APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1994, p. 763-770 Vol. 60, No. 30099-2240/94/$04.00+0Copyright © 1994, American Society for Microbiology

Characterization and Sequence of a Thermomonospora fuscaXylanase

DIANA IRWIN, ELYSE D. JUNG, AND DAVID B. WILSON*Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853

Received 3 December 1993/Accepted 17 December 1993

TfxA is a thermostable xylanase produced by the thermophilic soil bacterium Thermomonospora fusca. Theenzyme was purified to homogeneity from the culture supernatant of Streptomyces lividans transformed byplasmid pGG92, which carries the gene for TfxA, xynA. The molecular mass of TfxA by sodium dodecylsulfate-polyacrylamide gel electrophoresis is 32 kDa. TfxA is extremely stable, retaining 96% of its activity after18 h at 75°C. It has a broad pH optimum around pH 7 and retains 80% of its maximum activity between pH5 and 9. The native enzyme binds strongly to both cellulose and insoluble xylan even though it has no activityon cellulose. Treatment of TfxA with a T. fusca protease produced a 24-kDa catalytically active fragment thathad the same N-terminal sequence as TfxA. The fragment does not bind to cellulose and binds weakly to xylan.The Vmax values for TfxA and the fragment are 600 and 540 ,umol/min/mg, respectively, while the Kms are 1.1and 2.3 mg of xylan per ml, respectively. The DNA sequence of the xynA gene was determined, and it containsan open reading frame that codes for a 42-amino-acid (42-aa) actinomycete signal peptide followed by the32-kDa mature protein. There is a 21-aa Gly-Pro-rich region that separates the catalytic domain from an 86-aaC-terminal binding domain. The amino acid sequence of the catalytic domain of TfxA has from 40 to 72%identity with the sequences of 12 other xylanases from seven different organisms and belongs to family G. Twoglutamic acid residues, previously identified as essential for catalytic activity in BaciUlus pumilus XynA, areconserved in all 13 proteins. TfxA is the only thermophilic xylanase in family G as well as one of only twofamily-G xylanases to contain a binding domain.

Xylanases cleave the 3-1,4 linkage of xylan, a polymer witha linear backbone of P-1,4-D-xylopyranoside residues, whichare commonly substituted with acetyl, arabinosyl, and gluc-uronosyl residues. Xylan is a major component of hemicellu-lose from monocots and is usually associated with the celluloseand lignin components of plant cell walls. Numerous bacteriaand fungi grow on xylan as a carbon source by using a varietyof enzymes, including exoxylanases, endoxylanases, ,B-xylosi-dases, c-glucuronidases, a-arabinofuranosidases, and esterases(3, 5, 30, 33).Zymogram analysis of isoelectric focusing gels has revealed

six protein bands with xylanase activity from Thermomono-spora fusca BD21 (1) and four from T. fusca YX (9). The genethat codes for one of these, xynA, has been cloned andexpressed in Streptomyces lividans (9). The gene product is a32-kDa protein (TfxA) that is excreted into the culture super-natant. In this article, we report the purification, N-terminalamino acid sequence, and characterization of this xylanase aswell as the DNA sequence of the xynA gene. Most xylanasegenes that have been cloned and sequenced fit into eitherfamily F or G (11). A search of GenBank found 12 xylanasesthat show significant sequence identity to TfxA, and theybelong to family G (11). TfxA differs from all of the otherfamily-G xylanases in its thermostability, which is a usefulproperty for industrial purposes, and it is one of only twofamily-G xylanases that contain a substrate-binding domain.

* Corresponding author. Mailing address: Section of Biochemistry,Molecular and Cell Biology, Cornell University, 458 BiotechnologyBuilding, Ithaca, NY 14853. Phone: (607) 255-5706. Fax: (607) 255-2428.

