# Patent

## Title
GH8 xylanase variants

## Abstract

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 35 U.S.C. 371 national application of PCT/EP2010/069249 filed Dec. 9, 2010, which claims priority or the benefit under 35 U.S.C. 119 of European application no. 09178558.4 filed Dec. 9, 2009 and U.S. provisional application No. 61/329,362 filed Apr. 29, 2010 the contents of which are fully incorporated herein by reference.


REFERENCE TO A SEQUENCE LISTING
This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.
FIELD OF THE INVENTION
The present invention relates to methods of producing a variant of a parent GH8 xylanase polypeptide which retains xylanase activity. It also relates to isolated GH8 polypeptide variants having xylanase activity and isolated polynucleotides encoding the polypeptide variants. The invention further relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, such as, in the preparation of a dough or a baked product prepared from the dough. More particularly, it relates to such a process where the dough is less sticky than if prepared with the parent GH8 xylanase and/or where the bread has an increased volume.
BACKGROUND OF THE INVENTION
Carbohydrates and glycol-conjugates are substrates for glycosyl transferases (GTs) and glycoside hydrolases (GHs). The structure of glycoside hydrolases began to be solved starting from the 1980s. At the same time, new GH proteins were discovered and their amino acid sequence determined. Two main observations emerged from the new data. 1) The classical E.C. nomenclature system for naming enzyme families was not precise enough to classify the increasing number of enzymes that had different structure yet performed the same enzymatic reaction. 2) Enzymes related by homology could have different enzymatic activity thus also making the E.C. nomenclature system confusing for these related enzymes.
A new family based nomenclature system was proposed by Bernard Henrissat in 1991 based on the structure of the enzymes (Henrissat B., A classification of glycosyl hydrolases based on amino-acid sequence similarities. Biochem. J. 280:309-316 (1991); Henrissat B., Bairoch A. New families in the classification of glycosyl hydrolases based on amino-acid sequence similarities. Biochem. J. 293:781-788 (1993); Henrissat B., Bairoch A. Updating the sequence-based classification of glycosyl hydrolases. Biochem. J. 316:695-696 (1996) and Davies G., Henrissat B. Structures and mechanisms of glycosyl hydrolases. Structure 3:853-859 (1995).). Updated classifications are available on the Carbohydrate-Active EnZymes website (CAZy).
The classification of glycoside hydrolases in families based on amino acid sequence similarities was introduced because there is a direct relationship between sequence and folding similarities, and such a classification is expected to:

    
    
        (i) reflect the structural features of the enzymes, which cannot be reflected by the substrate specificity alone,
        (ii) help to reveal the evolutionary relationships between the enzymes, and
        (iii) provide a convenient tool to derive mechanistic information.
    
    


Amino acid sequences grouped by nature of their similarity to a particular GH family can give ideas as to the activity of the new hypothetical protein. Some of these amino acid sequences, grouped in a GH family by homology have later been suggested to have certain enzymatic activity. So, in short, grouping a new amino acid sequence in a GH family does not specifically indicate the exact enzymatic activity. The enzymatic activity must be demonstrated by an activity assay of the cloned or purified protein. If the assay is difficult determination of the proteins actual function can remain un-revealed for years.
Glycoside hydrolase family 8 (GH8) comprises enzymes with several known activities; endoglucanase (EC:3.2.1.4); lichenase (EC:3.2.1.73); chitosanase (EC:3.2.1.132).
Some xylanases are inhibited by components found in flour which makes them unsuitable for the manufacture of products based on a dough. GH8 xylanases are not inhibited by components in dough and they require only a very low dosage in the dough to provide a volume increase in the baked bread. Unfortunately, as is well-known in the art of baking, use of any of the naturally occurring GH8 xylanases identified to date results in a sticky dough which is problematic to handle.
SUMMARY OF THE INVENTION
The inventors identified a characteristic amino acid motif conserved throughout a diverse range of GH8 xylanases; it comprises the glutamic acid (E) nucleophile of the active site in those xylanases:









(SEQ ID NO: 1)



[ST]E[GAS]X[GAS][YFW].






Molecular modelling was employed and a structural loop lining the active site of the GH8 xylanases was identified (see FIG. 1). The loop comprised about 20 consecutive amino acids directly flanking the conserved GH8 motif on the N-terminal side (FIG. 1).
A synthetic expression construct (SEQ ID NO:2) was manufactured encoding the mature GH8 xylanase from Bacillus sp. KK-1 (UNIPROT accession number: O52730; SEQ ID NO:4). The synthetic construct comprised a sequence encoding a heterologous signal peptide fused to a mature GH8 xylanase polypeptide coding sequence; the nucleic acid sequence of the construct was optimized for Bacillus expression. The amino acid sequences of the encoded heterologous signal peptide and the mature GH8 xylanase are shown in SEQ ID NO:3. The amino acid sequences of the mature polypeptides in SEQ ID NO:3 and SEQ ID NO:4 are identical, of course.
The conserved GH8 motif is found in amino acid positions 65-70 of the mature polypeptide shown in SEQ ID NO:3 or 4.
The loop flanking the conserved GH8 motif in SEQ ID NO:3 was mutated by site-specific mutagenesis of amino acids comprised therein. Deletions, insertions and substitutions of single amino acids were made, as well as some combinations thereof, to produce a number of GH8 variants having xylanase activity, vide infra. The activity of the variants was investigated in baking applications and they showed surprisingly interesting and promising properties.
Accordingly, in a first aspect, the present invention relates to a method of producing a variant of a parent GH8 xylanase polypeptide, said method comprising the steps of:

    
    
        a) providing a polynucleotide encoding a mature parent GH8 xylanase polypeptide comprising a first conserved GH8 xylanase motif:
    
    











(SEQ ID NO: 1)



[ST]E[GAS]X[GAS][YFW],






    
    
        b) introducing at least one mutation into the encoding polynucleotide, whereby at least one alteration is introduced in the encoded polypeptide in one or more of the 20 amino acid positions flanking the first conserved GH8 xylanase motif on the N-terminal side, and
        c) expressing the mutated polynucleotide in a suitable host cell and under conditions conducive to producing the variant,
        
            wherein the variant has xylanase activity.
        
        
    
    


In a second aspect, the invention relates to an isolated variant of a parent GH8 xylanase polypeptide comprising a first conserved GH8 xylanase motif:









(SEQ ID NO: 1)



[ST]E[GAS]X[GAS][YFW],






wherein the variant comprises at least one amino acid alteration compared to the parent in one or more of the 20 amino acid positions flanking the first conserved GH8 motif on the N-terminal side, and wherein the variant has xylanase activity.

In a third aspect, the invention relates to an isolated polynucleotide comprising a nucleotide sequence that encodes the variant polypeptide of the second aspect.
A fourth aspect relates to a nucleic acid construct comprising the polynucleotide of the third aspect.
A fifth aspect relates to a recombinant expression vector comprising the nucleic acid construct of the fourth aspect.
A sixth aspect relates to a recombinant host cell comprising the recombinant expression vector of the previous aspect, the nucleic acid construct of the fourth aspect or the polynucleotide of the third aspect operably linked to one or more control sequences that direct the production of the variant polypeptide.
A seventh aspect relates to a method of producing the variant polypeptide of the second aspect, comprising: (a) cultivating a host cell comprising a nucleic acid construct comprising a nucleotide sequence encoding the variant polypeptide under conditions conducive for production of the variant; and (b) recovering the variant.
Other aspects relate to a composition comprising the variant polypeptide of the second aspect, a method for the preparation of a baked product, said method comprising the step of adding to a dough of said baked product the variant polypeptide of the second aspect or a bread improving composition comprising the variant polypeptide of the second aspect.
Another aspect of the invention relates to a method for increasing the loaf volume of a baked product, comprising the step of adding during the mixing of the dough of said baked product, a sufficient amount of the variant polypeptide of the second aspect or a composition comprising said polypeptide.
Yet another aspect of the invention relates to a method for increasing the loaf volume of a baked product or for increasing the width of cut on the surface of a baked product, comprising the step of adding during the mixing of the dough of said baked product, a sufficient amount the variant polypeptide of the second aspect or a composition comprising said polypeptide.
In a final aspect, the invention relates to a bread improver composition for increasing the loaf volume of a baked product or for increasing the width of cut on the surface of a baked product, characterised in that it comprises at least one variant polypeptide of the second aspect.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 (landscape) shows a section of a molecular model of the parent GH8 xylanase from Bacillus sp. KK-1 (UNIPROT accession number: O52730; SEQ ID NO:4). A close-up of the substrate binding pocket is provided (light grey net) with the active site (dark grey net) just below the center of the figure. The loop (dark grey coil) flanking the first conserved GH8 xylanase motif (not shown) on the N-terminal side is found just above the active site, may be protruding slightly into the substrate binding pocket. The three loop amino acid residues 53 (Asp), 55 (Asn) and 59 (Gly) are indicated (dark grey) and a stick-model is shown of a substrate molecule in the substrate pocket.
FIG. 2 (landscape) shows a xylanase activity standard curve calculated as shown in the definition section below; the x-axis is the xylanase activity in FXU/ml and the y-axis is the absorbance at 595 nm (Abs595 or OD595 nm).



DEFINITIONS
Xylanase activity: For the purposes of the present invention any of the commercially available xylanase activity measurement kits is suitable to determine xylanase activity. The sole purpose of the xylanase activity measurement in the present invention is as a primary screen to ensure that the GH8 xylanase variants of the invention have not inadvertently been inactivated by the amino acid alteration(s) introduced therein. One suitable way of measuring the xylanase activity is as follows:

Substrate:

AZCL-Arabinoxylan from wheat (Megazyme).
Assay Buffer:
50 mM B&R (50 mM H3PO4, 50 mM acetic acid, 50 mM H3BO3), 50 mM KCl, 1 mM CaCl2, 0.01% Triton X-100, pH adjusted to 6.0 using NaOH.
Enzyme:
All enzyme dilutions are made in distilled water with 0.01% Triton X-100.
Substrate Solution:
A 0.2% (w/v) slurry of AZCL-Arabinoxylan substrate is prepared in assay buffer. Cut the tip of a 1000 uL pipette tip to siphon off homogenous aliquots.
Standard:
BioFeed WheatTM (Novozymes A/S), diluted in 0.01% Triton X-100. FXU/ml: 0.05; 0.10; 0.15; 0.20; 0.25; 0.30; 0.40.
Analysis:


    1. Eppendorf termomixer at 37° C.
    2. 750 μl substrate solution is placed on ice for 5 minutes.
    3. 50 μl enzyme sample is added.
    4. Incubation for 15 min. at 1400 rpm, 37° C.
    5. Reaction is stopped in ice/waterbath.
    6. Centrifugation 5 min. at 10.000 rpm, 4° C.
    7. 200 μl supernatant is transferred to a microtiter plate
    8. Endpoint absorbance is measured, OD595 nm
    Note: The absorbance, OD595 nm, should be in the range of 0-1.5.
    Note: In this assay, the standard curve is made from a FXU standard, but the activity measured is xylanase units. FXU is measured on azo-xylan not azcl-xylan.

