A β-mannanase from Paenibacillus sp.
: Samriti Dhawan1
Optimization of production and its possible
prebiotic potential
1 Department of Biotechnology, GGDSD College, Chandigarh, India
2 Department of Biotechnology, Panjab University, Chandigarh, India
3 Department of Biotechnology, Guru Nanak Dev University, Amritsar, India
Abstract
A thermotolerant bacterium Paenibacillus thiaminolyticus with
an ability to produce extracellular β-mannanase was isolated
from a soil sample. Bacterium produced 45 U/mL
β-mannanase at 50 ◦ C. The culture conditions for high-level
production of β-mannanase were optimized. Optimized MS
medium [wheat bran 2% (w/v), ammonium sulfate 0.3% (w/v),
yeast extract, and peptone (0.025% each) pH 6.5] was
inoculated with 2% of 16 H old culture. The culture was
incubated at 50 ◦ C for 48 H resulting in 24-folds higher
β-mannanase production (1,100 ± 50 U/mL). Optimum pH and
temperature for enzyme activity of the crude enzyme was 6.0
and 60 ◦ C, respectively. The enzyme demonstrated 65%
relative enzyme activity at 37 ◦ C. The hydrolytic activity of the
Keywords: 4-β-D-mannanase, Endo-1, mannolytic,
manno-oligosaccharides, Paenibacillus thiaminolyticus, prebiotic
1. Introduction
Mannans and heteromannans are ubiquitous in nature as
part of the hemicellulose fraction in hardwoods and softwoods
[1, 2]. Mannans occur in different forms in plant cell walls
as acetylated galactoglucomannan, a primary component of
hemicellulose that has a heterogenous backbone of β-1,4-
linked mannose, and glucose residues, and galactomannan
that is found in the seeds of leguminous plants. Endo-1,4-β-d-
Abbreviations: GG, guar gum; KGM, Konjac Gluco Mannan; LBG, locust
bean gum; MOS, manno-oligosaccharides; MS, mineral salt; TLC, thin-layer
chromatography; WB, wheat bran..
∗ Address for correspondence: Professor Jagdeep Kaur, M.Sc., Ph.D,
Department of Biotechnology, Panjab University, Chandigarh, India.
Tel.: +91 0172 2534085; Fax: +91 0172 2541409;
e-mail: jagsekhon@yahoo.com.
Supporting Information is available in the online issue at
www.wileyonlinelibrary.com.
Received 27 January 2015; accepted 17 July 2015
DOI: 10.1002/bab.1419
Published online in Wiley Online Library
(wileyonlinelibrary.com)
Rajvinder Singh2
Ramandeep Kaur3
Jagdeep Kaur2
∗
crude enzymatic preparation was assessed on various agro
residues. Thin-layer chromatographic analysis showed that
the enzyme activity to saccharify heteromannans resulted in
production of a mixture of manno-oligosaccharides (MOS) and
enzyme exhibited classic endo-activity. To evaluate the
possible prebiotic potential of the MOS thus obtained, initial
screening for their ability to support the growth of probiotics
was carried out by the pure culture method. Bifidobacterium
and Lactobacillus sp. responded positively to the addition of
enzymatically derived oligosaccharides and their numbers
increased significantly. C 2015 International Union of Biochemistry
and Molecular Biology, Inc. Volume 00, Number 0, Pages 1–10, 2015
mannanase (EC 3.2.1.78) is a hydrolytic enzyme that catalyzes
the random cleavage of β-1,4-d-manno-pyranosyl linkages
within the main chain of galactomannan, glucomannan, galac-
toglucomannan, and mannan and releases linear or branched
oligosaccharides of various lengths. Mannanases possess po-
tential utility in several processes in the food, feed, pulp and
paper industry, coffee extraction, as well as oil and textile
industries [3, 4], which all require thermostable mannanases.
Although many microorganisms such as Bacillus subtilis WY34,
Bacillus circulans, Cellulomonas fimi, Sclerotium rolfsii, and
Aspergillus sp. [4–6] with optimum temperature at 50 ◦ C and
thermostability in the range 60–70 ◦ C have been reported as
potential microbial mannanase producers, there is always a
need for isolating new enzyme that can withstand harsh condi-
tions such as thermostability, acid stability, and salt tolerance.
Despite having high practical potentiality, the use of mannanase
is still limited because of the low yield and high-production cost
[7]. Owing to the increasing biotechnological importance of
mannanases, a thermostable mannanase from a newly isolated
strain of Paenibacillus thiaminolyticus is reported. This strain
has been found as an outstanding producer of mannanase
using cost effective medium. Colonic microflora has profound
1
 Biotechnology and
Applied Biochemistry

impact on a host’s health and their populations are influenced

immensely by the host’s diet [8]. The selective stimulation of the Morphological and biochemical characteristics of
growth and activity of beneficial microorganisms indigenous TABLE 1 Paenibacillus thiaminolyticus
to the gut (i.e., probiotics) by supplementing with prebiotics
has emerged as a relatively new field [9]. The identification of Morphological characteristics
potential prebiotics is of considerable scientific and industrial Colony
interest. As β-mannanase can be used for the generation of
manno-oligosaccharides (MOS) from heteromannans, attention Configuration Circular
was focused to the optimal production of β-mannanase and Margin Crenate
to explore the role of this enzyme for the production of MOS
having potential prebiotic property, which is evaluated with Elevation Faintly raised
respect to growth of selected probiotic strains by the pure Surface Shiny
culture method. As mannan and heteromannans are widely
distributed in nature, the conversion of low value products Gram’s reaction Positive
into valuable oligosaccharides of industrial importance will Cell shape Rods
enhance commercial implication of this enzyme.
Size (μm) 2.0–3.0 × 1.0

