Biotechnol Lett (2014) 36:2229–2237
DOI 10.1007/s10529-014-1595-1
ORIGINAL RESEARCH PAPER
Characterization of industrially-valuable xylano-
pectinolytic enzymes produced concurrently by a novel
isolate of Bacillus pumilus
Amanjot Kaur • Avtar Singh • Ritu Mahajan
Received: 19 May 2014 / Accepted: 19 June 2014 / Published online: 22 July 2014
Ó Springer Science+Business Media Dordrecht 2014
Abstract Xylanase and polygalacturonase were
concurrently produced by a novel alkalo-thermotoler-
ant Bacillus pumilus AJK. They were purified and
characterized to evaluate their potential for various
industrial applications. Xylanase was purified to
19-fold with 67 % recovery and polygalacturonase
up to 23-fold with 75 % recovery. Existence of
multiple forms of xylanase was indicated by its
elution-profile through Sephadex G-100 as two peaks,
xylanase-I and xylanase-II, with molecular weights of
*24.5 and *13 kDa, respectively, and by the
presence of two pH optima, one at pH 6.0 and other
at pH 8.5. The molecular weight of polygalacturonase
was *40 kDa by gel-filtration chromatography. Zy-
mographic studies confirmed the presence of seven
isozymic forms of xylanase. Xylanase and polygalac-
turonase are stable over a broad range of pH and
temperature.
Keywords Bacillus pumilus
Pectinase

