International Journal of Farming and Allied Sciences
Available online at www.ijfas.com
©2014 IJFAS Journal-2014-3-5/543-554/ 31 May, 2014
ISSN 2322-4134 ©2014 IJFAS
Gamma and UV radiation induced mutagenesis in
Trichoderma reesei to enhance cellulases enzyme
activity
Samira Shahbazi1*, Khodayar Ispareh2, Mahsa Karimi2, Hamed Askari1 and Mohammad
ali Ebrahimi1
1. Nuclear Agriculture Research School, Nuclear Science and Technology Research Institute (NSTRI),
Atomic Energy Organization of Iran (AEOI), P. O. Box: 31485-498, Karaj, Iran
2. Biotechnology Department, Faculty of Agriculture, Peyam e Noor University, P. O. Box 31578-36899,
Tehran, Iran
Corresponding author: Shahbazi samira
ABSTRACT: The need for renewable energy resources has focused attention on the renewable
polysaccharide cellulose, which can be enzymatically hydrolyzed to yield sugars. The filamentous fungus
Trichoderma reesei is an important host organism for industrial enzyme production producing large
amounts of hydrolytic enzymes. The aim of the present study was to improve the cellulase production of
T. reesei through a successive mutagenesis step. Trichoderma reesei (PTCC 5142) a cellulolytic
microorganism, was subjected to mutagenesis using gama and UV radiation individually as mutagens.
Successive mutants showed rapid growth on plates containing MYG medium, clear hydrolysis zone on
mandels medium and enhanced cellulase production. The mutant strains of T. reesei were isolated and
selected by mandel screening method. Results showed some mutant strains has had higher cellulases
activities in comper with parent strain. A mutant strain of Trichoderma reesei (PTCC 5142) with
maximum production potential of cellulase was finally selected. The developed mutant from gama
mutagenesis way is the best mutant for Ccase, CMCase, Avicellase and Fpase activities. This new
method could be applied to obtain potent fungal mutants for more enzymes production.
Keywords: Trichoderma reesei, cellulase, Gamma rays, Ultraviolet, Mutagenesis
INTRODUCTION
Agricultural wastes and in fact all lignocellulosics can be converted into products that are of commercial
interest such as ethanol, glucose and single-cell protein (Solomon, 1999). Cellulase enzymes have been reported
(Fan, 1987; Wu and Lee 1997; Kansoh, 1999; Solomon, 1999) for the bioconversion of lignocellulosics to useful
products.
The cellulolytic filamentous fungus Trichoderma reesei is one of the most widely investigated organisms for the
production of hydrolytic enzymes for applications in the food, feed, textile and pulp and paper industries
(Nevalainen, 1991; Nevalainen and Penttil, 1995). Furthermore, it has ability to secrete gram amounts of proteins
outside the cell, together with an eukaryotic post-translational protein (Uusitalo, 1988). The extracellular cellulolytic
system of T. reesei is composed of 60–80% cellobiohydrolases or exoglucanases (EC3.2.1.74), 20–36% of
endoglucanases (EC3.2.1.4) and1% of β-glucosidases (EC3.2.1.21), all of which act synergistically in the
conversion of cellulose into glucose (Chandra et al 2009, Gadgil , 1995).
Intl J Farm & Alli Sci. Vol., 3 (5): 543-554, 2014
It is a now subject of intensive research to the development of a large scale conversion process beneficial to
mankind (Kumakura 1997).
The strains have been mutagenized and genetically modified to obtain an organism capable of producing high
levels of cellulases (Mandels and Andreotti 1978; Nevalainen, 1980; Durand, 1988; Szengyel, 2000). The objective
of these efforts has been to obtain high levels of cellulases in order to degrade waste cellulose. However, despite
the effort of many laboratories, no commercially efficient enzyme complex has been produced. The high cost of
enzyme production limits the industrial use of the enzymes in the production of soluble sugars. In addition, the
Trichoderma cellulase systemis deficient in cellobiase, causing the accumulation of the disaccharide cellobiose,
which produces repression and end product inhibition of the enzymes, both of which limit enzyme synthesis and
activity. As a result, production of glucose from waste cellulose is still not commercially feasible. To resolve these
problems, many researches have been involved in isolating new microorganisms to produce cellulolytic enzymes
with higher specific activity and greater efficiency(Ståhlberg 1991). The bioconversion of cellulosic materials has
been receiving attention in recent years. Random mutagenesis has been applied to improve cellulolytic activity of
the strain for its industrial application. The creation of different mutant strains with several-fold increase in the
amount of secreted cellulolytic enzymes compared to the wild-type strain has been achieved by both academic and
industrial research programs (Atalla, 1998, Barr, 1996). In this study effects of induced mutation via gamma
irradiated with 250 Gry and UV irradiation as physicale mutagens on levels of cellulytic enzymes production in
Trichoderma reesei have been evaluated.
MATERIALS AND METHODS
Microorganism
Trichodermu reesei (PTCC 5142) was obtained from the Bacteri and fungus collection of Industrial and
scientific researchs center of Iran. It was maintained on slants of potato- dextrose agar (PDA) and subcultured at
monthly intervals.
Mutagenesis
Two methods of mutations were employed for strain improvement:
Mutagenesis with gamma rays treatment
In this way, mutagenesis was carried out using gamma rays produced from Cobalt-60 (Co60) as a source of
gamma radiation(by gama cell set in Agricultural and Medical Research School- Nuclear Science and Technology
Institute, Karaj).
