WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
Kaur et al.
World Journal of Pharmacy and Pharmaceutical Sciences
SJIF Impact Factor 2.786
Volume 4, Issue 02, 521-534.
Research Article
ISSN 2278 – 4357
OPTIMIZATION OF CELLULASE PRODUCED BY FUNGUS
ISOLATED FROM WATER
Harjot Pal Kaur* and Deepti Joshi
SUS College of Research and Technology, Tangori (Mohali) Punjab, India – 140306.
Article Received on
02 Jan 2015,
Revised on ---------------,
Accepted on 07 Jan 2015
ABSTRACT
Cellulases are a group of hydrolytic enzymes capable of degrading
cellulose to the smaller glucose units and have a wide range of
applications in textile industry, drug industry, detergent industry, foodfeed industry, bioethanol production and pulp-paper industry. This
*Correspondence for
Author
study was designed to assess the cellulase production of fungus
isolated from water. 5 fungal isolates obtained from water samples
Harjot Pal Kaur
SUS College of Research
were identified by morphological and microscopic features. These 5
and Technology, Tangori
isolates were screened for cellulase production and cellulase
(Mohali), Punjab, India.
production of two isolates that gave highest zone of hydrolysis (P.
chrysogenum - zone 18mm and T. reesei – zone 12mm) was estimated
by CMCase and FPase assay then compared with cellulase activity of respective standard
strains. Isolate 2 belonging to P. chrysogenum showed more enzyme activity (0.552 U/ml)
than the isolate 1 belonging to T. reesei (0.363 U/ml) and both the isolates showed greater
enzyme activity than their respective standard strains. Optimization of process parameters
(temperature, pH, incubation time, carbon and nitrogen source) was carried out for the
isolates and respective standard strains to maximize enzyme yield. On optimization T. reesei
showed maximum enzyme activity at temperature of 30°C, pH 4, 120 hours of incubation
with sucrose as carbon source and yeast extract as nitrogen source whereas P. chrysogenum
showed maximum cellulase production at temperature of 30°C, pH 5, incubation of 144
hours, CMC as carbon source and ammonium nitrate as nitrogen source. Results indicate that
fungi belonging to T. reesei and P. chrysogenum effectively produced cellulase and its yield
was increased by optimizing process parameters.
KEY WORDS: Cellulase, Morphological, Microscopic, Sucrose, Yeast Extract
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INTRODUCTION
Cellulose is the most common organic polymer, representing about 1.5 x 1012 tons tons of the
annual biomass production through photosynthesis, and is considered to be an almost
inexhaustible source of raw material for different products. It is the most abundant and
renewable biopolymer on earth and the dominating waste material from agriculture. Cellulose
is generally degraded by multi-complex enzyme called cellulases.[1] Cellulases are produced
by a number of microorganisms such as bacteria, yeast and fungi.[2] However, the most
extensively studied cellulases are those produced by efficient lignocellulose degrading fungi,
particularly Trichoderma.[3]
Fungi have been studied extensively because their elongated hyphen creates mechanical
pressure on the cellulose structure, causing them to produce large amounts of cellulase. Most
fungal cellulases have the capability to digest cellulose. Fungi such as Penicillium sp.,
Aspergillus sp.and Trichoderma sp. are cellulase producers.[4] Enzyme produced by these
microorganisms is commercially available for agricultural and industrial uses. Improvement
of microbial strains for the over-production of industrial products has been the hallmark of all
commercial fermentation processes. Such improved strains can reduce the cost of the process
and may also possess some specialized desirable characteristics.[5]
Cellulase enzyme has its importance due to major role in industrial applications. [6] It is used
for bioremediation, waste water treatment and also for single cell protein.[7] It has importance
in food sciences like food processing; drying of beans in coffee, efficient purification of
juices, paper and pulp industry and as a supplement in animal feed industry. This enzyme is
helpful for plant protoplast isolation, plant viruses investigations, metabolic and genetic
modification studies.[6,
8, 9]
Cellulases also have pharmaceutical importance, treatment of
phytobezons (a type of bezoar cellulose existing in humans stomach) and a key role in textile
industry especially as its detergent applications to recover properties of cellulose related
textiles and biofuels production from cellulosic biomass.[10] Enzymes are used as antitumor or
antimicrobial agents, treatment of blood clots and herniated discs and for the treatment of
enzyme deficiencies. Enzyme cellulase acts as digestive aid i.e. celulase digests fiber. It helps
in the remedy of digestive problems such as ‘malabsorption’. Since, humans poorly digest
cellulose fiber, taking a digestive enzyme product, like Digestin, that contains cellulase
enzymes is not only necessary, but also vital for healthy cells.[11] Main function of cellulase
enzyme in food industry is extraction, clarification and stabilization of fruit juices and
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vegetables, biostoning of jeans and biopolishing of cotton and other cellulosic fabrics and
also applied to remove rough protuberances for a smoother, glossier and brighter fabric.[12]
Certain fermentation parameters such as temperature, incubation period, carbon source, pH
etc. play an important role in obtaining good cellulase yield.[13] Taking into consideration the
importance and applications of the cellulases, this study was aimed to screen the indigenous
fungal isolates for the cellulytic ability. Furthermore, this study aims to provide better
understanding of conditions for the production and activity of cellulases by using fungal
cultures isolated from water.
