Chapter 6. Paenibacillus - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/7236/12/12... ·...

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………

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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).


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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


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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


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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


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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).


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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).


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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


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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


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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).


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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).


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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)


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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


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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


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.


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).


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.


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


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).


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


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


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


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.


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


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


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


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


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


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


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


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


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


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


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.


220

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