- Email: [email protected]
A kinetic study on pH-activity relationship of XynA from alkaliphilic Bacillus halodurans C-125 using aryl-xylobiosides
JOURNAL OF BIOSCIENCEAND BIOENGINEERING Vol. 93, NO. 4, 428430. 2002
A Kinetic Study on pH-Activity Relationship of XynA from Alkaliphilic Bacillus halodurans C-125 Using Aryl-Xylobiosides M A M O R U NI SHIMOTO, l YUJI H O N D A , I M O T O M I T S U K I T A O K A , I, AND K I Y O S H I H A Y A S H I 1 Enzyme Laboratory, National Food Resear«h Institute, Kannondai, Ibaraki 305-8642, Japan a
Received 19 November2001/Accepted27 December2001 Xylanase A from alkaliphilic Bacillus halodurans C-125 was expressed in Escherichia coli and purified by affinity and anion exchange chromatographies. It exhibited a strong substrate inhibition using xylan as the substrate. Its K i value increased with an increase in pH. The effeet of pH on the enzyme activity was determined using two aryl-xylobiosides as substrates, and it was found that the enzyme had a flat kcat-PH eurve in the pH range of 5.8-8.8. This range was different from that obtained with 0.45% xylan as previously reported (Honda, H. et al., Agric. Biol. Chem., 49, 3165-3169, 1985). The substrate inhibition was presumed to cause the difference. It has been darified that the use of aryl-xylobiosides as substrates yields m o l e aceurate kinetic results than that of xylan. [Key words: Bacillus halodurans C- 125, family 10 xylanase, pH-activity]
for various kinetic parameters using pNPX 2 and o-nitrophenyl-]3-D-xylobioside (oNPX2) as substrates. For the expression of XynA in E. coli BL21 (DE3) using the pET system (Novagen Inc., Darmstadt, Germany), the xynA was prepared by the polymerase chain reaction with KOD polymerase (Takara Biomedical, Kyoto) using genomic DNA from B. halodurans C-125 as the template. The primers used for amplification were 5'-CCATGGTTACAC TTTTTAGAAAGCCTT-3' and 5'-CTCGAGATCAATAAT TCTCCAGTAAGCAGG-3' (restriction endonuclease sites for cloning are underlined). The amplified fragment and the pET28a vector digested by NcoI and Xhol were ligated using a DNA ligation kit, Ligation high (Toyobo Co. Ltd., Osaka). To construct the expression plasmid, Ile 2 was replaced with Val in order to construct an additional an NcoI site. At the C-terminus, a Leu-Glu-His-His-His-His-His-His sequence was added for easy purification. The expression plasmid was used to transform E. coli BL21 (DE3) and the transformant was cultivated with shaking in 700 ml of LB medium that contained kanamycin (50 ~tg/ml) at 30°C and at an absorbance of 0.5 measured at a wavelength of 600 nm. After the addition of isopropyl-l-thio-]3-D-galactoside to a final concentration of 1 mM, the culture was incubated at 30°C, with shaking, for 20 h. The enzyme was secreted in the culture broth. The culture was centrifuged at 15,000 x g for 10 min, and 740 g of ammonium sulfate was added to the supernatant (640 tal). The precipitate was collected by centrifugation (17,000xg for 30min) and dissolved in 20 ml of 50 mM sodium dihydrogenphosphate buffer (pH 8.0) containing 0.3 M sodium chloride and 10mM imidazole. The enzyme was dialyzed against the buffer, adsorbed to a Ni-NTA agarose (Qiagen, Hilden, Germany) column equilibrated with the same buffer, and eluted with a 10-200 mM
Xylanase [EC 3.2.1.8] catalyzes the hydrolysis of the main chain ofxylan, the major component of hemicellulose, in an endowise manner. Xylanase is classified into two families (families 10 and 11) according to glycoside hydrolase classification (1). Xylanase A (XynA) is produced from alkaliphilic Bacillus halodurans C-125, the entire genome sequence of which has been reported (2). XynA belongs to family 10 on the basis of its primary structure. The gene encoding XynA (xynA) was cloned, and expressed in Escherichia coli, and some properties of XynA were determined, as reported previously (3). An interesting characteristic of XynA is its optimum pH that ranged from 6 to 10, indicating that XynA is an alkaliphilic xylanase that uses xylan as the substrate (4). Xylan consists of polymers of 13-1,4 l inked D-xylopyranosyl residues in the main chain branched with ct-L-arabinofuranosyl and 4-methyl-«-D-glucuronopyranosyl residues. The structure of the branched chain is heterogenous and of chemically diverse origins. Honda et al. reported that a significant substrate inhibition was observed in the hydrolysis of xylan by Cex, a family 10 xylanase from Cellulomonas fimi (5). They also reported that this inhibition became stronger with a decrease in pH. Therefore, the pH-activity curve for XynA using xylan as the substrate was not very reliable because the level of inhibition decreased with an increase in pH, causing a relatively high activity in the alkaline region. Kitaoka and coworkers reported that aryl-xylobiosides such as p-nitrophenyl-[3-Dxylobioside (pNPX2) and 4-methylumbelliferyl-]3-D-xylobioside were good substrates of family 10 xylanase (6, 7). We will show in detail the pH-activity relationship of XynA * Corresponding author, e-mail: [email protected] phone: +81-(0)298-38-8071 fax: +81-(0)298-38-7321 428
