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Chapter 6
Production, purification, properties of cellulase free thermostable xylanase
from Paenibacillus sp. ASCD2 and its application in prebleaching of
eucalyptus kraft pulp
Part of this chapter has been published as:
Digantkumar Chapla, Harshvadan Patel, Atmika Singh, Datta Madamwar,
Amita Shah. Production, purification and properties of a cellulase-free thermostable endoxylanase from newly isolated Paenibacillus sp. ASCD2. Annals of Microbiology, (2011). DOI: 10.1007/s13213-011-0323-5. (Italy).
Digantkumar Chapla, Harshvadan Patel, Datta Madamwar, Amita Shah.
Assessment of a thermostable xylanase from Paenibacillus sp. ASCD2 for application in prebleaching of eucalyptus kraft pulp. Waste and Biomass Valorization, (2012). DOI: 10.1007/s12649-012-9112-z. (Germany).
Chapter 6. Production, purification, properties………
187
6.1 Introduction
The increasing concern for preserving our resources and environment has initiated a
growing interest in producing microbial enzymes. Enzyme mediated reactions are
attractive alternatives to tedious, expensive and pollution prone chemical methods.
The demand for thermostable enzymes of microbial origin is gaining wide industrial
and biotechnological interest due to the fact that such enzymes are better suited for
harsh industrial processes (Techapun et al., 2003). Some of the valuable advantages
of conducting biotechnological processes at elevated temperature are reducing the risk
of contamination, faster reaction rates and efficient hydrolysis of substrates (Becker
1997; Haki and Rakshit 2003). The pulp and paper industry is one of the fastest
growing industries and the use of thermostable xylanases seems attractive since they
provide global environmental benefits. The pulping processes mostly run at high
temperature and alkaline pH. Therefore, enzymes functional at high temperature and
alkaline pH are preferable in order to make the process technically and economically
viable (Viikari, 1986). Many commercial xylanases can only partially fulfill these
requirements and hence search for such xylanases is still an area of intense research.
Thermophilic microorganisms are the major source of thermostable xylanases. Such
xylanases have been reported from bacteria and fungi like Bacillus, Streptomyces,
Clostridium, Thermotoga, Thermomyces spp. etc. (Techapun et al., 2003). However
yield of xylanases is many times lower from bacteria as compared to fungi, but
bacteria with high thermal and pH stability are of great importance as they possess
better stability as compared to enzymes from fungus. Large scale cultivation of
thermophiles for enzyme production remains an economical challenge because of the
complex nutritional requirements and low specific growth rates (Turner et al., 2007).
For commercial applications of xylanases efforts have been directed for isolation and
development of high yielding strains, production of novel and robust enzymes,
development of efficient fermentation processes and recovery systems. Solid state
fermentation is a method of choice for production of many hydrolytic enzymes as it
allows fermentative production of enzymes on moist low cost substrates like agro-
residues with less expenditure of energy, easy product recovery and high volumetric
productivity (Pandey et al., 2000; Haki and Rakshit, 2003).
Chapter 6. Production, purification, properties………
188
The use of thermostable xylanases in paper pulp industry seems attractive since they
provide global environmental benefits. The original concept of using hemicellulases
in pulp bleaching was given by Viikari (Viikari, 1986). Microbial xylanases (E.C.
3.2.1.8) are the preferred catalysts for xylan hydrolysis due to their high specificity,
mild reaction conditions, negligible substrate loss and no side product generation
(Kulkarni et al., 1999). The most important advantages of microbial xylanases in
paper and pulp industry are demonstrated by their bleach boosting ability, decreasing
the chlorine consumption and consequently lowering the discharge of hazardous
chemicals in the effluent thereby creating an eco-friendly technology (Beg et al.,
2001). Cellulase free thermostable xylanases are necessary for pulp bleaching. Such
xylanases accomplish the process by selective removal of xylan from kraft pulp prior
to pulp bleaching and thereby allowing easy action of chemical bleaching agents
(Buchert et al., 1994). As the pulping processes mostly run at high temperature and
pH, enzymes functional at high temperature and alkaline pH are preferable in order to
make the process technically and economically feasible. Moreover xylanases with low
molecular weight are also advantageous as they can easily diffuse in pulp fibres (Haki
and Rakshit, 2003).
In the context of the above discussion, the present study was aimed at exploiting the
newly isolated thermophilic Paenibacillus sp. ASCD2 from the hot compost for the
production of cellulase free thermostable xylanase under solid state fermentation
using low cost agro-residues. Purification and biochemical properties of xylanase
were also studied in order to predict its possible application. Applicability of an
indigenously produced cellulase free thermostable xylanase in prebleaching of kraft
pulp was also evaluated.
6.2 Materials and methods
6.2.1 Materials
Birchwood xylan was obtained from Sigma, Germany. Standard xylooligosaccharides
(xylobiose, xylotriose, xylotetrose, xylopentose) were purchased from Megazyme,
Ireland. Wheat bran, wheat straw, rice bran, rice straw and corn cobs were procured
from local farmers. Dialysis membrane was procured from Sigma, Germany. Resins
of Sephadex G-100 were procured from Pharmacia, Sweden. Compost samples were
Chapter 6. Production, purification, properties………
189
collected from the inner side of the heaps of compost which had temperature in the
range of 45-55ºC, from Sardar Patel Renewable Energy Research Institute, Vallabh
Vidyanagar, Gujarat, India. All the reagents, media, chemicals used under study were
of analytical grade (Sigma, Qualigens, Hi-Media, Merck, Loba, etc.). Unbleached
eucalyptus kraft pulp used for the present study was kindly provided by J.K. Paper
Mills, which prepares virgin pulp from the eucalyptus tree, Songadh, Surat, Gujarat,
India.
6.2.2 Isolation and identification of xylanase producing bacterial strain
Suspensions were prepared by mixing compost samples in sterilized distilled water.
Suitably diluted samples were spreaded on a medium containing (g/l): yeast extract, 2;
peptone, 5; MgSO4, 0.5; NaCl, 0.5; CaCl2, 15; agar, 30 and wheat bran, 20; pH 7.0.
Plates were incubated at 50ºC for two days. Isolates were transferred on xylan
containing agar medium and incubated at 50ºC. Xylanase producing ability was
confirmed by observing the clear zone of hydrolysis around the colonies and further
microscopic observation was also done by grams staining and endospore staining (see
Chapter 2).
The most promising thermostable isolate DK2 which was later designated as ASCD2
was identified on the basis of 16S rDNA approach and was used for further studies.
The Genomic DNA from this isolate was extracted as described by Ausubel et al.,
(1997). The genomic DNA was diluted appropriately and used as template in PCR
reactions using universal eubacterial primers 8F and 1492R. The amplified PCR
product was purified and subjected to sequencing. Forward and reverse DNA
sequencing reaction of PCR amplicon was carried out with 8F (5'-
AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-
3') primers using BDT v3.1 Cycle sequencing kit on ABI 3730xl Genetic Analyzer at
Xcelris laboratories, Ahmedabad. The 16S rDNA gene sequence was used to carry out
BLAST with the database of NCBI gene bank database. Based on maximum identity
score, first ten sequences were selected and aligned using multiple alignment software
program Clustal W. Distance matrix was generated using RDP database and the
phylogenetic tree was constructed using MEGA 4. The evolutionary distances were
computed using the Kimura 2-parameter method (Kimura, 1980) and are in the units
of the number of base substitutions per site. Phylogenetic analyses were conducted in
Chapter 6. Production, purification, properties………
190
MEGA 4 (Tamura et al., 2007). The nearly complete sequence (>95%) of bacterial
16S rRNA gene (1435 bp) has been submitted to GenBank at NCBI (Accession No.
HM452162).
6.2.3 Xylanase production using agro-residues by Paenibacillus sp. ASCD2 under solid state fermentation
Xylanase production under solid state fermentation was carried out in 250 ml
Erlenmeyer flasks using 5 g of washed dried and finely powdered (5-7 mm particle
size) agro-residues as substrates and moistened with the basal medium containing
(g/l): yeast extract, 2; peptone, 5; MgSO4, 0.5; NaCl, 0.5; CaCl2, 0.15; initial pH 7.0.
