Biofuels

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Purification and characterization of cellulase from

a novel isolate of Trichoderma longibrachiatum

Priyanka Pachauri, Aranganathan V., Sunil More, S. B. Sullia & Sudha

Deshmukh

To cite this article: Priyanka Pachauri, Aranganathan V., Sunil More, S. B. Sullia & Sudha
Deshmukh (2017): Purification and characterization of cellulase from a novel isolate of Trichoderma
longibrachiatum , Biofuels, DOI: 10.1080/17597269.2017.1345357

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Published online: 11 Jul 2017.

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BIOFUELS, 2017
https://doi.org/10.1080/17597269.2017.1345357

Purification and characterization of cellulase from a novel isolate of

Trichoderma longibrachiatum
Priyanka Pachauria, Aranganathan V.b, Sunil Morec, S. B. Sulliaa and Sudha Deshmukhd
a
Department of Biotechnology, Centre for Postgraduate Studies, Jain University, Bangalore 560011, India; bDepartment of Biochemistry,
Centre for Postgraduate Studies, Jain University, Bangalore 560011, India; cSchool of Basic and Applied Sciences, Dayanand Sagar
University, Bangalore 560078, India; dIndian Institute of Science Education and Research, Tirupati 517507, India

ABSTRACT ARTICLE HISTORY

In the present study, the isolation, purification and characterization of a complex enzyme, Received 11 October 2016
cellulase, was carried out using a new local isolate of the fungus Trichoderma longibrachiatum Accepted 19 May 2017
(KM274866) from wood chips, with a view to explore its utility in the biofuel industry. The KEYWORDS
fungus was grown on a selected natural substrate, sugarcane bagasse, based on cost Trichoderma longibrachiatum
considerations for enzyme production. The enzyme was purified 14.82 fold with a yield of (KM274866); cellulase,
25.8% and specific activity of 30 U/mg of protein. The molecular mass of the enzyme was found lignocellulose; purification
to be 67 § 1 KDa. The optimum pH was 4.8, but the enzyme was stable at a pH range of 3–6.
Optimum temperature was 45  C, but the stability range of the enzyme was 30–55  C. Metal
ions such as Ca2+, Na+, Mg2+, Zn2+ and Fe2+ enhanced enzyme activity. Triton X100 in the
medium resulted in a significant increase of enzyme activity compared to other group specific
reagents. KM and Vmax for the enzyme were found to be 0.121 mg/ml and 0.421 mmol/min,
respectively, against the substrate carboxy methyl cellulose. As the enzyme is from an
inexpensive source, it will be most useful in the preparation of bioethanol for the biofuel
industry.

Abbreviations These include both bacteria and fungi, with fungi

EDTA ethylenediaminetetraacetate
DTT dithiothreitol
PMSF phenyl methyl sulphonyl fluoride
SDS sodium dodecyl sulphate
SSF solid state fermentation
CMC carboxy methyl cellulose
Introduction
Lignocellulosic biomass such as sugarcane bagasse,
rice husk, straw, forestry waste and agricultural residue
are abundant in nature. It can be utilized as an inex-
pensive feedstock for the production of various
enzymes useful in industry, including those for bioe-
thanol production. Thus the agricultural and forestry
biomass which is surplus in fields and forests can be a
useful starting material for the production of several
value added products [1].
Lignocellulose is composed of cellulose, hemicellu-
lose and lignin [2]. The complex structure of the bio-
mass is a major barrier in its efficient conversion to the
simple sugars [3] needed for fermentation into ethanol.
Cellulose, which is a linear polymer of glucose, is made
up of cellobiose units with about 2000–27,000 glucose
residues [4,5]. It can be degraded by several microor-
ganisms capable of producing the enzyme cellulase.
being the most common source.
The genus Trichoderma is well known for the produc-
tion of cell wall degrading enzymes [6,7]. Trichoderma
species are the most sought after commercial source for
cellulase production [8,9]. Cellulases are enzymes, mostly
composed of endoglucanases, cellobiohydrolases and
b-glucosidases, currently used for hydrolysis of lignocellu-
losic biomass for bioethanol production [10]. Solid state
fermentation (SSF), a cost-effective method, can be
employed for this purpose with advantages such as low
capital cost, lower space requirement and the ease with
which the downstream process can be handled [11,12].
The objective of the present study was to look for effi-
cient cellulase producing fungal strains from local sources
which can be useful for industrial production of bioetha-
nol (biofuel) from an inexpensive starting material (sugar-
cane bagasse). The fungus in the present study was
isolated from wood chips on a lignocellulosic substrate
using SSF, and the enzyme has been characterized.
Materials and method
Isolation, screening and identification of
cellulolytic fungi
Fungi were isolated from wood chips collected from
Gandhi Krishi Vigyan Kendra (GKVK) campus,

