Advances in Bioscience and Biotechnology, 2013, 4, 941-944 ABB
http://dx.doi.org/10.4236/abb.2013.410124 Published Online October 2013 (http://www.scirp.org/journal/abb/)
Characterization of purified β-glucosidase produced from
Trichoderma viride through bio-processing of orange peel
waste
Muhammad Irshad1*, Zahid Anwar1, Muhammad Ramzan2, Zahed Mahmood2, Haq Nawaz3
1Department of Biochemistry, NSMC, University of Gujrat, Gujrat, Pakistan
2Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad, Pakistan
3Institute of Animal Nutrition & Feed Technology, University of Agriculture, Faisalabad, Pakistan
Received 22 June 2013; revised 23 July 2013; accepted 12 August 2013
Copyright © 2013 Muhammad Irshad et al. This is an open access article distributed under the Creative Commons Attribution Li-
cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
In the present study, solid state fermentation was car-
ried out using orange peel waste to produce β-gluco-
sidase from Trichoderma viride. A locally isolated
fungal strain T. viride was cultured in the solid state
medium of orange peel (50% w/w moisture) under
optimized fermentation conditions and maximum act-
ivity of 515 ± 12.4 U/mL was recorded after 4th day of
incubation at pH 5.5 and 30˚C. Indigenously pro-
duced β-glucosidase was subjected to the ammonium
sulfate precipitation and Sephadex-G-100 gel filtra-
tion chromatography. In comparison to the crude ex-
tract β-glucosidase was 5.1-fold purified with specific
activity of 758 U/mg. The enzyme was shown to have
a relative molecular weight of 62 kDa as evidenced by
sodium dodecyl sulphate polyacrylamide gel electro-
phoresis. The purified β-glucosidase displayed 6 and
60˚C as an optimum pH and temperature respect-
ively.
Keywords: Orange Peel Waste; β-Glucosidase; T. viride;
Purification; SDS-PAGE; Characterization
1. INTRODUCTION
The major components of plant cell walls are cellulose,
hemicellulose and lignin and among all of them, cellu-
lose is about 35% to 50% which is the most common and
most abundant component of all plant matter [1]. Among
many of the developing countries, it’s a routine practice
that such agricultural wastes have not been fully dis-
carded, which has become a major source of pollution. A
large variety of micro-organisms including Trichoderma,
Aspergillus, Penicillium, and Fusarium have the ability
to produce enzymes like cellulases, and under mild fer-
mentation environment to hydrolyze insoluble polysac-
charides to soluble sugars [1,2]. Trichoderma is one of
the most efficient cellulases producer organisms which is
being studied for the production of cellulose degrading
enzymes. Cellulose degrading enzymes system is a com-
plex of three major enzymes that can be divided into
three main types: 1) Endoglucanase, 2) Exoglucanases,
and 3) β-glucosidase [1,3-5].
From the last few years, cellulase is being used in
many of the industrial applications, especially in the field
of cotton processing; paper recycling, and animal feed
additives animal feed industry, agriculture as well as in
the field of research and development [4,5]. One of the
potential applications of cellulase is the production of
fuel ethanol from lignocellulosic biomass. The most pro-
mising technology for the conversion of the lignocellu-
losic biomass to fuel ethanol is based on the enzymatic
breakdown of cellulose using cellulase enzymes [6,7].
Pakistan is an agricultural land that produced abundant
magnitude of agricultural wastes that can be utilized for
the production of useful industrial enzymes. Enzymatic
hydrolysis of such wastes is one of the attractive solu-
tions of this problematic issue that also provides an en-
vironmentally friendly means of depolymerizing cellu-
lose and other carbohydrates at high yields. With respect
to the factors affecting culture conditions, productivity
and properties of enzymes (cellulase complex), it was
considered of significance to purify and characterize this
enzyme through kinetic studies to explore such factors.
Keeping in mind the broad range of industrial applica-
tions of cellulases, this study was performed to purify
and characterize the β-glucosidase from T. viridi to pre-
sent its potential application for industrial application.
*Corresponding author.

M. Irshad et al. / Advances in Bioscience and Biotechnology 4 (2013) 941-944 942
The present study was also focused on providing a po-
tential solution for the management of large magnitude
of solid wastes.