Selection of optimum dose for mutation
Seven different doses of gamma radiation were evaluted, which were 0, 50, 1500, 200,250, 300, 350, 400, 450
Gray. The 250 Gry dose were selected as optimum dose for Trichoderma mutagenesis.
Mutagenesis with ultraviolet treatment
Ultraviolet treatment (power: 20 W, wavelength:254 nm) was given to the spore suspension for 20 min with a
distance of 15 cm from the UV light to the screening medium plated with the single-spore suspension. 5 ml spore
7
suspension of isolate wild Trichoderma after counting the number of spores (1.4* 10 ) in plate poured into 15 cm
from the source of ultraviolet light for 20 min placed., After treatment, mutagenic, and culture MYG number 23
germinated spores that were different in terms of appearance with the rest have passed away MYG medium.
Isolation of mutanted isolates
º
After mutagenesis treatment, the spore suspension were plated on MYG agar medium and incubated at 28 C
for 48 h. Then measured morphological traits as growth rate, and spore production scale. Screening of mutants
was carried by the procedure of Mandels et al (1974). Mandels medium containing Urea 0.3 g/l, (NH4)2 SO4 1.4
g/l,, KH2PO4 2.0 g/l, CaCl2.2H2O 0.4 g /l, MgSO4.7H2O 0.3 g/l, Peptone 0.75 g/l, Yeast extract 0.25 g/l, Colloidi
Cellulose 2.0 g/l, FeSO4.7H2O 5.0 mg/l, MnSO4.7H2O 1.6 mg/l, ZnSO4.7H2O 1.4 mg/l, CoCl2.6H2O 2.0 mg/l. and
the pH was adjusted to 4.8.
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Screening of mutanted isolates
-1
This medium contains Mandels mineral salts solution(Mandels et al. 1974) with the addition of 17.5 g l of
-1
-1
agar, 10 g l of phosphoric acid-swollen cellulose (Tansey1971), 0.1% (v/v) Triton X-100 and 4 g l of sorbose an
inhibitor of P–l, 3-glucan biosynthesis, thus enabling a large number of strains to be screened. Plates were
°
°
incubated at 28 C for 3 days followed by 18 h at 50 C. Hypercellulolytic mutants were selected on the basis of the
diameter of the hydrolysis zone surrounding the colonies. Based upon the ratio between the clearing zone diameter
and colony zone diameter, 28 mutant isolates from mutations.This isolates mutant were chosen for enzyme
production. These mutant strains were used for the fermentation and for checking their cellulase production
abilities.
Swollen cellulose (PASC) production
Phosphoric acid swollen cellulose (PASC) was used as the main carbon source since it is known to induce the
production of cellulase enzyme. Colloidal cellulose was produced by pretreatment of microcrystalline cellulose
(Avicel) in ortho-phosphoric acid 85% (w/v) for 24 h at 4 ˚C to increasing of colloidal property in cellulose so that it
could easily be accessed by enzymes. After acid pretreatment, the materials were filtered through cheese cloth
filter. Then, the solid fraction was thoroughly washed by distilled water to neutralize the pH (~5.0), freezed at -70 ˚C
for 24 h, freeze dried for48 h and milled to mesh size 53-125 micron.
Cellulase production
Trichoderma strains were maintained on agar media (MYG agar medium) containing(g.l-1): malt extract, 5;
yeast extract, 2.5; glucose, 10; agar, 20. Washed spore suspensions were prepared from seven-day-old slant
7
cultures in sterile saline solution and used as an inoculum of 1×10 spores/ml of medium. The spores were pelleted
by centrifugation at 4000×g for 10 min, and washed twice in sterile saline solution. Seed cultures were produced in
Trichoderma complete medium (TCM) which contained(g.l-1): bactopeptone, 1.0; urea, 0.3; KH2PO4, 2.0;
(NH4)2SO4, 1.4; MgSO4 .7H2O, 0.3; CaCl2 .6H O, 0.3; FeSO4 .7H2O, 0.005; MnSO4, 0.002; ZnSO4, 0.002;
CoSO4.7H2O, 0.002 and 2 ml.l-1, Tween 80. The medium was adjusted to pH 4.8 and supplemented with 0.3%
w/v of glucose. Cultures were produced in 50 ml volumes of TCM in250ml Erlenmeyer flasks shaken at 180 rpm at
28 ˚C for 24 h. To induce production of cellulase enzymes washed mycelium was transferred to 25ml of
-1
Trichoderma fermentation medium (TFM) which contained (g.l ): urea, 0.3; KH2PO4, 2.0; (NH4)2SO4, 1.4; MgSO4
.7H2O, 0.3; CaCl2 .6H O, 0.3; FeSO4 .7H2O, 0.005; MnSO4, 0.002; ZnSO4, 0.002; CoSO4.7H2O, 0.002 and 2
ml.l-1, Tween 80. This medium was adjusted to pH 4.8 and supplemented with 0.5% w/v of Phosphoric Acid
Swollen cellulose (PASC). Growth conditions were as described previously. Triplicate flasks were harvested after
48 h. Estimation of protein and extracellular cellulase activitywere harvested after 48 h. Estimation of protein and
extracellular cellulase activity was assayed in Trichoderma fermentation medium after centrifugation at 4000 ×g
for10 min at 4 ˚C.
