International Journal of Applied Research in Natural Products Vol. 5 (1), pp. 7-11.

April-May 2012 Directory of Open Access Journals 2012. IJARNP-HS Publication

Original Article Partial purification and characterization of Xylanase from Trichoderma viride produced under SSF
Irfan M*, Syed Q
Food & Biotechnology Research Center (FBRC), Pakistan Council of Scientific & Industrial Research Laboratories Complex, Ferozpure road Lahore Pakistan 54600.
Summary: In the present study xylanase enzyme was produced from Trichoderma viride in solid state fermentation using sugarcane bagasse as a substrate. The whole fermentation process was carried out in 250ml Erlenmeyer flask at 30oC for seven days of fermentation period. The enzyme was partially purified by ammonium sulphate (60%) fractionation followed by dialysis. The partially purified enzyme was further characterized showing optimum pH and temperature of 5.0 and 50oC respectively. Metal profile of the enzyme showed that it was stimulated by FeSO 4 (134%), CaCl 2 (129%), BaCl 2 (105%), MgSO 4 (113%), MnCl 2 (102%) or AgCl (107%) and it was strongly inhibited by EDTA (26%) or HgSO 4 (32%). Industrial Relevance: In the present study, xylanase enzyme was produced and characterized from Trichoderma viride in solid state fermentation using cheap substrate. This enzyme is very helpful in industrial sector especially in pulp and paper industry, food industry and also in bioethanol production. Pilot scale production of this enzyme in industries can reduce the import cost of the enzyme and make the whole process cost effective. Keywords: Partial purification; Characterization; Xylanase; Trichoderma viride; SSF

INTRODUCTION Hemicelulose is the second most abundant component in plant cell wall and xylan is the major component of hemicelluose which is found in solid agricultural and agroindustrial residues, as well as in effluents released during wood processing (Collins et al., 2005; Anthony et al., 2005). Xylan is a heterogeneous carbohydrate, consisting of a homopolymeric backbone of -1, 4 linked D-xylopyranose units and short chain branches consisting of O-acetyl, -L-arabinofuranosyl and -Dglucuronyl residues (Nair et al., 2008). 20-35% of the total dry weight of plants accounts for xylans which can be used for production of fermentable sugars and fuels (Haltrich et al., 1996; Filho 1998). Xylanases are the group of enzymes that are involved in the hydrolysis of xylans and arabinoxylan polymers. These enzymes include endo-1,4--xylanase, xylosidase, a-arabinofuranosidase and acetylxylan esterase (Biely, 1993). Xylanases hydrolyze 1,4--Dxylosidic linkages in xylan to produce xylooligosaccharide. These enzymes are produced from various sources. Among the microbial sources, filamentous fungi are particularly interesting because they are non pathogenic, easily cultivated and secrete
______________________ *Corresponding Author: mirfanashraf@yahoo.com +XXX-XXX-XXXX. Available online http://www.ijarnp.org

their enzymes into the medium and have high xylanase activity in contrast to yeasts and bacteria (Kar et al., 2006, Krisana, et al., 2005). Xylanases are produced by either solid state or submerged fermentation. Enzyme production in solid state fermentation (SSF) is usually much higher than that of submerged fermentation (Haltrich et al., 1996). Therefore, solid state fermentation has gained interest from researchers in recent years and has often been employed for the production of xylanases because of economic and engineering advantages (Pandey et al., 1999). The present study was aimed to partially purify and characterized the xylanase enzyme produced by Trichoderma viride in solid state fermentation using sugarcane bagasse as a substrate. MATERIALS AND METHODS Reagents. All the reagents used in present study were of analytical grade and purchased from Sigma (USA), Merck (Germany), Fluka (Switzerland) and Acros (Belgium). Agricultural residues such as bagasse, corn cobs, soybean meal, rice husk, rice bran, wheat bran etc. were purchased from the local market of Lahore city.

