Some properties of thermostable xylanase from an Aspergillus niger
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Ann. Microbiol., 52, 299-306 (2002) Some properties of thermostable xylanase from an Aspergillus niger strain G. CORAL1*, B. ARIKAN2, M.N. ÜNALDI3, H. KORKMAZ GÜVENMEZ2 1Mersin University, Faculty of Arts and Sciences, Department of Biology, 33342 Çiftliköy-Mersin; 2Çukurova University, Faculty of Arts and Sciences, Department of Biology, 01330 Balcali-Adana; 3Mustafa Kemal University, Faculty of Arts and Sciences, Department of Biology, 31040 Hatay, Turkey Abstract - A thermostable xylanase was isolated from an Aspergillus niger wild type strain in a liquid Czapek Dox medium, containing oat spelts xylan as the sole carbon source. The molecular mass of the enzyme was estimated to be about 36 kDa by sodium dodecyl sul- fate-polyacrylamide gel electrophoresis. The optimum pH for activity was found to be 7.5. The temperature optimum of the enzyme was found to be 60 °C at pH 7.5. The enzyme remained stable up to 100 °C, yet lost about 50% of its activity after 15 min at this tem- perature. Key words: xylanase, Aspergillus niger. INTRODUCTION Xylan is the major constituent of hemicellulose and is the second most abundant renewable resource with a high potential for degradation into useful end products (Goheen, 1982). Microbial xylanases (1,4-β−D-xylan xylanohydrolase, EC 184.108.40.206) are the preferred catalysts for xylan hydrolysis due to their high speci- ficity, mild reaction conditions, negligible substrate loss, and side product gener- ation. However, the cost of the enzymatic hydrolysis of a biomass is one of the main factors limiting the economic feasibility of this process. Therefore, the pro- duction of xylanases must be improved by finding more potent fungal or bacteri- al strains or by inducing mutant strains that can excrete greater amounts of enzymes, or both (Dekker and Richards, 1976). Filamentous fungi are particular- ly interesting producers of xylanases since they excrete the enzymes into the medium and their enzyme levels are much higher than those of yeast and bacteria (Steiner et al., 1987). * Corresponding Author. E-mail: email@example.com 299 In recent years, important applications for xylanases in different industrial processes have been found. One major area of application is the bleaching of craft pulp in the paper industry (Zamost et al., 1991; Viikari et al., 1994). Most xylanas- es known to date are optimally active at or below 50 °C at an acidic or neutral pH. However, in the process of enzyme-assisted pulp bleaching, the incoming pulp has a higher temperature and alkaline pH (Zamost et al., 1991) making the use of ther- mostable alkaline xylanases very attractive (Gessesse, 1998). Accordingly, the current paper, reports on certain properties of a thermostable xylanase from the Aspergillus niger Z1 wild-type strain. MATERIALS AND METHODS Fungal strain and culture conditions. The Aspergillus niger Z1 wild-type strain was used as the enzyme source. This fungus was isolated from soil samples accord- ing to the method of Raper and Fennel (1977). The cultivation was carried out in a Czapek Dox liquid medium containing oat spelts xylan (Sigma) (1%) as the sole carbon source. A Erlenmayer flask (1 liter) containing 250 ml of the growth medi- um was cultured for 5 days at 28 °C on an orbital shaker set at 250 rev min-1. The mycelia were removed by filtration and the filtrate used for partial purification. Partial purification of xylanase. Ethanol previously chilled to –20 °C was added dropwise to the culture filtrate (250 ml) at 4 °C with continuous stirring to a final concentration of 75%, then the solution was left at –20 °C for 24 h. The resulting precipitate was collected by decantation and centrifugation. The precipitate was then dissolved in 30 ml of a phosphate buffer (50 mM, pH 5.0) and the concen- trated solution dialyzed against the same buffer (pH 5.0) overnight at 4 °C (Bhel- la and Altosaar, 1984). Enzyme assay. The xylanase was assayed based on the detection of reducing sug- ars from oat spelts xylan. A solution of 1 % (wt/vol) oat spelts xylan in a phos- phate buffer (50 mM, pH 5) was used. An aliquot (0.5 ml) of the partially purified enzyme