Novel Approaches for Identification of Streptomyces noboritoensis TBG-V20 with Cellulase Production
The present study was aimed at to identify the cellulase producing Actinomycetes from soil of Southwest ghats, Tamilnadu, India. The morphology, cultural, physiological and chemo taxonomical analysis and phylogenetic analysis of TBG-V20 were characterized. According to the morphological, isolate TBG-V20 was identified as a representative of the genus Streptomyces. The 16S rRNA region of this strain was amplified and sequenced. A Neighbor-Joining algorithm tree of 16S rRNA was constructed. In endocellulase activity, the Carboxy Methyl Cellulose (CMCase) activities of the strain TBG-V20 on eighth day an amount of 935 U L-1 of glucose, 225 μg mL-1 of protein and 870 mg/100 mL of growth (biomass) on tenth day were recorded. In exocellulase activity strain TBG-V20 on first day an amount of 500 μg mL-1 glucose was produced. According to the observation and phylogenetic analysis, the strain TBG-V20 was proved to belong to the species Streptomyces noboritoensis with cellulase production.
Streptomycetes are the largest and well-studied group of actinomycetes. A wide
variety of bacteria are known for their production of hydrolytic enzymes with
streptomycetes being the best known enzyme producers (Vinogradova
and Kushnir, 2003). Bacterial, fungal and actinomycete cellulase and xylanases
have attracted considerable research interest because of their potential applications
in recovery of fermentable sugars from hemicellulose, biobleaching of pulp and
paper industry and to other industrial applications (Rawashdeh
et al., 2005). Actinomycetes, one of the known cellulase-producers,
has attracted considerable research interest due to its potential applications
in recovery of fermentable sugars from cellulose that can be of benefit for
human consumption and to the ease of their growth (Jang
and Chen, 2003) compared to anerobic cellulase producers such as Paenibacillus
curdlanolyticus (Pason et al., 2006). The
biotechnology applications of cellulases began in the early 1980s in animal
feed followed by food applications (Harchand and Singh,
1997). Today, these enzymes account for approximately 20% of the worlds
enzyme market (Jaradat et al., 2008). The Streptomyces
are able to utilize a wide range of organic compounds as a carbon source, including
complex biological materials such as cellulose and lignin and can also utilize
an inorganic nitrogen source (Kutzner, 1986). Nowadays,
enzyme cost is the major impediment to commercialization of enzymatic hydrolysis;
the major challenge is to significantly increase the activity of cellulases
(Jaradat et al., 2008; Walker
and Wilson, 1991). Rawashdeh et al. (2005)
isolated several Streptomyces isolates, upon further characterization; these
isolates were able to produce cellulase, pectinase and relatively large amount
Determination of difference in the sequence of the 16S rRNA gene is well established
as a standard method for the identification and phylogenetic classification
of prokaryotic species, genera and families ( Michael and
Sharon, 2007), but more recently it has also become important as a means
to identify an unknown bacterium to the genus or species level. Thereby, in
this investigation cellulase producing strain TBG-V20 was isolated from soil
samples and characterized to belong to Streptomyces noboritoensis ( Shirling
and Gottlieb, 1969) with cellulase activity by morphology, biochemical and phylogenetic
MATERIALS AND METHODS
All the chemicals used in the analytical methods and media preparation were
of analytical grade with maximum available purity supplied by Hi-media (Mumbai),
SISCO (Chennai) and Sigma (USA). The 16S rRNA primers were from Genei (Bangalore)
and PCR reagents and Taq Polymerase from Biogene, USA. The Actinomycetes presence
soil samples were collected from Southwest ghats, Tirunelveli, Tamilnadu, India.
