WO1995012668A1

WO1995012668A1 - Thermostable xylanase from a thermomonospora fusca gene

Info

Publication number: WO1995012668A1
Application number: PCT/US1994/012743
Authority: WO
Inventors: David B. Wilson; Elyse D. Jung; Gurdev S. Changas; Diana C. Irvin
Original assignee: Cornell Research Foundation, Inc.
Priority date: 1993-11-05
Filing date: 1994-11-04
Publication date: 1995-05-11
Also published as: EP0728197A1; FI961885A0; FI961885A

Abstract

A xylanase encoded by a Thermomonospora fusca gene, wherein the enzyme is thermostable and has optimal activity in alkaline conditions. Also disclosed is the nucleotide sequence of the Thermomonospora fusca gene denoted xynA. The xylanase can be expressed recombinantly, and then purified. The purified xylanase can be proteolytically cleaved to produce a polypeptide fragment which retains about 90 % of the activity of the uncleaved xylanase.

Description

THERMOSTABLE XYLANASE FROM A THERMOMONOSPORA FUSCA GENE

This invention was made with government support under contract number DE-FG02-84-ER-13233 awarded by the United States Department of Energy- The U.S. government has certain rights in this invention.

Field of the Invention

The present invention relates to a gene encoding a xylan degrading enzyme. More particularly, the invention is directed to a Thermomonospora fusca gene encoding a xylanase that has several desirable properties including thermostability, activity in a high pH range, and lack of cellulase activity. The purified recombinant protein, and a polypeptide fragment derived therefrom by proteolysis, display xylanase activity and can be used in processes for delignifying and bleaching pulp.

Background of the Invention

Xylanases are hemicellulases that cleave the /?-l,4 linkage of xylan, which is a polymer of /3-1,4-D- xylopyranoside residues. Xylan is a major component of hemicelluloses from monocots, and is usually associated with the cellulose and lignin components of plant cell walls. One application for a xylanase is its usage in the pulp and paper industry for chemical pulp bleaching to reduce or replace the use of elemental chlorine. Xylanase treatment of pulp can specifically hydrolyze carbohydrate chain linkages between lignin and hemicelluloses, thereby increasing the permeability of the fiber surface, and facilitating removal of lignin. By using a xylanase pretreatment stage in the bleaching process of hardwood and softwood pulps, bleaching chemical usage can be significantly reduced, thereby improving the bleaching effluent (Forber, 1992, Pulp & Paper 66:90) . The environmental benefit of a reduction in chlorine usage in the bleaching process is reflected by the reduction in absorbable halogens (AOX) and other chlorinated organic compounds (including dioxin) in the effluent.

Numerous bacteria and fungi grow on xylan as a carbon source, using a number of enzymes, including exoxylanases, endoxylanases, -xylosidases, oi- glucuronidases, α;-arabinofuranosidases, and esterases (See for example, Biely, 1985, Trends Biotechnol . 3:286- 290) . Thus, several xylanases have been described in the art. U.S. Patent No. 5,116,746 (Bernier et al.) describes a vector having a Streptomyces gene encoding xylanase in a host microorganism comprising a mutant strain of Streptomyces lividans lacking cellulase and xylanase activity. Treatment of pulp with this extracellular xylanase is preferably at 50°C at a pH range of five to seven.

U.S. Patent No. 5,179,021 (du Manoir et al. ) discloses a process for bleaching lignocellulosic material comprising an oxygen bleaching treatment, and an enzymatic treatment with xylanase, preferably at a temperature of 50°C at a pH near five.

U.S. Patent No. 5,183,753 ( izani et al.) describes a process for producing xylanase from Thermomyces lanuginosus by cultivation of the fungus in a nutrient medium which contains corn cobs. Xylanase activity was optimum at a pH range of between five and seven, and a temperature range of 60-70°C. U.S. Patent No. 5,202,249 (Kluepfel et al. ) discloses a vector having Streptomyces gene xln C gene encoding xylanase in a host microorganism comprising a mutant strain of Streptomyces lividans lacking cellulase and xylanase activity. Treatment of pulp with this extracellular xylanase is preferably at 50°C at a pH of five to seven. It is desirable to have xylanase activity and enzyme stability at higher temperatures and higher pH ranges. Rather than having optimal activity at the current temperature range of about 55°C in slightly acidic conditions, a xylanase having optimal activity in a 70-80°C range, in alkaline conditions, would allow the dissolved hemicelluloses to be sent to the recovery cycle instead of to effluent treatment (Grant, 1991, Pulp & Paper 33:61). In this regard, actinomycete bacteria have been examined for thermostable xylanase activity (Davis et al., 1992, in Xylans and Xylanases. ed. J. Visser et al., Elsevier Science Publishers). Ωιe--rπjoιπonos[pora fusca is a filamentous soil thermophile that produces cellulolytic, xylanolytic, and pectinoltic enzymes. Extracellular fluids from medium containing T. fusca cultures are crude enzyme preparations that have exhibited optimal xylanase activity at temperatures greater than 70°C and at pH ranges between 7.0-9.0 (Davis et al. , supra) . The number of xylanases each T. fusca strain produces, varies amongst strains. Six proteins with xylanase activity have been identified from T. fusca strain BD21 by isoelectric focusing, followed by zymogram analysis (Bachmann, 1991) ; while four xylanases were detected from T. fusca strain YX (Ghangas et al. , 1989, J. Bacteriol. 171:2963-2969). A T. fusca gene encoding a xylanase was cloned and expressed in Escherichia coli and Streptoiηyces lividans, and the culture supernatant from the recombinant organisms exhibited xylanase activity not present from control transformants (Ghangas et al., supra) .

