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Journal of Medical Microbiology (2016), 65, 985–991 DOI 10.1099/jmm.0.

000321

An endolytic mutanase from novel strain


Paracoccus mutanolyticus: its application
potential in dentistry
Sudheer Kumar Buddana,1,2 Prakasham Reddy Shetty1 and
Sambasiva Rao Raja Surya Krothapalli 3
1
Correspondence Bioengineering and Environmental Sciences, CSIR-Indian Institute of Chemical Technology,
Prakasham Reddy Shetty Hyderabad, India
prakasam@iict.res.in 2
Academy of Scientific and Innovative Research, New Delhi, India
or 3
Department of Biotechnology, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur, India
prakashamr@gmail.com

Mutanase, a-(1fi3)-glucanase, belonging to the family glucanohydrolase, catalyses mutan


[a-(1fi3)-glucan] synthesized by cariogenic streptococci and hence has potential in caries
prophylaxis. A novel bacterial strain with potential to produce higher mutanase
(glucanohydrolase) activity was isolated from soils contaminated with cellulosic waste. One of
the isolated strains, RSP-02, was subjected to biochemical and 16S rRNA molecular analysis,
and we noticed that it belongs to the genus Paracoccus. The mutanase production (800–
1200 U l 1) in this strain was growth associated and substrate induced, and the activity was
comparable with the strains reported earlier. The enzyme displayed a molecular mass of 138 kDa
by native PAGE studies, showed endolytic activity and produced nigerose as end product.
In vitro studies revealed production of 140±2.82 µg of glucose equivalents in 30 min from the
biofilm formed on glass surface indicating its potentiality in dentistry. To the best of our
Received 9 May 2016 knowledge, this is the first report on the production of mutanase by Paracoccus sp.; hence, this
Accepted 22 July 2016 isolated bacterial strain is designated as Paracoccus mutanolyticus RSP-02.

INTRODUCTION sp.) metabolism-mediated production of lactic acid, which


drastically lowers the oral pH (4.0) leading to the deminerali-
Dental plaque in general consists of water-soluble and water-
zation of enamel (Balakrishnan et al., 2000). Thus, one of the
insoluble portions in the ratio of 30 % and 70 %, respectively.
strategies to reduce the disease potential of dental plaque could
The water-insoluble matrix polysaccharide predominantly
be the use of carbohydrate-degrading enzymes to disrupt the
contains a-(1fi3)-glucan and a-(1fi6)-linked dextran (Horz
molecular architecture of plaque. Polysaccharide-hydrolysing
et al., 1972). This insoluble glucan polymer (mutan) plays a
enzymes such as dextranase (EC 3.2.1.11) (Fitzgerald et al.,
pivotal role in the development of dental plaque and in the
1968) and mutanase (EC 3.2.1.59) (Guggenheim et al., 1980)
onset and progression of dental disease (Quivey & Kriger,
were used in the treatment of dental plaques. Between these
1993). Mutan, a polysaccharide majorly comprising a-(1fi3)-
two enzymes, mutanase plays a key role in dental caries treat-
glucan backbone with a-(1fi6)-glycosidic branching, is basi-
ment, as it was mainly associated with hydrolysing a-(1fi3)-
cally synthesized by glucosyltransferases of oral streptococcal
bonds present in the mutans. Various bacteria such as Bacillus
bacteria using dietary sucrose (Wiater et al., 2013). The a-
(1fi3)-linkage is mainly associated with water insolubility, circulans (Matsuda et al., 1997) and Paenibacillus spp. (Plesz-
whereas a-(1fi6)-linked side chains contribute to the adhe- czynska et al., 2007), fungi such as Trichoderma harzianum
sive properties of these biopolymers (Wiater et al., 2005). (Wiater et al., 2001) and Trichoderma asperellum (Sanz et al.,
Streptococcal bacteria, dietary sucrose and an exposed tooth 2005), and yeast (Schizosaccharomyces pombe) (García et al.,
surface are considered to be the important factors implicated 2005), were reported for mutanase production. Although fun-
in the development of caries. Caries formation is aggravated gal sources were elaborately evaluated for mutanases com-
by biopolymer-harboured cariogenic bacterial (Lactobacillus pared to bacterial sources, yields of mutanase were extremely
low in both the species. An extensive literature survey revealed
that the maximum enzyme production was observed from
Abbreviations: BHI, brain–heart infusion; MTCC, Microbial Type Culture Flavobacterium spp. EK-14 (Ebisu et al., 1975) and the min-
Collection. imum from T. harzianum CCM F-470 (Wiater et al., 2001).
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S. K. Buddana, P. Reddy Shetty and S. R. Raja Surya Krothapalli

