Advances in Bioscience and Biotechnology, 2012, 3, 580-584 ABB
http://dx.doi.org/10.4236/abb.2012.35075 Published Online September 2012 (http://www.SciRP.org/journal/abb/)
Characterization of Exo 1, 4-β glucanase produced from
Trichoderma Viridi through solid-state bio-processing of
orange peel waste
Muhammad Irshad1*, Zahid Anwar1, Amber Afroz2
1Department of Biochemistry, Nawaz Sharif Medical College NSMC, University of Gujrat, Gujrat, Pakistan
2Department of Biochemistry and Molecular Biology, University of Gujrat, Gujrat, Pakistan
Received 25 June 2012; revised 27 July 2012; accepted 10 August 2012
Agro-industrial residues are primarily composed of
complex polysaccharides that strengthen the micro-
bial growth for the production of industrially impor-
tant enzymes like cellulases. In the present study we
aimed to characterize the Exo 1, 4-β glucanase that
was indigenously produced from Trichoderma viride.
T. viride MBL was cultured in the Solid-State medium
of orange peel (50% w/w moisture) under optimized
fermentation conditions and maximum activity of 412
± 12 U/mL was recorded after 4th day of incubation at
pH 5.5 and 30˚C. Exo 1, 4-β glucanase was 4.17-fold
purified with specific activity of 642 U/mg in com-
parison to the crude extract. To confirm its purity
and molecular weight, sodium dodecyl sulphate poly
acrylamide gel electrophoresis (SDS-PAGE) was per-
formed. The enzyme was shown to have a molecular
weight of 60 kDa with an optimum pH and tempera-
ture of 5˚C and 50˚C, respectively. Lineweaver-Burk
reciprocal plot revealed that the kinetic constants Km
and Vmax of purified Exo 1, 4-β glucanase were 76 µM
and 240 U/mL.
Keywords: Orange Peel Waste; Exo 1, 4-β Glucanase;
T. Viride; Purification; SDS-PAGE
In nature, cellulose, hemicellulose and lignin are the ma-
jor components of plant cell walls and among all of them,
cellulose is the most common and abundant component
of all plant matter comprised on about 35% to 50% . It
has been reported in literature by many researchers that a
wide spectrum of micro-organisms mainly including
Trichoderma, Trametes, Pleurotus, Aspergillus, Penicil-
lium, and Fusarium has ability to produce enzymes hav-
ing industrial importance like cellulases, hemicellulases,
lassases, pectinases and proteases [1,2-6]. Trichoderma is
one of the most competent cellulases producer which is
being study extensively for the production of cellulose
degrading enzymes from various agro-industrial waste
materials and their by-products. Among many of the de-
veloping countries it’s a routine practice that such agri-
cultural wastes are not been fully discarded that has be-
come a major source of ecological pollution.
From the last several years, there is an increasing de-
mand for industrial important enzymes. In such scenario,
cellulase is being used in many of the industrial applica-
tions mainly but not limited to in the field of cotton
processing; paper recycling, agriculture and in the field
of research and development [7-9]. Beside all those ap-
plications, the production of fuel ethanol from lignocel-
lulosic biomass through cellulase hydrolysis is a promis-
ing tool of the modern world. The most promising tech-
nology for the conversion of the lignocellulosic biomass
to fuel ethanol is based on the enzymatic breakdown of
cellulose using cellulase enzymes .
Pakistan is an agricultural land that produced abundant
magnitude of agro-industrial wastes. However, such
wastes can be utilized for the production of useful Indus-
trial enzymes or enzyme based products. Enzymatic hy-
drolysis of such wastes provides an environmentally
friendly means of depolymerizing cellulose and other
carbohydrates at high yields . By keeping in mind
the ever increasing demand and broad range industrial
applications of cellulases, this study was performed to
purify and characterize the Exo 1, 4-β glucanase from
cellulase enzyme complex by T. viridi to present its po-
tential application for industrial application.
2. MATERIALS AND METHODS
2.1. Chemicals and Agro-Industrial Substrate
All the chemicals used were of analytical grade and
mainly purchased from Sigma-Aldrich (USA). Orange
M. Irshad et al. / Advances in Bioscience and Biotechnology 3 (2012) 580-584 581
peel waste was obtained from the local fruit market, Gu-
jrat, Pakistan and used as a growth supported solid sup-
port. Before to use substrate was first crushed into pieces,
oven dried and finally ground to fine particle size.
2.2. Fungal Culture and Inoculum Development
Fungal strain T. viride was available in the Molecular
Biotechnology Laboratory, Department of Biochemistry
and Molecular Biology, University of Gujrat, Pakistan. A
spore suspension inoculum of T. viride was developed in
an Erlenmeyer flask containing 30 mL of Potato Dex-
trose broth at 30˚C ± 1˚C for 5 days after sterilizing at 15
lbs/in2 pressure and 121˚C.
