5 Puji Rahayu-380.pmd Volume 3, Number1, April 2008 p 23-26 ISSN 1978-3477 *Corresponding author:, Phone: +62-21-3169503, Fax: +62-21-3169510, E-mail: siswa59@yahoo.com Production and Purification of Xylanase From Indonesian Isolate Bacillus sp. AQ-1 Grown on Bunch Palm Oil PUJI RAHAYU1, SISWA SETYAHADI2*, AND HARMITA1 1Pharmacy Department, Faculty of Mathematics and Natural Science, Universitas Indonesia, Depok 16424, Indonesia; 2Center for the Bioindustrial Technology, Badan Pengkajian dan Penerapan Teknologi, BPPT building 2, 15 floor, Jalan MH. Thamrin 8, Jakarta 10340, Indonesia Xylanase (endoxylanase, EC 3.2.1.8) is a commercial enzyme that has been applied in the industrial production of fuel, food, textiles and paper. Xylanase was isolated from the culture supernatant of Bacillus sp. AQ-1 grown on Nakamura medium containing 0.5% powder bunch palm oil. The optimum pH and temperature of xylanase activity were pH 7.0 and 60 °C, respectively. The enzyme was purified by anion exchange chromatography using DEAE-Sepharose-Fast-Flow column and gel filtration chromatography using Sephacryl S-300 column. The results showed that purification of xylanase produced two forms of xylanase, which were identified as xylanase A and xylanase B. Xylanase A can be separated from xylanase B by ultrafiltration using a 30 kDa polyethersulfone membrane. The molecular weight of xylanase A and B were 15.7 and 57.7 kDa, respectively. Key words: xylanase, Bacillus sp., purification Currently, xylanolytic enzymes (endoxylanase, EC 3.2.1.8) have been the focus of much attention in industry and agriculture. These enzymes have many important applications in the saccharification of agricultural wastes (Biely 1985), the improvement of bakery products (Gilbert and Hazlewood 1993), the food and feed industry (Kulkarni et al. 1999) as well as the textile industry (Querido et al. 2006). Xylanase can also be converted into liquid fuel and solvents (Woodward 1984; Biely 1985; George et al. 2001). Poutanen (1997) observed that both endogenous and added enzymes have an important effect on the quality of cereal foods. Xylanase is also required for the degradation of xylan (Sunna et al. 1997b). It is the second most abundant biopolymer (after cellulose) and the main component of hemicellulose, which is present in nature in large amounts and is a major by-product of the farming industry (Simpson et al. 1991). The hydrolysis of xylan to xylo-oligosaccaride and xylose is catalyzed by xylanases (Takahashi et al. 2000). Xylanases are also applied for the bleaching of kraft pulp, increasing the brightness of the pulp in the pulp and paper industry (Purkarthofer et al. 1993; Viikari et al. 1994; Kulkarni et al. 1999). In the process of pulp bleaching, the pulp is usually treated at high temperature at alkaline pH. Therefore, thermostable xylanase would be preferred in biotechnological application, giving the advantage of running xylan digestion at high temperature. Another problem in enzyme treatment in the pulp and paper industry is the availability and cost of the enzyme, due to the high cost of the substrate for enzyme production (Hinnman 1994). Therefore, the use of low cost substrates such as solid agricultural wastes is one of the ways to reduce production costs. Bunch palm oil is one of the many solid agricultural waste materials that can be used as substrate in xylanase production. The purity of xylanase is also very important to ensure stability of enzyme in order to prevent enzyme protein denaturation. Purified xylanases have possible applications in the chemical industry, nutritional industry, beverage industry, food industry and also in the production of alternative artificial sweetener that is low in calories (xylitol) (Bhat 2000; Goulart et al. 2005). Aims of the present study were to produce and purify xylanase from Bacilus sp. AQ-1 using bunch palm oil as the substrate in the fermentation medium. MATERIALS AND METHODS Enzyme Production. Bacillus sp. AQ-1 (culture collection of Bioindustry