CA2539902A1 - Method of enhancing degradation of chitin - Google Patents

Abstract

The present invention provide a method of enhancing chitin degradation or weakening the structure of a chitin substrate comprising exposing chitin to a non--hydrolytic chitin binding protein (CBP). The invention further provides a method of enhancing chitin degradation comprising exposing chitin to a non-hydrolytic CBP and a chitin hydrolase. Compositions, including fungicides, comprising non-hydrolytic CBPs and transgenic plants comprising exogenous nucleic acid molecules encoding a non-hydrolytic CBP are also provided.

Description

Method of enhancing degradation of chitin B ackkround Chitin is a linear insolUble pnlymer of 13() -4)-dinked N-acetylglucosamine, which is a common constituent of fungal cell walls, shells of crustaceans, and exoskeletons of insects. It is the second most abuiidant polymer in natiire and each year moxe than one billion tons of chitin is produced in the biosphere, mainly by insects, fiingi, crustaceans, and other marine organistns.

In nature, two major types of chitin occur that are characterized by an anti-parallel ((.r.-c}titin) cii a parallel (0-chitin) nrraneement of the polymer chains.

Despite its resilience, insolubility, d d abundant production, r.hitin does not accumulate in most ecosystems, suggesting that nature has developed efficient processes for chitin degradation. Chitin is degraded for exantplG by chitinnse:;.
Chitinase enzymes are found in plants, microorganisms, and animals. Bacterial chitinase helps to provide a carbon source for bacterial growth. Insects produce cltitinase to digest their ctiticle at eacla molt_ In plants, chitinase is thought to provide a protective role against. parasitic fungi.

Chitinases have been cloned from various species of microorganisms and have been categorised into two distinct families, designated tamilv 18 and family 19 of the g1Srcoside hydrolases, based on sequence similarities (Henrissat and Bairoch, Biochem, J. 293:78 ] -788 (1993)).

In addition. to being important for biolnass turnover, chitin degradation is essential in a variety of biuIugical procosaeE. For examplP, plants are known to produce chitinases in response to attack by chitin-containing fungi, whereas some non-pathogenic fungi such as Trichoderma species are considered as "biucontrol" agents because of their ability tn inhibit other, chitin-containing, fungi. While chitin-containing organisms may be inhibited by exogenous chitinolytic activity, they do need some enauge-ious chitin turnover as part of morphological and developmental processes. A
further example cnncerns insect viruses, whiclt produce chitinases to disintegrate the t peritrophic naatrix of their tar.get organism, during the tu s t stage of infection thereby increasing virus infectiousness.

Degradation of chitin has a number of important applications. For example, chitinases have been shown to have fungicidal activity, and to play a role in infectivity of insect v;ruses. There are also sevPral examples of transgenic plants which by expressing a foreign chitinase gene, have acquired increased resistance towards certain fungal pathogens.

Degradation products of chitin which include the monomeric sugar, N-acetyl~,=lticosatnine, or oligosacchdrides ofN-acetyfglucdsamine can also be used for various different applications.

It can be seen that chitin degradation is important in a number of different fields.
Although chitinase enzyrms have bccn known for some rime and have been studied ir+
various systems, methods of improving the efficiency or the rate of degradation of chitin would be desirable. The inventors' finding that the eff'ic;enc}= of chitin degradation can be influenced by a chitin binding protein provides a means by which the efficiency of chitin degradation can be modulated artificially, by exposing chitin to suc.h proteins Iti work leading up to the present invent.ien, it has surprisingly been shown that efficient chitin degradation by chitinases in the Gram neeative soil bacterium 5'erralia depends not only on the three.Yerrulia chitinascg, but also on the action nf a cmall non-catalytic protein, CBP21, which binds to the insoluble crystalline chitin substrate on which its effect was tested. Although not wishing to be bound by theuty, ihe binding of this non-catalytic protein is believed to lead to structural changes in the chitin substrate, which result in increased substrate accessibility for the chitinases, and hence to improved chitin degradation by chitinases.

AlthouDh C:BP21 was idcntified a number of years previolisly (by Suzuki el al, Biosci Biotechnol Biochem, 1998 Jan;62(I):128-35), no biological role had previously been identified for this protein. This property ofC.BP21 was fuithcr invest~gated by generating and testing CBP21 variants with single mutations on the largely polar

2 binding surfacc. Various mutants lost their ability to proijiutc chitin de.r~,radation, while retaining considerable affinity for the polymer. Thus, binding alone is not sufficient for CBP21 functionality, and this seems to depend on specific, mostly pular intei actions between the prnteitt and crystalline chitin. This is the first time that a secreted binding protein has been shown to assist in the enzymatic degradation of an insoluble carbohydrate vidnosa-hydrolytic disruption of the substrate (Vaaje-Kolstad el al (2005a) JBC 280 (31) 28492-7 which is incorporated herein by reference).
Various homologues of CBP21 have been shown to exist in other chitin-degrading microorganisms, suggesting a general mechanism by which chitin-binding pioteins function to enhance chitinolytic activity in nature. Homologues also occur in chitinase-containing insect viruses, whose infectiousness is known to depend on chitinase effxciency. No biological role had been previously identitied for these homologues.

The findings made by the present inventors thus provide a rneans by which chitin degradation may be promoted in an in viyn, in vat.ro or industrial contr.xt.

The invention thus provides a method of enhancing chitin degradation comprising expusing cbitin to a non-hydrnlytic chitin binding protein (CBP).

Alternatively stated, the present i,7vcntion provides a method nf Fnhancing chitin degradation comprisimg exposing chitin to anon-hydrolytic CBP and a chitin hydrolase, e.g. chitinase and a methed of degrading chitin compl-biug cxposina chitin to a non..-hvdrolytic CBP and a chiti.n hydrolase, e.g. chitina.se.

Chitin is dcfined herein as any rolymercontaining R(1-4) linked N-acetylglucosamine residues that are linked in a linear fashion. Crystalline chitin in the a form (where the chains nrn anti-parallel), [i form (where the chains run parallel) or y form (where there is a mixture o#'parallel and antiparallel chains), amorphous chitin, colloidal chitin, chitin forms in which part of the N-acet.ylglucosamine sugars are deacetylated &
chitosan (a linear soluble polysaccharide composed of 13-(1-4)-linlced D-glucosamine (deacetylated unit) and N-acetyl-D-glucosarnine (acetylated unit), which can be

3 produced commercially by deacetylation of chitan) are a)1 included within thc dpf nition of this term.

Oiiiei- forzns of chitin that are found in nature include copolymers with proteins and these copol_ymers, which include protein chitin matrices that are found in insect and crustacean cells, and any uther naturally occurring nr synthetic copolynters comprising chitin molecules as defined herein, are also included within the definition of "chitin".

The term "chitin" thus includes purified crystalline cx, 0 and y preparations, or chitin obtained or prepared from natural sources, or chitin that is present in natural sources.
Examples of such natural sources include squid pen, shrimp shells crab shells, insect cuticles and fungal cell wtt.lls. 1/xaanpieõ of commexeia.lly available chitins are those available from sources such as France Chitin, Hov-Bio, Sigma, Sekagaku Corp, amongst others.

Degradation of chitin is caused by the enzymatic hydrolysis of the P(1-4) bonds that link the N-acetyigiucosarnine residues present in chitin. Depending on the number of bonds that are hydrolysed, this results in the generation of smaller fragments of chitin, N-flcetylglucosaaiailic n-iultizners and N-acetylglunnsantine monomers.
Chitinases are in general responsible for this enzymatic hydrolvsis.

Irrespective of any influence that exposure to a non-hydrolytic CBP has on the bonds between chitin subunits and any other members of the copolymer that might bC
Preseent, degradation of chitin as referred to herein is confined to hydrolysis of the glycosidic bonds that connect the P(1-4) Ar acetylglucosamine bond units in a chitin substrate.

This can be carried out by a chitinase enzymC, a cl--itosanase (Glycoside hydrnla.ce families 46, 75 and 80), or a lysozyme (Glycoside hydroiase families 2 i and 24). The rate or degree of hydrolysis of the glycosidic bonds that connect the p(1-4) N-acetylghicosamine bond units in a chitin substrate by any of these enzymes may be

4 enhanced in the methods of the inver-ttcn. These enzyruCs arc refcrred to colleetively herein as chitin ktydrolases.

By "enhancing thP degradation of ch.itirk", it is meant promotino or increasing the degree or rate of hydrolysis of the glycosidic bonds that connect the 5(14) N-acetylglucosamine buncl units of a chitin substrate, e.u by chitinme, relative to the narmal or usual degree or rate of hydralvsis of the glycosidic bonds that connect the 0(1-4)1V-acetylglucosamine bond units ofa claitin substratC wiiich is acon when said chitin substrate has not been exposed to a non-hydroJytic CBP. This can readily be deteranined by measuring tbe hydrolysis product forrnation e.g, at certain defined time points or by measuring the amount of undegraded chitin substrate which remains e,g, at certain defined time points. This can be carried out using methods that are well known in the vi t, based Qn e.g_ determination nf liberated reducing sugars (Hom., c.~t al Carbohydrate Polyniers, 56 (1), 35-39. 2004 and references therein) or determination of liberated chitin fragments, e.g_ by quantita.Live analysis of chromatograrns nht.a.ined upon High Perfornn.ance Liquid Chromatography (Hoell, I. el cxl (2005) Biochim Biophys Acta 1748(2), 180-190 and Vaaje-Kolstad, G et al (2005a, supra.)) Zf the rate of hydrolysis, i,e. the number of bonds hydrolysed in a certain time period is -greater when the chitin subst.ra.te has been exposed to non-hydrolytic CBP
than when it has not, then the rate of degradation is considered to be enhanced_ Similazly, if the number of bonds that are hydrolysed in a certain time period is greater when the chitin substrate has been exposed to i,uii-li_ydrolytie CBP
than when it has not, then the degree of degradation is c.onsidercd to be enhanced.

ThP rate or degree of degradation is also considered to be enhanced when the time taken for complete degradation of the chitin substrate is reduced when the chitin substi ate lias becn exposed tc, non-hydrolytic CBP than when it has not. For example, preferably the use of a non-hydrolytic CBP in accordance with the present invention will reduce the time taken for complete deg-adation by at least 2, 3, 4, 5, 6, 7, X, 9 or fold. In addition, the use of a non-hydrolytic CBP in accordance with the present invention can enable the complete degradation of a chitin substrate under cuuditions

5 where previously compiete degradation was not pussibla. For cxaraple, in embodiments of the invention where non-hydrolytic CBP is used in conjunction with a chitin hydrolase such as a chitinase, a chitosanase or tvsozyme enzyme, then in caces where the chitin hydrolase (e.g. chitinase, chitosanase or lysozyme) enzyme alone cannot give rise to complete degradatiozt of the substrate, the described use of a nun-hydrolytic CBP can result in such cnmplete degradation. This effect is also considered to be an enhancement of degradation.

The degree of hyclrolysis is also considered to be enhanced if the overall amount of solubilisation of the chitin substrate based on exposure to a certain amount of enzyme or an enzyme cocktail is increased when said substrate has been exposed to non-hydrolytic CBP, compared to the amount that is achieved when the substrate has not been oxposcd to non-hydrolydc CRP

The rate of hydrolysis is furtherrnui G co sidercd to be enhanced if the rniximum de,,-ree of solublization of a certain eihitin-containing substrate based on exposure to a certain amount of enzyme or an enzyme cocktail is attained faster whexi L1ie substratc has been exposed to non-hydrolytic GBP than when it has not been exposed to non-hydrolytic CBP.

Preferabl,y, any such enhancement of degradation is a statistically significant one, more preferably with a 1 ubability valuc of r0.05. Appropri;ite methods of deterrni.ning statistical significance are well known and described in the art and any of these may be used..

As demonstrated in the Examples, the kinetics of chitin degradation by chitinase hydrolysis of P(1-4) N-acet.ylglucosaXnine bonds when assayed in in vitro experiments are not straightforward linear kinetics. There are several kinetic phases, which in the literature are ascribed to liydiolysis of subfractions of the substra.t.c with varying degrees of crystal linity and, thus, varying degrees of accessibility for chitinases.
Sometimes, individual phases can be observed in degradation progir.ss curves.
The fast first phase is often linear and can be used to calculate initial hydrolytic rates. This phase is believed to reflect hydrolysis of the easily accessible and amorphous (i.e, the

6 chitin chains are not orderly arranged a.s in a crystal, but rathrt disordercd and thus in more contact with the solvent) regions in the substrate. During subsequent slower phases it is believed that the more recalcit.rant crystalline regions of the substrate are degiadcd. Similar lCin6tics a.re likely to appiy in respect of other chitin hydrolases.
The non-hydrolytic CBP as used hcrein in the methods of the invention result in a minor enhancement in the degree or rate of 0(l -4)1V acetylglucosamine bond hydrolysis by the chitin hydrolase in the first, fast phase of the dCgradation process.
However, a large effect is observed on the second slower phase. It is therefore important that if such assays with chitin hydrolases such as chitinase are per*ormed (e.g. to detcrminc experimPnt.a.lly whether a non-hydrolytic CBP has in fact functioned under the particular experimental set up to enhance the degradation of the chitin substrate, according to the dcfinitions provided herein), a suitably long incubation time is used. As such, to detect the effect of a non-hydrolytic CBP
on the rate or degree of chitin hydrolase, e,g. chittnase, degraddtiuii it is ncccssary to nssay the rate or degree of chitin deVadation after sufficiently long incubation times such that the reaction enters the subsequent, slower phase. In Example 2(see Figure 2) tt,is subsequent phase is ohsPrved after the chitin (in this case f3 chitin), non-hydrolytic CBP (in this case CBP21) and chitinase (in this case ChiA, B, C and G) has been incubated together for approximatcly 16-24 or 50 hniirG.

The precise kinetics will depend on rnany factors, 3ucL as the type of chitin, the denree of amorphou.sness of the chitin, the amount of enzyme present, the temperature and the pH. The degree of amorphousness will vary with the substrate source and isnlation/purification process, but can be assessed by measuring the degree of crystallinity of the substrate (which is a method known in the art).
Furthennore, since a-chitin is a mUi e recalcitrant chitin form r.nmpared to the 0 -aariant, it is anticipated that longer incubation times will be necessary in order to observe chitin degradation and hence any effect ot'thc non-hydrolytiu CDP on the rate or degrPP nf chitin degradation.

Taking these considerations into account therefore by choosing an appropriate incubation tinae, the skilled person would readily be able to determine whether the

7 degree or rate of chitin degradation is eahancCd in accordanco with the present invention.

The ahnve considerations apply irrespective of the order in which the chitin is exposed to the non-hydrolytic CBP and the chitin hydrolase, e.g, chitinase, i,e. ifnon-h_ydrolytic CDP and chitin hydrolase, P.g chitinase are not adininistered to the chitin substrate sirnultaneously, or the chitin substrate is not exposed to non-hydrolytic CBP
and chitin hydrolase, e.g. chitinase, togethei uR rhe chitin substrate is exposed first t.n non-hydrolytic CBP and then to chitin hydrolase, e.g. chitinase (for example wherein the exposure to chitin hydrolase, e.g. chitinase occurs such that. the chitin hydrolasG, e g. chitinase is added to the chitin and non-hydrolytic CBP or the non-hydrolvtic CBP is first removed and the chitin substrate is then exposed to the chitin hydrolase, e.g. chitinase alone), the efficacy of the method can be determined in the same way.
Comparison ot'the rate or degree uf tiydrolysis of the glycosidic bonrlti connecting 0(1-4) N-acetylglucosarnine units by an appropriate chitin hydrolase, e.g, chitinase of a chitin substrate that has been exposed to non-hydrolytic Cl3P with the rate or elCgree of hydroly.sic of the glycosidic bonds connecting 0(1-4) N-acetylglucosamine units by an appropriate chitin hydrolase, e.g. chitinase of a chitin substrate that has not been exposed to nun-liydrolytic CBP witl similarly shnw whether or not the degradation of the chitin substrate is considered to have been enhanced in accordance with the present invention.

The methods of the invention can alternatively be viewed as a method of weakeiii,lg, e.g rii.arupting or interfering with or modulating the structure of a chitin substrate, comprising exposing chitin to a non-hydrolytic CBP. As noted above, and as described in the Examples, it is believed that the function of e.g. CBP21 (and other non-hydrolytic CBPs) is to interact with and affect the overall structure of chitin such that it becomes more sensitive to degradatiu)ilhydrolysis by chitinases, which niay be due to improved accessibility of the chitinase substrate of chitin.. The structure of chitin is preferably weakened without any hydrolysis of the (31-4 N-aceiylglucosamine bonds present in said chitin.

8 By weakened, it is meant simply that the chitin subst<atc is rcndcrcd more sensitive to chitin hydrolase, e.g_ chitinases, i.e. it is more easily digested or hydrolyssd by chitin hydrolases, e.g. chitinases. Alternatively stated, the structure of the chitin substrate is dismrt.ed so tlaat a structural change can be observed by electronrnicroscopy , or such that the mechanical strength of the chitin-containing structure is reduced, or such that a reciucliun in crystallinity can be observed using atomic force microscopy or electron scattering analysis.

By "non-hydrolytic CBP" (CBP = chitin binding protein) it is meant a pratein that binds to chitin but that itself has no hydrolytic enzyme activity, e a. no significdnt ur dPt.ectable chitin hvdrolase, e.g, chitinase activity (it cannot hydrolyse the (3-1,4- N-aeetylglucosamine linkages that are present in the chitin substrate), or no significant or detectable ariwnanasc activity (it cannot hydmlyse the 5-1.4- N-m annose linkages that are present in mannan). As such, the use of whole chitin hydrolase, e.g.
chitinase enzymes such as ChiA, ChiB and Chi C which havc chitinase activity and contain one or more chitin binding modules is explicitly excluded. Assays to detect chitinase activity (or m.annanase activity) are well known in the art. The absence of chitinasc activity or mannanase activity in a CBF can therefore readily be determined.

Ps oteins that have the ability to binrl to carbohydrates are widespread in nature. ln general, such proteins contain one or more carbohydrate binding modules as part of their overall structure. These protcins st-iay for example be structural or signalling molecules or they can be enzymes, and the overall function of the protein may be cletermined by domains that are present in addition to the carbohydrxtc Linding rnodule. A carbohydrate-binding module (CBM) is defined as contiguous amino acid sequence within a carbohydrate binding protein with a discreet fold having carbohydrate bin.ding sctivity Indeed, chitinases are known which contain one or more chitin bindin(i modules in addition to catalytic regions_ ChiA of Serrcrtia 1Vlarce,+=cens contains a libroriectin type IQ - type CBM, rhiB of RS'errcr/iulVlarcNscens contains a family 5 Ct3M and ChiC ofr,Serrafla Murcescens contains a famiSy 12 and a fibroraectin type III - like CBM. See Y. Hourne, B. Henrissat, C'++rr. OpOn.
.Strucl.
Biol.. 11, 593 (2001) for domain nomenclature.

