Abstract: Alkaliphilic and thermophilic Bacillus sp. DM-15 was isolated from ciftehan thermal spring, Turkey, produced thermostable xylanase at 55°C at pH 9. The molecular weight of the enzyme was estimated to be 95.6 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme showed broad working pH range of 4.5-10.0 with an optimum pH of 6.5. The temperature optimum of the enzyme was found to be 60-70°C. Xylanase production by thermophilic Bacillus sp. strain cultivated in liquid media reached a maximum at 36 h, with levels of 192.8 μmol mg^-1 protein/min. The enzyme was stable for 15 min at 60°C while 13, 86, 95 and 96% of the original activities were lost at 70, 80, 90 and 100°C, respectively. Enzyme activity was increased in the presence of 5 mM CaCl₂ (137%), CoCl₂ (133%), CuSO₄ (112%), MgCl₂ (106%) and KCl (105%) and inhibited in the presence of 5 mM EDTA, HgCl₂, FeSO₄, SDS (1%), MnCl₂, Triton X-100 (1%) up to 93, 89, 73, 41, 29 and 8%, respectively. The DM-15 thermostable xylanase may be suitable for several industrial applications, such as food, agricultural and biofuel.

INTRODUCTION

The major structure of hemicellulose is xylan which is the second most abundant polysaccharide after cellulose in nature (Whistler and Richards, 1970; Goheen, 1982; Coral et al., 2002; Collin et al., 2005; Faulet et al., 2006). It is found in large amounts in wood and plant material (Luthi et al., 1990). Xylan is a polymer β-1,4-linked xyloses with arabinosyl and/or 4-O-methylglucurosyl side chains (Faulet et al., 2006; Whistler and Richards, 1970).

Xylan can be degraded to xylose units by xylanolytic enzymes. These degredation requires the catalysis of both endo-xylanase (EC 3.2.1.8) and β-xylosidase (EC 3.2.1.37) (Faulet et al., 2006). In natural environment, xylanases are produced mainly by microorganisms, marine algae, protozoans, crustaceans, insects and snails (Ratanachomsri et al., 2006). Among the microorganisms, many reports on xylanases from Bacillus sp., Clostridium sp., Streptomyces sp., Aspergillus sp., Thermomyces lanuginosus, Thermoascus aurantiacus and other microorganisms are available (Wong et al., 1988; Bastawde, 1992; Alam et al., 1994; Faulet et al., 2006). Xylanases show potential applications in biotechnology include baking, fruit juice, paper, textile, pulp, ethanol production, aroma and animal feed (Bhat, 2000). In industrial applications, xylanases have to be alkalophilic and thermostable. However, most of the xylanases known are optimally active at temperatures below 50°C and acidic or neutral pH (Gessesse, 1998; Ryan et al., 2003). Only a few xylanases are reported to be active and stable at high temperature and alkaline pH (Dhillon et al., 2000; Rani and Nand, 2000; Khandeparkar and Bhosle, 2006a).

In this research, isolated thermostable microorganism, Bacillus sp. DM-15, from the thermal spring in Turkey, producing thermostable xylanase which active at broad range pH values and described partial characterization of the enzyme.

MATERIALS AND METHODS

Organisms and cultivation conditions: Bacillus sp. DM-15 was isolated from the thermal spring in Ciftehan, Nigde, Turkey. The samples were incubated at 80°C for 10 min for selection of gram-positive spore forming bacteria, Bacillus sp. The isolates screened for xylanase production on LB-xylan-agar plates containing (g L^-1) peptone 10, yeast extract 5, NaCl 5, xylan 1, agar 15 at 55°C. The pH was adjusted to 7.5 with 1N NaOH. Xylanase positive colonies were selected by Congo-red staining procedure (Teather and Wood, 1982).

Enzyme production: Strain DM-15 was grown up in LB medium at 55°C with shaking at 200 rpm for 24 h. After removal of cells by centrifugation (Hettich Universal EBA12) (5,000 rpm, 10 min), the supernatant was used for all enzyme assays.