Analytical methods
Estimation of protein
The protein content in the TFM supernatant after 48 h fermentation were estimated by the dye binding method
of Bradford (1976). The test was carried out using 150 µl of supernatant of TFM and 3 ml of Bradford reagent. The
absorbance was read at 595 nm on a spectrophotometer (Jenway, USA).
Enzymatic assays
Enzymatic activities were measured in culture supernatants obtained after centrifugation. Overall cellulase
activity of the samples was determined as filter paper activity (FPase) using Whatman no.1 filter paper strips as the
substrate [50]. Endoglucanase activity was measured as CMCase activity with CMC (carboxymethyl cellulose)
dissolved in 50 mM citrate buffer (pH 5.0) as the substrate. The assay was performed for 30 min at50°C. The
amount of released sugar was assayed via the dinitrosalicylic acid (DNS) method using glucose as the standard.
FPase
Filter paper activity was determined as described by Mandels, We added 1 ml of appropriately diluted culture
supematant to2 ml of 0.1 M citrate buffer (pH 4.8) containing 50 mg of Whatman no. 1 filter paper (1 . 6 cm). After
°
incubation for 1 h at 5O C, the reaction was terminated by adding 3 ml of 3,5-dinitrosalicylic acid (DNS) reagent
and boiling for 5 min. We added 1 ml of 40% sodium-potassium tartarate to the hot solution and, after cooling
absorbance was measured at 540 nm.
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CMCase
CMCase activities was determined according to Saddler.12 We incubated 1 ml of appropriately diluted enzyme
extract with 1 ml of 1% carboxymethyl cellulose and 1 ml of 0.1 M citrate buffer, pH 4.8. at 50°C for 30 min. The
reaction was terminated by adding 3 ml of 3,5-dinitrosalicylic acid (DNS). The mixtures were also mixed well, then
placed into a boiling-water bath for 5min, and cooled with cooled water temperature. The absorbance of the
reaction solutions was measured at 540 nm. One unit of CMCase, activity was expressed as 1 µmol glucose
-1
equivalents released min .
Avicelase, Cloied cellulase and Cellubiase
Avicelase, Cloied cellulase (CCase) and Cellubiase(Cease) activity were determined by measuring the amount
of glucose released from substrates by the DNS method with glucose as the standard. The reaction mixtures
contained 0.5 ml of 5 g.L-1 Avicel, CE and CC cellulase in 0.05M citrate buffer (pH 4.8) and 0.5ml of each
supernatant of TFM medium. After incubation at 50 ˚C for 60min, the controls and samples were taken out of the
50 ˚C bath. The International unit(IU) of activity is defined as the amount of enzyme that liberates l µmol of glucose
per hours in a standard assay (Gama & Mota, 1998).
RESULTS AND DISCUSSION
Mutation optimazation and muatants isolation
Irradiated specimens: comparison of spore germination after 24 hours of irradiation with different doses
showed that irradiation with gamma rays at 250 Gy dose range, no conflict for hyphal growth and %44.6
germination of fungal spores and their ability have held that the optimum dose to induce mutations, selection and
mutant strains were obtained from the dose.
Screening of gamma irradiated muatnts
Mutant screened by comparing ratio of clearing zone diameter (mm) appered in a Mandels medium containing
colloedei cellulose in all mutated T. reesei . Quality evaluation mutants was carry out by culture Mandels medium
modified based on the ratio of the clearing zone diameter. The ratio of the clearing zone diameter(cm) produced by
the fungus mutated T.reesei via gama radiation on medium Mandels containing cellulose colloid was compared.
Based on this analogy, mutant strains, T.r M21 and T.r M19 were shown a greater clearing zone diameter from
control strain.
Figure 1. Clearing zone diameter (mm) appered in a Mandels medium containing colloedei cellulose in T. reesei ,T.r M21 and
T.r M19 (left to right)
Figure 2. Comparison of the clearing zone diameter of control and mutant isolates (mutated via gama radiation) cultured in
Mandels medium
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Isolate T.r M21 strain with 15.9 mm in clearing zone diameter compared to the mycellium, were the best and
T.r M1 strain with 13.3 mm in clearing zone diameter had least diameter of clearing zone. Results statistic analysis
using software SPSS version 13 and one-way ANOVA was performed. To compare the mean using Duncan
multiple range test statistic p <0.05 was used.
Screening of UV irradiated muatnts
The ratio of the clearing zone diameter(mm) produced by the fungus mutated T.reesei via UV on medium
Mandels containing cellulose colloid was compared. Based on this analogy, mutant strains TR-UV21, TR-UV8, TRUV17, and TR-UV3, were shown a greater clearing zone from control and another strains, also they had significant
statistic in 0.05 level with control strain to isolate. isolate TR-UV21 strain with 16.4 cm in clearing zone diameter
compared to the mycellium, were the best and TR-UV16 strain with 12.5 cm in clearing zone diameter had smallest
diameter of clearing zone.
Based on the results, selected seven mutant strains TR.uv1, TR.uv6, TR.uv8, TR.uv11, TR.uv17, TR.uv21,
TR.uv23 for the next stage of assessment (measurement of enzyme and protein). Mutant enzyme activity
compared to controls is shown in Table 1.