2. Materials and Methods Arrangement Pairs/short chains/palisades

2.1. Materials Spores Present

Ivory nut mannan (INM) and β-1,4-MOS were purchased from
Position Central/sub terminal
Megazyme, Wicklow, Ireland. Konjac powder was procured
from Terravita Fine Herbs & Chemicals, Ontario, Canada. Silica Shape Oval
gel G-60 TLC plates were purchased from Merck, Damstadt,
Sporangia bulging Non-bulged
Germany. Agro-residues were obtained from local market.
Each of these raw materials was oven dried and then milled Motility +
and ground to pass through a 30 mm mesh sieve. All other
Biochemical characteristics
chemicals were of analytical grade. The cultures of bacteria
used were procured from Microbial Type Culture Collection Growth on MacConkey agar −
(MTCC)-Institute of Microbial Technology, Chandigarh, and
Indole test −
National Collection of Dairy Cultures (NCDC)-National Dairy
Research Institute, Karnal (India). Methyl red test +

2.2. Screening and identification of P. thiaminolyticus Voges Proskauer test −

A thermotolerant bacterium P. thiaminolyticus was isolated Citrate utilization −
from a soil sample collected from a sewage treatment plant
at Mohali, Punjab, India. The ability to produce β-mannanase H2 S production −
was qualitatively screened on modified mineral salt-ivory nut Gas production −
mannan (MS-INM) medium (g/L)—KH2 PO4 , 1.0; K2 HPO4 , 2.0;
MgSO4 7H2 O, 0.1; NH4 NO3 , 2.0; yeast extract, 0.5; INM, 10. Casein hydrolysis W
Initial pH was adjusted to 7.0 and medium was sterilized by au- Esculin hydrolysis +
toclaving. Mannan hydrolysis was observed by the appearance
of zone of clearance around the individual bacterial colonies Gelatin hydrolysis +
after staining the plates with congo red for 15 Min, and then Starch hydrolysis +
destaining with 1 M NaCl. On the basis of its morphological,
physiological, and biochemical characteristics as indicated in Urea hydrolysis +
Table 1, the isolate producing maximum β-mannanase was Nitrate reduction +
identified.
Catalase test +
2.3. Enzyme production and activity assay
The enzyme production was studied in shake flask. The mod- Oxidase test +
ified MS medium [10] supplemented with 1 % INM was used Lysine decarboxylase −
for mannanase production. After 2 days of growth at 50 ◦ C,
cell free culture supernatant was obtained by centrifugation Arginine dihydrolase W
Continued
of culture broth for 10 Min at 8,000g. Mannanase activity was
assayed by measuring the amount of reducing sugars released