VOL.93, 2002 imidazole linear gradient. The active fractions were collected and dialyzed against 20 mM phosphate buffer (pH 7.0). Then, the enzyme was loaded to a DEAE-TOYOPEARL 650 M (Tosoh, Tokyo) column equilibrated with the same buffer, followed by the elution with a 0-700 mM sodium chloride linear gradient. After these purification steps, recovery yield, purity, and specific activity of the enzyme were determined to be 62.3%, 12.8 fold, and 39.8 U/mg, respectively. The purified enzyme gave a single band by SDSPAGE (data not shown). The N-terminal amino acid sequence was Ala-Gln-Gly-Gly-Pro-, indicating that a leader peptide of 28 amino acids was removed. This result agrees with the report describing that the N-terminal residue of the mature XynA expressed in E. coli was 29th alanine (8), indicating that the mutation at the second amino acid residues did not affect the processing. Finally, the purified enzyme (6.3 mg) was obtained from a 700 ml culture and it was used throughout the following experiments. Beech wood xylan was purchased from Sigma. The soluble xylan was prepared as previously described (5). Arylxylobiosides, oNPX 2 and pNPX» were synthesized from a xylobiose mixture (Wako Pure Chemicals, Osaka) (6). Xylanase activity toward nitrophenyl glycosides was determined by measuring the amount of nitrophenol liberated as described below. The enzyme in 25 [al of 20 mM phosphate buffer (pH 7.0) containing 0.04% Triton X-100 was preincubated with 50 gl of various 0.2 M buffer solutions, as described below for 3 min, and incubated with 25 ~tl of substrates at various concentrations for 5 min. The reaction was stopped by the addition of 100 ~tl of 1 M sodium carbonate, and the amount of liberated nitrophenol was measured based on the absorbance at 400 nm. When the absorbance was 1.0, the concentrations of o- and p-nitrophenol were determined to be 481 and 55.1 gmol/ml, respectively. One unit of xylanase activity was defined as the amount of enzyme which liberated 1 gmol of nitrophenol per minute under the above conditions. Xylanase activity for soluble beech wood xylan was measured using the copper-bicinchoninie acid method (9). The enzyme in 25 lal of 20 mM phosphate buffer (pH 7.0) containing 0.04% Triton X-100 was preincubated with 50 gl of various 0.2 M buffer solutions, as described below for 3 min, and incubated with 25 gl of substrates at various concentrations for 5 min. The reaction was stopped by adding 100 p,1 of the copper-bicinchoninic acid reagent. The reaction mixture was incubated at 100°C for 15 min and kept on ice for 20 min. The reducing power was measured based on the absorbance at 560 nm using xylose as the standard. The enzyme reaction was performed at 40°C with acetate (pHs 3.9, 4.3, 4.8, 5.3, 5.8), MES (pHs 5.2, 5.6, 6.1, 6.6), HEPES (pHs 6.5, 6.9, 7.3, 7.8, 8.3), and CHES (pHs 7.7, 8.2, 8.8, 9.2, 9.7) buffer solutions. The kinetic parameters were calculated by a curve fit method, using a computer program, GraFit (10). The s-v plots at several pHs of 4.8, 7.3, and 8.8 in the hydrolysis of xylan are shown in Fig. 1. The substrate inhibition was observed in all the pH values measured, particularly at a low concentration ofxylan at pH 4.8. However, the inhibition was not so strong on the basic side at pH 8.8. The K~ values for xylan calculated at pHs of 4.8, 7.3, and 8.8 were 0.004, 0.07, and 0.11%, respectively (Table 1). These
NOTES
429
"~ 60
~~~- 40 o
-ff 20 [] 0 0
I
I
r
0.1
0.2
0.3
s (%) FIG. I. s-v plot ofXynA using xylan as a substrate. The initial rate was measured using various concentrations of xylan at pH 4.8 (open square), pH 7.3 (open triangle), and pH 8.8 (open circle). TABLE I. Kinetic parameters of XynA for soluble beech wood xylan as the substrate at three pH values pH kca,(S ') Km(%) 4.8 28.2_+2.8 0.0021+0.0005 7.3 73.7+-0.9 0.005l_+0.0001 8.8 46.5+1.3 0.0050-+0.0003 Kinetic parameters were calculated using the ( Vmax X I g ] ) / ( K n + IS] + [S]2/K).
