WO2013127069A1

WO2013127069A1 - Thermostable xylanase from thermobifida fusca and methods of use thereof

Info

Publication number: WO2013127069A1
Application number: PCT/CN2012/071762
Authority: WO
Inventors: Zheyong YU; Kun ZHONG; Ling Hua
Original assignee: Danisco Us Inc.
Priority date: 2012-02-29
Filing date: 2012-02-29
Publication date: 2013-09-06

Abstract

Aspects of the present compositions and methods relate to a thermostable and alkali tolerant xylanase derived from Thermobifida fusca, polynucleotides encoding the xylanase, and methods for the production and use thereof. Formulations containing the recombinant xylanase have a wide variety of uses, including in processes carried out at high temperature and/or under alkali conditions, e.g., in pulp and paper processing and treating foodstuffs.

Description

THERMOSTABLE XYLANASE FROM THERMOBIFIDA FUSCA

AND METHODS OF USE THEREOF

Technical Field

[001] The present compositions and methods relates to a thermostable and alkali tolerant xylanase derived from Thermobifida fusca, polynucleotides encoding the xylanase, and methods for the production and use thereof. Formulations containing the recombinant xylanase have a wide variety of uses, for instance, in pulp and paper processing and treating foodstuffs.

Background

[002] Xylanases hydrolyze the carbohydrate backbone of xylan to produce oligoxylanosides (e.g., endo- "Xylanases, E.C. 3.2.1.8). Xylan is found in natural materials such as wood and fibrous plants and foodstuffs, typically as a component of hemicellulose. A variety of microorganisms that live on or aid in the decay of wood or other fibrous materials produce xylanases. Typically, each microorganism produces several different xylanases, each of which can attack different portions of xylan.

[003] Xylanases derived from such microorganisms have several commercial uses. Xylanases are useful in animal feeds to enable the animals to digest the feeds more efficiently. More efficient digestion of the animal feed results in greater weight gain in an animal for a given amount of feed. (See, e.g., PCT publication WO 97/20920). Xylanases are useful in production of human foods as well. For example, xylanase improves properties of bread dough and the quality of the bread. Xylanase can also aid the brewing process by improving the filterability of wheat beers. (See, e.g., PCT publication WO 91/19782). The usefulness of xylanases extends to industrial processes. Xylanases can be employed to aid in the

decomposition and disposal of agricultural waste and waste resulting from processing of agricultural products, including production of fuel from biomass. (See, e.g., Coughlan, M.P. in Xylans and Xylanases, J. Visser et al. eds., (Elsevier Science Publishers B.V., Amsterdam) 1992). Production of paper from wood provides a major industrial use for xylanases.

[004] The complex structure of wood includes cellulose, hemicellulose and lignin, along with other minor components. Lignin is associated with cellulose and hemicellulose, and is probably covalently bound to both cellulose and hemicellulose. In the paper-making process, lignin is generally removed from the wood pulp since it lends a brownish color, reduces strength and imparts other undesirable characteristics to the finished product. Removal of lignin can be achieved in many ways.

[005] A majority of the lignin is initially removed from wood pulp through chemical pulping (e.g., the Kraft process). In the subsequent bleaching process, chemical pulp is routinely reacted with chlorine and other delignifying chemicals to further remove lignin and then reacted with bleaching agents to modify the lignin from pulp, providing a stable brightened pulp.

However, the treatment with chlorine is undesirable from an environmental standpoint because the resulting effluents contain a large number of toxic compounds (e.g. chlorinated phenolics). Concern about the environmental harmful effects caused by pulp bleaching with chlorine containing chemicals has driven the industry to seek alternative bleaching methods.

[006] Many of the applications in which xylanases can be employed are those that use harsh conditions, such as high temperature and/or pH, etc., which render the xylanases less effective than under normal physiological conditions. During pulp bleaching, for example, material that comes from an alkaline wash stage can have a high temperature, sometimes greater than 80 °C, and a high pH, such as a pH greater than 10. Since most known xylanases do not function well under those conditions, pulp must be cooled and the alkaline pH neutralized before such xylanase can function. Taking some of these steps into account, the process can become more expensive since it must be altered to suit the xylanase.

[007] In another example, some animal feed applications in which xylanases can be employed have high temperature conditions for a short time (e.g., 0.5 - 5 min at 95 °C or higher during feed preparation). Inactivation of the xylanase can occur under these temperature conditions, thereby being rendered useless to perform their function at a lower temperature (e.g., 37 °C) later in the process.

[008] Although numerous xylanases have been identified and characterized, the need still exists to identify novel xylanases that are more effective under harsh conditions, e.g., high temperature and/or alkaline conditions, as compared to conventional bleaching agents and xylanases now available.

Summary

[009] Aspects of the present compositions and methods are drawn to a xylanase belonging to glycosyl hydrolase family 10 derived from Thermobifida fusca, referred to herein as TfuXyn4, nucleic acids encoding the same, and methods of producing and employing the xylanase in numerous applications. As demonstrated herein, TfuXyn4 has improved performance at extreme conditions of pH and temperature, making it suitable for use in processes in which such conditions are employed. [0010] Aspects of the present compositions and methods include a recombinant polypeptide comprising an amino acid sequence that is at least 55% identical to the amino acid sequence of SEQ ID NO: 6, wherein the polypeptide has xylanase activity. In certain embodiments, the polypeptide retains greater than 70% of the xylanase activity when incubated at a pH range from pH 5.0 to pH 9.0. In certain embodiments, the polypeptide retains at least 80% of the xylanase activity when incubated at a temperature of between 66°C and 93°C. In certain embodiments, the polypeptide has optimum xylanase activity at a temperature of at least 75°C or above. In certain embodiments, the polypeptide retains at least 50% of the xylanase activity when incubated for about 2 hours at a temperature of about 69°C. In certain embodiments, the polypeptide retains at least 90% of the xylanase activity when incubated for about 2 hours at a temperature of between 45°C and 60 °C. In certain embodiments, the polypeptide retains at least 90% of the xylanase activity when incubated for about 2 hours at a temperature of between 50°C and 60 °C. In certain embodiments, the polypeptide retains at least 90% of the xylanase activity when incubated for about 2 hours at a temperature of between 55°C and 60 °C. In certain

embodiments, the polypeptide retains at least 90% of the xylanase activity when incubated for about 2 hours at a temperature of about 60 °C. In certain embodiments, the polypeptide has an improved thermostability. In certain embodiments, the polypeptide comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

6. In certain embodiments, the polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 6.

[0011] Aspects of the present compositions and methods include a composition comprising the recombinant polypeptide as detailed above and a chemical pulp. In certain embodiments, the chemical pulp is a Kraft pulp, soda pulp, or sulfite pulp. [0012] Aspects of the present compositions and methods include an isolated nucleic acid encoding the recombinant polypeptide as described above. In certain embodiments, the polypeptide further comprises a signal peptide sequence. In certain embodiments, the signal peptide sequence comprises SEQ ID NO: 5. [0013] Aspects of the present compositions and methods include an expression vector comprising the isolated nucleic acid as described above in operable combination with a regulatory sequence.

[0014] Aspects of the present compositions and methods include a host cell comprising the expression vector. In certain embodiments, the host cell is a bacterial cell or a fungal cell. [0015] Aspects of the present compositions and methods include a composition comprising the host cell described above and a culture medium.

[0016] Aspects of the present compositions and methods include a method of producing a xylanase, comprising: culturing the host cell described above in a culture medium, under suitable conditions to produce the xylanase. [0017] Aspects of the present compositions and methods include a composition comprising the xylanase produced in accordance with the method of Claim 25 in supernatant of the culture medium.

[0018] Aspects of the present compositions and methods include a method for enzymatic treatment of a pulp, the method comprising: contacting the pulp with the polypeptide of any one of Claims 1-14 under alkaline conditions to produce a mixture, wherein the xylanase activity of the polypeptide is greater than that observed for Optimase® CX 255L (SEQ ID NO: 12) under similar conditions. In certain embodiments, the pulp is a chemical pulp. In certain embodiments, the chemical pulp is a Kraft pulp, soda pulp, or sulfite pulp. In certain embodiments, the pH of the mixture is at least pH 8.2, at least pH 8.4, at least pH 8.6, at least pH 8.8, at least pH 9.0, pH 9.2, at least pH 9.4, at least pH 9.6, at least pH 9.8, at least pH 10.0, at least pH 10.2, at least pH 10.4, at least pH 10.6, at least pH 10.8, or at least pH 11.0. In certain embodiments, the contacting is performed at a temperature of about 50°C or above. In certain embodiments, the contacting is performed at a temperature of at least 55 °C, at least 60 °C, at least 65 °C, at least 69 °C, at least 70 °C, at least 75 °C, at least 80 °C, at least 85 °C, at least 89 °C, at least 90 °C, at least 95 °C, at least 100 °C, at least 110 °C, or at least 120 °C. In certain embodiments, the mixture is at least pH 10 and the contacting is performed at a temperature of at least 80 °C.

[0019] Aspects of the present compositions and methods include a method for enzymatic treatment of a pulp, the method comprising: contacting the pulp with the polypeptide of any one of Claims 1-14 at a temperature of about 50°C or above, wherein the xylanase activity of the polypeptide is greater than that observed for Optimase® CX 255L (SEQ ID NO: 12) under similar conditions. In certain embodiments, the contacting is performed under alkaline conditions. In certain embodiments, the pH of the mixture is at least pH 8.2, at least pH 8.4, at least pH 8.6, at least pH 8.8, at least pH 9.0, pH 9.2, at least pH 9.4, at least pH 9.6, at least pH 9.8, at least pH 10.0, at least pH 10.2, at least pH 10.4, at least pH 10.6, at least pH 10.8, or at least pH 11.0. In certain embodiments, the contacting is performed at a temperature of at least 55 °C, at least 60 °C, at least 65 °C, at least 69 °C, at least 70 °C, at least 75 °C, at least 80 °C, at least 85 °C, at least 89 °C, at least 90 °C, at least 95 °C, at least 100 °C, at least 110 °C, or at least 120 °C. In certain embodiments, the pulp is a chemical pulp. In certain embodiments, the chemical pulp is a Kraft pulp, soda pulp, or sulfite pulp. In certain embodiments, the method is employed to facilitate biobleaching of Kraft pulp.