2 A β-Mannanase from Paenibacillus sp.


Continued
TABLE 1
Ornithine decarboxylase
Tween 20, 40 hydrolysis
Tween 60 hydrolysis
Tyrosine test
+, positive; −, negative; W, weak.
by the enzyme using dinitro-salicylic acid (DNSA) method [11]
taking d-mannose as the standard. β-Mannanase activity was
measured in an assay mixture containing, 900 μL of locust bean
gum (LBG) (1%, w/v) prepared in 50 mM potassium phosphate
buffer (KPB) (pH 7.0) and 100 μL of suitably diluted enzyme.
The reaction mixture was incubated at 60 ◦ C for 5 Min. Then,
1 mL of DNSA reagent was added to the mixture and kept in
boiling water bath for 10 Min. The optical density was mea-
sured at 540 nm. One unit of enzyme activity was defined as
the amount of enzyme liberating 1 μmol of d-mannose, per
minute under the assay conditions. Appropriate controls of
enzyme solution and substrate were set up with each assay.
Protein content was measured by bicinchoninic acid protein
assay method using bovine serum albumin as the standard.
2.4. Optimization of β-mannanase production
To maximize mannanase production by P. thiaminolyticus,
the medium and process were optimized for shake flask
cultures. One-factor-at-a-time optimization approach was
used. The optimization studies were performed by altering the
composition of the medium.
Different C-sources at a concentration of 10 g/L were
added to the modified MS medium [10]. The C sources used
for the production of mannanase were polysaccharides [guar
gum (GG), konjac glucomannan (KGM), locust bean gum (LBG),
xylan, pectin, CM-cellulose and starch], monosaccharides
[d-glucose, d-galactose, d-mannose, d-xylose] and various
inexpensive agro residues used were [oat bran, wheat bran,
apple peel, orange peel, potato peel and copra meal]. The
selected C-source concentration was also optimized.
Effect of different N sources ammonium dihydrogen phos-
phate, diammonium hydrogen phosphate, ammonium sulfate,
sodium nitrate and ammonium chloride, peptone, yeast extract,
peptone + yeast extract, beef extract, tryptone, malt extract,
and urea on mannanase production was studied by replacing
ammonium nitrate and yeast extract used in the MS medium.
These nitrogen sources were added at concentration equiva-
lent to the amount of nitrogen in 0.2% ammonium nitrate. The
effect of different levels of selected N-source on production of
mannanase was studied in range of 0.1%–0.5 %. Different con-
centrations of MgSO4 7H2 O (0.1%–0.5%), K2 HPO4 (0.1%–2.0%),
and KH2 PO4 (0.1%–2.0%) were added in the MS medium to
study the effect on β-mannanase production. To optimize the
process, effects of different initial pH values (4–10), incubation
Biotechnology and Applied Biochemistry
temperature (25–55 ◦ C), initial inoculum (0.5%–4 %), and time
period (0–72 H) were studied.
2.5. Characterization of crude β-mannanase
− The effect of pH and temperature on the activity of crude
β-mannanase was determined using LBG as a substrate. To
+
determine the optimal temperature, the activity of the crude
− β-mannanase toward LBG was assayed in a range of 37–75 ◦ C.
Thermal stability was analyzed by incubating samples for 2 H
−
at 55–70 ◦ C in 0.1 M KPB and then the residual activity was
measured at pH 6.0 and 60 ◦ C. To determine the optimal pH,
the β-mannanase activity was measured at 60 ◦ C at pH levels
3–10 using 50 mM of citrate and sodium citrate buffer (pH
3.0–5.0), KPB (pH 6.0–7.0), tris–HCl buffer (pH 8.0–9.0), and
glycine–NaOH buffer (pH 9.0–10.0). To assay effect of pH on
the enzyme stability, β-mannanase was incubated in various
buffers of different pH values (as shown above) for 2 H, and
then the residual activity was measured at pH 6.0 and 60 ◦ C.
2.6. Evaluation of possible prebiotic potential of the
oligosaccharides
Production of MOS from various residues
The MOS fractions were obtained by enzymatic treatment of
agro-residues and heteromannans. WB, OB, LBG, GG, and KGM
(1%, w/v) were treated with one unit mannanase equivalent
activity in 50 mM KPB (pH 6.0) for 2 H at 55 ◦ C in an incubator.
The reaction was then terminated by heating in boiling water
bath for 10 Min. The mixture was centrifuged at 10,000g for
10 Min at 4 ◦ C, the supernatant was collected and filtered
through 0.22 μm filters and the soluble fraction was analyzed
by thin-layer chromatography (TLC).
Enzymatic hydrolysis product analysis by TLC.
The products of hydrolysis were separated by TLC carried out
on Silica gel G-60 (layer thickness of 0.2 mm) plates, using
1-butanol/ethanol/water (10:8:7) as mobile phase. Carbohy-
drates were detected with 0.2% (w/v) solution of orcinol in 10%
(v/v) sulfuric acid in ethanol.
Culture conditions for probiotic and pathogenic bacteria.
Bifidobacterium bifidum (NCDC-235) and Lactobacillus strains,
that is, L. rhamnosus (MTCC-1408), L. casei (MTCC-1423), and
L. acidophilus (MTCC-447) were grown aerobically in MRS
broth [Lactobacillus broth according to De Man, Rogosa, and
Sharpe (MRS)] at 37 ◦ C for 24–48 H. While Escherichia coli
(MTCC-1554) and Bacteroides sp. (MTCC-1045) were cultivated
in Nutrient Broth (NB) and Reinforced Clostridial (RC) medium
under aerobic and anaerobic conditions, respectively, seed
inoculum of probiotic and pathogenic bacteria (OD600 nm  0.2–
0.3) was prepared by their cultivation in respective media at
37 ◦ C for 16–24 H.
Evaluation of MOS for use as a C-source by probiotic
and pathogenic bacteria.
Oligosaccharide consumption by probiotics was determined
by standard plate count method. MRS broth was reconstituted
without glucose and filter sterilized (0.22 μm) oligosaccharide
3