2. MATERIALS AND METHODS
2.1. Agro-Industrial Substrate
Agro-industrial waste orange peel was obtained from
local fruit market, Gujrat, Pakistan. The collected sub-
strate crushed into pieces, oven dried and finally ground
to fine particle size before to use.
2.2. Fungal Culture and Inoculum Development
The pure culture of T. viride was obtained from the De-
partment of Biochemistry University of Gujrat, Pakistan.
A homogeneous 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 the
potato dextrose broth at 15 lbs/inch2 pressure and 121˚C
for 15 min, and incubated under stationary conditions for
the development of fungal spore suspension.
2.3. Pretreatment of Agro-Industrial Waste
10 g of moisture free orange peel was pretreated with 2%
HCl by adopting thermal treatment methodology as de-
scribed earlier [1]. After pretreatment the slurry of sub-
strate was filtered through four layers of muslin cloth,
residue were washed 4 to 5 times with distilled water to
remove extra acidity and used for production of β-glu-
cosidase under optimum fermentation conditions.
2.4. Solid-State Fermentation Strategy
Basel salt media was used to moist the pretreated orange
peel in an Erlenmeyer flask for β-glucosidase production.
The initial pH value of the medium was adjusted to 5
before sterilization at 121˚C and 15.0 lbs/inch2 pressure
for 15 min. The autoclaved medium was inoculated with
5 mL of freshly prepared fungal inoculum and incubated
at 30˚C ± 1˚C for stipulated fermentation time period.
2.5. Extraction of β-Glucosidase
β-glucosidase was extracted from the fermented biomass
by adding citrate buffer 0.05 M of pH 4.8 in 1:10 ratio
and the flasks were shaken at 120 rpm for 30 min. The
contents were filtered through muslin cloth and filtrates
were centrifuged at 10,000 g for 10 min. After that su-
pernatants were carefully collected and used to determine
enzyme activity and for purification purposes.
2.6. Determination of Activity & Protein
Contents
β-glucosidase activity was determined by the method of
Gielkens [8] while, the protein contents of the crude and
purified enzyme extracts were determined by following
the method of Bradford [9], with Bovine serum albumin
as standard.
2.7. Purification and SDS-PAGE of
β-Glucosidase
Crude extract of β-glucosidase obtained from T. viridi
was centrifuged (10,000 g) for 15 min followed by the
ammonium sulfate fractionation as described by Iqbal et
al. [1]. Total proteins and activity of partially purified β-
glucosidase were determined before and after dialysis of
ammonium sulfate precipitation. β-glucosidase was ly-
ophilized and subjected to gel filtration chromatography
using Sephadex-G-100 column [10]. The flow rate was
maintained at 0.5 mL·min−1 and up to 20 active fractions
were collected. To determine the molecular weight of
purified β-glucosidase SDS-PAGE was performed on a
5% stacking and a 12% resolving gel according to the
method of Laemmli [11].
2.8. Characterization of Purified β-Glucosidase
Characterization of purified β-glucosidase was performed
to investigate the effect of pH and temperature. β-gluco-
sidase was incubated in buffers of different pH (2 - 10),
followed by standard assay protocol. To determine the
thermal features β-glucosidase was incubated under dif-
ferent temperatures ranging from 25˚C to 70˚C for 1 h
time period followed by normal assay protocol as previ-
ously described.
3. RESULTS AND DISCUSSION
3.1. Production and Purification of
β-Glucosidase
A locally isolated fungal strain T. viride was cultured
under optimized fermentation conditions in the solid
state medium of orange peel (50% w/w moisture) and
maximum activity of 515 ± 12.4 U/mL was recorded
after 4th day of incubation at pH 5.5 and 30˚C. T. viridi
showed high levels of β-glucosidase production under
SSF as well as growth rate of cells. The eco-friendly pro-
cedure has been adopted to utilize low cost substrates to
induce enzymes production by T. viridi. The separated
cell free supernatant as crude enzyme solution containing
β-glucosidase with activity of 103,000 U/200 mL and
specific activity of 149 U/mg was subjected to partial
purification by ammonium sulfate precipitation. The cru-
de enzyme was maximally precipitated at 85% satura-
tion with specific activity of 208 U/mg and 1.4 fold puri-
fication. The optimally active fraction was loaded on
Sephadex G-100 column, and after gel filtration the en-
zyme was purified up to 5.1 fold with specific activity of

M. Irshad et al. / Advances in Bioscience and Biotechnology 4 (2013) 941-944 943
758 U/mg (Table 1). Previously Xue et al. [12] has also
used the Sephadex-G-100 gel filtration chromatographic
technique to purify β-glucosidase from R. flaviceps. β-
glucosidase was purified by gel filtration on a Sephadex
G-100 column [13].