2.3. Pretreatment of Orange Peel Waste
10 g of moisture free orange peel was pretreated with 2%
HCl by adopting thermal treatment methodology as de-
scribed previously . After pretreatment the slurry of
the substrate was filtered using Whatman No. 1 filter pa-
per. Residues were washed 3 times with distilled water to
remove extra acidity and used for production of Exo 1,
4-β glucanase under optimum fermentation conditions.
2.4. Solid-State Fermentation
For the production of Exo 1, 4-β glucanase 10 g pre-
treated orange peel was moist with Basel salt media in an
Erlenmeyer flask (250 mL) capacity. The major con-
stituents of the Basel media were: (NH4)2SO4, 10 g·l–1;
KH2PO4, 4 g·l–1; MgSO4·7H2O, 0.5 g·l–1 and CaCl2, 0.5
g·l–1. Orange peel based sterilized Solis-State medium
was inoculated with 5 mL of freshly prepared fungal spore
suspension and incubated at 30˚C ± 1˚C for stipulated
fermentation time period under still culture conditions.
2.5. Extraction of Exo 1, 4-β Glucanase
At the end of selected incubation period, Exo 1, 4-β glu-
canase was extracted from the fermented biomass by
adding 100 mL of 0.1 M succinate buffer of pH 5 and the
flasks were shaken at 120 rpm for 30 min. The contents
were filtered and filtrates were centrifuged at 10,000 × g
(4˚C) for 10 min. A carefully collected supernatants were
and used to determine enzyme activity and for purifica-
2.6. Determination of Exo 1, 4-β Glucanase
Activity and Protein Contents
Exo 1, 4-β glucanase was assayed according to the method
of Deshpande et al. , using 1% salicin as reaction
substrate with DNS as coupling reagent. The reaction
mixture contained 0.1 mL of enzyme extract with 1 mL
of 1% salicin and 1 mL of 0.1 M succinate buffer of pH 5.
The mixture was incubated for 30 min at 50˚C and the
reaction was then terminated by adding DNS reagent (2
mL). The reaction mixtures were heated for 15 min in a
boiling water bath followed by cooling in ice. The ab-
sorbance was measured at 540 nm against reagent blank.
One unit of enzyme activity was defined as the amount
of glucose (μmol) released by 1 mL of enzyme solution
per min. To determine the protein contents of the crude
and purified enzyme extracts bovine serum albumin was
used as standard.
2.7. Purification of Exo 1, 4-β Glucanase
To purify the crude extract of Exo 1, 4-β glucanase ob-
tained from T. viridi ammonium sulfate fractionation fol-
lowed by the Sephadex-G-100 (Sigma, USA) column
(120 × 2 cm) gel filtration chromatographic technique
was adopted as described by Iqbal et al. , for purifica-
tion purposes. Total proteins and activity of partially pu-
rified Exo 1, 4-β glucanase were determined before and
after each purification step as described earlier.
To determine the molecular weight of purified Exo 1, 4-β
glucanase sodium dodecyl sulphate poly acrylamide gel
electrophoresis (SDS-PAGE) was performed on a 5%
stacking and a 12% resolving gel according to the meth-
odology, as described previously .
2.9. Characterization of Purified Exo 1, 4-β
Characterization of purified Exo 1, 4-β glucanase was
done by studying the effect of various kinetic parameters
including pH, temperature and substrate concentration on
the Exo 1, 4-β glucanase activity. To investigate the ef-
fect of pH Exo 1, 4-β glucanase was incubated in buffers
of different pH (2 - 10), followed by standard assay pro-
tocol. To determine the thermal features Exo 1, 4-β glu-
canase was incubated without substrate under different
temperatures ranging from 25˚C to 70˚C for 1 h time
period followed by normal assay protocol as previously
described. The Michalis-Menten kinetic constants Km
and Vmax for Exo 1, 4-β glucanase were calculated from
Lineweaver-Burk reciprocal plots using varying concen-
trations of salicin as substrate.
2.10. Statistical Analysis
All the experimental data was conducted in triplicate and
presented as mean ± standard error (SE) and SE are
showed in figures as Y-error bars.
