ISOLATION, CHARACTERIZATION AND OPTIMIZATION OF
CELLULASE PRODUCING FUNGI FOR MAXIMIZING THE ENZYME
YIELD
Shelly Rana1, Kamaldeep2, Manjeet Kaur3
1 PG
Student
Department of Biotechnology
University Institute of Engineering and Technology,
Maharishi Dayanand University, Rohtak-124001 Haryana (INDIA)
[email protected]
2
PG Student
Department of Biotechnology
University Institute of Engineering and Technology,
Maharishi Dayanand University, Rohtak-124001 Haryana (INDIA)
[email protected]
3 AssistantProfessor
Department of Biotechnology
University Institute of Engineering and Technology,
Maharishi Dayanand University, Rohtak-124001Haryana (INDIA)
[email protected]
1. Introduction
Abstract
The main objective of this study is to isolate the novel
Cellulase producing microorganisms. The enzyme
production rate from fungi is higher as compared to other
microorganism. For this study soil samples were collected
from wood and cellulosic waste collected from dumping
areas. Microorganisms were isolated using Potato dextrose
agar medium. Presently, the work is aimed at identification
of the isolates based on staining and molecular
characterization and optimization of the cultural and
environmental conditions for maximizing the yield of the
enzyme. Identification of the fungal isolates was done
based on biochemical and molecular characterization by
sequencing the 18S rRNA coding gene. Crude enzyme was
extracted and purified by centrifugation. The optimization
of various different parameters of the medium was carried
out to maximise the yield of cellulase.
Keywords: cellulase, optimization, 18S rRNA
Cellulases are a group of hydrolytic enzymes
which are capable of degrading cellulose to
smaller glucose units. These enzymes are
produced mainly by fungi though some bacterial
Strains have also been found to produce
cellulases. Although a large number of
microorganisms are capable of degrading
cellulose, only few of these produce significant
quantities of cell free enzymes capable of
completely hydrolyzing crystalline cellulose the
solid waste of sugar and paper industry and
wood wastes.There has beenresearch carried out
aiming at obtaining new microorganisms
producing
cellulasewith
higher
specific
activities(Subramaniyan and Prema, 2000).
Cellulase refers to a group of enzymes which
hydrolyzes cellulose (Emert et al., 1974 and
Whitaker, 1971). Therefore they are hydrolytic
enzymes which hydrolyze the glycosidic bonds
of cellulose and its related oligosaccharides.
Cellulose is the most abundant renewable
organic compound in the biosphere which
accounts for 40-50% of plant composition and
its production is expected to be 1010 tonnes
from cell wall of plants per year (Thu et al.,
2008). Cellulase in the future may provide a
better answer to the problems related with
cellulosic waste disposal (Roy et al, 1990). A
cellulosic enzyme system consists of three major
components: endo-ß-glucanase, exo-ß-glucanase
and ß-glucosidase.
The exo-ß-glucanase causes a disruption in
cellulose hydrogen bonding, followed by
hydrolysis of the accessible cellulose with endoß-gucanase(Reese et al. 1950). The complete
enzymatic hydrolysis of cellulosic materials
needs different types of cellulase; endo-1,4-βglucanase,
also
referred
to
as
carboxymethylcellulase or CMCase, exo1,4-βglucanase and β-1,4-glucosidase (J.C. Yi et al.
1999).
In this study, we examined the potential of
Alternariaalternatato produce cellulase. The
fungi was isolated from soil near wood wastes
dumping sites.
2. Materials and method
2.1 Wood wastes
Sample of decomposing wood-wastes were
collected from soil near dump sites. Sample was
prepared by serial diluting 1 gm of the sample
by introducing it into 9 ml of distilled water in a
sterile tube.
2.2 Other subsrates and Chemicals.
Potato Dextrose Agar (PDA) medium was
prepared in the laboratory.Carboxymethylcellulose(CMC) was obtained from HiMedia
Laboratories Pvt. Ltd. All other chemicals and
reagents were of analytical grade.
2.3 Isolation of Cellulolytic Fungi
Microfungi capable of degrading cellulose and
other cellulolytic materials were isolated from
the soil using Potato Dextrose Agar (PDA)
medium. The PDA plates were inoculated with
the sample and incubated at 30ºC for 3–15 days.
Colonies of cellulolytic fungi was sub-cultured
on Potato Dextrose Media supplemented with
0.5% (w/v) Ampicillin and preserved at low
temperature until needed.
2.4 Screening for potent cellulase producing
Fungi
Carboxymethylcellulose (CMC) assay acts as a
good indicator of cellulolytic ability since
endoglucanase is generally produced in larger
titres by fungi than cellobiohydrolase(Cai,
Buswell and Chang, 1994; Buswell et al.,
1996; Pointing et al., 1999). Congo red dye was
used to test the cellulolytic activity. CMC
degradation around the colonies appears as an
opaque area against a red colour for undegraded
CMC.