K~(%) 0.038-+0.010 0.067-+0.002 0.109-+0.007 equation of v =
results were consistent with the observation for Cex reported by Honda et al. (5). Therefore, xylan is not a suitable substrate for the kinetic study of XynA. The aryl-xylobiosides, oNPX 2 and pNPX» were thus used for the assay of pH-activity instead of soluble xylan. No such substrate inhibition was observed using these substrates. The velocities were measured in the pH range of 3.9-9.7 using the buffer system of acetate, MES, HEPES, and CHES. The kcat values for both the substrates were constant in the pH range of 5.8-8.8 (Fig. 2). The change of the buffer system into the buffer system of phosphate (pHs 6.2, 6.7), MOPS (pHs 7.0, 7.5, 7.9), and imidazole (pHs 7.5, 8.0) did not significantly affect the results. The result obtained was different from that obtained in the previous study (4) describing a constant activity in the pH range of 6-10. This difference of 1 pH unit on the basic side is presumed to be due to the degree of substrate inhibition by 0.45% xylan used in the previous study (4), because the xylan concentration was much higher than the K~ values at any pH. It is clear that oNPX 2 or pNPX 2 yields rauch more accurate results in kinetic experiments of XynA than xylan. Honda et al. observed a flat pH-kcat relationship for Cex, a family 10 xylanase from C. fimi, between pH 5 and pH 10 using only oNPX 2, not phenyl-[3-D-xylobioside and pNPX2, and discussed the effect of the nitro group at the ortho position of oNPX 2 (5). They also described that no such phenomenon was observed with another family 10 xylanase, XynB, from Clostridium stercorarium F9. Our results resemble those for XynB because no significant difference in the pH-k~at curves of oNPX 2 and pNPX 2 was found. Both of the kcat values of XynA for oNPX 2 and
430
NISHIMOTOET AL.
J. BlOSCI.BIOENG.,
A 150 kl
3
g
100
2
50
0
REFERENCES
4
[]
«
1
3
i
i
i
i
J
i
4
5
6
7
8
9
i
10
0.6~ 0.4 ~~
5. Honda, Y., Kitaoka, M., Sakka, K., Ohmiya, K., and Hayashi, K.: An invesfigation of the pH-activity relafion-
0 11
1 oo°
0.8
20 10 ._. 0 3
' 4
' 5
' 6
' 7 pH
0.2 ' 8
~ 9
' 10
0 11
FIG. 2, Effect of pH on the kinetic parameters ofXynA using arylxylobiosides. (A) oNPX2; (B) pNPX 2. Open symbols, kcat;closed symbols, Km. Circle, acetate; diamond, MES; triangle, HEPES; square, CHES. pNPX 2 decreased at alkaline pH. It was suggested that the behavior o f X y n A for these substrates resembled that o f XynB more than that o f Cex. The amino acid sequence identity o f X y n A with XynB and Cex were 53% and 31%, respectively, also suggesting that X y n A was more likely
XynB.
