APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1998, p. 35333535 0099-2240/98/$04.00 0 Copyright 1998, American Society for Microbiology.

. All Rights Reserved.

Vol. 64, No. 9

Purication and Properties of Two Thermostable Alkaline Xylanases from an Alkaliphilic Bacillus sp.
AMARE GESSESSE* Department of Biology, Addis Ababa University, Addis Ababa, Ethiopia
Received 8 January 1998/Accepted 14 July 1998

Two xylanases, designated XylA and XylB, were puried from the culture supernatant of the alkaliphilic Bacillus sp. strain AR-009. The molecular masses of the two enzymes were estimated to be 23 kDa (XylA) and 48 kDa (XylB) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pHs for activity were 9 for XylA and 9 to 10 for XylB. The temperature optima for the activity of XylA were 60C at pH 9 and 70C at pH 8. XylB was optimally active at 75C at pH 9 and 70C at pH 8. Both enzymes were stable in a broad pH range and showed good stability when incubated at 60 and 65C in pH 8 and 9 buffers. A wide variety of microorganisms are known to produce xylan-degrading enzymes. In recent years, important applications for xylanases in different industrial processes have been found. One major area of application is for the bleaching of kraft pulp in the pulp and paper industries (13, 16). Most xylanases known to date are optimally active at or below 50C and at acidic or neutral pH. On the other hand, in the process of enzyme-assisted pulp bleaching, the incoming pulp has a higher temperature and an alkaline pH (16), making the use of thermostable alkaline xylanases very attractive. To date, few xylanases are reported to be active and stable at alkaline pH and elevated temperature (8). In this paper, the properties of two thermostable alkaline xylanases from an alkaliphilic Bacillus sp. are reported. Bacillus sp. strain AR-009, an alkaliphile isolated from an alkaline soda lake (3), was grown at 35C with rotary shaking in 500-ml bafed asks containing 100 ml of medium. The composition of the medium was as follows: xylan, 5 g/liter; peptone, 5 g/liter; yeast extract, 1 g/liter; NaCl, 5 g/liter; K2HPO4, 1 g/ liter; MgSO4, 0.2 g/liter; CaCl2, 0.1 g/liter; and Na2CO3, 10 g/liter. Sodium carbonate was sterilized separately and added to the rest of the medium after cooling. The cell-free culture supernatant from a 48-h culture was precipitated by using solid ammonium sulfate to 70% saturation. The pellet obtained after centrifugation was dissolved in 10 mM Tris-HCl buffer (pH 8) and dialyzed against three changes of the same buffer. The dialyzed enzyme preparation was applied to a DEAESepharose column (2.5 by 12 cm) equilibrated with 10 mM Tris-HCl buffer (pH 8). The column was eluted rst with buffer alone at a ow rate of 90 ml/h, followed by a linear gradient of 0 to 0.5 M NaCl. Fractions containing xylanase activity were pooled, concentrated, and dialyzed against 10 mM Tris-HCl buffer (pH 8). The concentrated enzyme preparation was applied to a Sephadex G-75 column (1.5 110 cm) equilibrated with 10 mM Tris-HCl buffer (pH 8) and eluted at a ow rate of 12 ml/h. Xylanase-containing fractions were pooled, concentrated, and reapplied to the Sephadex G-75 column and eluted as described above. An assay for xylanase activity was performed by the dinitrosalicylic acid method as described previously (3) at 50C with 1% xylan in 50 mM glycine NaOH buffer (pH 9). One unit of xylanase activity was dened as the amount of enzyme that released 1 mol of reducing sugar equivalent to xylose per min. The protein concentration was measured with the bicinchoninic acid reagent (Sigma, St. Louis, Mo.) according to the procedure of the manufacturer. Two xylanases, designated XylA and XylB, were puried from the culture supernatant of Bacillus sp. strain AR-009. The molecular masses of XylA and XylB were estimated to be 23 and 48 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 1). Of the two enzymes, only XylB was adsorbed to the DEAE-Sepharose column. The purication procedure is summarized in Table 1. The two enzymes were puried from a 12-h culture and a 24-h culture by the same procedure. The same result was also obtained whether a protease inhibitor (2 mM phenylmethylsulfonyl uoride) was included or not, suggesting that XylA and XylB are not proteolytic degradation products. Multiple-xylanase production has been reported for a wide variety of xylanolytic microorganisms (2, 5, 6, 13, 14). The different xylanase isoenzymes are expected to differ in their specicities (2) and to have a synergistic effect on the process of xylan hydrolysis. He et al. (5) showed synergism in the hydrolysis of oat spelt and birch wood xylan by two xylanase isoenzymes of Streptomyces sp. strain A451. Table 2 shows the activities of the two xylanases assayed in the presence of different metal ions. Both enzymes were inhibited in the presence of Hg2 , Fe2 , Fe3 , and Pb2 . Partial inhibition of activity was observed in the presence of Sn2 for XylA and Mn2 for XylB. Inhibition by Fe2 and Fe3 seems to be unique for the two xylanases of Bacillus sp. strain AR009. No other xylanase was reported previously to be completely inhibited by these ions. The mechanism of inhibition of these two enzymes by Fe2 and Fe3 remains to be determined. The mode of action of the two enzymes was determined by measuring the rate of reducing sugar formation and viscosity reduction of oat spelt xylan by the method of Khasin et al. (7). Both enzymes resulted in a rapid reduction of viscosity and a corresponding rapid rise in reducing sugar level, indicating that they are endoxylanases (Fig. 2). The effect of temperature on activity was determined at different temperatures with pH 8 and 9 buffers. At pH 8, XylA showed optimum activity at 70C, while at pH 9, its optimum was shifted to 60C. The optimum temperatures for the activity of XylB were 70C at pH 8 and 75C at pH 9. The stabilities of both enzymes were tested by heating at 60 and 65C in pH 8 and 9 buffers. After 3 h of incubation at 60C, XylA retained
3533