The medium and substrate were sterilized separately at 121ºC at 15 lbs for 15 minutes
and mixed at the time of inoculation with 1 ml of inoculum and were incubated at
50ºC under static condition with 50% relative humidity in humidity controlled
incubator. The flasks were shaken intermittently (twice a day) for homogeneous
mixing of contents. The enzyme was extracted from each flask at regular interval of
time (at every 24 h) and crude enzyme was used for further analysis.
Inoculum was prepared from overnight grown culture on Luria agar slants at 50ºC.
Suspension was prepared by scraping the slant with sterile distilled water in such a
way that it reached 1.5 Absorbance at 660 nm.
6.2.4 Enzyme extraction
The content of each flask was extracted using 30 ml of 50 mM sodium phosphate
buffer (pH 7.0) and filtered through wet muslin cloth by thorough squeezing. The
extract was centrifuged at 8500 x g for 20 min. The clear supernatant was used as
crude enzyme for further analysis.
6.2.5 Study of physicochemical factors on production of cellulase free thermostable xylanase under solid state fermentation by Paenibacillus sp. ASCD2
Xylanase production was carried out under solid state fermentation as described
earlier using 5 g of washed, dried and finely powdered (5-7 mm particle size) agro-
residues viz. wheat bran, rice bran, corncobs, wheat straw and rice straw. Influence of
nitrogen source was studied by using various moistening agents with varying
concentration of nitrogen sources using wheat straw as a substrate at 50ºC. Varying
concentration of nitrogen sources were added to the basal media, composition of
Chapter 6. Production, purification, properties………
191
moistening media used under study were as follows: Medium I (g/l): yeast extract, 2;
peptone, 5; MgSO4, 0.5; NaCl, 0.5; CaCl2, 0.15. Medium II (g/l): yeast extract, 4;
peptone, 10. Medium III (g/l): yeast extract, 8; peptone, 15. Medium IV (g/l): yeast
extract, 10; peptone, 20. Medium V (g/l): Medium III + aspargine, 4 + corn steep liquor
(CSL), 20. Medium VI (g/l): Medium III + corn steep liquor (CSL), 20. The
concentration of MgSO4, 0.5; NaCl, 0.5; CaCl2, 0.15 was kept constant in all the
media. The effect of moisture level on xylanase production was studied by varying the
ratios of substrate (wheat straw) to moistening medium from 1:4 to 1:6 (w/v).
Influence of temperature was studied by carrying out solid state fermentation (SSF) at
45, 50, 55ºC. Yield of xylanase production is reported in terms of U/g of dry substrate
during solid state fermentation.
6.2.6 Enzyme assays
Xylanase (E.C. 3.2.1.8) activity was measured using 1% birchwood xylan solution as
substrate (Bailey et al., 1992). The reaction mixture consisted of appropriately diluted
0.2 ml of enzyme and 1.8 ml of 1% birchwood xylan. The release of reducing sugars
in 10 min at 60°C, pH 7.0 (50 mM sodium phosphate buffer) was measured as xylose
equivalents using dinitrosalysilic acid method (Miller, 1959). One unit of xylanase
activity (U) was defined as the amount of enzyme liberating 1 µmole of xylose per
min under assay conditions. Filter paper activity was measured according to IUPAC
recommendations employing filter paper (Whatmann no.1) as substrate (Ghose,
1994). The reaction system for filter paper assay consisted of 1 ml buffer with 0.5 ml
of appropriately diluted enzyme and 50 mg of filter paper. The release of reducing
sugars in 60 min at 60°C, pH 7.0 (50 mM sodium phosphate buffer) was measured as
glucose equivalents using dinitrosalysilic acid method (Miller, 1959). One unit of
filter paper activity was defined as the amount of enzyme liberating 1 µmole of
glucose per min under assay condition.
6.2.7 Protein estimation
The soluble protein was determined by Folin’s method using bovine serum albumin as
standard (Lowry et al., 1951).
Chapter 6. Production, purification, properties………
192
6.2.8 Purification of xylanase from Paenibacillus sp. ASCD2
The calculated amount of solid ammonium sulphate was added to the culture
supernatant obtained from solid state fermentation with constant stirring at 10ºC to
achieve initially, 0–30% saturation. After centrifugation at 8500 x g for 30 min at
4ºC, the supernatant was discarded and the precipitates were dissolved in small
volume of buffer. The enzyme solution was subjected to dialysis for about 20–24 h at
10ºC against 50 mM sodium phosphate buffer pH 7.0 fortified with 1 mg% sodium
azide with three intermittent changes of the buffer. After dialysis further purification
was done by gel permeation chromatography. Dialyzed enzyme was loaded on the
column containing Sephadex G-100 as a matrix (1×10 cm) equilibrated with 50 mM
sodium phosphate buffer pH 7.0. The protein was eluted at the flow rate of 1 ml/min.
The active fractions with highest specific activity were combined together. Xylanase
activity and protein estimation were carried out at each step of purification.
6.2.9 SDS-Polyacrylamide gel electrophoresis and zymogram staining
Electrophoresis of proteins at different stages of purification was carried out in 10%
SDS-PAGE (Laemmli, 1970) in the gel casting unit provided by Genei, Bangalore,
India. The samples were loaded on gel after its pretreatment of boiling at 100°C for
10 min with the loading buffer. Samples for zymogram were treated at 60°C for 10
min with the loading buffer. The gel was run at 80 V for initial 30 min and then at 100
V till end of the run. Protein bands were observed using silver staining. PMWM 14.3
to 97.4 kDa (Genei, Bangalore, India) was used as the molecular weight marker.
Zymogram staining was performed by overlaying the electrophoresed gel on a 0.8
mm replica gel prepared in 50 mM buffer (sodium phosphate, pH 7.0) containing
0.1% birchwood xylan and 2% agar. The electrophoresed gel was washed thoroughly
in buffer containing 25% isopropanol to remove excess of SDS before overlaying on
replica. The system was incubated at 60ºC for 90 min. The replica gel was flooded in
0.1% congo red for 1 h followed by destaining with 1 M NaCl for 12 h. Contrast was
created using 5 % acetic acid solution. Active bands were observed as clear bands
against dark blue background (Beguin, 1983).
Chapter 6. Production, purification, properties………
193
6.2.10 Characterization of purified xylanase from Paenibacillus sp. ASCD2
6.2.10.1 Effect of temperature on xylanase activity and stability
The optimal temperature for purified xylanase was obtained by assaying the enzyme
activity at different temperatures ranging from 40 to 90ºC. The thermostability of
xylanase was monitored by incubating the enzyme solution at a fixed temperature, in
the range of 55 to 75ºC and measuring the residual activity at every 30 min interval
for 3 h.
6.2.10.2 Effect of pH on xylanase activity and stability
The relative xylanase activity was determined by using different buffers ranging from
pH 4 to 9.5. Buffers used for this study were, sodium citrate (50 mM) buffer pH 4,
4.5, 5 and 5.5; sodium phosphate (50 mM) buffer pH 6.0, 6.5, 7.0 and 7.5; and
Glycine-NaOH (50 mM) buffer pH 8.0, 8.5, 9.0 and 9.5. Xylanase activity was
assayed using different buffers at 60°C.
6.2.10.3 Determination of Kinetic parameters
Stock solution of birch wood xylan (1% w/v) was diluted with 50 mM sodium
phosphate buffer pH 7.0 to obtain the various concentration of substrate ranging from
1.0 to 10.0 mg/ml in assay mixture. The enzyme concentration was kept constant for
all the assays. Lineweaver-Burk plot was used to study km and Vmax. Kcat and catalytic
efficiency were also calculated.
