Characterization of a thermostable family 10 endo-xylanase (XynB) from Thermotoga maritima that cleaves p-nitrophenyl-β-d -xyloside

JOURNALOFBIOSCIENCE AND BIOENGINEERING Vol. 92, No. 5,423428.2001

Characterization of a Thermostable Family 10 Endo-Xylanase (XynB) from Thermotoga maritima That Cleaves p-Nitrophenyl-P-D-Xyloside JIANG ZHENGQIANG,’ ATSUSHI KOBAYASHL’MOHAMMAD MAINUL AHSAN,’ LI LITE,’ MOTOMITSU KITAOKA,’ AND KIYOSHI HAYASHI’* National Food Research Institute, 2-I-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan’ Received 2 July 2001/Accepted 16 August 200 1

Thermotoga maritima MSBS possesses two xylanase genes, xynA and qmB. The xynB gene was isolated from the genomic DNA of T. maritima, cloned, and expressed in Escherichia coli. XynB was purified to homogeneity by heat treatment, affinity chromatography and ion-exchange column chromatography. The purified enzyme produced a single band upon SDS-PAGE corresponding to a molecular mass of 42 kDa. At 70°C, the enzyme was stable between pH 5.0 and pH 11.4, and it was stable at temperatures of up to 100°C from pH 7.0 to pH 8.5. At 50°C, XynB displayed an optimum pH of 6.14 and at this pH the temperature for optimal enzyme activity was 90°C. XynB exhibited broad substrate specificity and was highly active towards p-nitrophenyl-/3D-xylobioside with K,,, and &at values of 0.0077 mM and 5.5 s-l, respectively, at 30°C. It was also active towardsp-nitrophenyl-P-D-xyloside. The initial product of the cleavage ofp-nitrophenyl-pD-xyloside was xylobiose, indicating that the major reaction in the cleavage was transglycosylation, not hydrolysis. [Key words: Thermotoga maritima, xylanase B, characterization,

transglycosylation]

The hyperthermophilic bacterium Thermotoga maritima, an extremely thermophilic anaerobe that grows at temperatures between 55 and 90°C and has an optimal temperature of 80°C, can utlilize xylan to generate metabolic energy. This bacterium is isolated from geothermally heated sea floors in Italy and the Azores (IO, 11). The entire genomic sequence of T. maritima MSB8 has already been reported (10). It is worth noting that some of the most thermostable xylanases are from Thermotoga species, such as those isolated from 7: maritima and T. neapolitana (12, 13). Most xylanolytic microorganisms produce more than one type of xylan-degrading enzyme to assist in the degradation of xylanosic materials. These enzymes often form complexes with each other and this makes it difficult to isolate them homogeneously. Recombinant DNA technology has proven to be very useful in that it allows the isolation of pure enzymes when they are expressed in bacterial hosts. Through the application of recombinant DNA techniques in the study of xylanases, numerous xylanase genes (about 270 so far) have been isolated, cloned and expressed in Escherichia coli; subsequently the recombinant enzymes have been purified for characterization (2, 5). In general, xylanases can be grouped into two families, family 10 and family 11, based on the sequence similarities of their respective catalytic domains. Family 10 is composed of high-molecularweight xylanases and includes some cellulolytic enzymes. However, family 11 consists of low-molecular-weight xylanases (14). T. maritima MSB8 has two xylanase genes, xynA and xynB (10) and both xylanases belong to the glycosyl hydrolase family 10 and the identity of their amino acid se-

Second only to cellulose, hemicellulose has immense potential foor use as an alternative source of energy. A major component of the hemicellulose fraction of plant cell walls is xylan, a complex molecule with a backbone of 1,Clinked P-D-xylopyranose residues (1). This backbone carries various substituents such as arabinose and glucuronic acid, depending on the source of hemicellulose. Typically, depolymerization of the backbone is accomplished by the action of endoxylanases (EC 3.2.1.8; 1,4-P-D-xylanase, xylanohydrolase) and P-xylosidases (EC 3.2.1.37; 1,4-p-D-xylan xylohydrolase). Xylanases can hydrolyze j3- 1,-l-glycosidic linkages of the xylan backbone to produce short chain xylo-oligosaccharides of varying lengths; therefore it is the key enzyme in the complete degradation of xylan (2). Over the past few decades, xylanases have received a great deal of attention, mainly due to their many and varied industrial applications such as in the pulp and paper industry, and in the food and feed industries. In addition, there has also been an interest in xylanases for use in the production of xylose, xylobiose and xylo-oligomers (3-5). Many industrial processes involving xylanases, across a diverse range of applications, are optimally performed at elevated temperatures, thus it is highly desirable to identify thermostable xylanases as industrial catalysts. The xylanases from thermophilic bacteria such as Rhodothermus marinus, Thermomonospora sp. and Thermotoga sp. show optimal temperatures in the range from 65-100°C (6-9). * Corresponding author. e-mail: [email protected] phone: +8 l-(0)298-38-8071fax: +8 l-(0)298-38-7321 423