Proteins lhal bi,id to carbohydrates may be classified baaed on function and also based on structural and/or sequence characteristics. The proteins that are af particular use in the present invention are chitin binding proteins that contain chitin binding modules that are members of family 33, accordin; to the CAZY classification system (htt :/p /www.cazv.or CA.ZYlfam/acc CBM.html), which is based on sequence similarities (Davies, G. J., and Henrissat, B. (2002) Biuchem Jnc T 30, 291-297 and Y. I3oume, B. Ifenrissat, (2001) supra). The non-hydrolytic CBP as defined herein gcncrally contains a carbohydrate binding module that is a member of familv'i'i, and is thus referred to as a family 33 CBP.

All members of family 33 bind to carbohydrate structures (primarily chitin) and contain a family 33 carbohydrate binding module. In several case,ti, the chitin binding module makes up the whole protein, i.e. the chitin binding protein consists of or consists essentially of a single family 33 chitin binding module, that is in nature synthesised and secreted as such. HowevPr cnme family 33 CBMs may be fused to one or more additional non-catalytic carbohydrate binding modules (e.g. CBM
family 2, CBM family 3 and CBM family 5 modules). These proteins are bi- or multi-modular non-catalytic chitin binding proteins. There is also one known example of a family 33 carbohydrate bin.ding module that is present as an individual module within a inuch larger catalytic protein. This is the 0 1,4-tnannanase protein of C'alclihctcillus cellulnvorans (Sunna A el aX (2000) Applied and Environmental Microbiology 66(2):
664-670).

The family 33 CBM is appioximately 150-250 amino acids, c.g_ 160 740, 170-230, 180-220, 190-210 amino acids in size and has a molecular weight of a approximately 20kDa, preferably 19-21kDa, 18-21 kDa, I 9-22kDa or 1 s-20kUa in size. The size of a protein can readily be determined by standard methods that are known in the art.

ThP nnn-hydrolytic CBP is thus preferably a. family 33 CBP. In general, the non -hydrolytic C:F3P contains a single family 33 chitin binding module.
Preferably, the nnn-hydiulylic CBP wnsists of a single family 33 chitin binding module, or concictc essentially of a family 33 chitin binding module.

if said nun41ydroly-tit CBT' "consists e.ssenti:;lly of' a family 33 chitin bin.ding, rx-adule, it is meant that additional amino acids may be present in the protein, in addition to those that make up the famAly 33 Li3M. Preferably there are 1-3, 1-5, 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90 or 90-100 or more additional atnino acids present. These additional arnino acids are in general present C
termiinAl to the family 33 CBM.

AlternativC)y, the non-hydrolytic CBP can comprise a family 33 CBM Additional modules or domains may thus be present in the protein. These additional modules or domains are non-catalytic or non-hydralytic modules or domains a,ncl a.s such tlie CDP
as a whole is non hydrolytic. The additional modules or domains can be CBMs.
Examples of such module5 are CBM family 2, CBM fainily 3 and CBM family 5 modules, if additional domains or modules are presPnt, they are in general found C-terminal to tiie family 33 CBM. The presence of additional domains or modules will of course increase the overall size of the protein.

The non-hydzolytic CBP can be or can comprise or correspond to a naturally occurring fainily 33 chitin binding module such as CBP21 (or to a homologue thereof from another species)- It can alternatively be or can comprise or correspond to a vlriant of a naturally occnrring non-hydrolytic fatnily 33 CBP.

Naturally occurring non-hyJ.iolytic CDPs that can be used in the invention inclurle.
m,icrobial (e.g. bacterial), eukaryotic (e.g. dictyostelium) or viral non-hydrolytic CBPs. Bacterial non-hydrolytic CBPs are however preferred.

Examples of suitable non-hydrolytic CBPs are set out in Table 1:
ll BACTERIA

PRbTEIN ORGANISM GENBANKIGENPEPT
Cbpl Alferarnonas sp, 0-7 A8063629 BAB79619.ti chitin binding proteio ChbA Sacillus arnylelr7uefaciens ALKO 2718 AF181997 AAG09957.1 BA_3348 @aciflus antryracis str, a2012 NC 003995 NP_656708.1 BA2827 Bacillus anihracis sfr. Ames AE017033 AAP26659.1 NC003997 NP_345173.'i BA2793 Bacillus anthracrs sfr, A-nes AE017032 - AAP25628.1 NC_003997 NP_845142.1 tD CBAA2827 Baciltus anfhraafs str. Ames 0581 AE017334 AAT31944.1 GBAA2793 Baciflus anfhracis sb: Arnes 0581 AE017334 AAT31910.1 t3AS2636 Bacfflus anthra,rs str. Sterne AE017225 AAT54946.1 BAS2604 Bacillus anfhracis slr. StFerne AE017225 AAT54914.1 BCE2855 Bacilfus cereus A TGC 10987 AE017273 AAS41767..
NC_003909 NP979159.1 8CE2824 Bacilfus cereus A TCC 10987 AE017273 AAS41736.1 n NC 003909 NP 979128.1 BC2827 BaciAous cereus ATCC 14579 AE017007 AAP09778,1 IVC_0C4722 NP 832577.1 3C2798 Bacrlfus cereus ATCC 14579 AEO17007 AAP09751.1 NC004722 NP 832550.1 pE33L466_027E (ChbA) Bacillus cereus E33L CP000840 AAY60428.1 BTZK2523 (Ch6) Bacillus cereus ZK CP000001 AAJ 17736.1 BTZK2552 (ChE) Bacillus cereus ZK CP000D01 AAJ17707.1 ABC 1161 Sacillusclaus'l KSM-K.;-6 AP006627 BAD636991 BH1303 Bacillas haloaurans C-126 AP00- 511 BAB050221 NC_002570 NP_242189.1 BL900521 or BL40145 Bacilfus Jtchertiformis CSM 13 ATCC 14580 CP000002 AAU22121.1 AE017333 AAU39477.1 3T9727 2586 (ChB) Baciflus fhuringiensis serovarF:onkukJan str. 97=27 AE017355 AAT6131 0.1 3T9727_2556 (ChB) Bacillus thurir-grerisis serovar a:onkukian str. 97-27 AE017355 AAT61323.7 3MAA1785 Burkholderia rtialfei A TCC 23344 CP000011 AAU45854.1 3MA2696 8arisholderla maltea ATCC 23344 CP000010 AAU48386.1 3URPS' 7i0b Oi14 Burkholderia pseudorrailel 17;Ob CP000124 ABA49030.1 3URP5:710b_P2047 Surkhofdertapseudorrailer 17;Ob CP00o125 ABA53645,1 BPSL3340 Surkitcfderia pseudorr,alfei 1t96243 BX571965 CAH37353.1 BPSSO493 Burknofderra pseudorr:alle+ K96243 BX571966 CAH37950.1 Bcep18' 94_C6726 Burfshaiderra sp. 383 CP0001 50 ABB05775.1 BTH_111925 Burkhoideria haiiandensfs E264; ATCC 700383 CP0fl0085 ABC34637.1 tD BTH_I3219 Burfrhaiderfa nailandensrs E264; ATCC 700389 CP008086 ABC38514.1 =1,4-mannanase (ManA) Cafdibaciffcs celluiovorans AF163837 AAF22274.1 CV0554 Chrornobacterium vioiaceumATCC 12472 AE01E=911 AAQ582301 o NC_005085 NP_9o0224.1 CV0553 CnrornobacfarrUm vroiaceUrnAFCC 12472 AE01E911 AAQ58229 1 o NC 005085 NP 900223.1 m CV2592(CpbD) Chrornobacferitlrn violaceum A.TCC 124 72 AE016919 AA060262 1 N NC_005085 NP_902262.1 CV3489 Chrornobacterium violaceum ATCC 12472 AE01 6922 AAQ61150 1 ~ htC_005085 NP_903159.1 CV3323 (CbpDl) Clirornobacteriurn vioiaceum A TCC 12472 AE016921 AAQ609871 NC_005085 NP_902993.1 EF0362 ' Enferococcus faecalis V583 AE016948 AA080225 1 NC_004668 NP 814154.1 Sequence 4287 froin pwenf US 6583275 Enferococcas iaecrum - AA0437291 FTL_1408 Frarrciseila tularensfs sobsA, horarctfca L VS AM233362 CA,J79847.1 FTT0813c Frarrcfseifa fuJarensfs 3ubsp. tularensfs Schu 4 AJ749949 CAG45449.1 HCH_00807 Hahella cheJu:rtsis KC TC 2396 CP0001 55 ABC27701.1 HCH_03973 Nahena chejuensis KGTC 2395 CP000155 ABC30692.1 p_1697 Lactobaciifus piantarum WCFS1 AL935256 CAD641261 NC_0U4567 NP_7B5278.1 _SA1003 Zactobacilfes sakei srabsp, sakei 23K CR936503 CA155310.' YucG Lactococcus iactis subsp, iacfis fL1403 AE006425 AAK06049.1 NC_002662 NP 26810b.1 Ipp0257 Legioneiia pneumophita Parrs CR628336 CAH1'f404,1 Lin2611 Listena ionocua AL59E173 CAC97838.1 NC 003212 NP_471941,1 Lmo2467 Listeria monocytogenes EGD-a AL591983 CAD00545.1 NC_003210 NP465990.1 tD
LMOf2335_2444 Lrsteria monocyfogenes sb: 4b F2365 AE017330 AAT05205.1 OB0610 aceanobacillas iheyensis HTE831 AP004595 BAC12766 1 tD NC_0041 g3 NP_691731.1 PBPRB0312 Photobacferiutt profundum SS9 CR37S676 CAG22165.1 pau2352 PhotarhabdUs iaminescens subsp. laurnondii 7'TOI BX57' S66 CAE146451 rn NC 0a5126 lJP929598.1 rn ~ Sequern,e 6555 from pa:ent US 6605709 ProfeJs miraWlis - AAR43285 1 chitin-binding protein ChiB Pseudoalteromonas sp. S9 AF007895 AAC796661 chitin-biiding proteEn (CbpD;PAG852) Pseudomonas aeruginosa PAO 9 AE004520 AAG04241.1 NC_002516 NP_249543.1 chitin-bi-icfing pr3tein (CbpD) Pseudornonas aerugrinosa PA D25 AF196565 AAF12807.9 PFL_2090 Pseudornonas fltiores.ens Pf-5 CP600076 AAY91365.1 PFl3569 Pseudomonas flUores.ens Pft)-f GPO00094 ABA75387,1 Psyr_2856 Pseudomonas syrtngce pv. syringae 8728a GP800075 AAY37892,1 PSPTO2978 Pseudomonas syring2e pv. tornato str. DC3000 AE016866 AA056470.1 NC0134578 NF_792775.1 RF 0708 Rickettsra Felis !lRRVbXCa12 CP000053 AAY61559,1 chitift-binding pratein (CjpA) Saccharophagus deg'radarrs 2-40 BK001045 ORF Sallnivibrio cosficola 5SM- I AY207003 AAP42509.1 chitin-binding p-otein (Cbp21) Serratia r=rraroescens 2170 A8015998 BAA31 569.1 chiiin-binding p-otein (Cbp2l) Serratia marcescens BJL200 AY665558 AA1188202.1 ORF2 Serratia marcescens KCTC2172 L38484 AAC37123.1 SO1072 Shewanella c=neidensis 14rR-1 AE015551 AAN54144.1 NC_U04347 NP_716699.i SG1515 (possible fragrnent) Sodslis glossinidiUs str. 7morsitans' AP008232 BAE74790_1 SAV6550 Streptomyces avermi!Ns Jl9A4680 AP005047 - BAC74271.1 NC_003155 NP_827736.1 SAV2169 Streptomyces avermPffis MA-4680 AP005029 BAC69879.1 tD NC_003155 NP_823344.1 AP005042 BAC72935_1 SAV5223 ~Chbl StreAtomyces averrni*ilis MA-4680 NC_003155 NP_8264001 o SAV2254 (CeIS2) Streptomyces avermr?i~is MA-4880 AP005030 BAC69965.1 ftiC_003155 N P_823430.1 SC07225 or SC2H92.24 Streptomyces coericolorA3(2) AL359215 CAB94648.1 NC 003888 NP-6312F1.1 N SCO6345 or SC3A7.13 Streptomyces coelicofor A3(2) AL031155 CAA20076.1 NC-003888 NP f304-7.1 ~ _ -SCO2533 (Chb) Strepfomyces coeircolor A3(2) AL136058 CAB65563.1 NC 0a3888 AJP_6270E2.1 SC00643 or SCF91.03: Strelo-fomyces coelicolor A3(2) AL132973 CAB67160.1 - NC_003888 NP_6249!2.1 SC00481 or SCF80.02 Streptomyces coelicolor,43(2) AB017013 BAA75647.1 AL121719 CAB57190.1 NC 003888 P1P_624799.1 SCO1734 or SC111.23 Str'epfomye,es caeRicorar A3(2) AL095849 CAB50949.1 NC C,03888 NP 6260C7.1 CeIS2 (SC01188 or SCG11A.19) Strepfornyces tflelfcolorA3(2) AL133210 CAE61600.1 NC_003888 NP 625478.1 chitin binding protein Sfrepfc-myces giriseus AB02371185 BAP.86267.1 oelluiose b9nding grote9n (ORF2) Str+eptomyces hal.sfeclii 1151222 AAC45430.' chitin-binding protein Sirept+o-rnyces olivacenviridfs ATCC 11238 X78535 CAA55284.i chitin binding protein (Chb2) Streptomyces reticuli Y14315 CA,474695.1 chitin-binding protein {Cba1} Strepfomyces jhermovWaceus OPC-520 AB1 10078 BAD01591.1 chitin-binding prtriein ce1S2 Sfraptomyces riridospcrus AF126376 AAb27623.1 Yfu_1665 (ES) Therrrobifida Pasca YX CP000088 - AAZ55700.1 -1 tu_7 263 {f=7} Therrnobihda iusca YX CP4000S8 AAZ55306.1 VCA0145 V;brio cholerae N16961 AE004355 AAF96053. i tD NC_0C2506 NP23254C.1 'JCA0811 Vbrio cho/erae N16961 AE004409 AA7=96709. S
NC 802306 NP 233197.1 to - -'dFA0143 Vrbria fsci~err E5 1 14 CP000021 AA'N87213.1 VFA0013 4lrbrio frscheri ES? 14 CP000021 AAd1/87083.1 WpA0092 Vibrio Aarahaemolyticus RIMt)2210533 AP005084 BA~B1435.1 iJC 084605 NP_799602.1 04 o VPA15~8 Vfbrro parahaernofyticus RlN1D 2210633 AP005069 BAC62941 1 NC 004605 NQ_801108.1 1N27258 Vibric vulO+ficas CMCP6 AE016812 AAOO8152.1 NC_004460 NF_763162.1 VV20044 Vrbrio vulnilFc!js CMCP6 AE016808 AA.007021.1 NC004460 NP_762031.1 VVA0086 Vibrio vulniflals YJ01S AP006344 BAC96112.1 NC_005140 NP_936142,1 VVA4551 Vibrio vulniffcas YJ096 AP005346 BRC96577.1 NC_005140 NP_936607.1 GhiY Yers;nia enterocolrtica (type 0;8) WR-394 A.i344214 CAC83040,2 "PU706 Yersinia pestis biovarl4edieva,is sfr. 91001 AED17129 AAS60972.1 NC 005810 NP 992095.1 YP03227 Yersinie pestis C092 AJ414158 CAC92462.1 lVC9C3143 NP 406699,1 Y0962 Yersinia pestis K;M AE013699 AAMS4543,1 NC_O04088 NP 668292.1 YPTB3366 YersiMa pseadotuberctllosis 1P 32953 BX938398 CAH22604.1 YPTB08n Yersirda psoudafuberr.ulosis !P 32953 BX936398 CAH2O139_1 tD
EUKARYOTA
tD
N PR47EIN flRGAWISh9 GENBANKIGENPEPT
ORF-26 Agrotfs seget+im nocJeopolyhedrovirus D0123841 AAZ3S192.1 spheroicin-Iike protein (Gp 37) Autog.apha califomrcanucieopolyhedrovfrus L22856 AA466694.1 LO D00583 BAA00461.1 N -0 NC_001623 NP 05409L.1 4 'Tusolin Bombyx morr nuclear p olyhedrosis virus U55071 AAB47606.1 L33180 AAC63737.1 NC001962 NP_047468.1 spheroidin Chorisforreura b+enrrisentomopoxvrrus M34140 AAA42887,1 VIRUSES

