Download Identification and characterisation of Bacillus subtilis as cellulase

J. Trop. Agric. and Fd. Sc. 41(2)(2013): 319 – 327
A.K.R. Emmyrafedziawati
Identification and characterisation of Bacillus subtilis as
cellulase-producing bacteria
(Pengenalpastian dan pencirian Bacillus subtilis sebagai bakteria penghasil selulase)
A.K.R. Emmyrafedziawati*
Keywords: carboxymethyl cellulose, filter paper assay, polymerase chain reaction, cloning
Abstract
Three cellulolytic bacteria were isolated from oil palm empty fruit bunch (EFB)
compost under aerobic conditions. The genomic DNA extracted from isolates C4,
C12 and EB6 were amplified using specific primers L15 and L73 to determine
the presence of genes encoding cellulase CelL15 and CelL73 respectively. The
presence of the expected lengths of nucleotide bands at 1,500 bp and 730 bp
respectively, indicated the presence of the putative cellulase genes in these
isolates. The genes encoding the cellulases CelL15 and CelL73 were cloned
into the Bacillus subtilis expression vector Escherichia coli strain JM107 to
identify the cellulase gene in the recombinant plasmids. The filter paper assay
(FPase) on C4, C12 and EB6 were determined after 48 h of incubation period at
37 °C. C12 isolate showed the highest FPase activity at 1.733 ± 0.023 FPU/ml
followed by C4 at 1.718 ± 0.006 FPU/ml. EB6 showed the lowest FPase activity
at 1.695 ± 0.006 FPU/ml. However, the ANOVA results showed that all three
isolates of B.subtilis had no significant differences (p >0.05) in the FPase activity.
The sequence alignment of the 3 isolates C4, C12 and EB6 showed that C4 and
EB6 were the same strain while C12 differed slightly and this was confirmed by
the presence of 2 cellulase genes in C12 compared to only 1 in C4 and EB6.
Introduction
Bacillus subtilis is a ubiquitous bacterium
commonly recovered from water, soil,
air and decomposing plant residues. The
bacterium produces an endospore that allows
it to endure extreme conditions of heat
and desiccation in the environment. The
genus Bacillus consists of a large number
of diverse, rod-shaped Gram positive (or
positive only in early stages of growth)
bacteria that are motile by peritrichous
flagella. Like most members of the genus,
B. subtilis is an aerobic bacterium, except
in the presence of glucose and nitrate,
some anaerobic growth can occur (Claus
and Berkeley 1986). The bacterium is one
of the most extensively studied model
microorganisms, displays superior ability
to produce various secretary enzymes. This
ability has been widely applied to produce
various useful enzymes in the industrial
fields (Westers et al. 2004).
Bacillus subtilis is considered a benign
organism as it does not possess traits
that cause diseases. It is not considered
pathogenic or toxigenic to humans, animals
or plants (U.S. EPA 2011). Extensive recent
studies on proteins (such as cellulase,
protease and amylase) secreted by Bacillus
species have shown that the following
*Strategic Resources Research Centre, MARDI Headquarters, P.O. Box 12301, 50774 Kuala Lumpur, Malaysia
Author’s full name: Emmyrafedziawati Aida Kamal Rafedzi
E-mail: [email protected]
©Malaysian Agricultural Research and Development Institute 2013
319
Bacillus subtilis as cellulase-producing bacteria
Bacillus species produce cellulases: B.
cereus, B. licheniformis, B. subtilis and B.
polymyxa (Sharma et al. 1990). Because
these strains did not produce all three
types of cellulase, they did not extensively
hydrolyze crystalline cellulose (Han et al.
1995).
Enzymes degrading (hemi) cellulose
are found in microbes, plants and the
digestive tracts of animals. Three classes of
cellulases, including endoglucanases (EC
3.2.1.4), exoglucanases (EC 3.2.1.91) and
β-glucosidases (EC 3.2.1.21) are involved
in the degradation of cellulose (Lynd et al.
2002). These enzymes, which cleave the
β-1,4 bond of cellulose, belong to the large
family of glycosyl hydrolases. Cellulolytic
microorganisms play an important role in
the biosphere by recycling cellulose, the
most abundant and renewable biopolymer on
earth. The demand for microbial cellulases
and related enzymes is growing more
rapidly nowadays.
Bacillus species in several respects
has both endo- and exoglucanase activity.
It degrades Cm-cellulose, cellotetraose and
cellopentraose as an endoglucanase and
cleaves aglycosidic bonds in p-nitrophenylbeta-D-cellobioside (pNPC) as an
exoglucanase (Han et al. 1995). Reports
on strains belonging to species such as
B. sphaericus and B. subtilis express high
cellulose degradation activities (Mawadza et
al. 1996; Singh et al. 2004).