Summary of the Invention

The present invention is directed to a T. fusca gene (xynA) encoding a xylanase with an apparent molecular mass of the mature protein being about 32,000 daltons. The nucleic acid sequence of the present invention can be incorporated into vectors to form recombinant vectors, and the recombinant vectors can be introduced into a host cell system for the expression of the gene product. The present invention is also directed to the purified recombinant xylanase, denoted TFXA, with an apparent molecular mass of the mature protein being about 32,000 daltons; and a polypeptide fragment, from proteolytic cleavage of the recombinant protein, having catalytic xylanase activity and an apparent molecular mass of 24,000 daltons. The recombinant xylanase, and the polypeptide derived by proteolysis, exhibit significant enzyme activity at temperatures greater than 75°C and at a pH range of from about 7.0-9.0. Thus, the gene, the gene product, and the polypeptide fragment of the present invention have applications in delignifying and bleaching pulp.

Brief Description of the Figures

FIG. la is a restriction map of pTXIOl and the strategy for sequencing the insert containing xynA.

FIG. lb is a restriction map of pYY02 containing xynA which results in increased production of TFXA.

FIG. 2a represents a Coomassie-stained acrylamide gel showing the molecular weight standard, TFXA in lane 1, and proteolyzed TXFA showing the resultant 24kD polypeptide fragment in lane 2.

FIG. 2b represents a Remazol brilliant blue (RBB) -xylan overlay of a similar acrylamide gel containing TFXA in lane 1, and proteolyzed TFXA in lane 2. For this gel, the protein samples were not boiled before loading.

FIG. 3 are graphs representing the binding activity of TFXA and the 24kD polypeptide.

FIG. 3a is a graph depicting xylan binding activity of TFXA (•) and the 24kD polypeptide (D ) . FIG. 3b is a graph depicting crystalline cellulose binding activity of TFXA and the 24kD polypeptide. FIG. 4 are graphs showing the thermostability and pH range of TFXA.

FIG. 4a is a graph which represents the activity of TFXA upon incubation for 18 hours at various temperatures ranging from 50°-90°C.

FIG. 4b is a graph which represents the activity of TFXA in the range pH 5 to pH 9.

FIG. 5 represents thin-layer chromatography of the products of hydrolysis of birchwood xylan by TFXA. The standards are xylose (XI) , xylobiose (X2) , glucose (Gl) , cellobiose (G2) , cellotriose (G3) , cellotetraose (G4) , and cellopentaose (G5) .

Detailed Description of the Invention The present invention is directed to compositions comprising a xylanase of bacterial origin, wherein the purified enzyme has been designated TFXA. In accordance with this invention, the nucleotide sequence of the gene encoding xylanase TFXA, xynA, is disclosed. The gene sequence described herein has been isolated from the thermophilic soil bacterium T. fusca. As indicated by the nucleotide sequence of the present invention, xynA reveals that the predicted amino acid sequence of the mature TFXA protein has a calculated molecular mass of about 32,167 daltons. According to one embodiment of the present invention, using recombinant DNA techniques, xynA or a gene fragment encoding the catalytic domain of TFXA, is incorporated into an expression vector, and the recombinant vector is introduced into an appropriate host cell thereby directing the expression of these sequences in that particular host cell. The expression system, comprising the recombinant vector introduced into the host cell, can be used to produce TFXA and TFXA catalytically active polypeptides in the extracellular fluid from the culture. According to the present invention, TFXA can be purified by methods known in the art including ion-exchange chromatography. Additionally, polypeptides containing xylanase activity can be synthesized chemically from the amino acid sequence disclosed in the present invention, or can be produced from enzymatic or chemical cleavage of the purified mature protein TFXA. The thermostability of the enzyme compositions described herein,^' and activity at various pH ranges are disclosed.

For purposes of the description, the following embodiments illustrate the manner and process of making and using the invention and set forth the best mode contemplated by the inventor for carrying out the invention, but are not to be construed as limiting: Embodiment A- Molecular cloning and sequencing of the T. fusca xynA gene encoding TFXA;

Embodiment B- Characterization of the T. fusca gene, xynA;

Embodiment C- Expression and purification of recombinant

TFXA and generation of a catalytically active polypeptide;

Embodiment D- Physicochemical characterization of recombinant TFXA and a catalytically active polypeptide.

