# Patent

## Title
Xylanase

## Abstract

The inventors have identified a xylanase from a bacterial strain of Paenibacillus pabuli and found that the xylanase can increase the shelf life of baked products. More specifically, the xylanase in combination with a maltogenic amylase further improves the softness of bread crumb without having detrimental effects on elasticity.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 35 U.S.C. 371 national application of PCT/DK2005/000034 filed Jan. 20, 2005, which claims priority or the benefit under 35 U.S.C. 119 of Danish application no. PA 2004 00263 filed Feb. 20, 2004 and U.S. provisional application No. 60/547,301 filed Feb. 23, 2004, the contents of which are fully incorporated herein by reference.


FIELD OF THE INVENTION
The present invention relates to a process for preparing a dough-based product and to a xylanase for use in the process.
BACKGROUND OF THE INVENTION
K. B. Kubata et al., Biosci. Biotech. Biochem., 56 (9), 1463-1464 (1992) describes Xylanase I of Aeromonas caviae ME-1. Its amino acid sequence was submitted by T. Suzuki et al. in 1994 to the EMBL/GenBank/DDBJ databases where it was given the accession number Q43993.
WO 0039289 describes a xylanase from Bacillus subtilis said to be suitable for preparing non-sticky dough.
SUMMARY OF THE INVENTION
The inventors have identified a xylanase from a bacterial strain of Paenibacillus pabuli and found that the xylanase can increase the shelf life of baked products. More specifically, the xylanase in combination with a maltogenic amylase further improves the softness of bread crumb without having detrimental effects on elasticity.
Accordingly, the invention provides a process for preparing a dough-based product, comprising adding a xylanase with a high identity to SEQ ID NO: 2 to a dough, leavening, and heating the dough. More specifically, the xylanase is a polypeptide having at least 83% identity to the amino acid sequence as shown in positions 1-182 of SEQ ID NO 2 or encoded by a nucleic acid sequence which hybridizes at 38° C. in 0.1×SSC with the complementary strand of nucleotides 85-630 of SEQ ID NO: 1.
The invention further provides a dough composition which comprises flour together with the xylanase and a dough and/or bread-improving additive comprising the xylanase in the form of a granulate or agglomerated powder.
The invention also provides a polypeptide having xylanase activity. It may be a polypeptide encoded by the genome present in Paenibacillus DSM 16232 that can be amplified with the primers (SEQ ID NO.: 3) and (SEQ ID NO.: 4) or having an amino acid sequence as shown in positions 1-182 of SEQ ID NO 2, or it may be at least 95% identical to one of these. The polypeptide may also be encoded by a nucleic acid sequence which hybridizes at 49° C. in 0.1×SSC with the complementary strand of nucleotides 85-630 of SEQ ID NO: 1.
Alternatively, the xylanase may be a polypeptide having an amino acid sequence which can be obtained from the mature polypeptide of SEQ ID NO: 2 by substitution, deletion, and/or insertion of one or more amino acids and a polynucleotide having a sequence that can be derived from SEQ ID NO: 1 by substitution, deletion, and/or insertion of one or more nucleotides.
The invention also provides a polynucleotide encoding the xylanase, an expression vector comprising the polynucleotide, a transformed host cell comprising the vector, as well as a method of producing the xylanase by cultivating the transformant.