(A) Zone of clearance of P. thiaminolyticus on
FIG. 1 mannanase screening medium. (B) Zone of
clearance of P. thiaminolyticus on optimized
medium.
solution was added at different concentrations 1%–10% (v/v).
Five milliliters of each medium was inoculated with 1% (v/v)
seed inoculum. Appropriately diluted culture was spread
on MRS plates supplemented with filter sterilized (0.2 μm)
oligosaccharide solution, containing 1.5% agar and grown
overnight at 37 ◦ C.
The growth of pathogenic strains on oligosaccharide
carbon source was assayed in appropriate reconstituted broth
without glucose and filter sterilized (0.22 μm) oligosaccharide
solution was added at different concentrations 1%–10 % (v/v).
Five milliliters of each medium was inoculated with 1% (v/v)
seed inoculum: in NB under aerobic conditions for E. coli,
RC medium for Bacteroides sp. under anaerobic conditions.
Growth parameters were also measured by estimation of
absorbance (OD600 nm ) and change in pH after 48 H at 37 ◦ C.
Experimental conditions were validated by checking with
positive and negative control experiments performed respec-
tively with modified media (MRS, NB, and RC medium) sup-
plemented with glucose or without any C-source. The cultures
were incubated at 37 ◦ C for 48 H.
Effect of oligosaccharide on growth of probiotic and
pathogenic bacteria.
The enhancing and inhibitory activity on growth of Lactobacil-
lus sp., Bifidobacterium sp., Bacteroides sp., and E. coli was
tested by comparing the difference in number of colony forming
units (log10 cfu/mL) between the medium supplemented with
oligosaccharide and the control media. Samples were removed
initially to determine the immediate effects of the oligosaccha-
rides and then after 24 H to observe the overall effect of the
oligosaccharide on the bacterial growth [12, 13].
3. Results and Discussion
3.1. Isolation and characterization of β-mannanase-
producing bacteria
An extensive screening was carried out to isolate mannanase-
producing microorganisms from diverse ecological habitats.
Typical colonies, exhibiting discrete zone of clearance around
them on YE-INM agar plates, were randomly selected. Based on
4 A β-Mannanase from Paenibacillus sp.
Biotechnology and
Applied Biochemistry
the size of zone of clearance, the bacterial isolate was selected
(Fig. 1a). Identification of strain was carried out based on
morphological, physiological, and biochemical characteristics
of the isolate (Table 1). The isolate was a Gram positive,
endospore forming, motile, rod shaped, facultative anaerobe,
and moderate thermophile with minimum and maximum
temperature for growth at 20 and 55 ◦ C, respectively. The
strain was capable of growing in the pH range from 5 to 10 and
can tolerate 2%–7% NaCl concentration. The isolated bacterium
was identified as P. thiaminolyticus according to the Bergey’s
Manual of Systematic Bacteriology [14, 15].
3.2. Optimization of mannanase production in
submerged fermentation
To deduce the optimal conditions for β-mannanase production,
effects of C-source, N-source, pH, temperature, and inocu-
lum were studied. Among the different carbon sources tested
(Table 2), nearly 10-fold higher β-mannanase production was
observed by supplementing MS medium with different hetero-
mannan substrates as compared with INM. Galactomannans
of LBG, GG, and glucomannan of konjac yield 530, 380, and
520 U/mL of mannanase, respectively. With sugars such as
d-mannose, d-glucose, d-galactose, d-xylose, and polysaccha-
rides such as xylan (oat spelts), pectin, CM-cellulose, and starch
mannanase production was less. It is evident from Table 2, P.
thiaminolyticus is utilizing heteromannans more efficiently as
compared with pure mannan. This significant difference in the
regulation of mannanase synthesis was utilized to economize
the production process. Amongst agro-residues evaluated,
mannanase production was highest when WB was used as C-
source. WB, OB, and CM in MS media were promising substrates
as they fulfilled the stimulatory effect of the pure inducer,
galactomannan (LBG). As cost-effective technologies are re-
quired, substitution of the rather costly galactomannan by
cheap, low cost, abundantly available agro-residue WB will
reduce the cost of production. Moreover, mannanase pro-
duction increased as the concentration of WB increased and
reached maximum activity at 20 g/L (Supplementary Fig. S1),
which appeared to be the optimal level. As further increase in
concentration of bran resulted in lower enzyme production.
This might be due to the release of large amount of hydrolysis
end products that may repress many catabolic operons. Re-
sults were concordant with the reports by earlier workers. Agro
residue WB was used for β-mannanase production by B. subtilis
 Effect of different carbon and inexpensive Effect of different nitrogen sources on the
TABLE 2 agro-residues on the β-mannanase production by P. TABLE 3 β-mannanase production by P. thiaminolyticus
thiaminolyticus
Carbon source Activity (U/mL) Nitrogen source Activity (U/mL)

Mannan (INM) 45.15 ± 3.67 Inorganic sources

Guar gum 380.54 ± 3.67 Ammonium dihydrogen phosphate 493.6 ± 3.14

Konjac Gluco mannan 520 ± 4.72 Diammonium hydrogen phosphate 546 ± 2.42

Locust bean gum 530 ± 0.014 Ammonium nitrate 317 ± 1.25

Xylan (oat spelts) 78 ± 2.56 Ammonium sulfate 643.5 ± 4.2

Pectin 15.9 ± 0.85 Sodium nitrate 440 ± 3.22

CM-cellulose ND Ammonium chloride 564.2 ± 2.14

Starch 132.5 ± 1.25 Organic sources

D-Glucose 60 ± 1.25 Peptone 581 ± 4.2

D-Galactose 144.16 ± 0.28 Yeast extract (YE) 625 ± 2.5

D-Mannose 221.54 ± 3.67 Beef extract 537.74 ± 1.65

D-Xylose 80 ± 2.82 Tryptone 599.4 ± 3.28

Apple peel 50 ± 1.50 Malt extract 520 ± 0.42

Orange peel 55 ± 0.55 Urea 600 ± 5.25

Potato peel 80 ± 2.50 Combined form

Copra meal 372 ± 3.80 Ammonium sulfate + YE 650.5 ± 3.75

Oat bran 527 ± 1.0 Ammonium sulfate + peptone 605 ± 4.60

Wheat bran 590 ± 2.56 Ammonium sulfate + YE + peptone 670 ± 5

Cells were grown on mineral salt (MS) medium with each tested sub-
strate (10 g/L) and incubated at 50 ◦ C for 48 H.
Values shown are the averages from triplicate experiments with each
source.
±Standard deviation.
[16] and reported inducible effect of WB at an optimal concen-
tration of 35 g/L, but in present study, higher β-mannanase
yield was obtained in the MS medium containing WB at an
optimal concentration of 20 g/L. Oven dry weight of WB was
used for chemical analysis. Chemical analysis revealed the
chemical composition/percentage of dry weight of WB (mois-
ture content—9.6; ash content—5.58; [17], soluble reducing
sugars—2.67; [11], total protein—17.2; [18], total carbohydrate
content—68.5; holocellulose—52; hemicellulose—35) [19]. The
data presented show that WB contain considerable amount
of carbohydrate which stimulate the cells to express many
hydrolytic enzymes, as it contained, a finest amount of soluble
reducing sugar (required for the initiation of growth and repli-
cation), hemicellulose (inducer of β-mannanase), and organic
nitrogen source (necessary for protein biosynthesis). Thus, in
Values shown are the averages from triplicate experiments with each
source.
±Standard deviation.
present study WB was found to be best inducer of mannanase
when used as carbon source as compared with LBG.
The effect of nitrogen sources on the production of man-
nanase was studied using the MS-medium supplemented with
WB (2%, w/v) as sole source of carbon (Table 3). However, all the
tested nitrogen sources showed an enhanced mannanase pro-
duction than that obtained with ammonium nitrate used in the
original medium. Among the inorganic nitrogen sources tested,
ammonium sulfate gave the highest mannanase production
(643.5 U/mL). Higher production of mannanase was obtained
in fermentation media having ammonium sulfate (0.3%) and a
combination of yeast extract and peptone (0.025%) as nitrogen
source yielding 670 U/mL of mannanase.
Effect of incubation temperature (25–55 ◦ C) was studied
on growth and mannanase production. Optimum incubation
temperature for enzyme production was 50 ◦ C (Supplementary
Fig. S2). Although maximum growth of the bacterium was
after 24 H at 50 ◦ C; however, maximum mannanase yield was