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J. BIOSCI.BIOENG.,

quences is 39%. Of the two genes, only xynA (TM0061, accession number AEO01693) (10) has been cloned and expressed in E. coli. The purified recombinant XynA is quite thermostable, and has a half-life of about 40 min at 90°C at its optimal pH of 6.2 (15, 16). According to homology searches of the amino acid sequences, the XynB of I: maritima MSB8 has 85% identity with the XynA of Thermotoga sp. FjSS3-B.1, and 83% with the XynB from 7Y neapolitana which is optimally active at 90°C (8, 17). In the present study, a second xylanase gene (xynB, TM0070, AEO01693) (10) from the genomic DNA of 7: maritima MSB8 was isolated, cloned and expressed in E. coli, and the purification and characterization of the translated xylanase was investigated.

MATERIALS Bacterial

AND METHODS

strains and plasmids

The genomic

DNA of 7:

maritima MSB8 was kindly provided by Professors Karl 0. Stetter and Robert Huber. Topo-XL TOP 10 [F-mcr AA(mrr-hsdRMSmcrBC)Q8OlacZAM 15 AlacX74 recAl deoR araD 139 A(araleu)7697 gaN guZK rpsL (strR) endA nupG] was used as the host for the XynB gene from 17 maritima (TOPO@XL PCR cloning kit, Invitrogen, USA). E. coli BL21 (DE3) [hsd (clts 857 indl sum 7 nin5Iac WY-T7 genel)] was used as the host for the recombinant plasmid harboring the pET28a (+) vector (Novagen, Madison, WI, USA) and for subsequent expression. Recombinant DNA techniques as described by Sambrook et al. (18) were employed to perform DNA manipulations. Construction of an expression vector carrying the qtiB gene For the amplification of the xynB gene, PCR reactions were performed using a Gene Amp PCR system 9600 (Perkin-Elmer, Norwalk, CT, USA). The PCR conditions employed were as follows: a hot start at 98°C for 5 min, 22 repeated cycles consisting of 98°C for 30 s, 62°C for 1 min and 68°C for 1 min; and a final extension at 68°C for 10 min. Two primers were designed on the basis of the nucleotide sequence of the xynB gene. Forward primer: 5’ CCATGGAAA TATTACCTTC TGTCBGAT (containing a NcoI site (underlined)); Reverse primer: 5’ AAGCTTT CTTTCTTCTA TCTTTTTCTC CA (containing a Hind111 site (underlined)). For this construction, a C-terminal His-tag was introduced, and a one codon mutation in the primer was generated with the introduction of a 5’ restriction site into the cloning step (ATGA*AA + ATGG*AA). For the translated amino acid, the presence of the NcoI site changed the second residue of XynB from K to E. The PCR products were purified from agarose gels using QIAquick gel extraction (QIAgen, Germany). The amplified DNA fragment (1 .O kb) of xynB was cloned into a pCR-TOPO-XL vector using a pCRTOP0 cloning kit according to the protocol of the supplier (Invitrogen, The Netherlands). The resulting recombinant plasmids, TM-xynB-TOPO-XL, were extracted using a QIAminiprep kit (QIAgen) and were sequenced to ensure the nucleotide sequences were those of the xynB gene. DNA sequencing was performed on an Applied Biosystems 3 10 Genetic Analyzer using a Dye Terminator Cycle sequencing kit (Perkin-Elmer). The sequence data were analyzed using the GENETYX program (Software Development Co., Tokyo). Cloning and expression of the xynB gene in E. coli The xynB gene was excised from the recombinant plasmid TM-xynBTOPO-XL using the restriction enzymes NcoI and HindIII, and then it was ligated with the pET28a (+) vector, which had been digested with the same pair of restriction enzymes. Ligation was conducted overnight at 16°C using a ligation kit, High T, DNA ligase (Toyobo Co., Osaka). E. coli BL21 competent cells were