6.2.10.4 Effect of metal ions and additives on xylanase activity
Influence of metal ions and additives on xylanase activity was determined by
incorporating metal ions such as MgSO4, MnSO4, FeSO4, CuSO4, ZnSO4, MgCl2,
HgCl2, NaCl, AgNO3, KCl and BaCl2 at concentration of 10 mM and additives like
Tween 80, EDTA at concentration of 0.1% in the reaction mixture. Relative xylanase
activity was analysed by comparing with control.
6.2.10.5 Mode of action of purified xylanase from Paenibacillus sp. ASCD2
Enzymatic hydrolysis of 1% birchwood xylan was carried out using purified xylanase
(100 U/g) at 60°C and pH 7.0, fortified with 10 ppm of sodium azide in the reaction
Chapter 6. Production, purification, properties………
194
mixture. At defined times, reaction mixtures were sampled, the enzyme activity was
stopped by incubating it in boiling water bath for 60 min, centrifuged and clear
supernatant was obtained. Samples were applied on TLC plates with the help of
capillary. The products were examined by ascending thin-layer chromatography
(TLC) on precoated silica gel plates of 60 F254 (Merck, Germany) using the mixture
of acetonitrile and water in the ratio of 85:15 as the solvent system. The separated
products were detected by spraying 0.2% orcinol in the mixture of methanol and
sulfuric acid (90:10) followed by heating at 100°C for 10 min in oven.
6.2.10.6 Storage stability of purified xylanase
The storage stability of purified xylanase was studied by keeping the membrane
sterilized enzyme solution of purified as well as crude xylanase in 0.05 M phospahte
buffer (pH 5.3) at deep freeze (-20°C), refrigeration temperature (6-8°C) and room
temperature (25–30°C). The enzyme solution was stored in sterile microcentrifuge
tubes. Xylanase activity was assayed at regular intervals.
6.2.11 Biobleaching of kraft pulp using crude xylanase from Paenibacillus sp. ASCD2
Unbleached kraft pulp was pretreated with crude xylanase at 5% consistency in
polyethylene bags by immersing it in a water bath at 60°C for 3 h. The filtrate was
analysed for reducing sugars and absorbance at 237 and 465 nm to determine the
release of chromophores and the removal of hydrophobic material respectively (Patel
et al., 1993). Increase in the absorbance at these wavelengths determines the
liberation of the chromophores and hydrophobic compounds due to the effect of
xylanases from the pulp during biobleaching. The washed pulp sample was dried till
constant weight for their further analysis. Kappa number of pulp was measured
according to TAPPI (T236 m-60). The controls were prepared as above, without the
addition of enzyme. Reduction in the kappa number indicates the delignification of
pulp fibers.
6.2.12 Optimization of enzyme dose and incubation time for biobleaching of kraft pulp
The optimization of enzyme dose for biobleaching of kraft pulp was carried out by
treating the pulp with varying doses of crude xylanase from Paenibacillus sp. ASCD2
Chapter 6. Production, purification, properties………
195
ranging between 10-50 U/g of moisture free pulp with 5% consistency in water bath at
60°C for 3 h. The optimization of reaction time for prebleaching was carried out by
treating the moisture free pulp with 40 U/g of crude xylanase with 5% consistency in
water bath at 60°C for variable time intervals up to 6 h. Kappa number of pulp and
filtrate were analysed at regular time intervals as described above in 6.2.11.
6.2.13 Chemical bleaching of eucalyptus kraft pulp
The unbleached kraft pulp and enzymatically pretreated kraft pulp were treated in
three sequential stages for chemical bleaching. At each step of bleaching, the pulp
was maintained at 5% consistency. In the first stage of bleaching, the pulp was treated
with 5% sodium hypochlorite (NaOCl) and incubated at 70°C for 1 h, in the second
stage, the pulp was treated with 0.4% sodium hydroxide (NaOH) and incubated at
55°C for 1 h and in the third stage, the pulp was treated with 0.5% hydrogen peroxide
(H2O2) and incubated at 60°C for 1 h. The pulp obtained at each step was washed with
distilled water and dried in oven and was further used for analysis.
6.2.14 Scanning electron microscopy
The effect of enzymatic treatment and enzymatic treatment followed by chemical
treatment on kraft pulp fibers of unbleached and bleached kraft pulps were studied by
performing scanning electron microscopy. The pulp samples after each treatment
were washed thoroughly with distilled water till neutrality and dried in oven. The pulp
fibers were directly examined under scanning electron microscope with accelerating
voltage of 5.0 kV.
6.3 Results and discussion
Enzymes are extremely efficient and highly specific biocatalysts. With the
advancement in biotechnology, the demand for enzyme application to amend
conventional processes is ever increasing in pulp and paper industry (Bajpai, 2004).
The suitability of xylanase for bleaching pretreatment is generally dependent on the
enzyme stability at high temperature and alkaline pH, and the absence of cellulase
activity (Subramaniyan and Prema, 2002).
Chapter 6. Production, purification, properties………
196
6.3.1 Identification of xylanase producing microorganism
The xylanase producing thermophilic strain DK2 (which was further named as
ASCD2) was isolated from the inner side of the hot compost which showed
significant xylan hydrolysis on solid media as well as it produced good amount of
xylanase in liquid media at 50ºC. The newly isolated xylanase producing strain was
found to be thermophilic in nature as it was able to grow only within the temperature
range of 45-70ºC. The colonial characteristics of this isolate were small, round, entire,
slightly raised, smooth, colorless and translucent. Microscopic studies revealed that
newly isolated strain was gram positive, catalase positive and endospore forming rod
shaped bacteria. Figure 6.1 shows the agarose gels of the bacterial DNA isolated from
strain ASCD2 and its 16S rRNA gene amplification of around 1500 bp using
universal bacterial primers 8F and 1492R. It was evident from the 16S rRNA analysis
that this microorganism was closely related to the members of Paenibacillus sp.
Further phylogenetic analysis indicated that the levels of similarity were around 99%
with all the other Paenibacillus sp. as shown in Fig. 6.2. The isolated strain ASCD2
forms a common cluster with Paenibacillus sp. RKJ14 (Accession No. GQ927307).
The nearly complete sequence of bacterial 16S rRNA gene (1435 bp) of newly
isolated Paenibacillus sp. ASCD2 has been submitted to GenBank at NCBI and its
Accession No. is HM452162. The consensus gene sequence of Paenibacillus sp.
ASCD2 is also given in 6.3.1.2.
Members of the genus Bacillus are common saprophytic components of soil
microbiota (Claus and Berkeley, 1986; Sanchez et al., 2005). Some species are known
to secrete a variety of extracellular enzymes, several of which have important
industrial applications (Outtrup and Jorgensen, 2002). Genomic analysis of the genus
Bacillus, using rRNA-DNA sequencing has led to its division into distinct genera.
One of these is the genus Paenibacillus (Ash et al., 1993) and belongs to the family
“Paenibacillaceae”. Members of the genus Paenibacillus are generally facultatively
anaerobic organisms and generally possess 45 to 54 mol% of DNA G+C content.
Some of these bacteria excrete diverse assortments of extracellular polysaccharide
hydrolyzing enzymes, including xylanases (Zamost et al., 1991; Nielsen and
Sorensen, 1997; Lee et al., 2000).