Standard Curve:



A standard solution is prepared from BioFeed WheatTM (Novozymes A/S) with a declared xylanase activity of 439 FXU(W)/g. 2.05 g BioFeed WheatTM is dissolved up to 100 mL in 0.9% NaCl, which provides a solution with an activity of 9.0 FXU/mL (frozen stock kept at −80° C.).
Immediately prior to carrying out the assay the stock is diluted 10 times to provide the assay stock solution (stock).


















std-1


Standard
μl stock
μl buffer
dilution
FXU(W)/ml



















1
—
180

0


2
20
180
10
0.09


3
60
180
4
0.225


4
180
180
2
0.45


5
540
180
1.33
0.675


6
60
—
1
0.9









Calculations:

The standard curve is made from A595 for the enzyme standard subtracted std1 (as shown below).
The activity is calculated from A595 for the enzyme subtracted a blank-value. Since the enzyme dilution often is without colour, std1 is used as blank value in these cases.
Example of a standard curve calculation, the curve is shown in FIG. 2:

















μl
μl

std-1




Standard
stock
buffer
dilution
FXU(W)/ml
A595
A595-std1





















1
—
180

0
1.1064
0


2
20
180
10
0.09
1.1588
0.0524


3
60
180
4
0.225
0.2472
0.1408


4
180
180
2
0.45
0.385
0.2786


5
540
180
1.33
0.675
0.5659
0.4595


6
60
—
1
0.9
0.7492
0.6428









The mature variant polypeptides of the present invention have at least 20%, preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 100% of the xylanase activity of their mature GH8 parent xylanase polypeptide.
Isolated polypeptide: The term “isolated polypeptide” as used herein refers to a polypeptide that is isolated from a source. In a preferred aspect, the polypeptide is at least 1% pure, preferably at least 5% pure, more preferably at least 10% pure, more preferably at least 20% pure, more preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, and most preferably at least 90% pure, as determined by SDS-PAGE.
Mature polypeptide: The term “mature polypeptide” is defined herein as a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one aspect, the mature polypeptide comprises or consists of the amino acid positions listed in table 1, vide infra.
Mature polypeptide coding sequence: The term “mature polypeptide coding sequence” is defined herein as a nucleotide sequence that encodes a mature polypeptide having xylanase activity. In one aspect, the mature polypeptide coding sequence comprises or consists of the nucleotides encoding the mature polypeptides comprising or consisting of the amino acid positions listed in table 1, vide infra.
Sequence ldentity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.
For purposes of the present invention, the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics 16: 276-277), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps in Alignment)

For purposes of the present invention, the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Number of Gaps in Alignment)

Homologous sequence: The term “homologous sequence” is defined herein as a predicted protein having an E value (or expectancy score) of less than 0.001 in a tfasty search (Pearson, W. R., 1999, in Bioinformatics Methods and Protocols, S. Misener and S. A. Krawetz, ed., pp. 185-219) with any of the polypeptides shown in any of SEQ ID NO's: 3-154 and 159 or the mature part thereof.
Polypeptide fragment: The term “polypeptide fragment” is defined herein as a polypeptide having one or more (several) amino acids deleted from the amino and/or carboxyl terminus of the mature polypeptide of any of SEQ ID NO's: 3-154 and 159; or a homologous sequence thereof; wherein the fragment has xylanase activity.
Subsequence: The term “subsequence” is defined herein as a nucleotide sequence having one or more (several) nucleotides deleted from the 5′ and/or 3′ end of the mature polypeptide coding sequence or a homologous sequence thereof; wherein the subsequence encodes a polypeptide fragment having xylanase activity.
Allelic variant: The term “allelic variant” denotes herein any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.
Isolated polynucleotide: The term “isolated polynucleotide” as used herein refers to a polynucleotide that is isolated from a source. In a preferred aspect, the polynucleotide is at least 1% pure, preferably at least 5% pure, more preferably at least 10% pure, more preferably at least 20% pure, more preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, and most preferably at least 90% pure, as determined by agarose electrophoresis.
Coding sequence: When used herein the term “coding sequence” means a nucleotide sequence, which directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA, synthetic, or recombinant nucleotide sequence.
cDNA: The term “cDNA” is defined herein as a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps before appearing as mature spliced mRNA. These steps include the removal of intron sequences by a process called splicing. cDNA derived from mRNA lacks, therefore, any intron sequences.
Nucleic Acid Construct: The term “nucleic acid construct” as used herein refers to a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic. The term nucleic acid construct is synonymous with the term “expression cassette” when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present invention.
Control sequences: The term “control sequences” is defined herein to include all components necessary for the expression of a polynucleotide encoding a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding a polypeptide.
Operably linked: The term “operably linked” denotes herein a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide sequence such that the control sequence directs the expression of the coding sequence of a polypeptide.
Expression: The term “expression” includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
Expression vector: The term “expression vector” is defined herein as a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide of the present invention and is operably linked to additional nucleotides that provide for its expression.
Host cell: The term “host cell”, as used herein, includes any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention.
Modification: The term “modification” means herein any chemical modification of the polypeptide comprising or consisting of the mature polypeptide of SEQ ID NO: 2; or a homologous sequence thereof; as well as genetic manipulation of the DNA encoding such a polypeptide. The modification can be a substitution, a deletion and/or an insertion of one or more (several) amino acids as well as replacements of one or more (several) amino acid side chains.
Artificial variant: When used herein, the term “artificial variant” means a polypeptide having xylanase activity produced by an organism expressing a modified polynucleotide sequence encoding a variant of any of the polypeptides shown in SEQ ID NO's: 3-154 and 159 having xylanase activity, or a variant of the mature parts thereof having xylanase activity. The modified nucleotide sequence is obtained through human intervention by modification of the encoding polynucleotide sequence or complete synthesis of an polynucleotide encoding the modified variant.
DETAILED DESCRIPTION OF THE INVENTION
In its most general form, the instant invention relates to a method of producing a variant of a parent GH8 xylanase polypeptide, said method comprising the steps of:

    
    
        a) providing a polynucleotide encoding a mature parent GH8 xylanase polypeptide comprising a first conserved GH8 xylanase motif:
    
    











(SEQ ID NO: 1)



[ST]E[GAS]X[GAS][YFW],






    
    
        b) introducing at least one mutation into the encoding polynucleotide, whereby at least one alteration is introduced in the encoded polypeptide in one or more of the 20 amino acid positions flanking the first conserved GH8 xylanase motif on the N-terminal side, and
        c) expressing the mutated polynucleotide in a suitable host cell and under conditions conducive to producing the variant,

wherein the variant has xylanase activity.

    
    


In a preferred embodiment of the first aspect, the mature parent GH8 xylanase polypeptide comprises an amino acid sequence at least 60% identical to the mature part of a GH8 xylanase selected from the group of GH8 xylanases shown in any of SEQ ID NO:3 to SEQ ID NO:154 and SEQ ID NO:159; preferably the amino acid sequence is at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% identical to the mature part of a GH8 xylanase selected from the group of GH8 xylanases shown in any of SEQ ID NO:3 to SEQ ID NO:154 and SEQ ID NO:159.
In another preferred embodiment of the first aspect, the mature parent GH8 xylanase comprises a second conserved GH8 xylanase motif:









(SEQ ID NO: 155)



[ND][AS]X[RLPQ].






Preferably, the mature parent GH8 xylanase comprises or consists of amino acids 28 to 433 of SEQ ID NO: 3.
In a preferred embodiment, the at least one alteration in the encoded polypeptide comprises a substitution, deletion, and/or insertion of one or more amino acids in one or more of the 20 amino acid positions flanking the first conserved GH8 xylanase motif on the N-terminal side.
It is also preferred that the at least one alteration in the encoded polypeptide in the first aspect of the invention comprises an insertion of one or more amino acids in one or more of the 15 amino acid positions flanking the first conserved GH8 xylanase motif on the N-terminal side; preferably an insertion of one or more alanine(s).
In another preferred embodiment of the first aspect, the variant has at least one bread or dough improving property when added to the dough before or during mixing in a sufficient amount.
Preferably, the variant is capable of increasing the loaf volume of a baked product or increasing the width of cut on the surface of a baked product, when added to the dough before or during mixing in a sufficient amount; such as, when added to the dough before or during mixing in a sufficient amount, the variant provides a less sticky dough compared to the parent GH8 xylanase, determined as in the examples herein, vide infra.
Polypeptides Having Xylanase Activity
In one aspect, the invention relates to an isolated variant of a parent GH8 xylanase polypeptide comprising a first conserved GH8 xylanase motif:









(SEQ ID NO: 1)



[ST]E[GAS]X[GAS][YFW],






wherein the variant comprises at least one amino acid alteration compared to the parent in one or more of the 20 amino acid positions flanking the first conserved GH8 motif on the N-terminal side, and wherein the variant has xylanase activity.

Preferably, the isolated polypeptide comprises an amino acid sequence having a degree of sequence identity to the mature polypeptide xylanase of any of SEQ ID NO's: 3-154 and 159 of at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% (hereinafter “homologous polypeptides”).
In a preferred embodiment, the variant of the second aspect is selected from the group of variants consisting of:
(a) the variant polypeptide comprising an amino acid sequence having at least 60% sequence identity to the mature part of a GH8 xylanase selected from the group of GH8 xylanases shown in SEQ ID NO: 3 to SEQ ID NO: 154 and SEQ ID NO:159; preferably the amino acid sequence is at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% identical to the mature part of a GH8 xylanase selected from the group of GH8 xylanases shown in any of SEQ ID NO:3 to SEQ ID NO:154 and SEQ ID NO:159;
(b) the variant polypeptide encoded by a polynucleotide that hybridizes under at least medium-high stringency conditions with (i) the mature polypeptide coding sequence shown in SEQ ID NO: 2, or (ii) a full-length complementary strand of (i);
(c) the variant polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 60% sequence identity to the mature polypeptide coding sequence shown in SEQ ID NO: 2; and
(d) the variant of (a), (b) or (c) comprising a substitution, deletion, and/or insertion of one or more amino acids.
In another preferred embodiment, the parent GH8 xylanase polypeptide comprises a second conserved GH8 xylanase motif: [ND][AS]X[RLPQ] (SEQ ID NO:155).
Yet another preferred embodiment relates to the variant of the second aspect, wherein the mature parent GH8 xylanase polypeptide comprises an amino acid sequence at least 60% identical to the mature part of a GH8 xylanase shown in SEQ ID NO:3, preferably the amino acid sequence is at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% identical to the mature part of a GH8 xylanase shown in SEQ ID NO:3.
It is also preferred that the variant of the second aspect is encoded by a polynucleotide that hybridizes under at least medium-high stringency conditions with (i) a polynucleotide comprising a nucleotide sequence of the mature polypeptide coding sequence of SEQ ID NO: 2 or (ii) a full-length complementary strand of (i).
Preferably, the variant of the second aspect is encoded by a polynucleotide comprising a nucleotide sequence having at least 60% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 2; preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% identity.
In an even more preferred embodiment, the variant of the second aspect is encoded by a polynucleotide comprising or consisting of the nucleotide sequence of SEQ ID NO: 2; or a subsequence thereof encoding a polypeptide fragment having xylanase activity.
Even more preferable, is where the mature polypeptide comprises or consists of amino acids 28 to 433 of SEQ ID NO: 3, or wherein the mature polypeptide is encoded by a polynucleotide comprising or consisting of nucleotides 82 to 1299 of SEQ ID NO: 2.
Another preferred embodiment relates to the variant of the second aspect, wherein the at least one amino acid alteration comprises substitution, deletion, and/or insertion of one or more amino acids; preferably the at least one amino acid alteration comprises substitution, deletion, and/or insertion of one or more amino acids at one or more position(s) 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 and/or 1 amino acid(s) away from the first conserved GH8 xylanase motif on the N-terminal side or at one or more position(s) corresponding to position(s) 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63 and/or 64 in the mature polypeptide of SEQ ID NO: 3.
In another preferred embodiment, the at least one amino acid alteration(s) in the variant of the second aspect is comprised in one or more of the 15 amino acid positions flanking the first conserved GH8 xylanase motif on the N-terminal side, preferably in one or more of the 12 amino acid positions flanking the first conserved GH8 xylanase motif on the N-terminal side.
It is also preferable that the at least one amino acid alteration(s) comprises one or more insertion(s) at a position corresponding to a position 12 amino acids away from the first conserved GH8 xylanase motif on the N-terminal side in the mature polypeptide of SEQ ID NO: 3 or at a position corresponding to position 53 in the mature polypeptide of SEQ ID NO: 3.
Preferably, the at least one amino acid alteration comprises one or more insertion(s) at a position corresponding to a position 10 amino acids away from the first conserved GH8 xylanase motif on the N-terminal side in the mature polypeptide of SEQ ID NO: 3 or at a position corresponding to position 55 in the mature polypeptide of SEQ ID NO: 3.
More preferably, the at least one amino acid alteration comprises one or more insertion(s) at a position corresponding to a position 8 amino acids away from the first conserved GH8 xylanase motif on the N-terminal side in the mature polypeptide of SEQ ID NO: 3 or at a position corresponding to position 57 in the mature polypeptide of SEQ ID NO: 3.
Even more preferably, the at least one amino acid alteration comprises one or more insertion(s) at a position corresponding to a position 6 amino acids away from the first conserved GH8 xylanase motif on the N-terminal side in the mature polypeptide of SEQ ID NO: 3 or at a position corresponding to position 59 in the mature polypeptide of SEQ ID NO: 3.
In another preferred embodiment, the at least one amino acid alteration comprises one or more insertion(s) at a position corresponding to a position 4 amino acids away from the first conserved GH8 xylanase motif on the N-terminal side in the mature polypeptide of SEQ ID NO: 3 or at a position corresponding to position 61 in the mature polypeptide of SEQ ID NO: 3.
Yet another preferred embodiment relates to the variant of the second aspect, wherein the one or more insertion(s) comprise the insertion of at least one alanine residue, at least one aspartic acid residue, at least one serine residue, at least one lysine residue, and/or at least one phenylalanine; preferably the one or more insertion(s) comprises the insertion of at least one alanine residue; preferably the one or more insertion(s) comprise the insertion of at least one amino acid residue and at least one additional but different amino acid residue at the same position or at a different position.
Other preferred embodiments relate to the variant of the second aspect, which has at least one bread or dough improving property when added to the dough before or during mixing in a sufficient amount; preferably it is capable of increasing the loaf volume of a baked product or increasing the width of cut on the surface of a baked product, when added to the dough before or during mixing in a sufficient amount, and more preferably it provides a less sticky dough when compared with the parent GH8 xylanase added in the same amount.
In a preferred aspect, the homologous polypeptides comprise amino acid sequences that differ by ten amino acids, preferably by five amino acids, more preferably by four amino acids, even more preferably by three amino acids, most preferably by two amino acids, and even most preferably by one amino acid from the mature polypeptide of any of SEQ ID NO's: 3-154 and 159. A polypeptide of the present invention preferably comprises the amino acid sequence of any of SEQ ID NO's: 3-154 and 159 or an allelic variant thereof; or a fragment thereof having xylanase activity. In a preferred aspect, the polypeptide comprises the amino acid sequence of any of SEQ ID NO's: 3-154 and 159. In another preferred aspect, the polypeptide comprises the mature polypeptide of any of SEQ ID NO's: 3-154 and 159. In another preferred aspect, the polypeptide consists of the amino acid sequence of any of SEQ ID NO's: 3-154 and 159. In another preferred aspect, the polypeptide consists of the mature polypeptide of any of SEQ ID NO's: 3-154 and 159.
In another aspect, the present invention relates to isolated polypeptides having xylanase activity that are encoded by polynucleotides that hybridize under preferably very low stringency conditions, more preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 2, (ii) the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 2, or (iii) a full-length complementary strand of (i) or (ii) (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.).
The nucleotide sequence of SEQ ID NO: 2; or a subsequence thereof; as well as the amino acid sequence of any of SEQ ID NO's: 3-154 and 159; or a fragment thereof; may be used to design nucleic acid probes to identify and clone DNA encoding polypeptides having xylanase activity from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 14, preferably at least 25, more preferably at least 35, and most preferably at least 70 nucleotides in length. It is, however, preferred that the nucleic acid probe is at least 100 nucleotides in length. For example, the nucleic acid probe may be at least 200 nucleotides, preferably at least 300 nucleotides, more preferably at least 400 nucleotides, or most preferably at least 500 nucleotides in length. Even longer probes may be used, e.g., nucleic acid probes that are preferably at least 600 nucleotides, more preferably at least 700 nucleotides, even more preferably at least 800 nucleotides, or most preferably at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with 32P, 3H, 35S, biotin, or avidin). Such probes are encompassed by the present invention.
A genomic DNA or cDNA library prepared from such other strains may, therefore, be screened for DNA that hybridizes with the probes described above and encodes a polypeptide having xylanase activity. Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA that is homologous with SEQ ID NO: 2, or a subsequence thereof, the carrier material is preferably used in a Southern blot.
For purposes of the present invention, hybridization indicates that the nucleotide sequence hybridizes to a labeled nucleic acid probe corresponding to the mature polypeptide coding sequence of SEQ ID NO: 2; the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 29; its full-length complementary strand; or a subsequence thereof; under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film.
In a preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 2. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of any of SEQ ID NO's: 3-154 and 159, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 2.
For long probes of at least 100 nucleotides in length, very low to very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and either 25% formamide for very low and low stringencies, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures for 12 to 24 hours optimally.
For long probes of at least 100 nucleotides in length, the carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SDS preferably at 45° C. (very low stringency), more preferably at 50° C. (low stringency), more preferably at 55° C. (medium stringency), more preferably at 60° C. (medium-high stringency), even more preferably at 65° C. (high stringency), and most preferably at 70° C. (very high stringency).
For short probes of about 15 nucleotides to about 70 nucleotides in length, stringency conditions are defined as prehybridization, hybridization, and washing post-hybridization at about 5° C. to about 10° C. below the calculated Tm using the calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southern blotting procedures for 12 to 24 hours optimally.
For short probes of about 15 nucleotides to about 70 nucleotides in length, the carrier material is washed once in 6×SCC plus 0.1% SDS for 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10° C. below the calculated Tm.
In a third aspect, the present invention relates to isolated polypeptides having xylanase activity encoded by polynucleotides comprising or consisting of nucleotide sequences having a degree of sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 2 of preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99%, which encode a polypeptide having xylanase activity. See polynucleotide section herein.
In a fourth aspect, the isolated polypeptides having xylanase activity, comprise the following 6 amino acid long motif:









(SEQ ID NO: 1)



[ST]E[GAS]X[GAS][YFW],






wherein X is any amino acid and the amino acids listed together in brackets are alternative amino acid residues that may be found in a single position, e.g., [ST] denotes that either serine or threonine may be present in the that position in the motif. In the above motif, the accepted IUPAC single letter amino acid abbreviation is employed.

In another aspect, the present invention relates to artificial variants comprising a substitution, deletion, and/or insertion of one or more (or several) amino acids of the mature polypeptide of any of SEQ ID NO's: 3-154 and 159, or a homologous sequence thereof. Preferably, amino acid changes are of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.
Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.
In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline, and alpha-methyl serine) may be substituted for amino acid residues of a wild-type polypeptide. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for amino acid residues. “Unnatural amino acids” have been modified after protein synthesis, and/or have a chemical structure in their side chain(s) different from that of the standard amino acids. Unnatural amino acids can be chemically synthesized, and preferably, are commercially available, and include pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.
Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.
Essential amino acids in the parent polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (i.e., xylanase activity) to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identities of essential amino acids can also be inferred from analysis of identities with polypeptides that are related to a polypeptide according to the invention.
Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochem. 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).
Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
The total number of amino acid substitutions, deletions and/or insertions of the mature polypeptide of any of SEQ ID NO's: 3-154 and 159 is 10, preferably 9, more preferably 8, more preferably 7, more preferably at most 6, more preferably 5, more preferably 4, even more preferably 3, most preferably 2, and even most preferably 1.
Sources of Parent GH8 Xylanase Polypeptides
A parent GH8 xylanase polypeptide of the present invention may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by a nucleotide sequence is produced by the source or by a strain in which the nucleotide sequence from the source has been inserted. In a preferred aspect, the polypeptide obtained from a given source is secreted extracellularly.
A polypeptide having xylanase activity of the present invention may be a bacterial polypeptide. For example, the polypeptide may be a gram positive bacterial polypeptide such as a Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, or Oceanobacillus polypeptide having xylanase activity, or a Gram negative bacterial polypeptide such as an E. coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma polypeptide having xylanase activity.
In a preferred aspect, the polypeptide is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis polypeptide having xylanase activity.
In another preferred aspect, the polypeptide is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus polypeptide having xylanase activity.
In another preferred aspect, the polypeptide is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans polypeptide having xylanase activity.
A polypeptide having xylanase activity of the present invention may also be a fungal polypeptide, and more preferably a yeast polypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide having xylanase activity; or more preferably a filamentous fungal polypeptide such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllium, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria polypeptide having xylanase activity.
In a preferred aspect, the polypeptide is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis polypeptide having xylanase activity.
In another preferred aspect, the polypeptide is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride polypeptide having xylanase activity.
It will be understood that for the aforementioned species the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.
Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).
Furthermore, such polypeptides may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms from natural habitats are well known in the art. The polynucleotide may then be obtained by similarly screening a genomic or cDNA library of such a microorganism. Once a polynucleotide encoding a polypeptide has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are well known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).
A non-exhaustive list of polypeptides having (or predicted to have) xylanase activity belonging to the GH8 family is provided in table 1, vide infra.
Polypeptides of the present invention also include fused polypeptides or cleavable fusion polypeptides in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide or fragment thereof. A fused polypeptide is produced by fusing a nucleotide sequence (or a portion thereof) encoding another polypeptide to a nucleotide sequence (or a portion thereof) of the present invention. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fused polypeptide is under control of the same promoter(s) and terminator.
A fusion polypeptide can further comprise a cleavage site. Upon secretion of the fusion protein, the site is cleaved releasing the polypeptide having xylanase activity from the fusion protein. Examples of cleavage sites include, but are not limited to, a Kex2 site that encodes the dipeptide Lys-Arg (Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991, Biotechnology 9: 378-381), an Ile-(Glu or Asp)-Gly-Arg site, which is cleaved by a Factor Xa protease after the arginine residue (Eaton et al., 1986, Biochem. 25: 505-512); a Asp-Asp-Asp-Asp-Lys site, which is cleaved by an enterokinase after the lysine (Collins-Racie et al., 1995, Biotechnology 13: 982-987); a His-Tyr-Glu site or His-Tyr-Asp site, which is cleaved by Genenase I (Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248); a Leu-Val-Pro-Arg-Gly-Ser site, which is cleaved by thrombin after the Arg (Stevens, 2003, Drug Discovery World 4: 35-48); a Glu-Asn-Leu-Tyr-Phe-Gln-Gly site, which is cleaved by TEV protease after the Gln (Stevens, 2003, supra); and a Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro site, which is cleaved by a genetically engineered form of human rhinovirus 3C protease after the Gln (Stevens, 2003, supra).
Polynucleotides
The present invention also relates to isolated polynucleotides comprising or consisting of nucleotide sequences that encode the variant GH8 xylanase polypeptides of the present invention.
In a preferred aspect, the nucleotide sequence comprises or consists of SEQ ID NO: 2, wherein one or more mutation(s) has been introduced according to the methods of the invention. In another preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding sequence of SEQ ID NO: 2, wherein one or more mutation(s) has been introduced according to the methods of the invention. The present invention also encompasses nucleotide sequences that encode variant polypeptides prepared from the parent GH8 xylanase comprising or consisting of the amino acid sequence of SEQ ID NO: 3 or the mature polypeptide thereof, which differ from SEQ ID NO: 2 or the mature polypeptide coding sequence thereof by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 2, wherein one or more mutation(s) has been introduced according to the methods of the invention, that encode fragments of variants polypeptides prepared from the parent GH8 xylanase comprising or consisting of the amino acid sequence of SEQ ID NO: 3 having xylanase activity.
The present invention also relates to mutant polynucleotides comprising or consisting of at least one mutation in the mature polypeptide coding sequence of SEQ ID NO: 2, in which the mutant nucleotide sequence encodes the mature variant polypeptide prepared from the parent GH8 xylanase comprising or consisting of the amino acid sequence of SEQ ID NO: 3.
The techniques used to isolate or clone a polynucleotide encoding a polypeptide are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the polynucleotides of the present invention from such genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleotide sequence-based amplification (NASBA) may be used.
The present invention also relates to isolated polynucleotides comprising or consisting of nucleotide sequences having a degree of sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 2 of preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99%, which encode a variant polypeptide having xylanase activity according to the present invention.
Modification of a nucleotide sequence encoding a polypeptide of the present invention may be necessary for the synthesis of polypeptides substantially similar to the polypeptide. The term “substantially similar” to the polypeptide refers to non-naturally occurring forms of the polypeptide. These polypeptides may differ in some engineered way from the polypeptide isolated from its native source, e.g., artificial variants that differ in specific activity, thermostability, pH optimum, or the like. The variant sequence may be constructed on the basis of the nucleotide sequence presented as the mature polypeptide coding sequence of SEQ ID NO: 2, e.g., a subsequence thereof, and/or by introduction of nucleotide substitutions that do not give rise to another amino acid sequence of the polypeptide encoded by the nucleotide sequence, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions that may give rise to a different amino acid sequence. For a general description of nucleotide substitution, see, e.g., Ford et al., 1991, Protein Expression and Purification 2: 95-107.
It will be apparent to those skilled in the art that such substitutions can be made outside the regions critical to the function of the molecule and still result in an active polypeptide. Amino acid residues essential to the activity of the polypeptide encoded by an isolated polynucleotide of the invention, and therefore preferably not subject to substitution, may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989, supra). In the latter technique, mutations are introduced at every positively charged residue in the molecule, and the resultant mutant molecules are tested for xylanase activity to identify amino acid residues that are critical to the activity of the molecule. Sites of substrate-enzyme interaction can also be determined by analysis of the three-dimensional structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photoaffinity labeling (see, e.g., de Vos et al., 1992, supra; Smith et al., 1992, supra; Wlodaver et al., 1992, supra).
The present invention also relates to isolated polynucleotides encoding polypeptides of the present invention, which hybridize under very low stringency conditions, preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 2, (ii) the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 2, or (iii) a full-length complementary strand of (i) or (ii); or allelic variants and subsequences thereof (Sambrook et al., 1989, supra), as defined herein.
The present invention also relates to isolated polynucleotides obtained by (a) hybridizing a population of DNA under very low, low, medium, medium-high, high, or very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 2, (ii) the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 2, or (iii) a full-length complementary strand of (i) or (ii); and (b) isolating the hybridizing polynucleotide, which encodes a polypeptide having xylanase activity.
Nucleic Acid Constructs
The present invention also relates to nucleic acid constructs comprising an isolated polynucleotide of the present invention operably linked to one or more (several) control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
An isolated polynucleotide encoding a polypeptide of the present invention may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the polynucleotide's sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotide sequences utilizing recombinant DNA methods are well known in the art.
The control sequence may be an appropriate promoter sequence, a nucleotide sequence that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter sequence contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any nucleotide sequence that shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention, especially in a bacterial host cell, are the promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proceedings of the National Academy of Sciences USA 80: 21-25). Further promoters are described in “Useful proteins from recombinant bacteria” in Scientific American, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.
Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporum trypsin-like protease (WO 96/00787), Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpi promoter (a modified promoter from the gene encoding neutral alpha-amylase in Aspergillus niger in which the untranslated leader has been replaced by an untranslated leader from the gene encoding triose phosphate isomerase in Aspergillus nidulans); and mutant, truncated, and hybrid promoters thereof.
In a yeast host, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488.
The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3′ terminus of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice may be used in the present invention.
Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.
Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.
The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5′ terminus of the nucleotide sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used in the present invention.
Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.
Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).
The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3′ terminus of the nucleotide sequence and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell of choice may be used in the present invention.
Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase.
Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.
The control sequence may also be a signal peptide coding sequence that encodes a signal peptide linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway. The 5′ end of the coding sequence of the nucleotide sequence may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the secreted polypeptide. Alternatively, the 5′ end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. The foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, the foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the polypeptide. However, any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell of choice, i.e., secreted into a culture medium, may be used in the present invention.
Effective signal peptide coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.
Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, Humicola insolens endoglucanase V, and Humicola lanuginosa lipase.
Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.
In a preferred aspect, the signal peptide comprises or consists of amino acids −27 to −1 of SEQ ID NO: 3 or −28 to −1 in SEQ ID NO:4. In another preferred aspect, the signal peptide coding sequence comprises or consists of nucleotides 1 to 81 of SEQ ID NO: 2.
The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the amino terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila laccase (WO 95/33836).
Where both signal peptide and propeptide sequences are present at the amino terminus of a polypeptide, the propeptide sequence is positioned next to the amino terminus of a polypeptide and the signal peptide sequence is positioned next to the amino terminus of the propeptide sequence.