MATERUILS AND METHODS

Bacterial strains and plasmids. A xylanase gene was previ-ously cloned from T. fusca YX, (4) first into XgtWES.AB andthen into pBR322, to yield plasmid pTX101 (9). The Esche-richia coli strain C600 (supE44 hsdR thi-1 thr-I leuB6 lacYltonA21) (26) that contains this plasmid is designated D467.The host strain for transformations and transfections was E.coli JM101 {rk' mk' supE thi A(lac-proAB)[F' traD36 proABlacIqZ M15]} (26). Plasmid pGG92 (9) consists of pTX101subcloned into the Streptomyces plasmid pIJ702, and S. lividansTK24 was the host strain.

Isolation of TfxA from T. fusca for N-terminal amino acidanalysis. ER1, a protease-minus mutant of T. fusca YX, wasgrown in 1 liter of Hagerdahl medium (14) with 1% xylan asthe carbon source. The culture was centrifuged after 24 h ofgrowth, and the supernatant was treated with 1 mM phenyl-methylsulfonyl fluoride (Sigma Chemical Co., St. Louis, Mo.).A stirred cell with a 30,000-molecular-weight-cutoff membrane(Millipore PTTK) was used to concentrate the supernatant. Aprecipitate formed during concentration, and the supernatantwas found to retain only 44% of the original xylanase activity.The precipitate was recovered by centrifugation and resus-pended in 50 mM Tris base (pH 9.5). After sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (9), awell-isolated 32-kDa protein band was visualized with Coom-assie blue stain.The resuspended material was run on an SDS-12% poly-

acrylamide minigel and electroblotted onto an Immobilon-Ptransfer membrane (Millipore) by using 10 mM CAPS (3-[cyclohexylamino]-1-propanesulfonic acid; Sigma)-10% meth-anol buffer (pH 11). The transfer membrane was stained by theprocedure described in the Immobilon instructions (23a). Thetransferred 32-kDa protein bands were compared with aknown amount of a reference protein standard (SDS7; Sigma)and estimated to contain about 0.5 ,ug per band. Seven of these

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FIG. 1. Plasmid and M13 subclone maps used for DNA sequenc-ing. On the plasmid map, DNA from T. fusca is indicated by theshaded portion of the circle; the unshaded portion is DNA frompBR322. Two SalI-PstI fragments of this insert were subcloned intoM13mpl8 and M13mpl9 vectors that had been digested with Sall andPstI. Arrows at either end of the insert DNA indicate that the DNAwas sequenced in both directions. The thin line represents M13 DNA.

bands were excised and used for determination of the N-terminal sequence on an Applied Biosystems model 470Agas-phase protein sequencer.

Purification of TfxA from S. lividans pGG92. S. lividansTK24 transformed by pGG92 (9) was grown in 1 liter oftryptone soy broth (Difco) containing 10 mg of thiostrepton at30°C for 36 h. The supernatant was harvested by centrifuga-tion, and phenylmethylsulfonyl fluoride (to 1 mM) and glycerol(to 10%) were added. The solution was concentrated from 800to 250 ml and dialyzed against 0.02 M Tris HCl (pH 9.0)-10%glycerol. The dialyzed material was loaded onto a 60-ml QSepharose (Pharmacia) column that was equilibrated with thesame buffer. Most of the protein bound to the column, but thexylanase activity passed through. The pass-through materialwas concentrated by using a stirred cell and a PTGC 10,000-molecular-weight-cutoff membrane (Millipore). The activity ofthe cleared supernatant was about 12 U/ml. The final purifiedprotein (1.2 mg) had a specific activity of 490 ,umol of xyloseper min per mg and represents about a 6% yield.

Xylanase activity assays. Assays were performed in KP,buffer (50 mM, pH 6) with 1% soluble birchwood xylan(Sigma). Reaction mixtures were incubated for 20 min at 50°C.To measure the reducing sugar produced, 1 ml of dinitrosali-cylic acid reagent (10) was added to the 400-jil assay mixture,the samples were boiled for 15 min, and the A600 was read. Thespecific activity was determined from the amount of enzyme

(micrograms) required to achieve 4% digestion of the xylansubstrate on the basis of a standard xylose curve. Km andmaximum velocity (Vmax) values were determined by usingsubstrate concentrations of from 0.1 to 1.2% xylan and 5.7pmol of enzyme per ml. Thermostability was tested by heatingenzyme samples for 18 h at various temperatures and thenassaying the activity at 50°C as described above. Assays at