Brief Description of the Drawings

[0020] Figure 1 shows a map of the expression plasmid for TfuXyn4.

[0021] Figure 2A shows an amino acid sequence alignment of TfuXyn4 and the protein encoded by a xylanase gene from Thermobifida fusca (NCBI Accession Number:YP_290979.1; SEQ ID NO: 9). TfuXyn4 has a 9 amino acid deletion as compared to YP_290979.1. Figure 2B shows an amino acid sequence alignment of TfuXyn4 (including the predicted signal peptide; SEQ ID NO: 4) and the protein encoded by a xylanase gene from Thermobifida alba (NCBI Accession Number: CAB02654). [0022] Figure 3 is a graph showing the effect of pH on TfuXyn4 xylanase activity. Xylanase activity was measured at 50°C for 10 min using 1% birch wood xylan as substrate in 25 mM Sodium Citrate/ 25 mM Sodium Phosphate buffer solution adjusted to pH values between 2 and 9. Activity is shown as relative activity, where the activity at the pH optimum was set to 100%.

[0023] Figure 4 is a graph showing the effect of temperature on TfuXyn4 xylanase activity. The assay was carried out at various temperatures from 45°C to 94°C for 10 minutes using 1% birch wood xylan as substrate in 50 mM sodium citrate buffer pH 5.3. Activity is shown as relative activity, where the activity at the temperature optimum was set to 100%.

[0024] Figure 5 is a graph showing the effect of temperature on the stability of TfuXyn4. The enzyme was incubated in 50 mM sodium citrate pH 5.3 buffer at the indicated temperature for 2 hours after which the xylanase activity was measured. The measured activity of the enzyme kept on ice was defined as 100% activity.

[0025] Figure 6 is a graph showing the release of reducing sugars from enzyme-treated Kraft pulp. TfuXyn4 (solid line) and a benchmark (purified xylanase from Optimase® CX 255L, dotted line) were added at 0-8 mg/g pulp, and incubated one hour in pH 9.0 buffer at 69 °C or pH10.5 buffer at 80 °C . The released reducing sugar was determined using PAHBAH reducing sugar assay.

[0026] Figure 7 is a graph showing the release of phenolic compounds from enzyme-treated Kraft pulp. TfuXyn4 (solid line) and the benchmark (purified xylanase from Optimase® CX 255L, dotted line) were added at 8 mg/g pulp, and incubated for one hour in pH 9.0 buffer at 69 °C or pH 10.5 buffer at 80 °C . The graph shows the absorbance reading from 220 nm to 400 nm of the supernatants from the enzyme-treated Kraft pulp.

Brief Description of the Sequences

[0027] SEQ ID NO: 1 is the nucleic acid sequence of the TfuXyn4 forward PCR primer employed in the Examples. [0028] SEQ ID NO: 2 is the nucleic acid sequence of the TfuXyn4 reverse PCR primer employed in the Examples.

[0029] SEQ ID NO: 3 is the nucleic acid sequence of the PCR product obtained from genomic derived from Thermobifida fusca using PCR primers designed to amplify the mature nucleic acid sequence of the TfuXyn4 gene (NCBI YP _.290979.1; see Examples). [0030] SEQ ID NO: 4 is the amino acid sequence of the predicted TfuXyn4 precursor protein; the signal peptide from NCBI YP_290979.1 has been added.

[0031] SEQ ID NO: 5 is the amino acid sequence of the predicted TfuXyn4 signal peptide based on the NCBI YP_290979.1 nucleic acid sequence. [0032] SEQ ID NO: 6 is the amino acid sequence of the TfuXyn4 mature protein (without the signal peptide).

[0033] SEQ ID NO: 7 is the nucleic acid sequence of the TfuXyn4 precursor gene in plasmid pDS270 (A4- CelA-TfuXyn4). [0034] SEQ ID NO: 8 is the amino acid sequence of the TfuXyn4 precursor protein translated from plasmid pDS270 (A4- CelA-TfuXyn4).

[0035] SEQ ID NO: 9 is the amino acid sequence of a xylanase gene from Thermobifida fusca as shown in Figure 2A (NCBI Accession Number:YP_290979.1).

[0036] SEQ ID NO: 10 is the amino acid sequence of a xylanase gene from Thermobifida alba as shown in Figure 2B (NCBI Accession Number: CAB02654).

[0037] SEQ ID NO: 11 is the amino acid sequence of the xylanase II enzyme from Trichoderma reesei, an endo-l,4- -xylanase (EC 3.2.1.8) (see US Patent 7,718,411, incorporated herein by reference).

[0038] SEQ ID NO: 12 is the amino acid sequence of Optimase® CX 255L, also referred to as the Y5 mutant of the xylanase II enzyme from Trichoderma reesei shown in SEQ ID NO: 11 (see US Patent 7,718,411, incorporated herein by reference).

DETAILED DESCRIPTION

1. Overview

[0039] Described herein are compositions and methods relating to a recombinant xylanase belonging to glycosyl hydrolase family 10 from Thermobifida fusca (TfuXyn4). The present compositions and methods are based, in part, on the observations that cloned and expressed TfuXyn4 is thermostable and retains xylanase activity under high pH (i.e., is alkali tolerant). These features of TfuXyn4 make it, or variants thereof, suitable for use in numerous processes, including pulp and paper processing and the treatment of foodstuffs.

[0040] Before the present compositions and methods are described in greater detail, it is to be understood that the present compositions and methods are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present compositions and methods will be limited only by the appended claims.

[0041] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the present compositions and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the present compositions and methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the present compositions and methods.

[0042] Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0043] The headings provided herein are not limitations of the various aspects or embodiments of the present compositions and methods which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0044] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present compositions and methods belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present compositions and methods, representative illustrative methods and materials are now described.

[0045] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present compositions and methods are not entitled to antedate such

publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. [0046] It is noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation. [0047] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present compositions and methods described herein. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

2. Definitions

[0048] "Xylanase" means a protein or polypeptide domain of a protein or polypeptide that has the ability to catalyze cleavage of xylan at one or more of various positions of xylan's carbohydrate backbone. [0049] As used herein, "TfuXyn4" refers to a xylanase belonging to glycosyl hydrolase family 10, e.g. a recombinant protein, derived from Thermobifida fusca (and variants thereof) that is thermostable and/or is alkali tolerant. According to aspects of the present compositions and methods, the TfuXyn4 includes proteins having the amino acid sequence depicted in SEQ. ID NO:6 as well as derivative or variant proteins having at least 55%, at least 60%, at least 65%, at least 70%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ. ID NO:6, or a derivative of the amino acid sequence of SEQ. ID NO:6, wherein the TfuXyn4 cleaves xylan.

[0050] TfuXyn4 proteins according to the present compositions and methods described herein are isolated or purified. By purification or isolation is meant that the TfuXyn4protein is altered from its natural state by virtue of separating the TfuXyn4from some or all of the naturally occurring constituents with which it is associated in nature. Such isolation or purification may be accomplished by art recognized separation techniques such as ion exchange chromatography, affinity chromatography, hydrophobic separation, dialysis, protease treatment, ammonium sulphate precipitation or other protein salt precipitation, centrifugation, size exclusion

chromatography, filtration, microfiltration, gel electrophoresis or separation on a gradient to remove whole cells, cell debris, impurities, extraneous proteins, or enzymes undesired in the final composition. It is further possible to then add constituents to the TfuXyn4-containing composition which provide additional benefits, for example, activating agents, anti-inhibition agents, desirable ions, compounds to control pH or other enzymes or chemicals. [0051] As used herein, "microorganism" refers to a bacterium, a fungus, a virus, a protozoan, and other microbes or microscopic organisms.

[0052] As used herein, a "derivative" or "variant" of a protein means a protein which is derived from a precursor protein (e.g., the native protein) by addition of one or more amino acids to either or both the C- and N-terminal end, substitution of one or more amino acids at one or a number of different sites in the amino acid sequence, deletion of one or more amino acids at either or both ends of the protein or at one or more sites in the amino acid sequence, or insertion of one or more amino acids at one or more sites in the amino acid sequence. The preparation of a TfuXyn4 derivative or variant may be achieved in any convenient manner, e.g., by modifying a DNA sequence which encodes the native protein, transformation of that DNA sequence into a suitable host, and expression of the modified DNA sequence to form the derivative/variant TfuXyn4. Derivatives or variants further include TfuXyn4 proteins that are chemically modified, e.g., to change a characteristic of the TfuXyn4. While derivatives and variants of TfuXyn4 are encompassed by the present compositions and methods, such derivates and variants will display one or both of thermostability and alkali tolerance. [0053] In certain aspects, a TfuXyn4 derivative/variant will have anywhere from 50% to 99% (or more) amino acid sequence identity with the amino acid sequence of SEQ. ID NO:6, e.g., 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the amino acid sequence of SEQ. ID NO:6. In some embodiments, amino acid substitutions are "conservative amino acid substitutions" using L-amino acids, wherein one amino acid is replaced by another biologically similar amino acid. Conservative amino acid substitutions are those that preserve the general charge, hydrophobicity/hydrophilicity, and/or steric bulk of the amino acid being substituted. Examples of conservative substitutions are those between the following groups: Gly/Ala, Val/Ile/Leu, Lys/Arg, Asn/Gln, Glu/Asp, Ser/Cys/Thr, and Phe/Trp/Tyr. A derivative may, for example, differ by as few as 1 to 10 amino acid residues, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue. In some embodiments, a TfuXyn4 derivative may have an N- terminal and/or C-terminal deletion, where the TfuXyn4 derivative excluding the deleted terminal portion(s) is identical to a contiguous sub-region in SEQ ID NO: 6.