Polygalacturonase
Purification
Ultrafiltration

Xylanase
Xylano-pectinolytic enzyme
Zymography
A. Kaur
A. Singh
R. Mahajan (&)
Department of Biotechnology, Kurukshetra University,
Kurukshetra 136119, India
e-mail: ritupanipat@rediffmail.com
A. Kaur
e-mail: amanjotbiotech@gmail.com
A. Singh
e-mail: avtarsingh87@gmail.com
Introduction
Xylanases and pectinases are produced by microorgan-
isms but there are only three reports, two concerning
fungi (Botella et al. 2007; Gomes et al. 1992) and one
concerning Streptomyces sp. (Beg et al. 2000), showing
the production of these two industrially-valuable
enzymes in combination from the same isolate, but these
workers have not reported the presence of acidic and
alkaline forms of xylanase. In this study, the industrially-
important Bacillus pumilus AJK produces xylanase and
polygalacturonase in combination. Purification of these
enzymes is necessary to know the various characteristics
of these enzymes so that they could be efficiently used for
various industrial purposes. This is the first report of the
purification of concurrently produced xylanase and
polygalacturonase from a bacterium.
The unique property of this multifunctional strain
(high concurrent producer of acidic xylanase, alkaline
xylanase and pectinase in the same production media,
is desirable from economic point of view) has not been
reported earlier in bacteria. Multiple forms of xylan-
ases (acidic and alkaline) show its suitability for wide
range of industrial applications. The xylanase and
polygalacturonase produced simultaneously were
purified and extensively characterized for various
properties like, pH optima, temperature optima, sta-
bility at different temperature and pH values, kinetic
properties, molecular weight, activities of these
enzymes in the presence of various additives etc. to
find their suitability in various industries.
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Materials and methods
Microorganism
A concurrent producer of xylanase and pectinase, B.
pumilus AJK, was isolated from soil contaminated
with the effluents of paper and pulp industry (Kaur
et al. 2011).
Production of xylano-pectinolytic enzymes
Xylano-pectinolytic enzymes were produced under
submerged fermentation in 50 ml basal medium (g/l:
peptone, 5; MgSO4
7H2O, 2.46; pH 7) supplemented
with 2 % (w/v) wheat bran and 2 % (w/v) citrus peel.
The flasks were inoculated with 2 % (v/v) inoculum
(21 h old, OD * 0.85) and incubated at 37 °C for
60 h at 200 rpm. Crude xylano-pectinolytic enzymes
were harvested by centrifugation.
Enzyme assay and protein estimation
The enzyme activities were determined using the
method of Miller (1959). Birchwood xylan, 2 % (w/v)
(prepared in 0.1 M glycine/NaOH buffer, pH 8.5) and
polygalacturonic acid, 0.5 % (w/v) (prepared in 0.1 M
glycine/NaOH buffer, pH 9.0) were used as substrates
for assaying the activity of xylanase and polygalactu-
ronase, respectively, as described by Kaur et al.
(2011). Protein was estimated by the Bradford method
using bovine serum albumin as standard. The protein
content of the chromatographic fractions was also
measured by monitoring the absorbance at 280 nm.
Purification of xylanase and polygalacturonase
The crude enzymes were purified using fast flow and
conventional purification techniques. All steps of
purification were carried out at 4 °C.
Microfiltration and ultrafiltration of crude extract
Clarification of the crude turbid extract was done by a
microfiltration unit (GE Healthcare Biosciences Ltd).
The extract obtained after microfiltration was ultrafil-
tered using 100 kDa nominal molecular weight cutoff
(NMWC) membrane. Discontinuous diafiltration was
done three times by adding an equal volume of
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Biotechnol Lett (2014) 36:2229–2237
glycine/NaOH buffer (10 mM, pH 8.5) to concen-
trated the retentate in order to get maximum recovery
of desired enzymes. In the second step, the permeate
(obtained above) was passed through a 3 kDa NMWC
membrane. The extract was concentrated tenfold
followed by discontinuous diafiltration, so that low
molecular weight impurities could get maximally
removed.
Ammonium sulphate fractionation
The ultrafiltered extract was subjected to (NH4)2SO4
precipitation to different cut-offs (0–25, 25–50, 0–50,
25–70, 50–70 % saturation). The protein precipitates
obtained after different cut offs were dissolved in a
minimum volume of 10 mM glycine/NaOH buffer
(pH 8.5). The precipitate sample and supernatant were
analyzed for xylanase and polygalacturonase activity
at each fractionation step. The ammonium sulphate
concentrated xylanase and polygalacturonase samples
were ultrafiltered using 1 kDa NMWC membrane
followed by discontinuous diafiltration for removing
the salt and then loaded onto gel filtration column.
Gel filtration chromatography and molecular weight
determination
A Sephadex G-100 column (95 9 0.8 cm) was pre-
equilibrated with 10 mM glycine/NaOH buffer, pH
8.5. The partially purified xylanase and polygalactu-
ronase obtained after ammonium sulfate fractionation
were loaded separately onto this column, which was
run at 15 ml/h. Fractions 1 ml were collected and
analyzed for protein at 280 nm as well as xylanase or
polygalacturonase activity. Active fractions were
pooled and concentrated using ultrafiltration (1 kDa
NMWC) membrane and stored at 4 °C till further use.
A mixture of gel filtration protein molecular weight
markers containing (1) cytochrome c (12.4 kDa) (2)
carbonic anhydrase (29 kDa) (3) albumin (66 kDa) (4)
alcohol dehydrogenase (150 kDa) and (5) b amylase
(200 kDa) was loaded on the Sephadex G-100 column
under the above identical conditions and their elution
volumes were determined. A standard graph was then
plotted between Ve/Vo on the x-axis and log molec-
ular weight on the y-axis for the calculation of the
molecular weight of the purified xylanase and
polygalacturonase.
Biotechnol Lett (2014) 36:2229–2237
Testing of enzymes purity
Purity was checked by native-PAGE. Xylanase and
polygalacturonase samples were run on 10 % native
PAGE gel. To avoid the merging of bands of isozymic
forms of xylanase, purified xylanase was run on 12 %
native PAGE gel (160 9 200 mm) in a long electro-
phoresis gel apparatus. Protein bands were visualized
by silver staining.
Zymography
For zymogram analysis, complete purified xylanase
(run on 12 % native PAGE gel) lane was overlayered
onto 1 % (w/v) agar gel containing 0.1 % (w/v) xylan
and incubated for 20 min at 37 °C. Another purified
xylanase lane of native PAGE gel was cut (corre-
sponding to the bands of silver stained purified
xylanase lane) into short gel pieces and all gel pieces
were overlayered far away from each other onto 1 %
(w/v) agar gel containing 0.1 % (w/v) xylan, and
incubated for 40 min at 37 °C to detect all the
isozymic forms of xylanase. After incubation, sub-
strate-agar gels were stained with 0.5 % (w/v) Congo
Red for 15 min, destained with 1 M NaCl, and the
zymogram was observed for clear halos. To confirm
the presence of isozymic forms of xylanase, the
purified xylanase was run on native 12 % PAGE and
then the complete purified xylanase lane (cut from rest
of the gel) was rotated and placed at an angle of 908 on
another native PAGE gel and again run to get the
bands separated in two dimensions. This gel was over-
layered onto 1 % (w/v) agar gel containing 0.1 % (w/
v) xylan, and incubated for 2 h at 37 °C. The halos
were observed after treating the gel with congo red.
The presence of isozymic forms of xylanase was
reconfirmed in another way, the purified xylanase was
run on native PAGE (12 %) and then the purified
xylanase lane of the polyacrylamide gel was cut
(corresponding to the bands of silver stained purified
xylanase lane) into short gel pieces. All gel pieces
were individually placed in separate wells one after the
other in the same dimension on another native PAGE
gel and again run to get the well separated bands. This
gel was overlayered onto 1 % (w/v) agar gel contain-
ing 0.1 % (w/v) xylan, and incubated for 2 h at 37 °C.
The isozymic forms were observed by the same congo
red method.
2231
The polygalacturonase clear halo was observed by
overlayering the complete purified polygalacturonase
lane of the native PAGE gel onto a 1 % (w/v) agar gel
containing 0.1 % (w/v) polygalacturonic acid and
incubated for 1 h at 37 °C. After incubation, agar-
polygalacturonic acid gel was stained with 1 % (w/v)
CTAB (Hadj-Taieb et al. 2011) for 30 min, washed
with deionised water, and the zymogram was observed
for clear halos.
Characterization of purified xylanase
and polygalacturonase
The effect of pH on enzymes activity was determined
by performing assays at different pH values (4–11)
using the following buffers (100 mM); citrate buffer
pH 4–6, phosphate buffer pH 6–8, Tris/HCl buffer pH
8–9, glycine/NaOH buffer pH 8.5–11 and the stability
of enzymes over wide range of pH was investigated by
mixing equal aliquots of purified enzyme and different
buffers (pH 4–11) followed by incubation at 37 °C.
The effect of temperature on xylanase and polygalac-
turonase activities was investigated by performing
enzymes assay at different temperatures (45, 50, 55,
60, 65, and 70 °C) and the temperature stability profile
was studied after pre-incubating these enzymes at
different temperatures (4–55 °C) for variable time
intervals. The effect of various metal ions and other
additives (see Table 2 below) on xylanase and poly-
galacturonase activity was also studied. The Linewe-
aver–Burk plot of the enzyme activity against
substrate concentration was plotted to determine the
Km and Vmax values. The enzyme activity was
estimated after incubating the optimally diluted
enzyme sample at 55 °C with different concentrations
of birchwood xylan (2.5–15 mg/ml) and polygalactu-
ronic acid (0.3–5 mg/ml) as substrates for xylanase
and polygalacturonase, respectively.
Results and discussion
Enzymes purification
After passing the crude enzyme from microfiltration
unit, the membrane based process of ultrafiltration,
using 100 and 3 kDa NMWC membrane followed by
discontinuous diafiltration, was used for the purifica-
tion of xylano-pectinolytic enzymes. The ultrafiltration
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Table 1 Summary of the purification of xylanase and polygalacturonase from Bacillus pumilus AJK
Purification steps Enzyme Total activity Total protein Specific activity Yield Fold
(IU) (mg) (IU/mg) (%) purification