1 2 1 2FIG. 2. (a) Coomassie blue-stained SDS-16% polyacrylamide gel

of xylanase TfxA before and after treatment with T. fusca protease.Lanes: 1, untreated xylanase; 2, xylanase treated with protease and0.1% SDS; mw, molecular mass standards (in kilodaltons; SigmaSDS7). (b) RBB-xylan overlay of proteolyzed and unproteolyzedxylanase after electrophoresis as described for panel a; except thatprotein samples were not boiled before loading the gel. Lanes 1 and 2are the same as those in panel a.

different pH values were performed as described above exceptthat the buffer was 0.05 M Na glycine at pH 8 to 11 and 0.05 Mcitric acid mixed with 0.05 M Na2HPO4 at pH 2 to 8, and theactivities were determined from the amount of enzyme re-quired to achieve 2% digestion. Thin-layer chromatograms ofthe products of xylan digestion were run as described previ-ously (19) with 10 [ul of the reaction mixtures. Xylose productswere red-brown in color, while glucose oligomers were blue-green when stained with p-anisaldehyde-sulfuric acid.

Xylan overlay. Samples containing 3 ,ul (2.8 ,ug) of eitherproteolyzed or nonproteolyzed TfxA were electrophoresed onan SDS-16% polyacrylamide gel. The gel was soaked, withgentle shaking, in 10% isopropanol-50 mM KPi (pH 6) for 30min and in 50 mM KPi (pH 6) for an additional 30 min toremove SDS and renature the protein. A xylan overlay wasprepared by pouring a boiling mixture of 0.15 g of RemazolBrilliant Blue-xylan (3), 0.25 g of agarose, and 30 ml of 0.05KPi (pH 6) buffer onto a sheet of Gel Bond (FMC Corp.) (9).The overlay was placed on the gel and was incubated for 75min at 50°C.

Proteolytic cleavage of TfxA. TfxA (74 jug) in 50 mM Trisbase-0.1% SDS (100 pul) was incubated for 18 h at 50°C with 3[1I (1.5 ,ug) of T. fusca protease (13). The products of prote-olysis were resolved on an SDS-16% polyacrylamide gel, andactivity was observed with a xylan overlay. Activity of theproteolyzed TfxA was also measured by the xylan hydrolysisassay. The N-terminal sequence of the 24-kDa proteolysisproduct was determined from an Immobilon blot as describedabove for intact TfxA.

Binding studies. Insoluble xylan was prepared from larch-wood xylan (Schweitzer Hall, Inc., Plainfield, N.J.) as follows.One gram of xylan was suspended in 20 ml of H20, and thesuspension was brought to pH 10 with 1 N NaOH and stirredgently at room temperature for 1 h. The xylan was centrifugedat 3,000 x g for 5 min and washed with H20. This wash wasrepeated twice with water and finally with 50 mM sodiumacetate (pH 5.5). The pellet was then suspended in 10 ml of95% ethanol and filtered on Whatman no. 1 paper. The pellet

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a100

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40

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0 20 40 60 80 1 00 1 20 1 40Insoluble Xylan (mg/ml)

b

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Avicel (mg/ml)

FIG. 3. Binding of TfxA to insoluble xylan (a) and Avicel (b). Symbols: 0, intact TfxA; Dl, proteolyzed TfxA. The xylanase concentration was

11 ,ug/ml, and the binding was done at room temperature for I h. Unbound xylanase was measured by the activity assay.

was dried in a desiccator and ground as finely as possible witha mortar and pestle. The yield of insoluble xylan was 0.6 g.

Binding experiments were run by adding 4.6 ,ug of protein tothe indicated amounts of Avicel or insoluble xylan in 400 p.l of0.05 M KPi (pH 6) buffer in 500-,ul Eppendorf tubes. Sampleswere rotated end-over-end for 1 h at 50°C and then centri-fuged. The amount of enzyme remaining in the supernatantwas determined by a xylanase activity assay.

