EXTRACTION OF XYLAN DEGRADING ENZYME FROM SOIL ISOLATES FOR ITS .....

EXTRACTION OF XYLAN DEGRADING ENZYME FROM SOIL ISOLATES FOR ITS APPLICATION AS BIOBLEACHING AGENT IN PAPER PULP PROCESSING

Mohan Gaanappriya and L. Vijaykumar*

Department of Biotechnology, Bannariamman Institute of Technology, Sathyamangalam – 638401, Tamil Nadu, India

* Author for correspondence – microlvk@yahoo.co.in

ABSTRACT

The use of hemicellulolytic enzymes has recently attracted considerable interest as a substitute for chlorine chemicals in pulp bleaching in view of the environmental concerns. Fungal biotechnology opens a potpourri of strains for identification and secondary metabolite extractions. The present work aims in extraction of xylanase from novel microorganism(s) for its commercial application as biobleaching agent in paper and pulp industries. Eight xylanolytic fungi were isolated for the enzyme production. The culture conditions for enzyme production were optimized. The optimal enzyme activity was assayed for the xylanse enzyme. The results of the work were discussed with related references.

Key Words: Xylanase, Fungal Biotechnology, Enzyme production, biobleaching

INTRODUCTION

            The past few decades witnessed a drastic change and advances in the lifestyle of human kind due to the conjoint formulations of science and the human demands. However, the rapid translation of laboratory findings into real time usage had posed a threat to the environmental safety and has brought considerable issues of concern to restore the pace of natural environment. Environmental concerns have drawn the interests of the biotechnologists to look out for an alternative way to chemical methods of processing. Advances  in  biotechnology  and process control,  for example,  offer opportunities  for responding to environmental  needs,  and  energy  considerations  will play  a  role  in  the development  of such control  technologies  as  bleach  effluent  recycling  by  concentration  and burning. This outcome has reflected in the field of chemical technology, where manufacture of a variety of products on large scale has resulted in serious effluent and hazardous waste disposal problems. Now, the need for safer and ‘environmental friendly’ technologies has become imminent. This has now started up the scientists to learn and identify the solutions from the nature. Finally, the newer field of microbial biotechnology has emerged to give remedies to the threatening issues. Microbes are capable of performing multifarious reactions that are biochemically driven, under ambient environmental conditions with the least or no hazardous effects. Thus, enzymes are becoming the cornerstones for the biotechnological research, becoming an alternative strategy to reduce or replace the hazardous polluting chemicals. (Srinivasan and Meenakshi, 2006)

            Xylanases (1,4-P-D-xylan xylanohydrolase; EC 3.2.1.8) are hemicellulases that hydrolyze xylan, which is a major constituent of the hemicellulose complex.(Browning, 1963). Viikari et al. (1986), for the first time, demonstrated the usefulness of xylanase in reducing the consumption of bleach chemicals. Today, the beneficial effects of xylanase have also been commercially demonstrated in the pulp and paper industry (Kulkarni et al., 1999, Techapun et al., 2003) The interest in xylan degrading enzyme and its application in pulp and paper industries has advanced significantly over the past few years. A survey of several microorganisms that produce xylanases that could be purified readily indicated that Fungal strains are potent producers of the enzyme. (Gaanappriya et al.,2011) The current research article explains the concept of biobleaching with the use of xyanolytic enzymes as an active bio agent, extracted from the fungal soil isolates, to replace the conventional bleaching of wood pulp by chlorine compounds.

The objectives of the present study were isolation, identification and characterization of xylanase producing fungi, optimization of cultural conditions for xylanase enzyme production. A variety of microorganisms were reported to produce endoxylanases, which can degrade ß -1, 4-xylan in a random fashion, yielding a series of linear and branched oligosaccharide fragments. The fugal strains were isolated from soil collected from effluent treatment area of paper mills. Eight strains were selected and optimized for the production of xylanase enzyme.