PROTEIN ORGANISM G ENBANKIGEl1PEPT

ORF-26 Agrotis segetam rruc-eoporyheadrovirtrs DQ12384'f AAZ38192,1 Spheroidin-Iike protein (Gp 37) Autoc.~rapha califomice nocle,oAolyl'tedrovfrvs D00583 BAA00461.1 1..22858 AAA66694,1 NC_OQ1623 NP 054094.1 fusolin Bombyx rrron noclearpolyhedrosis virus U55071 AAB47606.1 NC ~D1962 ~JP_Q47468.1 L331E0 AAC63737.1 tD
5pheroidin Chorfstonearnienrirs enfomopoxvirus M341140 AAA428S7.1 3RF60 Choristoneura fumiferana def+ectrve nu,~IeopolyhedrovirusAY327402 AA.Q91 667.1 t0 NG_005137 NP_932660.1 spindle-Iike protein Choristoneurs fumrferar+a nucJearpofy.hedrosis virus U28734 AAC55636.1 NC_0,?4778 NP_848371.1 GP37 (ORF-67 GP37) Chrysodeixis chalcites nucJeopofyhedr-3vFrus AY864330 AAY83998.1 rn ~ ORF57 Ep+jahyas postvttlarta r~ucleopolyl7edrovrrus AY043265 -~AfK85621.1 NC_003083 NP_203226.1 GP37 Helicoverpa amigera single rtucleocapsid poJy.hedrovirus4F266696 AAK57880.1 AF3d3045 AAK963051 NC_003094 NF_203613.1 ORF59 Heflceverpa zea nac'eopolyhedmylrus AF33q030 AAL56204.1 NC_003349 NP_542682.1 gp37 Nelrocoverpa armigere rnucfeopolyhedrovrrtrs G4 AF271059 AAG53801.1 NC_402654 NP_075127.1 fusolin HelroCais armigera entomopoxvirus L08077 AAA92858.1 HyrlVgpOB6 (sip) Hyphanfria cuaea nuc0opolyhedroviros AP009046 BAE72375.1 Gp37 protein Leocania separaha nuclear pol,rhedrosis virus A8009614 BAA24259.1 fusolin-like protein Lymaatria disparnijdeopofyhEdrovirus U38895 AAB07702.1 AF481810 AAC70254.1 NC_001973 NP047705.1 gp37 protein Mamestra brassicae nucfeopo.ryhedrovirus AF108960 AAD45231.1 ORF 37 (Gp37) Mamestra configurata nucleopoiyliedroviras A U59461 AAM09145.1 AF539999 -=AAQ11056.1 Gp37 Marzmsfra corrhgurafariucleopvlyhedruvirus B AY1262:75 AA.M95019.1 NC_004117 NP_689207.1 spheroidin-like protein (Gp 37) Orgyia pseudctsugata nuclearpoJyhearosis virrs U75930 - AA.C59068.1 D13366 - BpA02566.1 NC_001875 NP 046225.1 tD enhancing factor Pseudalefia s. .parafa entomopoxvirus D50590 Bf+A09138.1 ORF25 Spodoptera exigua nrucleopniyhecirovirois AF169823 AA.F33555.1 NC_002169 NP_037785.1 o gp37 (fragment) Spodaptera hvgiperda MNPV AY250076 A.A.P79107.1 ubiquitin GP37 fusion protein Spodoptera Mura nucaeoporyhadrovirus G2 AF325155 FlP.L01718.1 NC_003102 NP258300.1 gp37 Trichoplusia rni singie nucfeopoJyhedrovirus i7Q017380 AA.Z67435.1 N fusolin un+dentifed eatomopoxvirus X77616 CAA54706.1 ORF107 Xestia c-nrgrum granUiovirus AF162221 AP,F052-21.1 NG 002331 NP 059255.1 Rac,tPrial nnn-hyrlrnlytir, ('RPs nan he.f.rom any appropriate sourc.e but are preferably fmm a species selected from the group consisting of Bacillus, Chromobcrclerium, L= rllerUcuc:cus, 1='rurcusel.lu, Huyrellcr, Luc.labucill.zj.s, I,act.vcvccus.
Lcgionella, Ll.slerla, flceanohacxllus, I'hotobacteriunt, l"'hotothahdzr.s, Proreu.;-, P.;-etjd.oaltero zorias, 1'seudomonas, Rickettsia, .Sitccharophahus, Scrlimvihrio, ~Serraria, Aewctnella, .S'odali.s, Streptomyces, Therniobifida, Vihrzo and Yersinia.

Examples of specific non-hydrolytic CBPs are CBP 21 of Serratia Marescens (SEQ
[D NO: 1, M MCTSRTLLSLGLLSAAMFGVSQQANAT T(-,'S'VESPASRAY'QC'.Kl.QI.NTQCGS
V QYEP Q S VEGLKGFP QAGPAD GHIA S ADK S TFFLLDQ QTP TRVti'NKLNLKTGP
NSFTWKLTARHSTTSWRYFITTLPNWDASQPLTRASFDLTPFCQFNDGGAIPAA
QVTHQCNIPADRSGSHVILA-v'LVDIADTANAFYQAIDVNLSK, where amino acid residues 10 to 27 correspond to the leader peptide that is necessary for secretion of the protein in a natural system and amino acids 28-196 correspond to the mature protein), ChbA of 13.amyloliquefcrciens (Chu el al, Microbiology. 2001 Jul; 147(Pt 7):1793-803) CHB l, 2 & 3 of.Srreplorrryce.+ (Svergun .-r al., Biochemistr),. 2000 Sep 5;39(35):10677-83, Zeltins et al,, Eur r}3iochem. 1997 Jun 1;246(2);557-64, Zeltins el al., ,A.ztal Bxochezzt. 1995 Nov 1;231(2);287-94, Schnelrrnaztn er al., Mol Ivlicrobiol.
1994 Sep;13(5):807-19, Kolbe et al., Microbiology. 1998 May;144 (Pt 5):1291-7, Saito et al,, Appl Environ Microbiol, 2001 Mar;67(3): i268-73), CJ.3P 1 of Allerantryrras ((Tsujibo cyl al, Appl. Environm. Microbiol. 68:263-270 (2002)). All of these references are incorporated herein by reference, The non-hydrolytic CBP can thus be or correspond to or comprise a naturally occurring CBP (such as CBP21, CIibA, CHB1, 2&. 3 atttl CBPI) in thLtt it is a CBP
(e.g, a family 33 CBP) that is found in nature.

Alternatively, the non-hydrolytic CBP can be derived from a naturally occurring protein whicii contains an appropriate CBM, such as the p 1,4 mannanase referred to ahnve., nr any nther iamily 13 C'RP that r,ontaim rnnrhylPy nr ric7mains that srF nrecent in addition to the family 33 CBM. In the latter case, the appropriate CBM for use as a non-hydrolytic CBP in accordance with the present irtventiurt cart be obtained by any dppzvl-iiate method. For example, the s4quence that encodes the CBM can be determined and eapressed in isolation from the rest of the protein using, stan.dard molecular biology techniques that are known, in the art. Alternatively, for example, the CBM can be reni4ved from the native protein by for example proteo]ytic cleavage.
Variants, derivatives or fragments of naturally occurring CBPs or CBMs can also be used, providing that these variants, derivatives or fragments retain the functional propcrty ofthc CBP in that they bind to chitin and enhance the ciearaclatinn nf chitin or weaken chitin as described herein, for example by causing disruption to the cltitiu substrate, i.e. they should be considered to be functionally equivalent vrti-ant,. As noted elsewhere herein, the property can be tested for in a straightfotward manner_ The non-hydrolytic CBP as defined herein has the properfi> of being able to bind to at least cne type of chitin. It inust also have the property, which has been demonstrated for CBP21, of enhancing the degradation of chitin nr weakening chitin. Whether or not a protein has the ability to bind to chitin and to enhance the deruadation of chitin or weaken chitin by for example causing disruption tu tlic chitiri sufnstrate can rcadily be determined by straightforward assays that have been described in the art e.g. by using the assays that are set out in Examples 2-3 and Vaaje-Kolstad er ctl ((2005a) supra). For example the rate of chitin degradation by chitin hydrolase, e.g.
chitinase in the presence and absence of the putative non-hydrolvtic CBP or following exposure to the putative non-hydrnlytic CBP can be compared.

variants include or comprise iiatuially occun~ing variants of the non hydrolytic CBP
as defined herein such as comparable proteins or hornolopues found in other species or more particularly variants found within other micrUorganisms, which have the functional properties of a non-hydrolytic CBP as defined elsewhere hercin.

Variant_a of the nat.nral ly occurring non-hydrolytic CBPs as defined herein can also be generated syntbetically e.g. by using standard molecular biology techniques that are known in the art, foi cxnMple standard mutaf;cnc.sis techniques such as site directed or random mutagenesis. Such variants further include or comprise proteins having at ieast 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 991/o sequence similarity or identity with a naturally occurring CBP or CBM at the amino acid level.

When variants are generated, it should be noted that appropriate residues to modify depend on the propertics that arc bcing sougl-it in such a vaiia,a. lii tlic casc LhaL a variant having the same binding or enhancing or weakening properties of the non-hydrolytic CBl' is being sought, the residues are in general those residues that are not involved in t.he int.eraction of the non-hydrolytic CBP with the chitin substrate.
Preferably the residues that are modified are residues that are not involved with the property of enhancing or weaken.ine degradation exhibited by the non-hydrolytic CBP
(although of course it is possible that this property could be further improved by mutating these renidues), Un the other hand, it -ivill be appreciated by a person skilled in the art that residues that are crucial for the functionality of CBP21 (see Example 4), or analogous or corresponding residues in CBP21-like proteins may be mutated with the aim of further improving the function of tfie non-hydrolytic CBP. This could be done by standard protein engineering techniques or by techniques based on irandom rnutagenesis followed by screening, all techniques that are well known in the art.
Attempts to improve the function of a non-hydrolytic CBP may include improving the binding and disrupting ability of this non-hydrolytic CBP towards other types of insoluble carbohydrate polymers than the polymer(s) on which the CBP acts naturally.
Such alternative polymers include carbohydrate containing copolymers, e.g.
protein-carbohydrate co-polymers.

For Example, in the case ofCBP21. several residues have been shown to be iinpnrtant in the binding of CBP21 to chitin and more specifically to the ability of CBP21 to enl,nnce the degradation ofchitin. Scveral rnutations hlvc bccn shown not to affcct binding, but to affect the ability of CBP2] to enhance the degradation of chitin.
These residues are preferably not modified relative to the wild type CBP21 sequence as set out in SEQ ID NQ:1, if the aim is to niodify e.g. the 5tability of the non -hydrolytic CBP (for example under process conditions), but these residues may be targeted if one's aim is to improve or chanRe the non-hydrolytic CBP
functional properties.

ZL

A PPrcnn ckilled in the art will recctgnize the potential of using the CBP
Framewmk to create variants that are optim.ised for other insoluble polym-erie polysaccharide substrates (C.g. Utl1CI ful!!!s up4lJatlil ul cellulose), oj iirsolub-le crfbohyditate-containing co-polymers.

Pref.erred variants of CBP21 retain one or more and preferably all of: a tyrosine residue at position 54, a glutamic acid residue at position 55, a glutamic ac-id residue at position 60, a histidine residue at position 114, an aspartic acid residue at position 182 and an aspar'agine at position 185 (sequence numbering according to SEQ ID
NO.1).

Further preferred variants of CBP21 retain one or more and preferably all of:
a tyrosine residue at position 54, a glutatnic acid residue at position 55, aglutamic acid residue at position 60, a histidine residue at position 114 and an aspartic acid residue at position 182 (sequence numbering according to SEQ ID NO;1).

In connection with amino acid sequences, "sequence similarity", preferably "sequonee identity", refers to sequences which have the stated value w'hen assessed using e.g.
using the SW1SS-1'RU'1' protein sequence databank using FASTA pep-cmp with a variable pamfactor, and gap creation penalty set at 12.0 and gap extension penalty set at 4,0, and a window of 2 amino acids). Sequence identity at a pariicular residue is intended to include identical residues which have simply been derivatized, Preferred "variants" include those in .vhich instead of the naturally oc.curring amino acid the amino acid which appears in the sequence is a structural analogue t.hereof.
Amino acids used in the sequences may also be derivatized or moditied, e.g, labelled, glycosylated or methylated, providing the function of the non-hydrolytic CBP
is not significantly adven;ely affected.

Variants refers to peptides related to, or derived from the above described amino acid secluences, where the amino acid secluenc:e. hac heem mndified by single or multiPle amino acid (e.g. at 1 to 10, e.g. I to 5, preferably 1 or 2 residues) substitution, addition andior deletion or chemical modification, including deglycosylaliun ui glyrnsyla.tinn, hlit.which nnnPthelPCS rPtnin fimr.,tional activity, insofar as they bind to chitin and enhance the degradation of chitin.

Within the meaning of"addition" variants are included amino andior carhoayl terminal fusion proteins or polypeptides, comprising an additional protein or polypeptide or other rnolecule fused to the non-hydrolytic CBP sequence, Carboxyl terminal fusions are prefen-ed_ It must of course be ensured that any such fusion to non-hydrolytic CBP does not adversely affect the functional nroperties rerl7rirPCl for use in the methods of the invention as set out elsewhere herein, "Substitution" variants preferably involve the replacement of one or more a.mino acids with the same number of amino acids and making conservative substitutians.
Such functionally-ecluivalentvariants mentioned above include in particular naturally occurring biological variations (e.g. found in other microbial species) and derivatives prepared using known techniques. In particular functionally equivalent variants of the non-hydrol_ytic C:13Ps described herein extend to non-hydrolytic CBPs which are functional in (or prcscnt in), or dcrivcd from diffcrcnt gcncra or spccics than thc specific CBP molecules mentioned herein.

Vai-iants such as those described above can be generated in any appropriate manner using techniques which are known and described in the art, for example usini-7 standard recombinant TaNA, tech nology, Derivatives of non-hydrolytic CBPs as defined herein may also be used. Bv derivative it is meant a non-hydrolytic CBP or variant thereaf which instead of the naturally occurring amino acid, contains a structural analogue of that amino acid.
Derivatisation, modification (e.g. labelling, glycosylation, methylation) may also occur as long as the function of the non-hydrolytic CBP is not adversely affected.
Fragments of the non-hydrolytic CBPs rna.y also be used as long as the fiunction of the non-hydrolytic CBP required for use in the present invention is not adversely affected by the deletion of one or more amino acids from said non-hydrolytic CBP, i.e.
the ability of the non-hydrolytic CBP to bind to chitin and also to enhance the degradation of chitin for example by causing disruption to the chitin substrate.

The fragment may be a truncated version of a n.on-hydrolytic CBP, e,g, 03p 21 e.g, C

teTrnln2i.l deletion of 1, 2, 3, 4, 5, itlIllnu avids altllUugll i[1 gGllCl iLl N ICIIlIIIIY[l l1CICtAVIIs of CM1 should be avoided.

In the most preferred embodiment, the non-hydrolytic CBP is or comprises the CBP21 protein from Serratia marcescer-s having the annino acid sequence set out in SFQ TT) NO- I Funct.innal variants, derivatives and fragments of this protein are also preferred.

By "exposing" the chitin to the non-hydrolytic CBP, it is meant that the non-hydrolytic CBP and chitin are brought into contact in an appropriate context and under appropriate conditions so as to allow the two components to interact or bind to each other_ It has been shown for CBP21 that for this non-hydrolytic CBP, binding to chitin is necessary for its effect in enhancing chitin degradation (Vaaje-Kolstad ef al 2005a, supra) and it is believed that the enbancement of chitin degradation depends on the formation of the appropriatc bonds bctwccn these t.vo components.

The chitin substrate is thus mixed with, or contacted with the non-hydrolytic Llil' under suitable conditions so as to allow this interaction to take place.
Suitable contact must therefore be made between the non-hvdrolytic CBP and the chitin substrate so as to allow these two components to interact. Thus, the non-hydrolytic CBP may simply be brought into contact with the chitin substrate for example by adding it directly to the chitin substra.te. Conveniently, the noxx-hydrolytic CBP may be present in a liquid medium which is applied to the chitin substrate. In general, the non-hydrolytic CBP
will be present in an aqueous solution, although any appropriate conditions can be used. The following description sets out conditions that can be used, but it should be noted that any appropriate conditions can be used, and the step of "eaposing"
the chitin substrate to the non-hydrolytic CBP can take piace in a.ny appropriate context, e.g. in a transgenic plant as discussed in more detail below, or ln the context oli'a medical or agricultural treatment, where the non-hydrolytic CBP is administered to a patient or a plant.

In a preferred embodiment the non-hyydrolytic CBP is present in a buffer such as a phosphate butTer, e.g, a sodium phosphate buffer. Suitable concentration ranges for such a buffer are f-luOmM. The non-hydrolytic CBP can be present in the solution at any suitable concentration, such as a concentration of 0.001-1.0mg/ml, e.g.
0.01-0.1 mglml or 0.05-0.5mglml, Preferably the chitin substrate is exposed to the non-hydralytic CBP, e& the chitin substrate and the non-hydrolytic CBP are incubated with each other, for a period of 12 or 24 hours or more, e.g. 36 or 48 hours or more, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more_ 'Ibis incubation is in general carried out at or about 37 C, although appropriate temperatures for optimizing the enhaneetnent of chitin degradation can readily be determined by the skilled person in the art .~'or ex-ample, the temperature can be in tb,e range of36-38 C or 35-39 C.

It will be apprcciatcd by the person skilled in the art that the necessary incubation times, pH, temperature, substrate concentrations and CBP21 concentrations are not independent of each other. Thus, a large range oftondittons can be envisaged, which can easily be evaluated by a person skilled in the art.

Preferably the chitin substrat.e is exposed to the non-hydrolytic CBP, e.g, the chitin substrate and the non-hydrolytic CBP are incubated with each other, at an appropriate pH, for example at a pH in the range of 5.5 to 7, or 6 to 6.5, althourzh appropriate pHs for optimizing the enhancement of chitin degradation can readily be detetxnined by the skilled person in the art. Preferably the pH is in the range of 6.2-6.4 or 6.25-6.35.
The preferred pH is about pI-( 6.3.

Preferably the incubation is carried out without agitation, i.e. without stinina or disturbing the mixture. This may reduce any adherence of chitin substrate to the walls of the vessel.

The exposure of the chitin substrate to the non-hydrolytic CBP ca.n uccut in airru iri any appropriate vessel, e.g. by mixing together the chitin substrate and the non -hydrolytic CBP ir, an appropriate medium (e.g. a solution, such as an a:queous snlntinn) or hy applying the nnn-hydroly~.ic CRP to the chitin Guhstrat.tt (e 0 hy applying the non-hydrolytic CBP in a solution to a chitin substrate).

As indicated above, in an alternative embodirrreu.t of the invention a chitin hydrolase, e.g. chitinase, chitosanase or lysozyme may be used in combination with the non-hydrolvtie CBP under appropriate conditions to enhance chitin degradation.
Thus, the chitin substrate can be exposed simultaneously to the non-hydrolvtic CBP and the chitin hydrola.se, e.g. chitinase, chitosanase or lysozyme or else the chitin substrate is exposed to the nbn-hydrolytic CBP and the chitin hydrolase, e,g, chitin.ase ,uliitosanase or 1ysozyni.e,scquentially, in either order. It is preferred that the CDP is added before the chitin hydrolase, e.g. chitinase, chitosanase or lysozviiic.

Any chitin ltvdrolase, e.g. chitinase, chitosanase or lysozyme that is able to degrade the glycosidic bonds in the chitin substrate may be added. Such degradation may be complete or partial. For example, the activity of some chitin hydrolase, e.g.
chitinases on chitin substrates is not strong enough to result in complete degradation of the substrate. This is particularly thc casc for chitinnscs such as ChiG from SY.rcnton?} ces coelicolor that do not have their own CBM, or chitinases such as ChiB. In this case, the use of a non-hydrolytic U13Y in accordan.ce with the present invention can result in enhanced chitin degt'adation and preferentially result in complete degradation that was not previously possible. Examples of appropriate chitinases include all enzymes and putative enzymes listed in the CAZY database under glycoside hydrolase families 18 and 19.