CONTACT Sudha Deshmukh sudhadeshmukh@yahoo.com

© 2017 Informa UK Limited, trading as Taylor & Francis Group
2 P. PACHAURI ET AL.
Bangalore. The wood chips were immersed in distilled
water for 1 h and 0.1 ml of the liquid was inoculated
on to carboxy methyl cellulose (CMC) medium consist-
ing of 1% CMC, 0.5 g ammonium tartrate, 0.1 g
KH2PO4, 0.005 g MgSO4.7H2O, 0.1 g yeast extract,
0.0001 g CaCl2.2H2O, 1.6 g agar and 100 ml distilled
water. Fungi capable of producing cellulase were
screened by Congo red dye method and the isolates
were selected based on the width of zone of clearance
around the colonies on the fungal plates. The fungus
producing the broadest zone of clearance was selected
for the present work. Morphological and molecular
identification of the selected isolate was carried out
using ITS 1 sequencing. Sequence was submitted to
Gen Bank and accession number KM274866 was
obtained.
Substrate selection and preparation
Selecting the best substrate for the cultivation of the
fungus is a critical step for high enzyme yield. Different
lignocellulosic substrates such as ragi straw, rice straw,
pongamia cakes, neem cakes, pumpkin seeds, rice
husk, sawdust, ashgourd seeds and sugarcane bagasse
were screened for enzyme yield. Substrates were
washed in water, dried in sunlight for a week, crushed
and sieved through 1 mm mesh and used for further
studies.
Cellulase production and purification
The Erlenmeyer flask containing 40 g of substrate was
moistened with optimized minimal medium contain-
ing the following ingredients (g/l) – MnSO4: 0.05 g, FeS-
O4.7H2O: 0.05 g, CaCl2. 2H2O: 1.9 g, Lactose: 0.38 g, and
NH4Cl: 2 g. The pH was adjusted to 6.0 and the sub-
strate was autoclaved at 121  C for 45 min. After cool-
ing the substrate to room temperature, the flasks were
inoculated with 6 ml of fungal spore suspension (12 £
106 spores/ml). After an incubation period of 6 days,
the cells were harvested by centrifugation (8000 rpm
for 20 min at 4  C).
The cell-free clear supernatant was withdrawn and
purified using ammonium sulphate precipitation, ini-
tially with 0–40% and then 40–80% saturation at 4  C
with continuous stirring and left for 5 h. The precipi-
tated enzyme was centrifuged further at 8000 rpm for
20 min and re-dissolved in sodium citrate buffer (pH
4.8) and the sample was dialyzed against the same
buffer. The enzyme activity, protein content and spe-
cific activity were checked in both pellet and the super-
natant. After dialysis, the partially purified cellulase was
purified by gel filtration column chromatography using
Sephadex G-100, as per the method of Gomashe et al.
[13], with minor modifications. Acta prime GE FPLC col-
umn chromatography was used to elute the fractions
in 0.1 M acetate buffer (pH 5.5) with a flow rate of
1 ml/min. Ninety fractions of 1.5 ml each were col-
lected and stored at 4  C for further analysis. Protein
content, enzyme activity and specific activity were
determined for each fraction. The purified enzyme
sample was lyophilized using a freeze dryer (Model
LY3TTE, Snijders Scientific, Tilburg, Holland) which
served as the enzyme source for all further studies.
Enzyme assay and estimation of protein
Cellulase activity in the culture filtrate was determined
by filter paper assay (FPase). One unit of enzyme activ-
ity is defined as the amount of enzyme necessary to
release 1 mmol of glucose per ml per minute. Aliquots
of culture filtrate were added as enzyme source to
Whatman No. 1 filter paper strip immersed in 1 ml of
0.05 M sodium citrate buffer at pH 4.8. After incubation
at 50 § 2  C for 1 h, the amount of reducing sugar pro-
duced was determined by the dinitrosalicylic acid
(DNS) method [14,15]. Protein concentration was mea-
sured according to the method of Lowry et al. [16]
using BSA as a standard protein.
Molecular weight determination
Molecular weight of the enzyme was determined by
SDS-PAGE using appropriate protein markers of known
molecular weight. SDS PAGE was carried out with 8%
separating gel and 6% stacking gel stained with Coo-
massie blue. Cellulase molecular weight was quantified
in comparison with its mobility with that of the pro-
teins in the marker. Activity staining was also per-
formed to locate the cellulase in the gel. After running
the sample in polyacrylamide gel under similar condi-
tions, the gel was stained with 0.1% Congo red dye.
The gel was then de-stained with 1% NaOH. The activ-
ity was observed as a clearance zone around the band.
Enzyme characterization
Activity staining
For activity staining, 1% CMC (w/v) was added to 1%
agarose (w/v) dissolved in 25 ml of 0.05 M phosphate
buffer (pH 7.0), and poured on to the Petri-plates after
heating. Wells were punched and 0.05 ml enzyme solu-
tion was transferred to each well and incubated in a
moist chamber at 25  C for 24 h. The gel was stained
with 1% Congo red dye and the formation of a yellow
zone was an indication of cellulose degradation.
Effect of pH and temperature on enzyme production
The most suitable pH required for maximum cellulase
activity was determined by varying the pH from 3–9
using different buffer solutions. The optimal tempera-
ture was determined by checking the enzyme activity
at temperatures ranging from 5–50  C.