3.2. SDS-PAGE
β-glucosidase was further purified to homogeneity and to
confirm its purity, the purified enzyme was resolved on
5% stacking and 12% running gel and found to be a ho-
mogenous monomeric protein as evident by single band
corresponding to 62 kDa on SDS-PAGE (Figure 1). The
similarity in the molecular weights determined by dena-
turing SDS-PAGE suggested that β-glucosidase was
likely to be monomeric, as reported earlier by Kang et al.
[14]. Another study, conducted by Xue et al. [12] β-
glucosidase from R. flaviceps was purified to homogene-
ity by SDS-PAGE with a molecular mass of 93.6 kDa
while, β-glucosidase from Aspergillus glaucus (92.5 kDa)
[13].
[Lane M, Standard protein markers with
molecular weights in kDa; Lane 1 & 2,
purified β-glucosidase]
Figure 1. SDS-PAGE of purified β-
glucosidase produced from T. viridi.
3.3. Characterization of Purified β-Glucosidase
3.3.1. Effect of pH on β-Glucosidase
The pH-activity profile showed that β-glucosidase was
optimally active at a pH 6 (Figure 2). A further increase
in pH showed a sharp decreasing trend. The purified β-
glucosidase was stable in a large pH range (4.0 to 7.0)
for up to 1 h incubation time period. Earlier studies re-
ported optimum activities of β-glucosidase from different
enzyme sources in the pH range 5 to 6 [15]. Verma et al.
[16] reported that optimum pH of β-glucosidase was in
the range of 4.5 to 5.0 while, Xue et al. [12] reported that
β-Glucosidase was stable at pH ranging from 5.0 - 6.8.
Figure 2. Effect of pH on activity and stability of β-glucosi-
dase.
3.3.2. Effect of Temperature on β-Glucosidase
Figure 3 illustrated that the β-glucosidase from T. viridi
was heat-stable and optimally active up to 60˚C without
losing much of its original activity. A wide range of in-
dustrial applications required relatively high thermo-
stability as an attractive and desirable characteristic of an
enzyme [17,18]. In a recent study Verma et al. [16] re-
ported that thermal stability of enzyme β-glucosidase
was found to be 30˚C while, earlier reported β-Glucosi-
Figure 3. Effect of temperature on activity and stability of β-
glucosidase.
Table 1. Purification summary of β-glucosidase produced by T. viridi.
Sr. No. Purification Steps Volume (mL) Enzyme
Activity (IU)
Protein
Content (mg)
Specific
Activity (U/mg)Purification Fold % Yield
1 Crude Enzyme 200 103,000 690 149 1 100
2 (NH4)2SO4 Precipitation 25 14,375 69 208 1.4 13.9
3 Dialysis 20 12,360 47 263 1.8 12.0
4 Sephadex-G-100 12 8340 11 758 5.1 8.1

M. Irshad et al. / Advances in Bioscience and Biotechnology 4 (2013) 941-944 944
dase was stable above 30˚C and below 45˚C. In compari-
son the earlier reported the present β-glucosidase from T.
viridi was reasonably more stable and active for up to
one hour incubation at 60˚C that suggests its potential for
industrial applicability.
4. CONCLUSIONS
1) Bio-utilization and conversion of agro are based on
waste materials into useful products.
2) T. viridi produces high titers of β-glucosidase dur-
ing solid state bio-processing of an agro-industrial or-
ange peel waste material.
3) An extra thermo-stability feature of an indigenous T.
viride β-glucosidase suggests its potential for industrial
applicability and striking prospect for application of this
enzyme.
5. ACKNOWLEDGEMENTS
The authors are grateful to the Department of Biochemistry and Mo-
lecular Biology, University of Gujrat, Pakistan for providing financial
support and laboratory facilities.
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