In addition, chitosanases (glycoside hydrolase faa-nilies 46, 75 and 80) and lysozymes (glycoside hydrolase families 23 and 24) may be appropriate.

Other enzymes may also be added in addition to or as an alternative to the chitin hydyralyt.ic enzymes discussed above, depending on the nature of the chitin substrate that is to be degraded. For example, if the chitin to be degraded is a copolviater which contains protein, proteases may altn hP ;,rlclPrl S,ntahln cxamples include Alcalase, Neutrase, Papain and other broad-specificity proteolytic enzymes. In each experimental set-up the suitability of proteases will need to be checked, especially if other enzymes (e.g. chitinases), which may be dest-oyed by some of the available proteases, are present simultaneously. It miist he noted that disruption of chitinous parts of a chitin-cr,ntaining co-polymers may facilitate enzymatic decradation of the non-chitinous parts, even if no chitinase is prescnl yimutlaucously. If tlic non-chitinous parts consist of non-protein compounds, other appropriate enzvmes may be added In the embodiments including the use of chitin hydrolase, e.g. chitinases, the chitin substrate is mixed with, or~c.ontacteri with nnn-hydrolytic CBP and chitin hydrolase, e.g. chitinase utider suitable conditions so as to allow the appropriate interaction or interactions to take place between the cliitin Iiydrolase, c.g. chitinase enzymc and the chitin substrate and between the non-hydrolytic CBP and the chitin substrate the enzymatic hydroJysis of the 0 1-4 N-acetyl glucosamine bonds to occur. As discussed above, in relation to the contact between the CBP and the chitin substrate, suitable contact must be made between the components of this system. Thus, the non-hydrolytic CBP and chitin hyrlrnlase, e.g. chitinase may simply bebrought into contact with the chitin substrate for example by adding them direct.lv to the chitin substrate. Conveniently, the non-hydiulytic CBP arid the chitin hydrolasc, c,g.
chitinase may be present in a liquid medium which is applied to the chitin substrate.
In general, the non-hydrolvtic CBP and the chitin hydrolase, e.(-,. chitinase will be present in an aqueous solution, although any appropriate conditions can be used, In a preferred embodiment the non-hydrolytic CBP and the chitin hydrolase, e.g.
chitinase are present in a buffer such as a phosphate buffer, e.g. a sodium phosphate buffui. Suitable conccntration rangca for such a buffer are 1-100mM. The non-hydrolytic CBP can be present in the solution at any suitable concentration, such as a concentration of 0.001-1.0mg/ml, e.g. 0,01-0.1 mg/mi or 0.05 -0.5mg/ml, Preferably the chitin substrate is exposed to the non-hydrolytic CBP and the chitin hydrolase, e.g. chitinase e.g, the chitin substrate and the non-hydrolytic CBP
are incubated with each other, for a. period of 12 or?A hours or more, e_g. 36 or 48 hours or ntorc, or 3, 4, 5, 6, 7, 8, 9, 14, 11, 121, 13, 14 days or more. This incubation is in general carried out at or about 37 C, although appropriate temperatures for optimizing the enhancement of chitin degradation can readily be detennined bv the skilled person in the art. For example, the temperature can be in the range of 36-38 C or 35-39 C.

It will be appreciated by the person skilled in the art that the necessary incubation times, pH, temperature, substrate concentrations ancl CBP21 concentrations are not independent of each other. Thus, a large range of conditions can be envisaged, which can easily be evaluated by a person skilled in the art.

Preferably the chitin substrate is exposed to the non-hydrolytic CNP and the chitin hydrolase, e.g. chtttnase, e.g. the chitin substrate, the non-hydrolytic C:fiF
and the chitin hydrolase, e.g. chitinase arc incuuated with each other, at a pH in the range of 5,5 to 7, or 6 to 6.5, although appropriate pHs for optimizing the enhancement of chitin degradation can readily be determined by the skilled person in the art.
Preferably the pXd is in the range of 6.2-6.4 or 6.25-6.35, The preferred pFl is about pH 6,3.

Preferably the incubation is cazTied out without agitation, i.e. without stirrin:,Y or disturbing the mixture. This may reduce any adherence of chitin substrate to the walls of the vessel.

The exposure of the chitin substrate to the chitin hydrolase, e.g. chitinase and the non-hydrolytic C:RP can oecnr in vitro in any appropriate vessel, e.g. by mixinS
together the chitin substrate and the chitin hydrolase, e.g. chitinase and the non-hydrolytic CBP in an appropriate tttccliuitt (c,g. 4 stalution, such as an aqueous sulution) or by applying the chitin hvdrolase, c.Q. cliitinase and the non-hydrolytic CBP to the chitin substrate (e.g. by applying the cliitin hydrolase. e.g. chitinase and the non-hydrolytic CBP in a solution to a chitin substrate).

If the chitin substrate is exposed first to the non-hydrolytic CBP and then to the chitin hydrolase, e.g. chitinase (i.e. the chitin substrate is not sinaultaneously exposed to the nun-liydrolytic CBP and thc chitin hydrolase, e.g. chitinasc), the chitinasc mny sitnply be added to the chitin that has been exposed to the CBP, without the removal of the non-hydrolytic CBP, e.g. by supplementing the non-hydrolvtic CBP containing ;olution with chitinasc or mixing a chitinase containinS solution with the non-hydrolytic CBP containing solution.

Alternatively, the non-hydrolytic CBP, e.g. in a non-h,ydrolytic CBP
eontaining solution, may be removed from the chitin substrate and replaced with a chitin hydrolase, e.g. chitinase containing solution. In each case, the chitin is exposed first to the non-hydrolytic CBP and then to the chitinase Appropriate forms of non-hydrolytic CBP and variants etc,, thereof, for use in the methods of the present invention are desciibcd nLovc. Thus, the non-hydrolytic CBP
that is present, e.g. in the solution, can be in many different forms, and isolated, extracted or purified from various different sources or synthesised by various different means.

For example, the non-byrlrolvtic CBP (or variant, etc., thereof) can be a synthetic non-hydrolytic CBP that has been chemically syrtthesised. Chemical syntheses may be pertormed by methods well kiiuwn iii the art involving, in the case of peptides, cyclic sets of reactions of selection deprotection of the functional groups of a tertninal amino acid and coupling of selectively protected amino acid residues, followed finally by complete deprotection of all functional groups Synthesis may be performed in solution or on a solid support using suitable solid phases known in the art, such as the well known Nlerritield solid phase synthesis procedure. Preferably the non. -hydrolytic CBPs for use in the invention are substantially purified, e.g.
pyroeen- free, uiorc than 70%, cspccially preferably more than 40% pure (as assessed for example, in the case of peptides or protein5, by an appropriate technique such as peptide mapp)ng, sequencing or chromatography). Ptuili4atiun niay bc perforrncd for eaample by chromatoQraphy (e.g. HPLC, size-exclusion, ion-exchange, a#finity, hydrophobic interaction, reverse-phase) or capillary electrophoresis.

Recombinant expression of proteins is also well known in the art and an appropriate nuclcic acid sequence can be used to exprPqG s. non-hydrolytic CBP (or variant, etc., thereof) for subsequent expression and optional purification using techniques that are well known in the art. For example, an apprupiiatc i,ucleic acid sequence can be operabl_y linked to a promoter for expressic,n of the nun-hydrolytic CBP (or variant, etc., thereof) in bacterial cells, e v H: cnli. itmay be convenient to express the non-hydrolytic CBP under the control of an inducible promoter, in which case the expression of the non-hydrolytic C;SP will be iuduccd in culturc to cause expression.
As the non-hydrolytic Cb Ps are in general secreted, it is generally possible to purity them from the supernatant or periplasmic fractions of host cells using techniques that are known in the a.rt (e.g. as described in Vaaje-Kolstad ex al) to provide the source of the non-hydrolytir C'RP The purified protein can then be mixed with an appropriate liquid or solutiott for administration or addition to the chitin substrate that is to be exposed thereto. This approach is equally applicable to recombinant bacteria and to bacteria that naturaJly synthesise and secrete non-hydrolytic CBPs.

These proteins are secreted and in general contain a leader sequence that is responsible for secretion. Optionally therefore, genes encoding leader peptide-free non-hydrolvtic CBPs may be fused to DNA sequences endcoding leader peptides of choice to ensure efficient secretion by certain host bacteria. Alternatively, CBP genes may be clonod without leader peptide and produced intra.cellularly.

Alternatively, the supernatant or growth rrie<liuin in which the bactcria which naturally produces the non-hydrolytic CBP or which is expressing a recombinant nota-hydrolytic CBP can be used as the source of the non-hydrolytic CBP, lf the bacteria naturally express the non-hydrolytic CBP, it may be necessary to first induce the secretion of the CBP by growing or culturing the bacteria in the presence of a suitable inducer of synthesis such as chitin.

Partial purificativsi in concaitration of thc non-hydrolytic CBP from the stIpc.rnat;int or growth medium in which the bacteria which naturally produces the non -hydrolytic CBP or which is expressing a recombinant non-hydrolytic CBP is growing is also possible. Suitable examples of how this mi9ht be c.arri-ed out to achieve the required concentration or purity of non-hydrolytic CB P are well known in the art.
Chitin dP(rra.ding organisms can be grown in media containing chitin as the only carbon source. Secreted proteins can be harvested by precipitating all the proteins present in the inGClium, followed by rcnaturation and isolation through pgeneral rrntein.Separation methods (e.g. electrophoresis). A second option is to collect the chitin from the ~1 .-growth medium after a period of growth, and then elute all bound proteins (which should be chitinases and chitin binding proteins) off the cktitin. This can be done by ftrst washing the chitin w,>ith a tivash buffer (c.g. 50 mM eodium phosphate, pH 6.3) and then eluting the chitin bound proteins offtivith an elution buffer (a buffer either low or high in pH or with a high salt concentration). '1'he proteins eluted trom the chitin can then be separated by standard protein separation methods.

The appropriate non-hydrolytic CBP to be used in these methods depends on the particular type of chitin or chitin containing copolymer that is to be degraded and can readily be determined by a person skilled in the art. For example, CBP21 binds only to 0-chitin and would therefore be an appropriate non-hydrolytic CBP to use if the methods of the invention were to be applied to li-chitin, ChbB from B amylvliyz.rrfaciens as described in Chu el cxl (supra) also binds to (3-chitin and would therefore also be an appropriate non-hydrolytic CBP to use if the mctltods of the invention were to be applied to P-chitin.

CHB 1, CN]32 and CHB3 have all been isolated from S olivaceoviride.r (Svergun et al, Zeltins ei al, Schneliman el a! Kolbe er al, Saito ei crl supra). The binding preferences of these three proteins have been determined and CHBI and CHB2 bind preferably to a-chitin, whereas CHB3 binds to both a and p chitin. CS3Pl. from Alf ernrntmas as desr.,rihPrl by Tsnjihn 0 trl hindc t.n hoth rY. anrl (i nhitin, with aprefPrPnr.e fnr the n fonn.

It is clear that a. suitable ror approliriate ron-hydrolytic CBP to use in the methods of the invention has to he determined based on the ability of the non-hydrolytic CBP to interact with and disrupt the chitin substrate that is to be degraded, and that the correct choice of the non-hydrolytic CBP for a particular chitin substrate is important. As described above, different non-hydrolytic CBPs bind to different chitin substrates, and binding is essential for the non-hydrolytic CBP to enhance t.he delyradat.ion of chitin, A first step in identifying an appropriate non-hydrolytic CBP for use in the method is therefore to determine what non-hydrolytic CBPs bind to the chitin substrate of interest. This can be carried out by referring to binding studics that have been carried out in the prior art, e.g. if the nature of the chitin substrate that is to be degraded has bccn charactcriscd. Altcrnatively, appropriatc binding studics can readily br performed by the skilled man using techniques that are known in the art.

Such binding studies can also be performed if the nature of the chitin substrate that is to be degraded has not been fully characterised. Again, this can readily be performed by the skilled man using techniques that are known in the art.

Those non-hydrolytic CBPs that bind to the chitin substratc that is to bc dcgradcd carx then be tested for their ability to enhance the degradation of thA.t substrate e.()-. using assays as set out in the L;xamples. Moditications to the assays may he required depending on the particuiar chitin substrate that is to be degraded. For example, very long incubation times and/or higher enzyme concentrations are required if the substrate is a chitin, since the crystalline part of this chitin variant is very recalcitrant compared to (3 chitin or amorphous chitin.

It is similarly possible to use these techniques to identify whether a variant of a non-hydrolytic CBP would be suitable for use in the methods of the invention, As discussed above, i-nultipl.e chitinases exist in nature and they vary in their activity towards different chitin substrates. As such, the particular chitinase that is to be used in the methods of the invention can also be selected based on its ability to degrade the particular cb.itin substrate tbat is to be degraded. The properties of chitinases have been documented (e.g. Hollis, T el ra( (1997) Arch. Bi.ochem. Biophys. 344, and Suauki, K., et crl (1 9916) tiiosci. Biotech, 13 ioch, 02, I29- I35] atld chitinases can be tested based on routine assays that are well known to the person skilled in the art.
Any type of chitinase e.g. a naturally occurring, recombinant, or synthetic chitinase may be used. Preferred chitinases are microbial chitinases and plant chitinases.

It i.r, clear from the specific example of CBP21 that a single non hydrolytic C]3P cnn influence the degradation of a chitin substrate by multiple chitinases e.g.
chitinases belonging to family 1N of glycoside hvdrolases (e.g. C:hiA., B, C) or family 19 of '~3 gl_ycocide hyrirnlaspc (P L, f:hiCt) ancl thuc slrr.h varinits rliffPrent nhitinasp.c or conzbinations ot'chitinases can be used with any one non-hydrolytic CBP in the IIICtllUl1S Uf tlje !11'Yel7ttoII. Wltll any chltlll sul75tfatG that is to bC
dcgradcd, ollce a suitable non-hydrolytic CBP has been identified, one or more chitinases may be chosen to hydrolyse the glycosidic bonds connecting the p(1-4) N-acetylglucosamine sugars.

PrPfPrrPd enmhina.t.inns are CBP21 (or variants, fragments or derivatives thereof) with one or more of ChiA, ChiB, ChiC and ChiG. This is a preferred combination forthe degradation of chitin substrates that contain or consist of (3 chitin. For alpha chitin, a preferred combination would consist of CRB 1 or CRB2 (Svergun el ul, Zeltins el ca1., Schneliman et al Kolbe ef ul supra) or another alpha-chitin binding CBP21 analogue, combined with one or more of ChiA, ChiB, CbiC and ChiG. In principle, any chitinase could be tried in combination with any CBP that targets the "correct substrate".

lvlorz than one, i.e. multiple chitin hvdrolase, c.g. chitJZlflscs can bC uscd to hydrolysc the glycosidic bonds connecting the (3(1-4) X-acetylglucosamine sugars.
Multiple chitin hydrolase, e.g. cltitinases can be added simultaneously or sequentially to the chitin substrate. Preferably 2, 3, 4, 5 or 5 or more different chitin hydrolase, e.g.
chitinases can be used.

It is also possible to use more than one non-hydrolytic CBp in the methods of the invention. In other words, the chitin substrate that is to be deeraded can be exposed to one or more non-hydrolytic CBP or to multiple (e.g. 2, 3, 4 or more) non-hydrolytic CBPs. The non-hydrolytlc. CJ31's may have binding specificity to and function on the same type of chitin, or they may have different binding specificities and functions from each other. The latter may be appropriate if a chitin substrate that is to be de~raded contains more than one type of chitin (for example y chitin), Bnscd on the above considerations, it can be seen that a further elnbodinient of the invention relates to an assay method for identifying a non-hydrolytic CBP, or determining whether a protein is a non-hydrolytrc CI3Y. In such an assay method the putative non-hyLhulytic CBP is exposcd to chitin e.g. under conditions that are described above and the effect of exposure to this protein is assessed, e.g.
as described above. If chitin is weakened or an improvement in the rate or degree of chitin degradation is observed after exposure to this protein, compared to in the absence of the exposure to the protein, the protein is considered to be a non-hydrolytic CBP.

This invention has applications in a number of different fields and, due to the ability of the nun-hyclrolytic CDP to cnhance chitin degradation, cati be used to improvP a.ny method which is based on, the action of chitinolytic enzymes (chitin hydrolase enzymes).

Thus, the methods of the invention can be used to improve or enhance chitin de2ra.dation in the conversion of a chitin-containing biomass to chitin fragments or N-acetylglucosamine.

N-acetylglucosarn.ine has several applications (e.g. as discussed below) and thus the methods of the invention can be used to produce G1cNac 1'r uwe iii any ofthesc applications. Currently, G1cNAc is most often produced by acid hydrolysis of chitin (a linear polymer of C'rlcNAe.) extracted from crab and shrimp shells. The methnds of the present invention thus provide an alternative and improved method of producing GlcNac. Similarly, chitin fragments (chito-oligosaccharidcs) also have a variety of applications (e.g. as discussed below), and thus the methods of the i.nvention can be used to produce chitin fragments (chito-oligosaccharides) for use in any of these applications.

As indicated above, GIcNAc has several known applications, For example, N-acetylglucosamine and its derivative glucosamine are sold as a health produet, e.g, as a food supplement. Although the actions of supplemental glucosamine have yet to be clarified, it is thouuht to play a role in the promotion and maintenance of the structure and function of cartilage in the joints of the body. It may also have anti -inflammatory ,luci,saminc-contairaing glycosaminoglycan hyaluronic properties. For cxample, lhc p acid is vital for the function of articular cartilage.

In addition, during the progression of osteoarthritis, exogenous glucc7samine may have a beneficial role. Glucosamine has also been found to have antioxidant activity and to bc bcneficial in unimal models of experimental atthritis. Glucosamine may be ueed for the treatment and prevention o#'osteoarthritis, either by itself or in combination with chondroitin sulfate.

As referred to above, fragments of chitin and their derivatives have a variety of applications, (M.G. Peter, ,in Biopolymers, Vol. 6: Polysaccharides XX (A, Steinbuchel, Ed,), Weinheim: Wiley VCki, 2002, pp. 481-574). For example, they have been shown to act as iminune stimulants, and to cause chemotactic mi~ration oE"
polymorphonuclear cells_ They are also known to elicit defence responses in plants and to function as signalling molecules in ceirain cellular processes in humans (e.g.
stimulation of bone cell growth). See also Sven $ahrke el al, Biomacromolecules 3:696-704 (2002) and references therein.

