Purification and Properties of Two Thermostable Alkaline Xylanases from an Alkaliphilic Bacillus sp.

Abstract

Two xylanases, designated XylA and XylB, were purified 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 60°C at pH 9 and 70°C at pH 8. XylB was optimally active at 75°C at pH 9 and 70°C at pH 8. Both enzymes were stable in a broad pH range and showed good stability when incubated at 60 and 65°C 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 50°C 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 35°C with rotary shaking in 500-ml baffled flasks 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 DEAE-Sepharose column (2.5 by 12 cm) equilibrated with 10 mM Tris-HCl buffer (pH 8). The column was eluted first with buffer alone at a flow 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 flow 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 50°C with 1% xylan in 50 mM glycine NaOH buffer (pH 9). One unit of xylanase activity was defined as the amount of enzyme that released 1 μmol 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 purified 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 purification procedure is summarized in Table 1. The two enzymes were purified 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 fluoride) 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 specificities (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.

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.

TABLE 1.

Purification procedure of xylanases XylA and XylB from the cell-free culture supernatant of Bacillus sp. strain AR-009

Purification step	Total act (U)	Total protein (mg)	Sp act (U/mg)	Fold purifi- cation	% Recovery
Culture filtrate	67,243	1,729.0	3,829.0	1.0	100.0
Ammonium sulfate	51,342	322.0	159.4	4.1	76.4
Ion exchange
XylA	46,490	126.5	367.5	9.4	69.1
XylB	1,109	14.0	79.2	2.0	1.7
Gel filtration
1st
XylA	14,032	30.6	458.6	11.8	20.9
XylB	605	6.0	100.8	2.6	0.9
2nd
XylA	9,174	17.5	524.2	13.5	13.6
XylB	90	1.1	367.9	9.5	0.6

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 AR-009. 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.

TABLE 2.

Effect of different metal ions on activity of xylanases XylA and XylB from Bacillus sp. strain AR-009^a

Metal ion (1 mM)	% Activity
	XylA	XylB
None	100	100
NaCl	100	103
KCl	105	105
CaCl2	108	105
MgCl2	99	104
MgSO4	101	90
CuSO4	106	95
CoCl2	90	88
MnCl2	74	68
ZnSO4	101	105
Pb(CH3COO−)2	8	8
FeCl3	0	0
FeSO4	14	9
AlCl3	87	92
SnCl2	85	65
HgCl2	0	0

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

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).

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 50°C. Viscosity reduction was measured with an Oswald viscometer, and the amount of reducing sugar was determined by the dinitrosalicylic acid method. •, reducing sugar; ▴, relative viscosity.

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 70°C, while at pH 9, its optimum was shifted to 60°C. The optimum temperatures for the activity of XylB were 70°C at pH 8 and 75°C at pH 9. The stabilities of both enzymes were tested by heating at 60 and 65°C in pH 8 and 9 buffers. After 3 h of incubation at 60°C, XylA retained more than 95% of its original activity at both pHs. At 65°C, 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 60°C, it retained 51 and 74% of its original activity after 3 h of incubation at pHs 8 and 9, respectively. At 65°C, 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 50°C. 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 50°C 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 xylanases optimally active around neutrality (1, 6, 10–12). 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 55°C (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. 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 find 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 70°C at pH 9 and 75°C 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.