GB2392160A - Endo-b-1, 4- xylanase - Google Patents

Abstract

An endo- b -1, 4-xylanase enzyme represented by the amino acid sequence of SEQ ID No 5.

Description

PROTEINS

BACKGROUND OF THE PRESENT IN\/ENTIO

S The present invention relates to proteins.

In particular, the present invention relates to the isolation of and characterization of an endogenous endo-1,4xylanase inhibitor that is present in wheat flour and its effect on deferent xylanases. The present invention also relates to xylanases idenffled by a screen lo using the inhibitor and to novel xylanases denfffiod thereby.

BACKGROUND ART

Xylanases have been used in bakery for several years.

In this regard, it is known that wheat flour contains arabinoxylan onginating from the andosperm cdl walls. The amount of arabinoxylan in the flour differs depending on origin of the flour - for example, Bee Rounu et a/, Journal of Cereal Science (1994), 19, 25272 Exact of an Enzyme PrepetstJon Containing Pentosanases on the Bread making 20 C7ualty of Flour in Relation to Charges in Pentosen Properties; Finchor and Stone, (1986) Advances #7 Cema/ Technology,, Vol. Vlil (Why Pomeranz, Ed.) AACC, St Paul, Minnesota, 207-295; and Mouser and Suckow (1986), Chemistry and Physics of Belling (J.M.V. Blanchard, P J Frasier and T Glilard, Eds.) Royal Society of Chemistry, London, 421. Typically the amount of arabinoxylan can vary from 2-5% ((w/w) based on flour 2S dry weight).

Pincher and Stone (1986) report 70% of the polysaccharides in the endosperm cdl wall are arabinoxylan. A characteristic feature of arabinoxylan is its ability to bind to water.

Part of the arabinoxylan is water insoluble pentosan (VVIP) and part is water soluble 30 pentosan (WSP). Experimental results have shown a correlation between degradation of POP to high molecular weight (HOW) water soluble polymers and broad volume.

During the production of a bakery product, it is known that using a xylanase at a proper dosage may result in a more stable dough system (whim will typically comprise sad, flour, 3S yeast and water) and a better volume of, for example, raised bread.

l In this respect, a good xrlanaso for increasing bread volume should sohblli" WIP giving an increased viscosity in the dough liquid without furor degradation of VVSP into typos oligomere. This degradation of WIP into low molecular weight (LMW) WSP is believed to be detrimental for the dough properties and may GINO rise to stickirss (Rousu et al and S McCleary (1986) IntemationJoumal of Biology/ Macro Mdocules, 8, 3435.

US-A-5306633 discloses a xylanase obtained from a Becilus subtilis strain. Appa, this xyanase may improve the consistency and increase the volume of bmad Ad baked goods container the same.

Another xylana" from Bacillus subtDls has been isolated and sequenced (see Pa -, M.G., Bourbonnais, R., DearocJ,ors, M., Jurasek, L and Yaguchi, M. A - anese gone ham Bacillus subtilize nucwffde sequence and comparison With B. pumps gone, Arm.

Microbiol. 144, 201-206 (1986)).

IS It has been considered for some time now that bacterial xylanases would produce very sticky dough. Hence, one would norrnalty expect the xylanases of Bacillus subtftis- such as that of US-A-5306633 - to produce a very sticky dough.

20 Prior art enzymes which caused Stickiness had to be wed in carefully cordrolbd amounts

so that stickiness would not adversely affect handling to such a dorm that eff commercial handling wee hampered. However, the need to carefully control dosage prohibited the addition of xylanase directly to flour prior to production of the dough. It was ereforo necessary wffl prior art systems to add the xylano in a wry condoled

as manner during the production of No dough.

To d -, fungal xylanas" have been typically used in baking. For example, J Mast et al. (Xylans and Xylanases, edited by J - ser et 81, 3493SO, Xybneses UId Weir appikan in bakers teach a,B-1,4xylanase produced by an AsperI7/71us Niger var. awarmori sbain.

30 According to those authors, the fungal xyianaso is effective in increasing the specific volume of breads, without giving rise to a negative side Act on dough handling (stickiness of the dough) as can be observed with xylanasea derived from offer fungal or from bacterial sours -.

3S It has bean proposed by W Debyser at d, (J. Am. Soc. B. - w. Chain. 55(4), 153156, 1997, Ambinoen Solubilizetdon end Inhibition of the Barely Malt Xybnobvc Sylvan by

I/l/heat Dunng Mashing udth Street Wholemeal Adjunt. Evidence for a New Class of Enzyme Inhibitors in wheat), that xylanase inhibitors may be present in wheat. TO inhibitor discussed by W Debater et al. was not isolated. Furthermore, It is not disclosed by W Debyser at d whether the inhibitor is endogenous or microbiological. Moreover, no chemical data were presented for this inhibitor.

The presence of xylanase inhibitor in wheat flour has also recerdy been discussed by X Rouau and A Surget, (Joumal of Cereal Science, 28 (1998) 6370, Evidence for the Presence of a Pentosanase Inhibitor in Vmest FloursJ. Similar to Debar et d, Rousu lo and Surget believed that they have idenffled the existence of a thermolabile compound in the soluble fraction of wheat flours, which limited action of an added innate.

Also similarly to Debysor at so., these authors did not isolate an Inhibitor and wore unable to condude whether the inhibitor is endogenow or i. of microbial origin. ilc, no chemical data were presented for this inhibitor.

IS Thus, a known problem in the art is how to prepare baked goods from a dough which coos not have adverse handling properties. A more particular problem is how to provide a dough which is non-secky i.e. a dough that is not so sticky that It causes handling and PA probbma.

The present invention see Is to provide a solution to these probbrna.

SUMMARY ASPECTS OF THE P=ENT INVENTION

2s Aspects of the present invention are presented In the claims and in the following commentary. In br; - f, some aspects of the present inrenffon relate to: 30 1. An endogenous endo+1,xylanase inhibitor - induding nuckoWe sequences coding therefor and to amino acid sequences thereof, as well as variant, homologues, or*ag'' - rid thereof.

2. Assay methods for determining the effect of Me p-1,xylanase inhibitor on as different xylanases.

3. Assay methods for debrrnining effect of diflierent xylana - e in dough.

4. A - ay moods for determining the effect of glucana - (a) on different doughs containing xyhnas.

5. Nova xybnaeos including nucleoffde sequences coding erefor and amino acid sequences thereof. as wee as variant, homobuos, or fragrnords of.

B. Now use of qrlan.

7. Foock prepared we xylana_.

O-r asps cony To amino add sequo of pi indoor nuebodde sequence of the preadult invention include: a cony I or capable of is I ff-"quench of the presort irnnlon; a vector IBM or capable of - sequence of the present invasion; a pbamid cornprb or I of coequal of it irwonffon a moue oomprb or capable of I sequence of the it invenffon, an organ comprg or cay of I aequer of the prt inven00n; a banomtod host compress or 20 cay of express the quenoos of present irorondon; a but organism cornprich or capable of orsir the qwac of ff-present invention. The pyre invention also encompasses moods of engrossing same, such expansion in a melanism; include my for transecting same.

2S The present invention defers from the teachings of WA-88/49278 because moor aUe that POT patent application contain minimal sequence information regarding protfirdc inhibitor disbud thorn.

Aspects of the preserd invendon am now discussed under appropriate section headers.

30 For the sake of convenience, generally applicable teachings for aspect of the prey invention may be found in Go sections ffUod 'General Definition' and Gal T - chings.. However, the teachings under osah section are not nomesuily limited to earth puicubr soon.

s GEERAL DEFINITIONS

The term Wheat flour as used herein is a synonym for the finelyround meal of wheat.

Preferably, however, the temn means flour obtained from wheat per se and not from 5 another grain. Thus, and unless otherwise expressed, references to Wheat flours as used herein preferably mean references to wheat flour per se as well as to wheat flour when present in a medium, such as a dough.

The term xylanase. is used in its normal sense - e.. an enzyme that is inter alis capable to of catalysing the depoUmensation of arabinoxylan which may be present in wheat (e.. an enzyme that is inter era capable of catalysis the solubilisation of WIP and catalysing the depolymerisation of WSP which may be present in wheat).

An assay for determining endo-1,xylanase activity is presented later. For IS convenience, this assay is called the ÀXylanase Assays.

The term "nucleotide sequence. in relation to the present invention includes genomic DIVA, cDNA, recombinant DNA (i.e. DNA prepared by use of recombinant DNA techniques), synthetic DNA, and RNA - as well as combinations thereof.

Preferably, the term "nucleoffde sequencer moans DNA The nucleotide sequences of the present invent/on may be single or doubts stranded.

25 The nucleotide sequences of the present invention may indude within tom synthetic or modified nucleoffdos. A number of different types of modification to oligonucbotides are known in No am These include meylphosphonate and phosphorothbate backbones, Addison of acridine or polylysine chains at the 3' andtor 5' ends of the nmbcuh. For the purposes of the present invention, it is to be understood that the nucbode sequences 30 described herein may be modified by any method available in the art Such modifications may be carried out in to enhance the Volvo activity or life span of nuclsoWe sequences of the present invention.

The terms variant or Homology u ith respect to the nucleotide sequence of the present as invention and the amino acid sequence of the present invention are synonymous Ninth Alec varbdona of the sequeno -.

c In particular, the tend Homology as used herein may be equated with the term guidons.

Hore, sequence homology with respect to the nuchoffde sequence of to present imndon and the amino acid sequeno. of the present invention can be determined by a simple s eyeball. comparison (i.e. a strict comparison) of any one or more.of the sequences with another sequence to see if that other sequence has at best 75% idonffly to the sequence(s). Rustic sequence homology (i.e. sequence ider-) can also be determiner by commercially available computer programs that can calculate % homology been ho or more aeqwnoss. A typical example of such a computer program is CLUSTAL Hence, homology comparisons can be conducted by eye. However, more usually may are conducted with the aid of readily avaibbb sequence comparison programs. Them commercially available computer programs can calculate % homology bin halo or more "quench. IS % homology may be calculated over contiguous sequences, i.e. one "qwnce is aligned with the aver sequence and each amino acid in one sequence directly compared with the corresponding amino add in the over sequence, one residue at a time. This is called an ungappoa" alignment Typically, such ungapped alignments are performed only owr a 20 relatedly short number of residues (for example less man 50 congruous amino acids).

Although this is a very simple and consistent method, it fade to take into consideration that, fw example, in an otherwise identical pair of sequenc-, one insertion or deletion will cause e following amino acid residues to be put out of alignment thus potentially rmutting in a 2S large induction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are destined to produce optimal albnmer" that take into consideration possible insertions and deletions without penalsir unduly too overall homology acore. This is achieved by inserting gaps. in the sequel alignment to try to rnexim" Heal homology.

However, these more complex methods assign gap penalties to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence aligarnent wffl as few gaps as possible - reflecdr hther relatedness behHeen the halo compared sequenoss - wit achieve a higher Scone than one with many gaps. 'Alfinc gap cost as typically used that charge a Iy high cost for tt. of a gap and a Or port for each subsequent residue in the gap. This is the meal commonly used W acoring system. High gap penalties w 11 of coupe produce opffmid a0gnmon with S - r

gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons.

For example when using the GCG Wisconsin Beset package (see below) tl. default gap penalty for amino acid sequences is -12 for a gap and 4 for each extension.

S Calculation of rr aximum % homology therefore firstly requires the production of an optimal alignment, tapirs into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Besffit packed (University of Wisconsin, U.SA; Devereux eta/., 1984, NucJek Adds Research 12:387). Examples of 10 offer software than can perform sequer" comparisons indude, but am not limited to, Go BLAST package (see Ausubel et aL, 1999 ibid - Chapter 18), PASTA (Gaul et at., 1990, J. Me. Biol., 40310) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (son Ausubel at at., 1999 ibid, pages 7-58 to 70). However it is preferred to use the GOB Bestfit program.

IS Although the final % homology can be measured in terms of entity, ff alignment process itself is typically not based on an allor-notning pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwi" comparison based on chemical similarity or evolutionary distance. An example of such a 20 matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for furor details). It is preferred to use the public default values for the GCG package, or in the case of other sofhvaro, the default ma=, such as BLOSUM62.

IS, Once the software has produceci an optimal alignment, it is possible to calculate % homology, preferably % sequence icierdit,Y. The software typically does this as part of the sequence comparison and generates a numerical result.

30 Preferably, sequence comparisons are conduced using the simple BLAST search algorithm provided at httD:lAuwv.ncbi.nim.nih.aov/BIAST using the default parameters.

The present uvenbon also encompasses nucleotide sequences thad are complementary to tl. sequences presented herein, or any derivatne, fragmerd or denude tlof. If the 3S sequence is complementary to a Tent thereof then that sequence can be used a probe to identify simian coding sequenoss in- organisms etc.

The present invention also encompasses nudeotWe sequences that are capable of hybridizing to the sequences present - d herein, or any derive -, fragment or derivable of. S The present invention also encompasses nucleotide sequences Mat are capable of hybridizing to the sequences mat are complementary to sequences presented herein, or any derivative, fragment or defiv thereof.

Tte tenn Complementary also covers nudeotide sequences tot can hybridism to 10 nuoleo" sequent of me coding sequenoo.

The term Bavaria also encompasses sequences that are compbnntary to sequences that are capab e of hyd idising to the nucleotide "quenc" presonbd herein.

15 Preferably, the term variant encompasses sequences that are compbrnentary to sequences that are capable of hydrising under stringent condo (o.g. 6S C and I 0.1xSSC {1xSSC - 0.15 M NaCI, 0.015 Na3 cibab pH 7. 0}) to me nuce sequences pad herein.

20 1 present invention also relates to nuchotido sequences mat can hybrid) " to No I nucleode sequences of the present invention fincluding complemonry Query of i pow presented herein). I The present invention also relates to nudeotido sequences Cat ate conplomentary to 2S sequel that can hybrWise to the nucleoddo sequenc" present invention Excluding complementary sequences of the presented herein).

The Bern Hybridization a- used herein shall indude The process by which a strand of nucleic acid joins wffl a complementary strand through ba" pairing. (Coombe J (1994) 30 Dictionary of Biotechnology, Stockton Pross, New York NY) a. well as He process of amplification as carried out in polyrnerase chain reaction bchnolobe as described in Dieffenbach CW and GS Dvaksler (1995, PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Pbinviow NY).

as Ado induded within the scope of the preasat irn/eron ate polynudeoffdo "quenoos that ate capable of hybridizing to nucbotido sequeno" presonbd horde under conditions

of intermediate to maximal stnagency. HyUndhaffon conditions are based on the melting temperature (Tm) of the nucleic acid binding Compaq as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego CA), and confer a defined astringency' as explained below.

Maximum stringency typically occurs at about Tm-5 C (5 C below Tm of the pr -); high stnngency at about 5 C to 10 C below Tm; intern ediate stringency at about 10 C to 20 C below Tm; and low stringency at about 20 C to 25 C below Tm. As will be understood by Bose of skill in the art, a maximum stringency hybridization can be used to 10 ides or detect identical polynucleotide sequences white an interrnedbb (or low) stringency hybridization can be used to identify or detect similar or related polynuchoffde - sequences. In a prefened aspect, the present invention cowers nucleon sequences that can hybrid)" 15 to the nucleotide sequence of Me present invention under stringent condo (e.. 65 C and 0. 1xSSC).

ENDOGENOUS ENDO-1.^XYLANASE INHIBITOR

zo In one aspect the present invonffon provides an endonous endo+1, xylaneae inhibitor that is obtainable from vat flour.

In our studies, we have found that the inhibitor is a diptide, having a MW of about 40 kDa (as measured by SDS or MS) and that it has a pl of about 8 to about 9.5.

IS In one aspect of the present invention, the inhibitor is in an isolated form and/or in a substantially pure form. Here, the term Isolated means that the inhibitor is not in its nag env ronment 30 Sequence analysis to date has revealed the that the inhibitor has at best one or more of the sequences presented as SEQ ID No. 13, SEQ ID No. 14, SEQ ID No 16, SEQ ID Flo.