Biotechnology and Applied Biochemistry 5


Optimization of mannanase production.
FIG. 2 Comparison for growth (A600 ), mannanase
production, and extracellular protein concentration
of P. thiaminolyticus using optimized media and
process conditions with non-optimized MS-INM
media (Control).
obtained after 48 H at 50 ◦ C under shaking conditions. Although
cell biomass was not affected in temperature range between 37
and 50 ◦ C, but mannanase production was 458 and 720 U/mL,
respectively. Increasing the incubation temperature above
50 ◦ C decreased the mannanase production drastically and no
activity could be detected in the culture incubated at 55 ◦ C. It
seemed that at high temperature growth of bacteria was greatly
inhibited and hence effecting the enzyme production. Earlier
workers have reported the optimum mannanase production
from various Bacillus sp. [20, 21] at 50 ◦ C also, but not from
Paenibacillus sp. has been reported so far [22, 23].
Effect of pH indicated that medium adjusted at pH
6.5 favored maximum enzyme production of 740 U/mL
(Supplementary Fig. S3). Although the production of man-
nanase at pH 5–8 was also not severely affected, mannanase
production was favored by acidic and neutral conditions, but
markedly declined when the initial pH of the growth medium
exceeded 8.0.
Effect of inocula of different sizes (0.5%–4%) of the total
culture volume on β-mannanase production was studied
under shaking conditions at 50 ◦ C up to 48 H of incubation
(Supplementary Fig. S4). Maximal mannanase production
(840 U/mL) was observed in the medium provided with 2% (v/v)
inoculum size (OD600nm 1.5). An increase in the inoculum size
ensured increase in mannanase yield by P. thiaminolyticus.
However, it seemed after a certain limit the competition for the
6 A β-Mannanase from Paenibacillus sp.
Biotechnology and
Applied Biochemistry
nutrients may result in decrease of the metabolic activity of
the organism. As at 4% (v/v) inoculum size, both mannanase
production and growth (OD600nm 0.78) decreased. The inoculum
age (2, 8, 16, 24, and 36 H) too had a significant influence on
β-mannanase production. It was observed that 16 H old actively
growing culture (2%, v/v) resulted in maximum mannanase
activity (900 U/mL) after 48 H of incubation.
A typical time course of mannanase production by
P. thiaminolyticus using optimized medium was shown in
Fig. 2. The result indicates that a parallel increase in biomass,
protein concentration, and mannanase production occurred
with optimized medium. Growth of bacterium started just af-
ter inoculation, whereas extracellular mannanase production
can be observed after 2 H and reaching maximum at 48 H of
incubation. WB at a concentration of 2.0% along with ammo-
nium sulfate (0.3%), yeast extract, and peptone (0.025%) were
observed to be the best C-source and N-source respectively
for achieving the highest β-mannanase production (Fig. 1b).
The use of MgSO4 7H2 O, KH2 PO4 , K2 HPO4 at 0.2, 1.0, 1.5 g/L,
respectively (data not shown here), in MS-WB medium, resulted
in high mannanase production as compared with production
of enzyme (45 U/mL) in non-optimized MS-medium (con-
trol). β-Mannanase production increased by 24.4-fold yielding
1,100 ± 50 U/mL when the optimized MS-WB medium initially
adjusted to pH 6.5, inoculated with 2% of 16 H old culture was
incubated at 50 ◦ C for 48 H (Fig. 2).
Thus, in our study, a thermotolerant P. thiaminolyticus
with the ability to produce a high level of mannanase
(1,100 ± 50 U/mL) in just 48 H was isolated. Other Paenibacil-
lus sp. strains isolated so far [22–25] produced mannanase
below 50 ◦ C and after long incubation periods, and maximal
mannanase production level was also found to be much lower
than reported in the present study.

3.3. Characterization of the β-mannanase

The optimum temperature for β-mannanase activity of the
crude enzyme with LBG as substrate determined to be 60 ◦ C.
The enzyme demonstrated more than 90% activity at tem-
peratures between 55 and 65 ◦ C (Fig. 3a). The optimum pH
for mannanase activity (at 60 ◦ C) was determined to be 6.0
(Fig. 3b). Microbial mannanases were known to exhibit max-
imal activities in the diverse pH ranges of acidic 5.0–6.5 [26],
6.5–9.0 [3, 27], or alkaline 9.0–10.0 [28, 29].
To examine thermostability, the crude enzyme was incu-
bated at various temperatures and the residual activities were
then assayed. The enzyme was 100% stable up to 2 H at 55 ◦ C,
and retained more than 90 % of its activity up to 1 H and
80% of its activity up to 2 H at 65 ◦ C, but its stability rapidly
decreased at 70 ◦ C and above (Fig. 3c). This thermostability
was comparable to that reported for several mannanases from
the thermophilic fungi and significantly higher than that of
other Paenibacilli strains as previously described [22–25]. The
stability of β-mannanase for prolonged periods of time at ele-
vated temperature was a desirable property for enzyme to be
used in commercial processes involving either the paper and
pulp industry or the food and feed industry [30].