[0054] As used herein, "percent (%) sequence identity" with respect to the amino acid or nucleotide sequences identified herein is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical with the amino acid residues or nucleotides in a TfuXyn4 sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative

substitutions as part of the sequence identity. [0055] Methods for performing sequence alignment and determining sequence identity are known to the skilled artisan, may be performed without undue experimentation, and calculations of identity values may be obtained with definiteness. See, for example, Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley- Interscience, New York); and the ALIGN program (Dayhoff (1978) in Atlas of Protein Sequence and Structure 5:Suppl. 3 (National Biomedical Research Foundation, Washington, D.C.). A number of algorithms are available for aligning sequences and determining sequence identity and include, for example, the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the search for similarity method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith- Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997); and BLASTP, BLASTN, and

BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410).

[0056] Computerized programs using these algorithms are also available, and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym.. 266:460-480 (1996)); or GAP, BESTFIT, BLAST, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, California. Those skilled in the art can determine appropriate parameters for measuring alignment, including algorithms needed to achieve maximal alignment over the length of the sequences being compared. The sequence identity can be determined using the default parameters determined by the program. Specifically, sequence identity can determined by using Clustal W (Thompson J.D. et al. (1994) Nucleic Acids Res. 22:4673-4680) with default parameters, i.e.:

Gap opening penalty: 10.0

Gap extension penalty: 0.05

Protein weight matrix: BLOSUM series

DN A weight matrix: IUB

Delay divergent sequences %: 40

Gap separation distance: 8

DNA transitions weight: 0.50

List hydrophilic residues: GPSNDQEKR

Use negative matrix: OFF

Toggle Residue specific penalties: ON

Toggle hydrophilic penalties: ON

Toggle end gap separation penalty OFF [0057] As used herein, "expression vector" means a DNA construct including a DNA sequence which is operably linked to a suitable control sequence capable of affecting the expression of the DNA in a suitable host. Such control sequences may include a promoter to affect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome- binding sites on the mRNA, and sequences which control termination of transcription and translation. Different cell types may be used with different expression vectors. An exemplary promoter for vectors used in Bacillus subtilis is the AprE promoter; an exemplary promoter used in Streptomyces lividans is the A4 promoter (from Aspergillus Niger); an exemplary promoter used in E. coli is the Lac promoter, an exemplary promoter used in Saccharomyces cerevisiae is PGK1, an exemplary promoter used in Aspergillus niger is glaA, and an exemplary promoter for Trichoderma reesei is cbhl. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, under suitable conditions, integrate into the genome itself. In the present specification, plasmid and vector are sometimes used

interchangeably. However, the present compositions and methods are intended to include other forms of expression vectors which serve equivalent functions and which are, or become, known in the art. Thus, a wide variety of host/expression vector combinations may be employed in expressing the DNA sequences described herein. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences such as various known derivatives of SV40 and known bacterial plasmids, e.g., plasmids from E. coli including col El, pCRl, pBR322, pMb9, pUC 19 and their derivatives, wider host range plasmids, e.g., RP4, phage DNAs e.g., the numerous derivatives of phage λ, e.g., NM989, and other DNA phages, e.g., M13 and filamentous single stranded DNA phages, yeast plasmids such as the 2μ plasmid or derivatives thereof, vectors useful in eukaryotic cells, such as vectors useful in animal cells and vectors derived from combinations of plasmids and phage DNAs, such as plasmids which have been modified to employ phage DNA or other expression control sequences. Expression techniques using the expression vectors of the present compositions and methods are known in the art and are described generally in, for example, Sambrook et al.,

Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press (1989). Often, such expression vectors including the DNA sequences described herein are transformed into a unicellular host by direct insertion into the genome of a particular species through an integration event (see e.g., Bennett & Lasure, More Gene Manipulations in Fungi, Academic Press, San Diego, pp. 70-76 (1991) and articles cited therein describing targeted genomic insertion in fungal hosts).

[0058] As used herein, "host strain" or "host cell" means a suitable host for an expression vector including DNA according to the present compositions and methods. Host cells useful in the present compositions and methods are generally prokaryotic or eukaryotic hosts, including any transformable microorganism in which expression can be achieved. Specifically, host strains may be Bacillus subtilis, Streptomyces lividans, Escherichia coli, Trichoderma reesei,

Saccharomyces cerevisiae or Aspergillus niger. Host cells are transformed or transfected with vectors constructed using recombinant DNA techniques. Such transformed host cells may be capable of one or both of replicating the vectors encoding TfuXyn4 (and its derivatives or variants (mutants)) and expressing the desired peptide product. In certain embodiments according to the present compositions and methods, "host cell" means both the cells and protoplasts created from the cells of Trichoderma sp. [0059] As used herein, "signal sequence" means a sequence of amino acids bound to the N- terminal portion of a protein which facilitates the secretion of the mature form of the protein outside of the cell. This definition of a signal sequence is a functional one. The mature form of the extracellular protein lacks the signal sequence which is cleaved off during the secretion process. While the native signal sequence of TfuXyn4 (SEQ ID NO: 5) may be employed in aspects of the present compositions and methods, other non-native signal sequences may be employed.

[0060] As used herein, "functionally attached" or "operably linked" means that a regulatory region or functional domain having a known or desired activity, such as a promoter, terminator, signal sequence or enhancer region, is attached to or linked to a target (e.g., a gene or protein) in such a manner as to allow the regulatory region or functional domain to control the expression, secretion or function of that target according to its known or desired activity.

[0061] As used herein, the terms "polypeptide" and "protein" are used interchangeably to refer to polymers of any length comprising amino acid residues linked by peptide bonds. The conventional one-letter or three-letter codes for amino acid residues are used herein. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0062] As used herein, "wild-type" and "native" genes, proteins, or strains, are those found in nature. [0063] The xylanase II enzyme from Trichoderma reesei (SEQ ID NO: 11) has the following amino acid sequence:

QTIQPGTGYNNGYFYSYWNDGHGGVTYTNGPGGQFSVNWSNSGNFVGGKGWQPGTK NKVINFSGSYNPNGNSYLSVYGWSRNPLIEYYIVENFGTYNPSTGATKLGEVTSDGSVY DIYRTQRVNQPSIIGTATFYQYWSVRRNHRSSGSVNTANHFNAWAQQGLTLGTMDYQI VAVEGYFSSGSASrrVS [0064] Optimase® CX 255L (also referred to as the Y5 mutant of the the xylanase II enzyme from Trichoderma reesei), has the following amino acid sequence (SEQ ID NO: 12):

QCIQPGTGYNNGYFYSYWNDGHGGVTYCNGPGGQFSVNWSNSGNFVGGKGWQPGTK NRVINFSGSYNPNGNSYLSVYGWSRNPLIEYYIVENFGTYNPSTGATKLGEVTSDGSVY DIYRTQRVNQPSIIGTATFYQYWSVRRNHRSSGSVNTANHFNAWAQQGLTLGTMDYQI VAVEGYFSSGSASrrVSD

3. TfuXvn4, Derivatives and Preparation Thereof

[0065] The present compositions and methods relate to the expression, isolation and use of TfuXyn4 and derivatives of TfuXyn4. The TfuXyn4 or derivative may be prepared by recombinant methods. However, TfuXyn4 proteins for use in the present compositions and methods may be obtained by other convenient means such as purification from natural isolates or chemical synthesis.

A. TfuXyn4 Polypeptides

[0066] As discussed above, aspects of the present compositions and methods are drawn to TfuXyn4 polypeptides and derivatives or variants thereof.

[0067] The amino acid sequence of native TfuXyn4 derived from Thermobifida fusca (including the predicted native signal peptide in bold (SEQ ID NO:5; the signal peptide is based on the NCBI YP_290979.1 nucleic acid sequence)) is as follows in (SEQ ID NO: 4):

mtetrhrpsrrarrslsllltsaltaagllvtaapaqaestlrelaaqnggrhfgtaiayspln sdaqyrniaatqfsaithenemkweslepqrgqynwsqadniinfakannqivrghtlvwhsql pswlnnggfsgsqlrsimenhievvagryrgdvyawd vneafnedgtlrdsiwyrgmgrdyia hafrkahevdpdaklyindynieginaksnglynl vdllrdgvpihgigiqshlivgqvpstf qqniqrfadlgldvaiteldirmqmpadqyklqqqardyeavvnaclavtrcigitvwgidder swvpytfpgegapllydgqynrkpawya yealggdssgggpgepggpggpgepggpgdgtca nyt vndwghgmqgaitvsntgsspinnwtlqfsfsgvnisngwngewsqsgsqitvrapawns tlqpgqsvelgf adktgnvsppsqftlngatcs

[0068] The amino acid sequence of mature TfuXyn4 derived from Thermobifida fusca based on the PCR product obtained in the Examples (thus excluding the predicted native signal peptide) is as follows (SEQ ID NO: 6): estlrelaaqnggrhfgtaiaysplnsdaqyrniaatqfsaithenemkweslepqrgqynwsq adniinfakannqivrghtlvwhsqlpswlnnggfsgsqlrsimenhie vagryrgdvyawdv vneafnedgtlrdsiwyrgmgrdyiahafrkahevdpdaklyindynieginaksnglynl vd llrdgvpihgigiqshlivgqvpstfqqniqrfadlgldvaiteldirmqmpadqyklqqqard yea vnaclavtrcigitvwgidderswvpytfpgegapllydgqynrkpawyavyealggdss gggpgepggpggpgepggpgdgtcavnyt vndwghgmqgaitvsntgsspinnwtlqfsfsgv nisngwngewsqsgsqitvrapawnstlqpgqsvelgf adktgnvsppsqftlngatcs

[0069] Aspects of the present compositions and methods include a polypeptide (TfuXyn4 and derivatives) having an amino acid sequence that is at least 55% identical to the amino acid sequence of SEQ ID NO: 6, where the polypeptide has xylanase activity. As such, aspects of the present compositions and methods include TfuXyn4 polypeptides containing an amino acid sequence that is at least 60% identical to SEQ ID NO: 6, including at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 6. In some embodiments a TfuXyn4 polypeptide contains an amino acid sequence that is 100% identical to SEQ ID NO: 6.