18, SEQ ID No. 17, SEQ ID No. 18 and/or SEQ ID No. 19.

Thus, the present invention encompasses an endo-l3 1,4 xylanase inhibitor which as comprises has at best one or more of Me sequences presented as SEQ ID No. 13, SiEQ

ID No. 14, SEQ ID No 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18 and/or SEQ ID No. 19 or a variant, homologw, or fragment thereof.

The term. variant., homologue. or Fragment" in relation to the inhibitor of the present invention include any subsection of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acid from or to the sequence providing the rivulet amino acid sequence has xylanase inhibitory anon, preferably having at least the Mono acidity as an inhibitor that has at best one or more of the sequences presented as SEQ ID No. 13, SEQ ID No. 14, SEQ ID No 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. lo 18 and/or SEQ ID No. 19. In pacubr, the tone 'omologuo con homology Lath respect to uchre and/or function pleading the resultant inhibitor has Mane hhibRory action, preferably haAng at best the same ac0vibr of an inhibitor Mat has at least one or more of the sequences prosenW as SEQ ID No. 13, SEQ ID No. 14, SEQ ID No 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18 armor SEQ ID No. 19. with p to S sequence homology 0.e. sequence similarity or sequar identity), preferably Off k at least 75%, more preferably at least 80%, more preferably at best 85%, mom prothrab y at best 90% homology to the sequence shown in the attached sequer" lists. IUore preferably time at least 85%, more preferably at lent 98%, homology to Me sequence shown in the and sequence lishrp.

20. A putath/e example of a variant of the inhibitor of the present has at East one or more of the sequences presented as SEQ. ID No. 1 and SEQ. IC' No. 2.

The inhibitor aspect of the present invention is advantageous for a number of reasons.

2s By way of example, by now knowing the chemical identity of an ondogenom endo*1,^ Zymase inhibitor workers can now debrrnine the quantity of the inhibitor in, forexernple, a wheat thaw. For convenience, we shall call this method the InhlbTtor Amount Determination Melody.

The Inhibitor Amount Deterrninaffon Method would onabb workers to select one or more appropriate xyianases for addition to the Meat flour and/or select appropriate amounts of one or more xylanasos for addition to the wheat hour.

as Thus, the present invention provWes a method comprised: (9) tiabrminir the amount or type of inhibitor in a wheat flour; (b) socketing a Mumble xylanaso for acididon to wheat

flour and/or selecting a suitable amount of a xylanase for addition to the wheat flour; and (c) adding the suitable xylanase and/or suitable amount of the xylanase to the wheat flour.

The present invention also provides a method comprising: (a) determining the amount or typo of inhibitor in a wheat flour; (b) selecting a suitable xylanase inhibitor for addition to the wheat flour and/or selecting a suitable amount of a xylanase inhibitor for addiffon to the wheat flour; and (e) awing the suitable xylanase inhibitor andlor suitable amount of the xylanase inhibitor to the wheat flour.

10 The present invention also provides a method comprising: (a) determining amount or type of inhibitor in a wheat flour; (b) selecting a suitable xylanase and a suitable xylanase inhibitor for addition to the wheat flour andlor selecting a suitable amount of a xylanase inhibitor for addition to the wheat flour; and (e) adding the suitable xylanase and the suitable xylanase inhibitor and/or suitable amount of the xylanase inhibitor to the wheat IS flour.

Detection of the amount of inhibitor can be determined by standard chemical behniques, such as by analysis of solid state NMR spectra. The amount of inhibitor may even be determined by use of xylanase enzymes that are known to be detrimentally affixed by 20 the inhibitor. In this last aspect, it would be possible to bake a sample of the wheat flour and add it to a known quantity of such a xylanase. At a certain time point the acvTty of the xylanase can be determined, which resultant activity can then be Correlated to an amount of inhibitor in the wheat four.

2S Thus, the present invention also encompasses the use of the combination of a xylanase and the inhibitor as a means to calibrating and/or determining tl quantity of inhibitor in a wheat flow sarnph.

Antibodies to the inhibitor can be used to screen wheat flour samples for the presence of 30 the inhibitor of the present invention. The antibodies may even be used to isolate amounts of the inhibitor from a wheat flour sample.

ASSAy METHODS FOR DETERMINING THE EFFECT OF THE 1.4NASIi INHIBITOR ON DIFFtENT XIANASES There is an additional important use of the inhibitor of the present invention.

S In this respect, the inhibitor could be used in an assay/semen to identify xylanases that are affected by e inhibitor.

By way of example, in some circumstances, it may be desirable to screen for a xybna" 10 mat has a low residence - i.e. ate not that resistant - to the inhibitor.

In one asp -, the inhibitor can be used in an assay/screen to Wenfffy xylana hays a fair (medium) resistance - i.o. are reasonably resistant to inhibitor.

Is In one aspect, the inhibitor can be used in an assay/screen to identify xrianams that ha" a high resistance to the inhibitor.

A suitable Protocol for determining the degree of inhibition by the inhibitor is presented later on. For convenience, we shall call this Protocol Inhibitor Assay Protocol..

Thus, the present invention provides a mood for determining the degree of resistance of a xylanase to a xylanase inhibitor, wherein the method comprise-: (a) contacting a xylanase of interest web the inhibitor; and (b) determining whether the inhibitor inhibits the activity of the xylanase of interest. For convenience, we shall call this Synod the AS Inhibitor Assay Method.

Here, the term 'resistant. means that the activity of the xylanase is not tobily inhibited by the Inhibitor. In other wools, the inhibitor can be used in an assay/scroen to idorfy xylanases that are not detrimentally offend by tl" inhibitor.

Thus, the ban degr" of resistances in relation to the xylanase visvis to xylana" inhibitor is synonymous with the degree of nonnhibWon of the acidity of a xylanase by the xybna# inhibitor. Thus, a 1anase that has a high degree of resistance to the xybnase inhibitor is akin to a high degree of noninhibWon of a xylanase by the xylana" IS inhibitor.

The present invention also encompasses a process comprising the steps of (a) performing the Inhibitor Assay Method; (b) identifying one or more xylanases having a high (or medium or low) degree of resistance to the inhibitor; (c) preparing a quantity of those one or more identified xylanases.

Suitable identified xylanases can then be used to prepare a foodstuff, in particular a dough to make a bakery product In addition, by identifying a xylanase that is resistant to some extent to the inhibitor (i.o. a 10 xylanase that is not inhibited as much as other xylariases), it is possible to add Mu of that identified xylanase to a medium for subsequent ublisaffon thereof. End uses for the xylanases can include any one or more of the preparation of foodstuffs, protein and starch production, paper production and pulp processing etc. IS Thus, the present invention also encompasses a process comprising the steps of: (a) performing the Inhibitor Assay Method; (b) identifying one or more xylanases having a high (or medium or low) degree of resistance to the inhibitor; and (c) preparing a dough comprising the one or more identified xylanas -.

20 In the course of the experiments relating to the present invention, we surprisingly found that bacterial xylanases were ebb to be resistant to the inhibitor, in the sense that their act was not compeletly abolished. In some caves, the xylanases exhibited very favourabh resistance to the inhibitor.

2s ASSAY METHODS FOR DE I tRMINING THE EFFECT OF DIFFERENT XYLANASS IN DOUGHS

Wilson some bacterial xylanases that had been identified as being suitable by the Inhibitor Assay Method were present in a dough m xhro, we surposirly found that the dough 30 mixture was not as sticky as a dough mixture comprising a fungal xylanaso, Thee results were completely unexpected in view of ff - teachings of the prior am Thus, tlm present invention provides a further assay method for identifying a bacterial xylanase or mutant thereof suitable for use in the preparation of a balled foodstuff. 1 -

3S method comprises (a) incorporating a bacterial xylana" of interest in a dough mixture; and (a) Determining the stockings of the resultant dough mixups; such that the bacterial xylansse or mutant Hereof is suitable for use in the preparaffon of a baked foodstuff if

resultant dough mixture has a stickiness that is less than a similar dough mixture comprising a fungal xrlanase. For convenience, we shall call this method the Sffckiness Assay lUlethod..

s Thus, the present invention also provides a process comprisir the steps of: (a) performing the Stickiness Assay Method; (b) idening ore or more xylanases suibbk for use in the preparaffon of a baked foodstuff; (c) preparing a quantity of tho" one or more identffiod qrlanas.

0 A suibbb Protocol for determining the sticlaness of a dough is presented later on. For convenience, we shall call this Protocol the SffckinessProtocol.. In accordance with So present invention a dough comprising a xylana" according to the present inusnffon that is less sticky than a dough comprising a fungal xylaneso may be called, on occassbn, a non- city dough..

IS H a bacterial xylanase shows favourable properly" - in that it does not produce a dough that is as sticky as a dough comprising a fungal xylana" Upon that xylanase may be used to prepare a foodstuff, such as a dough for preparing a balcery product.

20 Thus, the present invention also provides a process comprising the stops of (a) performing the Stickineca Assay Method; (b) ideas - one or more xybroa suitable for use in the preparation of a baked foodshrff; and (c) preparing a dough comprising the one or more Wenfffiod Debases.

2S ASSAY 11 IETHOp$ FORETERMINING THE EFFECT 06 8L=LQ[d DOUGH PROPERTIES FOR DOUGHS THAT MAY C;)MPIE mAA In course of the experiment relating to the present invenffon, we also found that the presence of glucanase enzymes in certain amounts could have a defining effect on the 30 xylanasos.

Thw, in one aspect, it is advantageous not to have detrimental levels of glucana" ermines in the xylanase preparation - such as tt" medium wed to prepare or exact the Manage enyrnes. In addition, for some aspects, it is advantageous not to have 3S detrimental levels of glucanase enzymes in a medium Hat is to be "oaf to prepare a foodstuff which medium will contain the xWana -. Here, term Àdebimonbl 11.

means an amount of glucanase is present such that the benefits from tire xrlanase are masked by the adverse effect of the glucanase enzymes.

Thus, the present invention provides a further assay method for identifying a xylanase S composition (such as a xylanaso preparation) or a medium in which a xylanaso is to be prepared or a medium to which a xylanase is to be added that is to be suitable for use in the preparation of a baked foodstuff, the method comprising (a) providing a composition containing the xylanase of interest or a medium in which the xylanase is to be prepared or a medium to which the xylanase is to be added; and (b) determining the presence of 10 active glucanase ereyme(s) in the composition or medium; such that if there is at most a low level of active glucanaso enzyeT e(q) in the composition or medium tin that composition or medium is suibbb for the preparation of a baked foodstuff. For óonvenbneo, we shall call this method the GIucanase Assay Method..

IS The present invention also prwides a prwess comprising the steps of: (a) performing the Glucanase Assay Method; (b) Wentitnog one or more compositions or mediums suitable for use in the preparaffon of a baked foodstuff; (c) preparing a quantity of those one or more identified composWona or mediums.

20 A suitable Protocol for determining the activity of glucanases is preserved later on. For convenience, we shall call this Protocol the GIucanase Protocol..

If the composition or medium shows favourable properties - in the sense that the beneficial effects associated with the xylanase are not completely masked by the 2s presence of detrimental amounts of glucanase enzymes - then that composition or medium may be used to prepare a foodstuff, preferably dough that is used to make a bakery product À Thus, the present invention also encompasses a process comprising Me steps of: (a) 30 perfonning the Glucanase Assay Method; (b) idenfffinng on. or more idenffled compositions or mediums suitable for use in the preparation of a baked foodshff; and (c) preparing a dough composing Me one or more idend identified compositions or mediums. Is Thus, the present invention covers a xylanase preparation, wherein the xylanase preparation is substantially free of glucanase enzymes)

In this respect, the xylanase preparation can be prepared from an initial preparation from which at least substantially all of the glucanase enzyme(s) that may be present is(are) removed or even wherein the activity of the IJlucanase enzyrno(s) is suppressed or diminated. Techniques for achieving this could include using anffbodbs that recognize s and bind to the glucanase enzyme(s) and in doing so inactive the achier of the glucanaso enzyme(s). Altematively, glucanase enzyme(s) specific antibodies could be bound to a support such that passe" of the initial preparation past the bound antibodies would result in the glucanase enzyme(s) being remowd from K thereby forming a xylanase preparation being substantially free of glucanase enzyrno(s). In an aRema 10 embodiment, or even in an additional embodiment, the xylanase preparation can be prepared from a host organism that has minimal or no glucanase enzyme acthmy. In this aspect, the activity of the glucanase enzymes that are present in the host organism may be inacvabd. In an albmatiw aspect, the expression of the glucana" gongs can be silenced and/or knocked-out. Techniques for achieving this could include up ariense 5 sequences to the glucanase coding sequenc -. In a further embodiment, a host organism is used that has no or at most minimal expression of glucanase enzymes.

15 ASSAY

do In some cases, measurement of the value of a xrlanase (which we call here a K, assay.) may be useful. In this respect, we have found that in some cases the K, value is sometimes indicative of the suitability of the xylanase for certain applicaffon().

Knowledpo of the 1< value could be useful on its own.

2S QMBInON A,SSAY The present invention also encompasses suitable combinations of the assays of the present invention.

30 In this respect, the present invention indudes a combination method comprising two or more of the following sops: a step comprising the Inhibitor Amount Detcrminaffon Method, a step comprising the Inhibitor Assay Method, a step comprising the Sticidness Assay Method; a step comprising the Glucana" Assay Method; and a step comprising the assay. In combination method, the steps can occur in any order and need not 3S necessarily occur simulatancoualy or COAT.

NOVEL XYLANASE

As indicated above, the preserd invention provides a suitable assay for identifying xylanases that can be used in the preparation of foodstuffs, in particular doughs for me in S the preparation of bakery products.

In this respect, we have Wenffied tier" new xylanases that are suitable for the preparation of foodstuffs, in particular doughs for U80 in the prepaon of baked prods. SO Thus, the present invention also includes an amino sad sequence comprising any one of the amino acid sequences presented as SEQ ID No. 7, SEQ ID No. or SEQ ID No. 11, or a variant, homologuo or *segment thereof.

15 The temns Nevadan,, hornolow" or "fragment. in relation to Uxylaneso of the present nvendon Delude any subsffluffon of, variation of, modification of, rophcornont of, destain of or Addison of one (or more) amino acid from or to the sequence providing the resultant amino acid sequence has xylana activity, preferably haven at least ff-same act composing any one of the amino acid sequences presented as SEQ ID No. 7, SEQ ID 20 No. or SEQ ID No. 11. In Palomar, the term homologuo" covem homology with r to wu and/or function prov don the resultant protein has xWanaso ac-, pub y at bad same activity of any one of the amino acid sequence presumed SEQ ID No. 7, SEQ ID No or SEQ ID No. 11. With mspect to sequence homology 0.o.

sequence lumbar ty or "quenec ider-), prefey there b at hast 75%, more proforab y 25 at 1-st 8556, more preferably at best 90% homology to the sequin shown in the atactd sequence Ups. More Ably there at best 95%, mom prably at best 98%, homology to sequence shown in the attuned sequence listings.

Preferably, the lanaso comprises the sequence presented as SEQ ID No. 7 or SEQ ID 30 No. 11, or a variart, homologuo orfragmont tteraof.

The present invention also encompasses a nucleotido sequence encoding the amino acid sequence Tithe present invonffon.

as Preferably, nucodde sequence of the present invention seemed from:

(a) a nuchodde sequenec comprisTng any ono of the nuebodo sequencos presented as SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No. 12, or a variant, homologuo ar fragment theroof; s (b) any ono of tho nucleotWe aequences preaenbd as SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No. 12, or the complemont of; (c) a nuclootida sequence capablo of hdising any one of thc nusboti" sequencos presentod as SEQ ID No. 8, SEQ lD No. 10 or SEQ ID No 12' or a fragmont 10 of; (d) a nuchodde aequence capable of hybridbing to o comp ana ono of tho nudeodde sequenoes pnesenbd as SEQ ID No.8, SEO ID No.10 orSEQ ID No. 12,ora Sagmontthenok and (o) a nuclootido seq w nce vhichis degenorate as a rastcithe gnotic oode totho nuddotid4e donedin(a), Jb), (c)or(c).