In this study, the cellulolytic bacteria
were isolated from oil palm empty fruit
bunch (EFB) compost. Utilising the EFB as
compost was a way of managing agricultural
waste, besides it can also add value to EFB.
We tried to isolate these bacteria from EFB
because in theory, isolates from the original
high cellulose substrate can potentially
produce cellulolytic bacteria for industrial
use. As reported by Sun et al. (1999), EFB
are lignocellulosic composites which are
comprised of cellulose, hemicellulose and
lignin with 44% cellulose fraction. Cellulose
degrading microorganisms can convert
cellulose into soluble sugars either by acid
320
or enzymatic hydrolysis. Thus, microbial
cellulose utilisation is responsible for one of
the largest material flows in the biosphere
(Lynd et al. 2002). However, pure cellulases
from the cellulolytic bacteria are not
commercially produced yet (Shabeb et al.
2010). The most studied group of cellulosedegrading microorganisms is the fungi,
which are characterised by multicomponent,
synergistic cellulolytic enzyme systems
(Ulrich et. al 2008).
The objectives of this study were
to confirm the existence of the cellulase
genes in the C4, C12 and EB6 isolates of
B. subtilis and determine the isolates ability
to produce cellulase enzyme using the filter
paper assay or FPase (measures the total
cellulase activity). B. subtilis has agricultural
applications as a biopesticide and as a plant
stimulant on various crops (Eeden and
Korsten 2004). The Bacillus species showed
potential to convert cellulose into reducing
sugars which could be readily used in many
applications such as animal feeds and feed
stock for production of valuable organic
compounds (Niranjane et al. 2007).
Materials and methods
Isolation of cellulolytic bacteria
The B. subtilis used in this study were
isolated from EFB compost using
enrichment media in the pour plate
technique (Van Soestbergen and Lee 1969).
The main ingredients in the compost are
empty fruit bunches (EFB) and palm oil
mill effluent (POME). Ten-fold serial
dilutions of compost sample were prepared
in sterilised distilled water and 1.0 ml each
of the sample dilutions was spread onto
carboxymethyl cellulose (CMC) agar plates.
The CMC agar medium consisted of CMC
(10.0 g), (NH4)2HPO4 (2.0 g), K2HPO4
(0.5 g), MgSO4.7H2O (0.02 g), CaCl2.2H2O
(0.02 g), FeSO4.7H2O (0.02 g) and distilled
water up to 1,000 ml. The CMC was
supplemented to react as a sole carbon
source. The CMC medium plates were
incubated at 37 °C for 24 h. A single colony
was picked and streaked onto a new CMC
A.K.R. Emmyrafedziawati
medium plate. This procedure was repeated
until a pure culture was obtained.
Identification of bacterial isolates
After obtaining the pure bacterial cultures,
genomic DNA extraction was performed
using a Macharey Nagel Kit (Germany)
according to the manufacturer’s instructions.
The genomic DNA of bacterial isolates
was amplified using 16S rDNA universal
primers; COM1: 5’- CAG CAG CCG CGG
TAA TAC -3’ and COM2: 5’- CCG TCA
ATT CCT TTG AGT TT - 3’ (Schwieger
and Tebbe 1998). The PCR conditions
were as follow: initial denaturation at
94 °C for 3 min, 35 cycles of denaturation
at 94 °C for 45 s, annealing at 61 °C for
45 s, extension at 72 °C for 2 min and a
final extension at 72 °C for 10 min. The
successful bands obtained on 1.0% (w/v)
agarose gel (Figure 1) were outsourced to
First Base Laboratories Sdn. Bhd., Selangor,
for sequencing analysis. The nucleotide
sequences obtained were then matched to
sequences in the Genbank using the Basic
local alignment search tool (BLAST)
provided by the National Centre for
Biotechnology Information (NCBI, USA)
to identify the sequences. The nucleotide
sequences were then aligned using the
ClustalW2 program provided by the
European Bioinformatics Institute (EBI).
Amplification using specific primers to
obtain the cellulase genes CelL15 and
CelL73
Two pairs of DNA oligonucleotide primers
designed by Li et al. (2009), L15F: 5’- AGG
ATC CAT GAA ACG GTC AAT CT -3’
and L15R: 5’- CGA GCT CCT AAT TTG
GTT CTG TT -3’and L73F: 5’- GGA
TCC ATG CCT TAT CTG AAA C -3’ and
L73R: 5’- GGC GAG CTC TTA TTT TTT
TGT ATA - 3’ were used. These primer
sets amplified the entire cellulase genes
CelL15 and CelL73 with an expected size
of about 1,500 bp and 730 bp respectively.