2.5 Identification of the isolated fungi by
sequencing of the amplified 18S rRNA gene
The most powerful tool to identify the unknown
microorganism is to sequence the gene (DNA)
coding for 18S rRNA .The gene coding for the
18S rRNA was amplified using the PCR and the
amplified product was subjected to sequencing
and the sequence obtained was compared with
the sequence obtained from the Nucleotide
Database of NCBI.
Fungal DNA extraction:
DNA was extracted from fungus using fungal
genomic DNA extraction method in which
fungal mycelium was taken and ground with
liquid nitrogen. After then 15 ml extraction
buffer and 100µl of β mercaptoethanol was
added and mixture vortexed and incubated at
65ᵒC for 15 min. Then trisphenol,chloroform
and isoamyl alcohol were added and centrifuged
at 14,000 X rpm for 15 min. The aqueous
fraction was recovered and 100µL of RNAse
was added and incubated at 37 C for 30 min.
Again chloroform and isooamyl alcohol were
added and centrifuged followed by recovery of
the fraction. The DNA precipitate was incubated
at – 20 C for 30 min and centrifuged to get the
DNA pellet.
Cellulase Assay was done by DNS method (3, 5dinitrosalicylic acid)(Miller, 1959) and the
activity of the enzyme was expressed in
μmol/ml/min. It was calculated by the following
formula
OD of Unknown – OD of Test = to the OD of
the Colour intensity of the liberated product.
From the graph the concentration of the reducing
sugar liberated by the action of the enzyme was
determined and the enzyme activity is expressed,
Enzyme Activity = μ moles of the product
liberated per mole of enzyme per ml per minute
Enzyme Activity = Concentration of Glucose
Liberated/ Incubation time μg
PCR Amplification:
Amplification of the ITS region (ITS1, ITS2,
and 5.8 SrRNA Gene) was performed using
Universal primers ITS 1 and ITS 4. The ~600 bp
PCR product was purified by gel elution and
used for sequencing.The amplification was
carried out in a Master cycler® Thermocycler
(Eppendorf, Germany). PCR product was
purified to remove unincorporated dNTPS and
Primers before sequencing using PCR
purification kit (NorgenBiotek, Canada).
Sequencing:
Both strands of the rDNA region amplified by
PCR were sequenced by automated DNA
sequencer -3037xl DNA analyzer from Applied
Biosystems using BigDye® Terminator v3.1
cycle sequencing Kit (Applied Biosystems).
Sequence data were aligned and dendrograms
were generated using Sequence analysis
software version 5.2 from Applied Biosystems.
The sequence so obtained was compared with
already reported results from the public
databases (NCBI) and the assembled sequence
of the18S rRNA gene (DNA) of the unknown
fungi was determined.
3. Results and Discussion
3.1. Appearance of fungal colonies on Potato
dextrose agar plates
Colonies of cellulolytic fungi appeared on potato
dextrose agar plates. The number and size of the
colonies increased as incubation period
increased.
3.2. Screening for Cellulolytic Fungal Isolates
Cellulolytic activity on the CMC agar was
established by the appearance of clearance zone
around the fungi which showed that CMC was
degraded. The clearance zone was observed with
the help of Congo red dye.
Morphological identification of the fungal
isolate:
The isolated fungi was purified by repeated subculturing on the Potato Dextrose Agar medium
at regular intervals and incubating at 29°C. The
isolate was identified based on the colony
morphology, microscopic observation and
molecular identification (St-Germain et al.,
1996).
PCR amplification of 18S rRNA gene:
Systematic Position of Alternaria
Kingdom: Fungi
Phylum: Ascomycota
Order: Pleosporales
Family: Pleosporaceae
Genus: Alternaria
Figure 3: 1 % Agarose gel electrophoresis
showed PCR product of ~600bp.
Phylogenetic tree :
Figure 1: Alternariaalternata
Molecular characterization based on 18S
rRNAsequence
The DNA isolated from the desired
cellulaseproducing fungi tentatively identified as
species of Alternaria.
Sequence analysis of the ITS region of
the18SrRNA gene showed that the Sample
wasAlternariaalternata .
Optimization of physico-chemical parameters:
Figure 2:0.5% Agarose gel electrophoresis
showing band of genomic DNA
Conclusion
Cellulose is the most abundant organic
compound on Earth. It was first discovered in
1834 by the French chemist AnselmePayen
(1795–1871). Cellulase is mainly derived from
fungi that degrade cellulose by acting on the
oxygen bond between the individual cellulose
units.The fungi was isolated from the soil near
dump sites. Crude cellulase was isolated by
centrifugation of liquid medium. The fungi
capable of producingcellulase was identified as
Alternariaalternatabased on colony characters,
microscopic observation and identification at
molecular level based on DNA coding for ITS
region18S
rRNA.
To
maximize
the
cellulaseactivity the optimum conditions like
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Acknowledgment
We acknowledge the facility and support
provided by University Institute of
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