Chapter 6. Production, purification, properties………
197
DNA 1500 bp amplicon
The normal habitat of the paenibacilli is the soil, particularly soils rich in humus and
plant materials in which they presumably aid composting through the secretion of
extracellular carbohydrases and other enzymes (Whitman, 2009). Paenibacillus
species are generally capable to hydrolyze plant materials and are frequently isolated
and identified from soil and plant related sources (Rivas et al., 2006). Members of the
genus Paenibacillus are generally gram variable, spore forming, facultatively
anaerobic and mesophilic microorganisms (Velazquez et al., 2004). However,
recently a thermophilic cellulose degrading Paenibacillus sp. strain B39 was isolated
from manure heaps (Wang et al., 2008) and Thermobacillus xylanilyticus nov. sp. a
member of Paenibacilleacea family was also reported as thermophillic xylanolytic
microorganism (Touzel et al., 2000). Xylanase production by mesophilic
Paenibacillus sp. such as Paenibacillus favisporus, Paenibacillus sp. strain HC1 and
Paenibacillus campinasensis BL11 have been reported (Velazquez et al., 2004;
Harada et al., 2008; Ko et al., 2010a). In recent years the reports on xylanolytic
Paenibacillus spp. are increasing however, the reports on production of thermostable
xylanase by Paenibacillus sp. under solid state fermentation using low cost agro-
residues are very rare. Hence it was found interesting to exploit the newly isolated
thermophilic Paenibacillus sp. ASCD2 for the production of thermostable xylanase
using low cost agro-residues.
a. b.
Fig. 6.1 (a) Agarose gel (0.8%) showing bacterial DNA isolated from ASCD2 and (b) agarose gel (1.2%) showing PCR amplicon of 16S rRNA (~1500 bp) gene from ASCD2. (Marker was supermix DNA ladder (Genei, Bangalore)
Chapter 6. Production, purification, properties………
198
Paenibacillus alvei , strain AUG6 (AB377108)
Paenibacillus sp. enrichment culture clone S25 (GQ288407)
Paenibacillus sp. Dg-904 (EU497636)
Paenibacillus sp. L5 (EF426449)
Paenibacillus sp. GT08-03 (AM162320)
Paenibacillus alvei , isolate CCM2B (FN433032)
Paenibacillus sp. RKJ14 (GQ927307)
Paenibacillus alvei , strain V1 (EU435385)
Paenibacillus alvei (AB073200)
Paenibacillus alvei , strain DSM29T (AJ320491)
ASCD2
Fig. 6.2 Phylogenetic tree based on 16S rRNA gene sequence of Paenibacillus sp. ASCD2 (Gene bank Accession No. HM 452162) indicating the position of isolate among the sequences of closest phylogenetic neighbours obtained from NCBI BLAST analysis. The numbers in the parenthesis indicates the accession numbers of corresponding sequences. The scale bar indicates the evolutionary distances 6.3.1.2 Consensus Sequence of Paenibacillus sp. ASCD2 submitted to GenBank at NCBI with its Accession No. as HM452162
GTGCCTAATACATGCAAGTCGAGCGGACTTGATGGAGTGCTTGCACTCCTGATGGTTAGCGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCATAAGACTGGGATAACCCACGGAAACGTGAGCTAATACCAGATAGGCATTTTCCTCGCATGAGGGAAATGAGAAAGGCGGAGCAATCTGTCACTTATGGATGGACCTGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGCCAGGGAAGAACGCCTAGGAGAGTAACTGCTCTTAGGGTGACGGTACCTGAGAAGAAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCGAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGCAATGTAAGTTGGGTGTTTAAACCTAGGGCTCAACCTTGGGTCGCATCCAAAACTGCATAGCTTGAGTACAGAAGAGGAAAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGGCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGCTAGGTGTTAGGGGTTTCGATACCCTTGGTGCCGAAGTTAACACATTAAGCATTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCAGTGGAGTATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTGAATGACCGTCCTAGAGATAGGGCTTTCCTTCGGGACATTCAAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTAACTTTAGTTGCCAGCATTCAGTTGGGCACTCTAGAGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTACTACAATGGCTGGTACAACGGGAAGCGAAGCCGCGAGGTGGAGCCAATCCTAAAAAGCCAGTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGTCGGAATTGCTAGTCATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCACGAGAGTTTACAACACCCGAAGTCGGTGAGGTAACCGCAAGGAGCCAGCCGCCGAA
Chapter 6. Production, purification, properties………
199
6.3.2 Xylanase production under solid state fermentation by Paenibacillus sp. ASCD2 6.3.2.1 Xylanase production using different agro-residues by Paenibacillus sp. ASCD2 under solid state fermentation at 50°C
Xylanase production was initially carried out using various agro-residues viz. wheat
bran, rice bran, wheat straw, corncobs and rice straw as a substrate under SSF at 50ºC.
Maximum xylanase production upto 96±10 U/g of dry substrate (11.4±0.8 U/ml) was
obtained after 72 h of cultivation time when wheat straw was used as the substrate. No
detectable cellulase activity was found in any of the culture filtrates. Corncobs yielded
xylanase activity up to 42.2±5.3 U/g of dry substrate while rest other substrates did
not favor significant xylanase production (Table 6.1). Almost all the filtrates showed
absence of filter paper activity. Ko et al., (2010a) have reported maximum xylanase
production by Paenibacillus campinasensis BL11 upto 10.5 U/ml at 37ºC after 24 h
using pure birchwood xylan and also found that only same level of xylanase was
produced by Paenibacillus campinasensis BL11 using rice husk and rice straw as the
substrate.
Table 6.1 Xylanase production by Paenibacillus sp. ASCD2 using different agro-residues and mineral media under solid state fermentation at 50ºC
Time (h)
Wheat bran (U/g)
Wheat straw (U/g)
Rice bran (U/g)
Rice straw (U/g)
Corncobs (U/g)
24 1.25±0.05 35.05±0.2 0.2±0.1 0.5±0.2 1.65±0.4 48 2.29±0.03 73.61±12.8 1.2±0.8 1.69±0.2 15.78±1.8 72 3.24±0.6 96±10 1.49±0.4 2.68±0.8 28.74±2.5 96 2.56±0.2 82.06±8.5 1±0.3 1.59±0.3 42.2±5.3 120 1.69±.14 31.76±7.8 -- -- 34.25±8.9
Selection of appropriate substrates plays an important role during SSF. Various
substrates have been used by different researchers as per the need and availability of
substrate for the xylanase production under SSF. Wheat straw has been used
successfully for xylanase production by various research groups (Elegir et al., 1994;
Dhillon and Khanna 2000; Techapun et al., 2003). Higher xylanase production using
wheat straw may be attributed due to its high hemicellulosic content, favourable
degradability and the presence of suitable nutrients during production. Dhillon and
Khanna (2000) used wheat straw as a substrate for xylanase production by Bacillus
Chapter 6. Production, purification, properties………
200
circulans AB16. Elegir et al., (1994) also reported wheat straw as a good inducer for
xylanase production. Moreover various other physical properties of the substrate such
as crystalline or amorphous nature, the accessible surface area, porosity, and particle
size also plays the important role in xylanase production under SSF (Archana and
Satyanarayana, 1997; Pandey et al., 2000; Poorna and Prema, 2007).
6.3.2.2 Influence of moistening agents on xylanase production by Paenibacillus sp. ASCD2 under solid state fermentation at 50°C
Xylanase production was initially carried out using normal basal medium. To enhance
the xylanase yield further efforts were directed towards supplementation of basal
medium with increased concentration of nitrogen sources. Medium IV contained
higher concentration of nitrogen sources than Medium III but the xylanase production
was almost similar with both the moistening agents. Xylanase production increased up
to 389±18 U/g when moistened with medium IV and wheat straw as a substrate after
120 h, while xylanase production was achieved up to 356±19 U/g with medium III
after 72 h (Table 6.2). It is evident from the Table 6.2 that even addition of CSL and
aspargine + CSL to the normal basal medium did not increase the xylanase
production. Xylanase production was not supported without yeast extract and peptone
in moistening media. Cheap nitrogen sources such as ammonium sulphate and urea
were also used during preliminary studies which did not gave reasonable xylanase
production. Hence, Medium III was further used as the moistening agent for xylanase
production under SSF. Thus it was clear that concentration of nitrogen sources had
strong influence on xylanase production by Paenibacillus sp. ASCD2. The source of
nitrogen in the culture medium is one of the important parameter influencing the
growth and production of metabolites. Similar experiments were also carried out by
Virupakshi et al., (2005) and found that there was increase in xylanase production by
Bacillus sp. JB-99 with the increase in nitrogen sources in media.
