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Buddana 2016
Buddana 2016
000321
Although only few bacterial mutanases were reported, they were compared with the GenBank database through NCBI BLAST (http://
have beneficial properties, mainly because of the mode of www.ncbi.nlm.nih.gov). Alignment of nucleotide sequences was carried
catalysis and have more endolytic activity compared to fungal out using CLUSTAL W software. Data analysis was performed on a boot-
strapped dataset containing 1000 replicates. To determine the genetic
mutanases, and hence help in the rapid degradation of dental relationship between these strains, a phylogenetic tree was generated
plaques (Pleszczynska et al., 2007). Therefore, it is motivating based on the percentage difference between the sequences using the
to find new, efficient sources for mutanase production with neighbour-joining method and MEGA 5.05 software.
better yields. In this study, the authors report a new bacterial
strain that produces more mutanase enzyme inductively by Production and estimation of mutanase. The isolated Paracoccus
the supplementation of cell wall-associated a-(1fi3)-glucans sp. RSP-02 was used for the production of mutanase enzyme. Initially,
isolated from Streptococcus mutans (MTCC 497). the isolate was grown in 50 ml of nutrient broth as seed medium, and
later 4 ml of seed culture was transferred to 100 ml of mutanase pro-
duction medium (MPM) (pH 7.0±0.2) consisting of mutan 4.0 g l 1,
inositol 10.0 g l 1, peptone 5.0 g l 1, yeast extract 3.0 g l 1, KH2PO4
METHODS 2.0 g l 1, NH4Cl 2.0 g l 1, KNO3 2.0 g l 1 and MgSO47H2O 0.3 g
l 1. The production of mutanase enzyme was carried out at 30 C and
Preparation of mutan. Mutan [a-(1fi3)-glucan] used in this study 150 r.p.m. A 2 ml sample was withdrawn every 24 h, and mutanase
was produced from Streptococcus mutans (MTCC 497) according to activity was monitored by incubating 1.0 ml cell-free broth with 1.0 ml
Buddana et al. (2015). Briefly, Streptococcus mutans (MTCC 497) strain of 0.2 % (w/v) mutan suspended in 0.2 M sodium acetate buffer, pH
was grown in brain–heart infusion (BHI) medium supplemented with 5.5, at 50 C. The reducing sugars released in 30 min were estimated
5.0 % sucrose at 37 C at an agitation speed of 150 r.p.m. After by the Nelson–Somogyi method using D-glucose as a standard. One
72 h incubation, the cell mass was separated by centrifugation at unit of mutanase activity (U) is defined as the amount of enzyme that
15 000 r.p.m., and the pellet was treated with 2 M KOH for 2 h at 95 C catalysed the release of reducing sugars equivalent to 1.0 µmol min 1
for the extraction of cell-associated glucans. The alkali mixture was cen- under the defined conditions. Subsequently, a plate assay was per-
trifuged at 15 000 r.p.m. for 30 min, and supernatant was collected and formed to analyse the incubation period of maximum enzyme
neutralized to pH 7.0 with glacial acetic acid for the precipitation of production.
mutan. The precipitated mutan was collected and washed thrice with
deionized water, lyophilized and stored at room temperature until fur-
Hydrolysis of mutan and identification of degradation
ther use.
products. The reaction mixture (10 ml) consisted of 10 mg of mutan
in 9 ml of 0.2 M sodium acetate buffer (pH 5.5) and 1.0 ml of crude
Screening and isolation of mutanase-producing microbial
mutanase enzyme in 50 ml boiling tube. The tubes were incubated at
strains. To screen mutanase-producing bacteria, a serial dilution tech-
100 r.p.m. and at 50 C in a water bath shaker. After 24 h incubation,
nique was used. Soil samples were collected from wastes dumped at
reaction mixtures were subjected to 100 C for 5 min to stop the reac-
the Sirpur Paper Mills. One gram of soil sample was added to 100 ml
tion and analysed for total reducing sugars. The hydrolytic end products
distilled water and stirred for 30 min at 150 r.p.m. The resultant suspen-
were identified by TLC. TLC was performed on aluminium plates
sion was subjected to serial dilutions from 10 1 to 10 10. From each
coated with silica gel (Merck) using isopropanol, ethyl acetate, nitro
dilution, about 1 ml of sample was taken and inoculated into a medium
methane and water (6 : 1 : 1 : 2) as mobile phase. The reducing sugars
containing 1.0 % mutan as the sole carbon source and 2.0 % agar by
the pour plate method. The plates were incubated at 37 C. After 7 days were detected by spraying 0.2 % orcinol dissolved in sulfuric acid and
of incubation, the bacterial colonies that developed were picked carefully methanol (1 : 9). HPLC analysis of mutan-degraded products was per-
and maintained on nutrient agar slants at 4 C until further use. formed using a Waters Alliance HPLC system fitted with a phenomenex
H+ (monosaccharide) column and refractive index detector at 60 C
Secondary screening (Congo red test). Mutan-containing agar with a run time of 60 min. Five millimolar sulfuric-acid-supplemented
medium was used for secondary screening of selected bacterial strains water was used as mobile phase at a flow rate of 0.5 ml min 1.