Isolation and Screening of Cellulase Producing Actinomycetes
In this study, was conducted during the period of November 2007-March 2008,
for that, the soil samples were collected aseptically from Southwest ghats,
Kerala, India at a depth of 6-12 inches. The soil samples were air dried in
a hot air oven at 45°C for 1 h to reduce the proportion of bacteria other
than Actinomycetes (Williams et al., 1972). Standard
dilution plate technique was followed for the isolation of Actinomycetes (Kuster
and Williams, 1964). Ten gram each of the soil samples were added to 90
mL distilled water in a 250 mL Erlenmeyer flask under sterile condition and
kept in a rotary shaker (120 rpm) at room temperature for 30 min. Vacuum filtration
was used for collecting supernatant and then the supernatant was serially diluted
to obtain 10-4, 10-5 and 10-6 dilutions. Each
dilution was plated on sabourauds agar. After inoculation of 7-9 days
at 27±1°C the actinomycetes colonies were selected, counted and made
into pure culture following single spore culture technique. The culture was
maintained on sabourauds agar by periodical sub-culturing.
In screening, 24 colonies were used for screening; the inorganic salts cellulose agar was prepared. Twenty milliliter of the medium was poured in to the petriplates. The isolated Actinomycetes were inoculated into different petriplates and incubated at room temperature (27±1°C) for 7 days then the plates were taken and 0.1% congored was spread over the plates. After half hour incubation 0.5 M NaCl was spread. After the 0.5 M NaCl was discarded, the presence of a clear zone indicates the cellulolytic activity of the strain. The diameter of the zone is measured and based on this the activity of the organisms were categories as weak and promising ones.
The inorganic salts cellulose broth supplemented with 1% Carboxy Methyl
Cellulose (CMCase) and 1% yeast extract was prepared. One hundred milliliter
of the broth was taken in 250 mL Erlenmeyer flasks and inoculated heavily using
the individual stock cultures maintained on sabourauds slants. The flasks
in duplicate were incubated at room temperature at 120 rpm. During incubation,
every day 1 mL of the broth was collected up to 12th day for CMCase assay. In
endocellulase determination the β (1-4) glucanase was determined by measuring
the reducing sugar as glucose by dinitrosalicyclic acid (DNS) method (Miller,
1959). The reaction mixture was contained 1 mL of w/v CMC in citrate buffer
pH 5.0 and 1 mL of the broth, which was collected on first day to seven days.
The assay was started by incubating the reaction mixture at 50°C for 20
min. The reaction was stopped by the addition of 3 mL DNS reagent and boiling
for 5 min. After cooling the absorbance was read at 540 nm in a colorimeter.
Gluscose was used as the standard and the reducing sugar formed was calculated
as the glucose equivalent. One unit of CMCase was expressed as 1 μmol of
reducing sugar released per min per mL. In exocellulase activity, the Whatman
No.1 filter papers were cut into 4 mm dia pieces. A reaction mixture was contains
1 mL of 1% citrate buffer pH 5.0 and 1 mL of the broth was prepared. Thirty
milligram filter papers were added to it. Further assay was similar to the above.
For determining the amount of cellulase (protein estimation) produced by strain
TBG-V20 was performed by the method of Lowry et al.
Extraction of Protein
Extraction was carried out with buffers used for the enzyme assay. Five
hundred milligram of the sample was weighed, ground well and grinded well with
a mortar and pestle in 5-10 mL of the buffer. Protein estimation was carried
out based on method described by Lowry et al. (1951).
Standard graph was sketched and calculated the amount of protein in the sample.
Expressed the amount of protein in IU mL-1 of culture broth was used
for calculation. Estimating a biomass, 100 mL culture was filtered through a
pre-weighed and dried Whatman filter paper. The filter paper was dried at 40°C
for 24 h and the weight was taken in a weighing balance. The biomass was expressed
in mg mL-1. Based on the performance of cellulose activity out of
24 one colony was taken for further study.