preparation was incubated with 0.5 ml of the xylan suspension for 30 min at 37 °C. The reducing sugars were detected using the dinitrosalicylic acid (DNS) method of Miller (1959). Temperature, pH optima and heat stability. In all the determinations, the xylanase activity was measured using oat spelts xylan as the substrate. To esti- mate the temperature, pH optima and heat stability, the activity was determined by carrying out the above standard assay at several temperatures or pH values. After the incubating at different temperatures or pH’s, the xylanase activity was measured under standard conditions according to a previous paper (Coral and Çolak, 2000). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To determinate the homogenity and molecular weight, the enzyme preparations and known molecular weight markers were subjected to electrophoresis according to 300 G. CORAL et al. the method of Bollag and Edelstein (1991) using 10% acrylamide gel. Oat spelts xylan (0.2%) was incorporated into the separating gel prior to the addition of ammonium persulfate and polimerization. After the electrophoresis, the gel was stained for 1 h with Coomassie Blue R dye in a methanol-acetic acid-water solu- tion (4:1:5, by volume) and then destained in the same solution without the dye. Zymogram analysis. For the activity staining of the xylanase activity, The SDS was removed by washing the gel at room temperature in solutions containing 50 mM Na2HPO4, 50 mM NaH2PO4 (pH 7.2), isopropanol 40% for 1 h and 50 mM Na2HPO4, 50 mM NaH2PO4 (pH 7.2) for 1 h, respectively. The renaturation of the enzyme proteins was carried out by placing the gel overnight in a solution containing 50 mM Na2HPO4, 50 mM NaH2PO4 (pH 7.2), 5 mM β-mercap- toethanol, and 1 mM EDTA at 4 °C. The gel was then transferred onto a glass plate, sealed in film, and incubated at 37 °C for 4-5 h. The gel was stained in a solution of 1% Congo Red for 30 min, and destained in 1 M NaCl for 15 min. Any clear bands indicated the presence of xylanase activity (Saul et al., 1990; Ikeda et al., 1992; Lee et al., 1994). RESULTS AND DISCUSSION Molecular weight The molecular weight was determined by SDS-PAGE, as described in Materials and Methods above. The analyses of the enzyme by SDS-PAGE only revealed a single band indicating xylanolytic activity in the gel. The molecular weight of this band was estimated to be around 36 kDa (Fig. 1). Optimum temperature To estimate the optimum temperature of the enzyme, the activity was determined at several temperatures between 40 °C to 100 °C. The optimum temperature was observed to be around at 60 °C (Fig. 2). pH optimum The pH optimum was determined in two buffer systems. It was observed that the enzyme did show activity in an alkali pH. The optimum pH of the enzyme was found to be 7.5 (Fig. 3). Heat stability The thermostability of the enzyme was studied by heating the enzyme at different temperatures (60-100 °C) for 15 min. The enzyme remained stable up to 100 °C, with 49.2% of the original activity retained after heat treatment at 100 °C for 15 min (Fig. 4). Commercial applications of xylanases demand the identification of highly stable enzymes that remain active under routine handling conditions. Many advantages, such as reduced contamination risk and faster reaction rates, have been proposed for the use of thermophiles in biotechnology processes. In gener- al, parameters such as temperature, pH, and enzymatic stability are important for the industrial applicability of any enzyme (Kulkarni et al., 1999). Ann. Microbiol., 52, 299-306 (2002) 301 1 2 3 1 2 3 116 kD 66 kD 45 kD 29 kD A) B) FIG. 1 – Separation (A) and activity pattern (B) of Aspergillus niger Z1 xylanase in SDS-Polyacrylamide gel. The electrophoresis was carried out in an SDS-Poly- acrylamide gel containing 0.2 % oat spelts xylan (SIGMA). The gel was stained as described under Materials and Methods. Lane 1 and 2 contains the partially purified xylanase from Aspergillus niger Z1, lane 3 contains the molecular weight markers: 116000, 97400, 66000, 45000, and 29000 Dalton respectively. Relative activity (%) Temperature (°C) FIG. 2 – Optimal temperature range of xylanase from Aspergillus niger Z1. 