Crude microfiltereda Xylanase 560,000 2,000 280 100 1

supernatant Polygalacturonase 180,000 2,000 90 100 1
Ultrafiltrationb (cut off Xylanase 558,330 1,030 542 99.7 1.93
100 kDa) Polygalacturonase 176,794 1,030 172 98.2 1.9
Ultrafiltrationc (cut off Xylanase 552,882 306 1,807 98.7 6.45
3 kDa) Polygalacturonase 171,278 306 560 95.15 6.2
(NH4)2SO4 precipitation Xylanase 459,204 126 3,644 82 13
(0–50 %) Polygalacturonase 154,806 101 1,532 86 17
(50–70 %)
Ultrafiltration (cut off Xylanase 448,004 114 3,929 80 14
1 kDa) Polygalacturonase 149,460 92.4 1,618 83 18
Sephadex G-100 Xylanase 375,212 70 5,360 67 19
Polygalacturonase 135,013 65 2,077 75 23
a
Microfiltration (membrane with a pore size of 0.2 micron) was operated at 0.34 atm transmembrane pressure
b
Ultrafiltration was operated at 0.41 atm transmembrane pressure followed by discontinuous diafiltration
c
Ultrafiltration was operated at 0.51 atm transmembrane pressure followed by discontinuous diafiltration