Purification and characterization of Xylanase from Trichoderma viride

Microorganism and Maintenance. Trichoderma viride-IR05 was obtained from Microbiology laboratory, Food and Biotechnology Research center (FBRC), Pakistan Council of Scientific and Industrial Research (PCSIR) laboratories complex Ferozpur Road, Lahore Pakistan. The culture was maintained on slants containing potato-dextrose-agar (PDA, Oxoid) stored at 4C in a cold cabinet. Inoculum preparation. In present study, conidial inoculum was used. The spore suspension was prepared by adding 10 ml of sterile distilled water in to a 7 days old slant culture aseptically. Conidial clumps were broken using inoculation needle. The tube was shaken to make homogeneous mixture of conidial suspension. Fermentation technique. The production of xylanase was carried out using SSF in 250 ml Erlenmeyer flask. Ten ml of diluents (vogels media) was transferred into the flask containing 10 g of bagasse and mixed well. The flasks were cotton plugged and sterilized them in an autoclave at 121C for 15 min at 15 lbs/in2. After cooling the flasks at room temperature, inoculated them with 1.0 ml of fungal conidial suspension under aseptic condition. The flasks were kept at 30+1C for seven days in the incubator. All experiments were run parallel in duplicate. Extraction of enzyme. After seven days of fermentation, 50 ml of distilled water was added in to the each flask containing fermented mash and rotated them on rotary shaker at 150 rpm for 2 h at 30+1C for maximum enzyme extraction. Then filtered slurry through muslin cloth followed by centrifugation at 8000 rpm at 4C for 10 min to separate fungal spores and small particles. The clear supernatant was used as a crude xylanase source. Estimation of Xylanase activity. Xylanase activity was assayed as described earlier (Irfan et al., 2010). Reaction mixture containing 0.5 ml of appropriately

diluted culture filtrate with 0.5 ml of 1% birchwood xylan (Sigma) solution prepared in citrate buffer (0.05M, pH5.0) for 15min at 50C. After incubation the reaction was stopped by the addition of 1.75ml of 3, 5 dinitrosalicylic acid (Miller 1959) and heated for 10 min in boiling water bath. After cooling the reducing sugars liberated were measured by spectrophotometrically at 550nm and expressed as xylose equivalent. Xylose was taken as standard. One unit of activity was defined as the amount of enzyme, which liberates reducing sugar (equivalent to xylose) from 1.0 % Birch wood xylan under standard assay conditions. Partial Purification of Xylanase. Partial purification of xylanase enzyme was done by the procedure as described earlier (Irfan et al., 2008). The spore free filtrate was fractionated by precipitation with ammonium sulphate between 50-80% of saturation. The protein pellet obtained after ammonium sulphate precipitation was suspended in 0.05M citrate buffer pH 5 and dialyzed against the same buffer. All these subsequent steps were carried out at 4oC. Protein Determination. Total protein content in the fermented mash was estimated as described by the method of Lowery et al., (1951). RESULTS AND DISCUSSION Purification of Enzyme. The enzyme produced by Trichoderma viride-IR05 in solid state fermentation was purified by ammonium sulphate fractionation followed by dialysis. The enzyme was fractionated with different percentage of ammonium sulphate. Results (Table .1) indicated that maximum enzyme activity was observed with 60% ammonium sulphate precipitation with specific activity of 77.2 U/mg. The precipitated enzyme was further dialyzed against the same buffer showing purification fold of 1.6 with specific activity of 112.5 U/mg and 68.9% of enzyme yield.

Table 1. Partial Purification of Xylanase Produced by T. viride-IR05 Total volume (ml) 1000 25 10 Total Activity (U/ml) 65.3 53.5 45.0 Total Protein (mg/ml) 0.94 0.77 0.40 Specific activity (U/mg) 69.4 77.2 112.5 Purification (Fold) 1 1.1 1.6 Yield (%) 100 81.9 68.9