Morphology and Taxonomy
The determination of Aerial mass colour, reverse colony colour and the 7
to 14 days old cultures growth characters in different culture media which were
reported by International Streptomyces Project (ISP) (Shirling
and Gottlieb, 1966). Medium ISP-1 to ISP-7 was used according to description
of Shirling and Gottlieb (1966). The colour of soluble
pigments were noted and compared with Methuen hand book of colour which was
noted from Kornerup and Wanscher (1967). The microscopic
studies were prepared according to the method described by Chakrabarthi
(1998). The pure cultures of the Actinomycetes strains were inoculated to
the contract line of immersed cover slips at an angle of 45° on Sabourauds
agar plates prepared aseptically. The plates with cover slips were incubated
at 28°C for 4-8 days. After attaining suitable growth with spore chains
the cover slips were removed and placed on micro slides with the growth on the
upper surface. The slides were viewed under a phase contrast microscope (Nikon
Optiphot-II). The details of the conidial chain/sporangium, spores, Arial and
substrate mycelium and fragmentation of the mycelia were noted and the slides
were photographed at suitable magnifications. In physiological and biochemical
studies, melanin production was determined on ISP-6 media; after 4 days of growth
the colour change of media were noted by the methodology described by Shirling
and Gottlieb (1966).
Carbon Source Utilization
Different organic carbon sources were sterilized using di-ethy1 ether and
incorporated to carbon utilization agar (Shirling and Gottlieb,
1966). These include D-Fructose, meso-Inositol D-mannitol, Raffinose, L-Rhamnose,
Sucrose and D-Xylose at 1% w/v. Results were determined after 7, 14 and 21 days
by comparing with a negative control (without carbon source) and a positive
control containing D-Glucose. In Chemotaxonomical studies, the Actinomyces strains
were inoculated in 100 mL sabarauds broth in Erlenmeyer flask and incubated
for 5 days at room temperature in a rotary shaker (120 rmp). The cells were
killed with formalin (con.1%) for 24 h at room temperature and harvested by
centrifugation. The cells were washed once in double distilled water and once
in 95% ethanol and then dried by overnight heating in a hot air oven at 45°C.
The dried cells were analyzed for Diaminopimilic acid (DAP) and carbohydrates
(Staneck and Roberts, 1974).
Extraction of Genomic DNA
Samples used for DNA extraction were collected from a highly sproulated
growth on sabourauds agar were inoculated to 25 mL yeast extract malt
extract (YEME) broth supplemented with 0.5% glucose and 5 mM MgCl2
in a 100 mL Erlenmeyer flask, incubated for 2-3 days at room temperature at
10 rpm. Genomic DNA was isolated as described by Murray and
Thompson (1980). The integrity of the obtained genomic DNA was detected
by electrophoresis in 1% agarose gel (Sigma Aldrich, India) stained with ethidium
16S rRNA Amplification
The 16S rRNA was amplified using the bacterial universal primer pairs. The
reaction mixture for PCR amplification was prepared in a total volume of 50
μL with 10x PCR buffer, 10 mM of dNTPs, 1.0 μL of the taq polymerase
and 200 pmol of forward and reverse primers. The amplifications were performed
in a DNA thermal cycler 480 (Perkin Elorer, USA). The PCR reaction details were
as follows: 5 min at 95°C for initial denaturation, 1 min at 94°C for
denaturation, 2 min at 49°C for annealing, 1 min at 72°C for extension
with total 24 cycles of amplification and 7 min at 72°C for the final extension.
The 16S rRNA (EU857664) was purified using GenEluteTM Gel Extraction
Kit (Sigma Aldrich, USA) and sequencing of 16S rRNA gene was done in an automated
ABI-3100 Genetic Analyser (GeNei, India).
BLASTN (optimized for megablast) searches were manipulated with the sequences
of Streptomyces noboritoensis TBG-V20. The corresponding sequences of
representative species were used for phylogenetic analyses. Neighbor-Joining
(NJ) algorithm was used to construct a graphical phylogenetic tree using software
MEGA 4.1 (Tamura et al., 2007). The program was
starts with a set of aligned sequences using Clustal W and searches for phylogenetic
trees that are optimal according to NJ algorithm.
GenBank Accession Number
The GenBank accession number of the sequence reported in this study is EU857664.