Chapter 6. Production, purification, properties………
201
Table 6.2 Maximum xylanase production using various moistening agents and wheat straw as a substrate under solid state fermentation by Paenibacillus sp. ASCD2 at 50ºC
Moistening media with varying concentration of nitrogen sources (g/l)
Xylanase activity (U/g)
Medium I : yeast extract, 2; peptone, 5 94±10 (72 h)
Medium II : yeast extract, 4; peptone, 10 119.78±8.9 (72 h)
Medium III : yeast extract, 8; peptone, 15 356±19 (72 h)
Medium IV : yeast extract, 10; peptone, 20 389±18 (120 h)
Medium V: Medium III + aspargine, 4 + corn steep liquor (CSL), 20 335.28±12 (72 h)
Medium VI: Medium III + corn steep liquor (CSL), 20 348.54±16 (72 h)
6.3.2.3 Effect of moisture level on xylanase production by Paenibacillus sp. ASCD2 under solid state fermentation
The maximum xylanase yield upto 343.5±5.3 U/g was obtained with wheat straw to
moistening agent (Medium III) at a ratio of 1:5 (w/v) at 50ºC after 72 h. Xylanase
production decreased by 35 % below and 50 % above this moisture level (Fig. 6.3).
The xylanase activity declined drastically by 90 % at 1:6 (w/v) moisture level. The
moisture content in SSF is an important factor that determines the success of the
process. The role of moisture level in SSF and its effect on microbial growth and
biosynthesis of xylanase have been attributed to the impact of moisture on the
physical properties of the solid substratum. The moisture content beyond the optimum
level inhibits the enzyme production because the higher moisture levels decrease the
porosity due to gummy texture of the substrate, alters the wheat straw particle
structure, leads to poor oxygen transfer and decrease the diffusion. Lower moisture
levels than optimum may lead to poor solubility of the nutrients into substrate,
improper swelling and higher water tension thereby decreasing the product yield
(Shah and Madamwar, 2005a; Chapla et al., 2011).
Chapter 6. Production, purification, properties………
202
0
50
100
150
200
250
300
350
400
0 24 48 72 96 120
Time (h)
Xyl
anas
e ac
tivity
(U
/g)
1:4 (w /v) 1:4.5 (w /v) 1:5 (w /v) 1:5.5 (w /v) 1:6 (w /v)
Fig. 6.3 Effect of moisture level on xylanase production by Paenibacillus sp. ASCD2 under solid state fermentation at 50°C 6.3.2.4 Effect of temperature on xylanase production by Paenibacillus sp. ASCD2 under solid state fermentation
The optimum temperature for enzyme production is generally same as that of the
optimum temperature for growth of the microorganisms. Xylanase production was
carried out by Paenibacillus sp. ASCD2 at different temperatures (45, 50, 55ºC) under
SSF using wheat straw as a substrate with moistening medium III at a moisture level
of 1:5 (w/v). The maximium xylanase production upto 338±8.9 U/g was observed at
50ºC. However, fermentation process at 55ºC showed only 30% decrease in xylanase
production while incubation temperature lower than 50ºC showed drastic decline in
xylanase production (Fig. 6.4). Similar results were also achieved by Archana and
Satyanarayana (1997). They also found highest xylanase production at 50ºC by
Bacillus licheniformis A99. Virupakshi et al., (2005) also showed that 50ºC was the
best temperature for maximum xylanase production after 72 h by Bacillus sp. JB-99.
Chapter 6. Production, purification, properties………
203
0
50
100
150
200
250
300
350
400
0 24 48 72 96 120
Time (h)
Xyl
anas
e ac
tivi
ty (U
/g)
45ºC 50ºC 55ºC
A repeat fermentation set for xylanase production by Paenibacillus sp. ASCD2 under
optimal condition was also carried out using all its optimum parameters i.e., wheat
straw as a substrate (5 g), medium III as the moistening agent with 1:5 (w/v) of
moisture level at 50ºC and the enzyme was extracted after 72 h. The optimum
parameters used for xylanase production yielded around 343.52±6.8 U/g (35.2±0.16
U/ml) of xylanase with no cellulase activity. This is the first report for production of
cellulase free thermostable xylanase at 50ºC by Paenibacillus sp. using wheat straw as
a substrate under solid state fermentation. Moreover the xylanase yield by this strain
is comparatively higher than xylanase produced by other strains of Paenibacillus spp.
reported so far. (Harada et al., 2008; Ko et al., 2010a).
Fig. 6.4 Effect of temperature on xylanase production by Paenibacillus sp. ASCD2 using wheat straw as the substrate and moistening medium III under solid state fermentation
6.3.4 Purification of endoxylanase from Paenibacillus sp. ASCD2
Purification of an enzyme is very important aspect for studying the properties and
characteristic of any enzyme. Purification costs are becoming important issues in
modern biotechnology as an industrial matures and competitive products reach the
market place. Thus, new paths for successful and efficient enzyme recovery have to
Chapter 6. Production, purification, properties………
204
be followed. Ammonium sulphate precipitation is the widely used technique for initial
purification step in major enzyme purification strategies. Crude xylanase produced
from optimized parameters under SSF by Paenibacillus sp. ASCD2 was subjected to
ammonium sulphate saturation up to 0-30% followed by dialysis. After dialysis,
purification fold increased to 13.09 with 77.86 % xylanase recovery. In order to purify
further, xylanase obtained after dialysis was subjected to gel permeation
chromatography (GPC). Sephadex-G-100 was used as matrix. At the completion of
GPC, 16.59 fold purity was achieved with 38.33% xylanase recovery (Table 6.3). It
was found that there was about 50 % loss of enzyme activity during the third step of
purification and hence the rise in specific activity was not very high as compared to
the second step. Elution profile of xylanase in each fraction during gel permeation
chromatography is shown in Fig. 6.5. The crude extract and purified xylanase were
analysed by SDS-PAGE and activity staining (zymogram). Figure 6.6 shows the
photograph of SDS-PAGE with activity staining of purified xylanase at each step of
purification. It is evident from the Fig. 6.6 that xylanase was purified as a single
protein band on SDS-PAGE with molecular weight of 20.2 kDa from the crude
extract produced by Paenibacillus sp. ASCD2. Such low molecular weight xylanases
are preferable in paper pulp industry to facilitate diffusion in pulp fibers (Haki and
Rakshit, 2003). Similarly, gel permeation chromatography was also used by Milagres
et al., (2005) for purification of xylanase from Ceriporiopsis subvermispora having
molecular weight of 29 kDa but by combination of complex stages involving
ultrafiltration and anion exchange chromatography. Studies regarding purification of
xylanase from Paenibacillus sp. is not reported extensively but such low molecular
weight xylanases have also been reported by Harada et al., (2008). They purified two
xylanases with molecular masses of 30 and 18 kDa from Paenibacillus sp. strain
HC1. Similar to present study, presence of only a single component of xylanase (41
kDa) from Paenibacillus campinasensis BL11 have also been reported by Ko et al.,
(2010b).
Chapter 6. Production, purification, properties………
205
0
10
20
30
40
50
60
70
80
90
0 5 10 15 20 25Fraction No.