for mutanase production. Only those strains that showed substantial
growth in the initial screening were selected for the Congo red test. Purification of mutanase. The cell-free broth of Paracoccus sp. RSP-
Inoculation was carried out by using a nichrome loop by cross streaking 02 cultivated at 30 C for 72 h in 1 litre flask containing 250 ml of
on 1.0 % mutan-supplemented agar plates. Plates were incubated at mutanase production medium (as above) was used for harvesting
30 C for 96 h for developing the colonies followed by addition of 10 ml mutanase enzyme and its purification. The enzyme was precipitated by
of Congo red dye (2.5 g l 1) to each plate. After 15 min, the Congo red addition of ammonium sulfate from 20 % to 80 % saturation. The pre-
dye solution was discarded, and the plates were washed with 10 ml of cipitate was dissolved in 30 ml of 0.05 M sodium phosphate buffer (pH
1 M NaCl solution. Mutanase-positive strains were identified according 7.0), and the enzyme solution was dialysed against the same buffer over-
to halo zones produced around the colonies. The best isolate was night at 4 C. Later, the dialysed solution was subjected to DEAE-cellu-
selected for further studies based on the maximum zone of hydrolysis. lose ion-exchange chromatography, which was pre-equilibrated with
0.2 M Tris/HCl buffer pH 6.8, and the enzyme sample was eluted with
Identification and phylogenetic analysis of the organism. Gram gradient NaCl concentration ranging from 0.1 to 1.0 M. The collected
staining and morphology of the selected potential bacterial strain (RSP- fractions were quantified for enzyme and protein estimation. The
02) were studied by conducting biochemical tests according to Bergey’s enzyme-rich factions were pooled and ultracentrifuged using 100 kDa
Manual of Bacteriology. Specific tests such as indole production, Methyl molecular mass cut-off (Millipore) filters for concentrating enzyme
Red and Vogues Proskauer Test (MR-VP) test, gelatin hydrolysis, citrate solution and to remove unwanted low-molecular mass proteins. The
utilization, triple sugar iron agar, oxidase test, catalase production, concentrated enzyme sample was used for molecular mass determina-
nitrate reduction, urease test and starch hydrolysis were performed. Fer- tion by native PAGE analysis.
mentation efficiency of different sugars as sole carbon sources such as
arabinose, sucrose, glucose, fructose, rhamnose, xylose, raffinose, man- In vitro studies of mutanase on degradation of biofilm. All stud-
nitol, etc., was evaluated for the growth of the selected strain. Molecular ies were performed in sterile conditions. Initially, 10-cm-length glass
characterization based on ribotyping of 16S rRNA was performed at plates were placed (half immersed) in 20 ml boiling tubes containing
the Xcelris Genomics Limited, Ahmadabad, India. Nucleotide sequences BHI medium supplemented with sucrose. The tubes were then
Fig. 1. Congo red plate assay for secondary screening for mutanase-producing isolated microbial strain.