2. Takami, H., Nakasone, K., Takalä, Y., Maeno, G., Sasaki, R., Masui, N., Fuji, F., Hirama, C., Nakamura, Y., Ogasawara, N., Kuhara, S., and Horikoshi, K.: Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis. Nucleic Acids Res., 28, 4317-4331 (2000). 3. Honda, H., Kudo, T., and Horikoshi, K.: Molecular cloning and expression of the xylanase gene of alkalophilic Bacillus sp. strain C-125 in Escherichia coli. J. Bacteriol., 161, 784785 (1985). 4. Honda, H., Kudo, T., and Horikoshi, K.: Purification and partial characterization of alkaline xylanase from Escherichia coli carrying pCX311. Agric. Biol. Chem., 49, 3165-3169 (1985).
B
«
1. Henrissat, B. and Bairoch, A.: Updating the sequence-based classification of glycosyl hydrolases. Biochem. J., 316, 695696 (1996).
ships of Cex, a family 10 xylanase from Cellulomonasfimi: xylan inhibition and the influence of nitro-substituted arylq3D-xylobiosides on xylanase activity. J. Biosci. Bioeng., 93, 313-317 (2002).
6. Kitaoka, M., Haga, K., Kashiwagi, Y., Sasaki, T., Taniguchi, H., and Kusakabe, I.: Kinetic studies on p-nitrophenyl-cellobioside hydrolyzing xylanase from Cellvibrio gilvus. Biosci. Biotechnol. Biochem., 57, 1987-1989 (1993). 7. Kaneko, S., Kitaoka, M., Kuno, A., and Hayashi, K.: Syntheses of 4-methylumbelliferyl-13-D-xylobioside and 5bromo-3-indolyl-13-D-xylobioside for sensitive detection of xylanase activity on agar plates. Biosci. Biotechnol. Biochem., 64, 741-745 (2000).
8. Hamamoto, T., ttonda, tl., Kudo, T., and Horikoshi, K.: Nucleotide sequence of the xylanase A gene of alkalophilic Bacillus sp. strain C-125. Agric. Biol. Chem., 51, 953-955 (1987). 9. Waffensehmidt, S. and Jaenieke, L.: Assay of reducing sugars in the nanomole range with 2,2'-bicinchoninate. Anal. Biochem., 165, 337-340 (1987). 10. Leatherbarr°w' R" J': Using linear and n°n-linear regressi°n to fit biochemical data. Trends Biochem. Sci., 15, 455-458 (1990).
A Kinetic Study on pH-Activity Relationship of XynA from Alkaliphilic Bacillus halodurans C-125 Using Aryl-Xylobiosides M A M O R U NI SHIMOTO, l YUJI H O N D A , I M O T O M I T S U K I T A O K A , I, AND K I Y O S H I H A Y A S H I 1 Enzyme Laboratory, National Food Resear«h Institute, Kannondai, Ibaraki 305-8642, Japan a
Received 19 November2001/Accepted27 December2001 Xylanase A from alkaliphilic Bacillus halodurans C-125 was expressed in Escherichia coli and purified by affinity and anion exchange chromatographies. It exhibited a strong substrate inhibition using xylan as the substrate. Its K i value increased with an increase in pH. The effeet of pH on the enzyme activity was determined using two aryl-xylobiosides as substrates, and it was found that the enzyme had a flat kcat-PH eurve in the pH range of 5.8-8.8. This range was different from that obtained with 0.45% xylan as previously reported (Honda, H. et al., Agric. Biol. Chem., 49, 3165-3169, 1985). The substrate inhibition was presumed to cause the difference. It has been darified that the use of aryl-xylobiosides as substrates yields m o l e aceurate kinetic results than that of xylan. [Key words: Bacillus halodurans C- 125, family 10 xylanase, pH-activity]
for various kinetic parameters using pNPX 2 and o-nitrophenyl-]3-D-xylobioside (oNPX2) as substrates. For the expression of XynA in E. coli BL21 (DE3) using the pET system (Novagen Inc., Darmstadt, Germany), the xynA was prepared by the polymerase chain reaction with KOD polymerase (Takara Biomedical, Kyoto) using genomic DNA from B. halodurans C-125 as the template. The primers used for amplification were 5'-CCATGGTTACAC TTTTTAGAAAGCCTT-3' and 5'-CTCGAGATCAATAAT TCTCCAGTAAGCAGG-3' (restriction endonuclease sites for cloning are underlined). The amplified fragment and the pET28a vector digested by NcoI and Xhol were ligated using a DNA ligation kit, Ligation high (Toyobo Co. Ltd., Osaka). To construct the expression plasmid, Ile 2 was replaced with Val in order to construct an additional an NcoI site. At the C-terminus, a Leu-Glu-His-His-His-His-His-His sequence was added for easy purification. The expression plasmid was used to transform E. coli BL21 (DE3) and the transformant was cultivated with shaking in 700 ml of LB medium that