3.4. Product analysis of various heteromannan

residues
Product analysis of various residues by TLC reveals the pres-
ence of mannobiose and other higher MOS as the prime end
products. Mannobiose, mannotriose, and mannotetraose from
LBG, mannobiose, mannotriose, and mannotetraose were main
products from KGM and GG with diminutive amount of man-
nose (Fig. 4). WB and OB yield a mixture of MOS. This result
was an indication of that the crude enzymatic preparation
obtained from P. thiaminolyticus has higher activity of endo-
mannanase than that of β-mannosidase. It is worth mentioning
that the high β-mannanase activity was not a drawback of the
crude enzymatic preparation, since it was aimed to be used for
generating oligosaccharide-rich hydrolysate.

3.5. Evaluation of MOS for use as a C-source by
FIG. 3
bacteria
MOS formed by the hydrolysis of LBG, KGM, GG, WB, and OB
by P. thiaminolyticus mannanase were tested for their possible
prebiotic potential by the pure culture method. The effect on
growth of various probiotic strains, that is, Bifidobacterium
sp. and Lactobacillus sp. as well as on growth of E. coli and
Bacteroides sp. as an example of potentially pathogenic strain
was observed. The recorded change in number of selected
bacterial populations supplemented with the MOS tested along
with positive and negative control was presented. Bacterial
counts were determined and were expressed as log10 cfu/mL.
When negative control was compared with the oligosaccharide
supplemented sample, lactobacilli showed higher growth rates
than those of the other strains tested (Fig. 5). The results in the
(A–C) Characterization of crude mannanase. (A) pH
profile and pH stability. (B) Temperature profile.
(C) Thermal stability of β-mannanase. To assay the
enzyme pH stability, β-mannanase was incubated
in various buffers of different pH (3–10) for 2 H and
residual activities were measured at pH 6.0 and
60 ◦ C in KPB. To assay the enzyme thermal
stability, β-mannanase was incubated at different
temperatures (55–70 ◦ C) for 2 H, and aliquots were
removed at specific time points for the
measurement of residual activity at 60 ◦ C in KPB
(pH 6.0).

Biotechnology and Applied Biochemistry 7

 Biotechnology and
Applied Biochemistry

present study were discordant with the results reported ear-

Thin-layer chromatogram of hydrolysis products.
FIG. 4 Lane 1, Unhydrolyzed locust bean gum; lane 2,
locust bean gum oligosaccharides (LBGO); lane 3,
unhydrolyzed guar gum; lane 4, guar gum
oligosaccharides (GGO); lane 5, unhydrolyzed
Konjac-glucomannan; lane 6, Konjac-glucomannan
oligosaccharides (KGMO); lane 7, mannopentose
(M5); lane 8, mannose (M1); lane 9, mannobiose
(M2).
lier where most of the bifidobacteria tested have high growth
rates on oligosaccharides derived from plant oligosaccharides
[31]. Both E. coli and Bacteroides sp. showed low growth on
the oligosaccharide fractions (Fig. 6), whereas lactobacilli,
with the exception of L. rhamnosus, grew better than all the
E. coli or Bacteroides sp. tested. OBO (oat bran oligosac-
charides) showed a higher enhancing activity on growth of
L. rhamnosus as compared with L. casei. The result suggested
that the hydrolysate from this substrate may have stronger
prebiotic properties promoting growth of both L. casei and L.
rhamnosus. While considering the effect of MOS on L. casei,
the hydrolysate obtained from all substrates showed an en-
hancing activity, though to a considerably varying extent as
shown in Fig. 5. The number of bifidobacteria increased from
7.92 log10 cfu/mL to 9.14 log10 cfu/mL for oligosaccharides
from OB, but for other oligosaccharides, stimulatory ef-
fect was little less, depicting an enhancement of 0.24 to
1.22 log10 cfu/mL. It was known that different intestinal mi-
croorganisms can affect each other significantly, MOS have

Changes in bacterial population (log10 colony

FIG. 5 forming units/mL) of probiotic bacteria. Pure
cultures of Bifidobacterium sp., Lactobacillus casei
(LB-1), Lactobacillus rhamnosus (LB-2), and
Lactobacillus acidophilus (LB-3) were grown in
(MRS-medium) devoid of glucose and
supplemented with oligosaccharides [LBGO,
KGMO, GGO, WBO, and OBO (10%, v/v)] formed
by hydrolysis of locust bean gum, konjac
glucomannan, guar gum, wheat bran, and oat
bran, respectively, were used. MRS media with
glucose as positive control and devoid of any
C-source were used as negative control. Results
are given as the average value of three
replicates ± standard deviation.