Polymerase chain reaction (PCR) was
performed in an Eppendorf gradient thermal
cycler. The amplification was performed
as follows: initial denaturation for 2 min at
94 °C, 30 cycles each of denaturation for 2
min at 94 °C, annealing for 1 min at 54 °C,
and primer extension for 4 min at 72 °C
and a final extension for 15 min at 72 °C.
The PCR products were electrophoresed
on a 1.0% (w/v) agarose gel, stained with
0.1% (v/v) SybrSafe ® (Invitrogen, US) and
viewed using the Bio-Rad gel documentation
system.
Determination of cellulase activity
A volume of 100 µl pure culture of the
nutrient broth was inoculated into 5.0 ml
of 0.05M sodium citrate buffer (pH 4.75).
A Whatman number 1 filter paper strip
(1 cm x 3 cm) was soaked in this buffer and
two drops of glucose was added on it (Maki
et al. 2011). The sample was incubated
in a shaker incubator at 37 °C for 7 days
at 100 rpm. For observation, the enzyme
reaction was stopped immediately using 3,
5-dinitrosalicylic (DNS) acid. The mixture
was then boiled for 5 min, cooled for 10
min and analysed using the GENESYS
10S UV-VIS spectrophotometer (Thermo
Scientific, U.S.) at 540 nm against the blank.
Glucose equivalents (reducing sugars)
generated during the assay were estimated
by using the 3,5 dinitrosalicylic acid (DNS)
method (Miller 1959) with glucose as the
standard. One international Filter Paper
Unit (FPU) activity was defined as the
enzyme required for releasing one μmol of
glucose per min under the assay conditions
and activities were reported as FPU/ml
(Balamurugan et al. 2011). All samples were
analysed in triplicates and the mean values
were calculated.
Cloning of genomic DNA PCR products
The genomic DNA PCR products were
purified and cloned using the CloneJETTM
PCR cloning kit (Fermentas) with blue-white
screening. The pJET1.2 (50 ng/µl) provided
in the kit was used as the cloning vector.
Transformation was carried out using the
TransformAidTM Bacterial Transformation
321
Bacillus subtilis as cellulase-producing bacteria
Kit (Fermentas). The cellulase genes were
ligated into JM107 plasmid containing a
promoter upstream of the insert site and
digested with EcoRI to allow efficient
binding of blunt ends.
Statistical analysis
All data obtained were analysed using
Analysis Of Variance (ANOVA). The mean
values ± standard deviations (SD) are
presented in Tables 1 – 2.
Results and discussion
Thirteen isolates of the cellulase-producing
bacteria were screened previously on
carboxymethyl cellulose (CMC) plates
stained with Congo red to observe the
cellulolytic activity of the isolated strains
under aerobic conditions. Three Bacillus
isolates which produced the greatest clear
zone diameters were selected as indicated
in Table 1. Carboxymethyl cellulose (CMC)
in medium served as the sole carbon source
for the cellulolytic bacterial growth and the
results showed strong evidence that cellulase
was produced in order to degrade the
cellulose. Carboxymethyl cellulose (CMC)
as the endoglucanase substrate gave the
highest yield of cellulase enzyme.
Results showed that there were
significant differences among the means of
the clear zone diameters at the 0.05 level
(Table 1). EB6 showed the greatest clear
zone diameter (3.18 ± 0.1483 cm) with the
putative cellulase gene CelL73. However,
C12 which had the second greatest clear
zone diameter, showed amplification of
both specific products CelL15 (~1,500 bp)
and CelL73 (~730 bp). Not all 13 isolates
showed amplification of the ~500 bp product
using the 16S rDNA universal primers
COM1 and COM2 (Figure 1). Nucleotide
sequences of isolates which gave the ~500
bp band were blast to the National Centre
for Biotechnology Information (NCBI) gene
bank database to identify the species.
Three of the isolates with greatest
clear zone diameters were selected for
amplification using specific primers to
obtain the cellulase genes CelL15 and
CelL73. Isolates C4 and EB6 were identified
as B. subtilis strain BAB-2742 (gene bank
accession no. KF053069.1) and C12 was
identified as B. subtilis strain SBRh5 (gene
bank accession no. KF053071.1). The
multiple sequence alignment of genomic
Table 2. Mean FPase activity of cellulaseproducing bacterial isolates
Isolate
C4
C12
EB6
FPase activity (FPU/ml)
1.7182 ± 0.006a
1.7331 ± 0.023a
1.6951 ± 0.006a
The FPase activity values shown are the mean
± standard deviation (SD) of triplicates. Means
with the same letter are not significantly different
at p <0.05
Table 1. Screening of Bacillus isolates on CMC plate and PCR amplification using specific primer sets
Isolate
Identification
Diameter of clear
zone (cm)
PCR product obtained
using specific primers
GenBank
acession no.
