Purification and characterization of Laceyella sacchari strain B 42 xylanase and its potential for pulp biobleaching

Xylanase producing thermophilic actinomycetes strain B42 was isolated from bagasse. This strain was enriched on oat spelt xylan agar medium and screened onto xylan-congo red agar plate by the xylanolysis method. The Phylogenetic analysis using 16S rDNA sequence data showed that strain B42 had the highest homology (99.0%) with Laceyella sacchari and it was named as L. sacchari strain B42. L. sacchari strain B42 xylanase was purified to study its biochemical characteristics and its biobleaching efficiency. The partial purification of xylanase using acetone fractionation (at 1:3.0 ratio) gave 2.51 fold purification and the recovery of 88%. Further purification of the partially purified xylanase using DEAE-Sephadex A-50 and G-100 column chromatography gave 11.41 fold purification and 22.80% yield with the specific activity of 1750.0 U/mg. The molecular mass of the purified xylanase was ~30.0 kDa, as analyzed by SDS/PAGE and zymogram. The enzyme reactions followed Michaelis–Menten kinetics with Km and Vmax values of 4.166 mM and 3787.87μmole/min/ml/mg, respectively, as obtained from a Lineweaver–Burk plot. The optimal temperature of the enzyme was 70°C. The enzyme retained 72% of its activity at 70°C and 48% activity at 80°C after 6 h of incubation. The half life (t1/2) of purified xylanase was 6 h at 80°C. The optimal pH of xylanase activity was 10.0 and enzyme appeared to be stable over a broad pH range (pH, 11.0 to 12.0) under the assay conditions. Approximately 68 and 64% of the original activity was retained after 5 h of incubation at pH, 10.0 and 11.0, respectively. The enzymatic biobleaching of kraft pulp reduced ~26% kappa number, decreased 1.68% lignin content and released 24 fold reducing sugars. The enzyme also released sufficient amount of phenolic and hydrophobic compounds. The UV absorption spectrum of the compounds released by enzymatic treatment at 280 nm indicates the presence of lignin in the released coloring matter.


Xylan
is the most abundant non-cellulosic polysaccharide, which constitutes approximately one third of all renewable organic carbon sources on earth.Xylan *Corresponding author.E-mail: vikramsviking@rediffmail.com.Tel: +91-8688012725.
hydrolysis is important for proper utilization of lignocellulosic material.Two approaches are followed for xylan hydrolysis (I) conventional chlorine bleaching and (II) non-conventional total chlorine-free bleaching (TCF) (Subramaniyan and Prema, 2000).During conventional chlorine bleaching toxic byproducts get released which are harmful to the environment, so it is necessary to use such strategies which are environmentally safe (Biely, 1985).
One environmentally safe strategy is to use xylanase.Xylanase (EC 3.2.18)has great potential as a prebleaching agent in the pulp and paper industry.Biobleaching and bioprocessing of pulps using xylanases (Garg et al., 1998) is one of the most suitable biological applications to be used in the pulp and paper industry.Xylanase is used, primarily, for the removal of the lignincarbohydrate complex (LCC) that is generated in the kraft process and acts as physical barriers to the entry of bleaching chemicals (Paice et al., 1992).Other significant benefits of this enzyme include higher brightness ceilings, a reduction in the amounts of bleaching chemicals needed to achieve high brightness and to reduced amounts of organochlorine compounds in the bleach plant effluents (Ragauskas et al., 1994).The suitability of xylanase for biobleaching application is generally decided with respect to the enzyme stability at high optimum pH and temperature.
In order to minimize the risk of environmental pollution in pulp and paper industry, we undertook a study to isolate and identify the strain which secrete extracellular, thermostable, cellulase free xylanase active at alkaline pH.Further, the enzyme was purified, characterized and its potential for biobleaching of kraft pulp was studied.

MATERIALS AND METHODS
Thermophilic actinomycete was isolated from bagasse (collected from Century Paper Mill, Lalkuan).This isolate was grown on oat spelt xylan agar plate and screened by the xylanolysis method in xylan-congo red agar plate.