removed the high and low molecular weight proteins
producing the partially purified xylano-pectinolytic
retentate. Saturation of ultrafiltered enzyme extract
with ammonium sulfate to 0–50 and 50–70 % cut offs
precipitated xylanase and polygalacturonase, respec-
tively. These precipitates were dissolved individually
in glycine/NaOH buffer (0.01 M, pH 8.5) and desalted
by 1 kDa NMWC membrane. Partially-purified xy-
lanase and polygalacturonase were loaded separately
in Sephadex G-100 column. Xylanase was eluted as
two peaks from the Sephadex G-100 column, indicat-
ing the presence of at least two isozymic forms of
xylanase. Fractions containing xylanase activity were
pooled and concentrated using a 1 kDa NMWC
ultrafiltration membrane. The elution profile of poly-
galacturonase showed one peak. The polygalacturo-
nase fractions were also pooled and concentrated using
ultrafiltration (1 kDa NMWC membrane). The purifi-
cation fold, recovery and specific activity obtained
after each purification step are given in Table 1.
Xylanase was purified 19-fold with a 67 % recovery,
while polygalacturonase showed a 75 % recovery with
23-fold purification.
Xylanase by Bacillus sp. GRE7 was purified up to
28.5-fold with 27 % recovery using ammonium sul-
phate precipitation followed by anion exchange and
gel filtration chromatography (Kiddinamoorthy et al.
2008). Gomes et al. (2009) have reported the purifi-
cation of an exopolygalacturonase from Penicillium
viridicatum RFC3 by ultrafiltration, Sephadex G-75
gel filtration chromatography and Q Sepharose anion
exchanger with 37.7 fold purification and 3.4 %
recovery.
Molecular weight determination
Molecular weight of xylanase and polygalacturonase
was determined by gel filtration chromatography using
standard gel filtration markers. Xylanase I and xylan-
ase II (two peaks of xylanase eluted from Sephadex
G-100 column) have a molecular weight of *24.5 and
*13 kDa, respectively, while the molecular weight of
polygalacturonase was *40 kDa, as calculated from
the standard graph. Many xylanases (Kiddinamoorthy
et al. 2008) and pectinases (Kumar et al. 2012; Li et al.
2008) produced by the Bacillus genus have molecular
weights in the range of 20–45 kDa.
Testing of enzymes purity and zymography
Native PAGE (10 %) of xylanase obtained after
different purification steps is shown in Fig. 1a.
Initially, the two peaks of xylanase eluted from
Sephadex G-100 were concentrated and run

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Biotechnol Lett (2014) 36:2229–2237
Fig. 1 Native PAGE of a xylanase:- Lane C Crude xylanase;
Lane U Ultrafiltered xylanase; Lane A Ammonium sulfate
concentrated xylanase; Lane F1 Fractions from Sephadex G-100
covering peak I of xylanase were pooled, concentrated and run
and Lane F2 Fractions from Sephadex G-100 covering peak II of
xylanase were pooled, concentrated and run, b purified xylanase
(all active xylanase fractions covering peak I and peak II were
separately. The fractions covering peak I of purified
xylanase gave some merged bands of xylanase, while
the fractions covering peak II of xylanase showed two
bands on the native PAGE. Finally, all the fractions of
Sephadex G-100 column containing active xylanase
(covering peak I and peak II) were pooled, concen-
trated and run as purified xylanase on (12 %) native
PAGE in a long electrophoresis gel apparatus to avoid
merging of bands. The gel after staining with silver
nitrate showed the presence of seven bands of
xylanase (Fig. 1b). The purified polygalacturonase
eluted from Sephadex G-100 column showed a single
band on (10 %) native PAGE gel (Fig. 1c).
The seven bands of xylanase were confirmed as
isozymic forms of xylanase by zymography. Zymog-
raphy was performed in four different ways. Firstly,
by using complete purified xylanase lane and sec-
ondly, by using purified xylanase lane gel pieces. The
zymogram of complete purified xylanase lane gave a
2233
pooled, concentrated and run as purified xylanase on long
electrophoretic gel apparatus to avoid merging of bands)
showing seven bands of xylanase:- Lane A low sample loading
and Lane B heavy sample loading, and c purified polygalactu-
ronase:- Lane C Crude polygalacturonase; Lane U Ultrafiltered
polygalacturonase; Lane A Ammonium sulfate concentrated
polygalacturonase and Lane P Purified polygalacturonase
long continuous activity band (Fig. 2a), while the
zymogram of short gel pieces from the purified
xylanase lane (cut with respect to the bands observed
in stained purified xylanase lane) showed the presence
of seven prominent activity bands (Fig. 2b). Thirdly,
by using complete native PAGE gel, in which the
purified xylanase bands separated in one dimension
were again separated in second dimension (Fig. 2c),
its zymogram gave seven prominent activity bands
(Fig. 2d) and fourthly, by using complete native
PAGE gel, in which the purified xylanase bands were
again separated in same dimension (Fig. 2e). Its
zymogram also gave seven prominent activity bands
(Fig. 2f). Thus, confirming the presence of seven
isozymic forms of xylanase. Separation of xylanase
bands in two dimensions and then performing its
zymography facilitated a more complete examination
and confirmation of xylanase multiplicity. Graciliba-
cillus sp. TSCPVG secreted multiple xylanases in the
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Fig. 2 Zymogram of xylanase obtained by using a complete
purified xylanase lane of native PAGE gel, b short gel pieces of
the purified xylanase lane (cut with respect to the bands obtained
on similar stained gel), c Two-dimensional separation of
purified xylanase bands by using the complete purified xylanase
extracellular medium and its zymogram analysis on
native PAGE gel showed ten multiple bands associ-
ated with xylanase activity (Giridhar and Chandra
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Biotechnol Lett (2014) 36:2229–2237
lane of native PAGE gel and, d its zymogram, e separation of
purified xylanase bands by using the short gel pieces of purified
xylanase lane (cut with respect to the bands obtained on similar
stained gel), and f its zymogram, g zymogram of polygalactu-
ronase on polygalacturonic acid
2010). A zymogram of polygalacturonase showed a
single prominent activity band at position correspond-
ing to that of the band in native PAGE (Fig. 2g).
Biotechnol Lett (2014) 36:2229–2237 2235