Subcloning and sequencing ofxynA. The xylanase gene frompTX101 was subcloned in two segments into M13mpl8 andM13mpl9 phage vectors (Bethesda Research Laboratories,Gaithersburg, Md.) to allow sequencing in both orientations. Arestriction map of each subclone is shown in Fig. 1. Transfor-mation of JM101 and preparation of single-stranded DNAwere performed by using methods described previously (la).Modified T7 DNA polymerase and the dideoxy chain termina-tion method (27) were used to sequence single-strand tem-plates as described by Jung et al. (19). Computer analysis ofDNA sequence data was carried out as described previously(19).

Nucleotide sequence accession numbers and references. TheGenBank accession number of xynA is U01242. The accessionnumbers for the other sequences used are as follows: Clostrid-ium acetobutylichm xynB, M31726 (35); Bacillus pumilus xynA,X00660 (8, 21); Ruminococclus flavefaciens xynA, Z11127;Aspergillus kawachii xync, S45138 (18); Trichoderma reesei C30xynl and xyn2, S51973 and S51975 (31); Cochlioboluls carbo-numn xylJ, L13596; Bacilluts subtilis PAP115, M36648 (24);Bacillus circlulans xlnA, X07723 (34); S. lividans xynB and xynC,S68767 and S68769 (20, 28); Neocallimastix patriciarum xynA,X65526; Pseludomonas fluorescens subspecies cellulosa xylA,

X15429 (15); T. fusca El and E4, L20094 and L20093 (19); T.fusca E2 and E5, M73321 and L01577 (22). The alignment ofTfxA with similar proteins was generated by using the GCG(Genetics Computer Group, University of Wisconsin) (6)program Pileup with a gap weight of 3.0 and a gap lengthweight of 0.1.

RESULTS

Purification and proteolysis of TfxA. TfxA, purified tohomogeneity from S. Iividans(pGG92), was used to character-ize the properties of this xylanase. The molecular mass deter-mined by SDS-PAGE was about 32 kDa. Incubation of TfxAwith T. fusca protease resulted in the formation of a 24-kDafragment (Fig. 2a). A xylan overlay of an SDS-polyacrylamidegel containing both proteolyzed and nonproteolyzed xylanaseshowed that the 24-kDa product maintained catalytic activity(Fig. 2b). The xylanase isolated from T. fusca ERI was foundto have an N-terminal sequence of NH2-Ala-Val-Thr-Ser-Asn-Glu-Thr-Gly-Tyr-His-Asp-Gly, and the identical N terminuswas found for the 24-kDa fragment. These results show thatthe N-terminal domain is the catalytic domain since thefragment had nearly the same catalytic activity as that of theintact enzyme.

During the purification of TfxA, both from T. fusca and S.lividans, it became apparent that it is a hydrophobic protein.The addition of 10% glycerol was essential to avoid significantlosses due to precipitation. The purified protein, stored in 10%glycerol-0.01 M Tris (pH 8.5) buffer, was very stable and couldbe frozen and thawed many times. Precipitated TfxA could beresolubilized in Tris base but was no longer catalytically active.

TABLE 1. Xylanase activity

Sp act

Type of TfxA Molecular K,,, V,,, (pLmol ofmass (kDa) (mg/ml) xylose!min/mg) ([Lmol of xylose/ ([tmoI of

min/,umol) xylose/miim/mg)

TfxA 31.9 1.1 600 15,600 490Proteolyzed TfxA 24 2.3 540 14,200 440

" Determined at 4%' digestion of substrate in t).05 M KP1 (pH 6) buffer.

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TABLE 2. Activity of TfxA in the presence of metals

Buffer Sp act (,umol ofxylose/min/mg)a

NaAc" (0.05 M, pH 6) ......................................... 594NaAc + 1 mM EDTA......................................... 594NaAc + 1 mM HgCl2 ......................................... 211NaAc + 1mM CUS04 ............................... ........................ 411NaAc + 10 mM CaCl2 ......................................... 523

Determined at 2.7% digestion of substrate." NaAc, sodium acetate buffer.