PCR amplification of the 16S rDNA and sequence determination
For the 16S rDNA sequence analysis, bacterial genomic DNA was extracted.Further, the 16S rDNA gene was amplified by PCR using 5' AGAGTTTGATCCTGGCTCAG-3' and 5' AAGGAGGTGATCCAGCCGCA-3' as the forward and reverse primers, respectively (Edwards et al., 1989).
The amplification was carried out in 25 µl of reaction mixture containing 4.0 µl of DNA template, 2.5 µl of PCR buffer (10x) (Bangalore Genei, India), 1.5 µl of dNTP (10 mM) (Bangalore Genei, India), 2.0 µl of the primers FP (40 ng) and RP (40 ng), respectively, 1.5 µl of Taq polymerase (5 U/µl) (Bangalore Genei, India) and 11.5 µl of autoclaved Milli-Q water (Millipore).The PCR program was run for 35 cycles in thermal cycler (Eppendorf, Germany).The following thermal profile was used for PCR: denaturation at 94°C for 1 min, annealing at 64°C for 1 min, extension at 72°C for 1 min 30 s. Final cycle included extension for 10 min at 72°C to ensure full extension of product.The amplified PCR products were analyzed in a 1.0 % (w/v) agarose gel, excised from the gel and purified (by Spin gel extraction kit, Genei).The purified PCR product was sent for commercial sequencing (Bangalore Genei, India).Databases (GeneBank) were searched for sequences similarity analysis of the 16S rDNA sequence obtained.The 16S rDNA gene sequence of the isolate was aligned with reference 16S rDNA sequences of the European Microbiological Laboratory (EMBL), GenBank (gb, Germany) using the BLAST algorithm (Altschul et al., 1997) available in National Centre for Biotechnology information (NCBI) in internet.

Enzyme production
The xylanase production was done under solid state fermentation using wheat bran as carbon source.Flask containing 10 g wheat bran and 25 ml tap water was sterilized by autoclaving at 121°C, 15 psi for 45 min then inoculated with 10% (v/w) of 18 h old inoculum, and incubated at 60°C for 72 h.The flasks were tapped at regular intervals in order to mix the contents.

Preparation of crude extract
After 72 h of growth in the production medium, the enzyme from each flask was extracted with 0.1 M Glycine-NaOH buffer, pH 9.0 (100 ml for 10 g of wheat bran).The contents of flask were squeezed through a wet muslin cloth followed by centrifugation at 10,000 rpm for 20 min at 4°C.Clear supernatant (crude enzyme) was used for enzyme purification.

Partial purification of xylanase
The crude enzyme was partially purified using acetone fractionation method.In this procedure the chilled acetone was added to precooled cell free extract at different percent of saturation ranging from 1:0.5 to 1:3.0.

Column chromatography
The concentrated protein sample obtained after purification by acetone fractionation was further purified by DEAE-Sephadex A-50 column chromatography and Sephadex G-100 column chromatography.Here we used two strategies for xylanase purification.Strategy (I) in this strategy the acetone fractionated sample was first loaded to Sephadex G-100 column, followed by DEAE-Sephadex A-50 column chromatography.Strategy (II) in this strategy the first purification of acetone fractionated enzyme was done by DEAE-Sephadex A-50 column chromatography and the enzyme was further purified by Sephadex G-100 column chromatography.The final adoption of purification strategy depends upon the purification fold and yield.Here we are describing only strategy-II because it gave better results over strategy-I.

Ion exchange chromatography
Concentrated enzyme (1 ml) was loaded onto anion exchange DEAE-Sephadex A-50 column (2.5 × 25 cm).The sample was eluted first by buffer alone with the flow rate of 90 ml/h, followed by step wise elution with 0.1 to 0.5 M NaCl.The 1.5 ml fractions were collected and analyzed for protein at 280 nm under UV-visible spectrophotometer.The xylanase activity in each fraction was determined; the fractions showing good xylanase activity were pooled and concentrated.This pooled enzyme was further purified by Sephadex G-100 column (Sigma-Aldrich Co., USA) chromatography.

Gel filtration chromatography
The gel filtration chromatography was done on Sephadex G-100 column.The DEAE-Sephadex A-50 purified enzyme was applied to a Sephadex G-100 column (1 × 30 cm).Elution of the enzyme was carried out with 0.1 M Glycine-NaOH buffer (pH, 9.0) at a flow rate of 12 ml/h.Each fraction was analyzed for protein and xylanase activity.The active fractions were pooled and concentrated.