[0070] As described above and demonstrated in the Examples, TfuXyn4 is thermostable and demonstrates significant alkali tolerance, properties that are evident when compared to other known xylanases (e.g., OPTIMASE® CX225L; see Examples).

[0071] As such, certain embodiments of the present compositions and methods include TfuXyn4 polypeptides and derivatives that retain greater than 70% (e.g., about 80%) of their optimal xylanase activity when incubated at a pH range from pH 5.0 to pH 9.0.

[0072] In certain embodiments, the TfuXyn4polypeptide or derivative retains at least 80% of its optimal xylanase activity when incubated at a temperature of between 66°C and 93°C.

TfuXyn4 polypeptide or derivatives may have optimal xylanase activity at a temperature above 75°C and retain significant activity levels when incubated for prolonged periods at elevated temperatures. In some embodiments, a TfuXyn4 or derivative retains at least 50% xylanase activity when incubated for about 2 hours at a temperature of about 69°C and/or may retain at least 90% of its xylanase activity when incubated for about 2 hours at a temperature of about 45°C to 60 °C, e.g., about 50°C to 60 °C, about 55°C to 60 °C, including retaining at least 90% of its xylanase activity when incubated for about 2 hours at a temperature of about 60 °C.

[0073] Under Kraft pulp conditions in which the pH is 10.5 and the temperature is 80 °C, TfuXyn4 polypeptides can retain significant enzymatic activity, approaching that of benchmark xylanases at much lower pH and temperatures (e.g., at pH 9.0 and 69 °C).

B. TfuXyn4-Encoding Polynucleotides

[0074] Aspects of the preset compositions and methods include polynucleotides encoding TfuXyn4 polyculeotides and derivatives as described in detail above.

[0075] The nucleic acid sequence of mature TfuXyn4 derived from Thermobifida fusca is as follows in (SEQ ID NO: 3):

GAGTCGACCCTGCGGGAACTGGCTGCCCAGAACGGCGGCCGCCACT TCGGTACGGCTATCGCCT ACAGCCCGCTCAACAGTGACGCCCAGTACCGCAACATCGCGGCTACCCAGT TCAGCGCCATCAC CCACGAAAACGAGATGAAGTGGGAGTCGCTGGAGCCGCAGCGGGGCCAGTACAACTGGAGCCAG GCCGACAACATCATCAACT TCGCCAAGGCCAACAACCAGAT TGTGCGCGGCCACACCCTGGTCT GGCACAGCCAGCTGCCGTCCTGGCTGAACAACGGCGGCT TCTCCGGCAGCCAGCTCCGGTCCAT CATGGAGAACCACATCGAGGTGGTGGCCGGACGCTACCGGGGTGACGTCTACGCCTGGGACGTG GTCAACGAAGCGT TCAACGAGGACGGTACGCTCCGCGACTCGATCTGGTACCGCGGCATGGGTC GCGACTACATCGCCCACGCGT TCCGCAAGGCGCACGAGGTCGACCCCGACGCCAAGCTGTACAT CAACGACTACAACATCGAAGGCATCAACGCTAAGAGCAACGGCCTCTACAACCTGGTGGTCGAC CTGCTCCGCGACGGTGTGCCGATCCACGGTATCGGTATCCAGTCCCACCTGATCGTCGGCCAGG TGCCGTCCACGT TCCAGCAGAACATCCAGCGGT TCGCTGACCTCGGCCTGGACGTGGCCATCAC CGAGCTGGACATCCGCATGCAGATGCCGGCCGACCAGTACAAGCTCCAGCAGCAGGCCCGCGAC TACGAGGCCGTGGTCAACGCCTGCCTCGCGGTGACCCGCTGCATCGGTATCACCGTCTGGGGTA TCGACGACGAGCGCTCCTGGGTGCCCTACACCT TCCCGGGTGAAGGTGCTCCGCTGCTCTACGA CGGCCAGTACAACCGCAAGCCCGCCTGGTACGCGGTCTACGAGGCTCTCGGCGGCGACTCCTCC GGCGGCGGTCCGGGTGAGCCGGGCGGTCCTGGCGGTCCGGGTGAGCCGGGCGGCCCCGGTGACG GCACCTGCGCGGTGAACTACACCGTGGTCAATGACTGGGGTCACGGTATGCAGGGGGCGATCAC CGTCTCCAACACCGGATCCTCGCCCATCAACAACTGGACCCTGCAGT TCAGCT TCTCGGGTGTG AACATCTCCAACGGCTGGAACGGCGAGTGGAGCCAGAGCGGCTCGCAGATCACCGTCCGCGCTC CTGCCTGGAACTCCACGCTCCAGCCGGGCCAGAGCGTGGAACTGGGCT TCGT TGCTGACAAGAC CGGCAACGTCTCCCCGCCCTCCCAGT TCACCCTCAACGGAGCCACCTGCTCCTGA

[0076] As is well known to those of ordinary skill in the art, due to the degeneracy of the genetic code, polynucleotides having significantly different sequences can nonetheless encode identical, or nearly identical, polypeptides. As such, aspects of the present compositions and methods include polynucleotides encoding TfuXyn4 polypeptides or derivatives thereof that contain a nucleic acid sequence that is at least 35% identical to SEQ ID NO: 3, including at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:3. In some embodiments, TfuXyn4 polypeptides contain a nucleic acid sequence that is identical to SEQ ID NO: 3.

[0077] In some embodiments, polynucleotides may include a sequence encoding a signal peptide sequence, where the signal peptide may have the sequence of SEQ ID NO: 5. However, any other convenient signal sequence may be employed.

C. Purification from Natural Isolates

[0078] TfuXyn4 can be purified from natural isolates (e.g., Thermobifida fusca) by known and commonly employed methods. For example, cells containing TfuXyn4 can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents. The TfuXyn4 can be recovered from the medium by conventional techniques including separations of the cells from the medium by centrifugation, filtration, and precipitation of the proteins in the supernatant or filtrate with a salt, for example, ammonium sulphate. The TfuXyn4 can then be purified from the disrupted cells by procedures such as: fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS- PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; and affinity chromatography. Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer- Verlag, New York (1982).

D. Chemical Synthesis

[0079] Alternatively, the TfuXyn4 sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques (see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, CA (1969); Merrifield, J. Am. Chem. Soc, 85:2149-2154 (1963)). In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using manufacturer's instructions. Various portions of TfuXyn4 may be chemically synthesized separately and combined using chemical or enzymatic methods to produce a full-length TfuXyn4.

E. Recombinant Methods

Isolation of DNA Encoding the TfuXyn4

[0080] DNA encoding a TfuXyn4 may be obtained from a cDNA library prepared from a microorganism believed to possess the TfuXyn4 mRNA (e.g., Thermobifida fusca) and to express it at a detectable level. The TfuXyn4-encoding gene may also be obtained from a genomic library or by oligonucleotide synthesis.

[0081] Libraries can be screened with probes (such as antibodies to a TfuXyn4 or

oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative means to isolate the gene encoding TfuXyn4 is to use PCR methodology (Sambrook et al., supra; Dieffenbach et al., PCR PrimenA Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)).

[0082] In known techniques for screening a cDNA library, the oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized. The oligonucleotide may be labeled such that it can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art, and include the use of radiolabels like 32 P-labeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al., supra.

[0083] Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in

Sambrook et al., supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA.

Selection and Transformation of Host Cells

[0084] Host cells are transfected or transformed with expression or cloning vectors described herein for TfuXyn4 production. The host cells are cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. The culture conditions, such as media, temperature, pH and the like, can be selected by the ordinarily skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.

[0085] Methods of transfection are known to the ordinarily skilled artisan, for example, CaP0₄ and electroporation. Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described in Sambrook et al., supra, or electroporation is generally used for prokaryotes or other cells that contain substantial cell-wall barriers. Infection with

Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al., Gene, 23:315 (1983) and WO 89/05859 published 29 June 1989. Transformations into yeast can be carried out according to the method of Van Solingen et al., J. Bact, 130:946 (1977) and Hsiao et al, Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, microporation, biolistic bombardment, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene, polyornithine, may also be used.

[0086] Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or filamentous fungal cells. Suitable prokaryotes include but are not limited to eubacteria, such as Gram-negative or Gram-positive organisms, for example,

Enterobacteriaceae such as E. coli. Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC 53,635). In addition to prokaryotes, eukaryotic

microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding TfuXyn4. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism.

[0087] In some embodiments, the microorganism to be transformed includes a strain derived from Trichoderma spp. or Aspergillus spp. Exemplary strains include T. reesei which is useful for obtaining overexpressed protein or Aspergillus niger var. awamori. For example, Trichoderma strain RL-P37, described by Sheir-Neiss et al. in Appl. Microbiol. Biotechnology, 20 (1984) pp. 46-53 is known to secrete elevated amounts of cellulase enzymes. Functional equivalents of RL-P37 include Trichoderma reesei (longibrachiatum) strain RUT-C30 (ATCC No. 56765) and strain QM9414 (ATCC No. 26921). Another example includes

overproducing mutants as described in Ward et al. in Appl. Microbiol. Biotechnology 39:738- 743 (1993). It is contemplated that these strains would also be useful in overexpressing Thermobifida spp. TfuXyn4. The selection of the appropriate host cell is deemed to be within the skill in the art.

Preparation and Use of a Replicable Vector [0088] DNA encoding the TfuXyn4 protein or derivatives thereof (as described above) is prepared for insertion into an appropriate microorganism. According to the present

compositions and methods, DNA encoding a TfuXyn4 enzyme includes all of the DNA necessary to encode for a protein which has functional TfuXyn4 activity. As such,

embodiments of the present compositions and methods include DNA encoding a TfuXyn4 protein derived from Thermobifida sp., including Thermobifida fusca.

[0089] The DNA encoding TfuXyn4 may be prepared by the construction of an expression vector carrying the DNA encoding TfuXyn4. The expression vector carrying the inserted DNA fragment encoding the TfuXyn4 may be any vector which is capable of replicating autonomously in a given host organism or of integrating into the DNA of the host, typically a plasmid, cosmid, viral particle, or phage. Various vectors are publicly available. It is also contemplated that more than one copy of DNA encoding a TfuXyn4 may be recombined into the strain to facilitate overexpression.

