- Email: [email protected]
Xylanase of Bacillus pumilus
632
HEMICELLULOSE
[79]
from cotyledon embryo tissue, whereas peak II is endosperm-derived a-galactosidase. The combined recovery of a-galactosidases I plus II is greater than 70% of that present in the crude extract (Table I) and the extent of purification is in excess of 1000-fold. Both a-galactosidases I and II are devoid of other glycanase and glycosidase activities (<0.002%). Contamination of a-galactosidase I with endo-fl-mannanase was less than one part in 106 but complete removal of this activity from a-galactosidase II required a second chromatographic purification on the affinity column support. Properties. Affinity purified a-galactosidase II appears as a single band on isoelectric focusing (pI 4.6) and on SDS-gel electrophoresis (MW = 21,000). It shows optimal activity at pH 4.5-5.5 and is unstable at temperatures above 45 °. In contrast, a-galactosidase I appears as a number of protein bands on isoelectric focusing (pI values 5.8-6.6) but as a single broad band on SDS-gel electrophoresis (MW = 33,000). The enzymes catalyze the hydrolysis of a wide range of substrates with nonreducing terminal o-galactose, including melibiose, raffinose, stachyose, galactomannan, and p-nitrophenyl-a-o-galactopyranoside, but the relative initial rates of hydrolysis vary significantly. Both enzymes are very effective in the removal of o-galactose from galactomannans (Fig. 1). Galactomannans with widely different D-galactose contents and varying patterns of distribution of o-galactosyl residues along the D-mannan backbone are hydrolyzed at essentially the same rate.14
[79] X y l a n a s e o f Bacillus pumilus By
HIROSUKE
OKADA
and
ATSUHIKO
SHINMYO
Xylan, the primary component of pentosan, consists of a backbone chain of 1,4-1inked fl-o-xylopyranosyl residues and side chains of arabinose, glucuronic acid, or methylglucuronic acid. The backbone is hydrolyzed by endoxylanase or exoxylanase and the xylooligosaccharides formed are hydrolyzed to xylose by fl-xylosidase. Xylanase production has been reported in many microorganisms including fungi, yeasts, and bacteria. Since natural xylan is a heterogeneous polysaccharide, the hydrolysis of side chains is not well understood. Bacillus pumilus is a microbe producing potent xylan degrading enzymes. In our laboratory, endoxylanase (1,4-fl-o-xylan xylanohydrolase, EC 3.2.1.8) and fl-xylosidase (1,4-fl-o-xylan xylohydrolase, EC 3.2.1.37, xylan 1,4-fl-xylosidase) of B. pumilus IPO, 4 isolated from a rice field, were purified, and hydrolysis of xylan to xylose was found to be accomMETHODS IN ENZYMOLOGY, VOL. 160
Copyright © 1988by Academic Press, Inc. All rights of reproduction in any form reserved.
[79]
XYLANASEOF B. pumilus
633
plished by sequential reaction of these two enzymes.~ A chromosomal DNA fragment of B. p u m i l u s IPO containing genes of both enzymes was cloned in Escherichia coh ~ and the complete DNA sequences of both genes were determined. TM Here, we describe the purification and properties of the xylanase of B. p u m i l u s IPO.4 Assay Methods Principle. Xylanase activity is measured from the reducing sugars liberated from larchwood xylan (Sigma, St. Louis, MO, approximate molecular weight 20,000). Reducing sugar is measured by the dinitrosalicylic acid method. 5 Reagents
Potassium phosphate buffer, pH 6.5 Larchwood xylan (Sigma) Xylose 3,5-Dinitrosalicylic acid reagent P r o c e d u r e . Xylan is suspended in 50 mM potassium phosphate buffer at 1% (w/v) and boiled for 10 min to dissolve it. The reaction mixture, consisting of 1 ml of xylan solution and 0.5 ml of enzyme solution, is incubated at 40° for 10 rain. The reaction is stopped by adding 3 ml of 3,5dinitrosalicylic acid reagent. Reducing sugar present without incubation at 40° is subtracted from that with incubation. One unit of xylanase is defined as the amount of enzyme that liberates 1 ~mol of xylose equivalent in 1 rain. When a crude enzyme solution is used, liberation of reducing sugar without xylan must be checked. B. p u m i l u s IPO excretes xylanase in the culture medium, but accumulates fl-xylosidase in the cytoplasm. On the other hand, the E. coli clone which harbors the plasmid coding for the xylanase and ~-xylosidase genes accumulates both enzymes in its cells. Xylanase activity determined in E. coli cell extracts is overestimated 1w. Panbangred, A. Shinmyo, S. Kinoshita, and H. Okada, Agric. Biol. Chem. 47, 957 (1983). 2 W. Panbangred,T. Kondo,S. Negoro, A. Shinmyo,and H. Okada,Mol. Gen. Genet. 192, 335 (1983). 3 E. Fukusaki, W. Panbangred, A. Shinmyo,and H. Okada, FEBS Lett. 171, 197 (1984). 3~H. Moriyama, E. Fukusaki, J. Cabrera Crespo, A. Shinmyo, and H. Okada, Eur. J. Biochem. 166, 539 (1987). 4 B. pumulis IPO is depositedin the FermentationResearch InstituteYatabe-cho, Higashigun, Ibaraki, Japan, and is designated culture 4. E. coli plasmid pOXN391 in the same collection is designatedFerm P-6996. 5G. L. Miller,Anal. Chem. 31, 426 (1959).