Molecular mass determination
The molecular mass of the purified xylanase was estimated by SDS-PAGE electrophoresis (Laemmli, 1970).The SDS-PAGE (stacking gel 5% and resolving gel 15%) was performed using medium range (14.3 to 97.4 kDa) molecular weight markers (Banglore Genei, Pvt. Ltd.India).Proteins were visualized by staining with Coomassie brilliant blue (CBB).

Zymogram analysis
Zymogram analysis was done by Morag method (Morag et al., 1990).Electrophoresis was done on (15%) SDS-PAGE gel containing 0.1% oat spelt xylan.Later the gel was washed four times.The first two wash contained 25% [v/v] isopropyl alcohol in a 0.1M Glycine-NaOH buffer (pH, 9.0) for 30 min at 4°C to remove SDS and renature proteins in gel.The gel was further incubated in the buffer for 10 min at 37°C.The gel was stained with 0.1% congo red solution for 15 min at room temperature and washed with 1 M NaCl to remove out excess dye from the active band.The zymogram was prepared after incubation of the gel into 0.5% acetic acid.The background turned blue and clear zones in area exposed to xylanase activity were observed.

Enzyme assay
Xylanase activity was assayed by measuring the release of reducing sugar from oat spelt xylan.Reaction mixture consisted of 1% xylan in 0.1 M buffer and enzyme to give a final volume of 1.0 ml.After incubating for 10 min at 60C, the release of reducing sugar was determined by Nelson-Somogyi method (Nelson, 1944;Somogyi, 1952).One unit of xylanase is defined as the amount of enzyme required to release 1 µmol of xylose per min under above assay condition.

Protein estimation
The protein concentration was determined by the Lowry method Singh et al. 1399 using bovine serum albumin as standard (Lowry et al., 1951).

Effect of pH on activity and stability of purified xylanase
The xylanase activity was determined at various pH ranges varied from 5.0 to 11.0 using three different buffers-1.0.1 M Citrate-phosphate buffer (pH, 5.0 and 6.0) 2. 0.1 M Tris-HCl buffer (pH, 7.0 and 8.0) 3. 0.1 M Glycine-NaOH buffer (pH, 9.0 and 11.0) To test the pH stability, the purified enzyme was diluted using respective buffers having pH ranging from 5.0 to 11.0 as described above and was incubated for 5 h at room temperature.The residual enzyme activity was estimated at 1 h intervals during the 5 h of incubation period.

Effect of temperature on the activity and stability of purified xylanase
The optimal temperature for the purified xylanase was obtained by assaying the enzyme activity at different temperatures range from 40 to 80C at their optimum pH value.Thermostability studies were done by incubating xylanase at temperature ranging from 40 to 80°C for 1 to 6 h.The residual activity was then quantified, at optimum temperature and pH using Nelson-Somogyi method (Nelson, 1944;Somogyi, 1952).

Kinetic analysis
In order to determine the Km and Vmax, purified xylanase was added to the test tubes each containing various amounts of xylan (0 to 20.0 mM) in 0.1 M Glycine-NaOH buffer (pH, 10.0).The reaction mixture was incubated at 60°C for 10 min and the enzyme activity was measured.Graph was plotted between substrate concentration vs enzyme activity.The Km and Vmax values were determined using the Lineweaver-Burk double reciprocal plot (Lineweaver and Burk, 1934).

Application of L. sacchari strain B42 purified xylanase for biobleaching of kraft pulp
The biobleaching efficiency of L. sacchari strain B42 purified xylanase was studied by giving treatment to pulp sample with the enzyme.The optimum enzyme dose and optimum reaction time for biobleaching were also determined.Further the release in reducing sugars; decrease in kappa number of pulp, decrease in percentage residual lignin of pulp, release in hydrophobic compounds (λ465 nm) and release in phenolic compounds (λ237 nm) were also determined.

Pulp sample
Unbleached kraft pulp was kindly provided by Shivangi paper mill, Kashipur, India.Pulp samples were thoroughly washed before use and after each treatment step with water.All the studies were performed at optimum pH and temperature of enzyme.