MATERIALS AND METHODS
Isolation and Screening of Cellulase Producing Fungi
Water samples were collected from agricultural fields around SUS College of Research and
Technology, Tangori and Sukhna Lake, Chandigarh in the sterilized containers for the
isolation of fungus. Serially diluted water samples were spread on surface of potato dextrose
agar and incubated for 7 days at 28ºC. Colonies were picked and sub-cultured to obtain pure
cultures and stock cultures were maintained on potato dextrose agar at 4ºC. The isolated
strains were carefully identified by morphological characteristics such as color of the colony
and growth pattern studies, as well as their vegetative and reproductive structures observed
under the microscope. Further fungal isolates were screened for cellulase production.[14]
Cellulase Production
Fungal isolates harvested with sterile distilled water for inoculums preparation were used for
cellulase production. 100ml of Mendels enriched media (pH 5) in 250ml shake flask was
inoculated with 2 ml of fungal spore suspension containing 105 spores per ml. The flask was
incubated at 30°C for 5 days at 120 rpm. After 5 days, culture filtrate was collected,
centrifuged at 6000 rpm for 15 minutes and supernatant was used for cellulase assay. The
enzyme activity was determined by carboxymethyl cellulase assay (Endoglucanase activity)
and filterpaper assay (total cellulase activity).
Cellulase Activity by Carboxymethyl Cellulase (CMCase) Assay
1 ml of crude enzyme supernatant was incubated with 1ml of 1% (w/v) CMC (carboxy
methyl cellulose) in 0.1M sodium acetate buffer solution of pH 5 for 30 minutes at 63ºC. The
resulted reducing sugars were determined according to Miller’s modified method.[15]
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Cellulase Activity by Filter Paper (FPase) Assay
For FPase activity 1 ml of crude enzyme supernatant was incubated with 2ml of 0.1M citrate
buffer of pH 4.8 containing 50mg Whatman no. 1 filterpaper. After incubation of 1 hour at
50ºC, obtained reducing sugars were estimated by Miller’s modified method.[15]
Optimization of Parameters for Cellulase Production:
To
optimize
the
cellulase
production, effects of fermentation conditions such as the incubation period, pH, temperature,
carbon source and nitrogen source were studied.[16] To determine the effective temperature
for cellulase production, fermentation was carried out at different temperature (28°C, 30°C,
34°C and 37°C) for 5 days. Enzyme activity was determined by DNS method by taking the
OD at 540nm. Effect of pH on enzyme production was determined by producing the cellulase
at different pH range (3, 4, 5, 6 and 7) and pH of the medium was adjusted by using 1N HCL
or 1N NaOH. Cellulase production was estimated by DNS method.
Effect of incubation time on enzyme production was checked by incubating the fermentation
medium for different incubation time (24, 48, 72, 96 and 120 hours) and enzyme assay was
carried out at interval of 24 hours by DNS method. Effects of various carbon compounds
(CMC, glucose, sucrose and maltose) and nitrogen compounds (peptone, beef extract,
ammonium nitrate, sodium nitrate and yeast extract) was examined by producing the
cellulase with 1% carbon and nitrogen source and enzyme activity was determined by DNS
method.
RESULTS AND DISCUSSION
Five filamentous fungi were isolated from two water samples (Fig.1). Presumptive identity of
fungal isolate 1, 2, 3, 4, and 5 was Trichoderma reesei (T. reesei), Penicillium chrysogenum
(P. chrysogenum), Fusarium sp., Aspergillus niger (A. niger) and Aspergillus flavus (A.
flavus) respectively (Table 1). These five, probably identified fungal isolates were further
subcultured and screened for cellulase production.
Screening for Cellulase Enzyme Production
To check the enzyme production of fungal isolates, zone of hydrolysis were observed around
the well in which fungal spore suspension was poured. Zone of hydrolysis of Isolate 1 (T.
reesei) and isolate 2 (P. chrysogenum) were more (12 mm and 18 mm respectively) as
compared to isolate 3, 4 and 5 (8mm, 9mm and 11mm respectively) indicating that isolate 1
and 2 produced more cellulase than isolate 3, 4 and 5. Enzyme activity of isolate 1 and 2 (T.