634
HEMICELLULOSE
[79]
because of the presence of a/3-xylosidase which produces reducing sugars from the xylooligosaccharides formed by the xylanase reaction. When purified fl-xylosidase is added to xylanase solution at an activity ratio of 3: l, the apparent xylanase activity is about 1.2 times that without /3xylosidase. Purification Highly purified enzyme with a specific activity in the range of 17001900 units/rag protein can be obtained from the culture fluid by a few purification steps. Culture Conditions. The enzyme production medium consists of 0.5% larchwood xylan (Sigma), 0.5% Bacto-yeast extract (Difco), 0.2% NH4NO3, 0.2% KHEPO4, and 0.02% MgSO4.7H20, pH 6.8. B. pumilus IPO cells are grown in a test tube containing 10 ml L-broth [10 g Bactotryptone (Difco), 5 g Bacto-yeast extract, 5 g NaC1, and 1 g glucose in ! liter] overnight at 30 ° with shaking and the whole culture broth is transferred to 500 ml of production medium in a 3-liter Sakaguchi flask. The flask is incubated at 30 ° for 36 hr in a water bath with shaking. Then the culture fluid is obtained by centrifugation at 10,000 g for 15 min and used for xylanase purification. The following procedures are performed at <4 °. Step 1. The protein fraction in 2 liters of the culture fluid which precipitated in the ammonium sulfate concentration range between 0.2 and 0.6 saturation is collected as follows; solid ammonium sulfate (114 g/liter) is added to the culture fluid with stirring. After 3 hr, the supernatant is obtained by centrifugation at 15,000 g for 30 min, and further ammonium sulfate is added (262 g/liter). The precipitate is collected after 3 hr by centrifugation, dissolved in 200 ml of 50 mM potassium phosphate buffer, pH 6.5, and dialyzed for about 20 hr against several changes of 5 liters of the same buffer. The resultant insoluble materials, which contain residual xylan, are removed by centrifugation. Step 2. The dialyzed enzyme solution is applied to a DEAE-Sephadex column (5.5 x 25 cm) equilibrated with 50 mM phosphate buffer, pH 6.5 (the same phosphate buffer pH and composition are used in all other steps). On elution with the same buffer at a flow rate of 200-250 ml/hr, xylanase activity appears in the void volume, whereas most of the other proteins and colored materials are adsorbed onto the gel. Fractions of 50 ml are collected. The fractions containing specific activity greater than 100 units/mg protein are pooled to yield a total volume of about 500 ml with an enzyme activity recovery of 60% in this step. Step 3. The pooled active fraction in Step 2 is applied to a CM-Sephadex C-50 column (5.5 x 20 cm) which has been equilibrated with the
XYLANASEOF B. pumilus
[79]
635
buffer. The column is washed with 1 liter of the phosphate buffer and the enzyme is eluted from the column with a linear gradient established between 500 ml of the phosphate buffer and 500 ml of the buffer containing 0.6 M NaC1. Fractions of 20 ml are collected at the flow rate of 200 ml/hr. Xylanase activity is eluted at about 0.22 M NaCI concentration. The fractions containing specific activity greater than 800 units/mg protein are pooled to yield a total volume of 520 ml with 87% recovery of enzyme activity in this step. The solution is concentrated to a volume of 20 ml to 25 ml by ultrafiltration using a Toyo UM-10 membrane, and dialyzed against three changes of 2 liters of the phosphate buffer. Step 4. The dialyzate in Step 3 is added ammonium sulfate to 40% of saturation by adjusting the pH at 6.5 with 1 N KOH, and applied to a column (1.5 × 40 cm) o f T S K HW polyvinyl gel (Toyosoda, Tokyo) which has been equilibrated with the phosphate buffer containing 40% saturation of ammonium sulfate. The column is washed with 200 ml of the same equilibration buffer solution. Then the enzyme is eluted with a linear gradient of ammonium sulfate from 40% saturation to 0%, each in 200 ml of the phosphate buffer. The flow rate is 20 ml/hr and 5-ml fractions are collected. The enzyme activity is eluted at 13% saturation of ammonium sulfate with the specific activity of about 1800 units/mg protein. Recovery of the activity in this step is about 70%. Approximately 90-fold