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

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Mutanase from novel Paracoccus mutanolyticus RSP-02

RSP-02 RSP-04 RSP-09 RSP-02 RSP-03

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

Gram staining Negative


RESULTS AND DISCUSSION Motility Non-motile
Starch hydrolysis +
Mutan production Casein hydrolysis
Mutan is an insoluble polymer of glucose linked with Methyl red
(a-1fi3)-bonds. Commercial availability of this compound Voges–Proskaurer
is limited; hence, in this study, mutan was synthesized using Citrate utilization +
Streptococcus mutans (MTCC 497), which produces mutan Urease activity +
as an exo-polysaccharide attached to the cell walls (Buddana Phenylalanine deamination
et al., 2015). The bacterial strain was grown in BHI medium Nitrate reductase +
supplemented with 5.0 % sucrose. After incubation, the cell Catalase +
mass was collected by centrifugation and used for extraction Oxidase
of a-(1fi3)-D-glucans using 2 M KOH. The extracted a- Indole production
(1fi3)-D-glucans were used as substrate for screening of Gelatin liquefaction
mutanase-producing organisms. Cellobiose +
Esculin (b-glucosidase) +
Screening and isolation of mutanase-producing Salicin (b-glucosidase) +
microbial strains Carbohydrate fermentation Acid production
Fructose
Screening of about 10 soil samples collected from cellulosic
Galactose +
waste dumps resulted in the isolation of 20 bacterial and 12
Glucose +
fungal strains that are able to produce mutanase. Among
Inositol
the isolated strains, only two bacterial strains designated as
Maltose
RSP-02 and RSP-03 and three fungal strains designated as
RSP-02, RSP-04 and RSP-09 exhibited effective degradation Mannose
of streptococcal mutan. A similar study was performed by Sucrose
Pleszczynska et al. (2007), and they isolated four bacterial Xylose +
strains that are able to produce mutanase from 50 soil sam- Mannitol
ples screened. Rhamnose
Raffinose
All five selected isolates (two bacterial and three fungal) Ribose +
were subjected to secondary screening using mutan as
Arabinose
the sole carbon source and confirmed by Congo red dye test
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99 |AB934888.1|Paracoccus sp. RSP 02


99 |HM218000.1|Paracoccus yeei MGA26-2
88 |GQ158671.1|Uncultured bacterium 16sl P96-1a06.w2K
|DQ337586.1|Paracoccus sp. BBTR62
|Y16930.1|Paracoccus denificans ATCC 19367
61
96 |HQ005404.1|Paracoccus aminovorans CT
34
99 |NR 108224.1|Paracoccus huijuniae FLN-7
|NR 113864.1|Paracoccus aminovorans NBRC 16711
44 |AB680696.1|Paracoccus pantotrophus NBRC 14906
62
54
100 |NR 113662.1|Paracoccus versutus NBRC 14567
|KC355305.1|Paracoccus marinus KUDC1798
93 |NR 109093.1|Paracoccus limosus NB88
|NR 113663.1|Paracoccus thiocyanatus NBRC 14569
|NR 118463.1|Paracoccus siganidrum M26
100 |AJ294415.1|Paracoccus alcaliphilus KTC002
|KF311079.1|Paracoccus pharagmitetus MS23

Fig. 2. Phylogenetic tree of Paracoccus mutanolyticus RSP-02.

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.