Enzyme assay: Xylanase activity was assayed by adding 1 mL enzyme to 0.02 g xylan (2%, w/v) in 0.1M phosphate buffer, pH 6.5 and incubating at 55°C for 30 min. The reaction was stopped by the addition of 3 mL of 3,5-dinitrosalicylic acid reagent and A_{540 nm} was measured in a Pharmacia spectrophotometer (Miller, 1959). An enzyme unit is defined as the amount of enzyme releasing 1 mmol of glucose from the substrate in 1 min at 55°C.

Effect of pH and temperature on activity and stability: Temperature and pH effects on enzyme activity were assayed at various temperatures ranging from 40-100°C and at pH values ranging from 4-10 for 30 min. Following buffers were used in the reactions: 100 mM Na-acetate (pH 4-6), 100 mM Na-phosphate (pH 6-7) and 100 mM Tris (pH 7-10) (Ozcan et al., 1996).

For the measurement of thermal stability, the enzyme was pre-incubated at temperatures between 40-100°C for 15 min at optimum pH. The enzyme activity was then determined under standard enzyme assay condition (60°C, 30 min and pH 6.5).

Effect of various metals ions, surfactants, chelating agents and inhibitors on activity: The effect of metal ions, chelating agents, surfactants and inhibitors on the activity of xylanase were determined by pre-incubating the enzyme in the presence of EDTA (5 mM), SDS (1%), CaCl₂ (5 mM), MgCl₂ (5 mM), KCl (5 mM), FeSO₄ (5 mM), CuSO₄ (5 mM), CoCl₂ (5 mM), MnCl₂ (5 mM), HgCl₂ (5 mM) and TritonX-100 (1%) for 30 min at 55°C before adding the substrate. Subsequently relative xylanase activities were measured at standard enzyme assay conditions (Faulet et al., 2006; Egas et al., 1998; Lo et al., 2001). The control (without any additive) was taken as having 100% activity.

SDS-PAGE and zymogram analysis: SDS-PAGE and SDS-Xylan-PAGE (0.2% xylan) were done as described by Laemmli (1970) with slab gels (12% w/v acrylamide). For visualizing of total proteins, SDS-PAGE was stained for 1 h with the solution of 0.1% Coomassie blue R 250-40% methanol-10% glacial acetic acid and then destained overnight in the same solution without dye. For activity staining (zymogram) of xylanase by SDS-Xylan-PAGE, SDS was removed by washing the gel at room temperature in solution-A (50 mM Na₂HPO₄, 50 mM NaH₂PO₄ (pH 7.2), isopropanol) for 1 h and solution-B (50 mM Na₂HPO₄, 50 mM NaH₂PO₄ (pH 7.2)) for 1 h, respectively. The gel was kept overnight in solution-C (50 mM Na₂HPO₄, 50 mM NaH₂PO₄ (pH 7.2), 5 mM β-mercaptoethanol, 1 mM EDTA) at 4°C for renaturation of the enzyme. It was then sealed with film and incubated at 55°C for 4 h. After incubation, the gel was stained in 0.1% (w/v) Congo-red dye for 1 h and washed with 1% (w/v) NaCl for 30 min to visualize the clear band of xylanase activity (Saul et al., 1990; Lee et al., 1994; Coral et al., 2002).

RESULTS AND DISCUSSION

Bacillus sp. DM-15 was gram positive, rod shaped, spore forming and aerobic bacterium. It was identified as Bacillus sp. depending on various morphological and biochemical characteristics. The growth was observed between pH 6 and 10 in LB medium at 55°C. The maximum enzyme synthesis occurred 12 h later from inoculation in LB medium at 55°C.

Determination of molecular mass: Molecular mass determined by SDS-PAGE electrophoresis revealed single band showing xylanase activity in gel using BioCapt MW software with a molecular weight of 95.6 kDa (Fig. 1).