transformed by electroporation with the ligated plasmid. Positive colonies, selected from Luria-Bertani broth (LB)-plates containing kanamycin (50 ug/ml), were screened by colony PCR using the vector-specific primers (T7 promoter and T7 terminator primer) and the correct DNA sequence of the plasmid, xynB-pET28, was confirmed. Purification of XynB from E. coli Freshly prepared seed culture (100 ml) of E. coli BL2 1 harboring the recombinant xynBpET28 was added to LB medium (1000 ml) containing kanamycin (50 ug/ml) and was cultured at 30°C on a rotary shaker (150 revimin). Upon reaching an optical density of 0.5-0.6 at 600 nm, protein production was induced by the addition of 1 ml of I M IPTG to the culture medium. Cultivation was continued overnight at 30°C and cells were then harvested by centrifugation (1 l,OOOxg, 10 min at 4’C), and suspended in 50 mM Na-phosphate buffer, pH 8.0. Disruption of the cells was accomplished by sonication (Branson Sonifier 250D, USA) to extract the enzyme. The cell lysate was then centrifuged at 4°C (30,000 xg for IO min) and the supernatant collected. To denature some of the E. coli proteins, the crude cell extract was heated at 70°C for 10 min. The heat-treated crude cell extract was centrifuged at 4°C (30,OOOxg for 10 min). XynB remained in the clear supernatant. All column chromatography was performed at room temperature. The supernatant of the heat-treated crude cell extract (50 ml) was mixed with 2 ml of Ni-NTA agarose (QIAgen). The resin binding to the target protein was then packed into a 2-ml column and the subsequent washing and elution steps were performed with a linear gradient of 250 mM imidazole in 50 mM Na-phosphate buffer, pH 8.0, at a flow rate of 1 ml/min using a FPLC system (Amersham Pharmacia Biotech, Uppsala, Sweden). The active fractions were combined and dialyzed overnight at 4°C against 2 I of 5 mM MOPS buffer, pH 6.5. As a second purification step, the dialyzed solution was applied to a Q-Sepharose Fast Flow 16110 column (Amersham Pharmacia Biotech) which was pre-equilibrated with 25 mM MOPS buffer, pH 6.5, and the enzyme was eluted with a linear gradient of 0.5 M NaCl at a flow rate of 2 ml/min. Fractions were collected and assayed for xylanase activity and the active fractions were combined. For further purification, removal of salt from the combined active fractions was accomplished by dialysis against 5 mM MOPS buffer, pH 6.5. The dialyzed samples were reloaded onto an ion-exchange Mono Q HR5/5 column (Amersham Pharmacia Biotech) pre-equilibrated with 25 mM MOPS buffer, pH 6.5, and the adsorbed proteins were eluted with three successive gradients of O-100 mM, 100-300 mM and 300-500 mM NaCl(25 mM MOPS buffer, pH 6.5) at a flow rate of 0.5 mYmin. The active fractions eluted as a single protein peak and were subsequently used as purified XynB. The homogeneity of the purified enzyme was confirmed by SDS-PAGE (19). Enzyme assay Unless stated otherwise, xylanase was assayed by incubation for 20 min at 50°C in 50 mM MES buffer, pH 6.1, using 1.15% RBB-xylan (Remazol Brilliant Blue-R-D-xylan, Sigma, St. Louis, USA) as a substrate. Standard assay mixtures (100 ~1) consisted of 50 1.11 of 1.15% RBB-xylan, 25 ul of 200 mM MES buffer, pH 6.1, and 25 pl of enzyme solution. The reaction was stopped by the addition of 200 1.11 of 100% EtOH, which precipitated the residual substrate, and the mixture was kept at room temperature for at least 15 min. The mixture was then centrifuged at 21 ,OOOxg for 5 min and the absorbance of the supernatant was measured at 595 nm against the respective substrate blanks (20). One unit of xylanase activity is defined as the amount of enzyme required to produce 1 unit of absorbance per minute under the assay conditions described above. Determination of pH and thermal profiles For the determination of the optimum pH, various buffers at 50 mM, namely citrate (pH 2.2-4.1), sodium acetate (pH 3.8-5.7), MES (pH S.l-7.2), MOPS (pH 6.2-8.2) sodium phosphate (pH 6.2-8.2), HEPTOSE

THERMOSTABLE XYLANASE FROM T. MARITIMA

VOL.92,200l TABLE 1. Protein (mg) 255 195 12.3 2.00 0.64

Purification step Crude extract Heat precipitation Ni-NTA Q-Sepharose Mono-Q

425

Summary of xylanase B purification

Total activity* (H) 76.0 69.0 31.8 16.5 8.50

Purification factor (-fold)