Figure 3. Clearing zone diameter (mm) appered in a Mandels medium containing colloedei cellulose in T. reesei , TR-UV21 and
TR-UV16 (left to right)
Figure 4. Comparison of the clearing zone diameter of control and mutant isolates (mutated via UV) cultured in
Mandels medium
Cellulase enzymes activity of selected mutants
There are two basic methods for measuring cellulase activities; measurement of all cellulase activity
measurement, and evaluation activities of each component is cellulase systems (Zhang, 2006). Based more
cellulases measure by measurement of reducing sugers following the hydrolysis reaction (Anderson: 2007).
Cellulase activity in mutated T. reesei via gama radiation
after mutagenic treatment, the activity rate optimization method for measuring the activity of fungi cellulases
CCase, FPase, CMCase and avicellase and mutant proteins were measured . The results showed that isolates of
Trichoderma ressei mutations with gamma rays ,had increase in the rate of protein production and celullase
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cmcase(u/ml)
activities. Compared side controls protein production in all mutants have increased. protein production maximum
was of T. r M7 mutant that 3.8 fold more protein can be produced. as well as increased enzyme activity avicellase,
CCase, CMCase and FPase respectively 95 , 42 , 70 , and 95 % of the mutants were observed .
Activity endoglucanase often based on the hydrolysis of derivatives of cellulose solution, such as carboxymethyl
cellulose is measured. Based on statistical analysis, 9 isolates (45% of isolates) CMCase Activity was more and 11
isolates (55% of isolates) less active than the control showed. In related to carboxymethyl cellulase activity, the
isolates T. r M4 and T. r M21 in 0.05 level were significantly different with control strain. The highest CMCase
activity was of T. r M21 strain with 6.09(u/ ml) and the lowest activity related to isolates of T. r M1 (4.28 u / ml),
respectively.
Activity Avicellase 19 isolates (95% of isolates) were higher than the control strains, the strains of T. r M21 and
T. r M11 meaningful difference with control strains are high avicellase activity in these mutants is probably due to
the high crystalline regions is their ability to analyze. The only isolate Tr M9 is less Avicellase activity.
7
6.09
5.69
5.36
6 5.04 5.17 4.96 5.07
5.41
4.84.9 5.244.63 4.74.85.05
4.28
4.45
5
4.38
3.76
4
3
2
1
0
Figure 5. Cmcase activity (U/ml) of gamma irradiated mutants of T.reesei in TFM supernatant after 72 h incubation at 180 rpm
and 28 ˚C
7.22
8
6.66
7
6
5.85
5.53 5.5
5.175.45.165.5
6
4.77 5 4.37
4.7 4.5 4.55.1
4.22
5
3.94
3.31
4 3.63
3
2
1
0
T. reesei
T. r M1
T. r M2
T. r M3
T. r M4
T. r M5
T. r M6
T. r M7
T. r M8
T. r M9
T. r M10
T. r M11
T. r M12
T. r M13
T. r M14
T. r M15
T. r M16
T. r M17
T. r M18
T. r M19
T. r M20
T. r M21
Avicellase(u/ml)
Activity Avicellase 19 isolates (95% of isolates) were higher than the control strains, the strains of T. r M21 and
T. r M11 meaningful difference with control strains are high Vyslaz activity in these mutants is probably due to the
high crystalline regions is their ability to analyze. The only isolate Tr M9 is less Avicellase activity.
Avicel contains some amorphous regions and is soluble selowdextrynes that can act as a substrate for the exo
and endo-glucanase. Avicel highest proportion of β-glucoside chain gangs to end domestic cellulosic substrates
are available in the model. CBH I and CBH II enzymes can lead to the absorption of several bands before
separation of substrate and enzyme complex, break (Valjamae, 1998:469-475). CBH I and CBH II, so the result is
a gradual reduction in the degree of polymerization of cellulose (Srisodsuk, 1998:345).
Chart 4- Avicellase special activity of mutated T.reesei via gama radiation in fermentation medium containing
colloidal cellulose
Figure 6. Avicellase activity (U/ml) of gamma irradiated mutants of T.reesei in TFM supernatant after 72 h incubation at 180 rpm
and 28 ˚C
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Measurement CCase specific activity showed, of 14 isolates (70% of isolates) had higher CCase activity, and 6
isolates (30% of isolates) had shown lower activity than control strain. Having T.r M21 isolates 20.09 (u / ml)
showed the highest CCase activity, that along with T.rM20 were significantly different with control strain.
25
20.1
17.4
14.7 14.4 15.114.6 14.415.3
14.915.6
13.7
15 13.513.112.9 13.8
13.5
12.913.213.414.114.3
CCase(u/ml)
20
10
5
T. reesei
T. r M1
T. r M2
T. r M3
T. r M4
T. r M5
T. r M6
T. r M7
T. r M8
T. r M9
T. r M10
T. r M11
T. r M12
T. r M13
T. r M14
T. r M15
T. r M16
T. r M17
T. r M18
T. r M19
T. r M20
T. r M21
0
Figure 7. CCase activity (U/ml) of gamma irradiated mutants of T.reesei in TFM supernatant after 72 h incubation at 180 rpm
and 28 ˚C
Overall, the results indicate the amount of variation in enzyme activity in mutant strains. Based on the results of
T. r M21 in terms of all four enzyme activities Avicellase, CCase, CMCase and Fpase is better than the other
isolates. Strains of T. r M20 and T. r M11, respectively, followed by the position.
