Journal of Applied Microbiology 1997, 83, 402406

Production of alkaline xylanase by an alkaliphilic Bacillus sp.isolated from an alkaline soda lake

A. Gessesse* and B.A. Gashe1Department of Biology, Addis Ababa University, Addis Ababa, Ethiopia and 1Department of Biological Sciences,University of Botswana, Gaborone, Botswana5956/10/96: received 21 October 1996, revised 29 January 1997 and accepted 30 January 1997

A. GESSESSE AND B.A. GASHE. 1997. An alkaline xylanase-producing alkaliphilic Bacillussp. AR-009 was isolated from an alkaline soda lake in Ethiopia. The enzyme wasoptimally active at pH 9 and was stable over a broad pH range. The optimumtemperature for xylanase activity, assayed at pH 9, was 6065C. Measured atpH 8 and 9, the enzyme had good stability at 55 and 60C. At both pH values, over80% of its original activity was retained after heating for 25 h at 55C. At 60C,the enzyme maintained 63% of its original activity after 25 h incubation while at pH9 it retained 54% of its original activity after 1 h heating. These properties qualify the enzymeto be novel and potentially important for application in some industrial processes.

INTRODUCTION et al. 1995). Many xylanase-producing alkaliphilic microbialstrains have been reported from different laboratories.

Xylan, a b-1,4 linked polymer of D-xylose, is the majorHowever, the xylanases from most of these alkaliphilic strains

constituent of plant hemicelluloses. A wide variety of micro-have their optimum pH around neutrality (Okazaki et al.

organisms are known to produce xylanases, enzymes that are1984; Tsujibo et al. 1990; Dey et al. 1992; Ratto et al. 1992).

involved in the hydrolysis of xylan (Wong et al. 1988 ; Bas-The majority of alkaliphiles known so far were isolated from

tawde 1992). In recent years increasing attention has beenneutral soil samples. On the other hand, naturally occurring

given to the study of xylan-degrading enzymes because ofalkaline habitats are found scattered in different parts of the

their potential application in different industrial processes.world (Grant and Horikoshi 1992). Such habitats are expected

One area of application is in the pulp and paper industryto harbour novel micro-organisms that are adapted to living

where xylanase can be used for the bleaching of kraft pulpsat alkaline pH. Extracellular enzymes produced by such

(Senior et al. 1992; Viikari et al. 1994). The use of xylanaseorganisms are likely to have their optimum pH for activity

prior to the normal bleaching operation has been shownin the alkaline range. Such enzymes may find important

to significantly reduce the amount of chlorinated organicapplication in different industrial processes. Until now there

compounds formed during the bleaching process (Senior ethas been very little effort to isolate alkaliphiles from naturally

al. 1992), thus reducing the risk of environmental pollution.occurring alkaline habitats. In the present study, the pro-

Since the kraft process of pulp and paper making is carriedduction of an alkaline xylanase by an alkaliphilic Bacillus sp.

out at alkaline pH and high temperature, the use of alkalineisolated from an alkaline soda lake and the properties of the

xylanases with higher temperature optima is considered to becrude enzyme are reported.

advantageous (Zamost et al. 1991; Yang et al. 1995). Alkalinexylanases will also find a number of other applications. Forexample, because of the high solubility of xylan at alkaline MATERIALS AND METHODSpH (Grant and Horikoshi 1992) alkaline xylanases may havegood potential for the hydrolysis of hemicellulosic wastes to Isolation and screeningfermentable sugars.

Water samples were collected from lake Arenguadie, an alka-Until now there have been very few reports on the pro-line soda lake found near the town of Debre Zeit, centralduction of alkaline xylanses (Nakamura et al. 1993a, b; YangEthiopia. A loop full of the sample was streaked onto xylan-containing nutrient agar plates. The pH of the medium wasCorrespondence to : Dr A. Gessesse, Department of Biology, Addis Ababa

University, PO Box 1176, Addis Ababa, Ethiopia. adjusted to 103 by adding 1% sodium carbonate. After 48 h

1997 The Society for Applied Bacteriology

PRODUCTION OF ALKALINE XYLANASE 403

incubation at 35C, individual colonies were transferred tofresh xylan-containing nutrient agar plates and incubated asabove. Xylanase-producing strains were selected by floodingthe plates with 01% aqueous Congo red for 15 min followedby repeated washing with 1 mol l1 NaCl. All colonies show-ing a clear zone on agar plates were further screened bygrowing them in liquid medium and assaying enzyme activityfrom the cell-free culture supernatant fluid. One strain, desig-nated as AR-009, was selected for subsequent studies.