Specific activity Wmg) 0.30 0.35 2.59 8.30 13.3

1.0 1.2 8.6 27 44

Recovery W) 100 91 42 22 11

a One unit of xylanase activity was defined as the amount of enzyme necessary to cause I AAbsS,, per minute. (pH 6.5-8.6), Tris-WC1 (pH 7.0-9.0), CHES (pH 8.2-10.2) and CAPS (pH 9.3-10.9), were used to assay the activity of the enzyme. In order to determine the pH stability, the enzyme was preincubated with the above buffers (except that CAPS (pH 9.3-10.9) was substituted by piperidine (pH 9.8-12.9)) for 30 min at two different temperatures (70°C and lOO’C), and then the residual activities of the treated enzymes were determined using the standard assay procedure. In order to estimate the optimum temperatures, the activity of the purified enzyme was determined at pH 6.1 using the standard assay with selected temperatures between 20 and 110% The measurement at 110°C was done in an autoclave. For determinations of the thermal stability, the enzyme was pre-incubated for 30 min in 50 mM MES at pH 6.1, with the temperatures ranging from 20 to 110°C. The heating was done in an autoclave at 110°C. After cooling, the treated enzymes were kept on ice for 10 min, then the remaining activity was determined using the standard assay method. pSubstrate specificity and analysis of kinetic parameters Nitrophenyl-P-D-xylobioside (pNP-X2) was synthesized as described previously (21), and the other substrates were purchased from SIGMA. Activity towards the p-nitrophenyl-derivatives was measured at 30°C by the rate ofp-nitrophenol release during substrate cleavage in 50 mM MES buffer, pH 6.1. The increase in absorbance was measured at 405 nrn using a Beckman Spectrophotometer (Model DU640, Beckman, USA) with a temperaturecontrolled cell holder. Initial hydrolysis rates were determined at six different concentrations ranging from approximately 0.5 to 2.0 times the K,,, value. The kinetic parameters and their standard errors were calculated using the nonlinear regression analysis program “Grafit” (22). The Influence of metal ions and EDTA on XynB activity influence of metal ions and the chelator EDTA on XynB activity was investigated in the presence of 1 mM of metal ions or other agents, compared to a control without metal ions or EDTA added, employing the standard assay conditions. Analysis of the reaction products Thin-layer chromatography (TLC), performed on silica gel plates (Merck Silica Gel 60F 254; E. Merck, Darrnstadt, Germany), was used to analyze the reaction products from 5 mM pNP-X2 or 15 mM p-nitrophenyl-P-Dxylopyranoside (pNP-Xl) in 50 mM MES buffer (pH 6.1) at 30°C. Aliquots were withdrawn at selected time intervals. The reaction mixtures were deionized with Amberlite MB3, concentrated, and spotted on silica gel TLC plates, then developed twice in an acetonitrile-water (85 : 15 v:v) solvent system. Saccharides were detected by heating the TLC plate for a few minutes in a hot dry oven after dipping the plates in a methanol-concentrated sulfuric acid mixture (95 : 5, v: v). A xylo-oligosaccharide mixture (Suntory Co., Osaka) containing xylose, xylobiose and xylotriose was used as the standard.

with a recovery of 11%. Heat treatment of the crude lysate at 70°C for 10 min provided a simple means of eliminating 23% of the host proteins by precipitation, and was therefore used as a convenient primary enrichment step in the purification of XynB. After heat treatment, Ni-NTA agarose was employed to trap the His-tagged enzyme. Two subsequent purification steps were performed using anion-exchange chromatography; a Q-Sepharose column followed by a Mono Q column. The combined active fractions from the Mono Q column revealed a single protein band with an apparent molecular mass of 42 kDa by SDS-PAGE analysis (Fig. 1). This is in good agreement with the molecular mass of 42,333 Da that was deduced directly from the amino acid sequence of the enzyme. Characterization of XynB The pH optimum of XynB was pH 6.1 at 50°C and 50% of the maximum level of activity was retained between pH 5.1 and pH 8.2 (Fig. 2). The cloned XynB exhibited pH stability in the pH range from pH 5.0-I 1.5 at 70°C and pH 7.0-8.5 at 100°C (Fig. 3). Figure 4 shows that a maximum level of enzyme activity was observed at a temperature of 90°C at pH 6.1. The distinct properties of this enzyme are its high degree of thermostability combined with a wide pH stability range and these properties may prove advantageous in industrial applications. While it is generally known that enzymes can be inhibited or activated by metal ions or other reagents, the influence of metal ions and a chelator (EDTA) on XynB activity

1

2

RESULTS Purification

A summary of the purification of in Table 1. XynB was purified 44-fold

of XynB

XynB is presented

FIG I. SDS-PAGE of the purified xylanase B. Lane 1, 10 kDa ladder marker protein; lane 2, purified xylanase B.