Tho tenn4 vadan, 1homohw. or 1Yagmon in neNdon to the nuK*soad À quano cf tho o prnt innffon include any suhon of, variation of, modcn of, q of, delotion of or aWition of one (or more) nucbic acid tm or to tho noe provWho tho resul nucbotido seqwnce ood for an amino acid soclwnoo has xyl_o activHy, pferably having at best tho samo acr comprising any ono of tho ambno add sequencos pneaented as SEQ ID No. 7, SEQ ID No. 9 or SEQ ID No. 11. In r, tho 2s ban homologuo covers homology wffl respect to struc and/or fion provi resultant oxprd ptsin has xylar activity, prably at b mo acy of an one of tho amino acid sequences presentod as SEQ ID No. 7, SEQ ID No. or SEQ ID NQ 11. WW respect to quenoo homoby (i.e. quenoe SiT or).

preferably ere Is d best 75%, mom prehrably at best 85%. more py d best 9096 30 homology to sequence stn as SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No. 12 in o attached sequence ltirs. Moro preferably shore is at lt 95%, moro prefomby at best 98%, homology to e asquence stwwn in the ad seqwoo. F. Preferably, tho nudootide aquenos of o prnt inwnffon compr soquenoe 3s psacnW as SEQ ID No. 8 or SEQ ID No. 12, or a varbat. honwhuo or ffrnont thof

l9 NOVEL USES OF XYLANASES

As indicated above, the present invention also provides a suitable assay for identifying xylanases that can be used in the preparation of nonsticky doughs (as defined herein) for S use in the preparation of bakery products.

In this respect, we have idenffled certain xylanases, both known and new bacterial xylanases, that are suitable for the preparation of foodstuffs, in particular doggie for use in the preparation of bakery products.

Thus, the present invention covers a non-sticky dough (as herein defined) which dough comprises a xylanase identifiable by the assay of the present invention. Preferably, xylanase has an amino acid sequence presented as any one of SEQ ID No.s 3, 5, 7, 9, 11, or a variant, derivative or homologue thereof. More preferably, the xylanasc has an amino IS acid sequence presented as arty one of SEQ ID No.s 5, 7, 9, 11, or a variant, derivative or homologue thereof.

In contrast to U,e prior art systems, the present invention provides for the possibility of me

addition of xylanase directly to flour prior to production of the dough. Thus, a single batch 20 a flour/xylanase mixture may be delivered to the dough producer. Moreover, the dough producer does not require dosing equipment to be abh to obtain a readily handabh dough. FOODSTUFFS PREPAREI:) WITH XYLANASES

IS Clue present invention provides a means of identifying suitable xylanams for use in the manufacture of a foodstuff. Typical foodstuff, which also include animal feed, include dairy products, meat products, poultry products, fish products and bakery products.

10 Preferably, the foodstuff is a bakery product Typical bakery (baked) products incorporated within the scope of the present invention include bread - such as loaves, rolls, buns, pee bases etc. - pretzels, tortillas, calves, cookies, biscuits, crackers etc.

GENERAL TEACHINGS

In the following commentary references to nucleotide sequence of the present invention' and Amino acid sequence of the present invention. refer respectively to any one or more s of the nucleotide sequences presented herein and to any one or more of the amino acid sequences present heran.

Amino Acid Seauence/PoipeDtide Sequence lo The term Amino acid sequence of the present ieion. is synonyrnow with the phi polypepffde sequence of the present invention'. Here, the amino acid sequence may be that for the xybnaso or the xylanaso inhibitor.

Polypep of Ho present invention ago include fragments of the presented amino acid IS sequence and variants thereof. Suitable fragments mill be at roast 5, e.g. at least 10, 12, 15 or 20 amino acids in sko.

Polypaptides of the present invention may also be modified to contain on. or more beg.

d bet 2, 3, 5, or 10) substitutions, demons or insertions, includir conserved 20 sulked Conserved substitutions may be made according to Me following table which indicates corrvadve substitutions, whore amino acids on Do same bionic in the second column and preferably in the same line in the third column may be substituted for each other.

AUPHAnC Non-pobr G A P ILV 1 Poor- uncharged C S T M l Polar- charged K R AROMATIC H F W Y

OTHER _ N Q D E.

Pdypaptidos of present invention may be in a substantially isolated fonn. It will be understood that the polypeptide may be mind wffl, carriers or diluents which wTII not hbttere with the intended pUIpOSB of the pdypopffdo and sell be a as 30 substanJaly isolated. A polypopffde of the pedant invention may also be in a substanffaNy purified foml, In which cam it will generally comprise the polypop in a

2l preparation in which more than 90%, e.g. 95%, 98% or 99% of the polypeptide in the preparation is a polypepUde of the present invention. Polypeptides of the present invention may be modified for example by the addition of histidine residues to assist their purification or by the addition of a signal sequence to promote their secretion from a cdl s as discussed bdow.

Polypeptides of the present invention may be produced by synthetic moans (e.. as described by Geysen et at., 1996) or recombinantly, as described below.

10 The we of suitable host cells - such as yeast, fungal and Pliant host cells - may provide for such post-translational modifications (em. myristolaffon, glycosylation, truncation, lapidaffon and tyrosine, serino or threonine phosphorylation) as may be ridded to confer optimal biological activity on recombinant expression products of the present invention.

Nucleotide Seauence/Pdvnucleotide Sequence The term nudeotide sequence of the present invention' is synonymous ninth the phrase polynudeotide sequence of the present irnntion'.

20 Polynucisotid" of the present invention include nuclootide acid sequences encoding the polypeptidos of the present invention. It will appredaW that a range of different poiynucleoVdes encode a given amino acid sequence as a consequence of the degeneracy of the genetic code.

as By knowledge of the amino acid sequences set out herein it is posse to devise partial and fulbiength nucleic acid sequenced such as cDNA and/or genomie clones that encode the polypepffdes of the present invention. Fw example, pobnucleotides of the present invention may be obtained unwire degenerate PCR which will use primers designed to target sequences encoding the amino acid sequences presented herein. The primers will 30 typicalb contain mcitiple degenerate positions. However, to rninimise degeneracy, sequences will be chosen that encode regions of the amino acid sequences presented herein cortaining amino adds such as methionine which are coded for by only one triplet.

In addition, sequences will be chosen to take into account codon usage in tt. organarn whose nuciek acid is used as the template DNA for the PCR procedure. PCR will be 3S used at stringency conditions lower than those used for cloning sequences with single sequence (nondorerate) primers against known sequ8.

Nucleic acid sequences obtained by PCR that encode polypeptide fragments of tt.

present invention may then be used to obtain larger sequences using hybridization library screening techniques. For example a PCR clone may be labelled with radioac atoms and used to screen a cDNA or genomic library from other species, preferably our plant s species or fungal specks. Hybridization conditions will typically be conditions of medium to high stringency (for example 0.03M sodium chloride and 0.03M sodium citrate at from about 50 C to about 60 C).

Degenerate nucleic acid probes encoding all or part of the amino acid sequence may also 10 be used to probe cDNA and/or genomic libraries from other spectra, preferably other plant species or fungal species. However, it is preferred to carry out PCR technique initially to obtain a singe acquence for use in furs - r screening procedures.

Polynucleoffde sequences Of the present invention obtained using the techniques IS described above may be used to obtain further homologous sequences and variants using the techniques described above. Coy may also be modified for use in expreasing the polypeptides of the present invention in a varieW of host cells systems, for example to optimise cadon preferences for a particular host cell in which the polyoucleotide sequences are being expressed. Other sequence changes may be desired in order to 20 introduce restricken enzyme recognition sites, or to alter the property or function of the polypepffdes encoded by the polynucleotidos.

Polynucleotides of the present invention may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplNicatbn reaction, a probe e.. Iabelled with a 25 revealing label by conventional means using radioactive or nonradioactive labels, or the polynudeotides may be Coned into vectors. Such primers, probes and other frnents will be at least 15, preferably at least 20, for example at best 25, 30 or 40 nucleotides in length, and are also encompassed by Me term polynucleotides of the present invention as wed herein.

Polynucleotides or primers of the present invention may carry a revealing labs. Suitable labels include radioisotopes such as 82p or 35S, enzyme labels, or odor proton labels suah as biotin. Such labels may be added to polyoudeotides or primers of present invention and may be detected using by techniques known per -.

as

Polynucisotides such as a DNA polynucleoffde and primers according to tot present invention may be produced recombinantly, synthetically, or by any remans available to those of skill in the art. They may also be cloned by standard techniques.

S In general, primers will be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nudeotide at a time. Techniqu" for accomplishing this using automated techniques are readily available in the art Longer polynucleoWes will generally be produced using recombinant means, for example.

lo using a PCR (polyrnereae chain reaction) cdoning techniques. This will involve mailing a pair of primers (e.g. of about 130 nudeotides) to a region of the enJo-1,4-xybn inhibitor gene which it is desired to done, bringing the primers into contact with mRNA or cDNA obtained from a fungal, plant or prokaryotic cell, performing a polymorase chain reaction under conditions which bring about amplification of the desired region, isolating Is the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified GINA. The primers may be designed to contain suitable restriction eryme recognition sees so that the amplified DNA can be cloned into a auibble cloning vector.

20 E3eoulatorv Sequences Preferably, the polynucleotide of the present invention is operably libeled to a regulatory sequence which is capable of providing for the expression of Me coding sequence, such as by the chosen host cell. By way of example, the present invention covers a vector US comprising the polynucleotide of the present invention operably linked to such a regulatory sequence, i.e. the vector is an expression vector.

The tem, Operably linked. refers to a juxtaposition wherein tie component described are in a relet onship permitting them to function in their intended manurer. A regulatory 30 sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with tl. control sequences. The term Regulatory sequences includes promoters and enhancers and our exprmsJon 3S reduction signals.

The term promoter. is used in the normal sense of the art, e.g. an RNA polymerase binding sib. Enhanced expression of the polynucleotide encoding the polypeptide of the present invention may also be achieved by the selection of hebrotogous regulatory regions, e.g. promoter, secretion leader and terminator regions, which serve to increase expression and, if desired, secretion levels of the protein of interest from the chosen expression host andlor to provide for the indudbb control of the expression of the polypeptide of the present invonffon Preferably, the nucteotide sequence of the present invention may be operably linked to at bet a promoter.

Aside from the promoter native to the gene encoding the polypeptide of the present IS invention, other promoters may be used to direct expression of the polypeptide of the present invention. The promoter may be selected for its efficbacy in directing the expression of the polypeptide of the present invention in the desired expression hod In another embodiment, a constitutive promoter may be sebeted to direct the expression 20 of the desired polypeptide of the present invention. Such an expression construct may provide additional advantages since it circumvents the need to culture the expression hosts on a medium containing an inducing subseab.

Examples of strong constitutive and/or inducible promoters which are preferred for use in 2S fungal expression hosts are those which are obtainable from the fungal genes for xylanase (xinA), phytase, ATPsynthebae, subunit 9 (oliC), otiose phosphate isomers" (4D0, alcohol dehydrogenase (AdM), -amylase (amp, amyloglucosida" (AG- from the pad ger -), acetamidase (amdE;) and glyceraldehyd - phosphabc dehydrogenase (gem promoters. Examples of strong yeast promoters are those obtainable from the gen" for alcohol dehydrogenase, lactase, phosphoglycerate hnase and triosephosphate isomers -.

Examples of strong bacterial promoters are the a-amylase and SPO2 prornoRrs as well as as promoters from exbcelluhr propane gen-.

IS Hybrid promoters may also be used to improve inducible regulation of Me expression construct The promoter can additionally include features to ensure or to increase expression h a suitable host. For example, the features can be conserved regions such as a PriLnow Elex or a TATA bow The promoter may even contain other sequences to affect (such as to maintain, enhance, decrease) the levels of expression of the nucleotide sequence of tl.

present invention. For example, suitable other sequences include the Sh1intron or an ADH intros. Over sequences include inducible elements - such as temperature, chemical, light 10 or stress inducible elements. AJso, suitable elements to enhance transaipffon or hanalaffon may be present An example of the latter element is the TMV 5 signal sequence (see Sleat Gene 217 11987, 217-225; and Dawson Plant Mo. Biol. 23 [1993197).

Secretion IS Often, it is desirable for the polypeptide of the present invention to be secreted Mom the expression host into the culture medium from where the polypeptide of the present invention may be more easily recovered. According to the present invonffon, the secretion leader sequence may be affected on the basis of the desired expression host.

20 Hybrid signal sequences may also be used wffl the context of Do present invention.

Typical examples of heterologous secretion leader sequences are those originating from the fungal amyloglumsidase (AG) gene (g/aA - both 18 and 24 amino add versions e.g. from Aspergillus), the a-factor gene (yeasts e. g. Saccharomyces and K7vmrryces) or 2s the a-amyla" gene (Bacillus).

Corm The term "constructs - which is synonymous web terms such as conjugate-, " - etch and 30 Alp- includes the nucleotide sequence according to the present inversion dirt or indirectly attached to a promoter. An example of an indimct affaclment the provision of a suitable spacer group such as an intron sequence, such as the Shin or the ADH intron, intermediate the promoter and the nudeotide sequence of the present Munson.

The same is two for the term Fused in relaffon to the present invention which induebs 3S direct or indirect attachrner In each case, the terms do not cover tl. natural combination

of the nucleotide sequence coding for the protein ordinarily associated with the Mid type gene promoter and when they are both in their natural environment Else construct may even contain or express a marker which allows for the sebction of to s genetic construct in, for example, a bacterium, preferably of the genus Bacillus, such as Bedlus sublets, or plants, such as potatoes, sugar beet Tic, into which It has been transferred. Various markers exist which may be used, such as for example those encoding mannose phosphate isomerase (especially for plants) or those markers that provide for antibiotic resistance - e.g. resistance to G418, hygromycin, bleomycin, kanamycin and 10 gen - Am. Preferably the construct of the present invention composes at least the nudeotide sequence of the present inversion operably linked to a promoter.

IS Vet The term Rectors inch des expression vectors and transformation vectors and shuttle Cot. zo The tend Expression vectors means a construct capable of e, To or An expression.

The term Retransformation vector' means a conduct capabis of being transferred from one entry to another entity - which may be of the species or may be of a different species. new correct is capable of being bansford from one specie to another - such as from an as Ecus plasmid to a bactenum, preferably of the genus Bacillus, then the tranddrmabon vector someffrnes called a Shuttle vectors. It may even be a construct capable of beL transferred from an decals plasmid to an Agrobacterhm to a pore The vectors of the present invention may be transferred into a suitable host coil as 30 described below to provide for expression of a polypepffde of the present invention. Thus, in a furler aspect the invention provides a process for preparing polypeptides accordir to the present invention which comprises cultivating a host cell transformed or transfeebd with an expression vector as described above under conditions to provide for expression by Me vector of a coding sequence encoding polypeptides, and rnr the as exprd polypeptides.

The vectors may be for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter.

s The vectors of the present invention may contain one or more selectable marker gen -.

The most suitable selection systems for industrial micro-organisms are those formed by the group of selection markers which do not require a mutation in the host organism.

Examples of fungal selection markers are the genes for acebmida" (amd!;), ATP synthetase, subunit 9 (oliC), oroffdine-5'-phosphateiecarboxylase If), phbomycin 10 and benomyl resistance (benA). Examples of non-fungal selection mars are bacterial G418 resistance gene (this may also be used in yeast, but not in fungi), the ampicillin resistance germ (E cow, the neomycin resistance gene (Bacillus) and the E.colf up" gone, coding her glucuronidase (GUS).

lS Vectors may be used in v -, for example for the production of RNA or used to transfect or transform a host cell.

Thus, polynudeotides of the present invention can be incorporated into a recombinant vector (typically a replicable vector), for example a cloning or oxpreadon vector. TO 20 vector may be used to replicate the nucleic acid in a compatible host cdl. Thus in a furor embodiment, the invention provides a method of making polynuchotides of U. . present invention by introducing a polynucleotide of the present invention into a replicabbvector, introducing the vector into a compatible host cell, and growing the host cell under conditions wt ich bring about replication of the vector. The vector may be recovered from as the host cell. Suitable host cells are described below in connection Lath expression vectors r;8u. 30 The term Tissue" as used herein includes tissue per se and organ.