Embodiment A Molecular cloning and sequencing of the T. fusca xynA gene encoding TFXA.

Cloning the xylanase gene xynA from T. fusca YX into a lambda library was described previously (Ghangas et al., 1989, supra) . Briefly, T. fusca chromosomal DNA was partially digested using EcoRI, and fragments in the range of 4 to 14 kb were isolated. These fragments were ligated to lambda phage EcoRI arms, and the ligation mixture was packaged with phage in vitro. Upon screening and finding a plaque with xylanase activity, the DNA insert from this clone (λxyl-1) was further subcloned into phage by partially digesting the insert DNA with Sail and ligating the DNA from this partial digest with lambda phage Sail arms (clone λxyl-2) . Screening of the plaques for xylanase activity was done using a agarose- soluble xylan overlay with either Congo Red, or Remazol brilliant blue in the detection process. The xylanase gene from λxyl-2 was then cloned into pBR322 (Sail cut and dephosphorylated with calf intestine alkaline phosphatase) , yielding plasmid pTXIOl. E. coli C600 ( supE44 hsdR thi -1 thr-1 leuBβ lacYl tonA21) , transformed with pTXIOl, is designated D467, and was used to subclone the xynA into sequencing vectors. The host strain for subsequent transformations and transfections was E. coli JMlOl (r_k' ' supE thi Δ (lac-proAB) [F' traD36 proAB lac-FZ M15] ) . The xynA was subcloned in two segments both into M13mpl8 and M13mpl9 phage vectors (Bethesda Research Laboratories, Gaithersburg, Md.) to allow sequencing in both orientations. A restriction map of pTXIOl and the subclones is shown in Figure la. Transformation of JMlOl and preparation of single-strand DNA were performed using methods according to the directions provided by the manufacturer of the phage vectors.

Modified T7 DNA polymerase and the dideoxy chain-termination method were used to sequence single-strand templates. A commercially available kit that included both dGTP and dlTP labelling mixes (Sequenase ™, United States Biochemical Corporation, Cleveland, Ohio) was used to resolve band compressions in sequencing gels. The high G+C content of T. fusca DNA produces many areas of secondary structure in template DNA, and band compressions frequently complicated sequencing gels. The addition of formamide (to 20% vol/vol) to the 6% polyacrylamide gels helped reduce problems arising from secondary structure. Oligonucleotide primers, usually 15-17 mers, were synthesized to facilitate sequencing. A computer program (DNA Inspector lie™, TEXTCO, West Lebanon, NH) was used to determine the correct open reading frame encoding TFXA, codon usage, predicted molecular weight, and hydrophilicity of TFXA. The reading frame of SEQ ID No. l, coded for a protein having a molecular weight of 32,167 daltons; which is in good agreement with the apparent molecular mass of 32,000 daltons of TFXA, as determined by SDS-gel electrophoresis. Using methods known in the art of molecular biology, xynA may be inserted into various expression vectors to increase production of TFXA. One such plasmid was constructed by ligating parent plasmid pUC18 cut with Sail, and a Sail restriction fragment containing xynA in forming pYYOl. pYYOl, cut with Hindlll and

SphI, was ligated with the Hindlll-SphI fragment from pI5702 carrying the tsr gene forming pYY02 (Fig. lb) . S. lividans transformants containing pYY02 produced approximately twice as much TFXA as did transformants containing pTXIOl.

Embodiment B Characterization of the T. fusca gene, xynA.

To confirm that gene xynA encoding TFXA had been identified, the amino terminal sequence of TFXA was determined. For determining the N-terminal sequence of TFXl,

ER1, a protease-minus mutant of T. fusca YX, was grown as previously described (Hagerdal et al., 1978, Appl. Environ. Microbiol . 36:606-612) with 1% xylan as carbon source. The culture was centrifuged after 24 hours of growth, and the supernatant was treated with 1 mM phenylmethylsulfonyfluoride (PMSF, a protease- inhibitor). A stirred cell with a 30,000 molecular weight cutoff membrane was used to concentrate the supernatant. A precipitate formed during concentration, and the supernatant was found to retain only 44% of the original xylanase activity. The precipitate was recovered by centrifugation and resuspended in 50 mM unbuffered Tris. A portion of the resuspended material was subjected to SDS/ acrylamide gel electrophoresis, and a well-isolated 32 kD protein (TFXA) band was visualized by Coomassie blue stain.