C4
Bacillus subtilis
(strain BAB-2742)
2.2 ± 0.1871c
+ve L73 (~ 730 bp)
KF053069.1
2.6 ± 0.1225b
KF053071.1
Bacillus subtilis
(strain BAB-2742)
3.18 ± 0.1483a
+ve L15 and L73
(~ 730 bp and 1,500 bp)
C12
EB6
Bacillus subtilis
(strain SBRh5)
+ve L73 (~ 730 bp)
KF053069.1
Note: The values shown for diameter of clear zone are the mean values ± standard deviation (SD) of
triplicates. Means with different letters are significantly different at p <0.05
322
A.K.R. Emmyrafedziawati
Figure 1. PCR products of COM1 and COM2
16S rDNA of all cellulase-producing bacteria
isolates. Lanes 1 and 11 represent 1kb DNA
ladder (Invitrogen, USA). Lanes 2 – 9 (upper
gel) and lanes 12 – 16 (lower gel) show genomic
DNA bands of all isolates. Lane 10 is the
negative control. The genomic DNA amplified is
about 500 bp
DNA from all 3 isolates C4, C12 and EB6
are shown in Figure 2.
Figure 3 shows the gel images of PCR
products amplified by both L15 and L73
primer sets. Primer L15 encoded a cellulase
gene of about 1,500 bp while primer L73
encoded beta-glucan of about 730 bp (Li
et al. 2009). C12 gave a positive result for
both PCR products, CelL15 and CelL73
with an expected size of about 1,500 bp
and 730 bp respectively. The C4 and EB6
isolates amplified only the CelL73 PCR
product with an expected size of about 730
bp. As described by Li et al. (2009), the
C12 sequence contained both cellulase and
beta-glucan hydrolysing enzyme primer
sequences while C4 and EB6 sequences
contained only beta-glucan hydrolysing
enzyme primer sequence.
The sequence analysis of the
recombinants determined that C12 was 99%
identical to B. subtillis cellulase gene in
the Genbank with accession no: NC014976
under the reference genome sequences
database (refseq). Under the protein
cluster database, this sequence showed the
presence of the endo-1,4-beta-glucanase
enzyme (accession numbers: YP004203745,
YP003866220 and NP389695). C12 was
also 99% identical to B. subtilis ssp.
subtilis (accession no. NC017195) which
had the beta-glucanase gene (accession no.
YP005558949). C4 and EB6 showed 99%
identity to B. subtilis cellulase gene with
accession no. NC014976. A study by Li et
al. (2009) showed that the predicted amino
acid (protein) sequence of the CelL15 gene
was cellulase from sequence position 43 to
302 while sequence position from 355 to
437 was cellulose binding domain (CBM3).
Meanwhile for CelL73, gene sequence
position 32 to 242 was a multi-domain of
glycosyl-hydrolases 16 superfamily which is
a more complicated gene.
In this study, the total cellulolytic
activity was assayed by filter paper. Other
assays to determine the specific cellulase
activity (e.g.beta-glucosidase) were not done
since the endoglucanase or endo-1, 4-betaglucanase was the major cellulase found
in all 3 isolates C4, C12 and EB6. Results
in Table 2 shows that C12 isolate had the
highest mean FPase activity (1.733 ± 0.023
FPU/ml) followed by C4 isolate (1.718 ±
0.006 FPU/ml). EB6 showed the lowest
mean FPase activity (1.695 ± 0.006 FPU/
ml). However, ANOVA (Table 2) indicated
that there was no significant differences
in FPase activity amongst the 3 isolates
(p >0.05). Results from this analysis further
strengthened the report of Desvaux (2005)
who stated that a single cellulase gene is
difficult to degrade cellulose even though
it may have high activity under specific
conditions (e.g. clear zone, CMC activity).
Cellulase is an inducible enzyme and it
is affected by the nature of the substrates
used for their production (Haung and
Monk 2004). When filter paper was used
as a substrate, further incubation period
was found to decrease the enzyme activity.