* Mailing address: Department of Biology, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia. Fax: (2511) 552350 or 552112.

3534

GESSESSE

APPL. ENVIRON. MICROBIOL. TABLE 2. Effect of different metal ions on activity of xylanases XylA and XylB from Bacillus sp. strain AR-009a
Metal ion (1 mM) % Activity XylA XylB

FIG. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation of XylA and XylB with 12% polyacrylamide gel. Lanes: 1, molecular mass markers (kilodaltons); 2, XylA; 3, XylB.

None NaCl KCl CaCl2 MgCl2 MgSO4 CuSO4 CoCl2 MnCl2 ZnSO4 Pb(CH3COO )2 FeCl3 FeSO4 AlCl3 SnCl2 HgCl2

100 100 105 108 99 101 106 90 74 101 8 0 14 87 85 0

100 103 105 105 104 90 95 88 68 105 8 0 9 92 65 0

more than 95% of its original activity at both pHs. At 65C, more than 78% and 55% of its original activity was retained at pHs 8 and 9, respectively. XylB showed better stability at pH 9 than at pH 8. At 60C, it retained 51 and 74% of its original activity after 3 h of incubation at pHs 8 and 9, respectively. At 65C, over 54% and 67% of its original activity was retained after 1 h of incubation at pHs 8 and 9, respectively. The effect of pH on xylanase activity was determined in a range of buffers of various pHs at 50C. XylA was optimally active at pH 9, while XylB was active in a broad pH range, with an optimum at pHs 9 to 10. The effect of pH on stability was tested by incubating the enzyme at 50C for 1 h in different buffers of various pHs, and residual activity was measured by the standard assay procedure. Both enzymes retained full activity in the pH range of 5 to 11. The majority of xylanases reported to date are optimally active in the acidic or neutral pH range. From the application point of view, xylanases active and stable in the alkaline pH range and at elevated temperature are very important. Most alkaliphilic and alkalitolerant microorganisms produce xylaTABLE 1. Purication procedure of xylanases XylA and XylB from the cell-free culture supernatant of Bacillus sp. strain AR-009
Purication step Total act (U) Total protein (mg) Sp act (U/mg) Fold purication % Recovery

a The activity of each enzyme (0.5 U) was assayed in the presence of the different salts at a nal concentration of 1 mM. Values obtained for the control (no additive) were taken as 100%.

nases optimally active around neutrality (1, 6, 1012). Although some strains are known to produce xylanases having good activity at pHs greater than 8, the optimum temperature for activity and stability is at or below 50 to 55C (4, 9, 15). On the other hand, the great majority of thermostable xylanases produced by thermophilic microorganisms have optimum activity at neutral pH or below. The two xylanases from Bacillus sp. strain AR-009, which are active and stable at alkaline pH and elevated temperature, may have interesting potential applications in the process of enzyme-assisted pulp bleaching.

Culture ltrate Ammonium sulfate Ion exchange XylA XylB Gel ltration 1st XylA XylB 2nd XylA XylB

67,243 51,342

1,729.0 322.0

3,829.0 159.4

1.0 4.1

100.0 76.4

46,490 1,109

126.5 14.0

367.5 79.2

9.4 2.0

69.1 1.7

14,032 605 9,174 90

30.6 6.0 17.5 1.1

458.6 100.8 524.2 367.9

11.8 2.6 13.5 9.5

20.9 0.9 13.6 0.6

FIG. 2. Viscosity reduction and reducing sugar formation from oat spelt xylan by XylA (a) and XylB (b). Oat spelt xylan (0.5%) in glycine NaOH buffer (pH 9) was mixed with xylanase and incubated at 50C. Viscosity reduction was measured with an Oswald viscometer, and the amount of reducing sugar was determined by the dinitrosalicylic acid method. F, reducing sugar; , relative viscosity.

VOL. 64, 1998

ALKALINE XYLANASES FROM ALKALIPHILIC BACILLUS SP.