Effect of various metal ions and group specific
reagents on enzyme activity
The role of metal ions, if any, on the enzyme activity
was also checked. Metal ions such as Ca2+, Mg2+, Fe2+,
Na+, K+, Mn2+, Co2+, Hg2+, Cu2+, Fe3+ and Zn2+ were
tried at a concentration of 5 mM. Measurements were
carried out in the presence of different group specific
reagents like EDTA, SDS, b-mercaptoethanol, PMSF,
DTT, sodium azide and Triton X-100, pre-incubated in
the samples for half an hour. The activity was analyzed
by standard assay methods.
Determination of kinetic parameters
Purified enzyme was incubated with various concen-
trations of soluble pure CMC substrate in 4.8 mM
sodium citrate buffer pH 4.8, at temperature 45  C.
Kinetic parameters KM and Vmax were calculated by
Lineweaver-Burk plots.
Results and discussion
Cellulases are known to break down cellulose into
monomeric structures, i.e. glucose subunits. To test the
activity of the enzyme isolated from the fungus, pure
carboxymethyl cellulose (CMC, 1%) was used in plate
test. In the medium supplemented with the dye Congo
red and iodine, a zone of clearance was produced
around the fungal colony because of the enzyme
exuded. The width of the zone of clearance indicated
the ability of enzyme to hydrolyze cellulose. Work
done by Gohel et al. [17] suggests that the enzyme
degrades pure CMC and is known to show a degrada-
tion zone. The enzyme breaks down the polysaccha-
ride because of which the area surrounded by the
enzyme gets reduced to smaller monosaccharides. The
dye cannot bind effectively to the monosaccharide
Figure 1. Cellulose (1%) degradation using purified enzyme from Trichoderma longibrachiatum.
BIOFUELS 3
and which results in the formation of a clear zone. It
has been suggested by Gomashe et al. [13] that fungal
colonies showing wider zone of degradation are higher
cellulase producers. In the present study, Trichoderma
longibrachiatum (KM274866) proved to be the best cel-
lulose degrader among all the fungi tested, with maxi-
mum clearance zone as shown in Figure 1.
The phylogenetic relationship of the fungal isolate
showed 99% similarity with Trichoderma longibrachia-
tum. Hence it was considered as a new local isolate of
the species T. longibrachiatum. When the extent of cel-
lulase produced by Trichoderma longibrachiatum iso-
late on the natural substrate, i.e. sugarcane bagasse,
was compared with the amount produced in other
agricultural residues, the amount produced in bagasse
was the highest amongst all the substrates tried
(Figure 2). This is in conformity with the findings of Sin-
ghania et al. [18] and Cunha et al. [19] who reported
sugarcane bagasse to be the best inducer of cellulase
in fungi. Ojumu et al. [20] reported that cellulase activ-
ity varied with the substrate used for the cultivation of
the fungus.
The purification profile of cellulase by ammonium
sulphate purification and gel filtration chromatography
is shown in Table 1. There was a 14.82 fold increase in
the specific activity of the final purified enzyme. The
elution profile of cellulase from Trichoderma longibra-
chiatum by FPLC method is shown in Figure 3. The
sample showed one major peak at 23.8 ml/min and
one conductance peak at 24.69 ml/min.
The elution fractions were checked for purity of cel-
lulase by SDS-PAGE (Figure 4) and the molecular
weight of the enzyme was found to be 67 § 1 KDa.
The single band appearance on gel proves the purity
of cellulase. Yasmin et al. [21] have reported a molecu-
lar weight of 87 KDa for cellulase isolated from
4 P. PACHAURI ET AL.