purification is achieved with an overall yield of 22%. A summary of the purification procedures is presented in Table I. Preparation of Xylanase from E. coli Cells Harboring the Plasmid. Five hundreds milliliters of L-broth containing 50/xg/ml of ampicillin in a 3-liter Sakaguchi flask is inoculated with 5 ml of culture broth of E. coli C600 harboring the hybrid plasmid pOXN391 which codes for the xylanase gene of B. pumilus IPO and the ampicillin-resistance gene, 3 grown at 37° overnight in the same medium, and incubated for 16-20 hr at 37° with shaking. E. coli (pOXN391) produces xylanase constitutively, TABLE I PURIFICATION OF XYLANASE
Step 1. 2. 3. 4. 5.
Culture fluid 20-60% (NH4)2SO~ DEAE-Sephadex A-50 CM-Sephadex C-50 TSK HW-65
Volume (ml)
Total protein (mg)
2080 220 600 20 10
8740 1830 234 47 22
Total Specific activity activity (units × 103) (units/mg) 175 110 67.2 56.6 39.1
20 60 287 1200 1780
Purification (fold)
Yield (%)
1 3 14 60 89
100 63 38 32 22
636
HEMICELLULOSE
[79]
whereas B. pumilus IPO xylanase is induced by xylan and also by xylose. Cells are harvested from 2 liters of culture by centrifugation at 10,000 g for 10 rain and washed once with 200 ml of ice-cold 50 mM potassium phosphate buffer, pH 6.5. The following procedures are done at <4 °. Washed cells are suspended in 25 ml of the same buffer solution and disintegrated by passing through a French pressure cell at 450-500 kg/cm z. The cell lysate is centrifuged at 10,000 g for 30 min and supernatant is again centrifuged at 40,000 g for 1 hr to yield clear supernatant. Xylanase is purified from the clear supernatant by the above procedure. Properties
Homogeneity and Molecular Weight. The purified enzyme preparation gave a single protein band on polyacrylamide disc gel electrophoresis with or without sodium dodecyl sulfate (SDS). The purity was estimated at more than 95%. No carbohydrate was detected by acid fuchsin staining of the gel. The molecular weight of the purified xylanase was 24,000 by SDS-polyacrylamide gel electrophoresis and 20,000 by equilibrium sedimentation, suggesting a single polypeptide chain. From the nucleotide sequence of the cloned xylanase gene in E. coli it was deduced that xylanase consists of 201 amino acid residues with a molecular weight of 22,384. Stability and Activity. The purified xylanase had an activity and stability optimum at pH 6.5. The enzyme retained more than 85% of its activity after standing at 40 ° for 30 min at pH 8.5 or 5.0. The maximum xylanase activity was observed at temperatures between 45 and 50° over a period of I0 min. The enzyme lost half of its activity when kept at 50° for 30 min, and the decrease was in accord with monomolecular kinetics. All activity was lost at 60 ° in 15 min. The xylanase activity was influenced by the salt concentration of the reaction mixture. In l0 mM phosphate buffer the activity was about 20% of the maximum value, which was obtained in 50-60 mM phosphate buffer. Sodium chloride added to 10 mM phosphate buffer affected the activity similarly. Amino Acid Sequence. The amino acid sequence of prexylanase, deduced from the DNA sequence is shown in Fig. 1. This is the first example of the total amino acid sequence of a xylanase. The protein is probably synthesized in the form of a prexylanase with a signal sequence consisting of 27 amino acids, of which 3 are basic amino acid residues in the region near N-terminus and 18 are hydrophobic amino acid residues. The signal sequence might be processed between Ala -1 and Arg +. The N-terminal amino acid sequence of the purified xylanase was NH2-Arg-Thr-Ile-Thr
XYLANASEOF B. pumilus
[79]
-27
-20
637 -I0
-l
MNLRKLRLLFVMCIGLTLILTAVPAHA
1
i0
20
30
40
50
RTITNNEMGNHSGYDYELWKDYGNTSMTLNNGGAFSAGWNNIGNALFRKG
60
70
80
90
i00
KKFDSTRTHHQLGNISINYNASFNPSGNSYLCVYGWTQSPLAEYYIVDSW
ii0
120
130
140
150
GTYRPTGAYKGSFYADGGTYDIYETTRVNQPSIIGIATFKQYWSVRQTKR
160
170
180
190
200
TSGTVSVSAHFRKWESLGMPMGKMYETAFTVEGYQSSGSANVMTNQLFIG
201 N
FIG. 1. The complete amino acid sequence of the prexylanase. The 27 amino acid signal peptide indicated by minus numbers might be processed between Ala and Arg. Amino acids are indicated by one-letter symbols.