3535

The use of such enzymes may allow manufacturers to cut down the amount of acid required for pH readjustment and the need for cooling and reheating of the large pulp mass, thus saving both time and money. Such enzymes may also nd potential application in the hydrolysis of xylan-containing waste, both as a method of waste management and as a source of fermentable sugars. Several million tons of xylan-containing waste is released annually throughout the world in the form of agricultural, industrial, and municipal waste. Because xylan is soluble at alkaline pH, xylanases active and stable at alkaline pH and high temperature could be very important for such applications. Nakamura et al. (8) reported the production of an alkaline xylanase by Bacillus sp. strain TAR-1, with temperature optima of 70C at pH 9 and 75C at pH 7. The optimum pHs of almost all xylanases known to date drop with increasing temperature. XylB of Bacillus sp. strain AR-009 is probably unique in having an alkaline pH optimum with increasing temperature. Further study of this enzyme might give information about the molecular basis of stability and activity of xylanases at alkaline pH and elevated temperature.
I thank Yemisrach Mulugeta, Aster Mekasha, and Meseret Mengistu for excellent technical assistance and Gashaw Mamo for valuable discussion. This work was supported by the Swedish International Development Cooperation Agency (Sida/SAREC), ESTC, and the International Foundation for Science (IFS).
REFERENCES 1. Blanco, A., T. Vidal, J. F. Colom, and F. I. J. Pastor. 1995. Purication and properties of xylanase A from alkali-tolerant Bacillus sp. strain BP-23. Appl. Environ. Microbiol. 61:44684470. 2. Elegir, G., G. Szakacs, and T. W. Jeffries. 1994. Purication, characteriza tion, and substrate specicities of multiple xylanases from Streptomyces sp. strain B-12-2. Appl. Environ. Microbiol. 60:26092615.

3. Gessesse, A., and B. A. Gashe. 1997. Production of alkaline xylanase by an alkaliphilic Bacillus sp. isolated from an alkaline soda lake. J. Appl. Microbiol. 83:402406. 4. Gessesse, A., and G. Mamo. 1998. Purication and characterisation of an alkaline xylanase from alkaliphilic Micrococcus sp. AR-135. J. Ind. Microbiol. Biotechnol. 20:210214. 5. He, L., G. F. Bickerstaff, A. Paterson, and J. A. Buswell. 1994. Evaluation of catalytic activity and synergism between two xylanase isoenzymes in enzymatic hydrolysis of two separate xylans in different states of solubility. Enzyme Microb. Technol. 16:696702. 6. Kang, M. K., P. J. Maeng, and Y. H. Rhee. 1996. Purication and characterization of two xylanases from alkaliphilic Cephalosporium sp. strain RYM202. Appl. Environ. Microbiol. 62:34803482. 7. Khasin, A., I. Alchanati, and Y. Shoham. 1993. Purication and characterization of a thermostable xylanase from Bacillus stearothermophilus T-6. Appl. Environ. Microbiol. 59:17251730. 8. Nakamura, S., Y. Ishiguro, R. Nakai, K. Wakabayashi, R. Aono, and K. Horikoshi. 1995. Purication and characterization of a thermophilic alkaline xylanase from thermoalkaliphilic Bacillus sp. strain TAR-1. J. Mol. Catal. B Biocatal. 1:715. 9. Nakamura, S., K. Wakabayashi, R. Nakai, R. Aono, and K. Horikoshi. 1993. Purication and some properties of an alkaline xylanase from alkaliphilic Bacillus sp. strain 41M-1. Appl. Environ. Microbiol. 59:23112316. 10. Okazaki, W., T. Akiba, K. Horikoshi, and R. Akahoshi. 1985. Purication and characterisation of xylanases from alkaliphilic thermophilic Bacillus sp. Agric. Biol. Chem. 49:20332039. 11. Park, Y. S., D. Y. Yum, D. H. Bai, and J. H. Yu. 1992. Xylanase from alkaliphilic Bacillus sp. YC-335. Biosci. Biotechnol. Biochem. 56:13551356. 12. Tsujibo, H., T. Sakamoto, N. Nishino, T. Hasegawa, and Y. Inamor. 1990. Purication and properties of three types of xylanases produced by an alkaliphilic actinomycete. J. Appl. Bacteriol. 69:398405. 13. Viikari, L., A. Kantelinen, J. Sundquist, and M. Linko. 1994. Xylanases in bleaching: from an idea to industry. FEMS Microbiol. Rev. 13:335350. 14. Wong, K. K. Y., L. U. L. Tan, and J. N. Saddler. 1988. Multiplicity of -1,4-xylanase in microorganisms: functions and applications. Microbiol. Rev. 52:305317. 15. Yang, V. W., Z. Zhuang, G. Elegir, and T. W. Jeffries. 1995. Alkaline active xylanase produced by an alkaliphilic Bacillus sp. isolated from kraft pulp. J. Ind. Microbiol. 15:434441. 16. Zamost, B. L., H. K. Nielsen, and R. L. Starnes. 1991. Thermostable enzymes for industrial applications. J. Ind. Microbiol. 8:7182.