DETAILED DESCRIPTION OF THE INVENTION
Genomic DNA Source
The source organism of the xylanase of the invention is a bacterial strain isolated from soil samples collected in New Zealand in 1991. The strain was classified as belonging to Paenibacillus pabuli. The inventors have deposited the strain under the terms of the Budapest Treaty on 17 Feb. 2004 as DSM 16232 with the DSMZ—Deutsche Sammiung von Micro-organismen und Zelikulturen GmbH, MascheroderWeg 1b, D-38124 Braunschweig D E.
Recombinant Expression Vector
The expression vector of the invention typically includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a selectable marker, a transcription terminator, a repressor gene or various activator genes. The vector may be an autonomously replicating vector, or it may be integrated into the host cell genome.
Production by Cultivation of Transformant
The polypeptide of the invention may be produced by transforming a suitable host cell with a DNA sequence encoding the xylanase enzyme, cultivating the transformed organism under conditions permitting the production of the enzyme, and recovering the enzyme from the culture.
The host organism may particularly be a prokaryotic cell, in particular a bacterial cell, such as gram positive bacteria including a Bacillus cell, e.g., Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, Bacillus subtilis or an alkalophilic Bacillus, or a Streptomyces cell, or gram negative bacteria such as E. coli and Pseudomonas sp.
Alignment and Identity
The polypeptide and polynucleotide of the invention may have identities to the disclosed sequences of at least 85%, at least 90%, at least 95% or at least 98%.
For purposes of the present invention, the alignments and identities of the protein sequences are analysed by Vector NTI—program (Invitrogen Corporation). The alignments are created using the Clustal W algorithm (Nucleic Acid Research, 22 (22): 4673-4680, 1994).
Alignment Parameters used for polypeptide alignments are: penalty for the first residue in a gap 10; penalty for additional residues in a gap 0.1; no penalty for gaps introduced at the end of a sequence.
Hybridization
Suitable conditions for determining hybridization between a nucleotide probe and a homologous DNA or RNA sequence involve presoaking of the filter containing the DNA fragments or RNA to hybridize in 5×SSC (standard saline citrate) for 10 min, and prehybridization of the filter in a solution of 5×SSC (Sambrook et al. 1989), 5×Denhardt's solution (Sambrook et al. 1989), 0.5% SDS and 100 μg/ml of denatured sonicated salmon sperm DNA (Sambrook et al. 1989), followed by hybridization in the same solution containing a random-primed (Feinberg, A. P. and Vogelstein, B. (1983) Anal. Biochem. 132:6-13), 32P-dCTP-labeled (specific activity>1×109 cpm/μg) probe for 12 hours at approx. 45° C. The filter is then washed two times for 30 minutes in 0.1×SSC, 0.5% SDS at a temperature of 25° C., 30° C., 35° C., 40° C., 45° C., 49° C. or 55° C. Molecules to which the oligonucleotide probe hybridizes under these conditions may be detected using a x-ray film.
Flour Composition or Dough
The flour composition may particularly comprise wheat flour. It may a dry mixture comprising flour and the xylanase, particularly in the form of the additive described above. The flour composition may also be a dough, which may be fresh, frozen or par-baked. It may be a laminated dough.
The xylanase may be added to the flour composition or dough at a dosage of 0.1-10 mg enzyme protein per kg of flour, particularly 0.2-5 mg/kg.
The dough may also comprise other conventional dough ingredients, e.g. proteins, such as milk powder and gluten; eggs (either whole eggs, egg yolks or egg whites); an oxidant such as ascorbic acid, potassium bromate, potassium iodate, azodicarbonamide (ADA) or ammonium persulfate; an amino acid such as L-cysteine; a sugar; a salt such as sodium chloride, calcium acetate, sodium sulfate or calcium sulfate. The dough may comprise fat (triglyceride) such as granulated fat or shortening.
Additional Enzyme
Optionally, one or more additional enzymes may be added to the dough together with the xylanase of the invention. The additional enzyme may be an amylase, a lipolytic enzyme (e.g. as described in WO 9953769) or a second xylanase.
The amylase may be an exo-acting maltogenic alpha-amylase. An example is a maltogenic alpha-amylase from B. stearothermophilus strain NCIB 11837, available from Novozymes A/S under the tradename Novamyl®; described in WO 9104669 and having the amino acid sequence shown as SEQ ID NO: 1 of U.S. Pat. No. 6,162,628A1. Another example is a Novamyl variant, e.g. as described in WO 9943794. The maltogenic amylase may be added at a dosage of 100-1000 MANU per kg flour (MANU activity unit defined in WO 9104669).