RESULTS AND DISCUSSION
In this study, 24 strains were obtained and one colony was found to produce
cellulase which could be used for further studies and it was termed as TBG-V20.
Cellulase activity of strain TBG-V20 on inorganic salt cellulose agar was found
to be 26 mm diameter zone after 7 days of incubation at 28±1°C.
||Cellulase producing strain Streptomyces noboritoensis
TBG-V20. (A) Top morphology view of Aerial mycelium with conidial chains
(>50 spores), (B) Presesnce of LL-Diaminopimilic acid as major cell wall
peptidoglycan, (C) Predominantly white spore mass colour and (D) Reverse
colony colour is yellow brown; (E) Production of melanin (Aerial mass colour)
in ISP media
The isolate TBG-V20 gives morphology similar to that of a Streptomyces
isolate with a distinct substrate and aerial mycelium with conidial chains observed
on microscopic examination. The conidial chains with more than 50 spores were
observed and these were oblong in shape (Fig. 1A). The spore
chains of Streptomyces may be of different type, straight to flexuous
(Rectus-flexibilis), open loops (Relinaculam-apertum), pen of closed spirals
(spira) and verticillate (Pridham et al., 1958).
The microsopic examinations of the present isolate TBG-V20 shows that the conidial
chains are Rectus-flexibilis. The cell wall analysis shows the presence of LL-Diaminopimilic
acid (LL-DAP) as major cell wall peptidoglycan content (Fig. 1B).
There were no diagnostic sugars detected in cell wall. So the isolate TBG-V20
was identified as a representative of genus Streptomyces (Waksman
and Henrici, 1943).
Table 1 shows the cultural characteristic of the isolate
after an incubation period of one week at 28±1°C. The spore mass
color of the Streptomyces members may vary from white, gray, red, yellow,
green and blue to violet. Colour of the aerial mycelium is one of the prominent
identification characters of Streptomyces isolates at species level (Pridham
and Tresner, 1974). On further examination of the morphological characters
the isolate is having a spore mass colour of predominantly white (Fig.
1C) and occasionally grey. The International Streptomyces Project
(ISP) (Shirling and Gottlieb, 1966) has recommended recording
of aerial mycelial colour in different media for use as a taxonomic character,
which was also followed in the present study. ISP-6 and ISP-7 were showed the
reverse colony color is yellow brown and is not producing any diffusible pigments
(Fig. 1D). The isolate was showing good growth on ISP media
and other suggested media (Table 1). The above-mentioned characters
suggest that the isolate TBG-V20 can be identified as Streptomyces groups
of white series (Nonomura, 1974).
||Cultural characteristics of isolate TBG-V20
||Results of similarity searches between 16S rRNA genes isolated
in the present investigation and GenBank accessions using BLASTN Algorithm
(optimized for megablast)
The isolate was utilizing D-fructose, Meso-ionsitol, Mannitol, Raffinose, Rhamnose
and D-xylose as the carbon source. In addition, isolate TBG-V20 was not utilizing
sucrose as the carbon source. The utilization of carbon compounds is an important
aid for species determination among actinomycetes (Pridham
and Gottlieb, 1948). The isolate TBG-V20 utilized almost all carbohydrates,
but not sucrose respectively. Likewise, the pigment production stands out as
a characteristic tool in identifying the various microorganisms. However, TBG-V20
does not produce any of these pigments, these minor physiological characters
are not delaminating the identification of this strain as Streptomyces noboritoensis.
The keys of Kuster (1972), Nonomura
(1974), Szabo et al. (1975) and Shirling
and Gottlieb (1966) with based on ISP description giving emphasis to aerial
mass colour, melanin pigment production, spore chain morphology and carbon source
utilization pattern, the isolate TBG-V20 was identified as Streptomyces noboritoensis.