Thus, confirmed the presence of a single form of Table 2 Effect of metal ions and other additives on purified
polygalacturonase. xylanase and polygalacturonase produced by Bacillus pumilus
AJK

Characterization of the purified xylanase Metal ion Xylanase Polygalacturonase

and polygalacturonase (1 mM) activity (%) activity (%)

Control 100 100

The purified xylanase showed two pH optima, one at KCl 61 125
pH 6.0 and other at pH 8.5, indicating the presence of NaCl 75 100
isozymic forms of xylanases. Maximum polygalactu- CaCl2 85 80
ronase activity was obtained at pH 9.0. Many MgCl2 153 190
researchers have reported the optimum pH of xylan- CuCl2 89 90
ases in the range of 6.0–9.0 (Nagar et al. 2012), while
CoCl2 70 225
reports on pectinases having optimum pH in the range
BaCl2 106 140
of 8.0–9.0 are also available (Celestino et al. 2006).
HgCl2 16 103
The xylanase was most stable at pH 8.0 and retained
MnSO4 89 295
almost 100 % of its original activity from pH 6.0–11.0
FeSO4 66 103
up to 3 h, while the polygalacturonase was 100 %
ZnCl2 67 203
stable at pH 8–9 even after 48 h of incubation and
Urea 8 80
more than 80 % stable from pH 6.0–11.0 even after
EDTA 96 178
72 h of incubation. The concentrated form of the
SDS 25 34
enzyme was more stable at different pH values (data
b-mercaptoethanola 112 148
not shown). Purified xylanase from B. pumilus SV-85S
Tween 20 150 115
was fully stable from pH 5 to 11 after 1 h of incubation
(Nagar et al. 2012). Pectin lyase from Aspergillus Tween 80 121 243
flavus was stable for 24 h in the pH range 4.0–10.0 Triton X-100a 120 120
(Yadav et al. 2008). To evaluate the effect of various metal ions and additives on
Both purified xylanase and polygalacturonase gave xylanase and polygalacturonase activity, the enzyme sample
was incubated for 1 h at room temperature in the presence of
maximum activities at 55 °C, though they retained
different metal ions and additives. Thereafter, xylanase and
more than 85 % residual activity from 50 to 60 °C. polygalacturonase assays were performed. The 100 % values
Most of xylanolytic (Kamble and Jadhav 2012) and of xylanase and polygalacturonase activities were 280 and
pectinolytic (Celestino et al. 2006; Nadaroglu et al. 90 IU/ml, respectively. Values are given as the mean of three
replicates
2010) enzymes show maximum activity at 50–60 °C. a
The purified xylanase was moderately stable over a Final concentration 1 % (v/v)
wide range of temperatures, while polygalacturonase
showed higher stability. Both the enzymes were purified pectin lyase from B. pumilus (P9) appeared to
100 % stable at 4 °C for more than six months. The be stable and retained its full activity after 1 h
xylanase retained about 100, 85 and 58 % of its incubation from 40 to 50 °C, but the activity was
activity after 1, 2 and 3 days, respectively, whereas reduced to 20 % after 1 h at 60 °C (Nadaroglu et al.
polygalacturonase retained 100 and 56 % of its 2010).
activity after 20 and 25 days, respectively, when kept The effect of various metal ions and additives on
at 37 °C. The xylanase showed nearly 69 % activity at the activity of the purified enzymes was evaluated
50 °C after 1 h of incubation, while polygalacturonase (Table 2). Among the various metal ions tested, Mg2?
showed 98 and 62 % activity at 50 and 55 °C, enhanced the xylanase activity, whereas Ca2?, Mn2?
respectively even after 3 h of incubation. Stability and Cu2? brought slight fall in the activity. All other
was greatly enhanced, when enzyme was stored in metal ions such as Co2?, Zn2?, Hg2?, Fe3?, Na? and
concentrated form at different temperatures (data not K? were inhibitors of xylanase activity. Among the
shown). Xylanase from Cellulosimicrobium sp. various additives tested, b-mercaptoethanol, Tween
retained 86 % of its original activity at 40 °C after 20, Tween 80 and Triton X-100 slightly enhanced
4 h of incubation (Kamble and Jadhav 2012). The xylanase activity, whereas urea decreased the activity.