Figure 2. Selection of substrate for cellulase production.

Table 1. Summary of purification.

Purification step Volume (ml) Protein (mg) Total activity (U) Specific activity (U/mg) Purification fold Yield (%)
Culture filtrate 600 1400 2900 2.07 1 100
Ammonium sulfate fraction 35 750 2100 2.8 1.351 72.4
Sephadex G-100 chromatography 5 25 750 30 14.82 25.8

Manual Run 1:10_UV Manual Run 1:10_Cond Manual Run 1:10_Fractions Manual Run 1:10_Inject

mAu

300

250

200

150

100

50

0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 min

Figure 3. Elution profile for cellulase. Red line corresponds to protein concentration, blue line indicates cellulase activity, x-axis is
the fraction number (0–70.0) and y-axis is absorbance at 280 nm.
 BIOFUELS 5

Figure 4. SDS-PAGE. (A) Purified sample, (B) Molecular weight marker, (C) Activity staining with Congo red dye.

Trichoderma viride, which is very high, and this proves Table 2. Effect of metal ions and group specific reagents on
that fungi also produce enzymes with high molecular cellulase activity.
weight. The molecular weight obtained in the present Metal Cellulase activity Group specific Cellulase activity
ions (U/ml) reagents (U/ml)
study compares well with the values reported in litera-
Control 4.222 EDTA 3.970
ture for cellulase from several bacteria and fungi grown Na+ 8.877 Triton X 100 5.131
on different substrates [22]. K+ 2.879 SDS 3.996
Mg2+ 8.586 b-mercaptoethanol 3.675
It can be seen from Figure 5a that the enzyme is sta- Mn2+ 3.725 PMSF 3.636
ble over a wide range of pH with a maximum activity at Co2+ 3.830 DTT 3.744
Zn2+ 8.295 Sodium azide 3.609
pH 4.8. Results of present study are in conformity with Pb2+ 3.566 Control 4.222
the results of Li et al. [23], who found that pH between Hg2+ 4.332
Cu2+ 4.861
4.8 and 5.0 was suitable for fungi for producing cellu- Fe2+ 6.631
lase. With regard to the effect of temperature, the cur- Fe3+ 4.280
rent enzyme expressed maximum activity at Ca2+ 9.110

temperature 45  C (Figure 5b). Findings of Fayyaz et al.
[24] who reported an optimum temperature of 40–
50  C for the enzyme from Planococcus citri are in con-
formity with our results. The enzyme was stable at 30–
55  C which could be a positive factor from the point
of view of its commercial production.
Table 2 shows the effects of different metal ions on
the activity of the enzyme. Metals such as Ca2+, Na+,
Mg2+, Zn2+ and Fe2+ had a positive effect on the cellu-
lase activity, while K+, Mn2+, Co2+, Pb2+, Hg2+, Cu2+ and
Fe3+ had an inhibitory effect. Ca2+ resulted in the high-
est effect on enzyme production, i.e. 9.11 U/ml.
Enhanced cellulase activity was previously reported by
Singh et al. [25], with MgSO4 and ZnSO4 while Wang
et al. [26] reported inhibition in the presence of K+ ions.
Table 2 shows the effects of group specific reagents
on enzyme activity. In the presence of Triton X 100,
enhanced enzyme activity resulted in 5.131 U/ml of
enzyme. Triton X100 could have increased the perme-
ability of cell membrane leading to better membrane
EDTA: Ethylene diamine tetraacetate; DTT: Dithiothreitol; PMSF: Phenyl-
methylsulphonylflouride; SDS: Sodium dodecyl sulphate
transport, and thus higher cellulase activity. However,
the reagent EDTA showed negative effect on cellulase
activity. EDTA is a metal chelating agent and reduction
in enzyme activity suggests that activity may depend
on certain metal chelators which are required for spe-
cific chemical reactions. It is possible that it may
involve inorganic groups which are inactivated due to
EDTA. SDS, b-mercaptoethanol, PMSF, DTT and sodium
azide showed no significant effect on cellulase activity.
Similar observations were made by Callow et al. and
Danmek et al. [27,28].
The kinetic constants obtained from a Lineweaver-
Burk plot (Figure 6) for cellulase from Trichoderma long-
ibrachiatum suggested high affinity of the enzyme for
its substrate. KM and Vmax values for the enzyme were
found to be 0.121 mg/ml and 0.421 mmol/min,
6 P. PACHAURI ET AL.
Figure 5. Effect of (a) pH and (b) temperature on cellulase activity.
Figure 6. Lineweaver-Burk plots of cellulase activity at differ-
ent CMC concentrations (mg/ml) for Trichoderma longibrachia-
tum. The velocity v of product formation on the y-axis is given
in U/ml.
respectively. The difference in the KM value of the pres-
ently purified cellulase from Trichoderma longibrachia-
tum and other reported fungi may be due to the
genetic variability [29].
Conclusions
Screening of woodchips for cellulase producing fungi
resulted in a very efficient fungus isolate, identified as
Trichoderma longibrachiatum, which is a new isolate of
the species which is locally available. The search for a
natural and inexpensive substrate for the cultivation of
the fungus among locally available agricultural sources,
for the purpose of cellulase production, led us to