The resuspended material was run in seven lanes of a 12% SDS PAGE mini-gel and electroblotted to a transfer membrane using 10 mM CAPS (3- [cyclohexylamino] -1- propanesulfonic acid), 10% methanol buffer, pH 11. The transfer membrane was stained according to a procedure as provided by the manufacturer (for Immobilon^,M, Millipore Corporation, 1990) . The transferred 32 kD protein bands were compared with known protein standards, and estimated to contain about 0.5 micrograms per band. These bands were excised and used for determination of the N- erminal sequence on a gas phase protein sequencer, with a blank piece of the transfer blot used as a negative control. The experimentally determined N-terminal sequence of TFXA was:

NH₂-Ala-Val-Thr-Ser-Asn-Glu-Thr-Gly-Tyr-His-Asp-Arg This sequence is identical to the N-terminal sequence (amino acids 43 through 54) predicted by the open reading frame of xynA as indicated in SEQ ID No. 1. A signal sequence of 42 amino acids, which precedes the N-terminus of TFXA, resembles those seen in other actinomycetes (Hύtter and Eckhardt., 1988, in Actinomycetes in biotechnology, p.89-184, ed. by Goodfellow et al. , Academic Press, Inc.).

An analysis of the G+C content of the sequence of the insert encoding TFXA reveals that the insert DNA has G+C content of 88% in the third position of the codons, and a 65% overall G+C content. The high G+C content is typical of many thermophilic organisms; a related organism, T. curvata, has an overall G+C content of 67% (Petricek et al., 1989, J. Gen . Microbiol . , 135: 3303- 3309) . The G+C bias in the third position of the codons was used to identify the correct reading frame in the DNA sequence. A potential ribosome binding site, AAGGAGG is located 11 bases upstream of the initiation codon, ATG. This sequence is perfectly complementary to the 3' end of the 16S RNA of both S. lividens (Bibb, 1982) and E. coli (Shine, 1974) . The A+T-rich region commonly found upstream of the translational start codon in many actinomycete genes is not seen in this sequence.

A surprising finding is that the T. fusca xynA promoter region is not AT rich like most promotors including the four T. fusca cellulase gene promotor regions sequenced. Each of the cellulase genes contain a stretch of DNA that is 50-60% AT upstream of their regulatory sequences while the region preceding xynA is 30% AT, which is essentially the same as the rest of xynA. The 14 base inverted repeat present in the E2, E4, and E5 cellulases of T. fusca (Lao et al., 1991, J^". Bacteriol . 173:3397- 3407) is not present in xynA. This inverted repeat has been shown to be the binding site of a regulatory protein (Lin et al., 1988, J. Bacteriol . 170:3843-3846).

Embodiment C Expression and purification of recombinant TFXA and generation of a catalytically active polypeptide. Recombinant TFXA was expressed in an expression vector system comprising a recombinant plasmid containing xynA, which was introduced into a host microorganism. The pTXIOl insert containing xynA was further subcloned into shuttle plasmids capable of replicating in E. coli and S. lividans as described previously (Ghangas et al., 1989, supra) . One of the resulting recombinant plasmids, pGG92, was used to transform S. lividans , and the transformants were grown in one liter of tryptone soy broth with 10 μg thiostrepton/ ml at 30°C for 36 hours. Recombinant TFXA was purified from the culture supernatant by first harvesting the supernatant by centrifugation and then adding PMSF (to 1 mM) and glycerol (to 10%) . The solution was concentrated from 800 ml to 250 ml and dialyzed against 0.02M Tris HCI pH 9.0 + 10% glycerol. The dialyzed material was loaded on a 60 ml ion-exchange column (Q Sepharose™, Pharmacia) which was equilibrated with the same buffer. Most of the protein bound to the column but protein containing xylanase activity passed through. This material was concentrated using a stirred cell with a membrane having a 10,000 MW cut off. The final purified protein (1.2 mg) had a specific activity of 490 μmoles xylose/min-mg.

Purified TFXA was proteolytically cleaved into polypeptides. TFXA (100 μl) in 50 mM unbuffered Tris (approximately 74 ug protein) was incubated for 18 hours at 50°C with 3 μl ( 1.5μg) of a protease isolated from T. fusca (Gusek et al., 1987, Biochem. J. 246:511-517) . SDS at a final concentration of 0.1% or 1.0% was included in the reaction mixture. The products of proteolysis were observed on a 16% SDS/acrylamide gel

(Fig. 2a) . Incubation of xylan with T. fusca protease of 0.1% SDS resulted in the formation of a 24kD polypeptide fragment as seen on a Coomassie-stained 16% SDS/acrylamide gel (Fig. 2a, lane 2) . A fragment of this size would be expected if proteolysis of the mature xylanase enzyme occurs near the 3' end of the Gly-Pro-rich region of the protein. In fact, sequencing of the N-terminus of the 24kD polypeptide isolated from an SDS/acrylamide gel showed that it was the same as intact protein TFXA. Inclusion of 1.0% SDS in the proteolysis reaction prevented this cleavage. Using methods known in the art of molecular biology, the 24kD polypeptide may also be produced recombinantly by inserting a portion of the xynA gene into an expression vector. Since the 24kD polypeptide is a fragment of TFXA, and lacks the C-terminal amino acids of TFXA, a truncated xynA gene lacking the coding sequence for C- erminal amino acids but inserted adjacent to, or being ligated to a translational termination sequence, can be used for expression of the polypeptide in transformed host cells such as S. lividans. Recombinant 24kD polypeptide may be purified according to the method for purifying recombinant TFXA.