Laboratoy, BPPT) was grown at 37 °C in Luria Broth medium containing 1% (w/v) bactopeptone, 0.5% (w/v) yeast extract and 0.5% (w/v) sodium chloride. The culture of Bacillus sp. AQ-1 was incubated at 30 °C for 24 h in the Nakamura (1994) medium using powder bunch palm oil as the substrate. The substrate was prepared by sun drying for 2 days, and then was ground and sieved (35 mesh sieve). The composition of Nakamura (1994) medium was as follows: 1% (w/v) bactopeptone, 0.5% (w/v) yeast extract, 0.1% (w/v) K 2 HPO 4 .3H 2 O, 0.02% (w/v) MgSO 4 .7H 2 O and 0.5% (w/v) substrate (Nakamura 1994). After 24 h, the cells were removed by centrifugation at 6 000 rpm using High-Speed Refrigerated Centrifuge Himac CR 21G and R10A3 rotor for 15 min at 4 °C and the supernantant from extracellular secretion was used for estimation of xylanase activity and protein content and also for xylanase purification. Enzyme Activity and Protein Concentration. Xylanase activity was assayed by the dinitrosalicylic acid (DNS) method, according to Bailey et al. (1992). Oat spelt xylan was dissolved in 50 mM sodium phosphate buffer (pH 7.0). The reaction mixture containing 50 µL of crude enzyme and 450 µL of 1% (w/v) xylan was incubated for 5 min in the thermomixer at the optimum temperature for enzyme activity. The reaction was terminated by the addition of 750 µL of DNS reagent. The mixture was centrifuged at 14 000 rpm using High-Speed Refrigerated Centrifuge Himac CR 21G and R10A2 rotor for 2 min and the supernatant was boiled at 100 °C for 5 min. Reducing sugars released during incubation were measured as xylose equivalents by absorbance (A) at 540 nm. One unit of xylanase activity is defined as the amount of enzyme required to liberate 1 µmol of xylose per minute at the assay condition (Takahashi et al. 2000; Tanaka et al. 2005). Microbiol Indones24 RAHAYU ET AL. Protein concentration was determined by the folin- phenol method, with bovine serum albumin as a standard (Lowry et al. 1951). The protein content was measured at 750 nm. Effect of Temperature and pH on Enzyme Activity. Xylanase activities were measured at temperatures ranging from 30-100 °C under the standard assay conditions. Enzymes activities were also assayed at pH values from 4.0 to 9.0 (universal buffers) at the optimum temperature. Enzyme Purification. The cell-free supernantant of the culture was applied to the DEAE-Sepharose-Fast-Flow column (20×200 mm). Elution of the enzyme was carried out with 50 mM sodium phosphate buffer (pH 8.0) at a flow rate 1 mL min-1. Each fraction was analyzed for xylanase activity and protein content. The active fractions were pooled and loaded onto Sephacryl S-300 column (16×600 mm) at a flow rate 0.5 mL min-1. Equilibration and elution were performed with 50 mM sodium phosphate buffer (pH 8.0). Fractions were collected and analyzed for xylanase activity and protein content. The final steps of xylanase purification was carried out by ultrafiltration using a 30 kDa polyethersulfone membrane. Electrophoresis. The molecular weight of the purified xylanase was estimated by sodium dodecyl sulphate- polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE was performed in 12% (v/v) gels using low molecular weight markers (14.4-97.0 kDa). A suspension of beechwood xylan at a final concentration 0.1% (w/v) was incorporated into the separating gel before addition of ammonium persulphate. Proteins were visualized by silver staining with silver nitrate solution. Zymogram Analysis. The electrophoretic gels were washed in 2.5% (v/v) Triton X-100 for 60 min and then incubated at 60 °C for 15 min in 50 mM sodium phosphate buffer (pH 7.0). The reaction was stopped by incubation at 4 °C for 15 min. The gel was then incubated in 0.1% (w/v) congo red solution for 20 min at room temperature with gentle shaking and destaining was accomplished by washing the gel with 1 M NaCl until clear bands, indicating xylanase activity, became visible. The addition of 1 N HCl caused the background to turn dark blue, emphasizing the activity