[0090] In certain embodiments, DNA sequences for expressing TfuXyn4 include the promoter, gene coding region, and terminator sequence all originate from the native gene to be expressed. Gene truncation may be obtained by deleting away undesired DNA sequences (e.g., coding for unwanted domains) to leave the domain to be expressed under control of its native transcriptional and translational regulatory sequences. A selectable marker can also be present on the vector allowing the selection for integration into the host of multiple copies of the novel TfuXyn4 gene sequences. [0091] In other embodiments, the expression vector is preassembled and contains sequences required for high level transcription and, in some cases, a selectable marker. It is contemplated that the coding region for a gene or part thereof can be inserted into this general purpose expression vector such that it is under the transcriptional control of the expression cassette's promoter and terminator sequences. For example, pTEX is such a general purpose expression vector. Genes or part thereof can be inserted downstream of the strong cbhl promoter.

[0092] In the vector, the DNA sequence encoding the TfuXyn4 of the present compositions and methods should be operably linked to transcriptional and translational sequences, i.e., a suitable promoter sequence and signal sequence in reading frame to the structural gene. The promoter may be any DNA sequence which shows transcriptional activity in the host cell and may be derived from genes encoding proteins either homologous or heterologous to the host cell. The signal peptide provides for extracellular production of the TfuXyn4 or derivatives thereof. The DNA encoding the signal sequence may be that which is naturally associated with the gene to be expressed, however the signal sequence from any suitable source, for example an exo- cellobiohydrolases or endoglucanase from Trichoderma, is contemplated in the present compositions and methods.

[0093] The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan.

[0094] The desired TfuXyn4 may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector or it may be a part of the TfuXyn4-encoding DNA that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (including

Saccharomyces and Kluyveromyces a-factor leaders, the latter described in U.S. Patent No. 5,010,182), or acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179 published 4 April 1990), or the signal described in WO 90/13646 published 15 November 1990.

[0095] Both expression and cloning vectors may contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria and the 2μ plasmid origin is suitable for yeast.

[0096] Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli. A suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)). The trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). An exemplary selection gene for use in Trichoderma sp is the pyr4 gene.

[0097] Expression and cloning vectors usually contain a promoter operably linked to the TfuXyn4-encoding nucleic acid sequence. The promoter directs mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Exemplary promoters include a fungal promoter sequence, for example, the promoter of the cbhl or egll gene.

[0098] Promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoter systems (Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)), alkaline phosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776), and hybrid promoters such as the tac promoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)). Additional promoters, e.g., the A4 promoter from A niger, also find use in bacterial expression systems, e.g., in S. lividans. Promoters for use in bacterial systems also may contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the TfuXyn4.

[0099] Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem., 255:2073 (1980)) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900 (1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3- phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate

dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.

[00100] Expression vectors used in eukaryotic host cells (e.g. yeast, fungi, insect, plant) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding TfuXyn4.

Polypeptide Purification [00101] Forms of TfuXyn4 (or TfuXyn4 derivatives) may be recovered from culture medium or from host cell lysates by the methods described above for isolation and purification from natural isolates. Additional techniques can be used depending on the host cell employed and any variant structures in the recombinant enzyme. For example, if the recombinant enzyme is membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g. Triton-X 100) or by enzymatic cleavage. Purification of recombinant enzyme may also employ protein A Sepharose columns to remove contaminants such as IgG and metal chelating columns to bind epitope-tagged forms of the TfuXyn4. The purification step(s) selected will depend, for example, on the nature of the production process used, the particular TfuXyn4 produced, and any variant structure for the recombinant enzyme. Antibodies directed to

TfuXyn4 or epitope tags thereon may also be employed to purify the protein, e.g., anti- TfuXyn4 antibodies attached to a solid support.

4. Derivatives of TfuXvn4

[00102] As described above, in addition to the native sequence TfuXyn4 described herein (e.g., as depicted in SEQ ID NO: 6), it is contemplated that TfuXyn4 derivatives can be prepared with altered amino acid sequences. In general, TfuXyn4 derivatives should have either one or both of thermostability and alkali tolerance as detailed herein for native TfuXyn4. Such derivatives may be made, for example, to improve expression in a particular host, improve secretion (e.g., by altering the signal sequence), to introduce epitope tags or other sequences that can facilitate TfuXyn4 purification/isolation, etc. In some embodiments, derivatives may have improved thermostability and/or alkali tolerance as compared to native TfuXyn4.

[00103] TfuXyn4 derivatives can be prepared by introducing appropriate nucleotide changes into the TfuXyn4-encoding DNA, or by synthesis of the desired TfuXyn4. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the TfuXyn4, such as changing the number or position of glycosylation sites.

[00104] Derivatives of the native sequence TfuXyn4 or of various domains of the

TfuXyn4 described herein can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Patent No.

5,364,934. Sequence variations may be a substitution, deletion or insertion of one or more codons encoding the TfuXyn4 that results in a change in the amino acid sequence of the TfuXyn4 as compared with the native sequence TfuXyn4. Optionally, the sequence variation is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the TfuXyn4.

[00105] Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired TfuXyn4 activity may be found by comparing the sequence of the polypeptide with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting derivatives for functional activity using techniques known in the art.

[00106] The sequence variations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassette mutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)) or other known techniques can be performed on the cloned DNA to produce the TfuXyn4-encoding DNA with a variant sequence.

[00107] Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Scanning amino acids may be relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. In this group, Alanine is often used as a scanning amino acid because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the derivative. Alanine is also the most common amino acid. Further, it is frequently found in both buried and exposed positions (Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol, 150:1 (1976)). If alanine substitution does not yield adequate amounts of derivative, an isosteric amino acid can be used. 5. Anti-TfuXyn4 Antibodies [00108] The present compositions and methods further provides anti-TfuXyn4 antibodies.

Exemplary antibodies include polyclonal and monoclonal antibodies, including chimeric and humanized antibodies.

[00109] The anti-TfuXyn4 antibodies of the present compositions and methods may include polyclonal antibodies. Any convenient method for generating and preparing polyclonal and/or monoclonal antibodies may be employed, a number of which are known to those ordinarily skilled in the art.

[00110] Anti-TfuXyn4 antibodies may also be generated using recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. [00111] The antibodies may be monovalent antibodies, which may be generated by recombinant methods or by the digestion of antibodies to produce fragments thereof, particularly, Fab fragments.

6. Exemplary Methods of Employing TfuXvn4

[00112] As discussed above, TfuXyn4 and derivatives thereof have applications in enhancing the delignification and/or the bleaching of pulp according to art-recognized techniques (see, e.g., Subramaniyan et al., 2002 Critical Reviews in Biotechnology Vol. 22 (1), pp 33-46). The process includes contacting the pulp with TfuXyn4 (or a derivative thereof) and is dependent upon factors such as pH, temperature, treatment time, dosage of enzyme and the quantity and type of pulp. Because the TfuXyn4 (or derivative thereof) of the present compositions and methods is thermostable and/or alkali tolerant, it provides significant advantage over currently used xlanases in delignifying/treating chemical pulps (e.g., for biobleaching), processes in which high temperatures and alkaline conditions are routinely used and can significantly negatively impact the activity of other xylanases.

[00113] As such, in certain embodiments, the above process may be carried out at a temperature and pH in which the enzymatic activity of the TfuXyn4 is greater than that observed for a benchmark xylanase (e.g., Optimase® CX 225L).

[00114] In view of the above, aspects of the present compositions and methods include methods for the enzymatic treatment/delignification/biobleaching of a pulp, by contacting the pulp with the TfuXyn4 polypeptide or derivative under alkaline conditions to produce a mixture, where the xylanase activity of the TfuXyn4 polypeptide or derivative is greater than that observed for Optimase® CX 255L under similar conditions. Pulps that may be contacted with the inventive polypeptides include chemical pulps (e.g., Kraft pulp, soda pulp, or sulfite pulp). The pH of the mixture can be at least pH 8.2, at least pH 8.4, at least pH 8.6, at least pH 8.8, at least pH 9.0, pH 9.2, at least pH 9.4, at least pH 9.6, at least pH 9.8, at least pH 10.0, at least pH 10.2, at least pH 10.4, at least pH 10.6, at least pH 10.8, or at least pH 11.0.

[00115] In embodiments where the TfuXyn4 polypeptide or derivative is thermostable, the contacting can be performed at a temperature of about 50°C or above, e.g., at least 55 °C, at least 60 °C, at least 65 °C, at least 69 °C, at least 70 °C, at least 75 °C, at least 80 °C, at least 85 °C, at least 89 °C, at least 90 °C, at least 95 °C, at least 100 °C, at least 110 °C, or at least 120 °C. In one exemplary condition, the mixture is at least pH 10 and the contacting is performed at a temperature of at least 80 °C.

[00116] In other embodiments, the enzymatic treatment/delignification/biobleaching of a pulp includes contacting the pulp with a TfuXyn4 polypeptide or derivative at a temperature of about 50°C or above, wherein the xylanase activity of the polypeptide is greater than that observed for Optimase® CX 255L under similar conditions. In some embodiments, the contacting is performed under alkaline conditions (e.g., where the pH is at least pH 8.2, at least pH 8.4, at least pH 8.6, at least pH 8.8, at least pH 9.0, pH 9.2, at least pH 9.4, at least pH 9.6, at least pH 9.8, at least pH 10.0, at least pH 10.2, at least pH 10.4, at least pH 10.6, at least pH 10.8, or at least pH 11.0.)

[00117] In some embodiments, the contacting is performed at a temperature of at least

55 °C, at least 60 °C, at least 65 °C, at least 69 °C, at least 70 °C, at least 75 °C, at least 80 °C, at least 85 °C, at least 89 °C, at least 90 °C, at least 95 °C, at least 100 °C, at least 110 °C, or at least 120 °C. Pulps that may be contacted with the inventive polypeptides include chemical pulps (e.g., Kraft pulp, soda pulp, or sulfite pulp).