Fig. 1:	Zymogram analysis of xylanase on SDS-PAGE. The gel was cut into two pieces, the total protein and mekar was visualized with Coomassie Brillant Blue (Lane: 1) staining and the activity of anzyme relealed by Congo-red (Lane: 2)

Fig. 2:	Enzyme properties of Bacillus sp. DM-15 xylanase. a) Effect of temperature, b) Effect of pH and c) Thermal stability

Enzyme properties: The enzyme had a broad temperature range between 40-90°C and the optimum activity was observed at 60°C. The relative enzyme activities were 77, 91, 96, 68 and 57% at 40, 50, 70, 80 and 90°C, respectively, whereas only 17% activity was retained at 100°C for 30 min (Fig. 2a). The enzyme also showed a significant relative activity (73%) between pH 4.5 and 10 with an optimum pH of 6.5 (100%) (Fig. 2b). For thermal stability estimation, the retaining activity was determined at optimum pH and temperature (Fig. 2c). The enzyme was relatively thermostable at 70, 80, 90 and 100°C with, 13, 86, 95 and 96% lost of the original activities, respectively.

Effect on some metal ions, surfactants, chelating agents and inhibitors on enzyme activity: The enzyme was pre-incubated at 60°C for 30 min at different concentration of the metal ions and various chemicals prior to standard enzyme activity assay. The results for residual enzyme activity have shown in Table 1. The relative activity of enzyme was significantly increased to 137 and 133% by adding CaCl₂ and CoCl₂ (5 mM each), respectively.

Table 1:	Effect of different metal ions surfactans, chalating agents and inhibitors on xylanase from Bacillus sp. strain DM-15

Slightly stimulation was also observed by addition of CuSO₄ (112%), MgCl₂ (106%) or KCl (105%). On the other hand, partial inhibition of original enzyme activity was observed with use of SDS (1%), MnCl₂ (5 mM) and Triton X-100 (1%) up to 59, 71 and 92%, respectively. DM-15 xylanase enzyme activity was strongly inhibited up to 7, 11 and 27% by the use of EDTA (5 mM), HgCl₂ (5 mM) or FeSO₄ (5 mM), respectively (Table 1). It has been reported that there are many microorganisms producing xylanolytic enzymes. They include species such as Aspergillus niger, Marasmius sp., Termitomyces sp., Enterobacter sp., Bacillus sp. (Coral et al., 2002; Ratanachomsri et al., 2006; Faulet et al., 2006; Gessesse, 1998; Saleem et al., 2002; Anuradha et al., 2007). The xylanases from thermophilic bacteria such as Thermonospora fusca and thermophilic Bacillus species show an optimum temperature in the range of 65-85°C (Kulkarni et al., 1999).

We report here the partial characterization of thermostable xylanase from alkaliphilic and thermophilic bacterium Bacillus sp. DM-15. Bacillus sp. DM-15 showed growth at a wide range of pH from 6-10 with an optimum pH of 9. The bacterium is called typically alkaliphilic, as it grows optimally at pH values above 8 and grows poorly under neutral pH value of 7 (Horikoshi, 1999). The optimum temperature for enzyme production and growth was 55°C. Vieille and Zeikus (2001) reported that thermophilic organisms grow optimally between 50 and 80°C. This suggests that strain Bacillus sp. DM-15 could be thermophilic bacterium.

Xylanase from the isolate Bacillus sp. DM-15 exhibited a broad range temperature profile with a peak of maximal activity at 60°C (Fig. 2a). Enzyme activity at 60°C was 192.8 μmol mg^-1 protein/min and it showed good working temperature at 40-70°C at optimum pH value. It has been reported that fungal and bacterial xylanases are generally show temperature optimum between at 40 and 60°C (Butt et al., 2008). Enzyme activity was reduced to half at the temperature of 90°C with broad temperature interval. Also, enzyme thermostability was maintained until 70°C and then sharply reduced.