contained kanamycin (50 ~tg/ml) at 30°C and at an absorbance of 0.5 measured at a wavelength of 600 nm. After the addition of isopropyl-l-thio-]3-D-galactoside to a final concentration of 1 mM, the culture was incubated at 30°C, with shaking, for 20 h. The enzyme was secreted in the culture broth. The culture was centrifuged at 15,000 x g for 10 min, and 740 g of ammonium sulfate was added to the supernatant (640 tal). The precipitate was collected by centrifugation (17,000xg for 30min) and dissolved in 20 ml of 50 mM sodium dihydrogenphosphate buffer (pH 8.0) containing 0.3 M sodium chloride and 10mM imidazole. The enzyme was dialyzed against the buffer, adsorbed to a Ni-NTA agarose (Qiagen, Hilden, Germany) column equilibrated with the same buffer, and eluted with a 10-200 mM
Xylanase [EC 3.2.1.8] catalyzes the hydrolysis of the main chain ofxylan, the major component of hemicellulose, in an endowise manner. Xylanase is classified into two families (families 10 and 11) according to glycoside hydrolase classification (1). Xylanase A (XynA) is produced from alkaliphilic Bacillus halodurans C-125, the entire genome sequence of which has been reported (2). XynA belongs to family 10 on the basis of its primary structure. The gene encoding XynA (xynA) was cloned, and expressed in Escherichia coli, and some properties of XynA were determined, as reported previously (3). An interesting characteristic of XynA is its optimum pH that ranged from 6 to 10, indicating that XynA is an alkaliphilic xylanase that uses xylan as the substrate (4). Xylan consists of polymers of 13-1,4 l inked D-xylopyranosyl residues in the main chain branched with ct-L-arabinofuranosyl and 4-methyl-«-D-glucuronopyranosyl residues. The structure of the branched chain is heterogenous and of chemically diverse origins. Honda et al. reported that a significant substrate inhibition was observed in the hydrolysis of xylan by Cex, a family 10 xylanase from Cellulomonas fimi (5). They also reported that this inhibition became stronger with a decrease in pH. Therefore, the pH-activity curve for XynA using xylan as the substrate was not very reliable because the level of inhibition decreased with an increase in pH, causing a relatively high activity in the alkaline region. Kitaoka and coworkers reported that aryl-xylobiosides such as p-nitrophenyl-[3-Dxylobioside (pNPX2) and 4-methylumbelliferyl-]3-D-xylobioside were good substrates of family 10 xylanase (6, 7). We will show in detail the pH-activity relationship of XynA * Corresponding author, e-mail: [email protected] phone: +81-(0)298-38-8071 fax: +81-(0)298-38-7321 428
VOL.93, 2002 imidazole linear gradient. The active fractions were collected and dialyzed against 20 mM phosphate buffer (pH 7.0). Then, the enzyme was loaded to a DEAE-TOYOPEARL 650 M (Tosoh, Tokyo) column equilibrated with the same buffer, followed by the elution with a 0-700 mM sodium chloride linear gradient. After these purification steps, recovery yield, purity, and specific activity of the enzyme were determined to be 62.3%, 12.8 fold, and 39.8 U/mg, respectively. The purified enzyme gave a single band by SDSPAGE (data not shown). The N-terminal amino acid sequence was Ala-Gln-Gly-Gly-Pro-, indicating that a leader peptide of 28 amino acids was removed. This result agrees with the report describing that the N-terminal residue of the mature XynA expressed in E. coli was 29th alanine (8), indicating that the mutation at the second amino acid residues did not affect the processing. Finally, the purified enzyme (6.3 mg) was obtained from a 700 ml culture and it was used throughout the following experiments. Beech wood xylan was purchased from Sigma. The soluble xylan was prepared as previously described (5). Arylxylobiosides, oNPX 2 and pNPX» were synthesized from a xylobiose mixture (Wako Pure Chemicals, Osaka) (6). Xylanase activity toward nitrophenyl glycosides was determined by measuring the amount of nitrophenol liberated as described below. The enzyme in 25 [al of 20 mM phosphate buffer (pH 7.0) containing 0.04% Triton X-100 was preincubated with 50 gl of various 0.2 M buffer solutions, as described below for 3 min, and incubated with 25 ~tl of substrates at