It may also be desirable to add regulatory sequences that allow the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter may be used as regulatory sequences. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the nucleotide sequence encoding the polypeptide would be operably linked with the regulatory sequence.
Expression Vectors
The present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleic acids and control sequences described herein may be joined together to produce a recombinant expression vector that may include one or more (several) convenient restriction sites to allow for insertion or substitution of the nucleotide sequence encoding the polypeptide at such sites. Alternatively, a polynucleotide sequence of the present invention may be expressed by inserting the nucleotide sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the nucleotide sequence. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.
The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.
The vectors of the present invention preferably contain one or more (several) selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus. 
The vectors of the present invention preferably contain an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
For integration into the host cell genome, the vector may rely on the polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleotide sequences for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleotide sequences. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.
For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” is defined herein as a nucleotide sequence that enables a plasmid or vector to replicate in vivo.
Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permitting replication in Bacillus. 
Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.
Examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883.
More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of the gene product. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).
Host Cells
The present invention also relates to recombinant host cells, comprising an isolated polynucleotide of the present invention operably linked to one or more (several) control sequences that direct the production of a polypeptide having xylanase activity. A construct or vector comprising a polynucleotide of the present invention is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.
The host cell may be any cell useful in the recombinant production of a polypeptide of the present invention, e.g., a prokaryote or a eukaryote.
The prokaryotic host cell may be any Gram positive bacterium or a Gram negative bacterium. Gram positive bacteria include, but not limited to, Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, and Oceanobacillus. Gram negative bacteria include, but not limited to, E. coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, and Ureaplasma. 
The bacterial host cell may be any Bacillus cell. Bacillus cells useful in the practice of the present invention include, but are not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.
In a preferred aspect, the bacterial host cell is a Bacillus amyloliquefaciens, Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtilis cell. In a more preferred aspect, the bacterial host cell is a Bacillus amyloliquefaciens cell. In another more preferred aspect, the bacterial host cell is a Bacillus clausii cell. In another more preferred aspect, the bacterial host cell is a Bacillus licheniformis cell. In another more preferred aspect, the bacterial host cell is a Bacillus subtilis cell.
The bacterial host cell may also be any Streptococcus cell. Streptococcus cells useful in the practice of the present invention include, but are not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.
In a preferred aspect, the bacterial host cell is a Streptococcus equisimilis cell. In another preferred aspect, the bacterial host cell is a Streptococcus pyogenes cell. In another preferred aspect, the bacterial host cell is a Streptococcus uberis cell. In another preferred aspect, the bacterial host cell is a Streptococcus equi subsp. Zooepidemicus cell.
The bacterial host cell may also be any Streptomyces cell. Streptomyces cells useful in the practice of the present invention include, but are not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.
In a preferred aspect, the bacterial host cell is a Streptomyces achromogenes cell. In another preferred aspect, the bacterial host cell is a Streptomyces avermitilis cell. In another preferred aspect, the bacterial host cell is a Streptomyces coelicolor cell. In another preferred aspect, the bacterial host cell is a Streptomyces griseus cell. In another preferred aspect, the bacterial host cell is a Streptomyces lividans cell.
The introduction of DNA into a Bacillus cell may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), by using competent cells (see, e.g., Young and Spizizen, 1961, Journal of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169: 5271-5278). The introduction of DNA into an E coli cell may, for instance, be effected by protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNA into a Streptomyces cell may, for instance, be effected by protoplast transformation and electroporation (see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), by conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171: 3583-3585), or by transduction (see, e.g., Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into a Pseudomonas cell may, for instance, be effected by electroporation (see, e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or by conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cell may, for instance, be effected by natural competence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), by protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios. 68: 189-207, by electroporation (see, e.g., Buckley et al., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or by conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method known in the art for introducing DNA into a host cell can be used.
The host cell may also be a eukaryote, such as a mammalian, insect, plant, or fungal cell.
In a preferred aspect, the host cell is a fungal cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et al., 1995, supra).
In a more preferred aspect, the fungal host cell is a yeast cell. “Yeast” as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980).
In an even more preferred aspect, the yeast host cell is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell.
In a most preferred aspect, the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis cell. In another most preferred aspect, the yeast host cell is a Kluyveromyces lactis cell. In another most preferred aspect, the yeast host cell is a Yarrowia lipolytica cell.
In another more preferred aspect, the fungal host cell is a filamentous fungal cell. “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
In an even more preferred aspect, the filamentous fungal host cell is an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.
In a most preferred aspect, the filamentous fungal host cell is an Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. In another most preferred aspect, the filamentous fungal host cell is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum cell. In another most preferred aspect, the filamentous fungal host cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.
Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238 023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81: 1470-1474. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75: 1920.
Methods of Production
The present invention also relates to methods of producing a variant polypeptide of the present invention, comprising: (a) cultivating a cell, which produces the polypeptide, under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide. In a preferred aspect, the cell is of the genus Bacillus. In a more preferred aspect, the cell is Bacillus licheniformis. 
The present invention also relates to methods of producing a variant polypeptide of the present invention, comprising: (a) cultivating a recombinant host cell, as described herein, under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
The present invention also relates to methods of producing a variant polypeptide of the present invention, comprising: (a) cultivating a recombinant host cell under conditions conducive for production of the polypeptide, wherein the host cell comprises a mutant nucleotide sequence having at least one mutation in the mature polypeptide coding sequence of SEQ ID NO: 2, wherein the mutant nucleotide sequence encodes a variant of any of the mature polypeptides of SEQ ID NO's: 3-154 and 159 according to the invention; and (b) recovering the polypeptide.
In the production methods of the present invention, the cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods well known in the art. For example, the cell may be cultivated by shake flask cultivation, and small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted into the medium, it can be recovered from cell lysates.
The polypeptides may be detected using methods known in the art that are specific for the polypeptides. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide as described herein.
The resulting polypeptide may be recovered using methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
The polypeptides of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides.
Compositions
The present invention also relates to compositions comprising a variant polypeptide of the present invention. Preferably, the compositions are enriched in such a polypeptide. The term “enriched” indicates that the variant GH8 xylanase activity of the composition has been increased, e.g., with an enrichment factor of at least 1.1.
The composition may comprise a polypeptide of the present invention as the major enzymatic component, e.g., a mono-component composition. Alternatively, the composition may comprise multiple enzymatic activities, such as an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase. The additional enzyme(s) may be produced, for example, by a microorganism belonging to the genus Aspergillus, preferably Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae; Fusarium, preferably Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, or Fusarium venenatum; Humicola, preferably Humicola insolens or Humicola lanuginosa; or Trichoderma, preferably Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride. 
The polypeptide compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition. For instance, the polypeptide composition may be in the form of a granulate or a microgranulate. The polypeptide to be included in the composition may be stabilized in accordance with methods known in the art.
Examples are given below of preferred uses of the polypeptide compositions of the invention. The dosage of the polypeptide composition of the invention and other conditions under which the composition is used may be determined on the basis of methods known in the art.
Uses
The present invention is also directed to methods for using the variant polypeptides having GH8 xylanase activity, or compositions thereof, including baking applications.
In one aspect the invention relates to a method for the preparation of a baked product, said method comprising the step of adding to a dough of said baked product the variant polypeptide of the second aspect or a bread improving composition comprising said variant polypeptide.
In one preferred embodiment, said variant polypeptide or bread improving composition is added during the mixing of the dough.
In another preferred embodiment, said bread improving composition further comprises one or more bread-improving agent selected from the list consisting of enzymes, emulsifiers, oxidants, milk powder, fats, sugars, amino acids, salts, proteins (gluten, cellulose binding sites) or a mixture thereof; preferably said enzymes are selected from the list consisting of alpha-amylases, beta-amylases, maltogenic amylases, other xylanases, proteases, glucose oxidase, oxidoreductases, glucanases, cellulases, transglutaminases, isomerases, lipases, phospholipases, pectinases or a mixture thereof.
It is preferred that said variant polypeptide is present as a cell extract, a cell-free extract or as a purified protein.
In another preferred embodiment, said variant polypeptide is mixed with other ingredients in the form of a dry powder or a granulate, in particular a non-dusting granulate, or in the form of a liquid, preferably with one or more stabilizer(s) such as polyols, sugars, organic acids or sugar alcohols.
Baked Product
The process or the GH8 xylanase variants of the invention may be used for any kind of baked product pre-pared from dough, either of a soft or a crisp character, either of a white, light or dark type. Examples are bread (in particular white, whole-meal or rye bread), typically in the form of loaves or rolls, French baguette-type bread, flat bread, pita bread, tortillas, cakes, pancakes, biscuits, cookies, pie crusts, crisp bread, steamed bread, pizza and the like.
Pre-mix
The present invention further relates to a pre-mix comprising flour together with a GH8 variant xylanase polypeptide according to the invention. The pre-mix may contain other dough-improving and/or bread-improving additives, e.g. any of the additives, including enzymes, mentioned above.
Enzyme Preparation
The invention provides an enzyme preparation comprising a GH8 variant xylanase according to the invention, for use as a baking additive in the process of the invention. The enzyme preparation is preferably in the form of a granulate or agglomerated powder. It preferably has a narrow particle size distribution with more than 95% (by weight) of the particles in the range from 25 to 500 μm.
Granulates and agglomerated powders may be prepared by conventional methods, e.g. by spraying the xylanase onto a carrier in a fluid-bed granulator. The carrier may consist of particulate cores having a suitable particle size. The carrier may be soluble or insoluble, e.g. a salt (such as NaCl or sodium sulfate), a sugar (such as sucrose or lactose), a sugar alcohol (such as sorbitol), starch, rice, corn grits, or soy.
The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.
EXAMPLES
The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.
Chemicals used as buffers and substrates were commercial products of at least reagent grade.
Example 1
Parent GH8 Xylanases
A non-limiting and non-exhaustive list of diverse potential parent GH8 xylanases is provided in table 1 below. Each of the GH8 xylanases listed comprises the conserved GH8-motif of SEQ ID NO:1, the amino acid position of which is indicated in the rightmost column for each sequence. The sequence identifiers (SEQ ID NO's) are indicated in the leftmost column of the table, the organism name and the UniProtKB accession numbers are shown in the second and third columns, respectively. Where applicable, the amino acids predicted or shown to be part of a signal peptide are indicated, as are the amino acids predicted or shown as comprised in the mature GH8 polypeptide. The amino acids in a signal peptide are labeled with ‘-’ in the accompanying sequence list, but in the table below they are numbered from the start codon of the full polypeptide. Not all the listed polypeptides are secreted. A few of the listed polypeptides may also comprise a prepro sequence that is cleaved off to produce the mature polypeptide, but is shown here in most cases as part of the mature polypeptide—this may be determined by standard practices in the art, such as, N-terminal amino acid sequencing of the mature polypeptide.