Host Cd.

The Ann Host cell. - in relation to the present inventor includes any cell that owed 3S comprise the nucleoffde sequence coding for the recombinant protein according to present invention and/or products obtained therefrom, wherein a premolar can allow

expression of the nudeotide sequence according to the present invention when present in the host cdl.

Thus, a further embodiment of the present invention provides host celk transformed or transfected with a polynucleotide of the present invention. Preferably said polynucleo is carried in a vector for the replication and expression of said polynucleotidos. The cell.

will be chosen to be compatible with the said vector and may for example be prokayotic (for example bactenal), fungal, yeast or plant coils.

10 The gramnegative bacterium E cold is Widely used as a host for iebrologous "no expression. However, large amounts of heterologous protein tend to accumulate inside the cell. Subsequent purification of the desired protein from the bulk of E.coll intracellular proteins can sometimes he difficult.

IS In contrast to Scow, bacteria from the genus Bac'71us are very suitable as heterologous hosts because of their capability to secrete proteins ink the culture medium. OU - r bacteria suitable as hosts are those from the genera S - ptomyoes and Pseudomonas.

Depending on the nature of the polynucisotide encoding the polypepffde of the present 20 invention, andlor the desirability for further processing of the expressed protein, oukaryotic hosts such as yeasts or fungi may be preferred. In general, yeast cells are preferred over fungal celk because they are easier to manipulate. However, some proteins are either poorly secreted from the yeast cell, or in some eaves are not processed properly (e.. hyperglyeosylation in yeast). In these instances, a fungal host 2s organism should be solmted.

Examples of preferred expression hosts within the scope of Me present invention are fungi such as Aspergnius species (such as Hose described in EPA0184438 and 1EP-A 0284603) and Trichodems species; bacteria such as B. - flus species (such as those 30 described in EP-A-0134048 and EP-A253455) , Septomyces sums and Pseudomonas species; and yeasts such as Kluyveroms specie (such as tho" described in EP-A-0096430 and EP-A-0301670) and S xhammyces specks.

Typical expression hosts may be selected from AspergJllus niger, As, oerfilus Niger var.

3S tubbenis, AsperJ5lus near var. awamori. Asperglus access, Aspergus nlvJans,

Asperil/us orvzee, Tnchoderma reesei, Bacillus subtilis, Bacillus lichenifonnis, Bacillas amyloijquefaciens, Klayveromyces lactis and Saccharomyces cerevisiae.

Organism The krrn "organism. in relation to the present invention includes any organism Mat cou d comprise the nucleoffde sequence coding for the recombinant protein accordir to the present invention and/or products obtained therefrom, wherein a prnoler can allow expression of the nucteotide sequence according to the present invention when present in lo the organism. For the xianaso inhibitor aspect of the present invention, preferable organisms may include a fugue, yeast or a plans For the xianase aspect of present invention, a preferabe organism may be a bacterium, preferably of the genus Bactilua, more preferably Bacons babes.

Is The term transpenic organism. in relation to the present invention Indudes any organic that composes We nudeoffde sequence coding for the protein accolade to ff1o present invention tinder products obtained therefrom, wherein the promoter can allow expression of the nucleotide sequence according to the present invention unpin me organism. Prefeab y the nucleotide sequence is incorporated h the gonorne of the organism.

The term ansenic organism" does not cover the nature nucleoffdo coding aequor according to the present invention in its natural environment when it is ureter the corm of its native promoter which is also in its natural environment In addition, the present invention does not cover the native protein according to the present invention when it is in its as natural environment and when it has been expressed by its native ne Cody sequence wt Ah is also in its natural Environment and when mat nucleotide sequence is under the control of its native promoter Rich is also in its natural environment.

Therefore, the transpenic organism of the present invention includes an organian 30 composing any one of, or combinations of, the nucieoffde sequence coding for amino acid sequence according to the present invention, constructs according to the prowar.

invention (including combinations thereof,, vectors acting to the prt invention, plasmids according to the present invention, cells according to the present invoron, tissue according to me present invention or to products thereof. The ttansforrnod cdl or 3S orgarusm could prepare acceptable quantities of the desired compound which wand be easily retrievable from, the cell or organists.

Transfonnation of Host Cells/Host Organism.

As indicated earlier, the host organism can be a prokaryotic or a eukaryotic organism.

Examples of suitable prokayotic hosts include E cob and Beaus sambas. Tearings on s the transtonnation of prokaryotic hosts is wolf documented in the art, for ample see Sambrook et al (Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cow Spring Harbor Laboratory Press) and Ausubol et a/., Current Protocols in Molecular Biology (1995), John VVihy 8 Sons, Inn to If a pmkaryoc host is used then the nucleotide sequence may need to be suitably modified before kansforrnatbn - such as by removal of inborn As mentioned above, a preferred host organism is of the genus Bacillus, such as BeclBus sublets. In another embodiment the transpenic organism can be a yeast In this rewed, yeast have ado been widely used as a vehicle for heterologous yens expression. The species Sacchuornces crews-has a brig history of industrial use, include its use for heterologous gene expression. E=wsion of heterologous Arm in Sacchararnyces 20 cerevisiae has been red by Gooday et al (1987, Yeast Biotechnology, D R Betty et al, ads, pp 401 429, Allen and Unwin, London) and by King et al (198B, Molar and Cell Biology of Yeasts, E F Walton and G T Yarronton, eds, pp 107-133, Blackie, Glasgow).

For several reasons Sacchermnyces cereuisiae is well suited for homologous gene 2S expression First, it is nonathogenic to humans and it is incapable of producing certain endotoxins. Second, it has a long history of saw use follows censures of comer exploitation for various purposes. This has led to wee public accoptabili. Tl-, the extending commercial US6 and research devoted to the organism has resulted in a wed of know edge about the genetics and physiology as well as larscab fermentation 30 characteristics of Secchnyces cerevJse.

A review of He principles of heterologous gene expression In Saccharomyces oarwis and secretion of gene products is ghen by E HinQhcime E Kenny (1893, "Yeast as a vehicle for. the expression of heterobous gones-, Yeasts, Vol 5, Anthony H R-o md 3S J Stuart Hamson, ads, End Edison, Acaderok Press ltd.).

Several types of yeast vectors are available, including integrative vectors, which require recombination with the host genome for their maintenance, and autonomously replicating plasmid vectors.

5 In order to prepare the transgenic Saccharomyces, expression construct are prepared by inserting the nucleotde sequence of the present invention into a construct designed for expression in yeast Several types of constructs used for heterologous expression have been developed. The constructs contain a promoter active in yeast fused to the nudeotide sequence of the present invention, usually a promoter of yeast origin, such as the GAL1 lo promoter, is used. Usually a signal sequence of yeast origin, such as the sequence encoding the SUC2 signal peptize, is used. A terminator acme in yeast ends expression system.

For the transformation of yeast several transforrnabon protocols have been developed. For IS example, a transgenic Saccharomyces according to the present invention can be prepared by following the teachings of Hinrmn et al (1978, Proceedings of the National Academy of Sciences of the USA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Its, H et al (1983, J Bacteriology 153, 16168).

20 Else tansforrned yeast cells are selected using venous selective maricus. Amonl, the mariners used for transfommaffon are a number of auxotrophic markers such as LEU2, HIS4 and TRP1, and dominant antibiotic resistance markers such as aminoglycosWe anffbiodc markers, eg G418.

Is Another host organism is a Want The basic principle in the cony of genetically modified plants k to insert geneffc information in the plant genome so as to obtain a stabs maintenance of the inserted genetic material.

Several techniques exist for inserting the genetic infonnaffon, the two main principles being 30 direct introduction of the genetic infomnaffon and introduction of the genetic information by

use of a vector system. A review of the general techniques may be found h aria by Potrykw (Annu Rev Plant Physiol Plant Mol Biol 119911 42:20225) and Chfistou (prom Foodlndustry Hi-Tech MaraApril 1994 17-27).

35 Thus, in one aspect, the present invent on relates to a vector system which Cal' - a nucleotide sequence or cons according to the parent invention and capable of

introducing the nucleotide sequence or construct into the genome of an organism, such as a plant The vector system may comprise one vector, but it can compose two vectors. In the cam of S too vectors, the vector system is normally referred to as a binary vector An. Binary vector systems are described in furler detail in Gynheung An et al. (1980), Binary \lectom, Plant Molecular Biology Manual As, 1-19.

One extensively employed system for transforrnabon of plant cells wffl a goon nuebobd 10 sequence is based on the use of a n plasmid from Agmbacterlum turns or a Hi plasmidlm Agrobacterium rhizogenes An et al. (1986), Plant Physiol. 81, 301 and Butcher D.N. et al. (1980), Tissue Culture Moffods for Plant Paohid#, oafs.: AS.

Ingrains and J.P. Hebewn, 203208.

IS Severe deferent Ti and Hi plasmids have been conducted which are suitable for construction of the plant or plant cell constructs described above. A norlimiting ernph o such a Ti plaamid is pGV3850.

The nucleotide sequence or construct of the present inventor should preferably be inserted 20 into the plasmid between the Carnival asqueres of the TUNA or adjacent a T-DNA sequence so as to avow disruption of the sequences immediately surrounding TO borders, as at least one of these regions appear to be essential for insertion of modified T-

DNA into plant genomo.

as As In be understood horn the above explanation, if the organism is a plant then the vector system of the present invention is preferabb, one whim contains the "qwnces nectary to infect the plant.. the vir region) and at least one border part of a TUNA sequence, It border part being located on the same vector as the genetic construct inferably, vector system is an Agrobacterium tumefaciens plasmid or an Agrobactm rhkq 30 RiplasmW or a derivative thereof, as these plasmids are wellknown and widely employed in she construction of hanagenic plants, many vector systems exist which are based on these plasmids or derivatives Of.

In the consbuction of a banspenk plant tile nucleotide sequent orconstnuct of the present S invention may be first constructed in a microorganism in which He vector can replicate and which is easy to manipulate before insertion into the pbnL An example d a Ohs rnicroorganian is E. cap., but our microorganisms having above prop-es may be

used. When a vector of a vector system as defined above has been constructed in . coli. it is transferred, if necessary, into a suitable Agrobacterium strain, e.g. Agrobacterium tumefaciens. The Tplasmid harbouring the nucleoffde sequence or construe of the present invention is thus preferably transferred into a suitable Agrobactorum strain, e.. A tumefaciens, so as to obtain an Agrobactenum cell harbounn9 the nudeotide asquenco or construct of the present invention, which DNA is subsequery transferred into the plant cell to be modified.

In this way, tle nucleotide or construct of the present invention can be introduced into a 10 suitable restriction position in the vector. The contained plasmid is used for the transformation in tacos. The Ecoli cells are cultivated in a suitable nutrient medium and then harvested and Iysed. The plastnid is then recovered. As a method of analyze them is generally used sequence analysis, restriction analysis, ebckophoris and Ardor biochemicalnolecular biological methods. After each manipulation, the used DNA IS sequence can be restricted and connected with the next DIVA sequence. Each sequence can be Coned in the same or deferent plasmid.

After each introduction method of the desired nucleotide sequence according to the present

invention in the plants the presence andlor insertion of furler DNA sequences may he zo necessary. If, for example, for the transformation the T or Ri plasmid of the plant odds is used, at least the right boundary and often however the right and tit Oft boundary of Off To and Ri-plasmid T-DNA, as flanking areas of the introduced genes, can be connected. The use of T-DNA for the transformation of plant cells has been intensively studied send is desalbed in EP-A-120516; Hoekema, in: The Binary Plant Vector System Offsetdnuk :5 Kanters B.B., Alblasserdam, 1985, Chapter V; Fraley, et al., Crib Rev. PLant Sci., 4:1-46; and An et al., EM80.1. t1985) 4:277-284.

Direct infection of plant tssues by Agrobacterium is a simple technique which has been widely employed and which is described in Butcher D.N. et al. (1980), Tissue Culture 30 Methods for Plant Pathologists, eds.: D.S. Ingrams and J.P. Helgeson, 20208. For furff'or teachings on this topic see Potrykus (Annu Rev Plant Physiol Plant Mol Biol 11991] 42:2 -

225) arm Christou (AgroPood-lndustry Hi-Tech March/April 1994 17-27). AH this technique, infection of a plant may be done on a certain part or Ussw of the plant, i.e. on a part of a leaf, a root, a stern or another part of the plant 3S Typically, win direct infection of plant tissues by Agrobacterium carOnag nudeoffde sequence, a plant to be infected Is wounded, e.g. by a'ffing the plant with a razor or

puncturing the plant with a needle or rubbing the plant with an abrasive. The wound is then inoculated With the Agrobacterium. The inoculated plant or plant part is then grown on a suitable culture medium and allowed to develop ink mature plants.

When plant cells are constructed, these cells may be grown and maintained in accordance with wellknown tissue culturing methods such as by culturing the cells in a suitable culture medium supplied win the necessary growth factors such as amino acids, plant hormones, vitamins, etc. Regeneration of the transformed cells into genetically modified plants may be accomplished using known methods for the regeneration of plants from c H or tissue 10 cultures, for example by selecting transformed shoots using an arbbffc and by subculturing the shoots on a medium containing the appropriate nutr;ier -, plant hormones, Tie Further teachings on plant transformation may be found in EP - 0449375.

Production of the PolvDegffde According to the present invention, the production of the polypeptide of the present invention can be effected by the culturing of, for example, microbial expression hosts, 20 which have been transformed ninth one or more polynucleoffdes of the present invenffon, in a conventional nutrient fermentation medium. The selection of the appropriate medium may be based on the choice of expression hosts andlor based on the regulatory requirements of the expression constnucL Such media are well Icnown to those skilled in the art The medium may, If desired, contain additional composers favouring the as transformed expression hosts over other potentially conbminaffng microorganisms.

Artibos We amino acid sequence of the present invention can also be used to general 30 antibodies - such as by use of standard techniques - against me amino add sequence.

For the production of antibodies, various hosts induding goats, rabbits, rats, mice, -.

may be immunized by injection with the inhibitor or any portion, variant, homologue, figment of derivative thereof or olopepffde whist' repins immunogenic properUes.

Depending on the host species, venous adjwants may be used to incro 3S immunological response. Such adJuvants indude, but are not limited to, Fround%, mineral gem such as aluminium hydrodde, and surface acthsubstances such as It, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemonin, and

3S dinitrophenol. BCG (Bacilli Calmett - Guerin) and Corynebactenvm pablum are potentially useful human adjuvants which may be employed.

Monodonal antibodies to the amino acid sequence may be even prepared using any s technique which provides for the production of antibody molecules by continuous cell firms in culture. These include, but are not limited to, the hybridoma technique originally described by Koehler and Milstein (1975 Naturs 256:49S-497), the human Bowl hybridoma technique (Kosbor et a/ (1983) Immunol Today 4:72; Cote et a/ (1983) Proc Natl Acad Sal 80:2026-2030) and the EBV-hybridoma technique (Cob et a1(1985) 10 Monoclonal Antibodies and Cancer Therapy, Alan R Use Inc. pp 77-96). In addildon, techniques developed for the production of "chimenc anffbodbsn, the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can-be used (Morrison et a1(1984) Proc Natl Acad Sc 81:6851855; Neuberger et al (1984) Nahum 312:604608; Takeda et a/ (1985) Nature S 314:45254). Altematively, techniques described for the production of single chain antibodies (US-A - 946779J can be adapted to produce inhibitor specific single chain anffbodiea. Antibodies may also be produced by inducing in veto production in Me lymphocyte 20 population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et s1(1989, Proc Natl Acad Sci 86: 3833-

3837), and Winter G and Milstein C (1991; Nature 349:293-299).

*PROTOCOLS

PROTOCOL

s. XYLANASE ASSAY (Endo-1,4Xyhnaso ace -) Xylanase samples are diluted in citric acid (0.11\11) - dsodiumydrogen phosphate (0.2M) buffer, pH 5.0, to obtain approx. ED = 0.7 in the final assay. Three dilutions of tt.