Spe
cific
act
ivity
(U/m
g)
Table 6.3 Summary of purification of endoxylanase from Paenibacillus sp. ASCD2 at each step
Purification step Total xylanase units (U)
Total protein (mg)
Specific activity (U/mg)
Fold purification
Xylanase yield (%)
Crude 3358 640 5.24 1 100
0-30% Ammonium sulphate fraction after dialysis
2614.74
38.1
68.62
13.09
77.86
Gel Permeation Chromatography
1287.2
14.8
86.97
16.59
38.33
Note: All the experiments were done in triplicate and the standard deviation was within 5%
Fig. 6.5 Elution profile of xylanase from Paenibacillus sp. ASCD2 during gel permeation chromatography
Chapter 6. Production, purification, properties………
206
Fig. 6.6 SDS-PAGE along with zymogram staining showing purification of endoxylanase from Paenibacillus sp. ASCD2 at each stage. Lane M : molecular weight marker; Lane 1 : crude extract; Lane 2: dialysed fraction; Lane 3 : fraction after gel permeation chromatography; Lanes 4, 5, 6 : zymogram staining of crude extract, dialysed fraction, fraction after gel permeation chromatography respectively 6.3.4.1 Effect of temperature on xylanase activity and stability of purified xylanase from Paenibacillus sp. ASCD2
The influence of temperature on xylanase activity was evaluated in the range of 40-
90°C. The results revealed that the optimum temperature for xylanase activity was
60°C (Fig. 6.7). The xylanase activity was reduced only up to 22% and 28%
respectively at 55 and 65°C. Around 27% of xylanase activity was still retained even
at the 90°C temperature. Xylanase with similar temperature optima have been
reported from many Bacillus sp. (Techapun et al., 2003). Xylanase for industrial
application needs to be thermally stable for quite a long period of time as its thermal
inactivation usually hinders its utilization and decreases its importance in industry.
97.4 kDa
66 kDa
43 kDa
29 kDa
20.1 kDa
14.3 kDa
M 1 2 3 4 5 6
Chapter 6. Production, purification, properties………
207
0
20
40
60
80
100
120
35 40 45 50 55 60 65 70 75 80 85 90 95
Temperature (º C)
Rel
ativ
e ac
tivi
ty (
%)
0
10
20
30
40
50
60
70
80
90
100
0 30 60 90 120 150 180
Time (min)
Res
idu
al a
ctiv
ity
(%)
55°C 60°C 65°C 70°C 75°C
Fig. 6.7 Effect of temperature on xylanase activity of purified xylanase from Paenibacillus sp. ASCD2
Fig. 6.8 Thermal stability of purified xylanase from Paenibacillus sp. ASCD2 at different temperatures for different time intervals
Chapter 6. Production, purification, properties………
208
Thermostability studies were carried out by pre-incubating the purified xylanase up to
3 h at different temperatures (55, 60, 65, 70 and 75°C). It was observed that more than
90% of xylanase activity was retained even up to 3 h at 60°C. Moreover it was
revealed that the xylanase activity was retained upto 70% at 65°C and 35 % at 70°C
after 3 h, whereas it decreased drastically at 75°C after 2 h (Fig. 6.8). Half life of
purified xylanase from Paenibacillus sp. ASCD2 was found to be 415.12 min at 65°C
and 102.8 min at 70°C. This result indicates that the xylanase from Paenibacillus sp.
ASCD2 is capable for industrial application at the temperature range of 55 to 70°C.
Similar temperature optima and thermostability of purified XylX xylanase from
Paenibacillus campinasensis BL11 was also reported by Ko et al., (2010b).
6.3.4.2 Effect of pH on xylanase activity of purified xylanase from Paenibacillus sp. ASCD2
Xylanase activity at various pH was measured using birchwood xylan as a substrate at
60°C. The optimum pH for xylanase activity was found to be pH 7. The enzyme was
remarkably active even at pH 6.5 and 7.5 with loss of only 30 and 15% of xylanase
activity respectively (Fig. 6.9). The enzyme activity is markedly affected by variation
in pH range than its optimum pH. This may be due to substrate binding and catalysis
which is often affected by charge distribution on both substrate and particularly of
enzyme molecules (Shah and Madamwar, 2005b). This result indicates that xylanase
from Paenibacillus sp. ASCD2 can be applied in neutral to slightly alkaline
environment. pH stability for purified xylanase from Paenibacillus sp. ASCD2 was
also studied by incubating the purified xylanase in different pH and the residual
activity was measured after specific time intervals. It was observed that purified
xylanase was quiet stable in the range of pH 6 to 8.5 for almost three hours
(Fig. 6.10). These results indicate that xylanase from Paenibacillus sp. ASCD2 can be
applied in industry for large pH range from neutral to slightly alkaline for good period
of time for its easy action during application.
Chapter 6. Production, purification, properties………
209
0
20
40
60
80
100
120
3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10
pH
Rel
ativ
e ac
tivi
ty (
%)
0
20
40
60
80
100
6 6.5 7 7.5 8 8.5
pH
Res
idu
al a
ctiv
ity
(%)
Fig. 6.9 Effect of pH on xylanase activity of purified xylanase from Paenibacillus sp. ASCD2
Fig. 6.10 pH stability of purified xylanase from Paenibacillus sp. ASCD2 after 3 h
Chapter 6. Production, purification, properties………
210
6.3.4.3 Determination of kinetic parameters of purified xylanase from Paenibacillus sp. ASCD2
The effect of varying concentration of birchwood xylan as a substrate on xylanase
activity at 60°C and pH 7 was measured using double reciprocal plot. The Michaelis-
Menten constant (Km) was calculated as 2.5 mg/ml and maximum velocity (Vmax) as
571 µmoles/mg/min for the purified xylanase from Paenibacillus sp. ASCD2.
Kcat of the purified enzyme on birchwood xylan was 192 S-1 whereas its catalaytic
efficiency was 76.89 mg-1s-1ml. In comparison to this Ko et al., (2010b) has reported
higher Km of 6.78 mg/ml and Vmax of 4953 mol/min/mg for the purified XylX of
Paenibacillus campinasensis BL11. The lower Km value for Paenibacillus sp. ASCD2
xylanase suggests high affinity of xylanase towards birchwood xylan. Microbial
xylanases widely differ with respect to kinetic parameters. Lower Km values have also
been reported for xylanase from Ceriporiopsis subvermispora (Milagres et al., 2005).
Higher Km of 11.1 mg/ml and lower Vmax of 45.45 µmoles/min/mg for the xylanase
from Streptomyces cyaneus SN32 have also been reported by Ninawe et al., (2008).
6.3.4.4 Effect of metal ions and additives on xylanase activity of purified xylanase from Paenibacillus sp. ASCD2
Various metal ions, surfactant and additives were tested to study their influence on
xylanase activity under standard assay condition. Xylanase activity was enhanced in
presence of NaCl, KCl and MgSO4 by 14, 38 and 19 % respectively (Table 6.4) while
it was drastically reduced in presence of HgCl2, AgNO3 and CuSO4 by 74, 48 and
85 % respectively. It is believed that KCl increases the enzyme activity by altering the
conformation of inactive domains of enzyme thereby changing it in to more active
form (Palmer, 2000). Cu ions are known to catalyze auto oxidation of cysteine
molecules which lead to the formation of intra and inter moleculer di-sulfide bridge or
to the formation of sulfenic acid (Vieille and Ziekus, 2001). Moreover xylanase
activity was also decreased in the presence of MnSO4, MgCl2, BaCl2 and EDTA by
31, 28, 23 and 42% respectively. EDTA is a chelating agent used to scavenge
generally the heavy metal present as impurities. Here the presence of EDTA reduced
the xylanase activity by 42% which indicates the requirement of some metal ions for
its activity (Shah and Madamwar, 2005b). Xylanase activity was unaffected by
FeSO4, ZnSO4, and Tween 80. It is also thought that surfactant like Tween 80 plays
Chapter 6. Production, purification, properties………
211
an important role in preventing nonspecific binding of enzyme to substrate, allowing
more enzymes to be available for the conversion of substrate and results in a higher
conversion rate (Palmer, 2001).