TABLE 1







Potential parent GH8 xylanases; the G8 motif 1 is indicated in the mature


sequence numbering, where the signal has been included as a feature in


the sequence listing (with negative numbers), otherwise it is indicated


from the start of the signal.

















G8


SEQ ID
Organism
UniProt
Signal
Mature
Motif 1















3
Bacillus sp. KK-1 (artificial signal)

1-27
28-433
65-70


4
Bacillus sp. KK-1 (native signal)
O52730
1-28
29-434
65-70


5
Burkholderia phymatum
B2JFS8
1-39
40-388
81-86


6
Labrenzia aggregata
A0NZ93
1-21
22-400
79-84


7
Alteromonadales bacterium TW-7
A0XXD5
1-21
22-376
58-63


8
Yersinia enterocolitica
A1JST6
1-23
24-375
55-60


9
Mycobacterium vanbaalenii
A1TEN4
1-28
29-376
75-80


10
Lactococcus lactis
A2RKE9

 1-298
54-59


11
Burkholderia cenocepacia
A2VR29
1-40
41-409
86-91


12
Streptococcus sanguinis
A3CKD9

 1-342
42-47


13
Leptospirillum rubarum
A3ER07
1-23
24-366
55-60


14
Sagittula stellata
A3K4W8
1-25
26-416
99-104


15
Burkholderia mallei
A3MCA1
1-23
24-437
113-118


16
Rhodobacter sphaeroides
A3PL64
1-25
26-356
66-71


17
Rhodobacterales bacterium
A3VA52
1-27
28-342
58-63


18
Paenibacillus sp. W-61
A4F259

 1-380
69-74


19
Cronobacter sakazakii
A4H2S1
1-21
22-371
54-59


20
Burkholderia vietnamiensis
A4JDK7
1-32
33-407
84-89


21
Polynucleobacter necessarius
A4SY19
1-27
28-337
64-69


22
Enterobacter sp. 638
A4WFV3
1-13
14-360
56-61


23
Enterobacter sp. 638
A4WFW1
1-24
25-330
52-57


24
Rhodobacter sphaeroides
A4WR51
1-25
26-356
65-70


25
Rhodobacter sphaeroides
A4X062
1-24
25-362
67-72


26
Flavobacterium johnsoniae
A5FD37
1-33
34-430
100-105


27
Flavobacterium johnsoniae
A5FL64
1-19
20-465
75-80


28
Acidiphilium cryptum
A5FW12
1-26
27-376
60-65


29
Vibrio fischeri
A5JJ40
1-23
24-392
74-79


30
Vibrionales bacterium SWAT-3
A5L0Z5
1-18
19-369
51-56


31
Pseudomonas putida
A5W2B7
1-16
17-367
51-56


32
Vibrio harveyi
A6ARS3
1-18
19-369
51-56


33
Plesiocystis pacifica
A6FX48
1-8 
9-452
127-132


34
Limnobacter
A6GMN1
1-31
32-383
61-66


35
Limnobacter
A6GNP5
1-25
26-380
62-67


36
Klebsiella pneumoniae
A6TFD4
1-22
23-369
56-60


37
Klebsiella pneumoniae
A6TFE2
1-23
24-333
52-57


38
Sinorhizobium medicae
A6UK07
1-19
20-344
54-59


39
Marimonas
A6W1N6
1-27
27-417
91-96


40
Bifidobacterium adolescentis
A7A3Z7
1-22
23-379
65-70


41
Yersinia pseudotuberculosis
A7FP36
1-22
23-371
55-60


42
Asaia bogorensis
A7M789
1-25
26-345
60-65


43
Escherichia coli
A7ZT72
1-18
19-365
51-56


44
Citrobacter koseri
A8ARA5
1-22
23-369
55-60


45
Serratia proteamaculans
A8G820
1-23
24-370
55-60


46
Vibrio campbellii
A8T846
1-18
19-369
51-56


47
Burkholderia multivorans
A9ACB8
1-38
39-414
91-96


48
Herpetosiphon aurantiacus
A9AYU3
1-39
40-543
110-115


49
Herpetosiphon aurantiacus
A9B7H2
1-29
30-511
88-93


50
Herpetosiphon aurantiacus
A9B7H3
1-27
28-609
88-93


51
Agrobacterium tumefaciens
A9CEZ6
1-28
29-351
61-66


52
Sorangium cellulosum
A9G2N6

 1-383
58-62


53
Sorangium cellulosum
A9GFS5
1-22
23-467
134-139


54
Sorangium cellulosum
A9GJA6
1-23
24-497
142-147


55
Clostridium phytofermentans
A9KRR7

 1-381
65-70


56
Salmonella arizonae
A9MLJ1
1-22
23-369
55-60


57
Salmonella paratyphi
A9MUP9
1-21
22-368
54-59


58
Bacillus thuringiensis
A9P7F6
1-27
28-453
121-126


59
Lactococcus lactis
A9QS66

 1-377
54-59


60
Lactococcus lactis
A9QSM5
1-25
26-371
68-73


61
Methylobacterium extorquens
A9W2G4
1-29
30-419
124-129


62
Pseudomonas putida
B0KHK5
1-21
22-371
55-60


63
Methylobacterium sp. 4-46
B0UPS6
1-28
29-393
97-102


64
Escherichia albertii
B1EHK5
1-22
23-369
55-60


65
Burkholderia graminis
B1FXP2
1-58
59-476
152-157


66
Burkholderia pseudomallei
B1H9R8

 1-513
189-194


67
Yersinia pseudotuberculosis
B1JH76
1-22
23-371
55-60


68
Burkholderia cenocepacia
B1JZP0
1-40
41-409
86-91


69
Escherichia coli
B1LJ80
1-23
24-370
56-61


70
Methylobacterium radiotolerans
B1LS51
1-24
25-385
89-94


71
Methylobacterium radiotolerans
B1M836
1-29
30-367
71-76


72
Burkholderia ambifaria
B1TH37

 1-359
36-41


73
Leptothrix cholodnii
B1Y239

 1-379
48-53


74
Burkholderia ambifaria
B1YNG4
1-41
42-406
83-88


75
Methylobacterium populi
B1ZDE2
1-34
35-421
126-131


76
Opitutus terrae
B1ZP97
1-22
23-412
101-106


77
Opitutus terrae
B1ZP98

 1-445
121-126


78
Cupriavidus taiwanensis
B2AI90
1-23
24-393
72-77


79
Burkholderia phymatum
B2JFS8
1-40
41-388
71-76


80
Burkholderia phymatum
B2JS35
1-42
43-410
76-81


81
Escherichia coli
B2N7Y3
1-18
19-365
51-56


82
Burkholderia phytofirmans
B2TFR8
1-25
26-393
71-76


83
Shigella boydii
B2U4J9
1-18
19-365
51-56


84
Erwinia tasmaniensis
B2VCL4
1-23
24-333
52-57


85
Bacteroides intestinalis
B3C8W4
1-20
21-419
89-94


86
Bacteroides intestinalis
B3CES9
1-26
27-418
103-108


87
Geobacillus
B3K473
1-30
31-407
92-97


88
Rhizobium etli
B3PVE9
1-24
25-348
59-64


89
Escherichia coli
B3WP91
1-23
24-370
56-61


90
Lactobacillus reuteri
B3XPX3
1-23
24-380
78-83


91
Salmonella enterica
B3YCW2
1-22
23-369
55-60


92
Bacillus cereus
B3ZAY7
1-27
28-453
121-126


93
Bacillus cereus
B3ZAZ1

 1-412
95-99


94
Clostridium thermocellum
B4BE17
1-33
34-477
94-98


95
Burkholderia cenocepacia
B4E6B0
1-14
15-383
60-65


96
Proteus mirabilis
B4F0R7
1-31
32-345
63-68


97
Alteromonas macleodii
B4RSC3
1-26
27-366
59-64


98
Anaeromyxobacter
B4UAH9
1-29
30-1140
70-75


99
Microcoleus chthonoplastes
B4W0R5

 1-454
116-121


100
Microbacteriaceae
B5BUR0
1-14
15-326
44-49


101
Verrucomicrobiae
B5JFU0

 1-387
76-81


102
Verrucomicrobiae
B5JIL3
1-18
19-431
88-93


103
Methylobacterium chloromethanicum
B7KRP7
1-26
27-385
89-94


104
Clostridium cellulolyticum
B8I0N9

 1-376
65-70


105
Clostridium cellulolyticum
B8I0S8
1-26
27-477
92-97


106
Stenotrophomonas
B8L0W8
1-29
30-380
67-72


107
Rhodobacter sphaeroides
B9KK73
1-24
25-356
66-71


108
Bifidobacterium pseudocatenulatum
C0BQ52

 1-384
70-75


109
Subdoligranulum variabile
C0CBU5

 1-388
67-72


110
Halomonas
C0JSK6
1-32
33-350
66-71


111
Geodermatophilus obscurus
C0U1B3
1-22
23-390
88-93


112
Lactobacillus gasseri
C0XE89

 1-372
67-72


113
Azotobacter vinelandii
C1DJH4
1-24
25-337
54-59


114
Sulfurihydrogenibium azorense
C1DW55
1-19
20-320
50-55


115
Acidobacterium capsulatum
C1F831
1-29
30-388
60-65


116
Lactobacillus jensenii
C2DZT7

 1-381
71-76


117
Spirosoma linguale
C4CS14
1-31
32-432
101-106


118
Edwardsiella ictaluri
C5BB42
1-24
25-373
57-62


119
Teredinibacter turnerae
C5BJ89
1-29
30-436
109-114


120
Aquifex aeolicus
O67401
1-16
17-325
50-55


121
Acetobacter xylinus
O82857
1-16
17-341
55-60


122
Cellulomonas uda
P18336
1-23
24-359
52-57


123
Erwinia chrysanthemi
P27032
1-23
24-332
52-57


124
Bacillus sp. KSM-330
P29019
1-27
56-463
129-134


125
Acetobacter xylinus
P37696
1-20
21-342
56-61


126
Xanthomonas axonopodis
P58935
1-25
26-384
62-67


127
Fulvimarina pelagi
Q0FZX6
1-23
24-365
73-78


128
Chlorobium ferrooxidans
Q0YP21
1-23
24-376
59-64


129
Cytophaga hutchinsonii
Q11NQ3
1-25
26-1223
59-64


130
Pseudoalteromonas atlantica
Q15WZ3
1-44
45-912
585-590


131
Deinococcus geothermalis
Q1J317
1-26
27-292
101-106


132
Ralstonia metallidurans
Q1LL46
1-31
32-387
60-65


133
Candidatus Kuenenia stuttgartiensis
Q1Q2C1
1-24
25-344
55-60


134
Vibrio alginolyticus
Q1V8W2
1-18
19-368
51-56


135
Aurantimonas sp. SI85-9A1
Q1YMU9
1-25
26-364
72-77


136
Photobacterium profundum
Q1Z1X9
1-18
19-369
51-56


137
Lysobacter sp. IB-9374
Q25C15
1-33
34-414
98-103


138
Bordetella avium
Q2KWR4
1-31
32-398
67-72


139
Hahella chejuensis
Q2S7F9
1-30
31-555
127-132


140
Chlorobium chlorochromatii
Q3ATR3
1-22
23-398
60-65


141
Pelodictyon luteolum
Q3B476
1-23
24-376
56-61


142
Xanthomonas euvesicatoria
Q3BPE1
1-34
35-393
71-76


143
Pseudoalteromonas haloplanktis
Q3IER3
1-21
22-376
66-71


144
Cupriavidus pinatubonensis
Q46VF0
1-29
30-392
63-68


145
Ustilago maydis
Q4PG97

 1-361
37-42


146
Zymomonas mobilis
Q5NNK0
1-20
21-339
56-61


147
Erwinia carotovora
Q6CYY2
1-22
23-371
56-61


148
Photobacterium profundum
Q6LRE7
1-18
19-369
51-56


149
Acetobacter xylinus
Q76KK1
1-20
21-342
56-61


150
Chromobacterium violaceum
Q7NUM1
1-21
22-382
56-61


151
Pseudomonas syringae
Q888J5
1-29
30-403
67-72


152
Pseudomonas putida
Q88JL2
1-19
20-370
54-59


153
Bacillus circulans
Q93HV0
1-30
31-407
92-97


154
Paenibacillus fukuinensis
Q93IE7
1-41
42-797
114-119


159
Pseudoalteromonas haloplanktis

1-27
29-402
77-82









Example 2
Construction of Variants by Site-directed Mutagenesis
Site-directed variants were constructed of the Bacillus sp. KK-1 GH8 xylanase (SEQ ID NO:3 or 4) comprising specific insertions/deletions/substitutions in the loop flanking the conserved GH8 motif on the N-terminal side according to the invention. The variants were made by traditional cloning of DNA fragments (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989) using PCR together with properly designed mutagenic oligonucleotides that introduced the desired mutations in the resulting sequence.
Mutagenic oligos were synthesized corresponding to the DNA sequence flanking the desired site(s) of mutation, separated by the DNA base pairs defining the insertions/deletions/substitutions. In this manner, the variants listed in table 2 below were constructed and produced.
As an example, the mutagenic primers for the construction of variant LBei411, which has a single alanine amino acid insertion in position 53 of the mature polypeptide (*53aA), were designed as follows:







LBei411f (SEQ ID NO: 156):


5′-gcttctacaacagacgctgacaaccttggcaacggctattac


 


LBei411r (SEQ ID NO: 157):


5′-gtaatagccgttgccaaggttgtcagcgtctgttgtagaagc






In order to purify GH8 xylanase variants of the invention, the mutated DNA comprising a variant of the invention was transformed into a competent B. subtilis strain and fermented using standard protocols (PS-1 media, 3-4 days, 30° C.).







TABLE 2





Loop variants of Bacillus sp. KK-1 GH8 xylanase (SEQ ID NO: 3 or 4),


where the amino acid mutation(s) is listed for each variant.


















LBei386
D53*



LBei388
L56*



LBei390
N58*



LBei411
*53aA



LBei413
*55aA



LBei417
*59aA



LBei418