Colour removal from the kraft pulp
The kraft pulp was treated with xylanase at pH, 10.0 and 70°C with the enzyme doses of 0 to 100 U/ml and for the incubation time of 1 to 5 h.The pulp sample that contain only buffer (no enzyme added)  was used as control.After incubation, the pulp samples were washed with distilled water and the absorbance of the filtrate was determined spectrophotometrically from λ 200 nm to λ 400 nm.

Optimization of enzyme dose and reaction time for biobleaching
To optimize the enzyme dose the pulp was treated with the xylanase doses, ranging between 0 to 100 U/g for the incubation time of 3 h.Further, the incubation time was optimized by treating the pulp sample with the optimum enzyme dose for the time interval of 1 to 5 h.After incubation, the pulp was washed with ddw and filtered through separating funnel using Whatman No. 1 filter paper.
The filtrate was taken for the study of chromophore content (Hydrophobic compounds at λ 465nm and phenolic compounds at λ 237nm) and for estimation of reducing sugar (by Nelson-Somogyi method).

Determination of kappa number and residual lignin
The kappa number is defined as the amount (ml) of a 0.1 N KMnO4 solution consumed by 0.5 to 1.0 g of dried pulp under standard conditions.It was determined according to the standard methods of the Technical Association of the Pulp and Paper Industry (TAPPI, Atlanta, GA, USA) (T236-cm-85) (TAPPI, 1996).Percentage residual lignin content was calculated as kappa number × 0.15.

Screening of isolates
A total of 95 thermophilic isolates were screened from sugarcane field soil (Crop Research Center, Pantnagar), compost (MRDC, Pantnagar) and bagasse (collected from Century Paper Mill, Lalkuan).Of these 95 isolates, 26 isolates showing good clearing zone around their colonies on xylan-congo red agar media were selected as xylanase producers.Among these 26 isolates L. sacchari strain B42 produced largest zone of hydrolysis on xylancongo red agar media (Figure 1).Strain was further tested for xylanase production by growing them in oat spelt xylan agar medium.

Cultural and morphologic characteristics
Morphologic observations of L. sacchari strain B42 showed typical growth characteristics of actinomycetes on solid media.This strain produced characteristic dry, whitish, large, opaque, uneven, large-sized colonies with peculiar smell (Figure 2).Ball-shaped structures were formed in liquid media.

Strain B42 identification by 16S rDNA sequence
In order to confirm the identification of strain B42, the 16S rDNA was amplified after PCR amplification of genomic DNA.The amplified product was analyzed in 1.0% agarose gel (Figure 3).Sequencing result showed that 16S rDNA sequence from B42 was 1456 bp (Figure 4).Analysis of 16S rDNA gene revealed that this organism was closely related phylogenetically to the genus Laceyella rRNA group.Strain B42 showed highest nucleotide identity of 99% with L. sacchari strain VTT E-062990 (GenBank accession no.EU430566) (Figure 5).Therefore, on the basis of phylogenetic analysis, strain B42 was considered as one strain of L. sacchari and identified as L. sacchari strain B42.

Purification of Lsacchari strain B42 xylanase
The partial purification of xylanase using acetone fractionation (at the ratio of 1:3.0) gave 2.51 fold purification with the recovery of 88%, this resulted in the enhancement of specific activity from 153.30 to 385.70 U/mg.

Purification of partially purified xylanase by column chromatography
L. sacchari strain B42 partially purified xylanase was further purified using two strategies-strategy-I and strategy-II.Out of these two strategies, strategy-II gave better results over strategy-I.More yield and purification fold was obtained using strategy-II (Data shown only for strategy-II).Further, the characterization studies and application in biobleaching of kraft pulp was done only for strategy-II purified xylanase.

Ion exchange chromatography
The partially purified xylanase was further purified by DEAE-Sephadex A-50 column chromatography.This purification gave three peaks namely F D -1, F D -2 and F D -3 (Figure 6).The fraction F D -2 contained high xylanase activity while fraction F D -1 and F D -3 showed very low activity.Hence F D -1 and F D -3 were not used for further investigation.The active fraction F D -2 was pooled, concentrated and further purified by Sephadex G-100 column chromatography.The DEAE-Sephadex A-50 column chromatography gave 5.32 fold purification and yield of 23.04% with the specific activity of 815.38 U/mg (Table 1).