Enzyme production

The growth medium used for xylanase production was com-60

5

00

Time (h)

Bio

mas

s (O

.D. 6

00)

50

En

zym

e ac

tivi

ty (

U m

l1 )

35

0

10

4

3

2

1

10 20 30 40

30

25

20

15

5

posed of (g l1): xylan, 5; peptone, 5; yeast extract, 1; Fig. 1 Time course of growth and enzyme production by BacillusK2HPO4, 1; MgSO4 . 7H2O, 02; CaCl2, 01; and Na2CO3, 10. sp. AR-009. Cells were grown in a medium containing birch woodSodium carbonate was autoclaved separately and added to xylan, pH 103. Growth was monitored by measuring opticalthe rest of the medium after cooling. The above medium (100 density at 600 nm (O.D. 600). Xylanase activity was assayed

from the cell-free culture supernatant fluid at 50C, pH 9, ,ml in 500 ml baffled flasks) was inoculated with 2 ml of anO.D. 600; , enzyme activity (U ml1)overnight culture and incubated at 35C in a rotary incubator

shaker. The culture was harvested at the stationary growthphase and centrifuged at 5000 g. The cell-free supernatant produced a high level of xylanase activity both in solid andfluid was used as the enzyme source. liquid media. The organism was rod-shaped, Gram-positive,

aerobic, motile, an endospore-former, with terminally locatedspores and catalase-positive. On the basis of these propertiesPartial purification of the enzymeit was classified as a strain of the genus Bacillus (Claus and

The cell-free supernatant fluid was precipitated with the Berkeley 1986).addition of solid ammonium sulphate to 70% saturation.After centrifugation the pellet was suspended in a minimum Enzyme productionvolume of 50 mmol l1 glycine-NaOH buffer, pH 9, and

Xylanase production by AR-009 was growth-associated,dialysed against three changes of the same buffer. The dia-reaching a maximum after 20 h (Fig. 1). Enzyme productionlysed crude enzyme preparation was used for all subsequentremained more or less the same up to 56 h while biomassstudies.started to gradually decline after 40 h. A high level of enzymeproduction was observed when the organism was grown in

Enzyme assay media containing oat spelt xylan, wheat bran and birchwoodxylan as carbon sources (Table 1). A significant amount ofXylanase activity was assayed by measuring the release of

reducing sugar from birch wood xylan following the dinitro-salicylic acid (DNS) method (Miller 1959). To 09 ml of Table 1 Effect of different carbon sources on xylanasesubstrate in 50 mmol l1 glycine-NaOH buffer, pH 9, 100 ml production by Bacillus sp. AR-009of appropriately diluted enzyme was added and incubated at 50C. After 10 min, 15 ml of DNS solution was added to Sugar Xylanase activity (U ml1)

the reaction mixture and boiled for 5 min. Absorbance wasOat spelt xylan 452measured at 540 nm against a reagent blank. One unit ofBirchwood xylan 350xylanase activity was defined as the amount of enzyme thatWheat bran 446released 1 mmol reducing sugar equivalent to xylose per minStarch 210under the above assay conditions.Sucrose 146Arabinose 141Glucose 106RESULTSXylose 60The organismCells were grown for 40 h in the presence of 05% carbon source.

AR-009 was isolated from an alkaline soda lake water sample Xylanase activity was measured from the cell-free supernatant fluidat pH 9 and 50C.collected from lake Arenguadie. It grows at pH 10105 and

1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 83, 402406

404 A. GESSESSE AND B.A. GASHE

xylanase was also produced when starch, sucrose, arabinose,glucose and xylose were used as carbon sources (Table 1).However, compared with xylan and wheat bran, these sugarsinduced lower xylanase activity.