N55A, L56*, G57N, N58L



LBei440
*61aA



MDTo253
*60aA



MDTo254
*62aA



MDTo255
*53aS



MDTo256
*53aD



MDTo257
*53aK



MDTo258
*53aF



MDTo259
*53aL



MDTo260
*55aS



MDTo262
*55aK



MDTo263
*55aF



MDTo264
*55aL



MDTo265
*59aS



MDTo266
*59aD



MDTo267
*59aK



MDTo268
*59aF



MDTo269
*59aL



MDTo298
*53aA, *55aA



MDTo299
*55aA, *59aA



MDTo300
*53aA, *59aA



MDTO301
*53aA, *55aA, *59aA



MDTo323
*53aA, *55aL



MDTo324
*55aA, *59aD



MDTo325
*55aL, *59aA



MDTo326
*55aL, *59aS



MDTo327
*55aL, *59aD



MDTo328
*53aA, *59aS



MDTo329
*53aA, *59aD



MDTo330
*53aA, *55aL, *59aA



MDTo331
*53aA, *55aA, *59aS



MDTo332
*53aA, *55aA, *59aD



MDTo333
*53aA, *55aA, *59aF



MDTo334
*53aA, *55aL, *59aS



MDTo335
*53aA, *55aL, *59aD



MDTo336
*53aA, *55aL, *59aF







Amino acid position numbers 53-62 refer to the mature polypeptide of SEQ ID NO: 3 or 4.



An amino acid deletion is shown as the deleted amino acid, its position number and an asterisk, e.g., “D53*” denotes the deletion of an aspartic acid residue (D) from position 53.



An insertion is shown as an asterisk followed by the position number, a lowercase “a” to denote the amino acid already present in that position and finally the inserted amino acid, e.g., “*53aA” denotes the insertion of an alanine (A) residue in the polypeptide following the aspartic acid (D) in position 53.



An amino acid substitution is shown as the amino acid, its position number and the new amino acid replacing or substituting the first mentioned, e.g., “N55A” denotes that the asparagine (N) residue in position 55 was replaced with an alanine (A) residue.






Example 3
Straight Dough Procedure
Recipe











Dough
% on flour basis







Ascorbic acid
to be optimized for each flour (40 ppm in this trial)


Cream yeast
4 (73% water)


Salt
1.5


Sugar
1.5


Water
to be optimized for each flour (57% in this trial)


Wheat flour +
100 (Kolibri flour from Meneba or Kluut flour


Enzyme
with lower gluten content from Meneba)









Procedure

    
    
        1. Scaling of ingredients, addition of flour, yeast, salt, sugar, ascorbic acid and enzyme
        2. Enzyme was dosed from 0.02 to 0.2 mg protein enzyme/kg flour; excl. the negative control (no enzyme)
        3. Temperature adjustment of water in order to reach a dough temperature of 27° C. after mixing, scaling and addition of water into mixer bowl
        4. Mixing: 3 min at 78 rpm and 6 min at 138 rpm using a Diosna spiral mixer
        5. The dough is taken from the mixer bowl and the dough temperature is determined, the dough parameters are determined (dough evaluation after mixing) and the dough is molded by the baker
        6. The dough is given 20 min. floor-time under plastic cover and the second dough evaluation is performed (dough evaluation after floor time)
        7. The dough is scaled at 1500 g (30 rolls) and 6×350 g (bread)
        8. The molded rolls and bread dough is given 10 min bench-time covered in plastic
        9. The dough for rolls is formed to a  ̃34 cm round plate and put on a roll maker plate and rolls are formed in a rounder.
        
            a. The rolls are transferred to a silicone covered baking sheet.
            b. The dough for bread are shaped in a sheeter and transferred to pans which are put in baking sheet
        
        
        10. The bread and rolls are proofed at 32° C., 86% RH.
        
            a. The proofing time for rolls is 45 min. and 70 min. (overproofing)
            b. The proofing time for bread is 55 min and 80 min. (overproofing)
        
        
        11. The bread and rolls are baked at 230° C. with steam
        
            a. The rolls are baked for 22 min (damper opens after 12 min in order to let out the steam from the oven)
            b. The bread is baked for 35 min (damper opens after 25 min in order to let out the steam from the oven)
        
        
        12. The bread is taken out of the pan after baking and put on a baking sheet
        13. The bread and rolls are allowed to cool down
        14. The bread and rolls are evaluated
    
    


Example 4
Chorleywood Baking Procedure (CBP)
Recipe











Dough
% on flour basis







Ascorbic acid
to be optimized for each flour (150 ppm in this trial)


Cream yeast
4.5 (73% water)


Salt
2.0


Soy flour
0.5 (contains active enzymes)


Water
to be optimized for each flour (58% in this trial)


Wheat flour +
100 (Kolibri flour from Meneba)


Enzyme









Procedure

    
    
        1. Scaling of ingredients, addition of flour, water, cream yeast, salt, soy flour, ascorbic acid and enzyme
        2. Enzyme was dosed from 0.02 to 0.2 mg protein enzyme/kg flour; excl. the negative control (no enzyme)
        3. All ingredients is put in a Pentagon K5 High speed mixer
        4. The dough is mixed to a energy input of 11 w/kg dough at a speed of 425 rpm (30% of the mixing time with 0.6 bar atmosphere pressure and 70% of the mixing time at 18 hg vacuum)
        5. The dough parameters are evaluated (dough evaluation after mixing)
        6. The dough is resting (first) for 5 min. covered in plastic
        7. The dough is scaled at 4×600 g
        8. The dough is resting (second) for 10 min. covered in plastic
        9. The dough is moulded and shaped in a sheeter and transferred to lidded pans which are put in baking sheet
        10. The dough is proofed at 40° C., 80 RH in Termaks proof box
        
            a. The proofing time for pan bread is 55 min.
            b. The proofing time for 3-cut-bread is 45 min.
        
        
        11. The bread is baked at 220° C. in Reed Trap oven with no steam
        12. The bread is evaluated
    
    


Example 5
Dough and Bread Evaluation Parameters


    Stickiness: The dough stickiness is the degree to which a dough adheres to ones hands or other surfaces. This is a sensory evaluation performed by an experienced baker where the control dough without enzyme is given the value 5 and the other doughs are judged compared to the control on a scale from 0 to 10, where 0 is very little stickiness and 10 is very sticky.
    Softness: The dough softness is the ease with which a dough will compress. This is a sensory evaluation performed by an experienced baker where the control dough without enzyme is given the value 5 and the other doughs are judged compared to the control on a scale from 0 to 10, where 0 is less soft and 10 is more soft.
    Extensibility: The dough extensibility is the degree to which a dough can be stretched without tearing. This is a sensory evaluation performed by an experienced baker where the control dough without enzyme is given the value 5 and the other doughs are judged compared to the control on a scale from 0 to 10, where 0 is low/short extensibility and 10 is high/long extensibility.
    Elasticity: The dough elasticity is the ability of a dough to resist stretching as well as to return to its original size and shape when the force is removed. This is a sensory evaluation performed by an experienced baker where the control dough without enzyme is given the value 5 and the other doughs are judged compared to the control on a scale from 0 to 10, where 0 is low/weak extensibility and 10 is high/strong extensibility.
    Machinability: The machinability is the degree to which a dough tends to retain its original surface without damages after work up in machinery. This is evaluated by an experienced baker where the control dough without enzyme is given the value 5 and the other doughs are judged compared to the control on a scale from 0 to 10, where 0 is low machinability and 10 is high machinability.
    Volume: The volume of baked rolls and bread is determined through rape seed displacement. The specific volume index is calculated as follows:

Specific volume=specific volume of bread with enzyme (in ml/g)/specific volume of bread without enzyme (in ml/g)

The average specific volume of two control doughs is set to 100%. The specific volumes of bread or rolls prepared from dough with added enzyme are averages of double samples.



Example 6
Evaluating the Performance of the GH8 Xylanase Parent
Determining the dough and bread parameters described above, in most cases, involves a sensory evaluation by an experienced baker, but even so, there will likely be some natural variation, depending on which day the test is carried out and on which baker does the test etc.
The mature parent GH8 xylanase of Bacillus sp. KK-1 (SEQ ID NO:3 or 4) was tested in twelve experiments over several days by at least two bakers, using an enzyme dosage of 0.05 mg/kg in the straight dough procedure with Kolibri flour from Meneba. The dough was evaluated after floor time for stickiness, softness, extensability, elasticity and machinability. The specific volume of the resulting bread was also determined, both after normal proofing and long proofing. The results are shown in table 3 below.







TABLE 3







Dough evaluation of example 6; results.















Sticki-

Extens-
Elastic-
Machin-

Vol. long


Test
ness
Softness
ability
ity
ability
Vol.
proof

















1
7
7
6
4
4
108
112


2
7
7
6
4
4
106
108


3
6
6
5
5
5
105
106


4
6
5
5
5
5
98
99


5
7
6
6
4
4
118
114


6
7
7
6
4
4
115
111


7
6
6
6
4
4
106
116


8
6
6
5
4
5
110
113


9
6
6
6
5
5
110
113


10
6
6
6
5
5
109
109


11
7
7
6
4
4
110
115


12
6
6
5
5
5
111
116









Example 7
Stickiness of Doughs Prepared Using the GH8 Xylanase Variants
The stickiness of doughs prepared using the GH8 xylanase variants in the straight dough procedure with Kolibri flour from Meneba at an enzyme dosage of 0.05 mg/kg was evaluated after floor time. For some of the more interesting variants that provided a significantly less sticky dough, the evaluations after floor time were also done for higher enzyme dosages of 0.1 mg/kg, 4 times overdose (4×OD) and 10 times overdose (10×OD). The results are shown in table 4 below.
There is a trend that the GH8 variants of the invention provide less sticky doughs than the parent GH8 xylanase; some of them even at very high enzyme dosages.