Gel filtration chromatography
The active peak from DEAE-Sephadex chromatography F D -2 was applied to Sephadex G-100 column.Only single peak was obtained after purification by Sephadex G-100 column chromatography (Figure 7).G-100 purified enzyme gave 11.41 fold purification and 22.80% yield.
The specific activity of xylanase was increased at each purification step.The final specific activity of enzyme after Sephadex G-100 column chromatography was 1750.0U/mg (Table 1).The purity of finally purified enzyme was seen by electrophoresis analysis.
Low molecular weight xylanases are preferred for commercial application in paper and pulp industry as they    Zymogram analysis showed that the culture supernatant, acetone fractionated enzyme and purified enzyme gave single prominent activity band at the same position.The active band corresponds to ~30 kDa.

Effect of pH on the activity and stability for purified xylanase
The optimum pH of the purified xylanase from Laceyella sacchari strain B42 was 10.0.Enzyme retained good pH stability at broad pH range from pH, 8.0 to 11.0.Approx.68 and 64% of the original activity was retained after 5 h of incubation at pH, 10.0 and 11.0, respectively.Enzyme showed low activity at pH 8.0 and 9.0.Both enzyme activity and stability were decreased, and lost more than 60% in acidic pH, (5.0 & 6.0) after 5 h.In comparison to the activity retained after 5 h of incubation at optimum pH (10.0), ~23 and ~32% of the activity were retained at pH, 5.0 and 6.0, respectively, while ~89 and ~75% of activity were retained at pH, 9.0 and 11.0, respectively.
The wide range of activity of L. sacchari strain B42 xylanase, especially at alkaline pH is advantageous for application of the enzyme in biobleaching of kraft pulp.

Effect of temperature on the activity and stability for purified xylanase
The optimum temperature of Laceyella sacchari strain B42 purified xylanase was 70°C.At this temperature the enzyme was stable for 1 h and thereafter the stability decreased with the increase in the incubation period.At 70°C, 72% of the original activity and at 80°C, 48% of the original activity was retained after 6 h of incubation for purified enzyme.At 50 and 60°C, enzyme was quite stable and retained low activity.The half life (t 1/2 ) of purified xylanase was 6 h at 80°C (Figure 9).The observed stability of L. sacchari strain B42 xylanase is much better as reported by (Blanco et al., 1995;Sa-Pereira et al., 2002;Virupakshi et al., 2005).
The utilization of thermostable xylanase in industry could improve the technical and economic feasibility of industrial processes, specifically in the paper pulp industry (Gessesse and Mamo, 1999;Kohli et al., 2001).The process of enzyme assisted kraft pulp bleaching requires high temperature; hence a thermostable xylanase from L. sacchari strain B42 would fulfill these requirements.

Kinetic analysis
The reaction between xylanase and oat spelt xylan was found to follow Michaelis-Menten kinetics and showed hyperbolic curve (Figure 10).Lineweaver-Burk plot indicate that K m value was 4.166 mM (0.62 mg/ml) and V max value was 3787.87 µmole/min/ml/mg (Figure 11).The observed affinity of Laceyella sacchari strain B42 xylanase was similar to the xylanase II isolated from Streptomyces sp.PC22 (0.63 mg/ml) (Wateewuthajarn et al., 2008).

Colour removal from the kraft pulp
Colour from kraft pulp was removed by treatment with the enzyme.The amount of colour released was increased with the increase in enzyme dose.When the pulp was treated with the enzyme dose of 100 U/g the release of phenolic compounds (at λ 237 nm ) was observed from 0.25 to 2.51 and the release of hydrophobic compounds (at λ 465 nm ) was observed from 0.10 to 0.44 (Figure 12).At the optimum enzyme dose (60 U/g), the absorption at 280 nm was 1.57 for the release of lignin compounds (Figure 13).When the pulp was incubated for different time from 1 to 5 h with the optimized enzyme dose (60 U/g), the release in phenolic compounds (at λ 237 nm ) were observed from 1.8 to 2.67 and the release of hydrophobic compounds (at λ 465 nm ) were observed from 0.25 to 0.49 (Figure 14).The optimum enzyme dose (60 U/g) and optimum incubation time (4 h) gave the absorption of 1.88 at 280 nm (Figure 15).
It is interesting that with increase the enzyme dose and reaction time, the increase in release of phenolic compounds (at λ 237 nm ) and hydrophobic compounds (at λ 465 nm ) were observed.