Binding of TfxA and proteolyzed TfxA to xylan and Avicel.Both the native 32-kDa TfxA and the proteolyzed 24-kDafragment bound to xylan (Fig. 3a), although the proteolyzedenzyme bound much less strongly. The native enzyme alsobound to Avicel, but the proteolyzed TfxA did not bind toAvicel at all (Fig. 3b). These data prove that the C-terminaldomain removed by proteolysis increases the affinity of theenzyme for xylan and is essential for cellulose binding.

Activity of TfxA. The Vmax and Km values for both intact andproteolyzed TfxA are presented in Table 1. The Vmax valuesare very similar, but the Km exhibited by the proteolyzedenzyme is higher than that of the intact molecule. Xylan isheterogeneous, and the xylanase assay is not linear withenzyme or substrate. Therefore, in Table 1, we have alsopresented values for the specific activities which have beencalculated at the same arbitrary percentage of hydrolysis (4%)of the substrate. These values, as expected, are somewhatlower than the Vmax values, but the proteolyzed TfxA is nearlyas active as the native protein. Calculating the specific activityat a specified percentage of hydrolysis of the substrate hasproven very useful for the comparison of cellulases (10, 17),and it should also be useful for xylanases. The enzyme retainedfull activity in the presence of EDTA and calcium but was 31%inhibited by 1 mM CuS04 and 65% inhibited by HgCl2 (Table2). Mercury inhibition is commonly seen with cellulases andxylanases. Despite the ability of the xylanase to bind tocellulose, it had no activity on carboxymethyl cellulose, acid-swollen cellulose, filter paper, or Avicel even after an 18-hincubation. There was a small amount of reducing sugar

produced after incubation with Solka-flok; this suggests that asmall amount of xylan was present in the Solka-flok.Thermal stability and pH optimum of TfxA. TfxA was

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incubated for 18 h at temperatures from 50 to 90°C in itsnormal storage buffer which contained 10% glycerol, and theresults are shown in Fig. 4a. The activity of the samplesincubated at 50 and 65°C was actually higher than that of thesample kept overnight at - 20°C, and the sample incubated at75°C retained 96% of the original activity. When visualized on

SDS-polyacrylamide gels, these samples still gave 32-kDabands, showing that the protein had not been cleaved (data notshown). TfxA has a broad pH optimum (Fig. 4b), and it retainsgreater than 80% of its activity between pH 5 and 9.

Hydrolysis products. The products of xylan hydrolysis byTfxA are shown in Fig. 5. The smallest final product afterovernight incubation is xylobiose, and many higher oligomersare still present in the digest. This shows that TfxA is definitelyan endoxylanase. It is possible that these oligomers could notbe digested further because of the side chains on the xyloseresidues.DNA and protein sequence. The sequence of the xynA gene

is shown in Fig. 6. An open reading frame that coded for a

mature protein having a molecular weight of 31,890 was found;this is in good agreement with the molecular mass of 32 kDadetermined by SDS-PAGE. The N-terminal sequence of thexylanase protein isolated from T. fusca is identical to thatpredicted by the DNA sequence of the xynA gene. A signalsequence of 42 amino acids, which precedes the N terminus ofmature TfxA, resembles those seen in other actinomyceteproteins (16). The amino acid sequence from residues 230 to252, which contains mainly Pro, Gly, and Asn, is typical of thelinker sequences seen in T. fusca cellulases between thecatalytic domain and the cellulose-binding domain. Proteolysisnear the end of this linker region would produce a fragment ofabout 24 kDa. Computer analysis of the sequence predicts a plof 9.0 for the native protein and a pl of 7.4 for the proteolyzedprotein. This is in reasonable agreement with actual bands seenat pls of 10 and 7.5 on isoelectric focusing gels (PharmaciapHast gels, pH 3 to 10) overlaid with RBB-xylan (data notshown). The hydrophobic character of the protein was con-firmed by a Hopp Woods hydrophilicity plot of the amino acidsequence with the computer program DNA Inspecter Ile(TEXTCO).A second open reading frame was detected within the

PstI-SalI fragment of the plasmid (not shown), and the se-

quence is included in the GenBank file. A search of GenBankdid not reveal any DNA sequences similar to this sequence.