Fragments resulting from the degradation of chitin may be used directly or they may be used as building blocks to I;eneratP for examplP hiningicnlly a.ct.ive glycoconjugates. Biological activities of chitin fragments include roles as signalling rnolec:ulcs, C6, exaniple in plants and ritan,mals, c.g. humans. Other roles includc immune stimulation, promotion of bone cell (chondrocyte) growth, morphoRenetic activity and elicitation of defcnce responses in plants. Certain fragments may inhibit chitinases, before or after cbemical functionalization. Chitinase inhibitors are of interest because many plague organisms (malaria parasite, fungi, nematodes) need chitinases for growth.

Chitin frtLgmcnts n.re good building blocks for synthesizing compounds, that potentially interfere with enzymes involved in chitin catabolism or metabolism. Since many plague organisms contain chitin whereas humans do not, chitin catabolism and iiietabolism are very interesting target areas for development of drugs, fungicides, pesticides. The use as building blocks means that one mav couple fragments resulting from the degradation of chitin to other compounds in order to give them a desirable activity. One example is a synthetic chitinase inhibitor described in detail by Vaaje-Kolstad ct al. (Interactions of a familv 18 chitinase with. the designed inhibitor H?v1508 and its degradation product, chitobiono-delta-lactone; J$iol Chc;ni, '004 Jlan 30;2791;51:3612-91. Here, a dimer of GlcvAchas been made active as a chitinase inhibitor by coupling it to another chemical group.

As noted above, since many plague organisms contain chitin whereas humans do not, chitin catabolism and metabolism are very interest.ing target areas for development of drugs, fungicides, pesticidcs. Thus, the disruption of the cell walls and/or membranes and/or skeletons of appropriate pathogens and parasites has been a useful therapeutic strategy against pathogens (in particular fungi) and parasites. For example, Amphotericin B and fluconazole exert their anti-fungal activity by atTectinp membranc stcroxds. Various nnti-funga.l therapeutics have been developed but fungal infections of manimals, in particular humans, have increasingly become responsible for life-threateningy disorders. Furthermore, toxicity associated with known antifungal drugs can cause serious adverse side effects, and mortality rates of certain fungal infections such as systemic candidiasis remain high despite Amphotericin B
treatment.

Examples of fungal species and parasites include C'crndicZa, AspergilJiis, (_."r}~tnrnrr.~J.c, Hi,stopla.sma, (;nccid.ioid.es and Pneumncy.sti.s. These pathogens are particularly dangerous in immunocompromised individuals, such as patients with AIDS, patients undergoing chenaotherap,y, and immunosuppressed organ transplant patients.

Fungi also presezit a serious problem to farmers as they are very common causative agents of infectious disease of crop plants. Phytopathogenic fungi cause devastating epidemics, and chuse annual crop yield lnsses and crop failures. The FffPr,ts nf fiinUi are not specific to a few plant species, hut attack all of the known species of flowering plants, and also to attack cmp plants and trees.

Traditionally the strategies that are used to control plant disease include the use of disease resistant cultivars. Such cultivars can be selected or devtloped by plant breeders e.b_ by breeding to incorporate natural resistance mechanisms into the crops_ There are h.owever disadvanta-es with this approach as the genetic sources of this natural resistance are often associated with other undesirable properties and in ortler to recrea.te the desired background, extensive backcros5ing and introgression is needed. This is cUmpounded by the fact that the resistance inechanism(s) are often polygenic. In view of these problems, the steps of improving disease resistance by conventional breeding ai'e eypens'tve in terms of both time and rnon,ey.

As an alternative to conventional breeding techniques, the use of recombinant UNA
technology has provided the possibility of introducing disease resistance using transgenes. For exainple, an isolated resistance gene or other useful gene in plant defence mechanisms can be used to transform a plant.

This new tpchnnlocy hps nnt hnwPVPr prPvented crop disease epidemics and additional strategies should be contemplated to ,upplennent the conventional breeding iriethoc] aiid existirig genetic engineering strategies.

As noted above, chitin is present in fungal cell walls and the walls and membranes of other pathogens and parasites, and chitinases have antibiotic action against such organisms. It should therefore be possible to combat plant fungal infections and diseases in plants caused by other chitin containing pathogens, e.E> inspct.s or nernatodes by taking advantage of the newly identified properties of non-hydrolytic CBPs to iinprovc ur enhanue ul,iti~i dcV,addtiuxA ur to wcaken chitin.

Therefore in a further aspect, the present invention relates to a composition comprising a non-hydr.olytic CBP as defined herein and a pharmaceutically or agriculturally acceptable carrier, diluent or excipient. Said eompositions (and indeed other non-hydrolytic CBP containing compositions as defined herein) can he used to enhance the degradation of chitin or to weaken chitin. Sais uses can be carried out in vitro, c.g. in a bic~rcactor or tcst tubc, or in vivo, e.g. in a plant, auii,ial, rnicroorganism, etc, i.e. in any environzztent where chitin is present.

As the composition contains a non-hydrolytic CBP, the ability of this protein to enhance the degradation of chitin, which is key component of ftingi (and other plant pathogens such as nematodes and insects), will adversely affect the growth of said fungi (and other plant pathogens such as nematodes and insects).

;~

In addition to a non-hydrolytic CBP, the composition may further comprise other active ingredients such as a chitizt hydrolase, e,g. chitinase as defined herein and/or a fi,rther fungicidal or pa.thiciclal agent. Suitable agents for inclrrcinn in Pi r.nmPncitinn that is to be used in the treatment of mammals will be known to a person skilled in the ai t a,id can be selected' depeixdin g on the nature of ttie fungus ur odier pntlYuscõ wlrit3, is to be treated by the compostion. Suitable fungal agents include amphotericin B, nystatin, pimaricin, 5-fluorocytosine; azole derivatives such as fluconazole, ketoconazole, clotrimazole, miconazole, econazole, butoconaz..ole, oxiconazole, sulconazole, terGonazole, itraconazole and tioconazole; allylamines-thiocarbamates, such as to}naftate, naftifine and terbinafine; griseofulvin; ciclopirox olamine;
haloprogin; undecylenic acid; and benzcic acid. The fact that synergistic anti -fungal effects may be obtained by combining the application of chitinolytic enzymes with application of anti-fungal agents such as gliotoxin, flusilazole, miconazole, captan and benomyl is documented in the literature (Lorito M, el alMicrobiology. 1994 Mar; 14U
( Pt 3):623-9), Suitable additional firngicidal agents for inclusion i.n a composition that is to be used in the treatment of plants will be known to a person skilled in the art and can be selected depending on the nature of the fungus which is to he treated hy the compostion. Suitable agents include fungal cell membrane affecting compounds sclrctCd fiorõ t1rCg,uup uuu,,istiõy, ufstcrull5v,aLlrcais iu}-ib;ti,xK.fuzA~iciacs, az~lifunbal peptide antibiotics, zeamatin and proteins that are serologically related to zeamatin, and antifungal lipid lytic enzymes. Alternative additional components of the composition include antifungal polvene macrolide antibiotics, antifungal epithiodiketopiperizine antibiotics, fungal cell wall biosynthesis inhibitors such as chitin synthetase inhibitors and pglucan synthetase inhibitors. Such compounds are described in. US6,512,166 which is incorporated herein bv reference.
Such compositions can bc fornzulatcd according to any of the i,onvcntional mctliods known in the art and widely described in the literature. Thus, the active ingredient (i.e. a non-hydrolytic CBP) rnay be incorporated, optionally together with other active substances (such as chitin hydrolase, e.g. chitinases and/or other fungicidal or pathicidal agents, examples of which are as described above), with one or more conventional carriers, diluents and/or exripients appropriate for the particular use for ., agriculturally acceptable carriers for agricultural uses and a composition, e.v pharmaeeutically acceptable carriers for medicinal uses, to produce conventional preparations which are ::uitable or can be made suitable for administration s>>c:h ae powders, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, ointments, sterile injeciable solutions, sterile packaged powders, aaiel thc likG. TtLcy may be formulated as liquids (solutions or suspcnsiosis) or as solids.
Preferably the pharmaceutical composition cotxrnprising the non-hydrolytic CBP is prepared in a form appropriate for topical application .

Examples of suitable carriers, ekcipients, and diluentc arp lacl:ose, dextrose, sucrose, maltose, glucose, sorbitol, ryiannitol, starches, gum acacia, calcium phosphate, aglinates, tragacanth, gelatin, calciuni silicatc, l,olyvinylpyrrolidone, water syiup, water, water/etksanol, water/ glycol, water/polyethylene, glycol, propylene glycol, methyl cellulose, rxtethylhydroxvbenzoates, propyl hydroxybenzoates, talc, magnesium stearate, mineral oil or fatty substances such as hard fat or suitable xrtixtures thereof. The compositions may additionally include lubricating agents, wetting agents, emulsifying agents, suspending afients, preserving agents, sweetening agents, flavouring agents, and the like. The compositions of the invention may be formulatcd so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient or plant by employing procedures well known in the art.

As described above, preferably the composition is in a fonn suitable for topical administration and suitable carriers may be present at any appropriate concentration, but exernplary concentrations are from 1% to 20% and preferabl,y from 5% to 10%.
Suitable doses of the non-hydrolytic CBP and the other active ingredients (if included) wil I vary from plant to plant and fiusc- Natie,it to paticnt and will also depend on the nature of the particular infection (as the type of chitin or co-polymer may be different). Suitable doses can be determined by the person skilled in the art or the physician in accordance with the weight, age and sex of the patient, the severity of the fungal infection, and according to the nature and age of the plant to be treated.
F.xPn,rla.ry unit doses are- 0.1 to 50 mg. of ttte active ingredient. Unit doses will normally be a.dniinistered once or more times a day, for example 2, 3 or 4 times a day, more usually I to 5 times a day, such that the daily dose is norsnally in the rangc of 0. 1 to 50 m.g, for example 0.1 to 5 mg for a 70 kg adult, that is in the range of ilpNL uxiniatcJ.y 0.001 to I m_g/kg/day, more usually 0.005 to 0.2 mg/ku/day.

If chitin hydrolase, e.g. chitinase is present in the composition, since free watet is needed for their activity, water must be present at the time of function. This can be accomplished, for example, by making up the chitin hydrolase, e.g. chitinases as aqueous solutions or by making a formulation as a dry powder and applying the powder with the enzyme becoming active once water becomes available, e.g., from rain or saliva. WatNr is th c a l,referrPd vehicle where components are soluble in it.
Ortyanic solvents can also be used and may be required in some cases if a solution is desired. Suspensions can also be etttpluyCa. Most polycnc n,acrolide antibiotics havc poor water solubility and are therefore normally formulated as dispersions or suspensions for application or applied as a powder, The chitin synthetase inhibitors may also be applied as a powder.

The composition of the invention can be applied to all or part of a plant, e.g. the seeds, roots, stems, leaves, flowers and fruits. Alternatively or additionally the treatment can bc applied to the soil in which said plant is ero -ing or is to be !~rorvn or to the fungus itself. Normally, application is topical. However, other administration strategies can be used.

For medicinal purposes (i.e., human and veterinary therapy) the composition comprising a non-hydrolytic CBP is preferably administered topically e.g. to the skin of a human or non-humar- anitnal. Administration can also be, at least in some instanceF, via parenteral iajec.tion, e.g., intrapPritnne,ally; this administration routc is particularly useful where the immune system has been compromised since immune-deficient humans and indlviduals will inacUvatC c,icy,natii, protcins rnore slowly than normal individuals.

Thus, a vet further aspect of the invention provides a method for treatin," or preventing disease in a plant, wherein said disease is caused by a chitin containing rnie.roorganism or pathogen, for example a fiingus, insect or nematode, comprising contacting the chitin containing microorganism or pathogen or contactina the plant to be protected with an effective announ.t of a non-hydrolytic CBP as defined herein_ .A. prefenred embodiment of the invention provides a method for treating or preventing fungal infection in a plant, comprising contacting the fungus or a plant to be protected from the fungus v--ith an antifungal effective amount of a non-hydrolytic CF3P
as defined herein.

Anv of'the compositions that are described above can be used in such a method.
The compositions and method of the invention can be used to prevent or treat any cli:ea.sc in a p1dnt which is caused bv a chitin containing microorganism or pathogen.
A yet further aspect of the invention provides a method for treating or preventing disease in an animal, wherein said disease is caused by a chitin containing microorganisrn or pathopen, for example a fungus, insect or nematode, comprising administering to a subject in need of such a treatanent with an effective amount of a non-hydrolytic CBP as defined herein.

A preferred embodiment of the invention provides a method for treating or preventing fungal infection in an animal, comprising administering to a subject in need of such a treatment an antifungal effective amount of a non-hydrolytic CBP as defined herein.
Any of the compositions that are described above can be used in such a method.
The modes of administration of the non-hydrolytic CBP can be any appropriate mode as discussed above in connection with the composition.

7 he method is generally carried out in a mammal. Any mammal may be treated, for example humans and any livestock, domestic or laboratory animal, Specific examples include mice, rats, pigs, cats, dogs, sheep, rabbits, cows and monkeys.
Preferably however the mammal is a human.

The composition and method described above can be used to prevent or treat fungal infection wherein the fungal species is from genera including Fusarium, Gliociadium, Rhizoctonia, Trichoderina, Unciriula, Ustilago, Erysiphe, Botrvtis, Saccharomyces, Sclerotlun7., Candida, Aspergillus and Alternaria.

The composition and method described above can also be used to prevent infection or damage by insects and'nematodes, e.g. nematodes bel.onging to the genera C;=lobodera, Meloidogyne and Ideterodera. Nematode eggs are of special interest because they contain a chitin layer in their shell.s. Strategies aimed at insects should normally focus on chitinous layers in the gastrointestinal tract and on the cuticle.

As used herein "treating" refers to the reduction or alleviation, preferably to normal levels, of one or more of the symptoms of disease (e.g. fungal infection), For example, such treatment might result in e.g. the reduction of infectivity of the chitin containing microorganism or pathogen, for example a fungus, insect or nematode, or an overall reduction in the amount of detectable chitin containing microorganism or pathogen, for example a fungus, insect or nematode.. "Preventing" refers to absolute prevention, i.e, absence of detectable fungus and/or maintenance of normal levels of detectable chitin containing microorganism or pathogen, for example a fungus, insect or nematode, or reduction or alleviation of the extent or timing (e.s.
delaying) of the infcction with said chitin containing microorganism or pathogcn, for criample a fungus, insect or nematode.

An "effective amount" is an amount effective to inhibit the infection, germination or grotivth of a relevant chitin containing microorganism or pathogen, for example a fungus, insect or nematode, relative to the infection, germination or growth that is seen in the absence of any such treatment.

As the present ir-vention provides the first demonstration of the chitin degradation enhancing properties of non-hydrolytic Cli!'s, it can be seen that suctt non-hydrolytic CBPs as described herein are targets in the control of chitin metabolistn, for example in chitin containing plague organisms. It is known that several plague organisms (e.g.
insects, nematodes and malarial parasites) require chitin degradation during their life cycles. Thus, the present invention further provid~~ a method for cornbating or treating diseases caused by chitin containing plague organisms suc.h as those described above, which niethod comprises the administration to an organism of an agent wbich targets a non hyrlrc,lytic CBP as defined herein, Examples of suitable agents include antibodies directed to non hydrolytic CBPs as defined herein, or agents that serve to reduce the eapression of iiuii 1iyd,olyt.ic CDPs e.g, at thc tran3criptional or translational level (e.g. antisense molecules, ribozymes).

As noted above, chitin is present in fungal cell walls and the walls and membranes of other pathogens and parasites, and chitinases have antibiotic action against such organisms. Yt chciuld therefore be pessible to combat pfant fungal infections and diseases in plants caused by other chitin containing pathogens, e.g. insects or nematoclG; by ldkirig advantage o~thc newly identified properties of non-hydrolytic CBPs to improve, enhance or weaken chitin degradation. Thus, the present invention provides utility in such exemplary applications as enhancing disease re:qistance in plants, particularly crop plants such as maize.

Plants expressing chitinases have been previously shown to have improved resistance to infection with chitin containing pathogens (see for example, Benharnou, N,, K, Bj-oglie, et at. (1993). "Antifungal Effect ofBean Endochitinase on R.hi7nrtnniA-Solani - Ultrastructural-Changes and Cvtochemical Aspects of Chitin Breakdown."
Canadian Journal of Microbiology 39(3): 318-328, Bultu, J. P., J. L. Norclli., et al.
(2000). "Expression of endochitinase from Trichoderrna harzianum in transgenic apple increases resistance to apple scab and reduces vi.gor." PHYTOPATHOLOGY
90(1): 72-77, Herrera-Estrella, A. and I. Chet (1999). Chitinases in biological control.
Chitin and Chitinases. P. Jolles and R. A. A. Muzzarelli, Birkhauser: 171-84 and Ding, X., B. Gopalalcrishnan, etal (1998). "Insect resistance of transgenic tobacco expressing an insect chitinase ctene." Transgenic Res 7(2)- 77-84 Plants that have been engineered to express non-hydrolytic CBPs are therefore likelv to similarly have improved or probably even better resistance to chitin containing pathogens.

A fi-rrher enihodiment of the invention relates to a transl;enic plant comprising an exogenous nucleic acid molecule eomprising, a sequence encoding a non-hydrolytic CBP 4s dcfineil hercin.

The transgtnic plant may further comprise an exogenous nucleic acid molecule comprising a sequence encoding a chitin hydrolytic, e.g. chitinase enzyme, as defined hPrPin These plants preferably have improved or enhanced resistance to chitin containing pathogens in that they show reduced rates of infectivity when compared to non-transgenic or wild type plants of the same species (i.e. exposure to higher doses, or quantities or exposure to more virulent chitin containing pathogens are required in order for thepI,ants to become infected with chitin containing pathogens).
Alternatively, inaproved resistance to chitin containing pathogens can be apparent in diminishment in the disease symptoms and/or growth, viability, reproduction and dispersal of the pathogen when compartd to non-transgcnic or wild type plants of the same species.