The pH activity of the DM-15 xylanase was in the range 4.5-10 with an optimum pH of 6.5 (Fig. 2b). This pH values are similar to thermostable xylanase from Bacillus stearothermophilus T-6 in the range 5-11 with an optimum pH of 6.5-7 (Khasin et al., 1993). The bacterial growth was found to be the best at pH 9. However, the optimum pH activity of xylanase was lower then growth pH optimum of bacteria. D-Xylanases from different organisms show broad pH optima and are usually stable over a wide pH range (3-10) and show optimum pH in the range of 4-7 (Kulkarni et al., 1999). Xylanases from Aspergillus kawachii and Penicillium herquei exhibit an optimum pH of 2-6 (Ito et al., 1992; Funaguma et al., 1991). However, the optimal pH of the xylanases isolated from Bacillus sp. C-59-2 (Horikoshi and Atsukawa, 1973) and Bacillus sp NCL 87-6-10 (Srinivasan and Rele, 1999) was 9. Although, DM-15 xylanase has slightly acidic optimal pH value, it show some activity at alkaline pH values with optimum growth at pH 9.0 with almost 50% activity. Similarly, the xylanase from Bacillus sp. with optimum growth at pH 10.0 showed remarkable stability at pH 9-10 but was not highly active above pH 8.0 (Okazaki et al., 1984).

Enzymes with high thermostability and an ability to function at wide pH range are desirable for many industrial applications as many industrial processes take place at very high or low pH and high temperature (Ratanachomsri et al., 2006). With this respect, the strain could be a good source for biotechnological applications.

Molecular weight of DM-15 thermostable xylanase was 95.6 kDa. Molecular weights of xylanases vary from 8-145 kDa (Sunna and Antranikian, 1997; Kulkarni et al., 1999). However, it has been reported that a high molecular weight of 340 kDa xylanase was produced by Bacillus subtilis (Sa-Pereira et al., 2002).

The effect of metal ions, surfactants, chelating agents and inhibitors on enzyme activity have been investigated. EDTA, HgCl₂, FeSO₄ and detergent agents inhibited the activity of the DM-15 xylanase. So that these chemicals must be eliminated after treatment of the oligosaccharide, polysaccharide or glycoprotein substrate when these needs to be denatured before its hydrolysis by the enzyme (Faulet et al., 2006). Similar results have been also reported (Bastawde, 1992; Tatjana and Vladimir, 1996; Chivero et al., 2001; Khandeparkar and Bhosle, 2006b). It is reported that the inhibition of xylanase enzyme by Hg⁺² ions may be due to its interaction with sulphydryl groups of cysteine residue in or close to the active site of the enzyme (Khandeparkar and Bhosle, 2006b). The strong inhibition of DM-15 xylanase activity in the presence of EDTA suggests that metals are needed for the enzymatic reaction (Khandeparkar and Bhosle, 2006b). This was confirmed by the observed increase in xylanase activity in the presence of CaCl₂, CoCl₂, CuSO₄, MgCl₂ and KCl (Table 1). The stimulation of xylanases by CaCl₂ and CuSO₄ (Bacillus sp. strain AR-009, Gessesse, 1998), MgCl₂ (Khasin et al., 1993; Gessesse, 1998) and K⁺ (Khasin et al., 1993; Gessesse, 1998; Faulet et al., 2006) have previously been reported and is in accordance with the present study. On the other hand, the inhibition of xylanases by Ca²⁺ (Faulet et al., 2006), Cu²⁺ (Khasin et al., 1993; Faulet et al., 2006) and MgCl₂ (Gessesse, 1998) have also been reported.

However, to reveal the mode of action of xylanase, further research using more inhibitors and combinations would be necessary. The DM-15 xylanase is sensitive to SDS and triton X-100. It was reported that denaturant detergent agents must be eliminated after treatment of the oligosaccharide, polysaccharide or glycoprotein substrate when this needs to be denatured before its hydrolysis by the enzyme (Faulet et al., 2006).

CONCLUSION

The Bacillus sp. DM-15 strain produced high levels of thermostable xylanase. The DM-15 xylanase is a slightly acidophilic with wide range of pH (4.5-10), thermostable and resistant to most of chelators and metal ions. Hence, it is qualified for use in biotechnological applications and all its properties make it a useful tool for biobleaching in pulp and paper industry. The DM-15 xylanase production process can be commercialized after further optimization for enhanced enzyme production.

ACKNOWLEDGEMENTS

This reserch was supported by The Scientific and Technological Research Council of Turkey (TUBITAK) through Short-Term R and D Funding Programme (1002, Project 108T666).