10 sample and an internal standard with a defined activity are thermostated for 5 minus" at 40 C. To time = 5 minutes, 1 Xylazyrne tab (crosslinked, dyed xylan substrate) is added to the enzyme solution. To time Is 18 minutes the reaction is terminated, by adding 10 ml of 2% TRIS. The reaction mixture is centrifuged and tt" OC' of the supamatant is measured at 590nm. Taking into account the dilutions and the amount of xylanaso, the 1' acth/ity (TXU, TotaFXylanaseUnits) of the sample can be calculated relatively to the standard.

PROTOCOL 2

STICKINESS PROTOCOL

(Stickiness Determination) S Dough stickiness is measured on a TA-XT2 system (Stable Micro Systems) using a SMS Dough Stickiness Cell, The protocol is a modified version of the method described by Chen and Hoseney (199B). A dough is made from flour, 2% NaCI and water to 400 Brabender Units (BU) using a Farinograph (MCC method 54-21). The flour and NaCI is 10 dry mixed for 1 minute. Water is added and the dough is mixed for another 5 minus -.

The obtained dough could advantageously be rested for 10, 30 or 45 minutes in seated containers at 30 C.

Approx. 4 gram dough is placed in the Dough Stickiness Cell. 4 mm dough is extruded IS to obtain an uniform extrusion. Hereafter measurement are made according to Stabb Micro Systems protocol (TA-XT2 application study for measurement of dough stickiness).

In brief, 1mm dough is exuded. The probe (25 mm perspex cylinder probe), connected to the TA-m system, is pressed into the extended dough at a set for -. The probe is raised and the adhesion between the dough and the probe is recorded. The following TA 20 m sewing are used: Option: Adhesive test Pre-tost sped: 0 mm/s Test speed: 2.0 mm/e 2s Post- test speed: 10.0 mm/s Distance: 15 mm Force: 40 9 T'rno: 0.1 s Trigger Type: Auto - 5 9 30 Data Acquisition rate: 400 pps The results recorded from the test are peak force, meaning the force needed to raise the probe from the extruded dough. The distance, meaning the distance the dough attach to the probe. Area, meaning area below the obtained curve.

3S

Dough stickiness is depending on the quality of the flour used and the recipe. Therefore a non sticky dough is a dough differing in stickiness from 100% to 20096 (relative) compared to a reference dough, without the xylanase or having preferably less than 7096 (relathe) of the stickiness obtained with a eommercal fungal xylanase (i.e. Pontopan 5 mono BG, Novo Nordisk) when dosed at a levels giving the same volume increase in a baking trial.

pROTOCQ INHIBITOR ASSAY PROTOCOL

10 (Inhibitor assay) To detect the inhibitor during isolation and eharacterisaffon No following away is used.

100 inhibitor fraction, 250 pI xylanase solution (eordaining 12 TXU/mD and 650 Al buffer (0.1 M citric acid - 0.2M Rhodium hydrogen phosphate buffer, pH 5.0) is mined. The Is mixture is therrnostaW for 5 minutes at 40.0 C. At ffmo = 5 minutes one Xylayrne bib is added. At time = 15 minutes the reaction is terminated by adding 10 ml 2% TRIS. The reaction mixture is centrifuged (3500 a, 10 minutes. room bmperabn-) Ad the supomabot is measured at 590 nm. inhibition is calmiated rosin at compared to the blank. The bleak is prepared the same way, except that the 100 pi 20 inhibitor substituted wffl 100 pI buffer (0.1 M citric add - 0.2 M odian' hydra phosphate buffer, pH 5.0). By way of example. X1 may be considered to ha-a Oh degree of resistance to the inhibitor (see Few 20). Xhl-2 and XM-3 may be considered to have a medium degree of resistance to the inhibitor (see Figure 20).

Ro4 GLUCANASE PROTOCOL I

(Endo*1,41ucana" acUvity) Glucanase samples are diluted in 0.1M sodiumacetab - citric acid buffer, pH 5.0, h 30 obtain approve ED = 0.7 in the final assay. Three dilutions of sample and an internal standard win a defined activity ate ff,etmostated for minutes at 40 C. To time - 5 minarets, 1 Glucayme tab (crosslinked, dyed glucan substrate) is added to ff-enzyme solution. To time = 15 minus" reaction is teiminad, by adders 10 ml of 2% US.

reaction mixture is oontrifuged and the OD of the supematant measured at 590nrn.

as Taldng into account the dilutions and U. amount of glucan, act (ECU, ED GlucanaslJni*) of sarnpb can be caroused rebtivab' to the rd.

PROTOCOl 5 INHIBITOR ASSAY PROTOCOL 11

(Inhibitor Kinetics Assay) To study kinetics on the inhibitor a soluble substrate was used (Azmxylan, Megazyme). A 2% (win) solution of the substrate was prepared, according to manufacturers protocol, in 20 mM NaPi, pH 6.0. The assay was performed by preheating substrate, xylanase and inhibitor at 40 C for 5 minutes.

For a preliminary inhibitor characterization, the xylanase used is diluted to 40 TXUlml.

For K/determinaffons, the xylanases are diluted to approx. 40 T3CU/ml.

0.5 ml of substrata, 0.1 ml of xylanase and 0.1 ml of inhibitor was mixed at time = 0 IS minutes, 40 C. At time = 125 minutes, the reaction was terminated by adding 2 ml of ethanol (95%), followed by vortexing for 10 seconds. Precipitated unhydrolyseci substrata was removed by contrifugation (3500 x lo, 10 minutes, room temperature). OD in the supomatant was measured against wear at 590 nm.

20 A blank was prepared the same way. The only modification wee substitution of the inhibitor win 20 mM NaPi, pH 6.0.

For kinetic experiments with decreased substrate concentration, the following substrata concentrations were made by dilution in 20 mM NaPi, pH 6.0. 296, 1%, 0.% and 0.25% 2S scrubs azo-xlrian (wh).

For determinations the above mentioned xylanases and substrate concentrations were used. These Hero combined with the following concentrations of inhibitor extras in the assay: 0, 2, 5, 10, 25, 50 and 100 Ill in the assay. Using pi inhibitor and not a molar 30 concentration of the inhibitor, K, is expressed as pi inhibitor.

SUMS In summary the present invention provides Ingram:

a. The isolation of an endogenous endo-1,xylanase inhibitor from wheat noun b. The characterisaffon of an endogenous endo-1,4 xylanaso inhibitor isolated from wlNat flour.

10 c. The characterization of the effect of endogenous endo+1,4 xylana" Inhibitor on different xylanasos.

d. A means for selecting xanases not detrimentally affected by endogenous endo 1,4-xylaneae inhibitor.

IS e. A means for selecting xylanases which are not detrimentally affected by undo 1,xaneae Inhib.

f. Xylanases that provide dough exhibiting favourable volume and acceptable 20 sffcicinesa than when compared to doughs comprising fungal xy analogs.

g. A method for screening xylanases and/or mutating the same using an endogenous endo-,1,xanaso inhibitor, and the use of those xylana or mutants thereof in the manufacture of doughs.

2S h. A foodstuff prepared u ith the xylanas of the present invonon.

DEPOSITS

The following samples were deposKed in accordance Path the Budapest Treaty at tl.

S recognized depositary The National Collections of Industrial and Alanne Bacteria Limited (NClklB) at 23 St. Machar Drvo, Aberdeen. Scotland, United IGngdom, AB2 1RY on 22 December 1998: DH5a:pCR2.1_BS xylana" NCIMB number NCIMB 409 -

BL21(DE3)::pE z4A Xhl1 NCIMB number NCIMB 4t000 BL21fDE3)::pET24A XM3 NCIMB number NCIMB 41001 ls DH5:pCR2.1_BS xHanase compases wild typo xylana -.

BL21(DE3J::pET24A_XM1 comprs XMt xylana -.

BL21(DE3)::pET24A_)OU3 comprises XM3 xylanaso.

The preserd invention aho encompas sequences derrvabb andlor expressabh from 20 those deposits and embodTments compasing same,

PRODUCTION TO THE EXAMPLE SECTION AND THE FIGURE.

The present inversion - 11 now be described, by way of example only, with reference to the accompanying drawings in which: S Figure 1 chaws a graph; Figure 2 shows a graph: Figure 3 shows a graph; Ftwe 4 shows a gra-I; 10 Euro 5 shows a graph; Figure 6 a graph; Fkp" 7 shows graph; Figure shaea a graph; Figure 9 shows graph; IS Figure 10 shows a graph; Ague t1 shows an image reauK of an SDS PAGE experiment Figure 12 shows a graph; Figure 13 shows a graph; Few 14 a graph; 20 Figure 15 shows a graph; Figure 16 sham a graph; Fours 17 shows an image result of an IEF experiment, Figure 18 stem a graph; F 19 shows a graph; 2S Fame Z0 sham a graph; Figure 21 shows a graph; Figure Z2 dam a graph; Figure 23 shows a graph; Fours 24 shows a graph; 30 Figure 25 shown a graph; Figure 26 shows a graph; Fbum 27 shove a graph; Fours 28 shows a graph; Fit 29 shows a graph; 3S Flows 30 81s a graph; 1:bure 31 shot a graph.

In slightly more detaTI: Figure 1 - Stickiness as a function of xyJanases, dose and resting tim*.

s Fioure 2 - Stickiness as a function of xylanases, dose and resting time.

Figure 3 - Gel filtration chromatography of a 75 ml inhibitor extract sample Column: 500 ml Superdex G-25 F. Flow 10 mUmin, Fraction see: 30 ml.

10 Figure 4 - Cation exchange chromatography of a 240ml gel filtrated inhibitor extract sample. Column: 50 ml Sepharose SP, Flow 5.0 mUmin, Fraction size: 10ml.

Ours 5 - HIC chromatography of a 147 ml ion exchanged inhibitor exbct sample added (NH4)2SO4 to 1.01U. Column: 10 ml Phenyl HIC, Flow: 2.0mUmin, Fraction she: 2.5 ml.

IS Figure 6 Preparative gel filtration chromatography of 2 ml cancer traded inhibitor sample.

Inhibitor eluted at 176 ml. Column: 330 ml Superdex 75 PG (Pharmacia). Eluent So mM NaOAc, 200 mM NaCI, pH 5.0. Flow 1 ml/minute. Fraction size: 5.S ml.

20 Figure 7 - Cation exchange chromatogram of pure xylanase boiled inhibitor em.

Sample: 1 ml desalted 980601 + boiled inhibitor extract. Column: 1 ml Source S 15.

Buffer system: 50 mM NaOAc, pH 4.5, B.: A + 1 M NaCI. Flow: 2 mUminuto.

Figure 8 - Cation exchange chromatogram of pure xylaneae after three hours incubation as win inhibitor extract Sample: 1 ml desalted 980601 inhibitor. Column: 1 ml Source S 15. Buffer system: A: 50 mM NaOAc, pH 4. 5, B.: A + 1 M NaCI. Plow: 2 mUrninute.

Figure 9 - Analytical gel filtration chromatography of 100 Ill concantrabd inhibKor sample.

Inhibitor eluted at 10.81 ml. Column: 24 ml Superdex 75 10/30 (Pharrnaaa, Sudden).

30 Eluent: 50 mM NaOAc, 100 mM NaCI, pH 5.0, Flow 0.5 ml/minute. Fraction size: 2.0 ml.

I:igure 10 - Log(MV as function of Kav for standard proteins run on a Superdex 75 10/30.

Is Figure 11 - SDS PAGE of fraction 31, 32 and fraction 33 from Prepare gel filon.

Lane 1 and 3 are MW mariners (Pharrnaaa's LOW markers, Swan). Lane 2 md 4 are

frac. 32, loaded with 10 and 25 pi respectively. Lane 6 anti 8 are frac. 31, loaded vow 10 and 25. Lane 7 and 9 are frac. 33, loaded with 10 and 2S.

Figure 12 - Reverse Phase Chromatogram of fraction 33 from Gel Filtration s Chromatography. Chromatogram reveals four destinct peaks. Peak 3 is the xylanase inhibitor. Peak 4, 5 and 6 are sequenced and show very high homology to t}" Whost protein, Serpin.

Figure 13 - MS of fraction 3 from RP - chromatography. Spectra shows or, miniscule 10 having a molecular weight of 38503 Da Figure 14 - Reverse Phase Chromatography of carboxy methylabd f,racdon 3 from Reverse Phase Chromatogram of fraction 33 (a" Figure 12). The Chromatogram revealed hero destinct peaks (fraction 2 and 3), indicating a dpeptide.

Pigure 15 - MS of fraction 2 from carboxy methylated Reverse Phase Chromatography (see figure 14). Spectra indicate a peptide having a molecular weight of 12104 Da.

Euro 16 - MS of fraction 3 from carboxy meylated Reverse Phase Chromatography 20 (see Figure 14). Spectra indicate a peptide having a molecular weight of 2822Z Da Figure 17- IEF of fraction 33 and 34 from Preperative Gel Filtration Chromatography.

Lane 2 is pi 3 - 10 standards, lane 3 is pl 2.5 - 6.5 standards, lane 4 and 5 are faction 33 and 34 - respectively, lane 6 is Trysin Inhibitor (pl 4.55), lane 7 is actophbulin (pi S.20) 2S and lane 8 and 9 are are fraction 33 And 34 - respectholy. Arrows indicate destinct bands in fraction 33.

Figure 18 - pH and relative OD (from inhibitor assay) as function of fractions from Chromatofocusing Chromatography of xylanase inhibitor. As can be seen from the figure, 30 the relative OD decreases in fraction 7, indicating hbibitor activity. Allis correspond to phi 9.4. Figure 19 Residual ached, % of four xylanases as a function of inhibitor conoentradon.

The four xylanases used are - -a- X1, - - X3, - x - i8X, - - Novel.

as

4S Figure 20 - Residual activity of 980601 (coli-1), 980603 (Belase) and three mutants of 980601 (XM1, XM2 and XM3) after incubation with a flour enact Figure 21 - Linweaver- Burk plot of xylanase (980601) I- inhibitor. Subab ate 5 concentration is % azo-xan. V is relative OD 590 from assay (where 100 is S=2%).

Figure 22 - for different xylanases expressed as miuolir inhibitor.

Figure 23 - Inhibition of three xylanases (980601 = Bac. sub. wt. 980801 X1 and 10 980901 = Therrnomyces) as a function of pi 1. Flee data are obtained by substraffng relevant blanks, Figure 24 - pH optimum for three xylanases (980601 = BX, 980801 Is X1 and 980901 = Novo). Figure 25 - Spec. vol = f(xylanase x dose) Figure 26 - Spec. vol. increase = f(xylanase x dose) 20 Figure 27 - Stickiness = f(xylanase x dose) Figure 28 Stickiness as function of different xylanase preparations and control, measured after 10 L10) and 4B (_45) minutes resting. 980603 is purified R6hm xylana -, XM1 is xylanase mutant 1 and #2199 is Rohm's Veron Special product.

2S Figure 29 - SUciciness increase as function of three xWanase preparations, after 10 L10) and 45 L45) minutes resting. 980603 is purified ROhm xylanase, XIU1 is xylana" mutant 1 and #2199 is ROhm's Veron Special product 30 Figure 30 - Stickiness increase as function of Nvo xylanase preparations, aver 10 Q10) and 45 (_45) minutes resting. XM1 is xylanase mutant 1 and #2199 is R6hm's Veron Special product.

Figure 31 - Stickiness increase as function of added End - 1,4Glucanaso. 1: Cornell IS dough without xylanase, 2: 7500 TXU pure ROhm xylanasekg flow, 3: 7500 TXU pure Rohm xylanase/kg flour 158 BGUA Flour, 4: 15000 TXU pure R6hm xylana flow,

5: 15000 TXU pure Rohm xylanase/lca flour 31S BGUlkg Flour. Douch were measured after 10 (Stik_10) and 45 (Sffk_45) minutes.

EXAMPIES

ExamDb 1 Douch sffckiness as a function of different xYlanases. doses and resffna ffmo, The following xylanases abDity to give dough sffcidness were teated.