bands (Sunna et al. 1997a). RESULTS Enzyme Production. Microbial xylanases are usually inducible and secreted into culture medium containing xylan. The extracellular xylanase activity of Bacillus sp. AQ-1 grown on medium containing 0.5% (w/v) powder bunch palm oil in 24 h incubation was 65.90 U mL-1, while its protein content was 0.5 mg mL-1. Xylanase activity was determined at optimum temperature and pH. Effect of Temperature and pH. Fig 1 shows the effect of temperature and pH on xylanase activity. The optimum temperature of xylanase at pH 7.0 was observed at 60 °C (Fig 1a), while the optimum pH for xylanase activity was observed at pH 7.0 (Fig 1b). Enzyme Purification. Purification was carried out at room temperature (26-28 °C). Sodium phosphate buffer (50 mM, pH 8.0) was used throughout the purification procedure. The purification protocol involves two steps of column chromatography: ion exchange and gel filtration. Filtrate from the culture was applied onto a DEAE- Sepharose-FF column (20×200 mm). Xylanase was eluted from that column as a single protein peak that coincided with the peak of enzyme activity (Fig 2). The active fractions of anion exchange chromatography (fractions 1-30) were further purified by gel-filtration-chromatography using Sephacryl S-300 column (16×600 mm). Fig 3 shows the xylanase activity and protein content from the gel filtration chromatogram. The active fractions from that column (fractions 185-360) were subjected to ultrafiltration. Using this method, xylanase A had been separated from xylanase B. Xylanase A was in the permeate solution, while xylanase B was in the retentate solution. Table 1 summarizes the procedure for the purification of extracellular xylanase from Bacillus sp. AQ-1, indicating the degree of purification and yield for each step. Electrophoresis. Electrophoretic studies of crude extract and purified xylanase showed the presence of multiple bands (Fig 4). A low molecular weight ranging from 14.4 to 97.0 kDa was also observed. The molecular weight standards were α-lactalbumin (14.4 kDa), trypsin inhibitor (20.1 kDa), carbonic anhydrase (30 kDa), ovalbumin (45 kDa), albumin (66 kDa) and phophorylase b (97 kDa). Zymogram Analysis. The purity of xylanase was demonstrated by SDS-PAGE using 12% (v/v) gel containing 0.1% (w/v) beechwood xylan (Fig 5). The permeate and retentate solution of ultrafiltration produced single activity band on each of the two gels. The molecular weight of these purified enzymes were estimated to be about 15.7 kDa (xylanase A) and 57.7 kDa (xylanase B) by standard curve of molecular weight. Fig 1 Influence of temperature (a) and pH (b) on the activity of xylanase from Bacillus sp. AQ-1. Optimum activity temperature was measured at pH 7.0, while optimum activity pH was determined at the optimum temperature. Buffers used for optimum pH determination were 50 mM sodium acetate (pH 4.0), 50 mM sodium citrate (pH 5.0), 50 mM sodium phosphate (pH 6.0-7.0) and 50 mM Tris-HCl (pH 8.0-9.0) at final concentration. R e la ti v e e n z y m e a c ti v it y ( % ) 1 2 0 1 0 0 8 0 6 0 4 0 2 0 0 0 b p H 2 4 6 8 1 0 R e la ti v e e n z y m e a c ti v it y ( % ) 1 2 0 1 0 0 8 0 6 0 4 0 2 0 0 1 2 01 0 08 06 04 02 00 Temperature (°C) a Microbiol Indones 25Volume 3, 2009 Fig 2 Elution profile of enzyme activity (l) and protein content (�) of xylanase cell-free extract by anion exchange chromatography in DEAE-Sepharose-FF column (20×200 mm). The column was eluted with 50 mM sodium phosphate buffer (pH 8.0). Fig 3 Elution profile of enzyme activity (l) and protein content (�) of pooled fractions from the first column by gel filtration chromatography in Sephacryl S-300 column (16×600 mm). The column was eluted with 50 mM sodium phosphate buffer (pH 8.0). Table 1 Purification profile of xylanase from Bacillus sp. AQ-1 Purification steps Culture fluid DEAE Sepharose FF Sephacryl S-300 Ultrafiltration Permeate (Xyl A) Retentate (Xyl B) a recoveries are expressed as percentage of initial activity. Purification factors are calculated on the basis of specific activity 1 2 3 4kDa 9 7 . 