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M. Irshad et al. / Advances in Bioscience and Biotechnology 3 (2012) 580-584
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3. RESULTS AND DISCUSSION 3.3. Characterization of Purified Exo 1, 4-β
3.1. Production and Purification of Exo 1,
4-β Glucanase 3.3.1 . E f fect of pH on Exo 1, 4-β Glucanase
Activity & Stability
T. viride was cultured in Solid-State orange peel based
medium for the production of Exo 1, 4-β glucanase. Un-
der optimized fermentation conditions in the solid state
medium of orange peel (50% w/w moisture) the maxi-
mum activity of 412 ± 12 U/mL was recorded after 4th
day of incubation at pH 5.5 and 30˚C. T. viridi showed
high levels of Exo 1, 4-β glucanase production during
solid state bio-processing of orange peel waste. In the
present study, an eco-friendly procedure has been
adopted to utilize low cost substrates to induce enzymes
production by T. viridi. The separated cell free super-
natant crude enzyme solution containing Exo 1, 4-β glu-
canase was maximally precipitated at 70% saturation
with specific activity of 167 U/mg and 1.98-fold purifi-
cation. The optimally active fraction was loaded on to a
Sephadex G-100 column (Figure 1), and after gel filtra-
tion the enzyme was purified up to 4.17-fold with spe-
cific activity of 642 U/mg (Table 1). Previously we have
successfully developed and reported Sephadex G-100
column gel filtration technique to purify various fungal
enzymes mainly including cellulases from Trichoderma
harzianum & Trichoderma viridi, protease from Asper-
gillus niger, laccase and MnP from Trametes versicolor
IBL-04 [4-6,12]. In an earlier study, El-Gindy et al. 
has also used the Sephadex-G-100 gel filtration chroma-
tographic technique to purify Exo 1, 4-β glucanase pro-
duced from Chaetomiurn olivaceum.
The pH-activity profile showed that the present Exo 1,
4-β glucanase was optimally activity at a pH 5 (Figure 3).
It was also observed during the trial that any further in-
crease in the pH showed a sharp decreasing trend in the
activity of enzyme. Stability profile based on the 1 h in-
cubation time period revealed that the purified Exo 1, 4-β
glucanase was stable in a large pH range for up to 1 h.
Earlier studies reported that optimum activities of β-
glucosidase from different enzyme sources in the pH
range 5 to 6 .
3.3.2. E f fe ct of Temperature o n Exo 1, 4-β
Glucanase Activity and Stability
Figure 4 illustrated that the Exo 1, 4-β glucanase from T.
viridi retained its up to 75% of original activity and was
optimally active at temperature up to 60˚C. High activity
and extra thermo-stability are a desirable characteristic of
an enzyme for a wide range of industrial applications
[5,6]. In comparison the earlier reported the present Exo
1, 4-β glucanase from T. viridi was reasonably more sta-
ble and active for up to 1 h incubation at 60˚C. The
maximum activity of Exo 1, 4-β glucanase was obtained
at 45˚C .
Exo 1, 4-β glucanase was further purified to homogene-
ity and to confirm its purity, the purified Exo 1, 4-β glu-
canase was resolved on 5% stacking and 12% running
gel and found to be a homogenous monomeric protein as
evident by single band of 60 kDa on SDS-PAGE (Figure
2). Exo 1, 4-β glucanase from Chaetomiurn olivaceum
was purified to homogeneity by SDS-PAGE with a mo-
lecular mass of 88 kDa . While Talaromyces emersonii
exo-1,3-β-glucanase was a monomeric with a molecular
mass of 40 kDa .
Figure 1. Gel filtration chromatography of Exo 1, 4-β gluca-
Table 1. Purification summary of Exo 1, 4-β glucanase pro- duced by T. viridi.
Sr. No. Purification steps Volume (mL) Enzyme activity
(U/mg) Purification fold %
1 Crude Enzyme 200 82,400 535 154 1 100
2 (NH4)2SO4 Precipitation 25 10,875 65 167 1.08 13.2
3 Dialysis 20 9600 43 223 1.45 11.7
4 Sephadex-G-100 12 6420 10 642 4.17 7.8
M. Irshad et al. / Advances in Bioscience and Biotechnology 3 (2012) 580-584 583
Figure 2. SDS-PAGE of
purified Exo 1, 4-β glu-
canase produced from T.
Figure 3. Effect of pH on activity and stability of Exo 1, 4-β
Figure 4. Effect of temperature on activity and stability of Exo
1, 4-β glucanase.
3.3.3. Determination of Km and Vmax
Using varying concentrations of salicin as substrate, re-
sults obtained were plotted as activity against substrate
(µM). Lineweaver-Burk double reciprocal plot reflecting
substrate affinity and catalytic efficiency of present re-
ported Exo 1, 4-β glucanase with Km (76 µM) and Vmax
(240 U/mL) values as shown in the Figure 5. In this arti-
Figure 5. Lineweaver-Burk reciprocal plot: Km and Vmax for
Exo 1, 4-β glucanase.
cle and for the first time the kinetic constants of Exo 1,
4-β glucanase through Michalis-Menten transformation
Lineweaver-Burk double reciprocal plot has been inves-
T. viridi produces high titers of Exo 1, 4-β glucanase dur-
ing solid state bio-processing of an agro-industrial or-
ange peel waste material. In conclusion, the present re-
ported approach based on the bio-utilization and conver-
sion of agro-industrial orange peel waste into useful
products presenting a superior way out for proper waste
management for agro based waste materials.