Compare FPase enzyme activity showed 17 isolates (85% of isolates) had more enzyme activity than control
strain, but 13 isolates (65%mutants) had a significant difference with control strain. T.r M8 strain showed Maximum
enzyme specific activity (10U / ml), and T.rM2 with specific activity (4.81U / ml) have shown low enzymatic
activity(see Chart 6). amounts of activity FPase isolates recovered mutant T.r M8, T.r M11, T.r M16, T.r M7, and T.r
M21 due to the synergy of enzymes CBH and EG complete hydrolysis of cellulose to glucose.
12
10
9.37
9.28
8.92
8.77 8.46
7.48
7.6 7.46
7.56
7.39
8
6.84
6.69 7.37.11
6.67
6
5.71
5.78
5.77
5.38
6
4.81
Fpase(u/ml)
10
4
2
0
Figure 8. Fpase activity (U/ml) of gamma irradiated mutants of T.reesei in TFM supernatant after 72 h incubation at 180 rpm and
28 ˚C
Cellulase activity in UV mutated T. reesei
Comparison of CEase activity in the mutated fungus T. reesei showed that all isolates of mutant activity, had
lower CEase activity than control strain. and no one had a significant in 0.05 level. mutated strains demonstrated
at a rate of 2.6 to 4.5 fold decrease in CEase activity. (see Chart 7). CCase of enzyme activity in mutated T. reesei
by UV, were shown TR.uv8 strain had CCaseActivity more other mutants, but the control of its activity was lower.
and was observed decreased CCase activity all isolates mutant .
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CEase activity(u/ml)
10
8.58
8
6
2.92
4
3
3.27
2.27
1.88
2
2.88 2.58
CEase(U/ml)
0
Figure 9. CEase activity (U/ml) of UV mutants of T.reesei in TFM supernatant after 72 h incubation at 180 rpm and 28 ˚C
Statistical analysis of CMCase activity isolates in mutant and control strains showed that isolates TR.uv1 and
TR.uv6 had greater CMCase activity. increase activity of CMCase in these isolates mutants, respectively, 1.17 and
1.37 to control one. Remaining strains showed decreased CMCase .
16
CCase activity(u/ml)
14
13.54
12.69
12
10
8
6.77
5.31
6
3
4
2
0.65
0.5
0.77
0
TR.cont TR.uv1 TR.uv6 TR.uv8 TR.uv11 TR.uv17 TR.uv21 TR.uv23
Figure 10. CCase activity (U/ml) of UV mutants of T.reesei in TFM supernatant after 72 h incubation at 180 rpm and 28 ˚C
CMCase activity(u/ml)
8
6.91
7
6
5
4
5.92
5.04
3.38
3
2
1
0.54
0.5
0.96
1.15
0
TR.cont TR.uv1 TR.uv6 TR.uv8 TR.uv11TR.uv17TR.uv21TR.uv23
Figure 11. Cmcase activity (U/ml) of UV mutants of T.reesei in TFM supernatant after 72 h incubation at 180 rpm and 28 ˚C
Avicellase activities of TR.uv6 and TR.uv1 isolates was 1.34 and 1.45 fold to control strain respectively. other
isolates showed decreased avicellase activity.
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Avicellase activity(u/ml)
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7
6
4.85
5.27
5
4
3.63
3
1.42
1.24
2
0.73
0.69
1
1.15
0
TR.cont TR.uv1 TR.uv6 TR.uv8 TR.uv11TR.uv17TR.uv21TR.uv23
Figure 12. Avicellase activity (U/ml) of UV mutants of T.reesei in TFM supernatant after 72 h incubation at 180 rpm and 28 ˚C
Complete cellulase system is composed of endoglucanase, exoglucanase and beta- glucosidase that all of
which act synergistically in the conversion of cellulose into glucose [7, 11]. The most common measure for total
cellulase activity ,is measurement FPA. FPase activity in mutated T. reesei via UV showed that isolates TR.uv1
and TR.uv6 more FPase Activity than others. FPase Activity increase in the succesfull mutants were1.55 and 1.26
fold respectively more than control strain. other strains showed decreased Fpase activity.
Fpase(u/ml)
10
8.95
8
6
Fpase(U/ml)
7.28
5.77
4
2
0
1.24
0.5
1.19
0.5
0.67
TR.cont TR.uv1 TR.uv6 TR.uv8 TR.uv11TR.uv17TR.uv21TR.uv23
Figure 13. Fpase activity (U/ml) of UV mutants of T.reesei in TFM supernatant after 72 h incubation at 180 rpm and 28 ˚C
According to the analysis carried out, TR.uv1 and TR.uv6 mutant isolates the activity FPase, CMCase, and
avicellase isolates had significant control over the activities of their show. However Cease and CCase activity
compared to control strains It was a weakness. Other isolates showed a deficiency of the enzyme production.
Table 1) The mean specific activity of enzymes, CEase, CCase, Avicellase, CMCas and FPase the upper liquid
fermentation TFM medium containing colloidal cellulose of mutated T.reesei via UV radiation (All experiments were
performed in triplicate mean compared using Duncan's test statistic at p <0. 05 is).
Protein profile of the mutants
Extracellular protein profiles of mutant strains in the upper liquid fermentation medium TFM was assessed
using SDS-PAGE and the results in Figure 1 are shown . Several molecular bands in protein profiles were
observed , whereas the upper liquid fermentation medium not inoculated TFM was no specific protein binding .