Purification steps

Crude Enzyme 60%Amm.sulphate ppt. Dialyzed

Irfan, Sayed

Effect of pH on Xylanase activity and Stability Every enzyme had its specific pH for efficient working. To determine the optimum pH of xylanase produced by T.viride-IR05 the enzyme was assayed at different pH. Different Buffers such as Citrate Phosphate (pH= 4-5.5), Phosphate buffer (pH=67.5), Tris-HCl buffer (pH=8-9.5), Glycin-NaOH buffer (pH=10-11) were used to check optimum pH. Enzyme activity started increasing with increase in pH showing highest activity at pH 5 using citrate phosphate buffer as shown in figure 1. As the pH of the substrate was increased, the enzyme activity was decreased showing lowest activity at pH 11 which was 12.0 0.23 U/ml. Stability of the enzyme was also studied by incubating the enzyme solution with different pH of buffers at room temperature for 30 minutes. After incubation the enzyme activity was checked and results showed that the enzyme was stable over pH range of 4-7. Querido et al., 2006 purified extracellular xylanase from Penicillium expansum which showed optimum activity at pH of 5.5 and stable from pH 5.5-6.5. Optimum temperature was found 40oC and stability range of 20-40oC. Xylanase of Pleurotus ostreatus SYJ042 was stable at pH range (3.0-9.0) and temperature up to 40oC with incubation period of 15min (Qinnghe et al., 2004). Trichoderma harzianum 1073 D3 produced xylanase showing optimum activity at pH 5, and stable in pH range of 3-7 and also retained more than 50 % of its original activity after four months (Isil and Nilufer 2005). Bakir et al., 2001 reported that an endoxylanase from Rhizopus oryzae showing optimum pH and temperature of enzyme activity was 4.5 and 55oC respectively Effect of temperature on Xylanase activity and Stability. To check the optimum temperature of xylanase produced by Trichoderma viride-IR05 under solid state fermentation, experiments were performed by incubating the reaction mixtures at different incubating temperatures ranging from 30 to 90oC with regular interval of 5oC. Results in the figure 2 indicated that enzyme activity gradually increased by increasing temperature from 30oC (41.3 1.5 U/ml) to onward. As the incubation temperature reached to 40oC (56.7 1.8 U/ml) the

enzyme activity further increased as compared to 30oC and reached to peak at 50oC (67.4 2.1 U/ml). When the temperature was further increase from 50oC to 90oC there was gradual decrease in enzyme activity was observed yielding lowest activity at 90oC which was 11.6 0.6 U/ml. Thermal stability of the enzyme was also checked by incubating the enzyme solution at various temperature ranges (30 to 90oC) for 30 min, and it was observed that enzyme was stable in temperature range from 30oC to 60oC. Enzyme activity above 60oC was not stable which showed that the enzyme produced by Trichoderma viride IR05 in SSF was not thermophilic in nature. Effect of metal ions on Xylanase activity. Enzyme was further characterized to check the metal profile for activation. Results indicated that some metal ions like FeSO 4 (134%), CaCl 2 (129%), BaCl 2 (105%), MgSO 4 (113%), MnCl 2 (102%) and AgCl (107%) stimulated the xylanase activity. The enzyme activity was strongly inhibited by EDTA (26%) and HgSO 4 (32%), while NaCl (67%), CoCl 2 (87%), CrCl 2 (65%), ZnSO 4 (87%), CsCl 2 (64%) and CuSO 4 (90%) have slight inhibitory effect on enzyme activity as shown in Figure 3. Fengxia, et al., (2008), purified and characterize the xylanase from Aspergillus ficuum AF-98 by 5080% (NH 4 ) 2 SO 4 , DEAE-Sephadex A-50 ion exchange chromatography and Sephadex G-100 chromatography. The purified enzyme showed optimum pH and temperature of enzyme was 5.0 and 45oC respectively. Metal profile of the enzyme showed that the enzyme was activated by Cu2+ (up to 115.8% of activity) and was strongly inhibited by Hg2+, Pb2+ (up to 52.8% and 89%), respectively. Chivero et al., (2001) reported that xylanase from genus Bacillus with optimum pH of 8 and stable in wide range of pH 6.0-9.0. The optimum temperature was found 60oC. The endoxylanase from isolate SB-9a was stable at 50oC, maintaining over 50% of its activity for 1 h at pH 8. Enzyme from isolate SB-9a was inhibited by Hg2+, Ag+ and Mn2+ ions while Fe3+, K+, Na+, Ca2+, and Cu2+ ions stimulated xylanase activity.

Purification and characterization of Xylanase from Trichoderma viride

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Xylanase activity

Stability

Enzyme activity (U/ml)

70 60 50 40 30 20 10 0 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12

pH

Figure 1: Effect of different pH on activity and stability of xylanase produced from T.viride-IR05 under solid state fermentation.

80 70

Xylanase activity

Stability

Xylanase activity (IU/ml)

60 50 40 30 20 10 0 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 Tem perature ( oC)

Figure 2: Effect of different incubation temperatures on activity and stability of xylanase produced from T.viride-IR05 under solid state fermentation.