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reesei and P. chrysogenum) was estimated and compared with respective standard strains.
Standard strain of T. reesei (MTCC-7725) and P. chrysogenum (MTCC-2725) used for
comparative studies were obtained from IMTECH, Chandigarh.
Isolate 1 and 2
Isolate 4
Isolate 3
Isolate 5
Fig.1. Fungal Growth on PDA Plates
Table.1.Morphological and Microscopic Characterization of Fungal Isolates
Isolate
Morphological Observation
1
Greenish- white colonies
2
Dark green coloured growth
3
Pinkish- white growth
4
Black brown colonies
5
Greenish-yellow sporulated
growth
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Microscopic Observation
Conidia were present in long chains,
filamentous and brush like head
long chains of conidia with smooth,
brush shaped conidiophores
Conidiophores forming branches, septate
conidia and septate hyphae
Septate hyphae, non-septate
conidiophores, columnar head
Septate hyphae, non-septate
conidiophores, columnar head
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Probable Identity
Trichoderma
Reesei
Penicillium
chrysogenum
Fusarium sp.
Aspergillus niger
Aspergillus flavus
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Cellulase Enzyme Activity
CMCase and FPase assay were performed and reducing sugars released by cellulase enzyme
production was determined by glucose standard curve. CMCase and FPase assay showed that
isolate 2 (P. chrysogenum) produced more cellulase than isolate 1 belonging to T. reesei and
both the fungal isolates showed more enzyme activity than their respective standard strains
(Table 2).
Table.2. Cellulase Production by CMCase and FPase Assay
Fungal Strain
Isolate1 (T. reesei)
Standard Strain (T. reesei)
Isolate 2 (P.chrysogenum)
Standard Strain (P.chrysogenum)
Cellulase (U/ml) by CMCase
Assay
0.358
0.332
0.541
0.534
Cellulase (U/ml) by
FPase Assay
0.363
0.340
0.552
0.538
Optimization of Parameters for Enzyme Production
Medium parameters such as temperature, pH, incubation time, and carbon and nitrogen
source play very important role in production of enzyme and greatly influence the enzyme
activity. CMCase and FPase test were used to check the enzyme activity at variable
parameters.
Effect of Temperature
Temperature is the important parameter that effects the cellulase production. Optimum
temperature for CMCase and FPase activity was found to be 30°C as maximum enzyme
activity was observed for both the isolates and their respective standard strains. Enzyme
activity was decreased at 35°C for all the tested strains (Fig.2 and 3).
Fig.2. Effect of Temperature on Cellulase Activity of Fungal Isolates and Respective
Standard Strains by CMCase Assay
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Fig.3. Effect of Temperature on Cellulase Activity of Fungal Isolates and Respective
Standard Strains by FPase Assay
Fig.4. Effect of pH on Cellulase Activity of Fungal Isolates and Respective Standard
Strains by CMCase Assay
These results of present study are comparable with the results of Maurya et al., Shafique et al.
and Karthikeyan et al., who reported 30°C optimum temperature for cellulase production.[17,
18, 19]
Optimum yield of cellulase by Penicillium notatum was obtained at 30°C after 24 hours
of fermentation.[20] Cellulase is highly sensitive towards temperature and high temperature
decrease the growth of microorganisms and enzyme production.[21]
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Fig.5. Effect of pH on Cellulase Activity of Fungal Isolates and Respective Standard
Strains by FPase Assay
Effect of pH: Effect of pH on enzyme production was evaluated at pH values 3, 4, 5 and 6.
pH 4 was found to be optimum for isolate 1 belonging to T. reesei and its standard strain as
maximum cellulase activity was found at pH 4 but isolate 2 belonging to P. chrysogenum and
its standard strain produced more enzyme at pH 5 as maximum activity was recorded at this
pH (Fig.4 and 5).
Fig.6. Effect of Incubation Time on Cellulase Activity of Fungal Isolates and Respective
Standard Strains by CMCase Assay
The enzyme production was favored in acidic range of pH 4 to 6 and the results of present
study were considerably similar to Das et al., who reported optimum cellulase activity at pH
4.8 and Shafique et al., where Trichoderma reesei produced maximum cellulase at pH 4.