Species of Trichoderma least two exoglucanase(cellubiohydrolase) includes cel7A (CBHI) and cel6A (CBHII) and
five endoglucanase includes cel5A (EGII), Cel7B (EGI), cel12A (EGIII), (EGV) cel45A and (EGIV) cel61A, two βglucosidase includes cel1A (BGLII) and cel3A (BGLI) for the decomposition of cellulose are produced. Band range
20-80 kDa protein encoded by the endoglucanase enzymes and can be exoglucanase. mutant strains of the
enzyme is produced in the range of 20-80 kDa Endo and exoglucanase have specific bands were considered. As
Figure (14) is observed in all mutant strains T.reesei, band in this range (10.5 to 95 kD) with a molecular weight of
approximately 19.5, 24, 42, 51.66 and 80 kDa was observed in the previous studies that produce enzymes Endo
and exoglucanase with 29, 36, 76 kDa molecular weight shows have been confirmed to (El-katany 2000:173-180).
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Therefore we can conclude all mutant strains except isolates Tr.M1, Tr.M6, and Tr.M17 compared with control
strains containing double bonds are highlighted increasing cellulase enzymes from these isolates it is.
Comparative protein and enzyme activity was determined by the intensity glucanase, cellulase bands in all samples
except Tr.M1 isolates corresponded with increased and decreased enzyme activity.
Cel5A is an endoglucanase belonging to glucohydrolases family 5. This enzyme has an estimated molecular
weight KDa42. However, the apparent molecular weight on SDS-PAGE gel is 48 KDa. Isoelectric point of the
enzyme 5/5 is. The SDS-PAGE gel protein profiles of isolates of T. r M3, T. r M4, T. r M10, T. r M11 and T. r M13 a
strong band of the enzyme was observed(Fig. 14).
A weaknes band of these enzymes in isolates of T. reesei, T. r M1, T. r M2, and T. r M21 is visible, no bands
were observed in the other mutants. Cel7B is an endoglucanase belonging to glucohydrolases family 7. Cel7B
molecular weight is 48KDa estimated, and molecular weight on SDS-PAGE gel shows 50-55KDa. Cel7B
isoelectric point is 4 -6 glycosidic chains in the mechanism of cellulose hydrolyzes certain. The fungus T.reesei
Cel7B expression between 6-10% of the total cellulase expression are included (Ståhlberg 1991; Ilmen, 1997).
Cel7B enzyme band in the protein profiles of the fungus T. reesei strains of T. r M3, T. r M4, T. r M5, T. r M7, T. r
M8, T. r M9, T. r M10, T. r M11, T. r M13 and T.r M15, T. r M16, T. r M21, T. r M14, T. r M19, T. r M20 was seen.
Cel6A is a cellubiohydrolase belonging to glucohydrolases family 6. This enzyme has a molecular weight on
SDS-PAGE gel 47 KDa, and isoelectric point of the 5.9 is. Cel6A (CBH II) enzyme that break glycosidic bonds of
the chain is non-regenerative. And is also mentioned in some reports that is has some endoglucanase activity
(Nath, 1998). During SDS-PAGE gel protein profiles of all mutant strains, a band of these enzymes was observed.
Cel7A enzyme has an estimated molecular weight KDa52. However, the apparent molecular weight on SDSPAGE gel is 66 KDa. Isoelectric point of the enzyme 4.3 is.(Fagerstam 1977:256-271). The enzyme was observed
in molecular weight 63KDa on SDS-PAGE gel. Cel7A highest expression of cellulase by T. reesei 50-60% of the
total amount is cellulase expression are included (Stahlberg 1991:91). Cel7A or CBH I is a cellubiohydrolase
belonging to glucohydrolases family 7. And first cellulose of T. reesei is detected (way 1994:31-39). Cel7A is an
efficient enzyme in hydrolysis glycosidic bonds, and it hydrolysed cellulose is preferably carried out from the end of
the chain revival.
The results showed that the possibility of a change in the production of extracellular proteins specific cellulase
activity in Trichoderma application of gamma radiation in there. Fungal strains to produce enzymes with higher
power than wild strains ( non- mutant ) , mutation induction is possible by this method .
Most Active CCase, CMCase and avicellase observed that the mutant M21, respectively, 5.1, 1.2 and 2 times
the activity in the control strain. FPase maximum activity is also related to the M8 mutant compared with control
activities, 1.7 fold. The results of SDS-PAGE gel analysis indicates increased protein production and cellulose
activity in mutant strains. The protein profiles of SDS-PAGE gel spinning Trichoderma mutant gamma-ray bands ,
with either an enzymatic complex of cellulases was observed. Stronger bands of the mutant strains on SDS-PAGE
protein gel is a reason for effective induced mutations in increasing cellulase enzyme activity.
Since 100% of the isolates mutation of Trichoderma obtained from irradiated with gamma rays random
mutations favorable outcome in terms of protein production followed and also increased the enzyme cellulase 80%
isolates had been irradiated with gamma rays can be a successful tool in achieving success genetic resources
than recommended.