Embodiment D Physicochemical characterization of recombinant TFXA and a catalytically active polypeptide.

Xylanase binding assays were performed to examine the ability of TFXA and the 24kD polypeptide to bind to xylan. Insoluble xylan was used so that activity lost from the assay supernatant represents the activity bound to the xylan. Insoluble xylan was prepared from larchwood xylan as follows: One gram of xylan was suspended in 20 ml of H₂0 brought to pH 10 with IN NaOH and stirred gently at room temperature for one hour. The xylan was centrifuged at 3,000 x g for 5 min, washed with H₂0. This was repeated twice with water and finally with 50mM sodium acetate, pH 5.5. The pellet was then suspended in 10 ml of 95% ethanol and filtered on Whatman No.l paper. The pellet was dried in a desiccator and ground as finely as possible. The yield of insoluble xylan was 0.6g. Aliquots of insoluble xylan (0-25 g) were measured into 0.5ml microfuge tubes. Each tube contained 400ml of 50 mM sodium acetate buffer, pH 5.5 and 15ml (11 μg) of either TFXA or the 24kD polypeptide. Tubes were rotated end-over-end for one hour at room temperature. At 15 minute intervals the samples were briefly agitated with gently vortexing to ensure adequate mixing of enzyme and substrate. After incubation the samples were centrifuged at high speed for 5 minutes and the supernatants were transferred to clean tubes. Supernatants were again centrifuged before activity assays were run. Before being loaded onto SDS/ polyacrylamide gels, samples were concentrated by TCA precipitation; 120 μl of supernatant was treated with 5ml of 100% TCA and kept on ice for one hour. Samples were centrifuged for 5 minutes and the precipitated protein washed once with 150 μl of 70% ethanol. Samples were centrifuged, the pellets resuspended in 15-20 ml of 1.5X SDS gel-loading dye, and the samples boiled for 5 minutes prior to loading onto the gel. The results of the xylanase binding assays indicate that both TFXA (•) and the 24kD polypeptide (D) could be removed from solution by the addition of insoluble xylan followed by centrifugation (Fig. 3a) . Furthermore, TFXA (•) but not the proteolytic fragment (D) , could be removed from solution by the addition of Avicel

(crystalline cellulose) followed by centrifugation (Fig. 3b) . In each case the presence of protein in the supernatant was determined by SDS gel electrophoresis. The results of quantitative binding experiments of TFXA and the 24kD polypeptide to xylan and Avicel, shown in Figure 3, prove that the C- erminal domain removed by proteolysis is essential for cellulose binding and greatly increases the affinity of the enzyme for xylan. Despite the ability of the TFXA xylanase to bind to cellulose it had no activity on any cellulosic substrate tested (CMC, acid swollen cellulose, filter paper) . Thus, from the binding assays it appears that the C-terminal domain may be involved in both binding to xylan and to crystalline cellulose, although only xylan is a substrate. TFXA and the 24kD polypeptide were assayed for xylanase activity with soluble xylan, using both the DNS (dinitrosalicylic) assay (Wilson, 1988, Methods Enzymol . 160:314-323) and a xylan overlay of an SDS/polyacrylamide gel. The DNS assays of the 24kD polypeptide and TFXA were performed in sodium acetate buffer (50 mM, pH 5.5) with 2% soluble xylan. Reaction mixtures were incubated for 30 min at 50°C; 1 ml of DNS reagent was added to the 400 μl assay mixture and the samples were boiled for 15 min. The absorbance was read at 600 n . The specific activity was determined from the amount of enzyme (μg) required to achieve 5.6% digestion of the xylan substrate, and xylose was used to generate a standard curve. Thermostability was tested by heating enzyme samples for 18 hours at various temperatures and then assaying using DNS. Assays at different pH values were performed as above except that the buffer was 0.05M Na glycine at pH range of 8-11, and 0.05M citric acid mixed with 0.05 Na₂HP0₄ at pH range 2-8. Thin layer chromatograms of the products of xylan digestion were run as previously described (Jung et al., 1993, Appl . Env. Microbiol . 59:3032-3043) using lOμl of the reaction mixtures.

The results of the DNS assays, presented in Table 1, show the Vmax and Km values for both the TFXA and the 24 kD polypeptide.