(See also Chen, W. Z and Hosenoy, R. C. (1995). Development of an objecffve method for dough sticiciness. Lebensmittel Wl" u.- Technol., 2a, 467473.) Enzymes IS ÀX1. corresponds to a purified sampb of endo+1, 4xylanase from AspergHlus nir.

Thia xylanase haa an activity of 8400 TXU (15000 TXU/mg).

Novo. comsponds to Novo Nordisk's Pentopan Mono BG from Therrnornyces. This 24 xylanase has an acvib of 350.000 TXU (56000 TXU/mg).

BX. comsponds to a purlied samph of the new bacterial xylanase. This sample has an ac of 2000 TXU (25000 TXU/mg).

2s Rohm corresponds to Rohm GmbH's bacterial xylanase, Veron Specel. This sample h" an actvity of 10500 TXU (25000 T)CU/mg).

Xyna" Ay 30 Xylanase assays were performed according to Protocol 1 Flour Two kinds of flour have been used in thb teal: Danish flour, batch no 98022 and Gonnan 3s flour, batch no. 98048. llc water absorbffons, at 400 BU, of the hNo kinds of flour are 58 and 60% rpovdy.

Dough preparation Dough were prepared as described in Protocol 2. After mixing the dough rests for 10 S and 45 minutes respectively at 30 C in sealed containers.

Sticicinese measurement Stickiness measurements were perForrned according to Protocol 2 Results and discussion Fungal xylanas" versus new bacterial xylana" IS The following dough were made and tested for dough stickiness after 10 and 45 minutes in flour 98048.

Tabb. 1 Dough made with different doses of two fungal xylanases and one bacterial xylana -.

20 (Dose is ca culated per kg of flour.) Enzyme TXUJkg Btank O _ __

X11980801) 1500

_ i-oooo l Novo (#2165) 5 _. 50000

BX (980802)

The dough in Table 1 gave the dough stickiness results presented in Tabb 2 and hgum 1.

Table. 2

Dough made with dfflerent doses of dfflerent xylanasos vs.ank.

The dough was rested for 10 and 45 minutes, respectivoly.

S5ckiness is given as 9 x s, the stickiness figure is an aveme of 5 deterrninaffons.

Dough Stickiness, x s Std.Dev std.dov., % | Control, 10min 5.533 0.16_ 2. 89 Control, 45min 3.103 0.277 3.4Z 150_ X1, 10min 7.275: 0.204 2.80 1500 X1, 45min 3.675 0.14 _ 1.54 10000 X1, 10min 9.295 0.802 8.63 10000 X1, 45min 13.339 1.264 9.48 5000 Novo, 10min _ 5.757 _ 0.218 33 5000 Novo, 45min 7.23 0.337 4.13e 60000 Novo, 10min 10.872 0.519 4.73 18.559 1.626!9 82

1500 BX, 45min 4.372 0.58 8 19 15000 BX, 10 min 5.567 0.639 9.73 15000 BX, 45min 5.545 0.518 9.34 The data 1m Table 2 are illusbabd in uro 1.

10 As can be seen fhm Tabk 2 and Fqaure 1 the hJngal xylanase X1 and the xylana" in the Novo product ghe rise to dough stickinesa. The new bacterial xylanase do" not give rae to the same stickineas. In addition, o stickiness seema to decmeae compared with control. IS New bacterial xylanase va R8hm's bacterial xylano To test e functionaUty of the novel bacterial xylansse compared to the bacteHal xylana in the Rohm product: Veron Special, the followin,g dough wee made (see Table 3) using flour 98022.

Table 3

Dough mads u ith d'fferent doses of buo bacterial xylana..

(Dove is calculated per kg of flour.) S E ITXUnI TI Blank O BX 5000

= Rohrn 5000 lile dough In Tabis 3 gave the dough sticic noss results presented in Tabb 4 and ure 2. 10 Tabb 4 Dough made nth different doses of diflorent xylanas" vs. blanic Sffckiness is gen as 9 x s, the sticWness figure h an avera" of 5 determinatTons.

Dough Stickinees, x mm Std.Dev si.dev,. % Control 10mTn 5.269 0.16 3.04 Control 45min 5. o.m _ 5.05 5000 BX, 10mTn 4.443 0.204 4.59 000 BX 45min - : _ 4.474 0.184 3.00 15000 BX. 10rnin 4.791 0.352 7.35 15000 BX, 45mh B. 288 0.5 - 9.53 5000 Rohm,10mh 5077 0.218 4= 5000 Rohm, 45min 757 0.337 4. -

15000 Rohrn, 10min 7.749 0.519 6.70 15000 Rohm, 45rnin 10.98 0.907 8.2B 15 The data from Table 4 are illustrated in Fure 2.

The results show that 8X (the new bacterial xylanase) gives rise to much less sBckTnoss than the fungal xylanase tesW. Moreover, it is found that the new xylana" gn to much less dough sffc dMss than the R0hm bacte ial xylan.

so Example 2

Inhibitor Durlcaffon. charactensation and effect on xvianasos Flour S Three deferent kinds of flour was used in these experiments (bath 98002, 98026 and 98058). Flour batch 98002 and 88058 is Danish flour. Fiour batch 98026 is German flour. 10 Inhibitor extraction The inhibitor was extracted from the flour using ice cold distilled water and atirrir. One equivalent of flour was added two equivalents of ice cold distilled water. The mix was added a magnetic bar, placed in an ice bath and stirred for 20 minutes, After stirring the IS flour slurry was poured into centrifuge vials and centrifuged (10000g, 4 C and 10 minute.). The supematant contained the xybnase inhibitor.

Inhibitor # -

20 Inhibitor assays were performed according to Protocol 3 Inhibitor isolation A8u extraction of a 100 flour sample (98026) the xylanase inhibitor was purified by 2s following chrnmatographic technTqu -: Gel relation chromatoaraDhv (this Droceduro was run twice) 75 ml extract was applied to a 500 ml Superdex G-25 F {Pharmacia, Sweden) column at 30 10mUminute, calibrated with 20 mM NaOAc, pH 4.25. Eluent was collected In 30 ml fractions at the same flow. AJI fractions were spy for inhibitor.

Cation exchange chromatoaraDhv fthis Dromdure was run twice, as The inhibitor peak collected from the gel filtration run (240 ml) was applied to a 50 ml SP Sepharo" (Pharmacy, Swan) column at 5 mUmhutc. Atter badly, the column was

S1 washed to baseline with A buffer (20mM NaOAc, phi 4.25). The inhibitor was eluted by a linear gradient from A to B buffer (B.: A 350mM NaCI) over 10 column volumes at the same flow. The eluate was collected in fractions of 10ml. Every second fraction was spotted for xylanase inhibitor.

HvdroDhobic interaction ehromatonraDhv (this Drocedure was run twined Tl. inhibitor peak from the cation exchange chromatography (110 ml) was added (NH4)2S04 to 1.0 M and applied to a 10 ml Phenyl Sepharose HIC (Pharrnacia, Swan) 10 column at 2 ml/minute. The inhibitor was eluded from the column by a 12 column volume linear gradient from A (20mM NaPI, tM (NH4)2S04, pH 6.0) to B (20 mM NaPi, pH B.O).

The eluate was collected In fractions of 2.5 ml. Every second fraction was spotted for 3q please inhibitor.

15 PreDarate Gel filtration chromatoaraDhv 5 ml inhibitor peak from HIC non was up concentrated to 2 ml using a rotatory evaporator.

This sample was loaded to a 330 ml Superdex 75 PG (Pharmacia, Sweden) column at 1 milminub. The butter system used was 50 rnM NaOAc, 0.2 M NaCI, pH 5.0. The eludes 20 was collected in 5.5 ml fractions. Every second fraction was spotted for xylanaso Tnhlbitor. Analysis of prose acffvlb 2S To be abh to determine whether the found inhibitor effect was due to an inhibitor or a protease hydrolyzing the xylanase, tho following experiments ware carried out Incubation trials 30 2 ml of pure xylanase, 980601 (see Endo-,1,xylanases) was incubated with 0.25 ml of inhibitor extract for three hours at 40 degree C. As a control the same incubation was made with bolted (5 minutes) inhibitor extract. AKer incubation the samples were added 50 mM NaOAc, pH 4.6 to 2.5 ml and desalted by gel filtration on a PD-10 column (Pharrnacia, Sweden), obtaining 3.5 ml sample in 50 mM NaOAc, pH 4.5.

S2 Analvsis for hYdrolvei' The two sampks of pure xylanase hom the incubation triah were analysed on SOURCE 15 S column. 1 ml of the gel filtered sample was appliod to tho column s (calibrated with A buffor: 50 mM NaOAc, pH 4.5) at 2 ml/minute. The samph was elutod wi a linear gradient from A to B (B: A 1 M NaCI) ovor 20 column volumes and collected in 2 ml frabtions. The xylanaso was detected using OD 280 nm and spod for xylanase acvity in the fractions (100 p1 *action + 900 p1 buffer (0.1 M cibic acid - 0.2 M dsodium hydrogene phosphate buffer, pH 5.0) 1 Xylazyrne bb, 10 minutos, 40 dear" 10 C. Reacdon terminatod 10 ml 2% TRIS, blue colour = lanase acffvibr).

Inhlbitcr charactorinaflon Analvtical ael flltration chromatoaraDhv 100 p1 (concentrated two times on rotatory evaporator) of the inhibitor peak from HIC run was applied to a 24 ml Superdex 75 10/30 (Phammacia, Swn) d 0.5 milminub.

Running buf.fer used was 50 mM NaOAc, 0.1 M NaCI, pH 5.0. Eluate was colbd in fractions of 2 ml. I fractions wero spoNed for inhibitor.

To be abh to dstermim the ske of the inhibitor a ser of known protein. wore appd to the 24 ml Superclex 75 10130 column. The cond tions for this run were as deocribed above. The standard probin used woro: 2s Probin Size, KDa BSA 67

Ovalbumir. 43 Chymotrypdno 25 Ribonucbaso A 13.7 Tho proteira wore detd at 280 nm.

S3 SDS PAGE

Fractions from Preparative gel filtration chromatography were added SDS sample buffer s (prepared according to NOVEX protocol). boiled for three minutes and loaded on a 16% PAGE gel (NOVEX). The gel was stained according to NOVE)C's protocol for silver staining. As molecular weight markers, Pharmada's LMW markers were used.

Iso electric focusing (IIEF) To determine the pl of the native inhibitor, a sample of purified inhibitor (fradon 33 ftorn 330 ml Superciex 75 PG) was loaded on a pH 3 - 10 IEF GEL (NOVEL. The gol was non according to manufactors protocol. Using Pharmacia's (Sweden) Broad pi Idt 3. - 9.3 stanciards. The gel was stained with coomassie bniliant blue, accords to producers IS protocol.

Chromatofocsina chromatoorashv A sample of fraction 33 from Preperffive getfiltration chromatography, wee gelfiltramd to zo water. 1W desalbt sample was loaded on a Mono P HR 515 (Phannecia, Sweden).

Starting conditions was obtained with 25mM ethanolaminCI, pH S.4. The column was eluted mud Poly buffer 96: Water in a 1:10 ratio. pH adjusted to 6.0 (flow 0.5ml/min; fraction see: 0.5ml). After elusion with Poly buffer 96, the column was further eluded with Poly buffer 74: water in a 1:10 ratio. pH adjusted to 3.80 (nOW 0.5milmin; fraction at -: 25 O.Sm).

All fractions was pH measured and spotted for xylanase inhibitor, using Protocol 3.

Amino acid sequence A sample (obtained from fraction 33 from 330ml Superdex 75 PG) of pD inhibitor from preperatNe purification was used. 200W was loaded on a C4 Rever" Phase column (Applied Biosystems). The buffersysbm used was A 0.1% TFA in water and B: 0.1% TFA in 10096 Acetonitrib. Inhibitor peak from this run was carboxymeybted and rennin 3S on C4 column again. In this way two inhibitor peptides, of intern Ore obtained. These were N terminal sequenced. furffiermore, the pepffdes were digested wow L+C.

obtained peptides were recovered using reverse phase chromatography and amino acid sequenced. To verify sequnces obtained by amino acid sequencing, a small fraction of the sample of interest, was analysed using.MS (/oyagor).

Inhibitor Kince Inhibitor assays were performed according to Protocol 5. In this Impact, for the 10 preliminary inhibitor characterization studios, the xylana" used was 980601, diluted to 40 fXU/ml and tl. inhibitor was extracted from flour 98002. For to dotorminaions, the following xylanases were used: 980601, 980603, 980801, 980901, 980903, 980906 and 980907, diluted to approx 40 TXU/ml. The inhibitor used Or dobnninaUons w extracted from flour 98058.

Deannination of Inhlbfflon a Mention of pH Them exporuTents wore carried out as descried in Protocol 3 wffl the following modific. Asides urns 660 pI buffer (0.1 M citric acid - 0.2 M dlodium hycirone 20 phosphate) pH 5. 0 In Me assay, ma assay was ado carried out wing same buffer system at pH: 4, 6 and 7.

Endo*1,^bna IS The following lay anally preparations wow used: 980B01 (i : Purified preparation of Danisco's new bacterial xybna" rd in E colt (1225 TXUhnl) 30 980603 (Rohm): Purified preparation of Frimond's Belase xylana" 0 - Ritual to Rohm's) (1050 11CUJm 980801 (X1): Purified X1 freon A-en us rr(8400 TXUlg) as 980802 (Rohm): Purified preparation of Frimond's Bela" xylana" (identical to Rohm's) (5 TXU/m'

ss 980901 (Novo): Purified preparation of Therrnomyces xylanase from Novo's Pentopan mono BG (2900 TXUlml) s 980903 (XM1): Purifiod mutant of BacJllus sub. Id typo xylanaso expressed in colt (1375 TXU/mi) 980906 (XM3): Purified mutant of Bacllus suh nid type xylanaso expressed in E ooli.

(1775 TXUlml) 980907 (XM2): Purified mutant of Bacillus sub. unid typo xylanase ex in E colt (100 TXWml) 9535 (X3): PurHied xylanaso, X3 from Aspergillus nJger (6480 TXUlml) IS Results and discwslon Inhibitor extraction tor isolation and characteraffon zo 100 flour (98026) was extracted. Afbr centrifugabon a supematant of 150 ml ww obtained. Tho presence of inhibitor was checked in is extract (Tabb 5) and hund positiv. Tabb 5.

zs Residual activity as a function of /- addition of inhibitor extract from wheat hur (98026).

The xybnaso ud is 980601.

I 1- inhibitor |. inhibitor |Residual aciiv - ' % pOD 590 10.675]0. 1B5 1 24.44

Inhibitor isolation 75 ml of Do inhibitor extract was loaded on a 500 ml gel filtration column (Figure 3). After s spotting for the inhibitor, it could be located in fractions t4 - 111 (Tabb B).

Tabb 6, Fractions from gel tiltrabon chromatography of 75 ml inhibitor exit assayed for 10 xylanase inhibitor. OD run 1 respectively 2 correspond to two rem that were performed on Me column. Inhibitor was found present in fractions 14 - 11]. 11 fractions were pooled for each run, giving hero time. 240mL Fraction no. OD run 1 0.674 I

2 0.5 3 0.652 _

4 0.618 0 4

1 0.3 - 0.1

1 0.186 0.126

_ _. _

0.188 _ 0.18

3 on 3281 0 217 10 0.406 0.24ti 12 0 72S 0.435

13 0.683

14 0.762 __

15 0.737 _ _ _

IS The pool of the Inhibitor peak in both runs on the go filtration column, was approx. 240 ml. is Two times, a 240 ml pool from gettilVation was applied to Do cation exchanger. The flow through was found negated for inhibitor. AS can be "on from hiauro 4 and Tai" 7 the inhibitor bound to the column and eluted at apprise 750 mM NaCI.

S7 Tabh 7.