Properties of the crude enzyme

The effect of pH on xylanase activity was measured at 50Cby varying the pH of the assay medium. Over 90% of themaximum activity was in the pH range of 7595 with anoptimum at pH 9 (Fig. 2). At pH 10, 67% of the maximumactivity was retained. To study the effect of pH on stability,the crude enzyme was diluted in different buffers of varyingpH values and incubated at 50C for 1 h. Residual activity

11

100

05

pH

Res

idu

al a

ctiv

ity

(%)

80

60

40

20

6 9 1087

was measured following the standard assay method. The Fig. 3 Effect of pH on xylanase stability. The enzyme wasdiluted with different buffers of varying pH values and incubatedenzyme was very stable in the pH range of 610 (Fig. 3).at 50C for 1 h. Residual activity was assayed at pH 9 and 50C.The temperature profile of AR-009 xylanase was studiedBuffers used were: , citrate; , phosphate and t, glycine-NaOHby assaying enzyme activity at different temperatures using

pH 9 buffer. The optimum temperature was 6065C (Fig.4). The effect of temperature on enzyme stability was deter-mined at 55 and 60C by incubating the enzyme in pH 8and 9 buffer. At 55C, the enzyme was very stable at bothpH values maintaining over 80% of its original activity after25 h heating (Fig. 5). At 60C, 54% of its original activitywas retained after 1 h incubation at pH 9 while 63% of theoriginal activity was maintained after 25 h heating at pH 8(Fig. 5).

DISCUSSION

Bacillus sp. AR-009 produced a high level of xylanase in thepresence of xylan and wheat bran as carbon sources. An

70

100

030

Temperature (C)

Rel

ativ

e ac

tivi

ty (

%) 80

60

40

20

5040 60

Fig. 4 Effect of temperature on xylanase activity. The reactionwas carried out at pH 9 for 10 min

appreciable level of xylanase activity was also produced in thepresence of other carbon sources including xylose. This mayindicate that isolate AR-009 produced multiple forms of xyl-anase whereby one form is produced constitutively in smallamounts and the other is induced by the substrate xylan.Many xylanase-producing microbial strains have been shownto produce multiple forms of xylanase (Honda et al. 1985;Wong et al. 1988; Elegir et al. 1994; Flint et al. 1994).

Xylanases that are thermostable and have their optimum12

100

06

pH

Rel

ativ

e ac

tivi

ty (

%) 80

60

40

20

108

pH in the alkaline range are considered to have good potentialFig. 2 pH profile of Bacillus sp. AR-009 xylanase. Enzymefor application in the pulp and paper industry. This is becauseactivity was measured at 50C and different pH values. Thethe use of such enzymes is expected to greatly reduce thebuffers used, each at a concentration of 50 mmol l1, were : ,

citrate; , phosphate; t, Tris-HCl and T, glycine-NaOH need for costly pH and temperature readjustments before

1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 83, 402406

PRODUCTION OF ALKALINE XYLANASE 405

REFERENCES

Bastawde, K.B. (1992) Xylan structure, microbial xylanases, andtheir mode of action. World Journal of Microbiology and Biotech-nology 8, 353368.

Claus, D. and Berkeley, R.C.W. (1986) Genus Bacillus. In BergeysManual of Systematic Bacteriology Vol. 2. ed. Sneath, P.H.A. pp.11051138. London : Williams and Wilkins.

Dey, D., Hinge, J., Shendye, A. and Rao, M. (1992) Purificationand properties of extracellular endoxylanases from alkalophilicthermophilic Bacillus sp. Canadian Journal of Microbiology 38,436442.

Elegir, G., Szakacs, G. and Jefferies, T.W. (1994) Purification,characterization, and substrate specificities of multiple xylanasesfrom Streptomyces sp. strain B-12-2. Applied and Environmental

150

100

00

Time (min)

Res

idu

al a

ctiv

ity

(%)

80

60

40

20

50 100

Microbiology 60, 26092615.Fig. 5 Thermal stability of Bacillus sp. AR-009 xylanase. The Flint, H.J., Zhang, J.-X. and Martin, J. (1994) Multiplicity andcrude enzyme preparation was diluted in 50 mmol l1 Tris- expression of xylanases in the rumen cellulolytic bacterium Rum-HCl buffer, pH 8, (, t), or 50 mmol l1 glycine-NaOH inococcus flavefaciens. Current Microbiology 29, 139143.buffer, pH 9 (, T), and incubated at 55C (, ) and Grant, W.D. and Horikoshi, K. (1992) Alkaliphiles : ecology and60C (t, T). Samples were withdrawn at time intervals and biotechnological applications. In Molecular Biology and Biotech-residual activity measured at 50C, pH 9 nology of Extremophiles ed. Herbert, R.A. and Sharp, R.J. pp.

143162. New York : Chapman and Hall.Honda, H., Kudo, T., Ikura, Y. and Horikoshi, K. (1985) Two

types of xylanases of alkalophilic Bacillus sp. No. C-125. CanadianJournal of Microbiology 31, 538542.