TABLE 1. Xylanase Activity

Specific Activity

Molecular μτa xylose/ j μm xylose/ mass Km (mg/ml) Vmax^a min-μmole 1 min-mg

TFXA, 32kD 1.1 600 15600 490

24kD poly-peptide 2.3 540 14200 440 μm xylose/min-mg As shown in T-, ie 1, the Vmax for both TFXA and the 24kD polypeptide are very similar, but the Km exhibited by the 24kD polypeptide is higher. Since xylan is heterogeneous and the xylanase assay is not linear with enzyme or substrate, values for specific activity were also calculated for the percentage of hydrolysis (5.6%) of the substrate. These values, as expected, are somewhat lower than the Vmax values, but as shown in Table 1, the specific activity of the 24kD polypeptide is again as nearly as active as TFXA.

Using the above assay, TFXA was tested for thermal stability and pH optimum. Figure 4a shows activity of TFXA upon incubation for 18 hours at various temperatures ranging from 50°-90°C. As shown in Fig. 4a, the activity of the samples incubated at 50° and 65°C was optimal; and of particular interest, the sample incubated at 75°C retained 96% of the original activity. Analysis of these samples by SDS-PAGE showed 32kD bands, indicating that the protein had not been cleaved. Figure 4b shows that TFXA retains at least 67% of its activity in the range pH 5 to pH 9. Of particular importance, when the assay was performed in a Na-glycine buffer, rather than Na₂HP0₄-citric acid buffer or KPi buffer, the activity at pH 8 was significantly higher. For the xylan overlay, samples containing 3μl

(2.8μg) of either TFXA and the 24kD polypeptide were electrophoresed on a 16% SDS/polyacrylamide gel. The gel was soaked in 100 ml of 10% isopropanol/90% 50 mM KPi, pH6 for 30 minutes to remove SDS and allow renaturation of the protein. The gel was then rinsed for an additional 30 minutes in 50 mM KP_i# pH 6, with gentle shaking. In a modified procedure (Ghangas et al., 1989, supra) an overlay of RBB-xylan was prepared and bound to Gel Bond™ (FMC Corp.). The overlay was placed on the gel and incubated for 75 min at 50°C. Clearing of the blue RBB-xylan by the 32 kD intact xylanase and the 24 kD polypeptide demonstrated catalytic activity (Fig. 2b) . These results show that the N-terminal domain, common to both TFXA and the 24 kD polypeptide is the catalytic domain. Thin-layer chromatography of the products of hydrolysis of xylan by TFXA (Fig. 5) showed that many oligomers of xylose are produced and that the smallest product produced is xylobiose. The results show that TFXA is definitely an endoxylanase. At least twelve xylanase genes have been cloned and sequenced and they all fit into two families (Glikes 1991) .

A search of the Genbank sequence database revealed four xylanases all in family G (Glikes et al., 1991, Microbiol . Rev. 55:303-315) whose amino acid sequences are similar to that of TFXA. The greatest similarity occurs in the middle of the proteins, a region containing one of two Glu residues believed to be involved in active site catalysis. TFXA is approximately 30% larger than the other xylanases, and contains a 21 amino acid Gly-Pro rich region that separates the N-terminal catalytic domain from an 86 amino acid region that is involved in substrate binding. TFXA clearly belongs to family G, but unlike the other members of this family, it contains a binding domain that binds both cellulose and xylan, but only xylan is a substrate. Thus, the ability of TFXA to bind to both xylan and cellulose appears to be a novel property amongst xylanases. TFXA also exhibits thermostability and a wide pH profile, desirable features for use in the processes of delignifying and bleaching pulp.

It should be understood that while the invention has been described in detail herein, the examples were for illustrative purposes only. Other modifications of the embodiments of the present invention that are obvious to those skilled in the art of molecular biology, enzymology, industrial biotechnology, and related disciplines are intended to be within the scope of the appended claims.

What is claimed is:

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANTS: David B. Wilson et al . (ii) TITLE OF INVENTION: Thermostable Xylanase From a Thermomonospora fusca Gene (iii) NUMBER OF SEQUENCES: 1 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Hodgson, Russ, Andrews, Woods & Goodyear

(B) STREET: 1800 One M&T Plaza (C) CITY: Buffalo

(D) STATE: New York

(E) COUNTRY: United States

(F) ZIP: 14203-2391 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette, 3.5 inch, 720 Kb storage

(B) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: MS-DOS/ Microsoft Windows 3.1

(D) SOFTWARE: Wordperfect for Windows 5.1 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER:

(B) FILING DATE: November 4, 1994 (vii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Nelson, M. Bud

(B) REGISTRATION NUMBER: 35,300 (C) REFERENCE DOCKET NUMBER: 18617.0002 (viii) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (716) 856-4000

(B) TELEFAX: (716) 849-0349 (2) INFORMATION FOR SEQ ID NO. 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1273 nucleo ides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double-stranded (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: genomic DNA (iii) HYPOTHETICAL: yes (iv) IMMEDIATE SOURCE:

(A) LIBRARY: genomic (B) CLONE: lambda clone

(C) SUBCLONE: pTXIOl (v) ORIGINAL SOURCE:

(A) ORGANISM: Thermomonospora fusca

(B) STRAIN: YX36 (C) CELL TYPE: bacterium (vi) FEATURE:

(A) LOCATION: xy-na gene region

(B) IDENTIFICATION METHOD: by experiment

(C) NAME/KEY: signal sequence of encoded protein (D) LOCATION: -42 to -1

(vii) SEQUENCE DESCRIPTION: SEQ ID NO. 1:

GTCGACGACT CGACGTGTGG CTCACCTAGC CGCGTCATCC CCGACGCCGA 50 GGAGCGGCGA CCACTGACCC CGGTCATCGC CCCCCACCAG GTCGCAGGAT 100

GCTCCCTGGT CCCCTCCAGC CCGTACAAGG AGGAAACACC CACA ATG 147

Met -42

AAC CAT GCC CCC GCC AGT CTG AAG AGC CGG AGA CGC TTC CGG 189 Asn His Ala Pro Ala Ser Leu Lys Ser Arg Arg Arg Phe Arg

-30

CCC AGA CTG CTC ATC GGC AAG GCG TTC GCC GCG GCA CTC GTC 231 Pro Arg Leu Leu lie Gly Lys Ala Phe Ala Ala Ala Leu Val

-20 GCG GTC GTC ACG ATG ATC CCC AGT ACT GCC GCC CAC GCG GCC 273 Ala Val Val Thr Met lie Pro Ser Thr Ala Ala His Ala Ala -10 -1 1

GTG ACC TCC AAC GAG ACC GGG TAC CAC GAC GGG TAC TTC TAC 315 Val Thr Ser Asn Glu Thr Gly Tyr His Asp Gly Tyr Phe Tyr

5 10 15

TCG TTC TGG ACC GAC GCG CCT GGA ACG GTC TCC ATG GAG CTG 357 Ser Phe Trp Thr Asp Ala Pro Gly Thr Val Ser Met Glu Leu 20 25

GGC CCT GGC GGA AAC TAC AGC ACC TCC TGG CGG AAC ACC GGG 399 Gly Pro Gly Gly Asn Tyr Ser Thr Ser Trp Arg Asn Thr Gly 30 35 40

AAC TTC GTC GCC GGT AAG GGA TGG GCC ACC GGT GGC CGC CGG 441 Asn Phe Val Ala Gly Lys Gly Trp Ala Thr Gly Gly Arg Arg 45 50 55 ACC GTG ACC TAC TCC GCC AGC TTC AAC CCG TCG GGT AAC GCC 483 Thr Val Thr Tyr Ser Ala Ser Phe Asn Pro Ser Gly Asn Ala 60 65 70

TAC CTG ACC CTC TAC GGG TGG ACG CGG AAC CCG CTC GTG GAG 525 Tyr Leu Thr Leu Tyr Gly Trp Thr Arg Asn Pro Leu Val Glu

75 80 85

TAC TAC ATC GTC GAA AGC TGG GGC ACC TAC CGG CCC ACC GGT 567 Tyr Tyr lie Val Glu Ser Trp Gly Thr Tyr Arg Pro Thr Gly 90 95

ACC TAC ATG GGC ACG GTG ACC ACC GAC GGT GGT ACC TAC GAC 609 Thr Tyr Met Gly Thr Val Thr Thr Asp Gly Gly Thr Tyr Asp 100 105 110

ATC TAC AAG ACC ACG CGG TAC AAC GCG CCC TCC ATC GAA GGC 651 lie Tyr Lys Thr Thr Arg Tyr Asn Ala Pro Ser lie Glu Gly 115 120 125 ACC CGG ACC TTC GAC CAG TAC TGG AGC GTC CGC CAG TCC AAG 693 Thr Arg Thr Phe Asp Gin Tyr Trp Ser Val Arg Gin Ser Lys 130 135 140 CGG ACC AGC GGT ACC ATC ACC GCG GGG AAC CAC TTC GAC GCG 735 Arg Thr Ser Gly Thr lie Thr Ala Gly Asn His Phe Asp Ala 145 150 155

TGG GCC CGC CAC GGT ATG CAC CTC GGA ACC CAC GAC TAC ATG 777 Trp Ala Arg His Gly Met His Leu Gly Thr His Asp Tyr Met

160 165

ATC ATG GCG ACC GAG GGC TAC CAG AGC AGC GGA TCC TCC AAC 819 lie Met Ala Thr Glu Gly Tyr Gin Ser Ser Gly Ser Ser Asn 170 175 180

GTG ACG TTG GGC ACC AGC GGC GGT GGA AAC CCC GGT GGG GGC 861 Val Thr Leu Gly Thr Ser Gly Gly Gly Asn Pro Gly Gly Gly 185 190 195

AAC CCC CCC GGT GGC GGC AAC CCC CCC GGT GGC GGT GGC TGC 903 Asn Pro Pro Gly Gly Gly Asn Pro Pro Gly Gly Gly Gly Cys 200 205 210 ACG GCG ACG CTG TCC GCG GGC CAG CAG TGG AAC GAC CGC TAC 945 Thr Ala Thr Leu Ser Ala Gly Gin Gin Trp Asn Asp Arg Tyr 215 220 225