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Almost all the metal ions, except Ca2? and Cu2?,
increased the polygalacturonase activity. Similarly,
the majority of the additives such as EDTA, b-
mercaptoethanol, Triton X-100, Tween 20 and Tween
80 increased polygalacturonase activity, whereas urea
decreased activity. In case of both xylanase and
polygalacturonase, SDS was the most potent inhibitor
causing almost 75 and 66 % inhibition of activity,
respectively. The xylanase produced by Bacillus sp.
GRE7 was also inhibited with Cu2? and Fe2? but,
contrary to our data, showed an enhanced activity in
the presence of Mn2? and Co2? (Kiddinamoorthy
et al. 2008). The activities of three endopolygalactu-
ronases from Bacillus gibsonii were stimulated by
Mg2?, Tween 80 and Tween 20 (Li et al. 2008). The
Km and Vmax values of the purified xylanase were
calculated to be 4.7 mg/ml and 500 IU/ml, respec-
tively whereas purified polygalacturonase gave the
Km value 1.5 mg/ml and Vmax was 119 IU/ml. The
xylanase from Cellulosimicrobium sp. had Km and
Vmax values of 4.8 mg/ml and 233 lmol/min/mg,
respectively (Kamble and Jadhav 2012), where as the
three purified endopolygalacturonases in B. gibsonii
had Km values of 1.2, 0.9 and 1.1 mg/ml (Li et al.
2008).
Conclusion
The presence of multiple forms of xylanases (acidic
and alkaline) shows its suitability for wide range of
industrial application, where acidic or alkaline xylan-
ases are required. The low molecular weight of these
enzymes also offers another advantage to this micro-
bial isolate. These industrially-valuable xylanase and
polygalacturonase preparations are active over a wide
range of pH and temperature for long duration.
Enhancement in activity of both enzymes in the
presence of low concentration of Mg2?, shows that its
inclusion will further decrease the cost during appli-
cation of these enzymes. Since these xylano-pectino-
lytic enzymes are produced by an industrially suitable
bacterial isolate, and both are required together for
most industrial applications, their production in com-
bination using agricultural wastes will reduce the
overall cost to great extent. For industrial application,
where purified enzyme preparations are required, the
fast flow rate microfiltration and ultrafiltration tech-
niques reported in this study can be easily scaled up.
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Biotechnol Lett (2014) 36:2229–2237
The present characterization study indicates that these
xylanase and pectinase preparations have great
potential to be used for various industrial applications
at very low cost.
Acknowledgments The authors acknowledge the financial
support provided by University Grant Commission, India.
University Research Scholarship awarded to Amanjot Kaur by
Kurukshetra University is also kindly acknowledged.
Conflict of interest The authors declare that they have no
conflict of interest.
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