MATERIALS AND METHODS

Chemicals and Reagents

M9 Minimal medium was freshly prepared. Potato Dextrose Agar (PDA), Xylan – from Birch wood, D – xylose , Carboxy Methy cellulose sodium salt, 2- hydroxy-3,5-dinitrobenzoic acid (DNS), were obtained from Merk. The other reagents were of analytical grade while the buffers used were freshly prepared and of analytical grade. Sterilized double distilled water was used through out the experiment.

M₉ Minimal medium preparation

            The M9 Salts medium was prepared : 800mL H₂O was aliquoted and the following components were added. 64g Na₂HPO₄-7H₂O, 15g KH₂PO_4, 2.5g NaCl and 5.0g NH₄Cl. This solution was made up to 1000 mL and sterilized. The M9 Minimal medium, 200 mL of M9 salts solution, 2 mL of 1M MgSO₄ (sterile), 100ul of 1M CaCl₂ (sterile), 20 mL of 20% xylan were added and the final volume was made upto 1000 mL with sterilized, double distilled water. Increasing the concentration of the xylan in the medium was done to get more active strains.

Strain Isolation and identification

            To 500 mL each of M9 Minimal medium containing 20 % xylan and 10% xylan respectively, 1 g of soil sample was inoculated and incubated for three days at 37°C. These samples were then plated on M9 Minimal agar medium amended with xylan. Enumeration of microbes was done. Eight strains (X1 – X8) producing xylanase were isolated based on the zone of clearance formed in xylan – agar plates. The cultures were maintained in PDA slants.

Enzyme Production

Spores were obtained by culturing the organisms at 28°C in sporulating medium contained (g/L): Trisodium citrate 5,  KH₂PO₄ 5, NH₄NO₃ 2,  (NH₄)₂SO₄ 4g,  MgSO₄ 0.2, peptone 1, yeast extract 2, glucose 2 at pH of 5.5. The sporulating medium was taken as the inoculating medium. A loopful of cultures were transferred into the enzyme production medium(g/100mL): Glucose 3.0, Bactopeptone 1.0, Urea 0.3, (NH₄)₂SO₄  1.4, MgSO₄.7H₂O  0.3, CaC1₂.6H₂O 0.3 and Xylan 2.0 for enzyme production. These flasks were incubated at 28°C for 5 days in static condition

Culture Harvest

Liquid state cultures were harvested by centrifugation at 10,000 × g for 20 min. at 4°C and the resulting supernatant was called as crude enzyme preparation.  The crude enzyme was first saturated up to 30% with solid (NH4)₂SO₄ and then centrifuged at 5,000 × g for 15 min. The supernatant obtained was further saturated up to 70% with solid (NH₄)₂SO₄ and again centrifuged. The pellets obtained were dissolved in minimum volume of 0.1 M phosphate buffer, pH 6.0.

Partial Purification of Xylanases:

The enzyme was isolated, partially purified and characterized at room temperature (28 ± 2˚C). The extra cellular xylanase was partially purified from the culture filtrate when grown on oat spelt xylan. Solid ammonium sulphate was dissolved to attain initially 35% saturation at 0˚C centrifuged at 12,000 rpm for 30 min at 4˚C.(Jaganathan et al.,1956)

Total Protien Estimation

Total soluble protein was measured according to Lowry et al., (1951). Protein concentration was determined using bovine serum albumin (BSA) as a standard. The protein content of the chromatographic eluant was measured by monitoring the optical density at 280 nm.

Xylanase Assay

Xylanase activity was measured according to Bailey et al. [1992]. Xylanase activity was assayed using 1% (w/v) of xylan as a substrate. Reaction mixture contained I mL of appropriately diluted enzyme and 1% xylan in citrate phosphate buffer. The mixture was incubated at 50 °C for 30 min. The reaction was terminated by adding 3 mL of dinitrosalysilic acid (DNS) reagent.(Miller,1951). After heating for 5 min in a boiling water bath and cooling, the absorbance was noted at 550 nm.

