Characterization of purified <i>β</i>-glucosidase produced from <i>Trichoderma viride</i> through bio-processing of orange peel waste

doi:10.4236/abb.2013.410124

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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

Email: *[email protected]

Received 22 June 2013; revised 23 July 2013; accepted 12 August 2013

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.

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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

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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.

REFERENCES

[1] 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. Advan-

ces in Bioscience and Biotechnology, 2, 149-156.

[2] Ilyas, U., Ahmed, S., Majeed, A. and Nadeem, M. (2012)

Biohydrolysis of Saccharum spontaneum for cellulase

production by Aspergillus terreus. African Journal of

Biotechnology, 11, 4914-4920.

[3] 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-

471.

[4] Iqbal, H.M.N., Kamal, S., Ahmed, I. and Naveed, M.T.

(2012) Enhanced bio-catalytic and tolerance properties of

an indigenous cellulase through xerogel immobilization.

Advances in Bioscience and Biotechnology, 3, 308-313.

http://dx.doi.org/10.4236/abb.2012.34044

[5] 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 Tri-

choderma harzianum SKW-36. Advances in Bioscience

and Biotechnology, 3, 7-13.

http://dx.doi.org/10.4236/abb.2012.31002

[6] 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 for Agro Veterinary and Med-

ical Sciences, 4, 47-55.

[7] Iqbal, H.M.N., Kyazze, G. and Keshavarz, T. (2013)

Advances in the valorization of lignocellulosic materials

by biotechnology: An overview. BioResources, 8, 3157-

3176.

[8] Gielkens, M.M.C., Dekkers, E., Visser, J. and De-Graaff,

L.H. (1999) Two cellobiohydrolase-encoding genes from

Aspergillus niger require D-xylose and the xylanolytic

transcriptional activator XlnR for their expression. Ap-

plied and Environmental Microbiology, 65, 4340-4545.

[9] Bradford, M.M. (1976) A rapid and sensitive method for

the quantitation of microgram quantities of protein utiliz-

ing the principle of protein-dye binding. Analytical Bio-

chemistry, 72, 248-254.

http://dx.doi.org/10.1016/0003-2697(76)90527-3

[10] Ahmed, I., Zia, M.A., Iftikhar, T. and Iqbal, H.M.N.

(2011) Characterization and detergent compatibility of

purified protease produced from Aspergillus niger by

utilizing agro wastes. BioResources, 6, 4505-4522.

[11] Laemmli, U.K. (1970) Cleavage of structural proteins

during the assembly of the head of bacteriophage T4.

Nature, 227, 680-685.

http://dx.doi.org/10.1038/227680a0

[12] Xue, Y.P., Jin, L.Q., Liu, Z.Q., Zhang, J.F. and Zheng,

Y.G. (2008) Purification and characterization of betaglu-

cosidase from Reticulitermes flaviceps and its inhibition

by valienamine and validamine. African Journal of Bio-

technology, 7, 4595-601.

[13] Ma, S.J., Leng, B., Xu, X.Q., Zhu, X.Z., Shi, Y. and Tao,

Y.M. (2011) Puriﬁcation and characterization of b-1,4-

glucosidase from Aspergillus glaucus. African Journal of

Biotechnology, 10, 19607-19614.

[14] Kang, S.K., Cho, K.K., Ahn, J.K., Bok, J.D., Kang, S.H.

and Woo, J.H. (2005) Three forms of thermo-stable lac-

tose-hydrolase from Thermus sp. IB-21: Cloning, expres-

sion, and enzyme characterization. Journal of Biotech-

nology, 116, 337-346.

http://dx.doi.org/10.1016/j.jbiotec.2004.07.019

[15] Dharmawardhana, D.P., Ellis, B.E. and Carlson, J.E.

(1999) cDNA cloning and heterologous expression of co-

niferin β-glucosidase. Plant Molecular Biology, 40, 365-

372. http://dx.doi.org/10.1023/A:1006226931512

[16] Verma, O.P. (2011) Isolation, purification and charac-

terization of ß-glucosidase from Rauvolfia serpentine.

Journal of Chemical Engineering & Process Technology,

2, 119. http://dx.doi.org/10.4172/2157-7048.1000119

[17] Asgher, M. and Iqbal, H.M.N. (2011) Characterization of

a novel manganese peroxidase purified from solid state

culture of Trametes versicolor IBL-04. BioResources, 6,

4302-4315.

[18] 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. BioResources, 6, 1273-1287.