On providing financial support and laboratory facilities, authors are
grateful to the Department of Biochemistry, NSMC, University of
Gujrat, Gujrat Pakistan.
 Iqbal, H.M.N., Ahmed, I., Zia, M.A. and Irfan M. (2011)
Purification and characterization of the kinetic parameters
of cellulase produced from wheat straw by Trichoderma
viride under SSF and its detergent compatibility. Ad-
vances in Bioscience and Biotechnology, 2, 149-156.
 Iqbal, H.M.N., Asgher, M., Ahmed, I. and Hussain, S.
(2010) Media optimization for hyper-production of car-
boxymethyl cellulase using proximally analyzed agro-
industrial residue with Trichoderma harzianum under
SSF. International Journal of Agro Veterinary and Medi-
cak Sciences, 4, 47-55.
 Stoilova, I., Krastanov, A. and Stanchev, V. (2010) Prop-
erties of crude laccase from Trametes versicolor pro-
duced by solid-substrate fermentation. Advances in Bio-
science and Biotechnology, 1, 208-215.
 Ahmed, I., Zia, M.A., Iftikhar, T. and Iqbal, H.M.N.
(2011) Characterization and detergent compatibility of
Copyright © 2012 SciRes. OPEN ACCESS
M. Irshad et al. / Advances in Bioscience and Biotechnology 3 (2012) 580-584
purified protease produced from Aspergillus niger by
utilizing agro wastes. BioRes, 6, 4505-4522.
 Asgher, M. and Iqbal, H.M.N. (2011) Characterization of
a novel manganese peroxidase purified from solid state
culture of Trametes versicolor IBL-04. BioRes, 6, 4302-
 Iqbal, H.M.N., Asgher, M. and Bhatti, H.N. (2011b) Op-
timization of physical and nutritional factors for synthesis
of lignin degrading enzymes by a novel strain of Tram-
etes versicolor. BioRes, 6, 1273-1287.
 Yin, L.J., Lin, H.H. and Xiao, Z.R. (2010) Purification
and characterization of a cellulase from Bacillus subtilis
YJ1. Journal of Marine Science and Technology, 18, 466-
 Iqbal, H.M.N., Ahmed, I. and Naveed, M.T. (2012) En-
hanced bio-catalytic and tolerance properties of an in-
digenous cellulase through xerogel immobilization. Ad-
vances in Bioscience and Biotechnology, in press.
 Yano, S., Ozaki, H., Matsuo, S., Ito, M., Wakayama, M.
and Takagi, K. (2012) Production, purification and char-
acterization of D-aspartate oxidase from the fungus Tricho-
derma harzianum SKW-36. Advances in Bioscience and
Biotechnology, 3, 7-13. doi:10.4236/abb.2012.31002
 Yeh, A.I., Huang, Y.C. and Chen, S.H. (2010) Effect of
particle size on the rate of enzymatic hydrolysis of cellu-
lose. Carbohydrate Polymers, 79, 192-199.
 Deshpande, M.V., Eriksson, K.E. and Göran-Pettersson,
L. (1984) An assay for selective determination of exo-1,
4,-β-glucanases in a mixture of cellulolytic enzymes.
Analytical Biochemistry, 138, 481-487.
 Asgher, M., Iqbal, H.M.N. and Asad, M.J. (2012) Kinetic
characterization of purified laccase produced from Tram-
etes versicolor IBL-04 in solid state bio-processing of
corncobs. BioRes, 7, 1171-1188.
 El-Gindy, A.A., Saad, R.R. and Fawzi, E. (2003) Purifi-
cation and some properties of exo-1,4-beta-glucanase from
Chaetomium olivaceum. Acta Microbiologica Polonica,
 O’Connell, E., Piggott, C. and Tuohy, M. (2011) Purifi-
cation of exo-1,3-beta-glucanase, a new extracellular glu-
canolytic enzyme from Talaromyces emersonii. Applied
Microbiology and Biotechnology, 89, 3685-3696.
 Dharmawardhana, D.P., Ellis, B.E. and Carlson, J.E. (1999)
cDNA cloning and heterologous expression of coniferin
β-glucosidase. Plant Molecular Biology, 40, 365-372.
Copyright © 2012 SciRes. OPEN ACCESS