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FIG. 4. (a) Stability of TfxA after incubation at elevated temperatures; (b) activity of TfxA at different pH values in citric acid-sodiumphosphate buffer (0) and sodium-glycine buffer (O).

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DNA sequence features. An analysis of the protein sequence

encoded in the first open reading frame reveals that the insertDNA has a G+C content of 88% in the third position of thecodons and a 65% overall G+C content. The high G+Ccontent is typical of many thermophilic organisms, and a

related organism, Thermomonospora curvata, has an overallG+C content of 67% (25). The G+C bias in the third positionof the codons was used to check the DNA sequence.

A potential ribosome binding site, AAGGAGG, is located11 bases upstream of the potential initiation codon, ATG. Thissequence is perfectly complementary to the 3' end of the 16SRNA of both S. lividans (2) and E. coli (29). The A+T-richregion commonly found upstream of the translational startcodon in many actinomycete genes is not seen in the xynAsequence. The 14-base inverted repeat, TGGGAGCGCTCCCA, shown to be a binding site for a regulatory protein (23) inthe El, E2, E4, and E5 cellulase genes of T. fusca (22), is notpresent in the xylanase gene. There is a 12-base inverted repeatshortly after the stop codon of the first open reading frame(underlined in Fig. 6) that may function in the termination oftranscription.Comparison with other xylanases. (i) Catalytic domains. A

search of the GenBank sequence data base revealed 12 xyla-nases with amino acid sequences similar to that of the catalyticdomain of TfxA (Fig. 7). The identity between this region ofTfxA and the other xylanases ranges from 40 to 72%. The C.

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SerGlnThrleuValAlaArgProAsnGlyAsnGlyAsnAsnTrpGlyMetThrIleMetHisAsnGlyAsnTrpThrTrpProThrValP8tl .

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90

1803

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FIG. 6. T. fusca xynA DNA sequence and corresponding protein sequence. The mature N-terminal amino acid sequence, determined from the

purified enzyme, is underlined; vector DNA (M13) is italicized. A potential ribosome binding site (indicated by double underline) is 11 basesupstream from the translational start codon; there is a 12-base inverted repeat after the stop codon (indicated by dashed underline).

VOL. 60, 1994

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FIG. 7. Alignment of the TfxA catalytic domain with similar xylanases. Amino acid (AA) numbers begin with the start codon; the amino acidnumbers from the mature N termini are given in parentheses. The C terminus of the protein, if shown, is indicated by a colon (:), and residuesshown to be important for the catalytic activity of B. pumilus XynA by Ko et al. (21) are indicated by a plus (+). References and GenBank accessionnumbers are given in Materials and Methods.

acetobutylicum and the three Bacillus xylanases were classifiedby Gilkes et al. as belonging to family G (11), and by inference,all of these xylanases belong to family G. Two Glu (E-93 andE-182) were found to be essential for catalysis in the B. pumilus

xylanase by using site-directed mutagenesis (21). These resi-dues are conserved in all 13 proteins in Fig. 7. Mutation of a

third residue (D21E and D21S) had a less drastic effect but didlower the V,ax to less than half that of the wild type and

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CHARACTIERIZATION AND SEQUENCE OF A T FUSCA XYLANASE 769

Organism GeneT. fusca xynAS. fividans xInBT. fusca ElT. isca E4T. fusca E2T.fusca ESP. fluorescens xyIA

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FIG. 8. Comparison of the binding domains of bacterial cellulases and xylanases. The amino acid (AA) numbers given are from the start codon.Four or more aligned similar residues are boxed. GenBank accession numbers and references are given in Materials and Methods.

increased the Km by threefold (21). This residue is conserved in10 of the proteins.

(ii) Linker regions. The extensive Gly-Pro-rich region afterthe catalytic domain of TfxA is similar to short sequences thatare enriched in proline, serine, threonine, glutamine, or aspar-agine present in XynZ from Clostridium thermocellum (12),XylA from P. fluorescens (15), and in many cellulases. Thesesequences, also called hinge regions, are believed to separateconserved domains and facilitate the transfer of entire domainsbetween genes (11).