Dough-Based Product
The invention provides a method for preparing a dough-based product by leavening the dough and heating it, e.g. by baking or steaming. The dough may be leavened e.g. by adding chemical leavening agents or yeast, usually Saccharomyces cerevisiae (baker's yeast). The product may be of a soft or a crisp character, either of a white, light or dark type. Examples are steamed or baked bread (in particular white, whole-meal or rye bread), typically in the form of loaves or rolls.
Modified AZCL-arabinoxylan from Wheat Assay pH 6.0
Substrate:
0.2% AZCL-Arabinoxylan from wheat (Megazyme) in 0.2 M Na-phosphate buffer pH 6.0+0.01% Triton-x-100.
Standard:
BioFeed Wheat (product of 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.
Method:
1. 900 μl substrate is preheated to 37° C. in a thermomixer
2. 100 μl sample is added
3. Incubate for 15 min at 37° C. at maximum speed
4. on ice for 2 min
5. spin 1 min 20.000×G
6. 2×200 μl supernatant is transferred to a micro titter plate
7. Endpoint OD 590 nm is measured.
EXAMPLES
Example 1
Production of xylanase
Cloning of Paenibacillus Pabuli GH 11 Xylanase
Chromosomal DNA of Paenibacillus pabuli strain DSM 16232 was isolated by QIAmp Tissue Kit (Qiagen, Hilden, Germany). A linear integration vector-system was used for the expression cloning of the gene. The linear integration construct was a PCR fusion product made by fusion of the gene between two Bacillus subtils homologous chromosomal regions along with a strong promoter and a chloramphenicol resistance marker. The fusion was made by SOE PCR (Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K. and Pease, L. R. (1989), Engineering hybrid genes without the use of restriction enzymes, gene splicing by overlap extension, Gene 77: 61-68). The SOE PCR method is also described in WO 2003095658. The gene was expressed under the control of a triple promoter system (as described in WO 99/43835), consisting of the promoters from Bacillus licheniformis alpha-amylase gene (amyL), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), and the Bacillus thuringiensis cryIIIA promoter including stabilizing sequence. The gene coding for Chloramphenicol acetyl-transferase was used as marker. (Described in eg. Diderichsen, B.; Poulsen, G. B.; Joergensen, S. T.; A useful cloning vector for Bacillus subtilis. Plasmid 30:312 (1993)). The final gene construct was integrated on the Bacillus chromosome by homologous recombination into the pectate lyase locus.
First 3 fragments were PCR amplified: the gene fragment with specific primers oth62 (SEQ ID NO.: 3) and oth63 (SEQ ID NO.: 4) on genomic DNA from Paenibacillus pabuli. The upstream flanking fragment was amplified with the primers 260558 (SEQ ID NO.: 5) and UpN 1361 (SEQ ID NO.: 6) and the downstream flanking fragment was amplified with the primers 260559 (SEQ ID NO.: 7) and DwC 1361 (SEQ ID NO.: 8) from genomic DNA of the strain iMB1361 (described in WO 2003095658).
The DNA fragments were amplified with “Expand High Fidelity PCR System” (Boehringer Mannheim, Germany) using the following conditions: 94° C. for 2 min followed by 10 cycles of (94° C. for 15 sec, 55° C. for 30 sec, 68° C. for 4 min) followed by 20 cycles of (94° C. for 15 sec, 55° C. for 30 sec, 68° C. for 4 min (+20 sec pr cycle)) and ending with one cycle at 68° C. for 10 min. The 3 resulting fragments were mixed in equal molar ratios and a new PCR reaction was run under the following conditions: initial 2 min. at 94° C., followed by 10 cycles of (94° C. for 15 sec., 55° C. for 45 sec., 68° C. for 5 min.), 10 cycles of (94° C. for 15 sec., 55° C. for 45 sec., 68° C. for 8 min.), 15 cycles of (94° C. for 15 sec., 55° C. for 45 sec., 68° C. for 8 min. in addition 20 sec. extra pr cycle). After the 1st cycle the two end primers 260558 (SEQ ID NO.: 5) and 260559 (SEQ ID NO.: 7) were added (20 pMol of each). Two micro-I of the PCR product was transformed into Bacillus subtilis and transformants were selected on LB-plates containing chloramphenicol (6 μg/ml medium). A clone containing the construct without mutations leading to amino acid changes was selected for fermentation in liquid media.
The supernatant was analyzed for xylanase activity with modified AZCL-arabinoxylan as substrate by the assay described above and had around 150 FXU/ml:
Fermentation
The clone was streaked on an LB-agar plate with 6 micro g/ml chloramphenicol from −80° C. stock, and grown overnight at 37° C. The colonies were transferred to 100 ml PS-1 media supplemented with 6 micro g/ml chloramphenicol in a 500 ml shaking flask.
Composition of PS-1 medium:

