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Mutanase from novel Paracoccus mutanolyticus RSP-02

enzyme production data suggested that mutanase activity


could be noticed within 24 h of incubation (minimum incu-
bation time used for sampling). At 24 h, the strain produced
an enzyme activity of 0.14 U ml 1, which continued to
increase with an increase in fermentation time up to 72 h.
However, the extracellular mutanase production pattern
continued until 120 h with a maximum enzyme production
of 0.86 U ml 1 at 72 h incubation, and thereafter produc-
tion decreased. Growth analysis of the strain and its enzyme
production pattern further suggested that the enzyme pro-
duction by Paracoccus mutanolyticus RSP-02 was growth
associated (Fig. 3a). This was further confirmed by
the analysis of enzyme production by the plate method
using the Congo red test, which indicated that hydrolysis of
mutan (hydrolysis zone) increased with increased time of
incubation (Fig. 3b). Production of enzyme was not noticed
in the absence of substrate (mutan). This suggests that
mutanase enzyme production in this strain is highly indu-
cible. Our results confirm those obtained earlier by Plesz-
Fig. 4. TLC of mutanase-hydrolysed end products. Standards: G, czynska et al. (2007) where mutanase activity was reported
glucose; M, maltose; T, turanose; IM, isomaltose; N, nigerose. to increase up to 0.36 U ml 1 with a substrate concentration
Sample: Mutanase-hydrolysed product. of 2.5 g l 1. The specific activity of mutanase enzyme pro-
duced by the present strain is 1.18 U mg 1 protein, while
Pleszczynska et al. (2007) reported 0.09 U mg 1 by Paeniba-
negative (Table 1). Glucose, ribose, xylose and galactose were cillus strain MP-1. Matsuda et al. (1997) noticed specific
easily fermentable by the isolated strain, while maltose, man- activity of 0.039 U mg 1 for mutanase from B.
nose, raffinose, rhamnose and arabinose were non-ferment- circulans HU-M1. In other studies, Meyer & Phaff (1980)
able. The isolated RSP-02 was positive for b-glucosidase reported mutanase specific activity of 1.9 U mg 1 by B. cir-
production as observed by hydrolysis of cellobiose, salicin and culans WL-12, while Takehara et al. (1981) recorded
esculin. Further identification of this strain was performed by 0.15 U mg 1 protein by Streptomyces sp. These data further
ribotyping of 16S rRNA, and the data revealed that this strain emphasize the superiority of the present isolate for effective
corresponds to the Paracoccus genus. The 16S rRNA gene and economic production of mutanase.
sequence analysis of the isolate RSP-02 yielded 1196 bp, and
NCBI BLAST search showed that the sequence was 97 % similar Hydrolysis of mutan and identification of end
to the sequence of Paracoccus yeei MGA26-2. Because of its products
specific application for mutanase production, the name of the TLC analysis of the end products of mutan hydrolysis by
strain is designated as Paracoccus mutanolyticus RSP-02. Its mutanase enzyme revealed mainly nigerose as end product
distinct phylogenetic position further corroborated this obser- in the hydrolysed samples (Fig. 4). Mutanase catalyses
vation (Fig. 2). The obtained 16S rRNA sequence was submit-
ted to the DNA Databank of Japan (DDBJ) under the 8
accession number AB934888. Although bacterial mutanases 3.0
have been reported from different strains such as Flavobacte- 7
Protein (mg ml–1)
rium spp. (Ebisu et al., 1975), Streptomyces chartreusis (Take- 2.5 Mutanase (U ml–1) 6
hara et al., 1981), Bacillus circulans (Matsuda et al., 1997),
Mutanase (U ml–1)
Protein (mg ml–1)

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.