Fractions from cation exchange chromatography of 240 ml gel filtered inhibitor exact assayed for the presence of xylanase inhibitor. OD run 1 respectively 2 correspond to the S two runs that were performed on the column, Inhibitor was found present in fractiona.144 541. Fraction no. OD run 1 _ OD run 2 40 0.476 0.624

42 0.407 0.58

44 _ o.22 4 0 137 48 0.144. 0.107

50 0.198 0.126

52 0.302 0.208

54 0.395 0.435

56 0 457-- 0.485 i 58 - 0 463 0.606

The pool of inhibitor from the ion exchange rune was 110 ml from each run. These two lo pooled fractions were added (NH4)2S04 to 1.0 M and applied to the HIC column in two rune. The flow through was spotted for inhibitor and found negative. As can be aeon from Pleura 5 and Table 8 all inhibitor bound to the column and a good separation was obtained. IS The analysis of the fractions from Me HIC chromatography is shown in Tabb 8.

Tabb. 8.

iracdons from HIC chromatography of 147 ml inhibitor extract assayed for skylark inhibitor. OD run 1 respectively 2 corresponds to the two runs that were performed on the 20 column. Inhibitor was found present in *actions 115 - 23].

Fraction no. OD run 1 _ OD run 2 Blanic 0.469 0.658 12 0.4B2 0.622

_. _._.

14 _ 0.48B 0.555

16 0.202 0.188.

18 0.1 0.118

_. 20 0.102 0.146

22 0.242 0.193

_ 24 0.392 _ 0.502

26 0.485 DAI

S8 Fractions 17 and 18 *em the HIC chromatography were concentrated approx. two times i and applied to a preparative gel filtration column (Figure B).

The analysis of the fractions from the preparative gel filtration is shown in Tabk 9.

S Tabh 9.

Fractions from Preparative gel filbaffon chromatography of 2 ml concentrated inhibitor i sample assayed for the preasnce of xylanase inhibitor. Inhibitor was found present in fractions 131 - 331.

Inks-' I-

34 0 70!

0'749 _ 0.769 i Ane yew p-ALSO a Paid on the show away of the xylanase Inhibitor, it can not be rued out that the lS decree" in Manage ad, Men mime the flour e= is not due a o hydrolysis of the ylanase. Therefore, a purified xylana" was ink wffl an inhibitor extract As can be seen from Figure 7 and 8, no hydrolysis memo to occur.

There is a little mom background in the chromatogram with active inhibitor (Figure 8).

However, this background corresponds to the ahromatogram of 1ho inhibitor albino

20 (chromatogram of inhibitor not shown). The difference in background must be duo to

precipitation in the boiled inhibitor sample.

Inhibitor characteradon

s9 Analvtical Gel filtration chromatoaraDI 100 pI two times concentrated inhibitor sample from fraction 18 in the seconci HIC run was applied to a 24 ml analytical Superdex 75 10/30 (Pharmaca, Sweden) (Figure 9).

5 The eluate was collected in fractions of 2 ml. These fractions wore assayed for the xylanase inhibitor (Tabh 10). i Tabb 10.

Fractions from analytical gel Libation chromatography of 100 pi concentrated inhibitor 10 sample assayed for the presence of xyJanase inhibitor. Inhibitor was found present In fractions lie - 7;.

Fraction no. OD 590nm_ _ Biank 0 613 7 _ 0.30

0.51 0.569 10 D 565 -'

11 3.652

After As gel filtration of the up concentrated inhibitor sample a mbt of four standard IS molecular weight proteins was applied to the column, uain a me exactly same procedure (chromatogram not shown). In tabb 11 the mobcuiar weights and me ehfion Barnes for the proteins are summarized.

Table 11.

20 Standard proteins used for determination of the MW of To inhibitor. Abbrwlabons and equations used are expired below the tabs.

BSA I 9.45 0.059508; =11i Ovalbumin 10.38 0.119017 Chymobypsin 12.49 0. 255498 25 1.39794 RibonuJdease A 13.49 0.320181 13.7 1.136721 "V 4 - Vow Vo) Where: Ve = - Anne, me Vo vow vol., ml Is 8.54 Vt = 24ml 24 Ploffinq he (MOO] as a function of Kav. It is possible to obtain an equation and US estimate molecular size of an unknown modicum (Figure 10).

Using be equation obtained in Figure 10 and the retention time for the inhibitor, it is possible to calculate the molecular size of the inhibitor: (-2,4485 x bv 1.9602) MW, IcDa s 10 (-2,4485 x 0.173559; 1. 9802)

1.5352

Is 10 34.29 The molecular weight found for the inhibitor was Honshu than we oxpacW accordir to Rouau and Surget (1998. Evidence for the presence of a Pentosanase Inhibitor in Whew Flour. Joumal of Cereal Science. 28: 6370)), the MW of the molecule approve 8 KQa.

To MW obtained by gel fitrhation could be explained by aggregation of several inhibitor 10 molecube. To study this further an SDS PAGE gol was run of fractions 31, 32 and 33 from the preparative gel filtration chromatography (Figure 11). As can be soon from lob Up, tier" bands appears in the lanes loaded wffl purified inhibitor sample. Them band correspond to proteins wffl MW s of approx. 40, 30 and 10 kDa IS IUIW determination USiM MS A sample of fraction 33 from preparative gel filtration of the inhibitor was desalted using the Presorb system and 5 volumes of 20 mM Acetic acid. 2001 was loaded on a C4 Reverse Phase column (Applied Biosysbms). From this run, three posies was obtained.

20 One of these peaks speak 3) was cbarb dominating, and thought to be the inhibitor Amp 12). The Aver pat hem run ha" also An At. F - m the sequence obtained it can be concluded that they ate an originating from the same wheat protein, Serpin, and ate not Identical to the inhibitor (peak 3). Therefore peak 3 is concluded to be the xanase inhibitor of Inter - L This peak was furler chard 25 Desire MS tVoyagor).

MS spoca analysis revealed a signal corresponding to a protein of 39503 Da, udr sinspic acid me (Fu. 13).

AS mentioned above, the SDS PAGE gel indicated Knee bands. One band at approve 10 kDa, one at approx. 30 kDa and a band at approx. 40 kDa. To explain the results asen frwn SDS+AGE, the pure dominant fraction was collected, Iyophilisod and carboxymethylated and then rerun on the C4 column, using same conditions as s mentioned abow.

The fraction obtained by this rennin (Flume 14) was analysed using MS. As can be seen from Figure 15 and 16, the MW of these poly-peptides are 12104 and 28222 Da.

10 Without wishing to be bound by theory we believe that the xylanase inhibitor is oilier a nathe dlpeptide (MW 39503 Da) or it is denahrated and reduced (two peptic" with MW 12104 and 28Z2 Da - respectively) during the analytical process.

Detenninaffon of Dl for the xYlanase inhibitor using l,EF anal Chromatofocusim IS ChromatoaraDtrX The IEF gd showed three bands in the alkaline area (approx. 9.3, 8.6 and 8.2 -

reapecffvely) and three bands in the acidic area (approve 5.1, 5.3 and 5. 5 - respectably) (figure 17). Based on these results alone it may not foe feasible to debrrnine the pl of 20 the native xylanase inhibitor. In this regard, we knew from the uenar results, mat the sample only contained the xylanase inhibitor and three fragments of Serpin, of approx.

4500 Da. A theoritcal calculation of the pi for Serpin is 5.58 and the pl calcuiad on fragment we obtained by suenen gives pi 5.46 (using Swim Prot prograrnm -). This could indicate that the three acidic bands seen on the gel, are the three pealce of Serpin as son ninth Rever" Sham Chromatography (Figure 12), and the three alkaline bard use the tier" different forms of the xylanase}nhlbKor, i.e. the native dipffde form and halo pepffdes (as indicateci by sequencing).

As can be seen from the Chromatofocusing Chromatography results presenteci in figure 36 18, the xyianase inhibitor does not bind to the column under the given conditions. As could mean that the native xyianase inhibitor has a pi of 8.5 or even higher. H - Go, it would seem that the asumptions presented above, namely that there are three alla bands on the IEF gel and so there could be three possible forms of xyJana" inhibitor, may be cot

In conclusion, we believe that the native xylanase inhibitor has pi in ff - interval 8.0 - 0.5. i

Within this interval, there are three bands. These three bands probably corresponci to the xylanase inhibitor possibly existing in three forms (see the results dotennined using IEF).

In this respect, in using IEF, the protein runs as a nadve protein but that sort. did s proteins may be partly damaged by this technique, thereby giving rise to mom Man one band. Seawace dab 10 The two peptides forming the inhibitor, were sequenced giving N-terrninal and internal Sequences. JOB results are prssen in the attached sequence iistis as SEE ID No.s 1319. The sequences making up the first Main (chain A) are shown as SEQ ID No.s 13 and 14.

IS The sequences making up the second chain (chain B) are shown as SI5Q ID No.s 15 to 19. A data bad search for homology to the sequenced poly peptizes came out regauge.

Neither of Me polypepUdes have been sequenceci or described before. Effect of inhibitor on different xybn Several bnals have been carried out

to study me inhibition of did - rent xylanas -. First we beloved that the decrease in xylanase activity was duo to a proic at in as enact. Therefore, different xylanases were incubated different wlurn" of Inhibitor expect (Figure 19). The xylanases were found to be inhibited to different Ponds. What we also found was that there seemed to im an increase in inhibition # a fashion of ÀinhibW concentration.

30 The results illustrated in Euro 19 could indicate that the decease was duo to proteolyds or inhibitor. However, time course experiments wffl, constant xylanase and inhibitor i concentrations and the above mentioned results under An91"is for probe" acffviy, did not show decreased activity as a function of time. To ebb to distinguish On proteaso and inhibitor, real kinetics has to be made (see ÀinhibHor kinos3.

3S

Two Bacillus subtilis xylanases have been studied very closely regarding their baking performance. These xylanases differed a little in their functionality, meaning that one gab a slightly higher specific volume when baked in identical doses. One explanation could be different inhibition of their activity in the flow. An experiment was therefore S performed to examine this Who experiment has been repeated tunce, using two different kinds of flour as source for the inhibitor (Tabb 14).

Tabb 14.

Inhibition of two xy anases (980601 and 980603) by inhibitor extracted from two kinds of 10 flour (98002 and 98026). Inhibition is calculated as % inhibition and as % residual acffvi, computed to blank.

Flour 1 _ 98002 - 26 -

Inhibation, 96, 67.03 75 04 980603 60.76 B1.33

- - 98002 9 Avis Re act., % 980601 32.97 24- 28.-

980603 39.24 1 38.67 1 38.86

_._. -' 1

Differena _ 1 25.66 1 trial shows that the two xy anases are inhibited to different extents by the inhibitor.

Is The xylanases differ in only six amino acids.

Based on 980B01, three xylanase mutants have been mad. (XM1, CM2 and XM3).

These mutants have been analyzed for inhibition (Figure 20).

20 As can be seen from Figure 20 the three mutants diner in residual activity, meaning that they are inhibited to different dorms by the xylanaso inhibitor. Four (EIX, Rohrn, XM1 and XM3) of the five qrlanases have the same specific activity (approve 25000 TXUlmg protein). XM2 is expected to have the same specific acffvib.

The difference in inhibition between XM1 and XM2 is approve 250% (the residual activity of XM1 is 2.5 Ames higher Man the rest activity of XM2). This difference is duo to one amino add. Amino acid 1Z in XM2 is changed from arginine to asparagine, introducing less posit charm near the acme do.

Inhibitor kineffcc Simple preliminary kinetics were performed. Just to be able to determine whistler inhibitor is compete - or rcompvo.

S Different amounts of substrate were incubated with a constant xylanasa" and inhibitor concentration (Figure 21).

As can be seen from Figure 21, V,,,,,, for both qrtanase win and without inhibitor is approve 10 1.19. This indicates that Me Inhibition is compeffthe.

Since the preliminary inhibitor experiments describeci above, indicator diflinco h iron the xylanases studied. The real Kit for several xylansaea wore debrmird. As can be men Mom the data h Fgure 22, the values do differ significantly bdwn the IS xylanases. This confirms the resulb indicated by the simple preliminary inhibitor characterisalion. Inhlbfflon a function of pH 20 A Ample Spot for xylanase inhibitor at a different pH reward that there seemed to be an effect of pH on the inhibition of xylanaa. Therefore, an experiment was sot up to examine this effect As can be seen from Euro 23 the inhibition of xybna are influenced by pH. Figure 24 illustrates pH optima for the xylanasea. If to two curves are compared, we see the highest inhlbiffon at the pH optimum for xylanaso, US except forgo pH 4 measurement of Novo xylanaso (980901).

To determine whether the inhibition ratios measured in assays repotted here are relevant in the dough, some calculations can be mods:

Inhibitor extraction Gram 6 flour: ml water: 12 g 0.5 flour/ml: g flour in aaany: 0.05 Xylaneae solution TXO/1: 12

TXOJml in Albany: 3 InhLb$tor: xylansa.

rat$oa ' TXU/kg flour: 60000 in inhibitor assay TXOJkg flour: 3000 in bakery applications From the above calculaffona, the inhibitor xylanese ratio in the assay can be calculated to be 20 times bwor in the assay than In dough. This can only mean that the xylanaso must S be much more inhibited in dough. However, the mobility and water ace is much lower In dough and this might influence the inhibition.

Summary Dleculon

10 Wheat flour contains endogenocus endo-i1,xydanase inhibitor. The inhibitor can be enacted from wheat flour by a simple extraction using water, meaning Wet inhib tor Is water soluble. The inhibitor was purified using gel filtration-, ion enchant and hydrophobic irraction chromatographic techniqu -.

IS Characterisaffon of the purified inhibitor, using analytical gel filtration cluematography, SDS PAGE, reverse phase chromatography and MS, revealed a poy-pepffde of approve 40 KDa. This poly pepbde turned out to be a di-peptide, containing two pepdes with molecular weights of 12104 and 28?22 Da, respecffvy. The pureed inhibitor (more pry the two pepffdes) was N-terminal sequenced, followed by digestion and 20 sequence of peptides obtained..

6C Tl. preliminary experiment With the inhibitor indicated that the decease in xylana" acuity found could be due to probolysis. However, analysis of incubation trials (xylana" + inhibitor) and kinetics on the inhibitor indicated that the observed decrease in xylana" acffvib was due to a competitive inhibitor.

Inhibitor experiments using several xylanases indicate differences in sensibility towards the inhibitor. Some xylanases are inhibited aimed 100% by inhibitor (at a rawer inhibitor xylanase ratio than present in the flour), By varying pH in inhibitor away it turns out the inhibition is highly dependent on the pH in tl. any. Examining Me lo xylanase mutants revealed that changing one amino acid can mean a 250% dynamo in inhibldon. To continn tl" results described above, K/ values were determined for Naval xylanasos.

The results showed different 's depending on the xylana" used, confirming the IS difference in resistance towards the inhibitor as function of xylana" asen in preliminary reach EXa3 :0 Baking Dials, The data below are from a baking trail with the XM1 mustard The data show that novel xylanase mutant is clearU superior to BX (Bacillus subtt7is wild type) bawd on volume.

Based on stickiness measurement there are no significant diffenco between the him 2S Igrlanas Emyn 980902 (B)t): Pureed Bacillus sub. wild typo xylanase expressed in ú colt. (2000 30 T,CU/ml) 980903 Me): Purified mutant of Bacillus sub. mid type xylanase expound in E. cab.

(1375 mg

Flour Danish flour, batch 98022 S Baking test (hare) crust rolls) Flour 2WO a, dry yeast 40 9, sugar 32 0, salt 32 0, GRINDSTEDT Panodan A2020 4 it, water 400 Brabender Units 4% were kneaded in a Hobart mixer with hook for 2 minutes low speed and 9 minutes high speed. The dough temperature was 26 C. The dough was 10 scaled to 1360 gram. Resting 10 minutes at 30 C followed by Would on a Fortune moulder. Proofing 45 minutes at 34 C, 85 % RH. Baked in a Bagoovon 18 minutes 220 C and steamed 12 seconds.

Mer cools rolls were scaled and their volume measured by the rape Wed IS deplacemcnt method.

* Specific volume = volume of the bread. ml weight of broad, no Stockings measurement Stickiness measuremerd was perforrnocl according to Protocol 2.