sugarcane bagasse which is a lignocellulosic agricul-
tural residue. In the present study, sugarcane bagasse
was used as the sole carbon source for the cultivation
of the fungus. Cellulase produced from this new fungal
isolate showed all the characteristics of an efficient
enzyme with high substrate affinity and high cellulo-
lytic activity. The enzyme had an optimum pH of 4.8,
with enzyme stability over the range 3–6. The optimum
temperature was 45  C, with a tolerance range of 30–
55  C. The optimum pH and temperature are most suit-
able for the industry to mass produce the fungus.
Metal ions and the detergent Triton 100 improved the
enzyme activity. The purified enzyme had a molecular
weight of 67 § 1, KM 0.121 mg/ml, and Vmax
0.421 mmol/min. Its specific activity was 30 U/mg of
protein.
The above characteristics of the enzyme make it
very useful in commercial production of bioethanol
(biofuel) from an inexpensive starting material (sugar-
cane bagasse).
Disclosure statement
No potential conflict of interest was reported by the authors.
References
[1] Saini JK, Saini R, Tewari L. Lignocellulosic agriculture
wastes as biomass feedstocks for second-generation
bioethanol production: concepts and recent develop-
ments. 3 Biotech. 2015;5(4):337–353.
[2] Sjostrom E. In: Wood chemistry: fundamentals and
applications. USA: Gulf professional publishing; 1993.
[3] Himmel ME, Ding SY, Johnson DK, et al. Biomass recalci-
trance: engineering plants and enzymes for biofuels
production. Sci. 2007;315(5813):804–807.
[4] Delmer DP, Amor Y. Cellulose biosynthesis. Plant Cell.
1995;7(7):987–1000.
[5] Hon DNS, Shiraishi N. In: Wood and cellulosic chemistry,
revised, and expanded. USA: CRC Press, Taylor and fran-
cis; 2000.
[6] Ashfaque M, Solomon S, Pathak N. Kinetic study of
immobilized cellobiase produced from immobilized
wild-type trichoderma longibrachiatum. Sugar Tech.
2016;18(4):340–346.
[7] Singh A, Srivastva M, Pathak N, et al. Evaluation of anta-
gonastic activity and shelf life study of Trichoderma vir-
ide (01PP-8315/11). Adv Life Sci. 2012;1(2):138–140.
[8] Pandey S, Srivastava M, Shahid M, et al. Trichoderma
species cellulases produced by solid state fermentation.
J Data Mining Genom Proteom. 2015;6:170.
[9] Van Peij NN, Gielkens MM, De Vries RP, et al. The tran-
scriptional activator xlnR regulates both xylanolytic and
endoglucanase gene expression in Aspergillus niger.
Appl Environ Microbiol. 1998;64(10):3615–3619.
[10] Vitikainen M, Arvas M, Pakula T, et al. Array comparative
genomic hybridization analysis of Trichoderma reesei
strains with enhanced cellulase production properties.
BMC genomics. 2010;11(1):441.
[11] Bai H, Wang H, Sun J, et al. Production, purification and
characterization of novel beta glucosidase from newly
BIOFUELS 7
isolated Penicillium simplicissimum H-11 in submerged
fermentation. EXCLI J. 2013;12:528–540.
[12] Neagu D, Destain J, Thonart P, et al. trichoderma reesei
cellulase produced by submerged versus solid state fer-
mentations. Bull UASVM Agric. 2012; 69(2): 320–326.
[13] Gomashe AV, Gulhane PA, Bezalwar PM. Isolation and