[00118] Therefore, the present method may be applied to upgrade or assist in the upgrading of any of a wide variety of processed pulps, i.e., pulps which have been already previously treated in any of a variety of ways to reduce their lignin content and are treated in the process to further enhance the lignin removal by chemical methods. The present method may be applied to treat hardwood and softwood Kraft pulps to enhance lignin removal and brightening of the pulps. The method is particularly applicable to chemical pulps, i.e., those in which the lignin component has been chemically modified by various chemical treatments such as in the sulfate (Kraft) processes and oxygen delignification, for example being applied to Kraft pulps. In certain embodiments, the TfuXyn4 is applied to the pulp after Kraft digestion or oxygen delignification but prior to bleaching. In the case where both Kraft digestion and oxygen delignification are performed on the same pulp, the enzyme is applied after Kraft digestion, prior to oxygen delignification or after oxygen delignification. The present compositions and methods are also applicable to ozone bleached pulps.

[00119] The resulting pulp is treated to remove the releasable lignin component using an appropriate extractant. In another embodiment, pulp treated with the TfuXyn4 may be subsequently treated with lignin-degrading chemicals such as chlorine, chlorine dioxide and peroxide, and further extracted with an appropriate extractant. In yet another embodiment, the enzyme treated pulp may be treated with an appropriate extractant, followed by lignin degradation and a final treatment with an appropriate extractant. Such extractants essentially solubilize the affected lignin component and suitable extractants include but are not limited to bases such as alkali metal hydroxides (E), DMF, dioxane, acetone, and alcohol. Hydroxide extractions may be combined with hydrogen peroxide (E_p) or oxygen (E_p). The resulting pulp may then be further bleached by a chemical bleaching sequence such as chlorine dioxide (DED) or peroxide (P-P) to the desired brightness whereby substantial savings of chemicals are observed when compared to pulp bleached to the same brightness by the same sequence but without using the enzyme treatment. Reduction of chlorine containing chemicals or peroxide is achieved in such a way. In addition, by performing the present compositions and methods with the above presented enzymes, one may apply the same amount of bleaching chemicals to the pulp and yet achieve a greater brightness in the treated pulp. [00120] In another embodiment, the present compositions and methods provides for additional applications of the TfuXyn4 described above in a variety of industrial settings. For example, the TfuXyn4 may be used to enzymatically breakdown agricultural wastes for production of alcohol fuels and other important industrial chemicals, for production of animal or human foodstuffs, or as a component in a detergent composition. [00121] Based on the improved characteristic of TfuXyn4 detailed herein, this polypeptide and its derivatives find use in a wide variety of applications, and thus no limitation in this regard is intended. As such, TfuXyn4 may be employed in any application in which xylanase activity at increased temperature and/or highly alkaline pH is desired. [00122] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

EXAMPLES

[00123] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present compositions and methods, and are not intended to limit the scope of what the inventors regard as their inventive compositions and methods nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for.

[00124] As described below, a recombinant TfuXyn4 protein was successfully expressed in S. lividans, and the enzyme was purified to homogeneity from culture supernatant by hydrophobic interaction chromatography and gel filtration. The biochemical properties of TfuXyn4 were determined using birchwood xylan as substrate. The enzyme was optimally active at 79 °C, pH 7.0, and retained greater than 80% of optimum activity between pH5 and pH9. The enzyme showed high thermal stability, with 50% activity remained at 69 °C after 2 hours.

[00125] To evaluate the activity towards Kraft pulp, TfuXyn4 was compared with a benchmark, the purified xylanase from Optimase® CX 255L. At lower temperature (69°C) and pH (pH9.0), both enzymes showed similar activities (defined as release of reducing sugars) towards Kraft pulp. But at 80°C and pH10.5, TfuXyn4 still retained high activity, while

Optimase® CX 255L exhibited almost no activity. The release of lignin/phenolic compounds from Kraft pulp by TfuXyn4 or the benchmark was also evaluated using UV- Vis spectrometry, and the result was concordant with the activity under the same condition. These properties indicated that TfuXyn4 has properties that will greatly improve enzymatic processing in the paper and pulp industry, as well as other industries that would benefit from a thermostable and/or alkali tolerant xylanase.

EXAMPLE 1 Cloning of Thermobifida fusca xylanase TfuXyn4

[00126] Thermobifida fusca (isolate GICC 1481) genomic DNA was isolated as the same method for Streptomyces described in Kieser et al., Practical Streptomyces Genetics, The John Innes Foundation, Norwich, United Kingdom (2000). The full length sequence of TfuXyn4 was obtained by PCR from the genomic DNA. Two primers were designed based on the xylanase gene sequence from Thermobifida fusca YX (NCBI Accession No. YP_290979.1). The primers used were: TfuXyn4-FW5 '-

TGCGCTAGCCGGCCCCCCGGCACAGGCCGAGTCGACCCTGCGGGAACTGGC -3^' (SEQ ID NO:

1) , and TfuXyn4-RV 5 ^'- GCAGATCTAGGTCAGGAGCAGGTGGCTCCGT TGA-3^' (SEQ ID NO:

2) . The forward primer contains a Nhel restriction site, and the reverse primer contains a Bglll restriction site. The nucleic acid sequence of the PCR product (SEQ ID NO: 3) has been confirmed by sequencing.

[00127] The PCR product was digested with Nhel and Bglll and ligated into the pKB128 vector, and then digested with the Nhel and BamHI to obtain the expression plasmid pDS270 (Figure 1). The pKB128 plasmid is a derivative of the pKB105 plasmid (described in U.S. Pat. Application Publication No. 2006/0154843) and is the source of the A4 promoter. The ligation mixture was used to transform E. coli TOP10 chemically competent cells (Invitrogen Corp.) following the manufacturer's protocol. The transformed cells were plated on Luria Agar plates supplemented with 50 ppm ampicillin and incubated overnight at 37 °C. Three transformants were picked from the plate and inoculated into 5 ml Luria Broth supplemented with 50 ppm ampicillin. Cultures were grown overnight at 37°C, plasmid DNA extracted and the sequence of the TfuXyn4 gene was confirmed by DNA sequencing.

[00128] The nucleotide sequence of the PCR product encoding the mature TfuXyn4 is set forth as SEQ ID NO: 3:

GAGTCGACCCTGCGGGAACTGGCTGCCCAGAACGGCGGCCGCCACT TCGGTACGGCTATCGCCT ACAGCCCGCTCAACAGTGACGCCCAGTACCGCAACATCGCGGCTACCCAGTTCAGCGCCATCAC CCACGAAAACGAGATGAAGTGGGAGTCGCTGGAGCCGCAGCGGGGCCAGTACAACTGGAGCCAG GCCGACAACATCATCAACTTCGCCAAGGCCAACAACCAGATTGTGCGCGGCCACACCCTGGTCT GGCACAGCCAGCTGCCGTCCTGGCTGAACAACGGCGGCTTCTCCGGCAGCCAGCTCCGGTCCAT CATGGAGAACCACATCGAGGTGGTGGCCGGACGCTACCGGGGTGACGTCTACGCCTGGGACGTG GTCAACGAAGCGTTCAACGAGGACGGTACGCTCCGCGACTCGATCTGGTACCGCGGCATGGGTC GCGACTACATCGCCCACGCGTTCCGCAAGGCGCACGAGGTCGACCCCGACGCCAAGCTGTACAT CAACGACTACAACATCGAAGGCATCAACGCTAAGAGCAACGGCCTCTACAACCTGGTGGTCGAC CTGCTCCGCGACGGTGTGCCGATCCACGGTATCGGTATCCAGTCCCACCTGATCGTCGGCCAGG TGCCGTCCACGTTCCAGCAGAACATCCAGCGGTTCGCTGACCTCGGCCTGGACGTGGCCATCAC CGAGCTGGACATCCGCATGCAGATGCCGGCCGACCAGTACAAGCTCCAGCAGCAGGCCCGCGAC TACGAGGCCGTGGTCAACGCCTGCCTCGCGGTGACCCGCTGCATCGGTATCACCGTCTGGGGTA TCGACGACGAGCGCTCCTGGGTGCCCTACACCTTCCCGGGTGAAGGTGCTCCGCTGCTCTACGA CGGCCAGTACAACCGCAAGCCCGCCTGGTACGCGGTCTACGAGGCTCTCGGCGGCGACTCCTCC GGCGGCGGTCCGGGTGAGCCGGGCGGTCCTGGCGGTCCGGGTGAGCCGGGCGGCCCCGGTGACG GCACCTGCGCGGTGAACTACACCGTGGTCAATGACTGGGGTCACGGTATGCAGGGGGCGATCAC CGTCTCCAACACCGGATCCTCGCCCATCAACAACTGGACCCTGCAGTTCAGCTTCTCGGGTGTG AACATCTCCAACGGCTGGAACGGCGAGTGGAGCCAGAGCGGCTCGCAGATCACCGTCCGCGCTC CTGCCTGGAACTCCACGCTCCAGCCGGGCCAGAGCGTGGAACTGGGCTTCGTTGCTGACAAGAC CGGCAACGTCTCCCCGCCCTCCCAGTTCACCCTCAACGGAGCCACCTGCTCCTGA

[00129] The amino acid sequence of the TfuXyn4 precursor is set forth as SEQ ID NO: 4. The predicted native signal peptide (based on NCBI YP_290979.1) is shown in italics and bold (SEQ ID NO: 5).

mtetrhrpsrrarrslsllltsaltaagll vtaapaqaestlrelaaqnggrhfgtaiayspln sdaqyrniaatqfsaithenemkweslepqrgqynwsqadniinfakannqivrghtlvwhsql pswlnnggfsgsqlrsimenhievvagryrgdvyawd vneafnedgtlrdsiwyrgmgrdyia hafrkahevdpdaklyindynieginaksnglynl vdllrdgvpihgigiqshlivgqvpstf qqniqrfadlgldvaiteldirmqmpadqyklqqqardyeavvnaclavtrcigitvwgidder swvpytfpgegapllydgqynrkpawya yealggdssgggpgepggpggpgepggpgdgtca nyt vndwghgmqgaitvsntgsspinnwtlqfsfsgvnisngwngewsqsgsqitvrapawns tlqpgqsvelgf adktgnvsppsqftlngatcs [00130] The amino acid sequence of the mature form of TfuXyn4 is set forth as SEQ ID NO: 6: estlrelaaqnggrhfgtaiaysplnsdaqyrniaatqfsaithenemkweslepqrgqynwsq adniinfakannqivrghtlvwhsqlpswlnnggfsgsqlrsimenhie vagryrgdvyawdv vneafnedgtlrdsiwyrgmgrdyiahafrkahevdpdaklyindynieginaksnglynl vd llrdgvpihgigiqshlivgqvpstfqqniqrfadlgldvaiteldirmqmpadqyklqqqard yea vnaclavtrcigitvwgidderswvpytfpgegapllydgqynrkpawyavyealggdss gggpgepggpggpgepggpgdgtcavnyt vndwghgmqgaitvsntgsspinnwtlqfsfsgv nisngwngewsqsgsqitvrapawnstlqpgqsvelgf adktgnvsppsqftlngatcs

[00131] The nucleotide sequence of the TfuXyn4 gene in plasmid pDS270 (A4- CelA- TfuXyn4) is set forth as SEQ ID NO: 7. The sequence encoding CelA signal peptide is shown in italics and bold (SEQ ID NO: 7).