(iii) Binding domains. The apparent binding domain ofTfxA is from Cys-253 to Asn-338. This region has 64% identitywith the comparable region of S. lividans XynB, which wasreported to bind to xylan (28) but was not tested for binding tocellulose. The alignment of these xylan-binding domains withthe cellulose-binding domains of four T fusca cellulases (Fig.8) shows amino acid conservation of a number of hydrophobicresidues. T. fusca cellulase E5 was tested for binding to xylanand showed none. XylA of P. fluorescens, a member of familyF, has a binding domain encoded in the first 100 amino acids ofits N-terminal end which does not bind to xylan but does bindto cellulose (7).

DISCUSSION

The mechanism of regulation of xylanase production is notknown. Xylanase activity was shown to be induced in T. fuscacultures grown on either xylan or Solka-flok, while carboxy-methyl cellulase activity was induced only by Solka-flok (9). Wehave found that growth on Avicel also induces xylanase activityeven though Avicel does not contain any xylan. The absence ofxylan was shown by the lack of any reducing sugar after 18 h ofincubation with TfxA. Some Trichoderma strains produce highlevels of xylanase after growth on Solka-flok, while others donot (32). S. lividans has a host xylanase that was induced whencultures were grown on xylan, and cultures of S. lividanspGG92 grown on xylan showed greater xylanase activity thancultures grown in minimal medium plus glucose or cellobiose,but this increase could be attributed to the activity of the hostenzyme (9). There is no obvious A+T-rich promoter orregulatory region in the upstream DNA sequence of the xyn,Agene. Each T. fusca cellulase gene contains a stretch of DNAthat is 50 to 60% A+T upstream of their sequences, but theregion preceding the xylanase gene is only 30% A+T, which isessentially the same as that of the rest of the xylanase gene.

The four cloned T. fusca cellulases all contain an invertedrepeat upstream of their initiation codons that has beenidentified as the binding site for a protein that regulatesinduction, but such a sequence is not found in the xynA gene.A computer comparison of the upstream DNA of the homol-ogous xylanases did not reveal any common motifs such asinverted repeats. At this time, we do not know whether we aresimply unable to recognize a regulatory sequence or if xynzA isan internal gene in an operon.

It is interesting that the family of homologous catalyticdomains in Fig. 7 contains proteins from a wide range oforganisms with diverse properties. A. kawachii is a fungus usedin the fermentation of Shochu, a Japanese spirit, and its XynChas a pI of 3.5 (18). T. reesei Xynl and Xyn2 have pls of 5.2 and9.0 and V.ax values of 100 and 1,600 ,umol of xylose per minper ,umol (31). Reported Kms range from 0.14 mg/ml for T.reesei Xyn2 (31) to 6.3 mg/ml for B. pumilus XynA (8, 21).There is also a wide range of reported hydrophilicity.

It is also interesting that the TfxA binding domain can bindto cellulose as well as xylan. Since xylan is frequently associatedwith cellulose in plant cell walls, perhaps this ability is useful tothe organism and not just the result of random domainshuffling. An organism growing on a solid substrate (ratherthan in a solution) might find it useful to make a xylanase thatwould bind to a nearby solid substrate and produce solublesugars for its use. The only other family-G xylanase thatpossesses a binding domain is XylB from S. lividans. Indeed,the binding domain does not seem to be required for efficienthydrolysis of soluble xylan, as shown by the nearly identicalspecific activities of intact and proteolyzed TfxA. However, itmay play a role in the hydrolysis of the normal substrate of theenzyme, plant cell walls.There is currently much interest in the use of xylanases for

the prebleaching of kraft pulps (32). The thermostability andwide pH profile of TfxA make it an attractive candidate for thisfunction among the family-G xylanases and one of the best ofall reported xylanases.

ACKNOWLEDGMENT

This work was supported by grant FG02-84-ER13233 from theDepartment of Energy biosciences program.

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