426

ZHENGQIANG ET AL.

J. Bmsci.

BIOENF.,

5

:E 60

5

m

.g

40

% z =

20

oc---_,,---,-30

40

0

-- _ 60

70

80

90

100

3 110

Temperature (oC) 2

3

4

5

6

7

8

10

9

11

12

FIG. 2. Effect of pH on xylanase B activity. The influence of pH on xylanase B activity was determined at 50°C using different buffers at 50 mM. Buffers used: citrate (closed diamond), acetate (closed square), MES (open triangle), MOPS (time), phosphate (asterisk), HEPES (closed circle), Tris-HCI (open circle), CHES (minus), CAPS (plus).

#’ 2

50

3

4

5

6

7

8

9

10

11

12

PH FIG. 3. Effect of pH on the stability of xylanase B. The residual activity was measured after incubation at 70°C (dotted line) and 100°C (solid line) for 30min over various pH ranges. Buffers used: citrate (closed diamond), acetate (closed square), MES (open triangle), MOPS (time), phosphate (asterisk), HEPES (closed circle), Tris-HCI (open circle), CHES (minus), piperidine (plus). at 1 mM concentrations

was only minimal. The addition of EDTA inhibited the xylanase activity by only 6%, suggesting that no metals are needed in the enzymatic reaction of XynB. Fe3+ (126% of the control), Mn2’ (119%), Mg2+ (112%) Zn*+ (105%) and Ca” (102%) showed some stimulation, whereas the addition of Co2+ (90%), Cu*’ (72%) and Hg2+ (3 1%) inhibited the enzyme activity. Substrate specificity and kinetic parameters Kinetic TABLE 2. Substrate pNP-B-D-xylobioside pNP-b-D-xylopyranoside PNP-P-D-cellobioside pNP-o-L-arabinopyranoside pNP-b-D-fucopyranoside .__

FIG. 4. Effect of temperature on the activity and stability of xylanase B. The temperature profile was measured at different temperatures using the standard assay as described in the Materials and Methods section at the optimum pH6.1 (open circle), in 50mM MES buffer. For determination of thermal stability of the enzyme, the residual activity of the heat-treated enzyme was measured (closed circle).

parameters for various p-nitrophenyl-glycosides were measured at pH 6.1 and a temperature of 30°C and the results are given in Table 2. For p-nitrophenyl-disaccharide derivatives, XynB was active toward both pNP-X2 and pNP-BD-cellobioside (pNP-CG2). The K,,, value for pNP-X2 was very low (0.0077 mM) compared with that for pNP-CG2 (4.1 mM), whereas the kc, values for both the substrates were similar (5.5 and 4.1 s-l, respectively). XynB was also active toward some p-nitrophenyl-monosaccharide derivatives. The p-nitrophenyl-monosaccharide derivative for which XynB activity was highest was pNP-Xl followed by p-nitrophenyl-P-D-fucopyranoside and p-nitrophenyl-a-Larabinopyranoside. XynB exhibited low activity towards pnitrophenyl-P-D-galactopyranoside (5.2%, the relative rate for pNP-Xl hydrolysis at 1 mM was defined as lOO%), and p-nitrophenyl-P-D-glucopyranoside (0.5%). Additionally, XynB showed no activity towards p-nitrophenyl-a-D-arabinofuranoside, p-nitrophenyl-a-L-arabinofuranoside, p-nitrophenyl-P-L-arabinopyranoside, p-nitrophenyl+L-fucopyranoside or p-nitrophenyl-B-D-mannopyranoside. Time course of the reaction with pNP-X2 and pNP-Xl The time courses of the reaction with 5 mM pNP-X2 and 15 mM pNP-Xl were tracked by using TLC. As shown in Fig. 5A, the main product in the pNP-X2 reaction was xylobiose indicating that hydrolysis of the bond between pNP and xylobiose was the major reaction. Small amounts of higher oligosaccharides were also detectable on TLC plates, indicating that transglycosylation occurred to some extent. On the contrary, xylose was not detected in the early stage of the pNP-Xl reaction (Fig. 5B) and xylobiose was the main product in the reaction. This indicates that the cleavage of pNP-Xl was not caused by simple hydrolysis.