Figure 14. SDS-PAGE analysis of proteins present in the acetone precipitated TFM supernatants of M: protein marker, C: T.
reesei control, 1-9 irradiated mutants of T. reesei
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Intl J Farm & Alli Sci. Vol., 3 (5): 543-554, 2014
CONCLUSION
The T. reesei is a common soil fungus of the Deuteromycete family which has been widely used for the
commercial cellulases production. cellulases produced by fungi, especially by Trichoderma reesei have been most
extensively studied. The strain improvement by mutation is still a traditional and efficient method to enhance the
enzyme production. Random mutagenesis is an interesting tool to achieve genetic and functional modifications of
an organism. In this study, we adopted the mutation steps involving treatment of T. reesei spores with gama and
UV-irradiation. UV radiation induces two types of mutations, changes in the base pairs are replaced. pyrimidine
dimer formation in response to radiation and subsequent repair mechanisms lead to the spaces of single-stranded
DNA its The most important form of mutation induction by ultraviolet irradiation (Kahrizi, 1368). Ultraviolet
biologically-effective because of its range (250 to 290 nm) nucleic acids do maximum absorption (Farsi and
Bagheri, 1386).
Wavelength of 260 nm by purine and pyrimidine can be absorbed and structural changes, particularly the
formation of pyrimidine dimers between adjacent cyclobutyl especially between thymine and create. Dimers
induced causes togather bases remain attached and create in replication time deletion mutation. Germicide UV
lamps contain mercury vapor is produced (Brown, 1953).
In our study, mutagenesis was carried out by  irradiation and uv individully. After each mutagenic treatment,
was compred mutants by mandels way. Then number of best them, was selected to quantity asseys. In next step,
was assessed protein production and cellulitic activities in shake flask cultures, and was selected the most
promising strain.
During this mutagenesis processes, we screened a lot of mutants, and of these, 28 mutants were found
superior, where a mutant of each mutagenesis method was selected and compared on the basis to produce
enzyme and ability to produce enzyme.
The highest cellulases activities were observed from mutagenesis by gama rays. We successfully isolated
one mutant T.r M21, which showed a larger zone of cellulosehydrolysis. Enzyme assay verifi ed that the
mutant showed approximately 1.55 fold increases in activity of FPase and 1.21 fold increases of CMCase in
shake fl ask culture. Also it showed 1.99 and 1.5 fold increases in activity of avicellase and CCase
respectively.
However mutagenesis by uv is a good way but we coudnot successfully isolated one mutant that it showed
increase amounts all compenent of cellulose complex. Thus mutagenesis by gama rays is the better way in
comparation with ultra violet.
REFERENCES
Adsul MG, Bastawde KB, Varma AJ and Gokhale DV. 2007. Strain improvement of Penicillium janthinellum NCIM 1171 for increased cellulase
production. Bioresour Technol 98:1467–1473
Askari H, 1Shahbazi S, Moosavi-nasab M and Bakhtiyari M. 2012. »The synergistic interactions of Trichoderma spp. cellulase enzyme activities
in biomass conversion; Part1: comparison of cellulose Iá, Iâ and II«, Nuclear Science and Technology Research Institute, Agricultural,
Medical and Industrial Research School, Alborz, Iran
Atalla RH, Hackney JM, Uhlin I and Thompson NS. 1998. Hemicelluloses asstructure regolatores in the aggregation of native cellulose «
.Int.j.Biol.Macromol.15:109-112
Bailey MJ and Nevalainene KMH. 1981. Induction, isolation and testing of stable Trichoderma reesei mutants with improved production of
solubilizing cellulose. Enzyme MicrobTechnol 3: 153–157
Barr BK, Hsieh YL, Ganem B and Wilson DB. 1996. »Identification of Two Functionally Different Classes of Exocellulases«. Biochemistry 35:
586-592.
Bon EP, November S. 2007. Brazilian »Present and Future Ethanol Production. Biomass Ethanol Potential, 4th Biomass Asia Workshop«, ,
Dural Ehsan, Malaysia 20-22
Bradford MM. 1976. A rapid and sensitive method for the quantification of micro-organism quantities of proteins utilizing the principles of protein
dye-binding. Anal Biochem 72:248–254
Bradford MM. 1976. » A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye
binding. « Analytical Biochemistry, 72: 248-254
Chand P, Aruna A, Maqsood AM and Rao LV. 2005. Novel mutation method for increased cellulase production 98, 318–323
Chand P, Aruna A, Maqsood AM and Rao LV. 2005. Novel mutation method for increased cellulase production. J Appl Microbiol 98:318–323
Chandra M, Kalara A and Sangwan N. 2009. »Development of a mutant of Trichodermacitrinoviride
for enhanced production of
cellulases«.Bioresource Technology 100 (1659-1662)
Cherry JR and Fidantsef AL. 2003. Directed evolution of industrialenzymes: anupdate. Curr Opin Biotech 14:438–443
El-Katatny MH, Somitsch W, Robra KH, El-Katany MS and Gubitz GM. 2000. »“production of chitinase and beta-1, glucanase by T. harizianum
for control of the pHytopathogenic fungus Sclerotinum rolfsii«. Food Technology and Biotechnology, 38: 173-180
Fägerstam L, Håkansson U, Pettersson G and Andersson L. 1977. » Purification of three different cellulolutic enzymes from Trichoderma viride
QM 9414 on a large scale«. In Proceedings of Bioconversion Symposium, Feb 21-23. (ed. T. Gohose), pp. 165-178. Indian Institute of
Technology, New Delhi.618. American Chemical Society, Washington, pp. 256.271.