various concentrations for 5 min. The reaction was stopped by the addition of 100 ~tl of 1 M sodium carbonate, and the amount of liberated nitrophenol was measured based on the absorbance at 400 nm. When the absorbance was 1.0, the concentrations of o- and p-nitrophenol were determined to be 481 and 55.1 gmol/ml, respectively. One unit of xylanase activity was defined as the amount of enzyme which liberated 1 gmol of nitrophenol per minute under the above conditions. Xylanase activity for soluble beech wood xylan was measured using the copper-bicinchoninie acid method (9). The enzyme in 25 lal of 20 mM phosphate buffer (pH 7.0) containing 0.04% Triton X-100 was preincubated with 50 gl of various 0.2 M buffer solutions, as described below for 3 min, and incubated with 25 gl of substrates at various concentrations for 5 min. The reaction was stopped by adding 100 p,1 of the copper-bicinchoninic acid reagent. The reaction mixture was incubated at 100°C for 15 min and kept on ice for 20 min. The reducing power was measured based on the absorbance at 560 nm using xylose as the standard. The enzyme reaction was performed at 40°C with acetate (pHs 3.9, 4.3, 4.8, 5.3, 5.8), MES (pHs 5.2, 5.6, 6.1, 6.6), HEPES (pHs 6.5, 6.9, 7.3, 7.8, 8.3), and CHES (pHs 7.7, 8.2, 8.8, 9.2, 9.7) buffer solutions. The kinetic parameters were calculated by a curve fit method, using a computer program, GraFit (10). The s-v plots at several pHs of 4.8, 7.3, and 8.8 in the hydrolysis of xylan are shown in Fig. 1. The substrate inhibition was observed in all the pH values measured, particularly at a low concentration ofxylan at pH 4.8. However, the inhibition was not so strong on the basic side at pH 8.8. The K~ values for xylan calculated at pHs of 4.8, 7.3, and 8.8 were 0.004, 0.07, and 0.11%, respectively (Table 1). These
NOTES
429
"~ 60
~~~- 40 o
-ff 20 [] 0 0
I
I
r
0.1
0.2
0.3
s (%) FIG. I. s-v plot ofXynA using xylan as a substrate. The initial rate was measured using various concentrations of xylan at pH 4.8 (open square), pH 7.3 (open triangle), and pH 8.8 (open circle). TABLE I. Kinetic parameters of XynA for soluble beech wood xylan as the substrate at three pH values pH kca,(S ') Km(%) 4.8 28.2_+2.8 0.0021+0.0005 7.3 73.7+-0.9 0.005l_+0.0001 8.8 46.5+1.3 0.0050-+0.0003 Kinetic parameters were calculated using the ( Vmax X I g ] ) / ( K n + IS] + [S]2/K).
K~(%) 0.038-+0.010 0.067-+0.002 0.109-+0.007 equation of v =
results were consistent with the observation for Cex reported by Honda et al. (5). Therefore, xylan is not a suitable substrate for the kinetic study of XynA. The aryl-xylobiosides, oNPX 2 and pNPX» were thus used for the assay of pH-activity instead of soluble xylan. No such substrate inhibition was observed using these substrates. The velocities were measured in the pH range of 3.9-9.7 using the buffer system of acetate, MES, HEPES, and CHES. The kcat values for both the substrates were constant in the pH range of 5.8-8.8 (Fig. 2). The change of the buffer system into the buffer system of phosphate (pHs 6.2, 6.7), MOPS (pHs 7.0, 7.5, 7.9), and imidazole (pHs 7.5, 8.0) did not significantly affect the results. The result obtained was different from that obtained in the previous study (4) describing a constant activity in the pH range of 6-10. This difference of 1 pH unit on the basic side is presumed to be due to the degree of substrate inhibition by 0.45% xylan used in the previous study (4), because the xylan concentration was much higher than the K~ values at any pH. It is clear that oNPX 2 or pNPX 2 yields rauch more accurate results in kinetic experiments of XynA than xylan. Honda et al. observed a flat pH-kcat relationship for Cex, a family 10 xylanase from C. fimi, between pH 5 and pH 10 using only oNPX 2, not phenyl-[3-D-xylobioside and pNPX2, and discussed the effect of the nitro group at the ortho position of oNPX 2 (5). They also described that no such phenomenon was observed with another family 10 xylanase, XynB, from Clostridium stercorarium F9. Our results resemble those for XynB because no significant difference in the pH-k~at curves of oNPX 2 and pNPX 2 was found. Both of the kcat values of XynA for oNPX 2 and
430
NISHIMOTOET AL.