Kinetic parameters of xylanase B

K, (mM) 0.0077 f0.0007 7.750.8 4.IkO.7 5.3kI.3 8.5f0.4

kc, (0 5.5fl.l 2.8kO.2 4.1+0.3 0.59+0.01 0.88f0.02

k,,jK,

(mM_‘.s-‘)

7lOf50 0.37f0.02 1.OfO.l 0.11 f0.02 0.104+0.003

-

THERMOSTABLE XYLANASE FROM 7: MARITIM

VOL. 92,200l

pNP-X :2xylose

xylobiose xylotriose

h

min

PNP-Xl

xylose

xylobiose xylotriose

min

h

FIG. 5. Thin-layer chromatography - _ _ of the hydrolysis products formed from pNP-X2 (A) and pNP-Xl (B) during 8 h irkuba6on with xvlanase B. The substrates of nNP-X2 and oNP-XI are in lane X2 &d Xl. Incubation times (minbr h) are indicated. A mixture of xylose, xylobiose and xylotriose was used as the standard (lane Xn).

DISCUSSION In contrast to other Thermotoga species such as the Thermotoga sp. strain FjSS3-B.l and ?Yneapolitana which each possess three different xylanase genes, I: maritima MSB8 has only two xylanase genes (10, 23). According to a homology search of the deduced amino acid sequences, XynB is most closely related to XynA from the Thermotoga sp. strain FjSS3-B. 1 (85% identity, accession no. U33060) (17, 24), and shows the second highest degree of similarity (83% identity) with the XynB from T. neapolitana (accession no. 249961). The other xylanases showed identities of less than 44%. Thermostability is a desirable property for xylanases used in industrial processes, and from the characterization of XynB summarized above, it is apparent that the cloned enzyme is extremely thermophilic and thermostable, in common with other xylanases from the Thermotoga species (7, 8, 11, 15-17). The optimum pH of XynB is similar to that of the recombinant xylanase A from the Thermotoga sp. strain FjSS3-B. 1, the pH optimum of which is 6.3, with

427

50% of the maximum activity level being retained in the pH range from pH 5.1 to 8.1 (17, 24). Interestingly, XynB was stable in the neutral to alkaline pH region and was relatively less stable in the acid pH region; moreover, it was stable at 100°C over the range of pH from pH 7.0-8.5, and this represents an attractive feature with regard to potential industrial applications. It is notable that XynB cleaves pNP-Xl at a k,, value comparable to that for pNP-X2. Some of the family 10 xylanases have been reported to cleave pNP-Xl (13, 2529). Schofield and Daniel found that the cleavage of pNPX1 by XynA from Caldocellum saccharolyticum was not caused by a simple hydrolysis but by transglycosylation (25). Amano et al. also described the similar mechanism in the cleavage of pNP-Xl by xylanases from Irpex lacteus (26). The cleavage of pNP-Xl by XynB of E maritima was not caused by simple hydrolysis and a transglycosylation step must occur in the reaction. The fact that the main product of the reaction was xylobiose (Fig. 5b) suggests that the reaction proceeded in the following manner. A xylosyl unit of pNP-Xl was transferred to another pNP-Xl molecule to form pNP-X2 followed by the hydrolysis into p-nitrophenol and xylobiose. This speculation is supported by the finding that Xy& has a very low K, value (0.0077 mM) for pNP-X2 and the major reaction toward pNP-X2 is hydrolysis (Fig. 5A). XynB hardly hydrolyzed xylobiose even with a longer incubation (Fig. 5A). This fact strongly suggests that the pNPX1 cleaving activity is not related to P-xylosidase activity. The driving force of the cleavage of pNP-X 1 is possibly explained by the strong affinity to the p-nitrophenyl group of XynB, evidenced by the low K, value for pNP-X2. ACKNOWLEDGMENTS

This work was partially supported by a grant from the Program for the Promotion of Basic Research Activities for Innovative Biosciences. The authors would like to convey their sincere thanks to Professors Karl 0. Setter and Robert Huber for the supply of genomic DNA of Thermotoga maritima. The UNU-kirin Fellowship is gratefully acknowledged for a grant to Mr. Jiang Zhengqiang. We also thank Drs. P. Selvakumar and K. Goyal for useful discussions.

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