and the C-terminal sequence was Ile-Gly-Asn-COOH by sequential Edman degradation and carboxypeptidase digestion, respectively. The amino acid composition from the purified enzyme agreed well with that deduced from the DNA sequence. Hydrolysis of Xylan and Xylooligosaccharides. The maximum degree of hydrolysis of xylosidic linkage of larchwood xylan (molecular weight 20,000) by the purified xylanase was about 25%. The end products were oligosaccharides, corresponding xylobiose (X2), xylotriose (X3), xylotetraose (X4), and higher oligomers. At the early stages of the reaction, xylose oligomers higher than xylopentaose were the only products. Authentic xylobiose was not hydrolyzed. Incubation with X3 yielded X4 and X2, while with X4 as the substrate, X2, X3, X4, and higher oligomers were detected, although the reaction rate was much slower than that toward xylan. The production of X4 from X3, and of the higher oligomers from X4 indicates that xylanase has trans-xylosidation activity.
HEMICELLULOSE
[79]
from cotyledon embryo tissue, whereas peak II is endosperm-derived a-galactosidase. The combined recovery of a-galactosidases I plus II is greater than 70% of that present in the crude extract (Table I) and the extent of purification is in excess of 1000-fold. Both a-galactosidases I and II are devoid of other glycanase and glycosidase activities (<0.002%). Contamination of a-galactosidase I with endo-fl-mannanase was less than one part in 106 but complete removal of this activity from a-galactosidase II required a second chromatographic purification on the affinity column support. Properties. Affinity purified a-galactosidase II appears as a single band on isoelectric focusing (pI 4.6) and on SDS-gel electrophoresis (MW = 21,000). It shows optimal activity at pH 4.5-5.5 and is unstable at temperatures above 45 °. In contrast, a-galactosidase I appears as a number of protein bands on isoelectric focusing (pI values 5.8-6.6) but as a single broad band on SDS-gel electrophoresis (MW = 33,000). The enzymes catalyze the hydrolysis of a wide range of substrates with nonreducing terminal o-galactose, including melibiose, raffinose, stachyose, galactomannan, and p-nitrophenyl-a-o-galactopyranoside, but the relative initial rates of hydrolysis vary significantly. Both enzymes are very effective in the removal of o-galactose from galactomannans (Fig. 1). Galactomannans with widely different D-galactose contents and varying patterns of distribution of o-galactosyl residues along the D-mannan backbone are hydrolyzed at essentially the same rate.14
[79] X y l a n a s e o f Bacillus pumilus By
HIROSUKE
OKADA
and
ATSUHIKO
SHINMYO
Xylan, the primary component of pentosan, consists of a backbone chain of 1,4-1inked fl-o-xylopyranosyl residues and side chains of arabinose, glucuronic acid, or methylglucuronic acid. The backbone is hydrolyzed by endoxylanase or exoxylanase and the xylooligosaccharides formed are hydrolyzed to xylose by fl-xylosidase. Xylanase production has been reported in many microorganisms including fungi, yeasts, and bacteria. Since natural xylan is a heterogeneous polysaccharide, the hydrolysis of side chains is not well understood. Bacillus pumilus is a microbe producing potent xylan degrading enzymes. In our laboratory, endoxylanase (1,4-fl-o-xylan xylanohydrolase, EC 3.2.1.8) and fl-xylosidase (1,4-fl-o-xylan xylohydrolase, EC 3.2.1.37, xylan 1,4-fl-xylosidase) of B. pumilus IPO, 4 isolated from a rice field, were purified, and hydrolysis of xylan to xylose was found to be accomMETHODS IN ENZYMOLOGY, VOL. 160
Copyright © 1988by Academic Press, Inc. All rights of reproduction in any form reserved.
