoglucanase activity was recorded when chloroform was
used. Acetone and chloroform showed a decrease in
exoglucanase activity whereas ethanol and methanol did
not show any inhibitory effect on the enzyme. The tested
detergents inhibited the exoglucanase activity by 20%,
45% and 30% respectively. The detergents tested had
negative influence on the exoglucanase activity. Presence
of methanol and ethanol had no influence on the
hydrolytic activity of exoglucanase.

The authors are thankful to the Director, Central

Food Technological Research Institute, Mysore and
Head, Plantation products, Spices and Flavor Technology
Department for providing the facilities and support to
carry out this study. The authors also wish to
acknowledge the Council of Scientific and Industrial
Research, New Delhi for funding this project.
REFERENCES
1.

2.

3.

4.

5.

6.

7.

CONCLUSION
This is the first report on the production, purification
and characterization of exoglucanase from Rhizopus
stolonifer. Thus, the present investigations, highlights
use of Rhizopus stolonifer to synthesize high amount of
extracellular exoglucanase utilizing coffee husk and its
enzyme characters which enable their use in food and

8.

788

Wood, T.M. Sheila and I. McCrae, 1986. The

cellulase of Penicillium pinophilum, Biochem. J.,
234: 93-99.
Uhlig, H., 1998. Industrial enzymes and their
applications, New York: John Wiley and Sons, Inc.
USA, pp: 435.
Kim, K.C., S.S. Yoo, Y.A. Oh and S.J. Kim, 2003.
Isolation and characteristics of Trichoderma
harzianum FJ1 producing cellulases and xylanase,
J. Microbiol. Biotechnol., 13: 1-8.
Murthy, P.S. and H.K. Manonmani, 2008.
Bioconversion of coffee industry wastes with
white rot fungi Pleurotus florida, Res. J. Environ.
Sci., 2: 145-150.
Jecu, L., 2000. Solid state fermentation of agricultural
wastes for endoglucanase production, Ind. Crops
Prod., 11: 1-5.
Abo-State, M.A.M., A.I. Hammad, M. Swelim and
R.B. Gannam, 2010. Enhanced production of
Cellulase(S) by Aspergillus spp. isolated from
Agriculture wastes by Solid State Fermentation,
American-Eurasian J. Agric. and Environ. Sci.,
8(4): 402-410.
Umbrin Ilyas, Abdual Majeed, Khalid Hussain,
Khalid Nawaz, Shakil Ahmed and Muhammad
Nadeem, 2011. Solis state fermentation of Vigna
Mungo for cellulase production by Aspergillus
niger, W. Appl. Sci. J., 12(8): 1172-1178.
Muhammad Irfan, Muhammad Nadeem and
Quratualain Syed, 2012. Influence of nutritional
conditions for Endoglucanase production by
Trichoderma viride in SSF, Global J. Biotechnol.
Biochem., 7(1): 07-12.

World Appl. Sci. J., 20 (6): 781-789, 2012

9.

10.

11.
12.

13.

14.
15.

16.

17.

18.

Immanuel, G., R. Dhanusha, P. Prema and

A. Palavesam, 2006. Effect of different growth
parameters on endoglucanase enzyme activity by
bacteria isolated from coir retting effluents of
estuarine environment, Int. J. Environ. Sci. Technol.,
3: 25-34.
Mandels, M. and J. Weber, 1969. The production
of celluases. In: Cellulases and their applications,
G.J. Hajny and E.T. Resse, (Eds.). Amer. Chem. Soc.
Adv. Ser., 95: 391-414.
Ghose, T.K., 1987. Measurement of Cellulase
activities, Pure and App. Chem., 59: 257-268.
Miller, G.L., 1959. Use of DNS reagent for the
determination of reducing sugars, Analyt. Chem.,
31: 426-428.
Gupta, R., P. Gigras, H. Mohapatra, V.K. Goswamu
and B. Chauhan, 2003. Microbial -amylases: A
biotechnological perspective, Process Biochem.,
38: 1599-1616.
Gervais, P. and Molin, 2003. The role of water in
solid-state fermentation, Biochem. Eng. J., 13: 85-101.
Sahin, H.T. and M.B. Arslan, 2008. A Study on
physical and chemical properties of cellulose paper
immersed in various solvent mixtures, Int. J. Mol. Sci.,
9: 78-88.
Shah, A.R. and D. Madamwar, 2005. Xylanase
production under solid-state fermentation and its
characterization by an isolated strain of Aspergillus
foetida in India, World J. Microbiol. Biotechnol.,
21: 233-243.
Deshpande, P., S. Nair and S. Khedkar, 2008.
Water hyacinth as carbon source for the production
of cellulase by Trichoderma reesei, Appl. Biochem.
Biotechnol., 158: 552-560.
Moumita Karmakar and Rina Rani Ray, 2011.
Statistical optimization of FPase production from
water hyacinth using Rhizopus oryzae PR 7, J.
Biochem. Tech., 3: 225-229.

19. Yejun Han and Hongzhang Chen, 2010. Biochemical

characterization of a maize stover b-exoglucanase
and its use in lignocellulose conversion, Bioresour
Technol., 101: 6111-6117.
20. Mohammad Ishfaq Ghori, Sibtain Ahmed,
Mohammad Aslam Malana and Amer Jamil, 2012.
Kinetics of exoglucanase and endoglucanase
produced by Aspergillus niger NRRL 567, Afr. J.
Biotechnol., 11(28): 7227-7231.
21. Rajoka, M.I., A. Bashir, M.R.A. Hussain and
K.A. Malik, 1998. Mutagenesis of Cellulomonas
biazotea for improved production of cellulases, Folia
Microbiologica., 43(1): 15-22.
22. Peng, Y., Z.M. Chi, X.H. Wang and J. Li, 2009.
Purification and molecular characterization of exobeta-1, 3-glucanases from the marine yeast Williopsis
saturnus WC91-2, Appl. Microbiol. Biotechnol.,
85(1): 85-94.
23. Devendra Kumar, M. Muthukumar and Neelima Garg,
2012. Utilization of Mahua cake for cellulase
production by using Trichoderma harzianum
NAIMCC-F-02957 under submerged fermentation,
Academic J. Plant Sci., 5(1): 01-06.
24. Tao, Y. M., X.Z. Zhu, J.Z. Huang, S.J. Ma, X. Wu,
M.N. Long and Q.X. Chen, 2010. Purification and
properties of endoglucanase from a sugar cane
bagasse hydrolyzing strain, Aspergillus glaucus
XC9, J. Agric. Food Chem., 58(10): 6126-6130.
25. Gardner, R.M., K.C. Doerner and B.A. White, 1987.
Purification and characterization of an exo beta-1, 4glucanase from Ruminococcus flavefaciens FD-1,
J. Bacteriol., 169(10): 45-81.

789