8 A β-Mannanase from Paenibacillus sp.

 Changes in bacterial population (log10 colony
FIG. 6
forming units/mL) of pathogenic bacteria. Pure
cultures of (A) E. coli, (B) Bacteroides sp. were
grown. Respective medium (NB, RC medium)
devoid of glucose and supplemented with
oligosaccharides [LBGO, KGMO, GGO, WBO, and
OBO (10%, v/v)] formed by hydrolysis of locust
bean gum, konjac glucomannan, guar gum, wheat
bran, and oat bran, respectively, were used. NB
and RC media with glucose as positive control and
devoid of any C-source were used as negative
control. Results are given as the average value of
three replicates ± standard deviation.
been reported to reduce incidence of pathogens in animals
[8, 32]. Yet, few studies have investigated MOS as prebiotic
to check their inhibitory effect. Bacteroides sp. and E. coli
represent that group. Results with respect to growth of E. coli
were quite consistent (Fig. 6a). Low inhibitory activities of
0.08, 0.26, and 0.28 log10 cfu/mL were found for oligosaccha-
rides of LBG, GG, and OB, whereas the oligosaccharides from
KGM showed a bit enhancement in growth of E. coli and no
effect on growth was caused by WB oligosaccharides. Similar
kind of growth pattern was observed for Bacteroides sp.
as well (Fig. 6b). Figure 6 presents that growth of both
pathogenic strains on oligosaccharides did not increase beyond
the negative control average value. In fact, it was remark-
ably below the positive control value. This data demonstrated
Biotechnology and Applied Biochemistry
that both the strains were unable to metabolize MOS and
suggested a possible use of oligosaccharides as potential
prebiotics.
Measured growth parameters showed utilization of MOS
by probiotic bacterial strains, evident through increase in
absorbance and decrease in pH. Maximum absorbance and de-
crease in pH was recorded in medium supplemented with 10%
(v/v) MOS solution. The result indicated that negative control
had no detectable decrease in pH, whereas positive control had
decrease in pH up to 1.2 units after 48 H of incubation (data
not shown here). The concomitant pH reduction is indicating
the production of short-chain fatty acids, known to contribute
in establishment of probiotic bacteria and growth inhibition
of pathogenic bacteria. Each MOS produced was used by
Bifidobacterium sp., L. casei, L. rhamnosus, and L. acidophilus
but not used by harmful bacteria such as Bacteroides sp. and
E. coli. It provided a first level relevant selection criterion
highlighting the possible prebiotic potential of MOS. This facet
needed to be inveterate in gut models in vitro or in assays in
vivo in order to access the selective growth effects of the tested
MOS.
The prebiotic nature of MOS has been demonstrated
previously and has also been revealed to have a positive effect
on gut motility [21, 32]. Although there were few reports
on oligosaccharide production from KGM [33] and CM, and
copra mannan hydrolysates were used for prebiotic analysis,
9