Table 6.4 Effect of metal ions (10 mM) and additives (0.1%) on activity of purified xylanase from Paenibacillus sp. ASCD2
6.3.4.5 Mode of action of purified xylanase from Paenibacillus sp. ASCD2
The mode of action of purified xylanase from Paenibacillus sp. ASCD2 was studied
by hydrolyzing birchwood xylan and analysing the products by ascending TLC
analysis. Quantitative estimation of reducing sugar was also done at each time
interval. The amount of reducing sugar after 2 h of xylan hydrolysis was found to be
253.8 mg/g of substrate. Later on the rate of release in reducing sugar was almost
constant and maximum amount of reducing sugar was obtained up to 311 mg/g even
after 12 h. TLC analysis showed the presence of only xylooligosaccharides with
varying degree of polymerization and absence of xylose. Xylobiose appeared in the
hydrolysate after 4 h which was not further cleaved to xylose even after 10 h
(Fig. 6.11) which indicated that the purified enzyme was an endoxylanase (Chapla et
al., 2011). Similar results were reported by Zhiwei et al., (2008) for the purified
xylanase obtained from the microbial community EMSD5. Similarly, the release of
Metal ions and additives
Relative xylanase activity (%)
Control 100±0.02 MgSO4 119.32±5.8 MnSO4 69.57±3.7 FeSO4 100.62±7.8 CuSO4 15.08±6.8 ZnSO4 108.72±1.8 MgCl2 72.81±1.8 HgCl2 26.43±1.2 NaCl 114.21±1.4 AgNO3 52.74±2.3 KCl 138.9±1.6 BaCl2 87.28±2.2 Tween 80 101.2±1.3 EDTA 58.22±3.3
Chapter 6. Production, purification, properties………
212
S C 2 h 4 h 6 h 8 h 10 h
Xylose
Xylobiose
Xylotriose Xylotetraose
Xylopentaose
xylooligosaccharides from xylan using xylanase of Bacillus halodurans S7 is also
reported by Mamo et al., (2006). Ko et al., (2010b) also studied mode of action for
purified XylX Paenibacillus campinasensis BL11 cloned in E. coli. They found
xylobiose and xylotriose as the major end products during xylan hydrolysis and
determined that the purified XylX was an endoxylanase.
Fig. 6.11 Thin layer chromatography (TLC) plate showing xylan hydrolysis by purified xylanase from Paenibacillus sp. ASCD2. Xylose, xylobiose, xylotriose, xylotetraose, xylopentaose were used as standards, C: control; 2, 4, 6, 8,10 h are the respective time for xylan hydrolysis
Xylooligosaccharides are sugar oligomers produced during xylan hydrolysis having
various physiological importance such as reducing cholesterol level, maintaining the
gastrointestinal health, improving biological availability of calcium, reducing the risk
of colon cancer, and cytotoxic effects on human leukaemia cells (Akpinar et al.,
2009).
6.3.5 Storage stability of purified xylanase from Paenibacillus sp. ASCD2
Storage of any enzymes is an important criterion for their industrial applications.
Storage of the enzymes at room or refrigerated temperature, without appreciable loss
of activity is one of the most important and desirable characteristics. The purified
Chapter 6. Production, purification, properties………
213
xylanase from Paenibacillus sp. ASCD2 could retain its xylanase activity completely
upto 4 and half months when stored in deep freeze (-20°C). There was only 25-30%
decrease in xylanase activity after 5th month. At refrigeration temperature (6-8°C) no
loss of activity was found till 8 weeks but after 8th week there was slight decrease
(10–15%) in xylanase activity which was reduced further to 60% after 3 months.
Enzyme was quiet steady even at room temperature. There was only 10% decrease in
xylanase activity of purified xylanase at room temperature after 24 h. Compared to
this crude xylanase preparation obtained from Paenibacillus sp. ASCD2 using wheat
straw and under SSF was more stable than that of purified xylanase. The crude
xylanase preparation was found steady till one year and 8 months at deep freeze
temperature (-20°C) whereas it could maintain its activity upto 10 months at
refrigeration temperature (6-8°C) which than decreased by 25% after 12 months.
Crude xylanase preparation was also found to maintain its xylanase activity upto 15
days at room temperature (30°C).
6.3.6 Biobleaching of kraft pulp using crude xylanase from Paenibacillus sp. ASCD2
The properties of xylanase produced from Paenibacillus sp. ASCD2 under solid state
fermentation revealed that it was cellulase free, thermostable, and active in neutral to
alkaline pH and was of low molecular weight. It was also found that crude xylanase
could retain more than 90% its activity upto 4 h at 60ºC and upto 2 h at 70ºC. It is
well known that apart from cellulase free and thermostable nature, xylanases with low
molecular weight are preferable in pulp bleaching due to their good penetration power
in pulp fibers (Haki and Rakshit, 2003). As the crude xylanase from Paenibacillus sp.
ASCD2 was having all these properties its efficacy was checked for prebleaching of
eucalyptus kraft pulp which are later used to prepare paper in paper pulp industry.
6.3.6.1 Effect of enzyme dose and incubation time on biobleaching of kraft pulp
The biobleaching efficiency of crude xylanase from Paenibacillus sp. ASCD2 was
checked by subjecting the unbleached pulp sample to enzymatic prebleaching using
different xylanase doses (10-50 U/g) of crude xylanase at 60°C for 3 h. As the
xylanase dose was increased from 10 to 50 U per g of dry pulp, there was decrease in
kappa number and increase in free reducing sugars in the filtrate (Fig. 6.12). Xylanase
Chapter 6. Production, purification, properties………
214
0
5
10
15
20
25
0 10 20 30 40 50
Enzyme dose (U/g dry pulp)
Kap
pa
nu
mb
er
0
5
10
15
20
Red
uci
ng
su
gar
(m
g/g
dry
pu
lp)
Kappa number Reducing Sugar
dose beyond 40 U/g was not found to be advantageous whereas the lower enzyme
dose than 40 U did not gave desirable reduction in kappa number. When pulp was
treated with 40 U of xylanase per g of dry pulp at 60°C for 3 h, it liberated 20.31±0.36
mg/g of reducing sugars along with 21.83±0.04% reduction in kappa number of kraft
pulp. Decrease in kappa number clearly indicated the delignification of kraft pulp
(Chapla et al., 2012).
Optimization of incubation time for enzymatic pretreatment of pulp with crude
xylanase from Paenibacillus sp. ASCD2 was also carried out. The unbleached pulp
with 5% consistency was pretreated with enzyme dose of 40 U/g of moisture free pulp
for different time interval (2-6 h). Gradual reduction in kappa number and increase in
free reducing sugars in the filtrate were achieved up to 3 h. Prolonged incubation after
3 h did not helped enzymatic prebleaching of pulp (Fig. 6.13). Thus prolonged
incubation for xylanase pre-treatment was not necessary for its action. This result
indicates that the xylanase is effective in short time period and decreases the overall
time and cost for the process.
Fig. 6.12 Effect of xylanase dose (U/g dry pulp) from Paenibacillus sp. ASCD2 for biobleaching of kraft pulp
Chapter 6. Production, purification, properties………
215
0
5
10
15
20
25
0 2 3 4 5 6
Time (h)
Kap
pa
nu
mb
er
0
5
10
15
20
25
Red
uci
ng
su
gar
(m
g/g
dry
pu
lp)
Kappa number Reducing sugar
Fig. 6.13 Effect of incubation time on biobleaching of kraft pulp using crude xylanase (40 U/g of dry pulp) from Paenibacillus sp. ASCD2 During this prebleaching process it was observed that there was increase in
absorbance at 237 nm and 465 nm after enzymatic pretreatment of pulp which
determines the liberation of phenolics and hydrophobic compound respectively from
the kraft pulp (Khandeparker and Bhosle, 2007). Increase in absorbance was observed
from 3.987 to 4.912 and from 1.422 to 2.011 at 237 and 465 nm respectively
(Table 6.5). The correlation between the release of chromophores, hydrophobic
compounds, reduction in kappa number coupled with the release of reducing sugars
suggest the dissociation of lignin-carbohydrate complex from the pulp fibers (Patel et
al., 1993). In comparison to this Khandeparker and Bhosle (2007) achieved 20%
reduction in kappa number of unbleached pulp using xylanase from Arthrobacter sp.
MTCC 5214 with 20 U/g of dry pulp at 70°C. Gupta et al., (2000) also reported 25%
reduction in kappa number with optimum xylanase dose of 1.8 U/g of moisture free
eucalyptus kraft pulp at 50°C for 4 h. Saleem et al., (2009) reported 60% reduction in
kappa number of dry pulp at 55°C using 40 U/g xylanase obtained from Bacillus sp.