A 557 bp sequence was amplified from the genome DNA with the bacterial universal
primers, its sequence was submitted to GenBank (EU857664). As is shown in Table
2, strain TBG-V20 16S rRNA sequence was used for identity search, which
was made using BLASTN algorithm (optimized for megablast). The 16S rRNA gene
showed high similarity with 16S rRNA genes deposited in the GenBank (Table
2). TBG-V20 strain 16S rRNA had 100% identity (E value 0.0) with 16S rRNA
gene of S. noboritoensis (FJ769838) followed by 100% idenity (E value
0.0) with 16S rRNA gene of S. phaeochromogenes (FJ486378) and 100% identity
(E value 0.0) with 16S rRNA gene of S. melanogenes (AB184222). Moreover,
TBG-V20 had 99% identity (E value 0.0) with 16S rRNA gene of S. olivochromogenes
(EU841608) and S. crystallinus (AB184652) and also TBG-V20 had 98% identity
(E value 0.0) with S. flavotricini (FJ532405), S. polychromogenes
(FJ547116), S. erythrochromogenes (AB184746), S. globosus (EU196532),
S. lavendulae (EF371426), S. bikiniensis (EF620359), S. gobitricini
(AB184666), S. lavendofoliae (AB184217), S. herbaricolor (DQ442505),
S. lilaceus (AB184457), S. katrae (EF654092), S. racemochromogene
(AB184235), S. lavendulocolor (AB184216), S. luridus (AB184150),
S. toxytricini (EU841711), S. bikiniensis (EU560974), S. castaneus
(AB184453) and S. venezuelae (EU221352). In this study, 16S rRNA gene
of different Streptomyces species (different strains of a species) was
obtained by BLASTN search, however 23 strains of Streptomyces species
were selected on the basis of high identity (%) with good E value for phylogenetic
analysis. As shown in Fig. 3, two strains belonging to Streptomycetaceae
were relatively closely related to Streptomyces; strain TBG-V20 had a
clade supported with S. noboritoensis (FJ769838).
The cumulative results from a limited number of studies to date suggest that
16S rRNA gene sequencing provides genus identification in most cases (>90%)
but less so with regard to species (65-83%), with regard to species from 1-14%
of the isolates remaining unidentified after testing (Drancourt
et al., 2000; Mignard and Flandrosis, 2006;
Woo et al., 2003). Michael
and Sharon, 2007 reported that, the minimum 500-525 bp essential for phylogenetic
analysis and also for species identification minimum >99% similarity and
ideal >99.5% similarity should be desirable. <0.5% similarity and other
properties such as phenotype should be considered to final species identification.
E value is related to the probability that the observed degree of similarity
could have arisen by change: E is the number of sequences that would be expected
to match as well or better than the one being considered, if the same database
were probed with random sequences.
||Cellulase activities of strain TBG-V20. (A) CMCase activities
of the Streptomyces isolate TBG-V20; (B) Exocellulase activities
of Streptomyces isolate TBG-V20
||Phylogenetic tree is based on the nucleotide sequence of 16S
rRNA genes. The Neighbor-Joining algorithm tree was constructed by MEGA
Values of E below about 0.05 would be considered significant; at least they
might be worth considering (Arthur, 2005). According to
Michael and Sharon, 2007 and Arthur (2005) reports, in
this study, isolate TBG-V20 (557 bp) had (100%) ideal >99.5% similarity and
E value (0.0) <0.05 with S. noboritoensis, S. phaeochromogenes
and S. melanogenes. On the basis of BLASTN search and phenotypic
results TBG-V20 was identified as a strain of S. noboritoensis.
Comparison of 16S rRNA sequences has become the gold standard for
the elucidation of phylogenetic relationships among microorganisms. As the number
of sequences available for analysis continues to grow, the structure of phylogenetic
trees derived from these sequences becomes both more intricate and more accurate
(OConnor et al., 1991).