J. BlOSCI.BIOENG.,
A 150 kl
3
g
100
2
50
0
REFERENCES
4
[]
«
1
3
i
i
i
i
J
i
4
5
6
7
8
9
i
10
0.6~ 0.4 ~~
5. Honda, Y., Kitaoka, M., Sakka, K., Ohmiya, K., and Hayashi, K.: An invesfigation of the pH-activity relafion-
0 11
1 oo°
0.8
20 10 ._. 0 3
' 4
' 5
' 6
' 7 pH
0.2 ' 8
~ 9
' 10
0 11
FIG. 2, Effect of pH on the kinetic parameters ofXynA using arylxylobiosides. (A) oNPX2; (B) pNPX 2. Open symbols, kcat;closed symbols, Km. Circle, acetate; diamond, MES; triangle, HEPES; square, CHES. pNPX 2 decreased at alkaline pH. It was suggested that the behavior o f X y n A for these substrates resembled that o f XynB more than that o f Cex. The amino acid sequence identity o f X y n A with XynB and Cex were 53% and 31%, respectively, also suggesting that X y n A was more likely
XynB.
2. Takami, H., Nakasone, K., Takalä, Y., Maeno, G., Sasaki, R., Masui, N., Fuji, F., Hirama, C., Nakamura, Y., Ogasawara, N., Kuhara, S., and Horikoshi, K.: Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis. Nucleic Acids Res., 28, 4317-4331 (2000). 3. Honda, H., Kudo, T., and Horikoshi, K.: Molecular cloning and expression of the xylanase gene of alkalophilic Bacillus sp. strain C-125 in Escherichia coli. J. Bacteriol., 161, 784785 (1985). 4. Honda, H., Kudo, T., and Horikoshi, K.: Purification and partial characterization of alkaline xylanase from Escherichia coli carrying pCX311. Agric. Biol. Chem., 49, 3165-3169 (1985).
B
«
1. Henrissat, B. and Bairoch, A.: Updating the sequence-based classification of glycosyl hydrolases. Biochem. J., 316, 695696 (1996).
ships of Cex, a family 10 xylanase from Cellulomonasfimi: xylan inhibition and the influence of nitro-substituted arylq3D-xylobiosides on xylanase activity. J. Biosci. Bioeng., 93, 313-317 (2002).
6. Kitaoka, M., Haga, K., Kashiwagi, Y., Sasaki, T., Taniguchi, H., and Kusakabe, I.: Kinetic studies on p-nitrophenyl-cellobioside hydrolyzing xylanase from Cellvibrio gilvus. Biosci. Biotechnol. Biochem., 57, 1987-1989 (1993). 7. Kaneko, S., Kitaoka, M., Kuno, A., and Hayashi, K.: Syntheses of 4-methylumbelliferyl-13-D-xylobioside and 5bromo-3-indolyl-13-D-xylobioside for sensitive detection of xylanase activity on agar plates. Biosci. Biotechnol. Biochem., 64, 741-745 (2000).
8. Hamamoto, T., ttonda, tl., Kudo, T., and Horikoshi, K.: Nucleotide sequence of the xylanase A gene of alkalophilic Bacillus sp. strain C-125. Agric. Biol. Chem., 51, 953-955 (1987). 9. Waffensehmidt, S. and Jaenieke, L.: Assay of reducing sugars in the nanomole range with 2,2'-bicinchoninate. Anal. Biochem., 165, 337-340 (1987). 10. Leatherbarr°w' R" J': Using linear and n°n-linear regressi°n to fit biochemical data. Trends Biochem. Sci., 15, 455-458 (1990).