TABLE 4







Dough evaluation of example 7; results of stickiness score by bakers.














0.05
0.1
4 ×
10 ×


Variant
Mutation
mg/kg
mg/kg
OD
OD















LBei386
D53*
6





LBei388
L56*
5


LBei390
N58*
7


LBei411
*53aA
6


LBei411
*53aA
5
5
6
6


LBei411
*53aA
5


LBei413
*55aA
6
6


LBei413
*55aA
5
6
6
7


LBei413
*55aA
5


LBei417
*59aA
5
6


LBei417
*59aA
5
6
6
6


LBei417
*59aA
5


LBei418
N55A, L56*,
6



G57N, N58L


LBei440
*61aA
6


MDTo253
*60aA
6
7


MDTo254
*62aA
5
6


MDTo255
*53aS
6
7


MDTo256
*53aD
6
8


MDTo257
*53aK
6
7


MDTo258
*53aF
6
6


MDTo259
*53aL
7
7


MDTo260
*55aS
6
7


MDTo262
*55aK
6
8


MDTo263
*55aF
6
6


MDTo264
*55aL
5
5


MDTo264
*55aL
5

6
6


MDTo265
*59aS
5
6


MDTo265
*59aS
6

8
7


MDTo266
*59aD
5
6


MDTo266
*59aD
6

6
7


MDTo267
*59aK
5
6


MDTo268
*59aF
5
6


MDTo268
*59aF
6

8
9


MDTo269
*59aL
5
6


MDTo298
*53aA, *55aA
6


MDTo298
*53aA, *55aA
6

8
7


MDTo299
*55aA, *59aA
6


MDTo299
*55aA, *59aA
6

7
7


MDTo300
*53aA, *59aA
6


MDTo300
*53aA, *59aA
6

6
7


MDTO301
*53aA, *55aA, *59aA
6


MDTO301
*53aA, *55aA, *59aA
5

6
7


MDTo323
*53aA, *55aL
6
8


MDTo324
*55aA, *59aD
6
6


MDTo325
*55aL, *59aA
6
7


MDTo326
*55aL, *59aS
6
7


MDTo327
*55aL, *59aD
6
7
8


MDTo328
*53aA, *59aS
6
6


MDTo329
*53aA, *59aD
6
7


MDTo330
*53aA, *55aL, *59aA
6
7


MDTo331
*53aA, *55aA, *59aS
6
7


MDTo332
*53aA, *55aA, *59aD
6
7


MDTo333
*53aA, *55aA, *59aF
6
7


MDTo334
*53aA, *55aL, *59aS
6
7


MDTo335
*53aA, *55aL, *59aD
6
7


MDTo336
*53aA, *55aL, *59aF
6
7









Example 8
Volume Increase in Bread Made with GH8 Xylanase Variants
Breads were baked from doughs made with the GH8 xylanase variants in the straight dough procedure with Kolibri flour from Meneba at enzyme dosages of 0.05 mg/kg using both normal and long proofing. The specific volumes of the breads were determined.
For some of the more interesting variants baking tests were also done using dough with higher enzyme dosages of 0.1 mg/kg, 4 times overdose (4×OD) and 10 times overdose (10×OD) with both normal and long proofing. The results are shown in table 5 below.







TABLE 5







Specific bread volumes of example 8; results.












0.05 mg/kg
0.1 mg/kg
4 × OD
10 × OD



















Vol. long

Vol. long

Vol. long

Vol. long


Variant
Mutation
Vol
proof
Vol
proof
Vol.
proof
Vol.
prop



















LBei386
D53*
102
103








LBei388
L56*
108
117


LBei390
N58*
109
115


LBei411
*53aA
100
100


LBei411
*53aA
110
114
109
104
119
132
127
132


LBei411
*53aA
111
120


LBei413
*55aA
109
110
107
109


LBei413
*55aA
109
113
107
108
127
133
123
126


LBei413
*55aA
108
117


LBei417
*59aA
108
110
110
109


LBei417
*59aA
111
107
104
105
128
135
121
129


LBei417
*59aA
105
118


LBei418
N55A, L56*, G57N, N58L
97
98


LBei440
*61aA
97
99


MDTo253
*60aA
114
124
114
125


MDTo254
*62aA
102
102
115
122


MDTo255
*53aS
108
113
108
113


MDTo256
*53aD
107
112
106
108


MDTo257
*53aK
120
126
116
117


MDTo258
*53aF
113
112
118
120


MDTo259
*53aL
111
119
116
121


MDTo260
*55aS
110
118
114
124


MDTo262
*55aK
111
118
112
122


MDTo263
*55aF
101
94
99
104


MDTo264
*55aL
108
123
116
139


MDTo264
*55aL
110
125


117
133
124
130


MDTo265
*59aS
110
123
116
136


MDTo265
*59aS
110
120


117
127
129
131


MDTo266
*59aD
109
129
122
136


MDTo266
*59aD
109
116


120
122
126
128


MDTo267
*59aK
108
120
116
128


MDTo268
*59aF
115
128
122
144


MDTo268
*59aF
111
123


114
127
128
134


MDTo269
*59aL
104
115
110
118


MDTo298
*53aA, *55aA
110
125


MDTo298
*53aA, *55aA
111
126


125
134
123
137


MDTo299
*55aA, *59aA
115
124


MDTo299
*55aA, *59aA
112
131


119
134
124
137


MDTo300
*53aA, *59aA
111
123


MDTo300
*53aA, *59aA
113
130


118
137
121
137


MDTO301
*53aA, *55aA, *59aA
115
119


MDTO301
*53aA, *55aA, *59aA
114
115


123
128
119
136


MDTo323
*53aA, *55aL
108
121
109
130
111
123


MDTo324
*55aA, *59aD
113
124
114
127


MDTo325
*55aL, *59aA
107
114
111
117


MDTo326
*55aL, *59aS
107
107
107
114


MDTo327
*55aL, *59aD
113
115
113
115
111
126


MDTo328
*53aA, *59aS
106
122
110
130


MDTo329
*53aA, *59aD
111
126
114
125


MDTo330
*53aA, *55aL, *59aA
112
132
111
131


MDTo331
*53aA, *55aA, *59aS


115
126


MDTo332
*53aA, *55aA, *59aD
114
120
117
132


MDTo333
*53aA, *55aA, *59aF
115
129
118
126


MDTo334
*53aA, *55aL, *59aS
115
118
117
126


MDTo335
*53aA, *55aL, *59aD
110
127
117
126


MDTo336
*53aA, *55aL, *59aF
114
125
116
128









Example 9
Softness etc. of Doughs Prepared Using the GH8 Xylanase Variants
The softness, extensability, elasticity and machinability of doughs prepared using the GH8 xylanase variants in the straight dough procedure with Kolibri flour from Meneba at an enzyme dosage of 0.05 mg/kg was evaluated after floor time. The results are shown in table 6 below.







TABLE 6







Enzyme dosage of 0.05 mg/kg.














Soft-
Extens-
Elas-
Machin-


Variant
Mutation(s)
ness
ability
ticity
ability















LBei386
D53*
5
5
5
4


LBei388
L56*
5
5
5
5


LBei390
N58*
7
6
4
5


LBei411
*53aA
6
4
6
5


LBei411
*53aA
6
5
5
4


LBei411
*53aA
6
5
5
4


LBei413
*55aA
6
5
5
5


LBei413
*55aA
5
4
6
4


LBei413
*55aA
5
5
5
5


LBei417
*59aA
5
5
5
5


LBei417
*59aA
6
5
5
4


LBei417
*59aA
5
5
5
5


LBei418
N55A, L56*,
6
5
5
4



G57N, N58L


LBei440
*61aA
5
4
6
4


MDTo253
*60aA
6
5
5
5


MDTo254
*62aA
4
5
5
5


MDTo255
*53aS
6
5
4
5


MDTo256
*53aD
6
6
5
3


MDTo257
*53aK
6
6
4
4


MDTo258
*53aF
6
5
5
4


MDTo259
*53aL
7
6
4
5


MDTo260
*55aS
6
5
5
4


MDTo262
*55aK
6
7
4
5


MDTo263
*55aF
5
6
5
5


MDTo264
*55aL
5
5
5
5


MDTo264
*55aL
6
4
6
5


MDTo265
*59aS
5
5
5
5


MDTo265
*59aS
7
6
5
5


MDTo266
*59aD
6
6
6
4


MDTo266
*59aD
6
5
5
5


MDTo267
*59aK
5
5
5
5


MDTo268
*59aF
6
6
4
5


MDTo268
*59aF
7
5
4
5


MDTo269
*59aL
5
4
5
5


MDTo298
*53aA, *55aA
5
5
5
5


MDTo298
*53aA, *55aA
6
6
6
5


MDTo299
*55aA, *59aA
6
5
5
5


MDTo299
*55aA, *59aA
6
6
4
4


MDTo300
*53aA, *59aA
6
5
5
5


MDTo300
*53aA, *59aA
6
6
4
5


MDTO301
*53aA, *55aA, *59aA
5
5
5
5


MDTO301
*53aA, *55aA, *59aA
5
5
5
5


MDTo323
*53aA, *55aL
6
6
5
5


MDTo324
*55aA, *59aD
6
6
5
5


MDTo325
*55aL, *59aA
7
5
5
5


MDTo326
*55aL, *59aS
7
5
5
5


MDTo327
*55aL, *59aD
7
5
5
5


MDTo328
*53aA, *59aS
6
6
5
5


MDTo329
*53aA, *59aD
6
6
5
5


MDTo330
*53aA, *55aL, *59aA
6
6
4
5


MDTo331
*53aA, *55aA, *59aS
6
6
5
5


MDTo332
*53aA, *55aA, *59aD
6
5
5
5


MDTo333
*53aA, *55aA, *59aF
6
5
5
5


MDTo334
*53aA, *55aL, *59aS
6
6
5
5


MDTo335
*53aA, *55aL, *59aD
7
6
4
5


MDTo336
*53aA, *55aL, *59aF
7
6
5
5









As already mentioned, for some of the more interesting variants that provided a significantly less sticky dough, the evaluations after floor time were also done for higher enzyme dosages of 0.1 mg/kg, 4 times overdose (4×OD) and 10 times overdose (10×OD). Those results with respect to softness, extensability, elasticity and machinability are shown in tables 7-9 below.







TABLE 7







Enzyme dosage of 0.1 mg/kg.














Soft-
Extens-
Elas-
Machin-


Variant
Mutation(s)
ness
ability
ticity
ability















LBei411
*53aA
6
5
5
4


LBei413
*55aA
6
4
6
5


LBei413
*55aA
6
5
5
4


LBei417
*59aA
6
4
6
5


LBei417
*59aA
6
5
5
4


MDTo253
*60aA
7
6
4
4


MDTo254
*62aA
6
5
5
4


MDTo255
*53aS
7
5
5
5


MDTo256
*53aD
7
6
4
2


MDTo257
*53aK
6
6
4
4


MDTo258
*53aF
6
5
5
4


MDTo259
*53aL
7
6
4
4


MDTo260
*55aS
7
6
4
4


MDTo262
*55aK
8
7
4
4


MDTo263
*55aF
6
6
5
5


MDTo264
*55aL
6
6
4
5


MDTo265
*59aS
6
5
5
5


MDTo266
*59aD
6
6
4
5


MDTo267
*59aK
6
6
4
5


MDTo268
*59aF
6
6
4
5


MDTo269
*59aL
6
6
4
5


MDTo323
*53aA, *55aL
7
6
4
4


MDTo324
*55aA, *59aD
7
6
4
4


MDTo325
*55aL, *59aA
8
6
4
5


MDTo326
*55aL, *59aS
8
6
4
5


MDTo327
*55aL, *59aD
8
6
4
5


MDTo328
*53aA, *59aS
7
6
4
4


MDTo329
*53aA, *59aD
7
6
4
5


MDTo330
*53aA, *55aL, *59aA
7
6
4
5


MDTo331
*53aA, *55aA, *59aS
8
6
4
5


MDTo332
*53aA, *55aA, *59aD
8
6
4
5


MDTo333
*53aA, *55aA, *59aF
7
7
4
5


MDTo334
*53aA, *55aL, *59aS
8
7
4
5


MDTo335
*53aA, *55aL, *59aD
7
6
4
4


MDTo336
*53aA, *55aL, *59aF
7
6
4
4
















TABLE 8







Enzyme dosage of 4 times overdose (4 × OD).
