Pernodet et al. (1989), Chang et al. (1997)
and Fukushima et al. (2002) reported that 16S
rRNA, gyrB and 23S rRNA of various Streptomyces, Salmonella,
Shigella and Eshcerichia coli species were partially sequenced and
used for defining all members of the genus, groups of species or individual
species. As shown in Fig. 3, two strains belonging to Streptomycetaceae
were relatively closely related to Streptomyces; strain TBG-V20 had a
branch supported with S. noboritoensis (FJ769838). The results supported
that the phylogenic position of strain TBG-V20 in the genus Streptomyces
might belong to the family Streptomycetaceae (Kataoka et
al., 1997). The results of a phylogenetic analysis based on the 16S
rRNA sequences can be suggested strain TBG-V20 should be positioned in the genus
Streptomyces. In addition, the special characteristics mentioned in the
study suggest that novel strain TBG-V20 belong to the species S. noboritoensis
with high cellulose activity.
Cellulase Activity of the Isolate
Actinomyces have been always a source of thousand of bioactive compounds.
Enzymes are one of the important products of this unusual group of bacteria.
In endocellulase activity, the CMCase activites of the isolate TBG-V20 on different
days of incubation is shown in Fig. 2A. The isolate was showing
good CMCase activity on the eighth day. On the eighth day an amount of 935 U
L-1 of glucose and 225 μg mL-1 of protein were recorded
at pH 5 and 50°C temperature. The growth (biomass) was 870 on the tenth
day. In exocellulase activity of the isolate TBG-V20 on different days of incubation
are shown in Fig. 2B. The isolate was showing maximum exocellulase
activity on first day of incubation. On the first day an amount of 500 U L-1
glucose was produced. Jaradat et al., 2008 have
observed that Streptomyces strain J2 is showed the highest crude enzyme
activity (432 U L-1) after 3 days of incubation at pH 7 and 60°C
of temperature. This result is considerably similar to what was reported by
Theberge et al. (1992) who showed that the optimum
pH for endoglucanase from a strain of Streptomyces lividans was 5.5.
However, the results appeared to contradict previous results reported by Solingen
et al. (2001) of an alkaline novel Streptomyces species isolated
from east African soda lakes that have an optimal pH of 8, highlighting the
effect of alkaline environment on the adaptation of these Streptomyces. Furthermore,
the maximum CMCase activity of our isolate TBG-V20 was recorded at 50°C
with no significant difference between 50 and 60°C. These results are in
agreement with results reported by McCarthy (1987),
who reported an optimal temperature for cellulase activity in the range of 40-55°C
for several Streptomyces species including S. lividans, S. flavogrisus
and S. nitrosporus. Jang and Chen (2003)
described a CMCase produced by a Streptomyces T3-1 with optimum temperature
50°C, whereas Schrempf and Walter (1995) described
a CMCase production by a S. reticuli at an optimum temperature 55°C.
Schlochetermeier et al. (1992) and Wachinger
et al. (1989) reported that Actinomycetes are playing an important
role in degradation of cellulose and Streptomyces reticuli is a soil
bacterium, which hydrolyzes crystalline cellulose due to the action of exocglucanase.
Kamini et al. (1999) also reported Streptomyces
species with cellulose activity. The isolate TBG-V20 is showing high cellulolytic
activity in the preliminary screening. In this study the present strain TBG-V20
was showing good endocellulolytic activity in eighth day and exocellulolytic
activity in first day which were differentiate our isolate from other scientist
described strains. Isolate TBG-V20 had produced According to the above evidences,
the present strain TBG-V20 was identified as a high cellulose producing Streptomyces
According to the results, the strain Streptomyces noboritoensis TBG-V20 has potentiality for industrial production for the reason that of the high yield of cellulase. It has been reported that the biosynthesis of cellulase is induced during growth on cellulose or other cellulose derivatives. In all cases, it has been found that it is essential to keep the required nutrients at low level to insure maximum accumulation of fermentation products. Overall, the study indicated that cellulase production from TBG-V20 isolate has potentiality for industrially important in this world for the production of the high yield of cellulase.
Authors thanks to Manonmaniam Sundaranar University, Alwarkurichi, India and Muthayammal College of Arts and Science, Rasipuram, India for provided necessary facilities.
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