160 140 120 100 80 60 40 20 0

Relative activity (%)

Figure 3: Effect of different metal ions on activity of xylanase produced from T.viride-IR05 under solid state fermentation.*5mM concentration of metal ions were used

Co nt r Fe ol SO 4 Ca Cl 2 Na Cl Ba C Hg l2 SO 4 ED TA Co Cl 2 Cr Cl Zn 2 SO M 4 nC M l2 gS O 4 Cs Cl 2 Ag Cu Cl SO 4
Metals

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Irfan, Sayed

REFERENCES. Anthony L, Marcel A, Eric R. 2005. Overproduction and characterization of xylanase B from Aspergillus niger. Candian J. Microbiol, 51: 177-183. Bakir U, Yavascaoglu S, Guvenc F, Ersayin A. 2001. An endo--1,4-xylanase from Rhizopus oryzae: production, partial purification and biochemical characterization. Enzy Microb Technol, 29:328334. Biely P. 1993. Biochemical aspects of the microbial hemicellulases; in Hemicelluloses and Hemicellulase, Coughlan, M. and Hazlewood, G. (eds.), pp. 29-51, Portland Press, London, U. K. Chivero ET, Anthony N, Mutukumira N, Zvauya R. 2001. Partial purification and characterisation of a xylanase enzyme produced by a microorganism isolated from selected indigenous fruits of Zimbabwe. Food Chem, 72:179-185. Collins T, Gerday C, Feller G. 2005. Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiol Rev, 29: 3-23. Fengxia L, Mei L, Zhaoxin L, Xiaomei B, Haizhen Z, Yi W. 2008. Purification and characterization of xylanase from Aspergillus ficuum AF-98 Biores. Technol, 99 :59385941. Filho EXF. 1998. Hemicellulase and biotechnology. In: Pandalai, S.G. (Ed.), Recent Research Development in Microbiology. Research Signpost, Trivandrum, -India, pp. 165 176. Haltrich D, Nidetzky B, Kulbe KD, Steiner W, Zupancic S. 1996. Production of fungal xylanases. Biores Technol, 58:137161. Irfan M, Nadeem M, Syed QA, Baig S. 2010. Submerged cultivation of Aspergillus niger on pretreated sugarcane bagasse. World J Agric Sci 6:466-472. Irfan M, Nadeem M, Baig S, Syed Q, Qureshi AM. 2008. Purification and characterization of protease from Bacillus sp. Pak J Biochem Mol Biol, 41(3):129-132.

Isil S, Nilufer A. 2005. Xylanase Production from Trichoderma harzianum 1073 D3 with Alternative Carbon and Nitrogen Sources. Food Technol. Biotechnol. 43 (1): 3740. Kar S, Mandal A, Mohapatra PK, Mondal KC, Pati BR. 2006. Production of cellulase-free xylanase by Trichoderma reesei SAF3. Braz J Microbiol, 37(4): 462-464. Krisana A, Rutchadaporn S, Jarupan G, Lily E, Sutipa T, Kanyawim K. 2005. Endo-1,4-xylanase from Aspergillus cf. niger BCC14405 isolated in Thailand: purification, characterization and gene isolation. J. Biochem. Mol. Biol. 38:1723. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951. Protein measurement with Folin phenol reagent. J. Biol. Chem., 193:265- 275. Miller GL. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugars. Anal Chem, 31: 426428. Nair SG, Sindhu R, Shashidhar S. 2008. Fungal xylanase production under solid state and submerged fermentation conditions. Afr. J Microbiol. Res, 2:082-086. Pandey A, Selvakumar P, Soccol CR, Nigram P. 1999. Solid state fermentation for the production of industrial enzymes. Curr. Sci, 77:149-62. Qinnghe C, Xiaoyu Y, Tiangui N, Cheng J, Qiugang M. 2004. The screening of culture condition and properties of xylanase by white-rot fungus Pleurotus ostreatus. Process Biochem., 39:15611566. Querido ALS, Coelho JLC, Arajo EF, ChavesAlves VM. 2006. Partial Purification and Characterization of Xylanase Produced by Penicillium expansum. Braz. Arch. Biol.Technol., 49(3): 475-480.

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