Elas-
Machin-


Variant
Mutation(s)
Softness
Extensability
ticity
ability















LBei411
*53aA
6
5
5
4


LBei413
*55aA
6
4
6
5


LBei413
*55aA
6
5
5
4


LBei417
*59aA
6
4
6
5


LBei417
*59aA
6
5
5
4


MDTo253
*60aA
7
6
4
4


MDTo254
*62aA
6
5
5
4


MDTo255
*53aS
7
5
5
5


MDTo256
*53aD
7
6
4
2


MDTo257
*53aK
6
6
4
4


MDTo258
*53aF
6
5
5
4


MDTo259
*53aL
7
6
4
4


MDTo260
*55aS
7
6
4
4


MDTo262
*55aK
8
7
4
4


MDTo263
*55aF
6
6
5
5


MDTo264
*55aL
6
6
4
5


MDTo265
*59aS
6
5
5
5


MDTo266
*59aD
6
6
4
5


MDTo267
*59aK
6
6
4
5


MDTo268
*59aF
6
6
4
5


MDTo269
*59aL
6
6
4
5


MDTo323
*53aA, *55aL
8
6
4
4


MDTo327
*55aL, *59aD
8
6
4
4
















TABLE 9







Enzyme dosage of 10 times overdose (10 × OD).

















Machin-


Variant
Mutation(s)
Softness
Extensability
Elasticity
ability















LBei411
*53aA
6
5
5
4


LBei413
*55aA
6
4
6
5


LBei413
*55aA
6
5
5
4


LBei417
*59aA
6
4
6
5


LBei417
*59aA
6
5
5
4


MDTo253
*60aA
7
6
4
4


MDTo254
*62aA
6
5
5
4


MDTo255
*53aS
7
5
5
5


MDTo256
*53aD
7
6
4
2


MDTo257
*53aK
6
6
4
4


MDTo258
*53aF
6
5
5
4


MDTo259
*53aL
7
6
4
4


MDTo260
*55aS
7
6
4
4


MDTo262
*55aK
8
7
4
4


MDTo263
*55aF
6
6
5
5


MDTo264
*55aL
6
6
4
5


MDTo265
*59aS
6
5
5
5


MDTo266
*59aD
6
6
4
5


MDTo267
*59aK
6
6
4
5


MDTo268
*59aF
6
6
4
5


MDTo269
*59aL
6
6
4
5









Example 10
GH8 Variants in the Straight Dough Procedure Using Low Quality Flour
Ten GH8 variants were also tested in the straight dough procedure but with low quality flour. Enzyme dosages were 0.05 mg/kg or 0.1 mg/kg.
Bread was baked from both enzyme dosages using both normal and long proofing time and the specific volumes were determined. The results are summarized in tables 10 and 11 below.







TABLE 10







Dough stickiness after floor time and specific bread volumes


after normal and long proofing, respectively, at enzyme


dosages of 0.05 mg/kg and 0.1 mg/kg in the straight dough


procedure using flour with a lower gluten content.


















Vol.
Vol.
Vol.
Vol.



Muta-
Sticky
Sticky
0.05
0.05
0.1
0.1


Variant
tion(s)
0.05
0.1
norm
long
norm
long





LBei411
*53aA
6
6
110
114
115
114


LBei413
*55aA
5
5
110
112
112
114


LBei417
*59aA
5
5
112
112
113
115


MDTo264
*55aL
5
6
108
118
109
120


MDTo265
*59aS
6
7
107
118
111
125


MDTo266
*59aD
6
7
108
120
112
121


MDTo268
*59aF
6
7
113
119
112
123


MDTO298
*53aA,
6
6
109
114
114
123



*55aA


MDTO299
*55aA,
6
6
107
113
111
124



*59aA


MDTO300
*53aA,
5
6
109
116
116
126



*59aA


MDTO301
*53aA,
6
7
112
110
119
120



*55aA,



*59aA
















TABLE 11







Softness (Soft), Extensability (Ext), Elasticity (Elas) and Machinability


(Mach) of dough made with enzyme dosages of 0.05 mg/kg and 0.1 mg/kg in


the straight dough procedure using flour with a lower gluten content.










0.05 mg/kg
0.1 mg/kg
















Variant
Mutation(s)
Soft
Ext
Elas
Mach
Soft
Ext
Elas
Mach





LBei411
*53aA
6
4
6
4
7
5
5
3


LBei413
*55aA
6
5
5
5
6
5
5
5


LBei417
*59aA
5
5
5
5
6
5
5
5


MDTo264
*55aL
5
5
5
5
7
5
4
5


MDTo265
*59aS
7
6
4
5
7
6
4
4


MDTo266
*59aD
7
6
5
4
7
6
4
3


MDTo268
*59aF
7
6
5
4
7
6
4
3


MDTO298
*53aA, *55aA
6
6
5
5
6
6
5
5


MDTO299
*55aA, *59aA
6
6
5
5
6
6
5
5


MDTO300
*53aA, *59aA
5
5
5
5
6
6
5
5


MDTO301
*53aA, *55aA,
5
5
5
5
8
7
4
5



*59aA









Example 11
GH8 Variants in the Chorleywood Baking Procedure
Eleven GH8 variants of the invention were also tested in the Chorleywood baking procedure (CBP) for dough stickiness after mixing and specific volume increase of the baked bread after normal proofing using enzyme dosages of 0.05 and 0.1 mg/kg. Results are shown in table 12.







TABLE 12







Dough stickiness after mixing in the Chorleywood baking


procedure using 0.05 mg/kg (0.05) or 0.1 mg/kg (0.01)


enzyme dosage.














Sticky
Sticky
Vol.
Vol.


Variant
Mutation(s)
0.05
0.1
0.05
0.1















LBei411
*53aA
5
6
112
115


LBei413
*55aA
5
5
114
112


LBei417
*59aA
5
5
110
113


MDTo264
*55aL
5
5
113
113


MDTo265
*59aS
5
5
103
117


MDTo266
*59aD
5
5
115
115


MDTo268
*59aF
5
5
114
121


MDTO298
*53aA, *55aA
6
6
113
116


MDTO299
*55aA, *59aA
5
5
104
114


MDTO300
*53aA, *59aA
6
6
106
123


MDTO301
*53aA, *55aA, *59aA
5
5
115
125
















TABLE 13







Softness (Soft), Extensability (Ext), Elasticity (Elas) and


Machinability (Mach) of dough made with enzyme dosages of


0.05 mg/kg and 0.1 mg/kg in the Chorleywood procedure.










0.05 mg/kg
0.1 mg/kg
















Variant
Mutation(s)
Soft
Ext
Elas
Mach
Soft
Ext
Elas
Mach





LBei411
*53aA
5
5
5
5
6
5
5
4


LBei413
*55aA
5
5
5
5
5
5
5
5


LBei417
*59aA
5
5
5
5
5
5
5
5


MDTo264
*55aL
5
5
5
5
5
5
5
5


MDTo265
*59aS
5
5
5
5
5
5
5
5


MDTo266
*59aD
5
5
5
5
5
5
5
5


MDTo268
*59aF
5
5
5
5
5
5
5
5


MDTO298
*53aA, *55aA
6
5
5
5
7
6
4
5


MDTO299
*55aA, *59aA
5
5
5
5
5
5
5
5


MDTO300
*53aA, *59aA
6
5
5
5
6
5
5
5


MDTO301
*53aA, *55aA,
6
5
5
5
5
5
5
5



*59aA

## Claims

1. An isolated variant of a parent GH8 xylanase polypeptide, wherein said variant comprises a conserved GH8 xylanase motif [ST]E[GAS]X[GAS][YFW] (SEQ ID NO: 1); the variant comprises an amino acid sequence having at least 90% sequence identity to amino acids 1-406 of the GH8 xylanase of SEQ ID NO: 3; the variant comprises at least one amino acid alteration which is a substitution, deletion, and/or insertion of one or more amino acids at one or more position(s) corresponding to position(s) 53, 55, 56, 58, 59, 60, 61, and/or 62 of SEQ ID NO: 3, and the variant has xylanase activity.

2. The variant of claim 1, wherein the at least one amino acid alteration comprises one or more insertion(s) at a position corresponding to a position 12 amino acids away from the conserved GH8 xylanase motif on the N-terminal side in amino acids 1-406 of SEQ ID NO: 3 or at a position corresponding to position 53 of SEQ ID NO: 3.

3. The variant claim 1, wherein the at least one amino acid alteration comprises one or more insertion(s) at a position corresponding to a position 10 amino acids away from the conserved GH8 xylanase motif on the N-terminal side in amino acids 1-406 of SEQ ID NO: 3 or at a position corresponding to position 55 of SEQ ID NO: 3.

4. The variant of claim 1, wherein the at least one amino acid alteration comprises one or more insertion(s) at a position corresponding to a position 8 amino acids away from the conserved GH8 xylanase motif on the N-terminal side in amino acids 1-406 of SEQ ID NO: 3 or at a position corresponding to position 57 of SEQ ID NO: 3.

5. The variant of claim 1, wherein the at least one amino acid alteration comprises one or more insertion(s) at a position corresponding to a position 6 amino acids away from the conserved GH8 xylanase motif on the N-terminal side in amino acids 1-406 of SEQ ID NO: 3 or at a position corresponding to position 59 of SEQ ID NO: 3.

6. The variant of claim 1, wherein the at least one amino acid alteration comprises one or more insertion(s) at a position corresponding to a position 4 amino acids away from the conserved GH8 xylanase motif on the N-terminal side in amino acids 1-406 of SEQ ID NO: 3 or at a position corresponding to position 61 of SEQ ID NO: 3.

7. The variant of claim 1, wherein the one or more insertion(s) comprise the insertion of at least one alanine residue, at least one aspartic acid residue, at least one serine residue, at least one lysine residue, and/or at least one phenylalanine residue.

8. The variant of claim 1, which has at least one bread improving property when added to a dough before or during mixing in a sufficient amount.

9. The variant of claim 1, which is capable of increasing the loaf volume of a baked product or increasing the width of cut on the surface of a baked product, when added to a dough before or during mixing in a sufficient amount.

10. The variant of claim 9, which provides a less sticky dough when compared with a dough to which the parent GH8 xylanase has been added in the same amount.

11. The variant of claim 1, wherein said variant comprises one or more alterations selected from the group consisting of D53*, L56*, N58*, *53aA, *55aA, *59aA, N55A +L56*+G57N+N58L, *61aA, *60aA, *62aA, *53aS, *53aD, *53aK, *53aF, *53aL, *55aS, *55aK, *55aF, *55aL, *59aS, *59aD, *59aK, *59aF, *59aL, *53aA+*55aA, *55aA+*59aA, *53aA+*59aA, *53aA+*55aA+*59aA, *53aA+*55aL, *55aA+*59aD, *55aL+*59aA, *55aL +*59aS, *55aL+*59aD, *53aA+*59aS, *53aA+*59aD, *53aA+*55aL+*59aA, *53aA+*55aA+*59aS, *53aA+*55aA+*59aD, *53aA+*55aA+*59aF, *53aA+*55aL+*59aS, *53aA+*55aL+*59aD, and *53aA+*55aL+*59aF.

12. A composition comprising the variant of claim 1.

13. A method for the preparation of a baked product, said method comprising the step of adding to a dough of said baked product the composition of claim 12.

14. The method of claim 13, wherein said composition further comprises one or more bread-improving agents selected from the group consisting of enzymes, emulsifiers, oxidants, milk powder, fats, sugars, amino acids, salts, proteins, and a mixture thereof.

15. The method of claim 14, wherein said enzymes are selected from the group consisting of alpha-amylases, beta-amylases, maltogenic amylases, additional xylanases, proteases, glucose oxidases, oxidoreductases, glucanases, cellulases, transglutaminases, isomerases, lipases, phospholipases, pectinases, and a mixture thereof.

16. The method of claim 13, wherein said variant is mixed with other ingredients in the form of a dry powder, a granulate, or a liquid.

17. A method for increasing the loaf volume of a baked product, comprising the step of adding during the mixing of the dough of said baked product, a sufficient amount of the variant of claim 1.

18. A method for increasing the loaf volume of a baked product or for increasing the width of cut on the surface of a baked product, comprising the step of adding during the mixing of the dough of said baked product, a sufficient amount of the variant of claim 1.

19. A bread improver composition for increasing the loaf volume of a baked product or for increasing the width of cut on the surface of a baked product, wherein said composition comprises the variant of claim 1.

20. The variant of claim 1, comprising an amino acid sequence having at least 95% sequence identity to amino acids 1-406 of the GH8 xylanase of SEQ ID NO: 3.