ATGGGCTTTGGGAGCGCTCCCATCGCGTTGTGTCCGCTTCGCACGAGGAGGAACGCTTTGAAAC GCCTTTTGGCCCTGCTCGCGACCGGCGTGTCGATCGTCGGCCTGACTGCGCTAGCCGGCCCCCC

GGCACAGGCCGAGTCGACCCTGCGGGAACTGGCTGCCCAGAACGGCGGCCGCCACTTCGGTACG GCTATCGCCTACAGCCCGCTCAACAGTGACGCCCAGTACCGCAACATCGCGGCTACCCAGTTCA GCGCCATCACCCACGAAAACGAGATGAAGTGGGAGTCGCTGGAGCCGCAGCGGGGCCAGTACAA CTGGAGCCAGGCCGACAACATCATCAACTTCGCCAAGGCCAACAACCAGATTGTGCGCGGCCAC ACCCTGGTCTGGCACAGCCAGCTGCCGTCCTGGCTGAACAACGGCGGCTTCTCCGGCAGCCAGC TCCGGTCCATCATGGAGAACCACATCGAGGTGGTGGCCGGACGCTACCGGGGTGACGTCTACGC CTGGGACGTGGTCAACGAAGCGTTCAACGAGGACGGTACGCTCCGCGACTCGATCTGGTACCGC GGCATGGGTCGCGACTACATCGCCCACGCGTTCCGCAAGGCGCACGAGGTCGACCCCGACGCCA AGCTGTACATCAACGACTACAACATCGAAGGCATCAACGCTAAGAGCAACGGCCTCTACAACCT GGTGGTCGACCTGCTCCGCGACGGTGTGCCGATCCACGGTATCGGTATCCAGTCCCACCTGATC GTCGGCCAGGTGCCGTCCACGTTCCAGCAGAACATCCAGCGGTTCGCTGACCTCGGCCTGGACG TGGCCATCACCGAGCTGGACATCCGCATGCAGATGCCGGCCGACCAGTACAAGCTCCAGCAGCA GGCCCGCGACTACGAGGCCGTGGTCAACGCCTGCCTCGCGGTGACCCGCTGCATCGGTATCACC GTCTGGGGTATCGACGACGAGCGCTCCTGGGTGCCCTACACCTTCCCGGGTGAAGGTGCTCCGC TGCTCTACGACGGCCAGTACAACCGCAAGCCCGCCTGGTACGCGGTCTACGAGGCTCTCGGCGG CGACTCCTCCGGCGGCGGTCCGGGTGAGCCGGGCGGTCCTGGCGGTCCGGGTGAGCCGGGCGGC CCCGGTGACGGCACCTGCGCGGTGAACTACACCGTGGTCAATGACTGGGGTCACGGTATGCAGG GGGCGATCACCGTCTCCAACACCGGATCCTCGCCCATCAACAACTGGACCCTGCAGTTCAGCTT CTCGGGTGTGAACATCTCCAACGGCTGGAACGGCGAGTGGAGCCAGAGCGGCTCGCAGATCACC GTCCGCGCTCCTGCCTGGAACTCCACGCTCCAGCCGGGCCAGAGCGTGGAACTGGGCTTCGTTG CTGACAAGACCGGCAACGTCTCCCCGCCCTCCCAGTTCACCCTCAACGGAGCCACCTGCTCCTG

A

[00132] The amino acid sequence of the TfuXyn4 precursor that is translated from plasmid pDS270 (A4-CelA-TfuXyn4) is set forth as SEQ ID NO: 8. The CelA signal peptide sequence is shown in italics and bold (SEQ ID NO: 8). mgfgsapialcplrtrrnalkrllallatgvsivgltalagppaqaest1re1aaqnggrhfgt aiaysplnsdaqyrniaatqfsaithenemkweslepqrgqynwsqadniinfakannqivrgh tlvwhsqlpswlnnggfsgsqlrsimenhievvagryrgdvyawd vneafnedgtlrdsiwyr gmgrdyiahafrkahevdpdaklyindynieginaksnglynl vdllrdgvpihgigiqshli vgqvpstfqqniqrfadlgldvaiteldirmqmpadqyklqqqardyeavvnaclavtrcigit vwgidderswvpytfpgegap11ydgqynrkpawya yealggdssgggpgepggpggpgepgg pgdgtcavnytvvndwghgmqgaitvsntgsspinnwtlqfsfsgvnisngwngewsqsgsqit vrapawnstlqpgqsvelgfvadktgnvsppsqftlngatcs

EXAMPLE 2

Expression and purification of TfuXyn4

[00133] The pDS270 plasmid was used to transform S. lividans TK23 derived protoplasts, as described in U.S. Pat. Application Publication No. 2006/0154843. The transformation techniques used are described in Kieser et al., Practical Streptomyces Genetics, The John Innes Foundation, Norwich, United Kingdom (2000). Transformed cells were plated on R5 selection plates and incubated at 30°C for 3 days. One clone from the Streptomyces transformation plate was inoculated in TSG medium in shake flasks at 28 °C for 3 days. Cultures were then transferred to a Streptomyces 2 Modified Medium (described in U.S. Pat. Application Publication No.

2006/0154843) and incubated for an additional 4 days at 28°C.

[00134] R5 plates: 206 g sucrose, 0.5 g K₂S0₄, 20.24 g MgCl₂, 20 g glucose, 0.2 g Difco casamino acids, 10 g Difco yeast extracts, 11.46 g TES, 4 g L-Asp, 4 ml of trace elements, 44 g Difco agar, 20 ml 5% K₂HP0₄, 8 ml 5 M CaCl₂ ^»2H₂0 and 14 ml IN NaOH were added to a final volume of 1 liter after autoclaving. After 20 hours, a layer of thiostrepton (50 μg/ml final concentration) was plated on the top of the plates.

[00135] TSG medium: 16 g BD Difco tryptone, 4 g BD Bacto soytone, 20 g Sigma caseine (hydrolysate), and 10 g potassium phosphate, dibasic, brought to 1 liter. After autoclaving, 50% glucose was added to a final concentration of 1.5%.

[00136] TfuXyn4 was purified from 1L concentrated shake flask fermentation broth using two chromatography columns. Concentrated fermentation broth buffered in 20 mM sodium

phosphate buffer pH 6.0 containing 1 M ammonium sulfate was loaded on a hydrophobic interaction chromatography column (Sepharose Phenyl FF, 26/10). After washing the column in 20 mM sodium phosphate buffer pH 6.0, the protein was eluted from the column using 10% glycerol. The fraction containing TfuXyn4 was loaded onto a gel filtration column (HiLoad Superdex 75 pg 26/60), and the mobile phase used was 20 mM sodium phosphate, pH 7.0, containing 0.15 M NaCl. The purified protein was concentrated using a 3K Amicon Ultra-15 device and the concentrated protein fraction was used in further studies.

EXAMPLE 3

Xylanase Activity of TfuXyn4

[00137] TfuXyn4 belongs to the glycosyl hydrolase 10 family (GH10, CAZy number). The beta 1-4 xylanase activity of TfuXyn4 was measured using 1% xylan from birch wood (Sigma 95588) or 1% arabinoxylan from wheat flour (Megazyme P-WAXYM) as substrates. The assay was performed in 50 mM sodium citrate pH 5.3, 0.005% Tween-80 buffer at 50 °C for 10 minutes. The released reducing sugar was quantified in a DNS (dinitro salicylic acid) assay (G. L. Miller, Anal. Chem. 31: 426-428, 1959). A standard curve using xylose was created and used to calculate enzyme activity units. In this assay, one xylanase unit is defined as the amount of enzyme required to generate 1 micromole of xylose reducing sugar equivalents per minute under the conditions of the assay. The specific activity of purified TfuXyn4 was determined to be 97 units/mg towards xylan from birch wood, and 197 units/mg towards arabinoxylan from wheat flour using the above method. EXAMPLE 4

pH Profile of TfuXyn4

[00138] The pH profile of TfuXyn4 was determined using xylan from birch wood (Sigma 95588) as substrate. The assay was performed in Sodium Citrate/Sodium Phosphate buffer solution adjusted to pH values between 2 and 9. Birchwood xylan (2% solution) dissolved in water was mixed with equal volume of 50 mM Citrate/Phosphate buffer solution in a 96-well plate, and the substrate was equilibrated at 50 °C before adding enzyme. After 10 minutes, the enzyme reaction was stopped by transferring 60 microliters of reaction mixture to a 96-well PCR plate containing 100 microliters of DNS solution. The PCR plate was heated at 95 °C for 5 minutes in a Bio-Rad DNA Engine. Then plate was cooled to room temperature and 100 microliters were transferred from each well to a new 96-well plate. Release of reducing sugars from the substrate was quantified by measuring the optical density at 540 nm in a

spectrophotometer. Enzyme activity at each pH was reported as relative activity where the activity at the pH optimum was set to 100%. The pH profile of TfuXyn4 is shown in Figure 3. TfuXyn4 was found to have an optimum pH at about 7, and was found to retain greater than 80% of maximum activity between pH 5 and 9.