[79]
XYLANASEOF B. pumilus
633
plished by sequential reaction of these two enzymes.~ A chromosomal DNA fragment of B. p u m i l u s IPO containing genes of both enzymes was cloned in Escherichia coh ~ and the complete DNA sequences of both genes were determined. TM Here, we describe the purification and properties of the xylanase of B. p u m i l u s IPO.4 Assay Methods Principle. Xylanase activity is measured from the reducing sugars liberated from larchwood xylan (Sigma, St. Louis, MO, approximate molecular weight 20,000). Reducing sugar is measured by the dinitrosalicylic acid method. 5 Reagents
Potassium phosphate buffer, pH 6.5 Larchwood xylan (Sigma) Xylose 3,5-Dinitrosalicylic acid reagent P r o c e d u r e . Xylan is suspended in 50 mM potassium phosphate buffer at 1% (w/v) and boiled for 10 min to dissolve it. The reaction mixture, consisting of 1 ml of xylan solution and 0.5 ml of enzyme solution, is incubated at 40° for 10 rain. The reaction is stopped by adding 3 ml of 3,5dinitrosalicylic acid reagent. Reducing sugar present without incubation at 40° is subtracted from that with incubation. One unit of xylanase is defined as the amount of enzyme that liberates 1 ~mol of xylose equivalent in 1 rain. When a crude enzyme solution is used, liberation of reducing sugar without xylan must be checked. B. p u m i l u s IPO excretes xylanase in the culture medium, but accumulates fl-xylosidase in the cytoplasm. On the other hand, the E. coli clone which harbors the plasmid coding for the xylanase and ~-xylosidase genes accumulates both enzymes in its cells. Xylanase activity determined in E. coli cell extracts is overestimated 1w. Panbangred, A. Shinmyo, S. Kinoshita, and H. Okada, Agric. Biol. Chem. 47, 957 (1983). 2 W. Panbangred,T. Kondo,S. Negoro, A. Shinmyo,and H. Okada,Mol. Gen. Genet. 192, 335 (1983). 3 E. Fukusaki, W. Panbangred, A. Shinmyo,and H. Okada, FEBS Lett. 171, 197 (1984). 3~H. Moriyama, E. Fukusaki, J. Cabrera Crespo, A. Shinmyo, and H. Okada, Eur. J. Biochem. 166, 539 (1987). 4 B. pumulis IPO is depositedin the FermentationResearch InstituteYatabe-cho, Higashigun, Ibaraki, Japan, and is designated culture 4. E. coli plasmid pOXN391 in the same collection is designatedFerm P-6996. 5G. L. Miller,Anal. Chem. 31, 426 (1959).