autoclaved at 121 C for 20 min; after cooling, the tubes were inoculated similar to that reported by Florencio et al. (2012). This sim-
with a loopful of Streptococcus mutans culture and incubated at 37 C for ple and rapid method was highly effective for the selection
48 h; one tube without organism served as control. Later, the glass plates
were removed from the boiling tubes and washed with phosphate buffer of maximum mutanase and other polysaccharide-
(pH 6.2) for removal of excess cells attached to the biofilm. The biofilm-
attached glass plates were then treated with purified enzyme for 30 min. Table 1. Biochemical characterization of Paracoccus
Reducing sugars were estimated from the test and control samples. mutanolyticus RSP-02
Later, tested glass plates were dyed with crystal violet for visual observa-
tion of degradation of the biofilm. Biochemical test Result
degrading, enzyme-producing microbial strains (Florencio Identification and phylogenetic analysis of the
et al., 2012; Ten et al., 2004). Congo red interacts with a- organism
(1fi3)-glycosidic bonds giving an indication of red colour, Microscopic analysis of the selected microbial strain showed it
while the mutanase-hydrolysed portion of mutan remains to be cocci shaped, Gram-negative and non-motile. The strain
colourless. Thus, halo produced by hydrolysis of mutan was showed good growth under aerobic conditions and the cata-
directly related to the region of action of mutanolytic lase test was positive. Growth was optimal at 37 C. Biochemi-
enzyme (mutanase) as reported by Lamb & Toy (2005). cal tests such as starch hydrolysis, citrate utilization, urease
Among the five microbial strains tested, bacterial strain and nitrate reductase were positive for this strain, while casein
designated as RSP-02 exhibited maximum halo zone hydrolysis, MR-VP, phenylalanine deamination, indole pro-
(Fig. 1); hence, this strain was selected for further studies. duction, oxidase production and gelatin liquefaction were
(a) (b)
1 0.8
0.9
0.7
0.8
0.6
Log growth & protein
0.7
Enzyme (U ml–1)
0.6 0.5
0.5 0.4
0.4
0.3
0.3 Mutanase (U ml–1)
Log growth 0.2
0.2
0.1 Protein (mg ml–1) 0.1
0 0
0 24 48 72 96 120
Time (h)
Fig. 3. Production of mutanase enzyme by Paracoccus mutanolyticus RSP-02 by time profile. (a) Production of mutanase
enzyme is growth associated, indicating that enzyme production increased with increase of growth of organism as a function
of time. (b) Hydrolysis of mutanase enzyme against mutan [a-(1fi3)- glucan] by the Congo red plate method at different pro-
duction time intervals A: 0 h, B: 24 h, C: 48 h and D: 72 h.
2.0 5
Microbispora rosea (Chung et al., 2001) and Paneibacillus MP-
1 (Pleszczynska et al., 2007), no reports are available in the lit- 4
1.5
erature on the production of mutanase by Paracoccus sp.
Thus, to the best of our knowledge, Paracoccus mutanolyticus 3
1.0
RSP-02 is a new source of mutanase enzyme. This strain was 2
deposited in the Microbial Type Culture Collection (MTCC), 0.5
Chandigarh, India, with an accession number MTCC 12283. 1
0.0
0
0 20 40 60 80 100 120
Fraction
Production and estimation of mutanase
In view of the first report on mutanase production by Para-
coccus mutanolyticus RSP-02, the isolate was investigated for Fig. 5. DEAE-cellulose ion-exchange chromatography. Inset
its enzyme production under submerged fermentation image shows the plate activity of the obtained four peaks (F1–F4),
using production medium at 30 C and at 150 r.p.m. The with F1 showing mutanase activity.
on a glass surface, indicating its role in the prevention of Hotz, P., Guggenheim, B. & Schmid, R. (1972). Carbohydrates in pooled
streptococcal infection. However, in view of the presence of dental plaque. Caries Res 6, 103–121.
other polysaccharides in dental plaque, mutanase could be Lamb, J. & Loy, T. (2005). Seeing red: the use of Congo red dye to identify
one of the components which play a role in disrupting plaque. cooked and damaged starch grains in archaeological residues. J Archaeol Sci
32, 1433–1440.
This is the first report on the production of mutanase by Para-
coccus sp., and hence this strain is designated as Paracoccus Matsuda, S., Kawanami, Y., Takeda, H., Ooi, T. & Kinoshita, S. (1997).
Purification and properties of mutanase from Bacillus circulans. J Ferment
mutanolyticus.
Bioeng 83, 593–595.
Meyer, M. T. & Phaff, H. J. (1980). Purification and properties of
(1.3)-a-glucanases from Bacillus circulans WL-12. J Gen Microbiol 118,
ACKNOWLEDGEMENTS 197–208.
Pleszczyn ska, M., Marek-Kozaczuk, M., Wiater, A. & Szczodrak, J.
The authors thank the Director of CSIR-IICT, Hyderabad. Mr
(2007). Paenibacillus strain MP-1: a new source of mutanase. Biotechnol
S. K. B. gratefully acknowledges the CSIR, New Delhi, for providing
Lett 29, 755–759.
a senior research fellowship.
Quivey, R. G. & Kriger, P. S. (1993). Raffinose-induced mutanase
production from Trichoderma harzianum. FEMS Microbiol Lett 112,
307–312.
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