EXAMPLE 5

Temperature Profile of TfuXyn4

[00139] The temperature optimum of purified TfuXyn4 was determined by assaying for xylanase activity at temperatures varying between 45°C and 94°C for 10 minutes in 50mM sodium citrate buffer at pH 5.3. The activity was reported as relative activity where the activity at the temperature optimum was set to 100%. The temperature profile of TfuXyn4 is shown in Figure 4. TfuXyn4 was found to have an optimum temperature of 79°C, and was found to retain greater than 80% of maximum activity between 66°C and 93°C.

EXAMPLE 6

Thermostability of TfuXyn4

[00140] The thermostability of TfuXyn4 was determined in 0.05M sodium citrate buffer pH 5.3. The enzyme was incubated at desired temperature for 2 hours in a PCR machine. The remaining activity of the samples was measured as described in Example 3. The activity of the sample kept on ice was defined as 100% activity. As shown in Figure 5, TfuXyn4 retained about 50% activity over a 2-hour incubation period at 69°C. At temperature lower than 60 °C, no activity loss was detected after 2-hour of incubation.

EXAMPLE 7

Enzymatic Treatment of Kraft Pulp

[00141] Unbleached Kraft pulp used in present study was mixed pulp (Jinhai Pulp & Paper, Hainan Province, China) with 70% from hardwood and 30% from softwood. The sample contained 21.92% dry solid as measured using Halogen Moisture Analyzer. After blending in a kitchen blender, the Kraft pulp sample was suspended to a 1% solution in 50 mM glycine buffer pH 9.0 or pH10.5. The pulp slurry was adequately mixed and dispensed to each well of a 96-well plate using a repeater 1 ml pipette (with 11 mm of the tips removed using a tube cutter). Enzymes were added at 0-8 mg/g pulp, and mixtures incubated 1 hour at 69 °C or 80 °C with gentle shaking. Control incubations contained no enzyme. After the incubation, the plate was centrifuged at 3500 rpm for 2 min at 4°C. Twenty microliters of supernatant were analyzed immediately for reducing sugar assay. The remainder of the supernatant was filtered through 96- well filter plate (Corning 3505), and UV-vis absorbances from 220 nm to 400 nm were measured to detect phenolic compounds release. Figure 6 shows the reducing sugar released from the enzymatic treatment of Kraft pulp. Under the same conditions tested, TfuXyn4 showed higher activity and released more reducing sugar than the same dosage of the benchmark, the purified enzyme from Genencor product Optimase® CX 255L. At pH10.5 and 80 °C, TfuXyn4 released about 8 to 10 times more reducing sugars than Optimase® CX 255L at the same enzyme dosages. The released phenolic compounds from Kraft pulp were detected at UV-vis spectrometry. Figure 7 showed the absorbances between 220 nm and 400 nm of the supematants from TfuXyn4 and Optimase® CX 255L incubations at the same dosages of enzyme (both at 8 mg/g pulp). At pH9.0 and 69 °C there is very little difference between these enzymes. Increasing pH and temperature facilitate the release of phenolic compounds, and the absorbances between 220 nm and 400 nm increased. This phenomenon is much more significant for TfuXyn4 compared to Optimase® CX 255L, which is consistent with their enzymatic activities determined as reducing sugar release (Figure 5). These results indicate that TfuXyn4 is a suitable enzyme to facilitate biobleaching of Kraft paper under high temperature and alkaline conditions.

Although the foregoing compositions and methods has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings herein that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Accordingly, the preceding merely illustrates the principles of the present compositions and methods. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the present compositions and methods and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the present compositions and methods and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the present compositions and methods as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present compositions and methods, therefore, is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

WE CLAIM:

1. A recombinant polypeptide comprising an amino acid sequence that is at least 55% identical to the amino acid sequence of SEQ ID NO: 6, wherein the polypeptide has xylanase activity.

2. The recombinant polypeptide of Claim 1, wherein the polypeptide retains greater than 70% of the xylanase activity when incubated at a pH range from pH 5.0 to pH 9.0.

3. The recombinant polypeptide of Claim 1 or 2, wherein the polypeptide retains at least 80% of the xylanase activity when incubated at a temperature of between 66°C and 93°C.

4. The recombinant polypeptide of any one of Claims 1-3, wherein the polypeptide has optimum xylanase activity at a temperature of at least 75°C or above.

5. The recombinant polypeptide of any one of Claims 1-4, wherein the polypeptide retains at least 50% of the xylanase activity when incubated for about 2 hours at a temperature of about 69°C.

6. The recombinant polypeptide of any one of Claims 1-5, wherein the polypeptide retains at least 90% of the xylanase activity when incubated for about 2 hours at a temperature of between 45 °C and 60 °C.

7. The recombinant polypeptide of any one of Claims 1-5, wherein the polypeptide retains at least 90% of the xylanase activity when incubated for about 2 hours at a temperature of between 50°C and 60 °C.

8. The recombinant polypeptide of any one of Claims 1-5, wherein the polypeptide retains at least 90% of the xylanase activity when incubated for about 2 hours at a temperature of between 55°C and 60 °C.

9. The recombinant polypeptide of any one of Claims 1-5, wherein the polypeptide retains at least 90% of the xylanase activity when incubated for about 2 hours at a temperature of about 60 °C.

10. The recombinant polypeptide of any one of Claims 1-9, wherein the polypeptide has an improved thermostability.

11. The recombinant polypeptide of any one of Claims 1-10, wherein the polypeptide comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 6.

12. The recombinant polypeptide of any one of Claims 1-10, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 6.

13. The recombinant polypeptide of any one of Claims 1-10, wherein the polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 6.

14. The recombinant polypeptide of any one of Claims 1-10, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 6.

15. A composition comprising the recombinant polypeptide of any one of Claims 1- 14 and a chemical pulp.

16. The composition of Claim 15, wherein the chemical pulp is a Kraft pulp, soda pulp, or sulfite pulp.

17. An isolated nucleic acid encoding the recombinant polypeptide of any Claims 1-14.

18. The isolated nucleic acid of Claim 17, wherein the polypeptide further comprises a signal peptide sequence.

19. The isolated nucleic acid of Claim 18, wherein the signal peptide sequence comprises SEQ ID NO: 5.

20. An expression vector comprising the isolated nucleic acid of any one of Claims 18-20 in operable combination with a regulatory sequence.

21. A host cell comprising the expression vector of Claim 20.

22. The host cell of Claim 21, wherein the host cell is a bacterial cell or a fungal cell.

23. A composition comprising the host cell of Claim 21 or 22 and a culture medium.

24. A method of producing a xylanase, comprising: culturing the host cell of Claim 22 or 23 in a culture medium, under suitable conditions to produce the xylanase.

25. A composition comprising the xylanase produced in accordance with the method of Claim 25 in supernatant of the culture medium.

26. A method for enzymatic treatment of a pulp, the method comprising:

contacting the pulp with the polypeptide of any one of Claims 1-14 under alkaline conditions to produce a mixture, wherein the xylanase activity of the polypeptide is greater than that observed for Optimase® CX 255L under similar conditions.

27. The method of Claim 26, wherein the pulp is a chemical pulp.

28. The method of Claim 27, wherein the chemical pulp is a Kraft pulp, soda pulp, or sulfite pulp.

29. The method of any one of Claims 26-28, wherein the pH of the mixture is at least pH 8.2, at least pH 8.4, at least pH 8.6, at least pH 8.8, at least pH 9.0, pH 9.2, at least pH 9.4, at least pH 9.6, at least pH 9.8, at least pH 10.0, at least pH 10.2, at least pH 10.4, at least pH 10.6, at least pH 10.8, or at least pH 11.0.

30. The method of any one of Claims 26-29, wherein the contacting is performed at a temperature of about 50°C or above.

31. The method of Claim 30, wherein the contacting is performed at a temperature of at least 55 °C, at least 60 °C, at least 65 °C, at least 69 °C, at least 70 °C, at least 75 °C, at least 80 °C, at least 85 °C, at least 89 °C, at least 90 °C, at least 95 °C, at least 100 °C, at least 110 °C, or at least 120 °C.

32. The method of any one of Claims 26, wherein the mixture is at least pH 10 and the contacting is performed at a temperature of at least 80 °C.

33. A method for enzymatic treatment of a pulp, the method comprising:

contacting the pulp with the polypeptide of any one of Claims 1-14 at a temperature of about 50°C or above, wherein the xylanase activity of the polypeptide is greater than that observed for Optimase® CX 255L under similar conditions.

34. The method of Claim 33, wherein the contacting is performed under alkaline conditions.

35. The method of Claim 33 or 34, wherein the pH of the mixture is at least pH 8.2, at least pH 8.4, at least pH 8.6, at least pH 8.8, at least pH 9.0, pH 9.2, at least pH 9.4, at least pH 9.6, at least pH 9.8, at least pH 10.0, at least pH 10.2, at least pH 10.4, at least pH 10.6, at least pH 10.8, or at least pH 11.0.

36. The method of any one of Claims 33-35, wherein the contacting is performed at a temperature of at least 55 °C, at least 60 °C, at least 65 °C, at least 69 °C, at least 70 °C, at least 75 °C, at least 80 °C, at least 85 °C, at least 89 °C, at least 90 °C, at least 95 °C, at least 100 °C, at least 110 °C, or at least 120 °C.

37. The method of any one of Claims 33-36, wherein the pulp is a chemical pulp.

38. The method of Claim 37, wherein the chemical pulp is a Kraft pulp, soda pulp, or sulfite pulp.

39. The method of any one of Claims 26-38, wherein the method is employed to facilitate biobleaching of a Kraft pulp or a chemical pulp.