634
HEMICELLULOSE
[79]
because of the presence of a/3-xylosidase which produces reducing sugars from the xylooligosaccharides formed by the xylanase reaction. When purified fl-xylosidase is added to xylanase solution at an activity ratio of 3: l, the apparent xylanase activity is about 1.2 times that without /3xylosidase. Purification Highly purified enzyme with a specific activity in the range of 17001900 units/rag protein can be obtained from the culture fluid by a few purification steps. Culture Conditions. The enzyme production medium consists of 0.5% larchwood xylan (Sigma), 0.5% Bacto-yeast extract (Difco), 0.2% NH4NO3, 0.2% KHEPO4, and 0.02% MgSO4.7H20, pH 6.8. B. pumilus IPO cells are grown in a test tube containing 10 ml L-broth [10 g Bactotryptone (Difco), 5 g Bacto-yeast extract, 5 g NaC1, and 1 g glucose in ! liter] overnight at 30 ° with shaking and the whole culture broth is transferred to 500 ml of production medium in a 3-liter Sakaguchi flask. The flask is incubated at 30 ° for 36 hr in a water bath with shaking. Then the culture fluid is obtained by centrifugation at 10,000 g for 15 min and used for xylanase purification. The following procedures are performed at <4 °. Step 1. The protein fraction in 2 liters of the culture fluid which precipitated in the ammonium sulfate concentration range between 0.2 and 0.6 saturation is collected as follows; solid ammonium sulfate (114 g/liter) is added to the culture fluid with stirring. After 3 hr, the supernatant is obtained by centrifugation at 15,000 g for 30 min, and further ammonium sulfate is added (262 g/liter). The precipitate is collected after 3 hr by centrifugation, dissolved in 200 ml of 50 mM potassium phosphate buffer, pH 6.5, and dialyzed for about 20 hr against several changes of 5 liters of the same buffer. The resultant insoluble materials, which contain residual xylan, are removed by centrifugation. Step 2. The dialyzed enzyme solution is applied to a DEAE-Sephadex column (5.5 x 25 cm) equilibrated with 50 mM phosphate buffer, pH 6.5 (the same phosphate buffer pH and composition are used in all other steps). On elution with the same buffer at a flow rate of 200-250 ml/hr, xylanase activity appears in the void volume, whereas most of the other proteins and colored materials are adsorbed onto the gel. Fractions of 50 ml are collected. The fractions containing specific activity greater than 100 units/mg protein are pooled to yield a total volume of about 500 ml with an enzyme activity recovery of 60% in this step. Step 3. The pooled active fraction in Step 2 is applied to a CM-Sephadex C-50 column (5.5 x 20 cm) which has been equilibrated with the
XYLANASEOF B. pumilus
[79]
635
buffer. The column is washed with 1 liter of the phosphate buffer and the enzyme is eluted from the column with a linear gradient established between 500 ml of the phosphate buffer and 500 ml of the buffer containing 0.6 M NaC1. Fractions of 20 ml are collected at the flow rate of 200 ml/hr. Xylanase activity is eluted at about 0.22 M NaCI concentration. The fractions containing specific activity greater than 800 units/mg protein are pooled to yield a total volume of 520 ml with 87% recovery of enzyme activity in this step. The solution is concentrated to a volume of 20 ml to 25 ml by ultrafiltration using a Toyo UM-10 membrane, and dialyzed against three changes of 2 liters of the phosphate buffer. Step 4. The dialyzate in Step 3 is added ammonium sulfate to 40% of saturation by adjusting the pH at 6.5 with 1 N KOH, and applied to a column (1.5 × 40 cm) o f T S K HW polyvinyl gel (Toyosoda, Tokyo) which has been equilibrated with the phosphate buffer containing 40% saturation of ammonium sulfate. The column is washed with 200 ml of the same equilibration buffer solution. Then the enzyme is eluted with a linear gradient of ammonium sulfate from 40% saturation to 0%, each in 200 ml of the phosphate buffer. The flow rate is 20 ml/hr and 5-ml fractions are collected. The enzyme activity is eluted at 13% saturation of ammonium sulfate with the specific activity of about 1800 units/mg protein. Recovery of the activity in this step is about 70%. Approximately 90-fold purification is achieved with an overall yield of 22%. A summary of the purification procedures is presented in Table I. Preparation of Xylanase from E. coli Cells Harboring the Plasmid. Five hundreds milliliters of L-broth containing 50/xg/ml of ampicillin in a 3-liter Sakaguchi flask is inoculated with 5 ml of culture broth of E. coli C600 harboring the hybrid plasmid pOXN391 which codes for the xylanase gene of B. pumilus IPO and the ampicillin-resistance gene, 3 grown at 37° overnight in the same medium, and incubated for 16-20 hr at 37° with shaking. E. coli (pOXN391) produces xylanase constitutively, TABLE I PURIFICATION OF XYLANASE
Step 1. 2. 3. 4. 5.