302 G. CORAL et al. Relative activity (%) pH FIG. 3 – Optimal pH range of xylanase from Aspergillus niger Z1. Two buffer systems were used to determine the optimal pH range of the enzyme: Citrate-Phosphate Buffer (0.1 M Citric acid, 0.2 M Na2HPO4, pH 3.0 to 7.0) and Tris Buffer (0.08 M Tris, 0.1 M HCl, pH 7.5 to 9.0). Relative activity (%) Temperature (°C) FIG. 4 – Heat stability of xylanase from Aspergillus niger Z1. From the electrophoresis of the partially purified enzyme solution, two pro- tein bands were observed after the gel was stained with Coomassie Brillant Blue, whereas, only a single band showing xylanolytic activity was detected after activ- ity staining with Kongo Red (Fig. 1). The molecular weight of the single protein Ann. Microbiol., 52, 299-306 (2002) 303 band was calculated to be about 36 kDa. Microbial xylanases are single subunit proteins with molecular masses whitin a the range of 8 - 145 kDa (Kulkarni et al., 1999). In a previous study, a purified xylanase enzyme preparation also showed a single protein band on SDS-PAGE, and the molecular weight of this enzyme was found to be 24 kDa (Sardar et al., 2000). According to the existing data, the molecular weights of the endo-1,4-β-xylanase (EC 220.127.116.11) and exo-1,4-β-xylosi- dase (EC 18.104.22.168) of Aspergillus niger were found to be 14 and 122 kD respec- tively (Frederick et al., 1981; Uhlig, 1998). The optimum temperature of the current enzyme was found to be 60 °C at pH 7.5. (Fig. 2). This value agrees well with previous the literature. The optimum temperature for xylanases from bacterial and fungal sources has been found to vary between 40 and 60 °C (Kulkarni et al., 1999). The heat stability of the cur- rent enzyme matched that of a mesophilic organism. Fungal xylanases are gener- ally less thermostable than bacterial xylanases. Yet, fungi that are mesophilic in origin and produce thermostable xylanases include Ceratocystis paradoxa, a xylanase that is stable at 80 °C for 1 h (Dekker and Richards, 1975) and Tricho- derma harzianum, a xylanase that can retain 52% of its optimum activity for 4 h at 100 °C (Fadel, 2001). The thermostable xylanase previously identified from Aspergillus is the only one reported from a thermotolerant Aspergillus strain at 37 °C showing a maximum activity at 80 °C (Mendicuti et al., 1997). It was found that the xylanase of Aspergillus niger Z1 exhibited the activity within a neutral to alkaline pH range. The optimum pH of the enzyme was found to be 7.5 at 37 °C. (Fig. 3) Other xylanases from different organisms show an optimum pH within a range of 4.0-7.0. However, certain xylanases from Aspergillus kawachii and Penicillium herque exhibit an optimum pH more on the acidic side (pH 2.0 - 6.0) (Funaguma et al., 1991; Ito et al., 1992). Endoxylanase I and II from Aspergillus awamori show an optimum pH at 5.5 – 6.0 and 5.0, respectively (Kormelink et al., 1992). The optimal pH value for the activity of a commercial xylanase from an Aspergillus niger strain was found to be 4.0 (Fred- erick et al., 1981). From the enzyme data on Aspergillus niger, the optimum pH’s for the xylanases are within a range of 4.0 - 5.0 (Uhlig, 1998). Ideally, for indus- trial application, xylanases should have a neutral to alkaline pH optimum and good thermal stability. Alkaline xylanases have gained importance due to their application in the development of eco-friendly technologies used in the paper and pulp industries as these enzymes are able to hydrolyse xylan, which is solu- ble in alkaline solutions (Horikoshi, 1996). The xylanases from Cephalosporium are the only ones reported from an alkaliphilic fungus with activity within a board pH range of 6.5 - 9.0 (Bansod et al., 1993). The current results showed that, the Z1 xylanase had a low activity in an acidic pH range yet, a good activity within a board pH range from 6.5 - 9.0. The xylanase enzyme in the present stduy has properties quite different from those produced by the other Aspergillus sp. So, the significance of this work is that, certain properties of the present xylanase are appropriate in different industrial applications, especially in the process of enzyme-assisted pulp bleaching. 