resulting in 0.09–2.15 log cfu/mL enhancing activity [34], but
no report was available with LBG, GG, WB, and OB. The effect
of plant polysaccharides on microbial population dynamics in
the intestinal tract has not been intensively studied, but a few
reports have indicated their prebiotic potential [9, 35].
If one seeks for effective prebiotics through hydrolytic
degradation of polysaccharides, it was necessary to analyze
the evolution of the individual and mixed bacterial population
in the presence of the carbohydrate source being tested. The
effect of the resulting oligosaccharides on growth of target
probiotic bacteria can be evaluated. These microbial targets
can grow on the medium by utilizing the oligosaccharide degra-
dation products as a carbon source [36–38]. This will plausibly
result in enzyme preparations that were more suitable for the
formation of the oligomeric products. In our study, various
oligosaccharides resulting from the degradation of substrate
added were evaluated for their possible prebiotic potential by
the pure culture method using bacteria which represent human
fecal populations. Although it was not feasible to fully char-
acterize the changes occurring in the colonic micro flora, but
it was possible to monitor the populations of selected species
indicative of bacteria inhabiting gut [12]. Although the pure
culture method for evaluation of the human gut micro flora
were useful for the early development of potentially prebiotic
oligosaccharides and in comparing prebiotic capabilities of dif-
ferent oligosaccharides under controlled laboratory conditions,
the final step in the validation of prebiotic activity can only
be derived from human/animal feeding studies which will be
carried out in future.
Enzymatic hydrolysis of mannans using mannanases is
essential for the production of potentially health-promoting
MOS [39]. Thus, P. thiaminolyticus is a potential source for
a novel, cheap β-mannanase as it is produced using cost-
effective medium. This study is providing basic data to the
capability of this β-mannanase application for MOS preparation
and improving the nutrient value of low value substrates to
utilize for their possible prebiotic potential. Alternatively, MOS
produced could be substituted for expensive growth media
to decrease the cost of commercial production of probiotic
cultures of Lactobacillus and Bifidobacterium.
4. References
[1] Pham, T. A., Berrin, J. G., Record, E., To, K. A., and Sigoillot, J. C. (2010) J.
Biotechnol. 148(4), 163–170.
[2] Yamabhai, M., Sak-Ubol, S., Sril, W., and Haltrich, D. (2014) Crit. Rev.
Biotechnol. 0, 1–11.
[3] Zhang, M., Chen, X. L., Zhang, Z. H., Sun, C. Y., Chen, L. L., He, H. L., Zhou, B.
C., and Zhang, Y. Z. (2009) Appl. Microbiol. Biotechnol. 83, 865–873.
[4] Dhawan, S., and Kaur, J. (2007) Crit. Rev. Biotechnol. 27, 197–216.
[5] Heck, J. X., Soares, H. B., and Ayub, M. A. Z. (2005) Enzym. Microb. Technol.
37, 417–423.
[6] Liao, H., Li, S., Zheng, H., Wei, Z., Liu, D., Raza, W., Shen, Q., and Xu, Y. (2014)
BMC Biotechnol. 14, 90.
[7] Lu, H., Zhang, H., Huang, H., Shi, P., Wang, Y., Peilong, Y., and Bin, Y. (2013)
Appl. Microbiol. Biotechnol. 97, 8121–8128.
10
Biotechnology and
Applied Biochemistry
[8] Fuller, R., and Gibson, G. R. (1997) Gastroenterology 32 (Suppl. 222),
28–31.
[9] Gibson, G. R., and Roberfroid, M. B. (1995) J. Nutr. 125, 1401–1412.
[10] Jones, G. H., and Ballou, C. E. (1969) J. Biol. Chem. 244, 1043–1045.
[11] Miller, G. L. (1959) Anal. Chem. 31, 426–428.
[12] Roberfroid, M. (2007) J. Nutr. 137(3), 830S-837S.
[13] Rycroft, C. E., Jones, M. R., Gibson, G. R., and Rastall, R. A. (2001) J. Appl.
Microbiol. 91, 878–887.
[14] Sneath, P. H. A., Holt, J. G., Krieg, N. R., Staley, J. T., and Williams, S. T. (1994)
In Bergey’s Manual of Systematic Bacteriology, 9th ed. (Hensyl, W. M., ed.).
pp. 559–571, Williams and Wilkins Publishers, Baltimore Maryland, USA.
[15] Chow, V., Nong, G., John, F. J., Rice, J. D., Dickstein, E., Chertkov, O., Bruce,
D., Detter, C., Brettin, T., Han, J., Woyke, T., Pitluck, S., Nolan, M., Pati, A.,
Martin, J., Copeland, A., Land, M. L., Goodwin, L., Jones, J. B., Ingram, L.
O., Shanmugam, K. T., and Preston, J. F. (2012) Stand. Genomic Sci. 19, 6(1),
1–10.
[16] Helow, E. R., Sabry, S. A., and Khattab, A. A. (1997) Anton. Van. Leeuwen. 71,
189–193.
[17] Browne, C. A., and Zerban, F. W. (1948) In Methods of Sugar Analysis. p.
1020, Wiley J, Sons, New York.
[18] Daughton, C. G., Jones, B. M., and Sakaji, R. H. (1984) In A Manual of
Analytical Methods for Wastewaters, 2nd ed. (Daughton, C. G., ed); Lawrence
Berkeley Laboratory, CA, Chapter VII, LBL-17421 (NTIS DE84015967).
[19] Dubois, M., Gillis, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F. (1956)
Anal. Chem. 28, 350–356.
[20] Li, Y. N., Yang, P. L., Meng, K., Wang, Y. R., Luo, H. Y., Wu, N. F., Fan, Y. L., and
Yao, B. (2008) J. Microbiol. Biotechnol. 18, 160–166.
[21] Tuohy, K. M., Kolida, S., Lustenberger, A. M., and Gibson, G. R. (2001) Brit. J.
Nutr. 86, 341–348.
[22] Chandra, M. R. S., Lee, Y. S., Park, I. H., Zhou, Y., Kim, K. K., and Choi, Y. L.
(2011) J. Korean. Soc. Appl. Biol. Chem. 54(3), 325–331.
[23] Fu, X., Huang, X., Liu, P., Lin, L., Wu, G., Li, C., Feng, C., and Hong, Y. (2010)
J. Microbiol. Biotechnol. 20(3), 518–524.
[24] Han, Y., Kye, C., Hong, S., Sun, L., Yong, K., Goon, K., and Hoon, K. (2006)
Appl. Microbiol. Biotechnol. 73(3), 618–630.
[25] Zhoua, Y., Leeb, Y. S., Parkb, I. H., Suna, Z., Yanga, T., Yanga, P., Choib, Y. L.,
and Ming, S. (2012) Enzym. Microb. Technol. 50, 151–157.
[26] Jiang, Z., Wei, Y., Li, D., Li, L., Chai, P., and Kusakabe, I. (2006) Carbohydr.
Polym. 66, 68–96.
[27] Liu, H. X, Gong, J. S., Heng, Li., Lu, Z. M., Hui, Li., Qian, J. Y., Zheng-Hong
Xu, Z. H., and Shi, J. S. (2015) Proc. Biochem. 50(5), 712–721.
[28] Hatada, Y., Takeda, N., Hirasawa, K., Ohta, Y., Usami, R., Yoshida, Y., Ito, S.,
and Horikoshi, K. (2005) Extremophile 9, 497–500.
[29] Luo, H., Wang, K., Huang, H., Shi, P., Yang, P., and Bin, Y. (2012). J. Ind.
Microbiol. Biotechnol. 39(4), 547–555.
[30] Vanzyl, W. H., Rose, S. H., Trollope, K., Gorgens, J. F. (2010). Proc. Biochem.
45(8), 1203–1213.
[31] Olano, M. E., Mountzouris, K. C., Gibson, G. R., and Rastall, R. A. (2000) Brit.
J. Nutr. 83, 247–255.
[32] Ohh, S. J. (2011) Asian-Aust J. Anim. Sci. 24(4), 573–586.
[33] Titapoka, S., Keawsompong, S., Haltrich, D., and Nitisinprasert, S. (2008)
World. J. Microbiol. Biotechnol. 24, 1425–1433.
[34] Rastall, R. A., Gibson, G. R., Gill, H. S., Guarner, F., Klaenhammer, T. R., Pot,
B., Reid, G., Rowland, I. R., Sanders, M. E. (2005) FEMS. Microbiol. Ecol.
52(2), 145–152.
[35] Steer, T., Carpenter, H., Tuohy, K., and Gibson, G. R. (2000) Nutr. Res. Rev.
13, 229–254.
[36] Gibson, G. R., McCartney, A. L., and Rastall, R. A. (2005) Brit. J. Nutr. 93(Suppl
1), S31–34.
[37] Palframan, R., Gibson, G. R., and Rastall, R. A. (2003) Lett. Appl. Microbiol.
37, 281–284.
[38] Roberfroid, M. B. (2000) Am. J. Clin. Nutr. 71, 1682s–1687s.
[39] Yamabhai, M., Sak-Ubol, S., Srila, W., and Haltrich, D. (2014) Crit. Rev.
Biotechnol. 1–11 [Epub: Jul 15].
A β-Mannanase from Paenibacillus sp.