AAC CTC AAC GTC AAC GTC AGC GGC TCC AAC AAC TGG ACC GTG 987 Asn Leu Asn Val Asn Val Ser Gly Ser Asn Asn Trp Thr Val

230 235

ACC GTG AAC GTT CCG TGG CCG GCG AGG ATC ATC GCC ACC TGG 1029 Thr Val Asn Val Pro Trp Pro Ala Arg lie lie Ala Thr Trp 240 245 250

AAC ATC CAC GCC AGC TAC CCG GAC TCC CAG ACC TTG GTT GCC 1071 Asn lie His Ala Ser Tyr Pro Asp Ser Gin Thr Leu Val Ala 255 260 265

CGG CCT AAC GGC AAC GGC AAC AAC TGG GGC ATG ACG ATC ATG 1113 Arg Pro Asn Gly Asn Gly Asn Asn Trp Gly Met Thr lie Met 270 275 280 CAC AAC GGC AAC TGG ACG TGG CCC ACG GTG TCC TGC AGC GCC 1155 His Asn Gly Asn Trp Thr Trp Pro Thr Val Ser Cys Ser Ala 285 290 295

AAC TAGTCC TGCCGGATAA CCCCAGCTGT GGCGCGGCGG TTCAGGGGGC 1204 Asn

CGCCGCGCCA CACCCGCGAG CAGAGCACTC CGCTCGCGGC GATTGGTCCG 1254 GGAATGCGGT GTGCGCTGG 1273

Claims

1. A purified xylanase which has thermostability characterized by retaining greater than 90% of its activity at 75°C, a wide pH range characterized by retaining greater than 65% of its activity in a range of pH 5 to pH 9, an apparent molecular mass of about 32,000 daltons, and encoded by Thermomonospora fusca gene xynA having a nucleotide sequence consisting essentially of the nucleotide sequence of SEQ ID No. 1.

2. The xylanase of claim 1, further characterized as having an optimum pH in the range pH 7 to pH 9.

3. The xylanase of claim l, further characterized as having a binding domain for cellulose but lacking cellulase activity.

4. The xylanase according to claim 1, wherein the xylanase is produced recombinantly from a host cell genetically engineered to include a vector containing xynA.

5. The xylanase of claim 4, wherein the host cell is Streptomyces lividans.

6. A purified and isolated polypeptide representing a fragment of the xylanase according to claim 1, wherein the polypeptide is characterized as having an apparent molecular mass of about 24,000 daltons, and retaining about 90% of the activity of the xylanase according to claim 1.

7. The polypeptide of claim 6, wherein the polypeptide is the product of proteolytic cleavage of the xylanase.

8. The polypeptide according to claim 6, wherein the polypeptide is produced recombinantly from a genetically engineered host cell.

9. The polypeptide of claim 8, wherein the host cell is Streptomyces lividans.

10. The polypeptide of claim 6, further characterized as lacking a binding domain for cellulose.

11. A method of hydrolyzing the 0-1,4 linkage of xylan thereby facilitating removal of lignin, said method comprising subjecting the xylan to hydrolysis with the xylanase according to claim l.

12. A method of hydrolyzing the 0-1,4 linkage of xylan thereby facilitating removal of lignin, said method comprising subjecting the xylan to hydrolysis with the xylanase according to claim 4.

13. A method of hydrolyzing the 0-1,4 linkage of xylan thereby facilitating removal of lignin, said method comprising subjecting the xylan to hydrolysis with the xylanase according to claim 6.

14. An isolated and purified nucleic acid molecule consisting essentially of a nucleotide sequence of SEQ ID No. 1 and which encodes the xylanase according to claim 1.

15. An isolated and purified polypeptide consisting of an endoxylanase, said endoxylanase having:

(a) an apparent molecular mass of about 32,000 daltons in its mature form; (b) catalytic activity of a xylanase, wherein at least 65% of its maximum activity is observed in a reaction with xylan substrate performed in a range of pH 5 to pH 9;

(c) optimal catalytic activity which is observed in a temperature range of 50°C to 65°C, and wherein at least 90% of the optimal activity is retained at temperatures greater than 70°C;

(d) binding domains for both xylan and cellulose, with the xylan binding domain located in the N-terminal portion of the endoxylanase, and the cellulose binding domain located in the C-terminal portion of the endoxylanase; and

(e) a specific activity in a range of about 15,000 to about 16,000 μm xylose/min-μmole.

16. A purified and isolated polypeptide, wherein the polypeptide is characterized as having an apparent molecular mass of about 24,000 daltons and having the N-terminal binding domain but lacking the C-terminal binding domain of the endoxylanase according to claim 15; and retaining at least 90% of the activity of the endoxylanase according to claim 15.