304 G. CORAL et al. REFERENCES Bansod S.M., Dutta -Choudhary M., Srinivasan M.C., Rele M.V. (1993). Xylanase active at pH from an alkatolerant Cephalosporium sp. Biotechnol. Lett., 15: 965-970. Bhella R.S., Altosaar I. (1984). Purification and some properties of extracellular α-amy- lase from Aspergillus awamori. Can. J. Microbiol., 31:149-154. Bollag D.M., Edelstein S.J. (1991). Protein methods. Wiley-Liss Publication. Coral G., Çolak Ö. (2000). The isolation and characterization of glucoamylase enzyme of an Aspergillus niger natural isolate. Turk. J. Biol., 24: 601-609. Dekker R.F.H., Richards G.N. (1975). Purification, properties and mode of action of hemicellulase-produced by Ceratocystis paradoxa. Carbohydr. Res., 39: 97-114. Dekker R.F.H., Richards G.N. (1976). Hemicellulases: their occurrence, purification, properties and mode of action. Adv. Carbohydr. Chem. Biochem., 32: 277-352. Fadel M. (2001). High level xylanase production from sorghum flour by a newly isolate of Trichoderma harzianum cultivated under solid state fermentation. Ann. Microbiol., 51: 61-78. Frederick M.M., Frederick J.R., Fratzke A.R., Reilly P.J. (1981). Purification and char- acterization of a xylobiose and xylose producing endo-xylanase from Aspergillus niger. Carbohydr. Res., 97(1): 87-103. Funaguma T., Naito S., Morita M., Okumara M., Sugiura M., Hara A. (1991). Purifica- tion and some properties of xylanase from Penicillium herquei Banier and Sartory. Agric. Biol. Chem., 55: 1163-1165. Gessesse A. (1998). Purification and properties of two thermostable alkaline xylanases from an alkaliphilic Bacillus sp. Appl. Environ. Microbiol., 64(9):3533-3535. Goheen D.W. (1982). Chemicals from wood and other biomass. Part I: Future supply of organic chemicals. J. Chem. Educ., 58: 465-468. Horikoshi K. (1996). Alkaliphiles: from an industrial point of view. FEMS Microbiol. Rev., 18: 259-270. Ikeda T., Yamazaki H., Yamashita K., Shinke R. (1992). The tetracycline inducible expression of α-amylase in Bacillus subtilis. J. Ferment. Bioeng., 74: 58-60. Ito K., Ogassawara J., Sugimoto T., Ishikawa T. (1992). Purification and properties of acid stable xylanases from Aspergillus kawachii. Biosci. Biotechnol. Biochem., 56: 547-550. Kormelink F.J.M., Gruppen H., Wood T.M., Beldman G. (1992). Mode of action of xylan degrading enzymes from Aspergillus awamori. In: Visser J., Beldman G., Kusters- van-Someren M.A., Varagen A.G.J., eds, Xylans and Xylanases. Elsevier, Amster- dam, London, New York,Tokyo, pp. 141-147. Kulkarni N., Shendye A., Rao M. (1999). Molecular and biotechnological aspects of xylanases. FEMS. Microbiol. Rev., 23(4): 411-456. Lee S., Morikawa M., Takagi M., Imankawa T. (1994). Cloning aapT gene and charac- terization of its product, α-amylase, pullulanase (aapT) from thermophilic and alka- liphilic Bacillus sp. strain XAL601. Appl. Environ. Microbiol., 60: 3761-3773. Mendicuti C., Laura P., Trejo-Aguilar B.A., Aguilar O.G. (1997). Thermostable xylanas- es produced at 37 °C and 45 °C by a thermotolerant Aspergillus strain. FEMS Microbiol. Lett., 146: 97-102. Miller G.L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem., 31: 426-428. Raper K.B., Fennel D.I. (1977). The Genus Aspergillus. Krieger R.E. Publishing Com., Huntington, New York. Ann. Microbiol., 52, 299-306 (2002) 305 Sardar M., Roy I., Gupta M.N. (2000). Simultaneous purification and immobilization of Aspergillus niger xylanase on the reversibly soluble polymer EudragitTM L-100. Enzyme Microbial. Tech., 27: 672-679. Saul D.J., Williams L.C., Grayling R.A., Chamley L.W., Love D.R., Berquist P.L. (1990). celB, a gene coding for a bifunctional cellulase from the extreme thermophile “Cal- docellum saccharolyticum”. Appl. Environ. Microbiol., 56(10): 3117-3124. Steiner W., Lafferty R.M., Gomes I., Esterbauer H. (1987). Studies on a wild type strain of Schizophyllum commune: Cellulase and xylanase production and formation of the extracellular polysaccharide schizophyllan. Biotechnol. Bioeng., 30: 169-170. Uhlig H. (1998). Industrial Enzymes and Their Applications. John Willey and Sons INC., pp.142-143. Viikari L., Kantelinen A., Sundquist J., Linko M. (1994). Xylanases in bleaching: from an idea to industry. FEMS Microbiol. Rev., 13: 335-350. Zamost B.L., Nielsen H.K., Starnes R.L. (1991). Thermostable enzymes for industrial applications. J. Ind. Microbiol., 8: 71-82. 306 G. CORAL et al.