The filter paper assay (FPA) has been
widely used to measure total cellulase
activity because it is readily available and
inexpensive. Coward-Kelly et al. (2003)
reported that assay performed with filter
323
Bacillus subtilis as cellulase-producing bacteria
Figure 2. The multiple sequence alignment of the 16S rDNA (amplified from isolates C4, C12 and EB6
using universal primers COM1/COM2) obtained using ClustalW2 program. C4 and EB6 have the same
sequence (480 bp) while C12 sequence differs slightly (443 bp)
paper provided reliable and reproducible
results. The degradation of the filter paper
would imply multiple cellulase activities
including exoglucanase activities because
these enzymes work in crystalline regions
(Dashtban et al. 2010).
Several studies have shown that B.
subtilis can be used as a host to secrete
heterologous cellulases, and naturally
occurring strains have been identified
that secrete cellulases (Joliff et al. 1989).
Cellulase enzymes have also been targeted
324
to the membrane, enabling protoplasts of B.
subtilis to degrade carboxymethylcellulose
(Kim et al. 2005).
Conclusion
The presence of the cellulase genes was
successfully demonstrated in three locally
isolated cellulolytic bacterial strains (C4,
C12 and EB6) from palm oil empty fruit
bunch (EFB) through amplification using
specific primers and were identified as
Bacillus subtilis. Two of these isolates,
A.K.R. Emmyrafedziawati
a)
730 bp
b)
1,500 bp
Figure 3. a) 1% agarose gel image of PCR product 730 bp amplified using L73 pair of primers in
isolates C4 (lane 2), C12 (lane 6) and EB6 (lane 8). Isolates C6 (lane 3), C7 (lane 4), C10 (lane 5)
and EB4 (lane 7) were not amplified by L73. Lane 1 is 1 kb DNA ladder (Invitrogen,US) and lane 9 is
the negative control. b) 1% agarose gel image of PCR product 1,500 bp amplified using L15 pair of
primers in isolate C12 (lane 4). Isolates C4 (lane 2) and EB6 (lane 3) were not amplified by L15 pair of
primers. Lane 1 is 1 kb DNA ladder (Invitrogen, US)
C4 and EB6, were identified to be the
same strain based on the 16S rDNA gene
sequences. Amplification using two primer
sets encoding CelL15 and CelL73 genes
showed that only C12 contained both the
cellulase genes while C4 and EB6 contained
only one cellulase gene. Assays on their
individual enzyme activities (total cellulase
activity) were not significant. These findings
suggest that combination of the 3 isolates
may be capable of producing higher enzyme
activities. Further studies have to be carried
out to confirm the capability of the mixed
culture by quantification of their enzyme
activities.
Acknowledgment
The author would like to thank Ms Noor
Shita Desa for her assistance in this
project. This study was funded by RMK-10
development fund under White Agriculture
project, No.148-2012.
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A.K.R. Emmyrafedziawati
Abstrak
Tiga bakteria selulosa telah dipencilkan daripada kompos tandan kelapa sawit
(EFB) dalam keadaan aerob. DNA genomik yang diekstrak daripada pencilan
C4, C12 dan EB6 telah diamplifikasi menggunakan primer spesifik L15 dan
L73 untuk menentukan kehadiran gen pengekodan selulase CelL15 dan CelL73.
Kehadiran band nukleotida pada 1,500 dan 730 bp menunjukkan kehadiran gen
selulase berpontensi dalam setiap pencilan. Gen pengekodan selulase CelL15
dan CelL73 telah diklon dalam Bacillus subtilis dengan vektor Escherichia coli
strain JM107 untuk mengenal pasti gen selulase yang terdapat dalam plasmid
rekombinan. Cerakin kertas penapis (FPase) dalam C4, C12 dan EB6 ditentukan
selepas 48 jam tempoh pengeraman pada 37 °C. Pencilan C12 menunjukkan
aktiviti FPase tertinggi iaitu 1.733 ± 0.023 FPU/ml diikuti oleh pencilan C4
pada 1,718 ± 0.006 FPU/ml. EB6 menunjukkan aktiviti FPase yang lebih rendah
pada 1.695 ± 0.006 FPU/ml. Bagaimanapun, keputusan ANOVA menunjukkan
bahawa ketiga-tiga strain B. subtilis tidak mempunyai perbezaan yang signifikan
(p >0.05) dalam aktiviti FPase. Penjajaran jujukan bagi ketiga-tiga pencilan (C4,
C12 dan EB6) menunjukkan C4 dan EB6 adalah strain yang sama manakala C12
berbeza sedikit dan ini disahkan dengan kehadiran 2 gen selulase dalam C12
berbanding dengan hanya satu yang hadir dalam C4 dan EB6.
Accepted for publication on 10 September 2013
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