Determination of optimal pH, temperature on enzyme activity

The enzymatic reactions were carried out for 5 min in 3 different buffers (50 mM): citric acid-Na₂HPO₄ (pH 5 to 6), phosphate buffer (pH 6 to 8), Glycine-NaOH buffer (pH 8 to 9.5). The actual pH in the assay mixture was determined at the reaction temperature. The effect of temperature on the reaction rate was determined by performing the standard reaction for 5 min at a temperature range of 55 to 80°C.

RESULTS AND DISCUSSION

Isolation and identification of strains:

            The soil sample was inoculated in M9 Minimal medium enriched with xylan (10% and 20%). The samples were then plated on M9 Minimal agar medium amended with xylan for the isolation of xylanolytic fungal strains(Fig 1). From the microbial diversity, eight strains were screened for the production of xylanase enzyme by checking for the zone of clearance in xylan agar medium. Totally 8 strains were isolated. These strains were named as X1 – X8.(Fig.2). The strains were identified based on their morphological characters as follows. However these identifications do not intend to identify the organisms at the species level.

Purification of Xylanase

The culture filtrate was precipitated by fractional (35–80%) ammonium sulphate saturation. Proteins precipitated within this range had maximum xylanase activity and was used for purification. (Table 2)

Effect of pH on enzyme activity

Fig.3 shows the pH effect on enzyme activity. It is clear from the figure that enzymes from Aspergillus strains were most active in the neutral pH range, between pH 6 to 7.0. For Trichoderma species, the enzymes were found to be active in ph between 6 to 7, and for Fusarium , pH was 6.5 and for the Penicilium species, the optimal pH was identified in the range of 5 -5.5. At pH above 7.5, the enzyme tends to precipitate in our assay conditions. Most xylanases known today are active at acidic (John et,al., 1979) or neutral pHs (Berenger et.al,1985). Hence the pH of the medium was adjusted to in accord for the growth of the fungal strains respectively in Vogel’s medium and also in all subsequent experiments. The optimal pH for fungal enzymes varies from species to species, though in most cases the optimum pH ranges from 3.0 to 8.0 (Subramaniyan, S and   Prema, P, 2002). A similar observation was reported by Alves-Prado et al., (2010) for the production of celluloytic enzymes from  Neosartorya spinosa.

Effect of Temperature on enzyme activity

Like most chemical reactions, the rate of an enzyme-catalyzed reaction alters as the temperature is altered. many enzymes are adversely affected by high temperatures too. Fig. 4 depicts the temperature effect on xylanse production. A maximum production of xylanase was achieved in the range of 50-55°C for Aspergillus spp. and Fusarium sp, 55 – 65 °C for Penicillium spp. and below 55°C for Aspergillus species  The optimum temperature for celluloses production by T. harzianum is in accordance with earlier research is 28°C. But in our study a optimum temperature of 55°C was achieved, which could be reason that the isolated strain was from a environmental sample. This is in contradictory with those of earlier worker conducted on C. thermophile (Ganju et al.,1989), and Myceliopthera thermophila (Zamost et al.,1991) whose reported the optimum temperature for xylanases produced from these species was 70ºC.

Effect of Time on enzyme activity

The Time factor study showed that a time period of 72 h - 120 h for an optimum xylanase production by Aspergillus spp, 144 h for Fusarium sp, 96 – 120 h for Penicillium spp and120 h for Trichoderma spp. (Fig. 5). The obtained finding was in accordance with earlier reports. The xylanase from Aspergillus niger recorded the optimal production between 120 – 168h. (Anthony et al., 2005) Similarly A. fumigatus showed maximum  production at 120 h of cultivation.( Ancharida Svarachorn, 1999).

CONCLUSION

Xylanase secretion often associates with low or high amount of cellulases. Haltrich et al.(1996) also reported that xylanases were always associated with cellulose. To use xylanase for pulp treatment, it is preferable to use cellulose-free xylanases, since the cellulase may adversely affect the quality of the paper pulp. (Ninawe et al., 2008). Upon eight strains, the strains X4, X5 and X6 were stable at a higher temperature and neutral pH, enabling the usage of these enzymes for biobleaching applications. The rest could be used for other applications in textile industries and food processing industries wherein the process conditions are at a relatively lower temperature.