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S. K. Buddana, P. Reddy Shetty and S. R. Raja Surya Krothapalli

mutanase is produced in the present study by Paracoccus


mutanolyticus RSP-02.
Catalase
(240 kDa) Purification of mutanase
Native PAGE
Mutanase enzyme produced by Paracoccus mutanolyticus
Mutanase RSP-02 was partially purified using ammonium sulfate
(138 kDa) precipitation. Maximum activity was noticed at 60 %
Bovine albumin
(67 kDa) ammonium sulfate saturation. Further purification using
ion-exchange chromatography by DEAE-cellulose resulted
Egg albumin in four peaks (F1–F4); among them, the first peak con-
Trypsin
(43 kDa) tained enzyme activity which was confirmed by the plate
Soyabean Inhibitor method (Fig. 5). Similar studies were carried out by Wiater
(20.1 kDa) et al. (2001) where they used DEAE-sepharose for initial
purification. Later, the protein sample obtained from ion-
Lactoglobulin
(18.4 kDa)
exchange chromatography was ultra-filtered through
100 kDa cut-off filters (Millipore) for removal of low-
molecular-mass proteins. This was performed since the
native molecular mass of mutanase was reported to range
Fig. 6. Native PAGE of mutanase enzyme. from 140 to 300 kDa (http://www.brenda-enzymes.org/).
Native PAGE studies revealed an approximate molecular
mass of 138 kDa for mutanase produced by Paracoccus
mutanolyticus RSP-02 (Fig. 6), which is comparable to that
mutan to isomaltose, nigerose and glucose together with of mutanase produced from B. circulans WL-12 (Mayer &
oligomers. To confirm the identity of the mutanase cata- Phaff 1980). Identical studies were performed by Waiter
lysed products, standard isomaltose, nigerose, turanose, et al. (2001) using size-exclusion HPLC from T. harzianum,
maltose and glucose were eluted along with mutanase- who reported a native molecular mass of 274 kDa, while
digested samples in the present study. The data based on Rf Takehara et al. (1981) observed a molecular mass of more
values suggested that the mutanase cleaves the mutan to than 300 kDa for mutanase isolated from Streptomyces
produce nigerose as the end product. Conflicting reports chartreusis.
are available on the multifunctional activity of mutanase.
To confirm further, the mutanase-catalysed reaction mix- In vitro studies of mutanase on degradation of
ture was analysed using HPLC, and data indicated that the biofilm
reaction mixture contains nigerose, which was eluted at Mutanase enzyme produced from Paracoccus mutanolyticus
11.2 min. Consistent with this, Guggenheim & Haller RSP-02 effectively degraded the streptococcal biofilm
(1972) reported only glucose as the end product with the formed on glass plates (Fig. 7). Ultrafiltered purified
mutanase produced by T. harzianum, while Wiater et al. enzyme was used in the experiments for the degradation of
(2001) reported glucose and its dimer as the end products biofilm. Analysis of mutanase enzyme-treated tubes
for mutanase from T. harzianum. However, Matsuda et al. revealed the release of 140±2.82 µg of glucose equivalents
(1997) noticed dimer, trimer and tetramer of glucose as end (reducing sugars) in 30 min, indicating enzyme action on
products of mutan hydrolysis in B. circulans, whereas Ebisu the streptococcal biofilm (mutan). Studies using crystal
et al. (1975) reported isomaltose, nigerose and nigerotriose violet also confirmed the loosening of biofilm on the
as end products of mutan hydrolysis by Flavobacterium enzyme-treated glass plate (Fig. 7). However, Wiater et al.
mutanase. These data further support the evidence that (2008) reported 95.3 % degradation of mixed oral biofilms
adhered to saliva-coated glass plates by mutanase (T. harzia-
num) and commercial dextranase after 24 h of treatment.
A new strain of bacterium having the potential to produce
mutanase [a-(1fi3)-glucanase] was isolated, characterized at
T2 Plate treated molecular and biochemical levels and studied for its mutanase
with enzyme
enzyme production. Mutanase enzyme showed a molecular
mass of 138 kDa. Mutanase production by this strain is
growth associated and highly substrate inducible. Nigerose as
end product has been detected when the enzyme-catalysed
reaction mixture was analysed. Crude mutanase enzyme was
purified by ammonium sulfate precipitation, DEAE-cellulose
ion-exchange chromatography and ultra-centrifugation by
Fig. 7. In vitro application of mutanase enzyme on biofilm degra- molecular mass cut-off filters. In vitro application of this
dation formed on glass plate. C, control; T2, test. enzyme effectively degraded the streptococcal biofilms formed
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Mutanase from novel Paracoccus mutanolyticus RSP-02

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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.
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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
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