Saccharose
100
g/l



Soy Bean Meal
40
g/l



Na2HPO4, 12 H2O
10
g/l



Pluronic TM
0.1
ml/l










The culture was shaken at 30° C. at 275 rpm for 3 days. The cells were spun down and the enzyme purified from the supernatant.
Example 2
Purification of xylanase
The fermentation broth from Example 1 was filtered through filter paper and finally a blank filtration. Ammonium sulphate was added to a final concentration of 3.0 M and incubated for 30 min at room temperature. After 30 min centrifugation at 10000 g, the precipitate was solubilized with 10 mM sodium acetate pH 5.0 and dialyzed against 10 mM sodium acetate pH 5.0. After dialysis the sample was applied on a S-Sepharose column (Amersham Pharmacia Biotech 2.6 cm×10 cm) equilibrated with 10 mM sodium acetate pH 5.0. Elution was performed with a linear gradient from 0-1 M NaCl in 10 mM sodium acetate pH 5.0. Fractions containing xylanase activity were pooled and stored at −20° C. This preparation was used in Example 3.
Example 3
Effect of xylanase on Freshness of Bread
Bread were baked according to the sponge & dough method.










Recipes









% on flour basis














Sponge




Soya oil
2.5



SSL
0.38



Yeast
5



Wheat flour
60



Water
62



Dough



Ascorbic acid
optimized for each flour



ADA
20 ppm



Salt
2



Syrup
7 (dry substance)



Water
to be optimized for each flour



Wheat flour
40



Calcium propionate
025










Sponge
Scaling of ingredients, addition of yeast, water, flour, SSL and oil into mixer bowl Mixing 90 rpm for 1 minutes, 150 rpm for 4 minutes. The sponge is weighted, the temperature is measured and the sponge is placed in a bowl and fermented for 3 hours at 27° C., 86% RH.
Dough
Addition of ingredients and the sponge into the mixer bowl. The sponge and ingredients are mixed together 90 rpm for 9 minutes
The temperature is measured, dough characteristics are evaluated, the dough is scaled into smaller pieces of 435 g each.
The dough rests on the table for 10 minutes
Doughs are sheeted and molded.
Fermentation for 55 minutes at 42° C. and 86% RH.
Bread are baked at 200° C. for 22 minutes
Enzymes were dosed at 2 mg of xylanase of the invention per kg of flour together with 400 MANU/kg of Novamyl. A control was made with 200 FXU/kg of the prior-art xylanase Shearzyme (product of Novozymes A/S) together with 400 MANU/kg of Novamyl.
Bread was stored at room temperature until analysis.
Texture and water migration by NMR were measured on day 7, 14 and 21. A small sensory evaluation of softness and moistness was performed on day 21.
Results
Firmness of the loaves was measured as described in WO 9953769 The results were as follows:
















Xylanase

Firmness
Firmness
Firmness


Novamyl
of
Prior-art
after
after
after


dosage
invention
xylanase
7 days
14 days
21 days


MANU/kg
mg/kg
FXU/kg
g
g
g




















400
2
0
460
663
922


400
0
200
475
754
1058









Elasticity of the loaves was measured as described in U.S. Pat. No. 6,162,628. The results were as follows:
















Xylanase






Novamyl
of
Prior-art
Elasticity
Elasticity
Elasticity


dosage
invention
xylanase
after
after
after


MANU/kg
mg/kg
FXU/kg
7 days %
14 days %
21 days %




















400
2
0
48.5
46.8
43.1


400
0
200
44.9
44.9
42.2









The data show that the effect of the xylanase of the invention together with Novamyl on firmness and elasticity outperforms the combination the prior-art xylanase and Novamyl.
The mobility of free water was determined as described by P. L. Chen, Z. Long, R. Ruan and T. P. Labuza, Nuclear Magnetic Resonance Studies of water Mobility in Bread during Storage. Lebensmittel Wissenschaft und Technologie 30, 178-183 (1997). The results were as follows:


















Free water
Free water
Free water



Xylanase

after
after
after


Novamyl
of
Prior-art
7 days
14 days
21 days


dosage
invention
xylanase
micro-
micro-
micro-


MANU/kg
mg/kg
FXU/kg
seconds
seconds
seconds




















400
2
0
7632
6800
6450


400
0
200
7725
6438
6034









The data show that the xylanase of the invention increases the amount of free water more than the prior-art xylanase. The amount of free water has been described in literature to correlate to moistness of bread crumb.
The ranking from the small sensory evaluation of softness and moistness on day 21 showed that bread crumb made with the xylanase of the invention together with Novamyl was perceived as more moist than bread made with the prior-art xylanase and Novamyl.

## Claims

1. A process for preparing a dough-based product, comprising adding a polypeptide having xylanase activity, which has at least 98% identity to the sequence of amino acids 1-182 of SEQ ID NO: 2, to a dough in an amount effective for increasing the shelf-life of the dough-based product prepared from the dough, leavening, and baking the dough.

2. The process of claim 1, wherein the polypeptide comprises the sequence of amino acids 1-182 of SEQ ID NO: 2.

3. The process of claim 1, which further comprises adding an exo-acting maltogenic alpha-amylase to the dough.

4. An isolated polypeptide having xylanase activity, which has at least 98% identity to the sequence of amino acids 1-182 of SEQ ID NO: 2.

5. The polypeptide of claim 4, which comprises the sequence of amino acids 1-182 of SEQ ID NO: 2.

6. A composition which comprises flour and a polypeptide of claim 4.

7. The composition of claim 6 which is a dough.

8. A granulate or agglomerated powder comprising a polypeptide of claim 4.