screening of cellulose degrading microbes from nagpur
region soil. Int J lif Sci. 2013;1:291–293.
[14] Miller GL. Use of dinitrosalicylic acid reagent for determi-
nation of reducing sugar. Anal Chem. 1959;31(3):426–428.
[15] Das A, Bhattacharya S, Roopa KS, et al. Microbial utiliza-
tion of agronomic wastes for cellulase production by
Aspergillus niger and Trichoderma viride using solid state
fermentation. Dyn Biochem Proces Biotech Mol Biol.
2011;5:18–22.
[16] Lowry OH, Rosebrough NJ, Farr AL, et al. Protein mea-
surement with the Folin phenol reagent. J Biol Chem.
1951;193:265–275.
[17] Gohel HR., Contractor CN, Ghosh SK, et al. A compara-
tive study of various staining techniques for determina-
tion of extra cellular cellulase activity on Carboxy
Methyl Cellulose (CMC) agar plates. Int J Curr Microbiol
App Sci. 2014;3:261–266.
[18] Singhania RR, Sukumaran RK, Pillai A, et al. A. solid-state
fermentation of lignocellulosic substrates for cellulase
production by Trichoderma reesei NRRL 11460. Indian J
Biotechnol. 2006;5(3):332–336.
[19] Cunha FM, Esperanca MN, Zangirolami TC, et al. Sequen-
tial solid-state and submerged cultivation of Aspergillus
niger on sugarcane bagasse for the production of cellu-
lase. Bioresource Technol. 2012;112:270–274.
[20] Ojumu TV, Solomon BO, Betiku E, et al. Cellulase produc-
tion by Aspergillus flavus linn isolate NSPR 101 fer-
mented in sawdust, bagasse and corncob. Afr J
Biotechnol. 2003;2(6):150–152.
[21] Yasmin S, Mattoo RL, Nehvi FA. Isolation, characterization
and molecular weight determination of Cellulase from Tri-
choderma viride. Afr J Biotechnol. 2013;12(28):4512–4518.
[22] Rizzatti ACS, Jorge JA, Terenzi HF, et al. Purification and
properties of a thermostable extracellular b-D-xylosi-
dase produced by a thermotolerant Aspergillus phoeni-
cis. J Ind Microbiol Biotechnol. 2001;26(3):156–160.
[23] Li XH, Yang HJ, Roy B, et al. Enhanced cellulase produc-
tion of the Trichoderma viride mutated by microwave
and ultraviolet. Microbiol Res. 2010;165(3):190–198.
[24] Fayyaz-ur-Rehman M, Tariq MI, Aslam M, et al. Inhibition
studies of cellulolytic activities isolated from Planococ-
cus citri. Open Enz Inhib J. 2009;2:8–11.
[25] Singh A, Gupta P, Wadhwa N. Properties of cellulolytic
enzymes from peel of Amorphophallus paeonifolius. Int J
Pharm Pharmace Sci. 2014;6:333–336.
[26] Wang G, Zhang X, Wang L, et al. The activity and kinetic
properties of cellulases in substrates containing metal
ions and acid radicals. Adv Biol Chem. 2012;2:390–395.
doi: 10.4236/abc.2012.24048
[27] Callow NV, Ju LK. Promoting pellet growth of Tricho-
derma reesei Rut C30 by surfactants for easy separation
and enhanced cellulase production. Enzyme Microb
Technol. 2012;50(6):311–317.
[28] Danmek K, Intawicha P, Thana S, et al. Characterization
of cellulase producing from Aspergillus melleus by solid
state fermentation using maize crop residues. Afr J
Microbiol Res. 2014;8(24):2397–2404.
[29] Ijaz A, Anwar Z, Irshad M, et al. Purification and kinetic
characterization of statistically optimized cellulase pro-
duced from Aspergillus niger. Romanian Biotechnol Lett.
2014;19(6):9835–9845.