Culture fluid 20-60% (NH4)2SO~ DEAE-Sephadex A-50 CM-Sephadex C-50 TSK HW-65
Volume (ml)
Total protein (mg)
2080 220 600 20 10
8740 1830 234 47 22
Total Specific activity activity (units × 103) (units/mg) 175 110 67.2 56.6 39.1
20 60 287 1200 1780
Purification (fold)
Yield (%)
1 3 14 60 89
100 63 38 32 22
636
HEMICELLULOSE
[79]
whereas B. pumilus IPO xylanase is induced by xylan and also by xylose. Cells are harvested from 2 liters of culture by centrifugation at 10,000 g for 10 rain and washed once with 200 ml of ice-cold 50 mM potassium phosphate buffer, pH 6.5. The following procedures are done at <4 °. Washed cells are suspended in 25 ml of the same buffer solution and disintegrated by passing through a French pressure cell at 450-500 kg/cm z. The cell lysate is centrifuged at 10,000 g for 30 min and supernatant is again centrifuged at 40,000 g for 1 hr to yield clear supernatant. Xylanase is purified from the clear supernatant by the above procedure. Properties
Homogeneity and Molecular Weight. The purified enzyme preparation gave a single protein band on polyacrylamide disc gel electrophoresis with or without sodium dodecyl sulfate (SDS). The purity was estimated at more than 95%. No carbohydrate was detected by acid fuchsin staining of the gel. The molecular weight of the purified xylanase was 24,000 by SDS-polyacrylamide gel electrophoresis and 20,000 by equilibrium sedimentation, suggesting a single polypeptide chain. From the nucleotide sequence of the cloned xylanase gene in E. coli it was deduced that xylanase consists of 201 amino acid residues with a molecular weight of 22,384. Stability and Activity. The purified xylanase had an activity and stability optimum at pH 6.5. The enzyme retained more than 85% of its activity after standing at 40 ° for 30 min at pH 8.5 or 5.0. The maximum xylanase activity was observed at temperatures between 45 and 50° over a period of I0 min. The enzyme lost half of its activity when kept at 50° for 30 min, and the decrease was in accord with monomolecular kinetics. All activity was lost at 60 ° in 15 min. The xylanase activity was influenced by the salt concentration of the reaction mixture. In l0 mM phosphate buffer the activity was about 20% of the maximum value, which was obtained in 50-60 mM phosphate buffer. Sodium chloride added to 10 mM phosphate buffer affected the activity similarly. Amino Acid Sequence. The amino acid sequence of prexylanase, deduced from the DNA sequence is shown in Fig. 1. This is the first example of the total amino acid sequence of a xylanase. The protein is probably synthesized in the form of a prexylanase with a signal sequence consisting of 27 amino acids, of which 3 are basic amino acid residues in the region near N-terminus and 18 are hydrophobic amino acid residues. The signal sequence might be processed between Ala -1 and Arg +. The N-terminal amino acid sequence of the purified xylanase was NH2-Arg-Thr-Ile-Thr
XYLANASEOF B. pumilus
[79]
-27
-20
637 -I0
-l
MNLRKLRLLFVMCIGLTLILTAVPAHA
1
i0
20
30
40
50
RTITNNEMGNHSGYDYELWKDYGNTSMTLNNGGAFSAGWNNIGNALFRKG
60
70
80
90
i00
KKFDSTRTHHQLGNISINYNASFNPSGNSYLCVYGWTQSPLAEYYIVDSW
ii0
120
130
140
150
GTYRPTGAYKGSFYADGGTYDIYETTRVNQPSIIGIATFKQYWSVRQTKR
160
170
180
190
200
TSGTVSVSAHFRKWESLGMPMGKMYETAFTVEGYQSSGSANVMTNQLFIG
201 N
FIG. 1. The complete amino acid sequence of the prexylanase. The 27 amino acid signal peptide indicated by minus numbers might be processed between Ala and Arg. Amino acids are indicated by one-letter symbols.
and the C-terminal sequence was Ile-Gly-Asn-COOH by sequential Edman degradation and carboxypeptidase digestion, respectively. The amino acid composition from the purified enzyme agreed well with that deduced from the DNA sequence. Hydrolysis of Xylan and Xylooligosaccharides. The maximum degree of hydrolysis of xylosidic linkage of larchwood xylan (molecular weight 20,000) by the purified xylanase was about 25%. The end products were oligosaccharides, corresponding xylobiose (X2), xylotriose (X3), xylotetraose (X4), and higher oligomers. At the early stages of the reaction, xylose oligomers higher than xylopentaose were the only products. Authentic xylobiose was not hydrolyzed. Incubation with X3 yielded X4 and X2, while with X4 as the substrate, X2, X3, X4, and higher oligomers were detected, although the reaction rate was much slower than that toward xylan. The production of X4 from X3, and of the higher oligomers from X4 indicates that xylanase has trans-xylosidation activity.