XTR-10. However, the pre-treatment time was 8 h in their study. Similarly various
reports are available which determines the delignification of pulp as indicated by
Chapter 6. Production, purification, properties………
216
decrease in the Kappa number of pulp after enzymatic bleaching in their studies
(Chauhan et al., 2006; Sindhu et al., 2006; Battan et al., 2007).
Table 6.5 Influence of xylanase pre-treatment on kappa number, release of chromophores, hydrophobic compounds and reducing sugars from eucalyptus kraft pulp under optimum conditions during biobleaching
Till date the mechanism of xylanase action on pulp fibers remains a mystery. It is
hypothesized that xylanases cleaves the bonds near point of attachment between
lignin and hemicellulose and this leads to the improved solubilization of lignin. The
enzymatic pretreatment renders the easy extract of lignin in the subsequent chemical
step during bleaching. Moreover it is also stated that addition of xylanase reduces the
chlorine consumption without reducing the brightness or strength of the final paper
quality (Buchert et al., 1994).
6.3.6.2 Chemical bleaching of kraft pulp
The conventional bleaching sequence contains prebleaching stages with various ratio
of chlorine dioxide and chlorine gas in paper pulp industry. But the use of such highly
toxic and hazardous chemicals has lead serious problems in the environment. Use of
microbial xylanases can reduce chlorine consumption and thereby decrease the
environmental pollution (Ninawe and Kuhad, 2006; Techapun et al., 2003). An
attempt was also made to study the effect of xylanase pretreatment on kraft pulp
bleaching. Comparison was made between, direct chemical bleaching of unbleached
eucalyptus kraft pulp and chemical bleaching of xylanase pretreated eucalyptus kraft
pulp. Kappa number was determined at each stage of chemical bleaching as shown in
Table 6.6. It was observed that there was drastic decrease in kappa number of
xylanase pretreated pulp after first stage of chemical bleaching while in subsequent
stages there was marginal decrease in kappa number of this pulp. During direct
chemical bleaching of the unbleached pulp, the gradual reduction in kappa number
Sample Kappa No.
% Reduction in Kappa No.
Release of chromophores A237 nm
Release of hydrophobic compounds A465 nm
Reducing sugar (mg/g)
Control 21.16±0.08 - 3.98±0.17 1.42±0.41 04.40±0.13 Xylanase pretreated pulp 16.54±0.05 21.83±0.04 4.91±0.29 2.01±0.28 17.40±0.36
Chapter 6. Production, purification, properties………
217
was observed after each stage of chemical bleaching. The results clearly established
the potential of xylanase pretreatment which allow exclusion of chemical bleaching
agents used in second and third stage. It was also observed that xylanase pretreated
pulp had lower kappa number (14.0) than untreated pulp (15.2) after chemical
bleaching. This result strongly indicates the potential of xylanase pretreatment in
reduction of toxic and hazardous chemicals. The effectiveness of xylanase treatment
before hypochlorite application may be because of cleavage of residual lignin to
hemicellulose leading to increased accessibility of pulp to bleaching chemical and
enhance extraction of lignin or target substrate modification during subsequent
bleaching stages (Viikari, 1986). One hypothesis says that xylanase depolymerises
hemicellulose precipitated on the surface of fiber, thereby opening the pulp structure
for access of bleaching chemical in the successive stage (Paice et al., 1992). Since
xylanase degrade xylan chain in unbleached pulp, the chromophores associated with
lignin-carbohydrate linkage could be more easily removed and oxidized by chlorine
dioxide (Wong et al., 1997). Ninawe and Kuhad (2006) observed the similar benefits
of microbial xylanase from Streptomyces Cyaneus SN32 on wheat straw rich soda
pulp. Similarly Ko et al., (2010a) also reported that pretreatment with crude xylanase
from Paenibacillus campinasensis BL11 was a better option prior to chemical
bleaching. The use of xylanases constitutes a very important technological
improvement in the bleaching effects of chemical reagents, thereby affording
substantial savings and more importantly diminishing the production of pollutants
during bleaching. This makes process more economic and environmentally
eco-friendly (Valls and Roncero, 2009).
Table 6.6 Effect of xylanase pre-treatment on kraft pulp prior to chemical bleaching
a Kappa no of unbleached pulp : 21.17±0.05 b Kappa no of pulp after xylanase pretreatment : 16.94±0.08
Sequence of chemical bleaching
a Kappa no of Untreated kraft pulp
b Kappa no of xylanase pretreated kraft pulp
Stage 1. 5% NaOCl 19.58±0.89 14.37±0.29
Stage 2. 0.4% NaOH 15.39±0.73 14.50±0.33
Stage 3. 0.5% H2O2 15.20±0.50 14.00±0.23
Chapter 6. Production, purification, properties………
218
6.3.6.3 Scanning electron microscopy
To understand the changes occurring in the fiber structure of pulp after xylanase
treatment and chemical treatment, pulp at different stages was studied using scanning
electron microscopy (SEM). Unbleached pulp, xylanase pretreated pulp and xylanase
pretreated chemically bleached pulp were analyzed by SEM. The mechanism by
which xylanases facilitate bleaching is not yet fully understood. The enzyme does not
bleach pulp, but rather changes the pulp structure. One hypothesis is that xylanases
depolymerise hemicellulose precipitated on the surface of the fiber, thereby help in
opening of the pulp structure which can later easily be accessed by bleaching
chemicals in the successive steps. The second belief is that, xylanases releases
chromophores associated with carbohydrates in the pulp fibers. This creates a
cleavage in the carbohydrate portion of lignin-carbohydrate complex to produce small
residual lignin molecules, which are easier to remove from the pulp fibers and there
by help in boosting up the bleaching process (Beg et al., 2001). The SEM study
revealed that the xylanase action introduced greater porosity, swelling up and
separation of pulp microfibrils and pulp fiber compared to the smooth surface of the
untreated pulp. After enzymatic treatment and chemical bleaching swelling,
separation and loss in packness of the pulp fiber were increased (Fig. 6.14). Such
effects were also observed by Ahlawat et al., in 2007 using cocktails of pectinase and
xylanase after prebleaching of kraft pulp.
Fig. 6.14 Scanning electron micrograph of kraft pulp (A) Unbleached kraft pulp (control); (B) xylanase pretreated kraft pulp; (C) xylanase pretreated chemically bleached kraft pulp
Chapter 6. Production, purification, properties………
219
6.4 Conclusions
The present work has established the potential of a novel strain of Paenibacillus sp.
for the production of cellulase free thermostable xylanase under SSF using low cost
agro-residue such as wheat straw. This is the first report showing production of
cellulase free thermostable xylanase at 50ºC under solid state fermentation by
Paenibacillus sp. Moreover, the present study states the higher xylanase yield
amongst the xylanase production by Paenibacillus sp. reported so far. Biochemical
characteristics viz. high thermal stability and low molecular weight as well as
cellulase free nature of this xylanase makes it attractive with respect to
biotechnological application in prebleaching of pulp in paper pulp industry. Many
cellulase free thermo and alkali stable xylanases have been reported so far. However,
xylanase from this Paenibacillus sp. is superior and valuable for industrial
applications due to its low cost production, reasonably high thermostability at 65 and
70ºC temperature and ability to produce xylooligosaccharides. The crude cellulase
free thermostable xylanase from Paenibacillus sp. ASCD2 was found beneficial in
prebleaching of eucalyptus kraft pulp. The prebleaching with xylanase was
advantageous in delignification of eucalyptus kraft pulp and thereby played an
important role in decreasing the use of hazardous chemicals like chlorine for
successive steps in bleaching of kraft pulp. The present study has established the
potential of enzymatic prebleaching of pulp at lab scale. However, scale up for this
technology is necessary to make the process applicable in paper pulp industry.
Chapter 6. Production, purification, properties………
220
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