18]
[22,
Adelke determined maximum enzyme production by Penicillium atroventum at pH 5. [ 23]
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Effect of Incubation Time
Time of fermentation has an important impact on the product formation. Enzyme production
is related to time of incubation.[24] Time course for production of cellulase enzyme was
investigated and fermentation for a period of 7 days was carried out. Enzyme activity was
investigated after interval of 24 hours. The results showed that incubation time of 144 hours
was optimum for isolate 1 as well as its standard strain and 120 hours for isolate 2 as well as
its standard strain. Decrease in cellulase activity was recorded with further incubation (Fig. 6
and 7).
This result of incubation time for isolate 1 was found comparable with Maurya et al., who
reported incubation time of 144 hours optimum for the production of cellulase. [17] For isolate
2 the result of incubation time was found to be similar with the reports of Karthikeyan et al.,
who reported 120 hours to be optimum incubation time for the production of cellulase
whereas Jayant et al., reported co-culture of Aspergillus niger and Penicillium chrysogenum
showed maximum enzyme activity at 192 hours. [19, 25]
Fig.7. Effect of Incubation Time on Cellulase Activity of Fungal Isolates and Respective
Standard Strains by FPase Assay
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Fig.8. Effect of Carbon Source on Cellulase Activity of Fungal Isolates and Respective
Standard Strains by CMCase Assay
Effect of Carbon and Nitrogen Sources
To check the effect of carbon sources, both the isolates and standard strains were grown in
Mendel’s media with various carbon sources (1% w/v). Isolate 1 and its standard strain
produced maximum cellulase enzyme when sucrose was used as carbon source and minimum
with glucose but in case of isolate 2 and its standard strain maximum cellulase enzyme
production was calculated with CMC as carbon source and minimum with glucose (Fig.8 and
9). Isolate 1 and 2 showed highest activities when sucrose and CMC respectively were used
as carbon source but enzyme production was decreased when glucose was used as carbon
source for both the isolates. Use of glucose as a carbon source results in less amount of
cellulase because of end product inhibition. Cellulase acts on cellulose producing glucose as
the end product. Presence of glucose in the medium inhibits cellulase production.[26]
Fig.9. Effect of Carbon Source on Cellulase Activity of Fungal Isolates and Respective
Standard Strains by FPase Assay
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Fig.10. Effect of Nitrogen Source on Cellulase Activity of Fungal Isolates and
Respective Standard Strains by CMCase Assay
The results of present study are comparable with previous studies where 1% sucrose was
reported to be best carbon source for cellulase production by Trichoderma sp. and CMC was
the best carbon source for Penicillium for enzyme production.
[24]
Strains of Penicillium
chrysogenum has also been reported to produce significant levels of cellulolytic enzymes. [27]
From CMCase and FPase assay, it was observed that yeast extract was optimum nitrogen
source for isolate 1 and its standard strain and ammonium nitrate was for Isolate 2 and its
standard strain for cellulase production (Fig. 10 and 11).
Fig.11. Effect of Nitrogen Source on Cellulase Activity of Fungal Isolates and Respective
Standard Strains by FPase Assay
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The results of present study regarding best nitrogen source were found to be similar with the
results of the previous studies where yeast extract was the best nitrogen source for the
production of cellulase by Trichoderma and ammonium nitrate by Penicillium. [24, 19]
In the present study temperature of 30°C, pH 4, incubation time of 144 hours, sucrose as
carbon source and yeast extract as nitrogen source were found to be optimum for isolate 1
belonging to T. reesei and its standard strain (T. reesei MTCC 7725) as maximum enzyme
activity was observed with these parameters. For isolate 2 belonging to P. chrysogenum and
its standard strain (P. chrysogenum MTCC 2725) temperature of 30°C, pH 5, incubation time
of 120 hours, CMC as carbon source and ammonium nitrate as nitrogen source were found to
be optimum for cellulase production. Results showed that isolate 2 produced more cellulase
as compared to isolate 1 indicating that P. chrysogenum had greater potential for cellulase
production as compared to T. reesei.
CONCLUSIONS
This study demonstrates that fungi belonging to Trichoderma reesei and Penicillium
chrysogenum effectively produced cellulase and can be used for industrial production of
cellulases. Comparative analysis of enzyme production by isolated fungi and their standard
strains revealed that fungal isolates exhibited better activity than their standard strains. It is
possible that still there may be many more isolates to be discovered which have enormous
potential to produce cellulase. Isolates exhibiting high cellulase activity can prove to be of
immense help to humans in production of pharmaceutical and many more products. Further
genetic, biochemical and microbial engineering techniques are required to make use of full
potential of these fungal isolates for cellulase production.
ACKNOWLEDGEMENTS
The authors acknowledge the management of SUS Group of Institutions, Tangori for
providing the required research facilities.
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