Optimization of enzyme dose and reaction time for biobleaching
The biobleaching efficiency of purified xylanase was studied by treatment of pulp with the varying enzyme dose of 0 to 100 U/g for the incubation time of 0 to 5 h.
The optimum enzyme dose for biobleaching of pulp was found to be 60 U/g.At this optimized enzyme dose ~20% reduction of kappa number and 24 fold release of reducing sugars was observed (Figure 16).At their  optimum reaction time (i.e. 4 h) and enzyme dose (60 U/g) purified xylanase produced ~26% reduction in kappa number with 24.0 fold increase the release of reducing sugars (Figure 17).

Kappa number of pulp
Kappa number is the measure of the amount of lignin present in the pulp.Kappa number of untreated pulp was  15.0.After treating the pulp with the 100 U/g of enzyme for 3 h kappa number was decreased to 11.1 (~26%) (Figure 16) while treatment of pulp for 5 h at the optimum enzyme dose (60 U/g) kappa number was reduced to 10.8 (~28%) (Figure 17).At their optimum enzyme dose (60 U/g) and incubation time (4 h) kappa number was reduced by ~26%.

Lignin content
Lignin content of the pulp was decreased after treatment with enzyme.Initial lignin content in untreated pulp was 2.25%.At the optimized enzyme dose 60 U /g and incubation time 5 h the lignin content was decreased to 1.68.

Conclusion
Xylanase which are active and stable at alkaline pH and elevated temperature have tremendous potential for application in enzyme-assisted bleaching of kraft pulps and other biotechnological process (Daneault et al., 1994;Zamost et al., 1991).The use of thermostable, alkaline xylanase for enzyme-assisted pulp bleaching  could greatly reduce the need for pH and temperature readjustment, thus, offering technical and economic advantages (Harris et al., 1997).In the present investigation, we have isolated a L. sacchari strain B42, which produced thermostable, cellulase poor, alkaline xylanase.Xylanase from this strain showed high temperature optima of 70°C with good thermostability at 60 to 80°C for 6 h.Xylanase from this source also showed good activity at alkaline pH 10.0 to 11.0.Since thermal stability of xylanase is very important property in pulp and paper industry, the xylanase from L. sacchari strain B42 could be a good candidate for biotechnological application.It is important to highlight that L. sacchari strain B42 also showed minimal cellulase activity which is advantageous in pulping operation.The biobleaching of kraft pulp by L. sacchari strain B42 xylanase causes reduction of kappa number, decreased in lignin content and releases sufficient amount of reducing sugars.The enzyme also releases phenolic and hydrophobic compounds.All of these properties of L.sacchari strain B42 xylanase make the applicability of this strain in pulp and paper industry.

Figure 5 .
Figure 5. Phylogenetic analysis of partial 16S rDNA gene sequence of Laceyella sachhari strain B42 and related microorganism.Bootstrap values (1,000 replicate runs, shown as percent).GenBank accession numbers are listed after species names.

Figure 10 .
Figure 10.Michaelis-Menten plot for the effect of oat spelt xylan concentration on xylanase activity for L. sacchari strain B42 purified xylanase.

Figure 11 .
Figure 11.Lineweaver-Burk plot for the effect of oat spelt xylan concentration on xylanase activity for Laceyella sacchari strain B42 purified xylanase.

Figure 12 .
Figure 12.Release of phenolic and hydrophobic compounds at different enzyme dose (U/g pulp).

Figure 13 .
Figure 13.UV spectrum of coloured compounds released during purified xylanase treatment at different enzyme dose U/g oven dry pulp.

Figure 14 .
Figure 14.Release of phenolic and hydrophobic compounds at different time of incubation.

Figure 15 .
Figure 15.UV spectrum of coloured compounds released during purified xylanase treatment at different time interval.

Figure 16 .
Figure 16.Kappa number and reducing sugars estimation by purified xylanase dose (U/g pulp) for biobleaching of pulp.

Figure 17 .
Figure 17.Kappa number and reducing sugars estimation by purified xylanase with 60 (U/g pulp) at different incubation time for biobleaching of pulp.