AS can be son from Tabb 15 We novel xylanase mutant ('Clf1) gives rise to significant 25 higher broad volume increase Wan B)C

À Table 1S

Bread volume increase (mUgram) and stickiness (9 x s) as function of two xylanasos (BX and XM1) applied at different dosage.

Sample Doso, silckin SpecHic Spoc. vol. XU/k x inercas % BX 2000 6.00 6. 03 2 5 '- -

l BX 5000 6.60 6.4SI 10.37 BX _ 8000 5.00 e.n-15 14 BX 12WO _ 7.00 47 14 29

XM1 2000 4.30 em 12.24 XM1 5000 6.20 6.88 _ 17.01

XM1 8000 6.20 7.06 20 07

XM1 12000 6.90_ 7.32 24 49

Control O 4.50 5.88 The data are shown in Fracas 25, 26 and 27.

ExamDb4 Douq shckiness as a funcffon of XM1. e Rshm Venon seciaixHanage nqLa DU46.d vemion of the R0hm Veon,Spedatdanase.

To determino whether novel xylanase, XM1 gh moro or b" sc dough an IS Rohm's Voron Special xylanase (and a pudfied veraion lrof) dough wero prepamd and adckinees es function of xHanaseraa datennined.

Fbur 20 Danah flow,batch 98022 we used.

Dough preparedon Dough weno prepared as described h Pnocol2. Aflor m Wng the dough ndod for 10 2S and45 minudos,respecffve,in sea W contairs befone sffckiness nneeaunent SUckineas measurcnnont Stiddn6# masuremont. woP. pr*UT to ltoool 2.

do Enzym" 980903 (XM1): Purpled mutant of Bec/llus sub. wild type xylanase expressed in E. coil.

S (1375 TXUlml) #2199: the R5hm Veron Spedal xylana" (10500 TXU/g) 980603 (Rohm): PurMed preparation of Frim ond's Be lase xyla n a se (idendcal to R6hm 's) 10 (1050 T)(U/m' The follounng doughs were made (Tablo 16): Tabb 18 IS Dough made for determination of sffckiness Xylan 1 Dwage, IXU/kg 980603 (Purified Rohm xylanaseL l 1500 #2199 (Rohm'a Veron Speciar) I 15 000 The dough in Tae 16 gave tte sdckinesa results in Tabb 17.

20 Tabb 17 Results from stickinces measurements on dough prepared with Purifbd ROhm xana -, À conbol, XM1 and o R81un Varon Spocial xylana -.

Xybne TXWkfiour j Leavening Sffckin, | Sffckin _,min. xe incre, x 980603 15.000 "iO-7.22 2.22 980603_ 15.000 45 10.15 4.08

Co _ O _-iO O Conl O 45 - _ _ XiU1 15.000 10 6.81 1.B1 1 XiU1 15.000 45 9. 64 i 3.55 1 #21 - 15.000 _ 1 8.57 1 3 57 1

#21 - 15.000 45 _ 12.14 1 6 05 1

The data are shown in Figure 28, 29 and 30.

The increase in stickiness using tile XM1 is lower than the stickiness Base with the purified ROhm xylana -. The stickiness increase obtained using the unpurified ROhm S lanase is much higher.

Example 5

Dounh stickiness as a function of bacterial Endo-B-1.4-G1ugana" 10 The resume h the following are from an experiment destined to Rudy the abUlty of bacterial Endo-1,4G1ucana" to gore sticicins.

Emym" 15 981102-1 (Xyl): Correspond to a purified preparation of R6hm's bacterial xylanase from product Voron Specie. The preparation Is pure xylanase and do not corin any Endo-1,4-Glucanase (350 TXUlml) 981102-2 (Xyl Gluc): Correspond to a purified preparation of R6hm's bacterial Dylan go from the product Voron Special, containing Endo+1,G1ucana" (900 TXU/ml 19 BGW6nl) Xylane Amy 2s Xylana assays were performed according to Protocol 1 Glucana" assay Glucanasc assays were performed according to Protocol 4 Four Danish flour, batch no 98058 was used. The water absorbtions, at 400 BU is 609,

7t Dough preparation Dough were prepared as described in Protocol 2. Alter mixing the dough rested for 10 and 45 minutes respectively at 30 C in sealed containers.

S SUckTness measurement SUckiness measurements were perforrnod according to Protocol 2 10 The dough listed in Table 18 were prepared and examined for sticidn.

Tabb 18 Dough prepared for examining stickiness : Dough No. Dough TXU/kg flour BGUlko flour 2 Corol 7500 = 3 TXU + BGU 7500 158

4 TXU 16000 0

5 TXU I BGU 16000 316

The dough listed in Table 18 gave the stickiness result in Table 19.

Tabb 19 Stickiness results from dough With xylanase and xylana" gIwanaso 20 Dough No. refers to the dough No. in Tabh 18 Sdk_10 indicate results from stickiness measurements aver 10 mint Stik_45 indicate measurements actor 45 minutes of resew Dough No. SiIk_10, 9 x s std.dev Sffk_45, x s std.dev 1 4.5 0.342 5.11 0.552

2 5.29 Q619 8.62 0.607

3 5.47 0.663 9.38 0.832

8.61 0.408 _ 9.15 0.418

5 8.73 0.35 10.19 0.857

2S As can be seen from Tabb 19, Me Endo-1,4-G1ucanaso addition to dough increases We stickiness of We dough. The results Rom Table 19 are illustrated in Fbum 31.

SUMMARY

In summary the present invention provides and the Examples show inter Fit.

S a. The isolation of an endogenous endo+1,xylanase inhibitor from wheat flour.

b. The characterisaffon of an endogenow endo-1,4.xylanase inhibitor isolated Mom wheat four.

c The characterization of the effect of endogenoua endo-,1,4xylanase inhibitor on dilldrent xylanas -.

10 d. A means for selecting xylanases not detrimentally affected by endogenow ends 1,4xylanase inhibitor.

e. A moans for seducing xylanasea which are not detrimenbily affected by endow 1,4-xylanase inhibitors.

f. Xylanasea that provide dough exhibiting favourabb volume and acceptable IS stickiness than when compared to doughs comprising fungal xylanas -.

g. A method for screening xylanasos and/or mutating the aa using an endogenous endo-1,xylanaso inhibitor, and the use of those xylaneaea or mutants thereof in the manufacture of doughe.

h. A foodstuff prepared with the xylanasoa of the present invention.

All publications mentioned in the above specification are herein incorporated by

2S reference. Various modifications and variations of the described methods and system of the present invention van be apparent to those sicilied in the art without departing Am Me scope and spirit of the present invention. Although th present invention has ban described in connect on wffl specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments.

30 Indeed, various modifications of the described modes for carrying out the invendon which are obvious to those skilled in bioclemiy and biotechnology or related fields are

intended to be within Me scope of following claims.

IYDIC.TIO'iS RELTI.'G TO DEPOSITED '&IICROORG^.NIS.\I A. Sac indons e2dc below.sc lo tic moor rc.ct to In hc descnpoun i one.41,1'c2 - 20 n I I- r 1: A_ IDE.YTIFIGT10N Of DEPOSIT Funhcr tcgosits arc iambi:' - on so additions -:: O Sync ot tepodc.Y uaaon The.3lational Collections of Indus. rial and Marine Bacteria Loiter (NCIH8, ._ Tress ot d posits." matucon (impose co" elf COJ 23 St Hachar Drive Aberdeen ABE lay United l('ingdon . _. D-c of depot Amazon Numb" 2LDecember 1998 NCIMS 40999 NCIMB 41000 NCIMB 41 W1 C DOITIONAt. tNDICATlOl1S blood / opal; a mt'oon "ndn _. In respect of those designations in which a European patent is sought, und any other designated state having equivalent legislation, a sample of the deposited mioroorgenis will only be made available either until the publication O. the mention of the greet of the patent or after twenty years From the data of filing if the application has been refused or withdrawn or la deemed to be withdrawn, only by the issue of such a sample to an expert nominated by the person re:ueatin _ - D. DESIC.aI^TED ST4 FOR WHI I.DICTtONS ARE M^DE tht o "otf ttH SEPARATE FUEWtSlil;YC OFtl4lCTtOlYSr ffrsotopp The inou list bclov will oc spiced al the on31 6we:1u leery-r,lewef At Off _ 'I _ For Aids Otiose Us utly For taonal Burmu arc only O Thushcarm;edheinenadormloppli l-crec:eved *Eliot ilurco _. _ ^_Om o

SEQUENCE LISTINGS

The following pages present a series of sequence listings - which include the follows sequences: SEQ ID NO. 1 - amino add seqwnco hom the inhibitor i SEQ ID NO. 2 - amino acid sequence from tl inhidior . SEQ ID NO. 3 amino add sequence of a wild type xybn#o (R) SEQ ID NO. 4 - nucleoffde sequence d a wild type xybn_ (R) lo SEQ ID NO. 5 - amino add sequer" of a wild type xy4n (D) SEQ ID NO. 6 - nudeoffde sequence of a wild typo xybn (D) SEQ ID NO. 7 - amino add sequence of 8 mutant Xyl81_ pai SEQ ID NO. 8 - nudeotido enhance of a mutant xybn (X141) SEQ ID NO. 0 - amino acid sequence of mubrt xylan Is SEQ ID NO. 10 - nudeotide sequence of a mutant xylems QW SEQ ID NO. 11 - amino acid sequence of 8 mubat xybr QEU3) SEQ ID NO. 12 - nucleodde sequence of mutard xybn pUd3) SEQ ID NO. 13 - amino aced seqw Am the inhibitor! SEQ ID NO. 14 - amino acid sequence hom the it 20 SEQ ID NO. 1 amino add sequence Born the TO SEQ ID NO. 16 - amino acid seqwnos Mom hhlbitor SEQ ID NO. 17 - amino acid sequel horn inhibitor SEQ ID NO. 18 - amino acid sequence from the ironer SEQ ID NO. 19 - arnino acid aequenos *em ma TrbRor 35, -

Noble: For XM1, XM2 and XM3 - we introduced chang" (as shriven) in nueboffde "queries and smirk acid sequence id sib direct mutation of the gene. The mutated 9 m" be expressed in E. cdl, in BacTlius or in any organism a, choice...DTD: 30,. .

SEQID NO.1

1 2 3 4 5 6 7 18 19 |10|11|12|13|14|15|16|17|18|19|20|21 122 I23I24I25I

L V V A R A IV IK ID IV 1A IP |F 1G IV IX TY 1D IT IK IT IL IG IN 1

_ _ _ _ _ _ _ _ _ _ _ I-I l _ _ I I I i _ _ _ _ _ _ _ _. _ _ I _ I I _ _ _ _I T Wrl 26 27 28 29 30 31 32 33 34 35 36 37138 39 40141 142 43 44 45 46147 48 49150

N L G G Y A V P N b L G L L D G G X D W T M r x K _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

= _ _ = = _ _ _ _ _ = = _ _ = = = = = _ _ _ _ _ _

51 52;3 54 55 56 57 58 59 6C 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

N S U V D K _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

SEQ ID NQ 2

. 1 12 13 14 15 16 17 18 |9 110111|12|13|14|15|16|17|18|19|20|222|23 24125

G IP |P IL ||P IT 116 IP IT IS IL IY IT Il IP IF IH |H1G A IA I I I Ll I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1---

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1;1''--

26127 128 129 130131 132133134 135 136 137 138 139 140141 142 143 144 145 14B 147 L48 49T50

X |V IL ID IV IX PS IS IX IL IL IW1X I I I I I l I I I

3! ax. hr Hi SLAV TS S SA = -

TG:IF;N "GSp== " Dot TYDG GYDm..S QYW5VR QS=P5C - A AmS W}CSE GSGS

Q tt.4..

Am I, 1 J1513TSZ A." A5SúT=OlT GGA5hlAl:W CFI;TO,S t21 =.T " 181,=,

241 GGel "= = 301 lU;ASt=C=C S=UCDA&= 5TaTt:ZW" CFaSTJW GT APACE 361.U.5 CG-' GAG. GcZACh AS]= 421 -.

401. 601 "A3:t a=r,D rit:w. A - Ace JU.

54aQ,. 5 77 sacillu aubtila wild type sylvan AA: v oS,qsrs "SOUSA" ToYW< GGTv='rNGs ac=SS SGNFW STGS,FRT Y2=G-NG GYIlYGn RSPD;YYW DSWGTYS D;5VXSDC G=Dmr ma TIFTO=SVR OSGS rrrSs wits trove eYcssGsnr S52. { À '

Doll.:: 1 ASCITS lGLllA ssn:TloTr G - I=I IS Ch6ShTD" À1 Tt5STIT CK.AC=C SW.l:CZ7.0C accacr cozo' =1 GG=:G= caA= CCQASZ zesma T: - it :81 lLcous: TT{;TT aim - ITOG AC at eAl 241 =3A CA.CST!W3C GCOCAa5WC AACZ: =,Acm&=, _ 301 AGF, TCA.D.CXA.D 55PCOSC CaSTC=OX { A..

3411 aCCAC GD.CZG: AC:G S 421.C C=S" TCOCCAX ASSD. aODCZO CACAO= d l1 GIC10 AACC:crr TOS" 5 541 T.Cm ATgsC =:a:co.OT AAIIeO=SS IS #01 CCa3A 0:5G"S SO== oswr AL ...

J4Utt Alll s hip em, it. "wF.,r tars FSASSaS ll3YiWIDC GV=V.JJGS CGllYS"R" VVOI TOGS = "

ADO GYD==I. YS:CDGOR mSQYWSV CSUS AS.

GYXSSGSSV SV11

Shy, cow ., ma. s 1 S6S SWL"A"A SSSCSWI G=50t5 cccmius csc I'- TIC

24: 301 ACC talTA5a.S1L SG;O GAIT=5C" C 34S A CSACTCa" AAC:GASCIW Scam C At 481, À41 SWC ASG.lA SO "5SS ache 601 "

Mutest X say \ -. q ma I(FFLV GS:UlISIS tF = -

T SP" = RS= DS

TYVSW G7YD:Ym2 YS=GD QS NlCSrGS NWAYQVL GYJSSGSSHV al-

\.lO "a. . A=s,Gr S.aMiB3l.IL T:Z:ZTI' GGF.SúW GAOAC 61 SSGSITS=O t:AACCOCCZC TCCAOC.C 121 COGG=.=

181 Acetic TSG:55TS" SA".CeSSC ACZ SrD.G' 301 ACASZC SCAN 7W C:5" IS CQ

. S41 1XC ASXAACA.A T =or A=TOWC" ASC 601 "

- mat SN3 =O e'\t ' ' AK t HFI,V GDSAPLB5SSS FSAWWlS 1:D:W SVAV-S GIIYS SCI 1GSP= "" DS

S4v^S" G!YDI S Q UXSIS lillKYOV== G''Q8S -

SO - A=

: ' S Asc.a IT 61,--WO GCC AT hAaASW 121 ThAA. CRISES BASS AO=GSZ" SWSIIS 181 A.

301 Y.SO.C 5= 51CDG" Ois O ACCI 433 = US

A5 5 "5 US

A CHAIN of inhibitor Sequence source: Wheat flour xylanase inhibitor Nterrninal: GAPVARAVEAVAPFGVCYDTICTLGNNLGGYAVPNV (35aa) SEQ ID NO. 13 Chrminal: KRLGFSRLPHFTGCGGL (17aa) SEQ ID NO. 14 15 B CHAIN of inhibitor Sequence source: Wheat flour xylanase inhibitor N-terminal: LPVPAPVTKDPATSLYTiPFH (21aa) SEQ ID NO. 15 Lys-C digested Chain B.: 2S LLASURGSTGVAGLANSGIAUAQVASAQK (31aa) SEQ ID NO. 16 GGSPAHYISARFIEVGDTRVPSVE (24as) SEQ ID NO. 17 VNVGVLAACAPSK (13aa) SEQ ID NO. 18 VANRFLLCLPTGGPGVAIFGGGPVPWPQFTQSIUPYTLVVVK SEQ ID NO. 19

as

Claims (1)

1. t CLAIMS

   1. A xylanase comprising the amino acid sequence presented as SEQ ID No. 5.