0 6 6 . 0 4 5 . 0 3 0 . 0 2 0 . 1 1 4 . 4 Fig 4 SDS-PAGE, of crude xylanase. Lane 1, protein standards; lane 2, xylanase crude extract; lane 3, permeate ultrafiltration; lane 4, retentate ultrafiltration. E n z y m e a c ti v it y ( U m L -1 ) 4 5 4 0 3 5 3 0 2 5 2 0 1 5 1 0 5 0 - 5 1 4 6 9 1 3 1 8 2 2 2 4 2 6 2 8 3 0 3 2 3 4 Fractions P ro te in c o n te n t (m g m L -1 ) 0 . 5 0 0 . 4 5 0 . 4 0 0 . 3 5 0 . 3 0 0 . 2 5 0 . 2 0 0 . 1 5 0 . 1 0 0 . 0 5 0 . 0 0 -0.05 E n z y m e a c ti v it y ( U m L -1 ) 7 0 6 0 5 0 4 0 3 0 2 0 1 0 0 P ro te in c o n te n t (m g m L -1 ) 0 . 3 0 0 . 2 5 0 . 2 0 0 . 1 5 0 . 1 0 0 . 0 5 0 . 0 0 -0.05 Volume (mL) 5 0 5 0 4 0 2 0 1 Total activity (U) 3 2 3 8 . 1 9 2 7 3 6 . 0 6 2 2 7 0 . 2 0 2 3 0 . 0 8 6 . 1 4 Protein (mg) 26.90 13.60 10.77 2 . 7 9 0 . 0 4 Specific activity (U mg-1) 1 2 0 . 3 7 2 0 1 . 2 3 2 1 0 . 7 3 82.41 1 6 5 . 9 6 Purification factor (fold) 1 . 0 0 1 . 6 7 1 . 7 5 0 . 6 8 1 . 3 8 Recoverya (%) 1 0 0 . 0 0 84.49 70.11 7 . 1 1 0 . 1 9 Fractions- 1 0 1 4 5 8 5 1 2 5 1 6 5 1 9 0 2 0 0 2 1 0 2 2 5 2 5 0 2 7 0 2 9 0 3 1 5 3 3 0 3 4 0 3 5 0 3 6 0 Xylanase B 57.7 kDa 1 2 3 kDa 9 7 . 0 6 6 . 0 4 5 . 0 3 0 . 0 2 0 . 1 1 4 . 4 Fig 5 SDS-PAGE of purified xylanase. Lane 1, permeate solution; lane 2, retentate solution of ultrafiltration; lane 3, protein standards. Microbiol Indones26 RAHAYU ET AL. DISCUSSION Hydrolysis of xylan requires multiple enzymes with different modes of action acting in cooperation. Different microorganisms produce different kinds of xylanolytic enzymes. In this work, we have produced xylanase from Bacillus sp. AQ-1 at an activity level of 65.90 U mL-1 using bunch palm oil as the xylan substrate. As shown in Fig 1a, the xylanolytic enzyme produced by Bacillus sp. AQ-1 was optimally active at 60 °C. Similar results were observed for other microorganism, an optimal temperature of xylanase produced by Aspergilus oryzae was at 60 °C (Kitamoto et al. 1999). The enzymes reported to date are optimally active in neutral pH. The highest activity was observed at pH 7.0 when incubated at the optimum temperature (60 °C). Although some microorganisms produce xylanases that have optimum activity at pH above 8, most alkaliphilic and alkali-tolerant microorganisms produce xylanases that are optimally active around neutral pH (Blanco et al. 1995). Using ion exchange chromatography, gel filtration chromatography and ultrafiltration, purified enzymes can be separated from other proteins. Some proteins at a low concentration and lacking assayable xylanase activity, may be coeluted with the enzyme. Two forms of xylanases, produced from ion exchange chromatography and gel filtration chromatography, were identified as xylanase A and xylanase B. Ultrafiltration using a 30 kDa polyethersulfone membrane separated both of them, one enzyme was in the permeate solution (xylanase A) and the other enzyme was in the retentate solution (xylanase B). A summary of purification steps is presented in Table 1. The overall levels of recovery were 7.11 and 0.19% (xylanase A and B), with 0.68- and 1.38-fold purification of xylanase A and B, and with specific activity of 82.41 and 165.96 U mg- 1 protein, respectively. The purification factor of xylanase A was lower than expected (less than 1). The high specific activity of xylanase B suggests that it is the major extracellular xylanase produced by Bacillus sp. AQ-1. The apparent purity of the enzyme was demonstrated by SDS- PAGE and zymogram analysis with gel containing 0.1% (w/v) beechwood xylan, which showed the molecular weight of xylanase A and B were 15.7 and 57.7 kDa, respectively. In conclusion, we report the production of xylanase from Bacillus sp. AQ-1 grown on medium containing powder bunch palm oil gave high assayable enzyme activity. Even though various materials can be used as a substrate in the xylanase production, bunch palm oil is a potential substrate to produce the enzyme. 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