Without being bound by theory, this may come about by any means, including, but not limited to improved lysis of the pathogen (e.g. fungal pathogen) through weakening of the cell wall, By "transgenic plant" it is meant a plant vvhich comprises within its igenome an exogenous polynucleotide. C7enerally, the exogenous polynucleotide is integrated, preferably stably integrated within the genanie such that the polynucleotide is passed on to successive generations. The exogenous polynucleotide may be int.egrated into the genome alone or as part of a recombinant expression cassette. "Transgenic"
is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of exogenous nucleic acid inchiding thnse t.ransgenics initially sn altPrPri as .vell as those created bv sexual crosses or asexual propagation from the initial transgenic. The term "transgenic" as used hereii, duCS javt encuiupass tlie alteratiun of the genome (chromosornal or Extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recornbinant transposition, or spontaneous mutation.

By "encoding" it iz meant that the nunlPir; aeicl nnmprises the necessary information for translation into the specified protein to be carried out. A nucleic acid molecule encoding a protein may contain a.rlaitiuiial uuõ-u-alyslated scquczrccs (e.g.
introns).
Exogenous in reference to a nucleic acid or protein molecule is one that originates from a different species, or if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
For Pxa.mple, a microbial sequence is an exogenous sequence when found in a transgenic plant.

Generally the exogenous nucleic acid sequence will be operably lin.ked, i.e.
functionally linked to a regulatory sequence.

The term "regulatory sequence" refers to nucleotide sequences located upstream (S' non-coding sequences), within, or downstream (3' non-codin_g sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the ns3ociated coding sequence. Regulatory sequences may inc.hide promoters, translation leader sequences, introns, and polyadenvlation recognition sequences. f'referably, the regulatory elements are ones that upn-ntiui,al in plants, for example, a plant promoter and plant polyadenylation recognition sequence.

As used herein, the term "promoter" refers to a nucleotide sequence capable of controlling the expression of a coding sequence or RNA. In szerteral, a coding sequence is located 3' to a PrnrnntPr sPnuence. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancCt" i, a IIuclaotiilz scqucnce that can stianulAtc promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-spec!,ificity of a promoter.

Promoters may be derived in their entirety from a native gene, or be composed of rlit'f'erent elements derived from ditTerent promoters found in nature, or even comprise synthetic nucleotide segments. It is understood by those skilled in the art that different piumoters mav dircct the expression of a gena in different tissues or cell types, or at different stages of development, or in response to different environmental conditions.

promoters that ca.use a nucleic acid fragment to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". Exemplary plant promoters includG, l-ut ai-e not limitcd to, those that are obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells such Agrobacterium or Rhizobium. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds.

New promoters of various types useful in plant cells are constantly being discovered;
numerous extuai}rles inay bc found in the compilation by Okamuro aDd Goldbcrrn (1989) Biochemistry of Plants 15-1-82, It is iiuiher recognized that since in most cases the exact boundaries of regulatory sequences have not been wnzl~lC~~ly defincd, nucleic acid fragments of different lengths rnay have identical prornoter activity.
Preferably, the plant is a monocot or dicot. Most preferably, the plant is a crop plant, e.g. Arahid.opsis .s.sp., Luphorhitc .ssp, particularly Fuphorhia pulcherrirrma, 13egarlia r 41zci1nar7tha, Begonia x hiemadi.s,l3egiuniaY hvbnrhynridtr, Regonia semperflo>retts, Campanula.ssp., Brassica ,s,sp., or Lycopersium escialentum.

The nucleic acid sequence as referred to herein encodes any non-hydrolytic CBP
as defined herein. Most of the appropriate non-hydrolytic CBPs are microbial in origin and it is well known that expression of mierobial sequences can be enhanced by modifying the codons that are used such that expression is enhanced in plants.
This is particularly usefiil in thP r.a.ce nf expressing a microbial sequence such as the sequence encoding CBP21 in a plant in view of the fact that codon preferences differ in microurga.ni.ins and plants. In a prcfcrrcd embodirnent therefore, the nucleie ae.ir_i sequence contains codons that are preferred for expression in plants (plant high use codons).

Methods for making transgenic plants are well known in the art. 7'he nucleic acid mofPr,,dP containing the exogenous sequence is typically inserted into a vector e,g, with a regulatory sequence. Typical vectors that are useful for expression of genes in plants ttre well known in the airt and include vectors derived from the tumor inr.limPrl (Ti) plasmid of A.grobacterium tumefaciens. The non-hydrolytic CBP and the chitin hydrolytic eTl'LyllAe, e.g. chitnase can bc on the same or different vector, under the control of the same or different regulatory sequences.

The vector comprising. the sequences frcm a polynucleotide of the present invention wil) typically also comprise a marker gene, which confers a selectable phenotype on plant c-r.lls. Usually, the selectable marker gene will encode antibiotic resistance, with suitable genes including genes coding for resistance to the antibiotic spectinomycin (e.g_, the aacla gene), the strcptomycin phosphotransferase (SPT) gene coding for streptorrxycin resistance, the neomycin phosphotransferase ('FTII) gene encoding kanamycin or geneticin resistance, the hygromycin phosphotransferase (HPT) gr-nC
coding for hygromycin resistance, genes coding for resistance to herbicides which act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides (e,g., the acetolactate synthase (ALS) gene containing mutations leading to such resistance in particular the S4 and/or Hra mutations), genes coding fvr resistance to hcrbicides which act to inhibit action of gh,tamine synthase, such as phosphinothricin or basta (e.g., the bar gene), or other such genes known in the art.
The bar gene encodes resistance to the hczl,ic.ide basta, the nptli gcnc oncodes resistan,ce to the antibiotics kanamycin and geneticin, and the ALS gene encodes resistance to the herbicide chlorsulfuron.

The method of transformationltransfection is not critical to the instant invention;
various methods nf transformation or transfection are currentiv available, As newer methods are available to transform crops they may be directly applied.
Accordingly, a wide variety of inethnd5 have been developed to insert a DNA sequence into the genome of a plant. Thus, any method which provides for effective transformation/transfection may be employed.

Any plant may be transformed. In this manner, genetically modified plants, plant r'Plls, plant tissue, seed, and the like can be obtained. Transformation protocols may vary depending on the tvpe of plant cell, i.e, monocot or dicot, targeted for Li ausfoniaation. Suitnble metbods of transforming plant cells inclnde microinjection (Crossway et al., (1986) BioTechniques 4;320-334), electroporation (Riggs et al.
(1986) Proc. Natl, Acad. Sci, USA 83:J602-5606, Agrobacterium mediat.ed transformation (Hinchee et al., (1988) Biotechnology 6:915-921), direct gene transfer (Paszkowski et al., (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al,, "Direct DNA
Tiairsfei inLu Iritacl Pl-arit Cells via Miuiupiujnaile BuulUaielrr'eril" In.
Gisinbui, and Phillips (.Eds.) Plant Cell, Tissue and Organ Culture: Fundamental Methods, Springer-Verlag, Berlin (1995); 'and McCabe et al., (1988) Biotechnology 6:923-926), Also see, Weissinger et aI., (1988) Annual Rev. Genet. 22:421-477; Saniord et al., (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al., (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al.. (1988) Si.olTechnology 6:923-926 (soybean); Datta et al., (1990) Biotechnology 8:736-740 (rice); Klein et al., (1988) Proc. Natl. Acad. Sci. USA E5:4305-4309 (maizc); Klein ot al., (1988) Biotachnology 6:559-563 (maize); Tomes et al., "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment" in (iamborg and Phillips (Eds.) Plant Cell, Tissue and Organ Culture: Fundamental Methods, Springer-Verlag, Berlin (1995) (maize);
Klein et al., (1988) Plant Physiol. 91:440-444 (maize) Fromm et al., (1990)BiotechnQlogy 8:833-839 (mai.ze); Hooydaas-Van Slogteren & Hooykaas (19S4) Nature (London) 311:763-764; Bytebier et al., (1987) Proc, Natl. Acad. Sci. USA 84:5345-5349 (Lilia.ceae); DeWet et al., (1985) In The Experimental Manipulation of Ovule Tissues ed. G. P. Chapman et al., pp. 197-209. Longman, N.Y. (pollen); Kaeppler et al., (1990) Plant Cell Reports 9:415-418; and Kaeppler et al., (1992) Theor, A.ppl, Genet.
84:560-566 (whisker-rneditated transformation); D'Halluin et alõ (1992) Plant Cell 4:1495-1505 (electroporation); LI et al., (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated b_y reference.

The cells, which have been transformed. may be grown into plants in accordance with ccynvCrttiurral ways. See, tur exartrple, 1Vli;Curuiiuk et al. (1986) Plarrt Cell ReuurLs, 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that the subject phenotvpic characteristics is stable maintained and inherited and then seeds harvested to ensure the desired phenotype or other property has been achieved.
One of skill wi11 recognize that after the exogenous nucleic acid molecule is stably incorporated in transgcnic pJants and confarmcd to bc opc.rablc, it can bc introduccd into other plants by sexual crossing. Any of number of standard breeding techniques can he used, depending upon the species to be crossed.

Li vegetatively piropagatcd crops, niature transgenic p+la.nts can bc propagatcd by thc taking of cuttings or by tissue culture techniques to produce multiple identical plants.
Selection of desirable transgenics is made and new varieties are obtained and propagated vegetatively for commercial use. In seed propagated crops, mature transgenic plants can be self crossed to produce a homozygous inbred plant.
The inbred plant produces seed containing the newly introduced heterologous nucleic acid.
These seeds can be grown to produce plans that would produce the selected phenotypc, Parts obtained from the regenerated plant, such as flowers, seeds, leaves, branches, fruit, and the like are included in the invention, provided that these parts comprise cells comprising the exogenous nucleic acid molecule. Progeny and variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts compromise the introduced nucleic acid sequences, A preferred embodiment is a transgenic plant that is homozygous for the added exogenous nucleic acid molecule; i.e., atransgenic plant that contains two added nucleic acid sequences, one gene at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by sexually mating (selfing) a heterozygous transgenic plant that contains a single added heterologous nucleic acid, germinating some of the seed produced and analyzing the resulting plants produced for alter(-il Pxrressinn nf a pnlynucleotide of the present invention relative to a control plant. (i.e., native, non-transgenic). Backcrossing to a parental plant and uut-cau:sing wiLli a iioii-tiaiisgeilic plant arc also contcmplated.

The. particular non-hydrolytic CBP encoding, sequence that is introduced can be chosen based on the chitin composition of the pathogen to which it is desired to improve resistance. Approaches for identifying appropriate non-hydrolytic CBPs are discussed elsewhere herein.

AlICi rlaLivclV, LlIC }Jl-esellt illvention i-elatcs to nicthods of modulating the level of non-hydr.olytic CBP in a plant by a) introducing an expression cassette containing a l,olynucleotide encoding a non-hvdrolytic CBP, b) culturing the plant cell under plant cell growing conditions, and c) inducing ex.pression of the polynucleotide for a time sul'i"icicuL tu ii,oaulatc tl-ic lcvel of non-hydrolytic CBP in the plant, The method can also he viewed as a method of improving the resistance of a plant to a chitin containing pathogen by a) introducing an cxpression cassette containing a polynucleotide encoding a non-hydrolytic CBP, b) culturing the plant cell under plant cell growing conditions, and c) inducing expression of the polynucleotide for a time sufficient to modulate the level of non-hydrolytic CBP in the plant.

Pesticide use is now in general criticised as a result of increased environmental and health awar.eness. There is therefore pressure to pursue alternative strategies in pest management. Insect viruses such as baculovirus can also be used as biological pest control systems, in vievv of the fact that they are able to kill specific insect pests and as such they thus represent an attractive alternative to the use of pesticides as biological pest control systems. These insect viruses are excellent candidates for spHcies-specific, narrow spectrum insecticidal applic:atinns. Raculovinases in particular have been shown to have no negative impacts on plants, mammals, birds, fish, or even on non-target insects.

lt would be of interest to increase or enhance the infectiousness of insect viruses and one possible way of doing this would be to improve or enhance the ability of such viruses to degrade chitin. The genome of at least some insect viruses contain c.hitina.se genes and genes encoding family 33 non-hvdrolytic CBPs (see Table 1) and chitinase activity has been associated with insect virus infectivity. The observation thi+l nuii-h,ydrolytic CDPs have a function which is to weaken the sarueture of chitin and hence to enhance the degradation of chitin thus provides a means for improving the ability of such viruses to weaken or degrade chitin, and a means to enhance the infectivity of such viruses.

The invention thus further provides a recombinant insect virus comprising a nucleic acid molecule that encodes an exogenous non-hydrolyt.ic CBP, wherein said non-hvdrolytic CI3P is as dcf~nad herein.

As used herein "recorabinant" includes reference to a virus that has been modified by the introduction of a heterologous or exogenous nucleic acid. Thus, for example, recombrnant viruses express genes that are not fbund in identical form within the native (non-recombinant) form of the virus or express native genes as a result of deliberate human intervention that are otherwise abnormally expressed, under-expressed or not cxpressed at all. The term "recombinant" as used herein does not encompass the alteration of the virus by naturally occurring events (e.g., spontaneous mutation, natural transfonnation/transduction/transposition) such as those or.nnrrinR
without deliber~te human interventi.on, The nucleie acid molecule that encodes the exogenous non-hydrolytic CBP can be placed under the control of appropriate promoters that are known in the art for expression in this biological context. Such promoters may be e.g. inducible or con,stitutive.

By "exogenous" non-hydrolytic CBP it is meant a non-hydrolytic CBP that is not encoded by tlie gciiwatC uf tlie naturally occurring insect virus, i.c, it originatcs frdm a different species, or it has been artificially synthesised, As discussed above, family 33 CBPs such as fusolin are found in insect viruses and the invention excludes naturally occurring viruses.

The nucleic acid molecule that contains the sequence encoding the non-hydrolytie CBP is thus introduced into the insect virus using standard techniques that are well known in the art. Thc resultant recombinant virus may then also be selected and cultured according to methods that are known in the art.

Alternatively, the invention provides a recombinant insect virus that expresses a naturally occurring non-hydrolytic CBP, under the control of an exogenous promoter, so as to enhance or improve the expression thereof, or to change the expression profile of the protein. By "exogenous" promoter it is meant a promoter that does not normally control the expression of the non-hydrolytic CBP. It may hP an insPr.t vinis promoter but it is considered to be exogenous if it is not found in its normal context in the recombinant insect virus.

The person skilled in the art can make *e appropriate choice of virus for the particular pest or insect, in view of the fact that these viruses are in gert.eral host specific.

Similarly, the choice of the non-hydrolytic CBP that is introduced can be made based on the compoRition of the chitin substrate in the insect that is to be infected.

The invention thus provides a method of increasing the infectivity of an insect virus comprising introducing a nucleic acid molecule that encodes an exogenous non-hydrolytic CBP into the -genome of said insect virus.

By improved infectivity it is meant that the insect virus infects its host more efficientlv or at lower con'centrations ofvinas than a wild type or unmodified virus does.

In addition to introducing an exogenous nucleic acid sequence encoding a non-liydiulylii; CBP into the i-cconibinant virus, it is also possiblc to introducc a scqucncc encoding a chitin hydrolytic enzvme such as a chitinase, The invention further provides a composition comprising a recombinant insect virus comprising a nucleic acid molecule that encodes an exogenous non-hydrolytic CBP
together with an appropriate carrier or diluent, or a recombinant insect virus that comprises a non-hydrolytic CBP under the control of an exogenous promoter.

L1se of such recombinant insect viruses or compositions in pest control is also provided, as is a method of treating a plant or animal comprising administering to said plant or animal an effective amount of a composition comprising a recombinant insect virus comprising a nucleic acid molecule that encodes an exogenous non-hydrolytic CBP together with an appropriate carrier or diluent.

As the present invention provides thefrst demonstration ofthc chitin riPgrs,rlatinn enhancinf,y properties of non-hydrolytic CBps as defined herein, and assays in which this activity can be assessed and measured, it can be seen that the methods and assays of the present invention can be used to identify residues of non-hydrolytic CBPs which are important for the functiort in enhancing chitin degradation.

Thus, a yet further aspect of the invention provides a method for identifying residues in a non-hydrolytic CBP as defined herein which are intportant for enabling an cnhaocement in chitin degradation or for weakening chitin, said method comprising the steps of:
(i.) mutating or otherwise altering one or more amino acid residues in the non -hydrolytic CBP, and (ii) testing the effect of the mutations or alteratione on the property of enhancing chitin degradation or weakening chitizr.

In such a method, step (ii) can be performed by assessing the ability of the mutated or otherwise altered non-hydrolytic CBP to enhance the degradation of chitin or weaken chitin, e.g. by using the methods and assays as described elsewhere herein. A
comparison of the ability of the mutated or otherwise altered non-hydrolytic CBP to enhance the degradation of chitin or weaken chitin can then be made relative to the non- mutated or otherwise altered non-hydrolytic CBP. The properties of the mutated or otherwise altered non-hydrolytic CBP can thus be cuinpaied diicctly tu llic wild typc, or parent sequence from which mutated or otherwise altered non-hydrolytic CBP
is derived, In such methods, the mutations may have no effect on the enhancement of degradation or may improve or reduce or inhibit the effect on enhancement of degradation. The residues whic}t when mulated have a positive or negative effect on the enhancemCnt of chitin degradation or for weakening chitin arc thcn idcntificd as being itnportant for function. Depending on the proposed use for the non -hydrolytic CBPs such residues are either targets for further mutation (see below) or are identified as residues which should be retained in an unmodified form in order to preserve the important functional property of enhancing chitin degradation.

Alternatively viewed therefore, this provides a method for generating a nov-hydrdlytic CBP with improved or increased ability to enhance the dearadation of chitin or for weakening chitin.

The present invention further provides a method of identifying, developing or producing other non-hydrolyt.lc C$Pti which can en.ll'di1uC JIAt~it 41ugi adaLiuj1 ui i all weaken chitxn., or variants thereof which have the ability to act on other sub strates/polym ers, said method comprising the steps of, (i) obtaining or othen-vise providing a non-hydrolytic CBP as defined herein, (ii) mutating or otherwise altering one or more amino acid residues in the non -hydrolvtic. CBP, (iii) assessing tkae ability of said mutated or altered non-hydrolytic CBP to degrade ol LL) cxxl,niicG the degradation of the substratc of intcrest or wca.kcn the substrate of interest, (iv) identifying mutated or altered non-hydrolytic CBPs which display the desired properties.

Appropriate substrates for use in the abave methods could be any substrate or polymer of interest, in particular any carbohydrate based structure or polymer.
Specific and prcfcrrcd cxamples include any of the forms of chitin described elsewhere herein (in particular chitin copolymers), cellulose and lignocellulose. As indicated above, such methods can be used to identify, develop or produce binding proteins which weaken a different substrate or enhance the degradation of a different.
substrate to the starting non-hydrolytic CBP, e.g, a different form of chitin or copolynler or a different substrate such as cellulose or lignocellulose.