553
Intl J Farm & Alli Sci. Vol., 3 (5): 543-554, 2014
Fan LT, Gharpuray MM and Lee YH. 1987. Cellulose Hydrolysis. Berlin: Springer-Verlag. pp. 1–68.
Gadgil NJ, Daginwala HF, Chakrabarti T, Khanna P. 1995. » Enhanced cellulaseproduction by a mutant of Trichodermareesei . Enzyme and
Microbial Technology 17 :942-946
Ilmen M, Saloheimo A, Onnela M-L and Penttila ME. 1997. » Regulation of cellulase gene expression in the filamentous fungus Trichoderma
reesei«. Appl Environ Microbiol 1997; 63:1298–306.
Johnwesly B, Virupakshi S, Patil GN and Ramalingam Naik GR. 2002. Cellulase-free thermostable alkaline xylanases from thermophillic and
alkalophillic Bacillus sp. JB-99. J Microbiol Biotechnol 12:153–156 1873–1881
Kansoh AL, Essam SA and Zeinat AN. 1999. Biodegradation and utilization of bagasse with Trichoderma reesei. Polymer Degradation and
Stability 62, 273–278.
La Grange DC, Pretorius IS and Van Zyl WH. 1996. Expression of a Trichoderma reesei β-xylanase gene (XYN2) in Saccharomyces cerevisiae.
Appl Environ Microbiol 62:
Miller GL. 1959. Use of dinitrosalicylic reagent for determination of reducing sugars. Anal Chem 31:426–42
Miller GL, Blum R, Glennon WE and Burton AL. 1960. Measurement of carboxymethylcellulase activity. Anal Biochem 1960, 2:127-132..
Mohammadi E, Zabihi M, Shahbazi S, Ahari Mostafavi H, Askari H and Abraham MA. 1390. "The use of gamma radiation to induce mutations
increase enzyme activity of chitinase in Trichoderma reesei isolates "Yazd, Iran's nuclear XVIII Conference, page 119.
Montenecourt BS, Eveleigh DE. 1977. Semiquantitative plate assay for determination of cellulase production by Trichoderma viride. Appl
Environ Microbiol1977;33:178–83.
Muthuvelayudham R and Viruthagiri T. 2006. Fermentativeproduction and kinetics of cellulose protein on Trichoderma reesei using sugar cane
bagasse and rice straw. Afri J Biotechnol
Nevalainen H and Penttila M. 1995. Molecular biology of cellulolytic fungi. In: Esser, K. and Kiick, U. (Eds.) The Mycota, Vol. III, Genetics and
Biotechnology, Springer Verlag, pp, 303-319
Nevalainen H, Penttill M, Harkki A, Teeri T and Knowles J. 1991. The molecular biology of Trichoderma and its application to the expression of
both homologous and heterologous genes. In: Leong, S.A. and Berka, R. (Eds.), Molecular Industrial Mycology, Systems and
Applications for Filamentous Fungi. Marcel Dekker Inc., New York, pp. 129-148
Oksanen T, Pere J, Paavilainen L, Buchert J and Viilkari L. 2000. Treatment of recycled kraft pulps with Trichoderma reesei hemicellulases and
cellulases. J Biotechnol 78:39–48
Schuchardt U, Ribeiro ML, Goncalves A and Anind R. 2001, » striapetroquimica no proximoseculo:Comosubstituir petroleocomomateriaprima?Qu?micaNova«, ,24(2);247-5
Shahbazi S, Ahari Mostafavi H and Askari H. 2011. "in 2011 to determine the optimum dose of gamma radiation to induce mutations in
Trichoderma Virida", Yazd, Iran's nuclear XVIII Conference, page 120.
Shin CS, Lee JP, Lee JS and Park SC. 2000. Enzyme production of Trichoderma reesei Rut C-30 on various lignocellulosic substrates. Appl
Biochem Biotechnol 84-86:237–245
Solomon BO, Amigun B, Betiku E, Ojumu TV and Layokun SK. 1999. Optimization of cellulase production by Aspergillus flavus Linn isolate
NSPR 101 grown on bagasse. JNSChE 16, 61–68
Srisodsuk M, Kleman-Leyer K, Keranen S, Kirk TK and Teeri TT. 1998». Modes of action on cotton and bacterial cellulose of a homologous
endoglucanase-exoglucanase pair from Trichoderma reesei«. Eur
Ståhlberg J. 1991. »Functional organization of cellulases from Trichoderma reesei. In Doctoral thesis. Acta Universitatis Upsaliensis«.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science 344. 45pp, Uppsala. ISBN 91-554-2800-2. Uppsala
University
Valjamae P, Sild V, Pettersson G and Johansson G. 1998. » The initial kinetics of hydrolysis by cellobiohydrolases I and II is consistent with a
cellulose surface - erosion model«. Eur J Biochem 253:469 – 475
Wey TT, Hseu TH and Huang L. 1994. »Molecular cloning and sequence analysis of the cellobiohydrolase I gene from Trichoderma koningii G39«. Curr. Microbiol. 28: 31-39.
Wu, Z. and Lee YY. 1997. Inhibition of the enzymatic hydrolysis of cellulose by ethanol. Biotechnology Letters 19, 977–979
Zaldivar M, Velasquez JC, Contreras I and Perez LM. 2001. Trichoderma aureoviride 7-121, a mutant with enhancedproduction of lytic
enzymes: its potential use in waste cellulose degradation and biocontral. Ele J Biotechnol 1–7.
554