Miller, G.L. (1959) Use of dinitrosalicylic acid reagent for deter-enzyme addition. Most xylanases known so far have their mination of reducing sugar. Analytical Chemistry 31, 426428.

Nakamura, S., Wakabayashi, K., Nakai, R., Aono, R. and Horikoshi,optimum pH around neutrality. Even xylanases produced byK. (1993a) Purification and some properties of an alkaline xylanasemost alkaliphiles reported to date have their optimum pHfrom alkaliphilic Bacillus sp Strain 41M-1. Applied and Environ-around neutrality (Okazaki et al. 1984; Rajarama and Varmamental Microbiology 59, 23112316.1990; Tsujibo et al. 1990; Dey et al. 1992; Park et al. 1992).

Nakamura, S., Wakabayashi, K., Nakai, R. and Horikoshi, K.Nakamura et al. (1993a, b) reported the first alkaline xylanase(1993b) Production of alkaline xylanase by a newly isolated alkali-

produced by Bacillus sp. strain 41M-1 which had an optimum philic Bacillus sp. strain 41M-1. World Journal of Microbiologytemperature and pH of 50C and 9, respectively. The thermal and Biotechnology 3, 221224.stability of this enzyme was low, losing about 90% of its Okazaki, W., Akiba, T., Horikoshi, K. and Akahoshi, R. (1984)activity after 30 min heating at 60C, pH 9. Yang et al. (1995) Production and properties of two types of xylanases from alkalo-

philic thermophilic Bacillus sp. Applied Microbiology and Biotech-isolated an alkaliphilic Bacillus sp. V1-4 from a hard woodnology 19, 335340.kraft pulp which produced a xylanase having a pH optimum

Park, Y.S., Yum, D.Y., Bai, D.H. and Yu, J.H. (1992) Xylanaseof 685 and a temperature optimum of 55C. At 60C andfrom alkaliphilic Bacillus sp. YC-335. Bioscience BiotechnologypH 9, this enzyme was reported to retain 1520% of itsBiochemistry 56, 13551356.original activity after 30 min incubation. The xylanase from

Rajarama, S. and Varma, A. (1990) Production and characterizationBacillus sp. AR-009 was a novel enzyme, being active at of xylanase from Bacillus thermoalkalophilus grown on agriculturalalkaline pH with an optimum at pH 9 and was stable over a wastes. Applied Microbiology and Biotechnology 34, 141144.broad pH range. It showed optimum activity at 6065C Ratto, M., Poutman, K. and Viikari, L. (1992) Production of xyl-and good stability at 55 and 60C at alkaline pH values. anolytic enzymes by an alkalitolerant Bacillus cirulans strain.

Applied Microbiology and Biotechnology 37, 470473.These are desirable properties for application in the pulp andSenior, D.J., Hamilton, J., Bernier, R.L. and Manoir, J. (1992)paper industry as well as in other industrial processes.

Reduction in chlorine use during bleaching of kraft pulp followingxylanase treatment. Tappi Journal 75, 125130.

Tsujibo, H., Sakamoto, T., Nishino, N., Hasegawa, T. and Inamori,Y. (1990) Purification and properties of three types of xylanasesACKNOWLEDGEMENTSproduced by an alkaliphilic actinomycete. Journal of Applied Bac-

This work was supported by a grant from Sida-SAREC teriology 69, 398405.(Sweden) administered through the Ethiopian Science and Viikari, L., Kantelinen, A., Sundquist, J. and Linko, M. (1994)Technology Commission. The authors thank Ato Gashaw Xylanases in bleaching : from an idea to the industry. FEMS

Microbiology Review 13, 335350.Mamo for valuable discussions.

1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 83, 402406

406 A. GESSESSE AND B.A. GASHE

Wong, K.K.Y., Tan, L.U.L. and Saddler, J.N. (1988) Multiplicity sp isolated from kraft pulp. Journal of Industrial Microbiology 15,434441.of b-1,4-xylanase in microorganisms : functions and applications.

Microbiological Reviews 52, 305317. Zamost, B.L., Nielsen, H.K. and Starnes, R.L. (1991) Thermostableenzymes for industrial applications. Journal of Industrial Micro-Yang, V.W., Zhuang, Z., Elegir, G. and Jeffries, T.W. (1995)

Alkaline-active xylanase produced by an alkaliphilic Bacillus biology 8, 7182.

1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 83, 402406