The non-h.ydrolvtic CBP used in thPCP rnPthnrlc has the properties as defined elsewhere herein and may be a newly identified non-hydrolytic CBP or may be a known molecule whose properties of enklauccilicia vf cllitin degradation or wcakoning of chitin have only become known by use of the methods and assays of the invention as defined elsewhere herein, If it is the aim of ihese methods to target a new or different substrate from the one that thP ctarting non-hydrolytic CBP acts on, then preferred residues for mutation or alteration are those. which have been identified as being important for binding to chit.in and prei'ela,bly tlause which have bccn idcntifxed as bcing important or esscntial for the enhancement of chitin degradation or weakening of chitin. Preferred residues for mutation are thus those which are exposed at the surface of the hinding prmteinc and in particular those present on the surface which binds to the substrate (the binding surfacc). I.n thc casc of non-itydrolytic CDPs as descril,ed herein, preferled lcslduG;i for mutation in the binding surface are residues which form part of the polar surface pat.ch which is involved in substrate binding (as opposed to a binding surface dominated by aromatic residues such as those found in cellulose binding domains/modules). Preferred residues are thus polar residues (as opposed to aromatic residues) which are capab~e of forming specific polar interactions, e.i;, hydrogen bonds, with the~substrate.

In the case of non-hydrolytic CBP21 particularly preferred residues for mutation are at amino acid positions 54, 55, 60, 114, 182 and 185 (i.e. Y54, E55, E60, Hl 14, lllh2 and N185) which have been shown to be important in the binding of CBP21 to 0 chitin. Especially preferred residues for mutation are those which have been shown to be important in the ability of CBP21 to enhance chitin degradation or weaken chitin, i.e. at amino acid positions 54, 55, 60, 114 and 182 (i,e, Y54, E55, E60, PT114 and 0182), In the caso ofothcr non-hydrolytic CBP3, prcfcrrcd re.iducs for mutation arc the corresponding residues to those givea above for CBP21. Methods of determining the corresponding residues in other non-hydrolytic CBPs are readily available to a person skilled in the art and conveniently will involve sequence alignments using publicly available databases as described elsewhere herein and databases such as CiustalX and T-COFFEE, which will allow the identification of corresponding residues. Examples of alignments of other bacteria] CBPs with CBP21 and disclosure of corresponding re.sidues and the amino acid position thereof are shown in F'it;ure. 7 (Fi_g 2 of Vaaje-Kolstad et al 2005, supra). As more structures of non -h.ydrolytic CBP-like proteins become available, structural comparisons and smacture-based sequence alignments (methods which are readily available to the person skilled in the art), will be of increased utility in the process of identifying mutation targets in the binding surface area. A particularly preferred residue for mutation is the Y54 residue in CBP21 and its corresponding residue in CBP homologues (see for example the corresponding residues shown in Figure 7). The corresponding residue in CHB 1 is Trp57.

Mutation at the residues which are important fnr thP fimr..tion of enhancing chitin degradation or weakening chitin are likely to result in a modified fianction, for cxamplc an ability to Uind to aitd pi r,fciab3y enhance degradation of or weaken another type of chitin from the original CBP or a different substrate such as those out.liited above.

The present invention further provides a method of identifying non hydrolytic CBP
homologues or related proteins from other organisms or the same organism, said method comprising the steps of:

(i) obtaining or otherwise providinL:, onc or morc moiccLllcs of interest which are candidate homologue or related protein molecules, and ( ) testing or screening to assess whether said molecule of interest is a non-hydrolytic CBP which enhances chitin degradation or weakens chitin as defined above, or is a related protein with activities toward other substrates.

Appropriate substrates for use in the above methods could be any substrate or polymer of intereet, in particula.r any carbohydrate based structure or polymer.
Specific and preferred examples include any of the forms of chitin described elsewhere herein (in particular chitin copolymers), cellulose and lignocellulose. As indicated above, such methods can be used to identify homologues or related proteins which enhance the degradation of the same substrate to the known or characterised non-hydrolytic CBP or a different substrate to the known or characterised non-hydrolytic CBP, e.g. a different ~om of chitin or copolymer or a different substrate such as cellulose or lignocellulose.

The moleculcs of interest uscd in step (i) of the ri7ctliorl i;uuld hC ;
iraglr tnulecules, or could be a library of molecules derived or cloned from one or more species of organism. For example, the molecules could be a library of genes which have been cloned and expressed for testing in accordance with the above method. Any appropriate library of molecules may be used, e.g. cDNA librarv, genomic fragments, etc. In addition, the library of molecules could represent a library of non-hydrolytic CBP variants, produced e.g. by rnutagenesis, which are to be tested for their activities on various substrntcs.

Although thc initial librn.ryo of molecules may be in the form of nucleic acid sequences, for the testing or screening step it is required that the molecules are in the form of proteins (i.e, the nucleic acid molecules need to be expressed).
Appropriate methods of expression are well known and described in the art and an appropriate one can be selected. The molecules to be expressed can then be cloned into appropriate expression vectors based on the host cell which is to be used. Preferably for example the molecules to be tested can be cloned into appropriate vectors for expression and preferably secretion from bacterial hosts such as E. coli, Equally however, eukaryotir;
host cells could be used.

The molecules used in step (i) of the method could be identified or obtained in any appropriate way, e.g. by genonae mining, sequence comparison searches, e.g.
using sequence databases, etc.

Preferably the bacte.rial host (or other host) will have some (but preferably a limited) ability to degrade the substrate of interest alone. Optionally the bacterial host can also be engineered to express and secrelc tui aNNiuNliate substratc dcgrading enzymc, c.g.
an appropriate chitinase or cellulase (depending on the substrate of interest). One could then transform these bacteria with a library of protein molecules of interest, e.a.
a library of CBP variants, and plate these bacteria on substrate containing plates. An increased halo size would then show the presence of an effective binding protein.
Alternatively, any appropriate reaction vessel can be used to test the protein ,-wleculcs, if a library of molcculcs arc to be tested then conveniently each individual protein molecule to be tested is present in an individual reaction vessel, e.g, an assay tube or well. The assays for non-hydrolytic CLiYs or related proteins which can enhance the degradation of chitin (or other substrates) can be carried out in such reaction vessels in accordance with the methods of the invention as described herein.
For example a certain substrate can be brought into contact with the CBPS or related proteins under test (which can either be purified or present in culture supernatants), optionally in combination with an appropriate hydrol_ytic, en7ymP, P g a chitinase or a cellulase. The deuree of substrate dearadation can then be monitored, for example by measurtng the turbidity in the vessel. Preferred conditions for carrying out tl-csc methods to assess substrate degradation are as described elsewhere herein.

In an alternative to the method described above, it would be possible to use a microorgansim that can utilize (ilcNAc2 as a source of energy, a slow working chitinase and pure crystalline chitin in combination with the various CBPs to be tested. After suitable incubation the mixtures can be analysed for any increase in cell density (turbidity). If the CBP variant is etTectivc, cell growth will be observed, If not, the cells won't grow since the chitinase alone will not produce G1cNAe2 sufficiently rapidly.

'I'hus, methods of testing or assessing the abilities as desc,ibcd -n t1,G
above metliods can be carried out in accordance with the methods of the invention, in the presence or absence of appropriate chitin hydrolase, e.g. chitinases, as described elsewhere herein.
If the methods are designed to identify molecules which are reactive to a non-chitin substrate then appropriate enzymes which facilitate the degradation of these substrates (e.g. appropriatP hyrlrnlytic enzymes) can be added in order to assess whether the identified molecule has an effect on enhancing the degradation, For example in the case of cellulose, appiupi iate ccllulascs can be added.

In any of the above methods where mutation steps are involved, then any appropriate methods of mutagenesis could be used, for example site directed mutagenesis, random mutagenesis or directed evolution, e.g, random muta~enesis followed by screening.
Such techniques are wcll known to a person skilled in the art.

Substrate binding proteins and proteins which degrade or enhance the dPUrariatinn of substrate that are identified, developed or produced by any of the above methods and the use thereof to enhance the degradation of substrate form yet fhrther aspects ofthe invention. These proteins can optionally be manufactured and optionally formulated into compositions for various uses.

In all the above described embodiments, the non-hydrolytic CBPs, and preferred non-hydrolytic CBPs for use in these methocis arP a.-; defined elsewhere herein.

This invention will now be described in more detail in tlie fullowina non-limiting Examples with reference to the drawines in which:

5y Fi,~ure t shows sc.anninY G1C4uun niicrograpbs of 0-chitin particles. The figure shows representative pictures of structures observed in the absence or presence of CBP21. Control particles (tio CBP21 added) are shown in panels A and B(400x, magnification) with elpse ups (5000x magnification) of the respective particles shown in panel E and F, respectively, Particles inctibated with CBP21 are shown in panels C
and D, witl, close ups (5000x ma2nitication) shown in panels C7 and H.
respectively.
The black frame drawn on, the 400x magnified images indicate the area tar-eted for the pictures taken at 5000x inaguiGcdtiuii. ?he scalc bars in pancls A-D and B-H
represent 50 and 5 rn, respectively.
Fi ru~, re 2 shows degradation of 0-chitin in the absence or presence of CklP21.
Reaction mixtures contained 0.1 mg/ml 0-chitin, 0.2 M enzyme and 5 M CBP21, added at t = 0, unless stated othenvise. The lines connecting the points are drawn for illustration purposes only. (A) ChiA (squares), ChiA + (='BP71 (c;lnsPd diamonds) or ChiA + CBP21 added at t= 48h (open diamonds). (B) ChiB (squares), ChiB + CBP21 (closed diamonds), Chili + C:til'l 1 added at t=48h (open diamonds) or ChiD t CBP21 added at t = 216h (squares connected by a dashed line; see text for details). C) C:hiC (squares), ChiC + CBP21 (closed diamonds) or ChiC + CBP21 added at t=
48h (open diamonds) and D) 0,3 M ChiG (squares) or 0.3 t.tM ChiG + 5}.dVl C:BP21 (diamonds).
Fi#;urc 3 shows dose response effects for ChiC. Reaction mixtures containPd 0.1 mti/ml 0-chitin, 50 nM (A) or 5 nM (B) ChiC and 500 (diamonds), 50 (squares), 5 (triangles), 0.5 (crosses), 0,05 (hollow squares), 0.005 (hollow triangles) or 0 nM
CBP21 (dotted line).
Firure 4 shows synergistic effects in the degradation of 0-ehitin. The curves 5hnw progress in degradation of 0-chitin with various combinations of chilinascs (as indicated by combinations of the letters A, B and C) and CBP21. The total enzyme concentratiuii wa; always 50 nM, mear.ing that thc roactions mixtures with one, tuio or three chitinases contained 50. 25 or 16,7 nM of eaeh enzyme, respectively.
The CBP21 concentration was 50 nM. For illustration purposes, the points are connected by dotted lines (single enzyme reactions), dashed lines (two-enzyme reactions) or solid lines (three-enzyme reactions), The effect of CBP21 may be evaluated by comparinu r,urve.e with solid symbols (,with CBP21) with curves with corresponding hollow symbols (same enzyme combination, no CBP21), Fieure 5 shows the structure of CBP21. The side chains of al l mut.a:t.Prl rPCiriues are shown as sticks. Note that A1a152 and G1n161 were not expected to be involved in chitin binding.

Figure 6 shows the degradation of 0-chitin by ChiC in the presence of CBP21 mutants. Degradation of 0.1 mg/ml 0-chitin with 50 nM ChiC in the presence of nM CBP21 wild-type, Y54A, E55A, E60A, HI I4A, D182A,, N185A, A152Ror no CBP21 (indicated by a hyphen). Total product release is shown as black bars (24 h), grey bars (48 h) and light grey bars (120 h).
Figure !'shows multiple alignmentofbacterial CBPs (QSGBD4, Yersinia enterncnlitica; [i8ES33,~Uceonnhacillu.s ihqVensis; Q8EHY2, Shetivanella nneid.ensrs;
Q87FTO,Uil)rio parahaemolylicv4s; Q838S1, i:~verocvccu.s fectcctlix; Q88WE3, Lactnhacill.us plantarum; Q7N415, Phntorhahdu.s luminescens (mihsp.
laumondii);
Q9CE94, Lactococcus laclis (suhsp. lacli.s); Q9F9Q5*, Bcacllus amyJ,oliquefaciens;
Q8Y4H4, Listeria mnnocyt.ngenes; Q54501 *, Streplomyce.c nlivacc.~oviridis (CHB1);
C_]R3009*, ,SPrrntin fltiarre.cr.en.e (C:BP?.1)). CB Ps markPd with asterisks have. been shown to bind chitin. The secondary structure elements of CBP21 are also shown and labelled. Arrows with residue numbers indicate residues mutated in Vaaje-Kolstad et a1. (2005) JBC 280(12), 11313-11319. The multiple alignment was created with ClustalX and edited with T-COFFEE, EXAMPLES
Example I

Scunn.ing elec(run micrusculiy uf )%chiiin, fruKmenl,Y

A 0.1 mglml J3-chitin (France Chitin,lVlarseille) suspension in 50 M
phosphate buffer, pH 6.3 was pre-incubated for 48 hours at 37 C in Eppendorf tubes with either 0.1 mg/ml BSA or 0,1 mg/m1 BSA and 0.1 m',.',!ml CBP21, applied onto an object glass (10 mm; 25 l drops), and dried at 37 C in order to fix the sample.
f"BP21 iuas produced as described in Vaaje Kolstad at al. 2005, JBC 280 (12) 11313-9 which is incorporated herein, by reference, The object glasses containing the samples were glued onto SEM aiuminium studs with carbon tape and sputter-coated with gold-palladium. Scanning vvan performed in a JEOL JSM 6400 scanning electron microscope at 5 W.

The edges and surfaces of the untreated particles are discrete in shape, with smooth surfaces (Fig. 1. panels A, B, E and F). Jn contrast, the edges and surfaces of the CBP2 I-treated particles showed an amorphous and porous character (Fig. 1, panels C
and G), along with areas of disassembled chitin fibrils (Fig. 1, panels D and H).
Example 2 1)e;;rarlntion nf&hitin tvith rlifferenr chirinrcces ChiA, ChIB and ChiC (Synstad et al., Genbank accession number AJ630582) from SerraticY rnarcescens BJL200 were overexpressed in E.colr and purified from periplasmic extracts using a two-step procedure. The first step consisted of standard ion-exchange chronnatography using Q-qerharnse p'ast Flow (Amcrsham Pharmacia Biotech AB) at pH 9.4 to separate the chitinases from the majority of proteins in the periplasmic extracts. The second step consistCd uf ]sydrophobic intcraction chromatography using a phenyl superpose 5/5 column (Amersham J'hannacia Biotech AB, Uppsala, Sweden), as described elsewhere (Brurberg et al.(] 996) Microbiology-i.IK 142, 1581-1589). His-tagged ChiG from. Slreptomyce.s coelicolor A3(2) (Genbank AB017013) was cloned behind a T7 promoter into the pETMI 1(Gutater Stier, EMBL
1+]PiriPlhPrg, Cerrnany) expression vector. The protein was produced in isopropyl-0-D-thiogalactopyranosid (IPTG) induced Lcnli BL21 DE3 cells; cells were lysed by sonication and il-G uiuteiil was purified using a nickcl column (5x2 cm, Qi:tgen), under standard conditions. All proteins were dialysed into 20 mM Tris-HCI, pH
8.0 before use and stored at 4 C, Determination of chitinolytic activity was done using 0-chitin from squid pen (France Chitin, Marseille), a-chitin isolated from shrimp shPlic (Flov-Bio, Tromso, Norway) or crab shells (Sigma) or naicroparticula.te P-chitin (Seika-gaku Corp., .lapan) as substrate. Standard reaction mixtures contained varying concentrations ui'cflitiiia,se and CBP21, 0, J mg/ml purified BSA, 0.1 rng/ml chitin powder (unless stated otherwise), in 50 mM sodium phosphate buffer, pH 6.3. Reaction mixtures were incubated at 37 C for up to two tveeks. No agitation was use.d since the insoluble substrate easily adheres to the dry inner walls of the Eppendorf tubes, which would affect the substrate concentration. At time points ranging from 2 hours to 400 hours, 60 l of the reaction mixture was transferred to an Eppendorf tube containing 60 l 70% acetonitrile, to stop the reaction. Before taking samples, reaction mixtures were resuspended by gentle pipetting in order to leave the chitin concentration unaltered.
All reactions were run in triplicate and all samples were stored at -20 C
until further analysis.

Samples were analysed bv isocratic high performance liquid chromatography (HPLC) using an Amide-80 column (Tosoh Bioscience, Montgomeryville, PA, USA), coupled to a Gilson Unipoint HPLC system (GilEon). The liquid phase consxsted of 70%

acetonitrile, with a flow rate of 0.7 ml/mira. 20 l samples were injected using a Gilson 123 a.utoinjector. Eluted oligosaccharides were monitored by recording absorption at 210 nm. Chromatograms were celiecte.d and analysed using the Gilson Unipoint software (Gilson). Since in all cases (G1cNAc)2 represented more than 95 %
of the total amount of degradation products on a molar basis, only (GIcNAc)2 peaks were subject for data analysis and used for quantification of the extent of chitin degradation. A standard solution contaiinirtg 0.25 mM (G1cNAc)2 was analyzed at the start, in the middle and at the end of each series of samples, and the resulting average value (displaying standard deviations of less than 3 %) was used for calibration.
Degradation of P-chitin with the family 18 chitinases ChiA, ChiB or ChiC
showed biphasic kinetics, with an initial fast linear phase, fullowCCl by a slowci, hypCivuliu phase (Fig, 2, panels A, B and C). Initial degradation rates were determined by linear regression, whereas the hyperbolic second phase only allowed endpoint analysis as a rate descriptor (Table 2). In the absence of CBP21 ChiA and ChiC had similar activities towards chitin, both in terms of initial rate and the time needed to fully degrade the substrate (trin), whereas ChiB displayed a -3-fold slower initial rate and never managed to fully digest the substrate (Fig. 2, Table 2). The addition of liail oiily iniiiur effccts on the initial rates but large effccts on the slower second phase (that is, on tt;,ii). For ChIA and ChiC Cj;, decreased approximately 7-fold, while, for ChiD, addition of CBP21 led to complete degradation of the substrate, albeit still at a slower rate than in the case of ChiA and ChiC (Tablc 2).

Table 2. Initial rates (yalculated for the first four timepoints: 2, 4. 6 and 8 hours) and reaction end points (trt,ii) for the degradation of (3-chitin with 0.2 M
ChiA, ChiB or ChiC, in the absence or presence of CBP2l .'The R-sduare values from the linear regression analvses are indicated in brackets. The data are derived from the curves shown in Fig. 2. N.d., "not determined".

lnitiAl rate Endpoint (tn~v) 1 1V[ (GIcNAc)2/h] (1i) Chitinase -CBP21 +CBP21 -CBP21 -1-CBP21 ChiA 22,7 (0,95) 3,4 (0.98) -360 -45 ChiB 0,9 (0.98) 1.3 (0.99) n.d. -200 ChiC 2.3 (0.93) 33 (0.99) -360 -48 In order to verify that the enzymes remained active during the reactions, a series of experiments were carried out, in which CBP21 was added after 48 hours pre-incubation with the chitinases. Figure 2 shows that addition of CBP21 increased reaction rates to levels comparable to those observed in reactions where the was present from t = 0. In an additional control experiment with ChiB, CBP21 was added after 216 hours, which led to full degradation of the substrate (Fig. 2, panel B).
Control reactions containing CBP2.1 wi.thout Pn7.yme did nnt yield detectable amounts of soluble chitooligosaccharides. Taken together, these results demonstrate that U13P21 facilitates the degradation of P-chitin by family 18 chitinases in a non-enzymatic manner.

To investigate whether the effects of CBl?21 on the efficiency of family 18 chitinases from S rrmarGe.scens were due to specific enzyme-CBP2a interac.tions, we also conducted experiments iuith ChiG, a family 19 r.hitinace from .S'lreplomyies coeticnlor. The results (Fig. 2, panel D) show that C13P21 increased ChiG
efficiency, suggesting that CBP21 has a general ef'fect on substrate availability and ducs nul acl through specific interactions with particular enzymes.

Dose-response studies of the effect of. CBP21 on ChiC efficiency showed that ChiC
displays maximum degradation rates at CBP21 eoncentzations ? 50 nM, regardless of the cnzynzc concentration (Fig. 3). Tlrus, the liene-fi4ial CfiCU[ of CBP21 dues not seem.
to be caused by a stoichiometric interaction with the enzyme.

E.XAIVI.PLE 3 &hitin rlegrttdntion using combinations of ChiA, -B, -C and CBP21 It is generally~assuzxi.ed that ChiA and ChiB are exochitinases, while ChiC is an endochitinase and syner,gistic effects between thcse enzymes havc hccn aLscrvcd in studies with colloidal chitin and a-chitin. In agreement with previous experiments, tigure 4 shows that the three S. marcescens chitinases act synergistically on j3-chitin too. In ail cases, CBP21 in.creased the degradation efficiency. The highest efficiency was obtained when combining all three enzymes in the presence of CBP21.

Hydrolysis uf Achitin ivirh ChiC in the presence n f C'SPZ1' ntutants CBP21 mutants were produced as described in Vaaje kolstad et al. 2005 JBC
280(] 2) 11313-9. Combination of the structure of CBP21 with a multiple sequence alignment of bacterial CBPs has previously revealed conserved surface residues, whose mutation to alanine decreased chitin affinity 2- to 8-fold, These mutants, as well as two control C$P21 variants with wild type binding characteristics (A152R and Q161A), were uscd in hydrolysis studies with ChiC. E=xperiments with a CBP21 concentration of 50 nM, which gives maximum efTects on ChiC efficiency in the case of wild type (Fig. 3), showed that CBP21 mutants Y54A, E55A, E60A, H114A and D182A had lost their functionality, while the N1 85A, A152R and Q161 A mutants showed wild type-like functionality (Fig. 6). The deleterious effects of the Y54A, E55A
and Hi 14A mutations on CBP21 function were only sli.ghtly negated by increasing the CBP21 concentration as much as 1.00-fold (results not shown; E60A and D182A
were not tested).

1N:JCAMYLL 5 Hydrolysis of other chitin fr,rmy Thc tlncc family 18 chitinases from .Serraria marcescens can degrade several chitin forms for which CBP21 has low affinity, for example a-chitin from crab shells and shrimp shells. Experiments similar to the ones described above showed that addition of CBP21 at concentrations up to as high as 5 M did not affect of the efficiency of ChiA, Chig and ChiC towards these substrates (results not shown). 66

Claims (76)

Claims

1. A method of enhancing chitin degradation comprising exposing chitin to a non-hydrolytic chitin binding protein (CBP).

2. A method of weakening the structure of a chitin substrate, comprising exposing chitin to a non-hydrolytic CBP.

3. The method of claim 1 or claim 2 wherein said chitin is .alpha. chitin, .beta. chitin, .gamma. chitin, amorphous chitin, colloidal chitin, chitin forms in which part of the N-acetylglucosamine sugars are deacetylated, chitosan, or a copolymer of chitin.

4. The method of claim 1 or claim 2which method comprises the steps of:
(i) contacting said non-hydrolytic CBP and chitin under appropriate conditions so as to allow said non-hydrolytic CBP and chitin to interact or bind to each other, (ii) incubating for sufficient time and under appropriate conditions to allow chitin weakening or enhancement of degradation to occur.

5. The method of claim 4 wherein the incubation step is carried out for 12 hours or more.

6. The method of claim 4 wherein said method is carried out at an appropriate temperature and pH.

7. The method of claim 4 wherein steps (i) and/or (ii) are carried out at pH 6 to 6.5.

8. The method of claim 4 wherein said method is carried out without agitation (p.16)

9. The method of claim 1 or claim 2 wherein the non-hydrolytic CBP is, or corresponds to, or comprises a naturally occurring non-hydrolytic CBP derived from a microbial, eukaryotic or viral source, or is a functional variant thereof.

10. The method of claim 1 or claim 2 wherein the non-hydrolytic CBP consists of or consists essentially of, or corresponds to, or comprises a family 33 carbohydrate binding module, or a functional variant thereof, or consists of, or consists essentially of, or corresponds to, or comprises a carbohydrate binding module from a family 33 carbohydrate binding molecule, or a functional vanant thereof.

11. The method of claim 1 or claim 2 wherein the non-hydrolytic CBP is, or corresponds to, or comprises CBP21 of Serratia Marescens (SEQ ID NO: 1), ChbA
of .beta..amyloliquefaciens, CHB1, 2 or 3 of Streptomyces, or CBP1 of Alteramonas, or a functional variant thereof.

12. The method of claim 11 wherein said functional variants display at least 70, 80, 85, 90, 91, 92, 93, 94, 95,,96, 97, 98 or 99% sequence similarity or identity with a naturally occurring non-hydrolytic CBP or CBM at the amino acid level.

13. The method of claim 9 wherein when said non-hydrolytic CBP is CBP21, then said functional variants retain one or more and preferably all of a tyrosine residue at position 54, a glutamic acid residue at position 55, a glutamic acid residue at position 60, a histidine residue at position 114, an aspartic acid residue at position 182 and an asparagine residue at position 185.

14. The method of claim 9 wherein when the non-hydrolytic CBP is not CBP21, then said functional variants retain one or more of the residues at the positions corresponding to the CBP21 residues as defined in claim 13.

15. A method of enhancing chitin degradation comprising exposing chitin to a non hydrolytic CBP and a chitin hydrolase.

16. A method of degrading chitin comprising exposing chitin to a non-hydrolytic CBP and a chitin hydrolase.

17. The method of claim 15 or claim 16 wherein said chitin hydrolase is a chitinase enzyme.

18. The method of claim 17 wherein said chitinase enzyme is one or more of ChiA, ChiB, ChiC or ChiG.

19. The method of claim 15 or claim 16 wherein said chitin is .alpha. chitin, .beta. chitin, .gamma.
chitin, amorphous chitin, colloidal chitin, chitin forms in which part of the N-acetylglucosamine sugars are deacetylated, chitosan, or a copolymer of chitin.

20. The method of claim 15 or claim 16 which method comprises the steps of:
(i) contacting said non-hydrolytic CBP and chitin under appropriate conditions so as to allow said non-hydrolytic CBP and chitin to interact or bind to each other, (ii) incubating for sufficient time and under appropriate conditions to allow enhancement of degradation to occur.

21. The method of claim 20 wherein the incubation step is carried out for 12 hours or more.

22. The method of claim 20 wherein said method is carried out at an appropriate temperature and pH.

23. The method of claim 20 wherein steps (i) and/or (ii) are carried out at pH
6 to 6.5.

24. The method of claim 20 wherein said method is carried out without agitation.

25. The method of claim 15 or claim 16 wherein the non-hydrolytic CBP is, or corresponds to, or comprises a naturally occurring non-hydrolytic CBP derived from a microbial, eukaryotic or viral source, or is a functional variant thereof.

26. The method of claim 15 or claim 16 wherein the non-hydrolytic CBP consists of, or consists essentially of, or corresponds to, or comprises a family 33 carbohydrate binding module, or a functional variant thereof, or consists of, or consists essentially of, or corresponds to, or comprises a carbohydrate binding module from a family 33 carbohydrate binding molecule, or a functional variant thereof

27. The method of claim 15 or claim 16 wherein the non-hydrolytic CBP is, or corresponds to, or comprises CBP21 of Serratia Marescens (SEQ ID NO: 1), ChbA
of .beta.amyloliquefaciens,CHB 1, 2 or 3 of Streptomyces, or CBP 1 of Alteramonas, or a functional variant thereof.

28. The method of claim 27 wherein said functional variants display at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence similarity or identity with a naturally occurring non-hydrolytic CBP or CBM at the amino acid level.

29. The method of claim 25 wherein when said non-hydrolytic CBP is CBP21, then said functional variants retain one or more and preferably all of: a tyrosine residue at position 54, a glutamic acid residue at position 55, a glutamic acid residue at position 60, a histidine residue at position 114, an aspartic acid residue at position 182 and an asparagine residue at position 185.

30. The method of claim 25 wherein when the non-hydrolytic CBP is not CBP21, then said functional variants retain one or more of the residues at the positions corresponding to the CBP21 residues as defined in claim 29.

31. A method for identifying a non-hydrolytic CBP, or determining whether a protein is a non-hydrolytic CBP, wherein said putative non-hydrolytic CBP is exposed to chitin and the effect of this exposure on the putative non-hydrolytic CBP
protein is assessed, and wherein if a weakening in chitin is observed or an enhancement in chitin degradation is observed, the protein is identified as being a non-hydrolytic CBP.

32. Use of the method of claim 1 or claim 2 to improve or enhance any method involving the action of chitinolytic enzymes.

33. Use of the method of claim 1 or claim 2 to enhance chitin degradation in the conversion of a chitin-containing biomass to chitin fragments or N-acetylglucosamine.

34. Use of the method of claim 15 or claim 16 to improve or enhance any method involving the action of chitinolytic enzymes.

35. Use of the method of claim 15 or claim 16 to enhance chitin degradation in the conversion of a chitin-containing biomass to chitin fragments or N-acetylglucosamine.

36. A composition comprising a non-hydrolytic CBP and a pharmaceutically or agriculturally acceptable carrier, diluent or excipient.

37. The composition of claim 36, wherein said composition is a fungicide, nematocide, or an insecticide.

38. The composition of claim 36 further comprising other active ingredients such as a chitin hydrolase and/or a further fungicidal or pathicidal agent.

39. A method for treating or preventing disease in a plant, wherein said disease is caused by a chitin containing microorganism or pathogen, comprising contacting the chitin containing microorganism or pathogen or contacting the plant to be protected with an effective amount of a non-hydrolytic CBP. p30

40. A method for treating or preventing disease in an animal, wherein said disease is caused by a chitin containing microorganism or pathogen, comprising administering to a subject in need of such a treatment an effective amount of a non-hydrolytic CBP.

41. The method of claim 39 or 40, wherein said chitin containing microorganism or pathogen is a fungus, insect or nematode.

42. A method for combating or treating diseases caused by chitin containing plague organisms, which method comprises the administration to an organism of an agent which targets a non hydrolytic CBP.

43. The method of claim 42, wherein said organism is a plant or animal.

44. The method of claim 42, wherein the composition of claim 36 or claim 38 is administered.

45. A method for identifying residues in a non-hydrolytic CBP which are important for enabling an enhancement in chitin degradation or for weakening chitin, said method comprising the steps of:
(i) ~mutating or otherwise altering one or more amino acid residues in the non -hydrolytic CBP, and (ii) ~testing the effect of the mutations or alterations on the property of enhancing chitin degradation or for weakening chitin.

46. The method of claim 45, wherein said method provides a method for generating a non-hydrolytic CBP with improved or increased ability to enhance the degradation of chitin. p34

47. The method of claim 45 or claim 45, wherein said chitin is a chitin, .beta. chitin, .gamma.
chitin, amorphous chitin, colloidal chitin, chitin forms in which part of the N-acetylglucosamine sugars are deacetylated, chitosan, or a copolymer of chitin.

48. A method of identifying, developing or producing a non-hydrolytic CBPs which can enhance chitin degradation or can weaken chitin, or variants thereof which have the ability to act on other substrates/polymers, said method comprising the steps of:
(i) ~obtaining or otherwise providing a non-hydrolytic CBP, (ii) ~mutating or other wise altering one or more amino acid residues in the non -hydrolytic CBP, (iii) ~assessing the ability of said mutated or altered non-hydrolytic CBP to degrade or to enhance the degradation of the substrate of interest or weaken the substrate of interest, (iv) ~identifying mutated or altered non-hydrolytic CBPs which display the desired properties.

49. The method of claim 48, wherein said substrate is a carbohydrate based structure or polymer.

50. The method of claim 49, wherein said substrate is a chitin, .beta. chitin, .gamma. chitin, amorphous chitin, colloidal chitin, chitin forms in which part of the N-acetylglucosamine sugars are deacetylated, chitosan, a copolymer of chitin, cellulose or lignocellulose

51. The method of claim 48, wherein said method is used to identify, develop or produce binding proteins which degrade or enhance the degradation of a different substrate to the, non-hydrolytic CBP of step (i).

52. The method of claim 48 wherein the residues which are subjected to mutation or alteration are those which have been identified as being important for binding to chitin

53. The method of claim 48 wherein the residues which are subjected to mutation or alteration are those which have been identified as being important or essential for the enhancement of chitin degradation.

54. The method of claim 48 wherein the residues which are subjected to mutation or alteration are those present on the surface which binds to the substrate (the binding surface).

55. The method of claim 48 wherein the residues which are subjected to mutation or alteration are polar residues which are capable of forming specific polar interactions with the substrate.

56. The method of claim 48 wherein when said non-hydrolytic CBP is CBP21, then the residues which are subjected to mutation or alteration are one or more of;
a tyrosine residue at position 54, a glutamic acid residue at position 55, a glutamic acid residue at position 60, a histidine residue at position 114, an asparagine residue at position 182 and an arginine residue at position 185.

57.The method of claim 48 wherein when the non-hydrolytic CBP is not CBP2l, then the residues which are subjected to mutation or alteration are the residues at the position corresponding to the CBP21 residues as defined in claim 56.

58. The method of claim 48, wherein the Y54 residue in CBP21 or its corresponding residue in non-hydrolytic CBPs that are not CBP21 is subject to mutation or alteration.

59. A method of identifying non hydrolytic CBP homologues or related proteins from other organisms or the same organism, said method comprising the steps of:
(i) ~obtaining or otherwise providing one or more molecules of interest which are candidate homologue or related protein molecules, and (ii)~testing or screening to assess whether said molecule of interest is a non-hydrolytic CBP which enhances chitin degradation or weakens chitin, or is a related protein with activities toward other substrates.

60. The method of claim 59, wherein said substrate is .alpha. chitin, .beta.
chitin, .gamma. chitin, amorphous chitin, colloidal chitin, chitin forms in which part of the N-acetylglucosamine sugars are deacetylated, chitosan, a copolymer of chitin, cellulose or lignocellulose.

61. The method of claim 59, wherein said molecules of interest used in step (i) are a library of molecules.

62. The method of claim 59, wherein said library of molecules is a library of non -hydrolytic CBP variants, which are to be tested for their activities on various substrates.

63. The method of claim 59, wherein step (ii) is carried out in accordance with the method of claim 1 or claim 2.

63. The method of claim 59, wherein step (ii) is carried out in accordance with the method of claim 3 or claim 15 or claim 16.

64. Substrate binding proteins or proteins which degrade or enhance the degradation of substrate identified, developed or produced by the method of claim 59.

65. The protein of claim 64, wherein said protein is manufactured and optionally formulated into compositions for various uses.

66. The use of a molecule as defined in claim 64 to enhance the degradation of substrate.

67. A recombinant insect virus comprising a nucleic acid molecule that encodes an exogenous non-hydrolytic CBP or a recombinant insect virus that expresses a naturally occurring non-hydrolytic CBP, under the control of an exogenous promoter.

68. The virus of claim 67 further comprising a nucleic acid sequence encoding a chitin hydrolytic enzyme such as a chitinase.

69. A method of increasing the infectivity of an insect virus comprising introducing a nucleic acid molecule that encodes an exogenous non-hydrolytic CBP into the genome of said insect virus and optionally introducing a nucleic acid molecule that encodes a chitin hydrolytic enzyme.

70. A composition comprising a recombinant insect virus of claim 67 or 68, together with an appropriatc carrier or diluent.

71. Use of a recombinant insect virus of claim 67 or 68 or a composition of claim 70 in pest control.

72. A method of treating a plant or animal comprising administering to said plant or animal an effective amount of the composition of claim 70.

73. A transgenic plant or parts thereof comprising an exogenous nucleic acid molecule comprising a sequence encoding a non-hydrolytic CBP.

74. The transgenic plant of claim 73, further comprising an exogenous nucleic acid molecule comprising a sequence encoding a chitin hydrolytic enzyme.

75. A method of modulating the level of non-hydrolytic CBP in a plant or a method of improving the resistance of a plant to a chitin containing pathogen comprising:

a) ~introducing an expression cassette containing a polynucleotide encoding a non-hydrolytic CBP, b) ~culturing the plant cell under plant cell growing conditions, and c) ~inducing expression of the polynucleotide for a time sufficient to modulate the level of non-hydrolytic CBP in the plant.

76. The method of claim 75, wherein an expression cassette containing a polynucleotide encoding a chitin hydrolytic enzyme is also introduced.