GB2376017A

GB2376017A - Cellulose binding domain conjugates linked or coupled to a polysaccharide backbone

Info

Publication number: GB2376017A
Application number: GB0129606A
Authority: GB
Inventors: Neil James Parry; Paul James David
Original assignee: Unilever PLC
Current assignee: Unilever PLC
Priority date: 2000-12-20
Filing date: 2001-12-11
Publication date: 2002-12-04
Also published as: GB0129606D0

Abstract

A cellulose binding domain (CBD) conjugate, comprises at least two CBDs attached to a polysaccharide. The polysaccharide may be capable of binding to cellulose, and is conveniently locust bean gum (LBG). Conjugates increase the strength and wear of the fabric by crosslinking fibres. The CBD conjugates may also be used as delivery vehicles to deposit materials in any stage of the laundering process. This latter application can be achieved by coating the benefit agent (either directly by chemical means or indirectly via a compound associated with the benefit agent e.g. capsule). Where the polysaccharide is not capable of binding to cellulose at least 2 preferably more) attached CBDs are required to produce a conjugate having 2 separate sites capable of binding to cellulose. In a variant of the invention, at least three CBDs may be attached to a protein or polypeptide, which supports the CBDs and acts as a "scaffold".

Description

IMPROVEMENTS IN OR RELATING TO CELLULOSE BINDING DOMAINS Field of the Invention This invention concerns cellulose binding domains (CBDs) and relates to novel chemical entities including CBDs and novel uses of CBDs.

Background CBDs are protein sequences that form part of naturally occurring cellulose-binding enzymes such as cellulases produced by various microbial organisms. Such enzymes are multifunctional protein species that are capable of binding to cellulose and catalysing important naturally occurring reactions, e. g. decomposition of plant materials. The function of binding to cellulose is typically performed by a CBD, that constitutes a distinct structural protein domain of the enzyme and that is connected to the bulk of the enzyme (including the active site) by an amine rich linker. The CBD is responsible for attachment to polyglucose chains present in cellulose and allows the active site to co-ordinate towards the cellulosic surface. The CBD has been specifically evolved in nature for this purpose. The CBDs are capable of binding to a range of polysaccharide based polymers. For example some CBD's are also capable of binding to chitin.

A common example of a fungal CBD is that of the cellobiohydrolase enzyme from Trichoderma reesei. See the discussion in van Tilbeurgh, H. et al. (1986) Limited proteolysis of the cellobiohydrolase 1 from Trichoderma reesei : Separation of functional domains. FEBS Letters 204,223-227, and Reinikainen, et al. (1992) Investigation of the function of modified cellulose binding domains of Trichoderma reesei cellobiohydrolase 1. Proteins: Structure, Function and Genetics. 14,475-482. For further discussion of CBDs and cellulases, see also P. Beguin and LP. Aubert (1994) The biological degradation of cellulose. Microbiology Reviews. 13, 25-28, and M. K. Bothwell et al. (1997) Binding capacities for

Thermomonospora fusca E3, E4 and E5, the E3 cellulose binding domain, and Trichoderma reesei CBH 1 on Avicel and bacterial microcrystalline cellulose (Bioresource Technology 60,169- 178.) Fungal CBDs comprise hydrophilic and hydrophobic surface regions forming a wedge-like structure. The hydrophilic portion (conferred by tyrosine residues) promotes cellulose binding, whereas the hydrophobic region may be used to disrupt cellulose fibres and direct them towards the active site.

Bacterial CBDs are generally much larger proteins than fungal CBDs, and are commonly produced as distinct, separate proteins that are responsible for the attachment of a cellulase complex rather than one enzyme. See Beguin, P. and Aubert, LP. (1994) The biological degradation of cellulose. Microbiology Reviews 13,25-28.

CBDs from a number of bacterial species including Clostridium and Cellulomonas have been investigated and cloned into a number of expression hosts. These CBDs generally have a size of about 17kDa.

WO-A-93/21331 discloses fusion proteins including CBDs from Cellumonas fimi and proposes uses including in purification, diagnostics etc.

The present invention concerns novel chemical entities including CBDs and novel uses of CBDs. The term cellulose binding domain (or CBD) is used broadly to refer to any protein sequence capable of binding to cellulose or sugar based polysaccharides, including, but not limited to, naturally occurring CBDs and modified CBDs (modified chemically or altered by genetic changes such as additions, substitutions and deletions in the amino acid sequence) that are still capable of binding to cellulose or sugar polysaccharides with appropriate affinity. The CBD may be on its own, or may be linked to more of the native cellulases or glycosyl hydrolases from which it

is naturally derived. The term CBD as used herein is to be interpreted as covering all such variants.

Summary of the Invention In one aspect, the present invention provides a cellulose binding domain (CBD) conjugate, whereby at least two CBDs are attached to a polysaccharide.

The term "polysaccharide" is intended to cover polysaccharides and oligosaccharides, and references to"polysaccharide"and "polysaccharide conjugate"should be considered accordingly.

The polysaccharide may be naturally occurring or synthetic. The polysaccharide may be one that binds naturally to cellulose or has been derivatised or otherwise modified to bind to cellulose.

In this case, the polysaccharide desirably has a 1-4 linked glycan (generalised sugar) backbone structure, which is stereochemically compatible with cellulose, such as a glucan backbone (consisting of ss 1-4 linked glucose residues), a

mannan backbone (consisting of fui 1-4 linked mannose residues) or a xylan backbone (consisting of ss 1-4 linked xylose residues). Suitable polysaccharides include xyloglucans, glucomannans, mannans, galactomannans, ss (1-3), (1-4) glucan and the xylan family incorporating glucurono-, arabino-and glucuronoarabinoxylan. See"Physiology and Biochemistry of Plant Cell Walls" (1990) by C. Brett and K. Waldron for a discussion of these materials.

The minimum chain length requirement for cellulose oligomers to bind to cellulose is 4 glucose units. For xyloglucans, the side chains make the binding less efficient and 12 backbone glucose units (i. e. about 25 total sugar units) are required for binding to cellulose. Structural considerations suggest galactomannans are intermediate in binding efficiency, and about 6 to 8 backbone residues are expected to be required for binding to cellulose. The polysaccharide should thus have at

least 4, and preferably at least 10, backbone residues, which are preferably 1-4 linked.

Naturally occurring polysaccharides that bind rapidly and strongly to cellulose by polysaccharide: polysaccharide interaction include xyloglucans such as pea xyloglucan and tamarind seed xyloglucan (TXG) (which has a ss 1-4 linked glucan backbone with side chains of a-D xylopyranose and P-D- galactopyranosyl- (1, 2)-a-D-xylopyranose, both 1-6 linked to the backbone: see Gidley et al. Carbohydrate Research, 214 (1991) 200-314 for a discussion of the structure of tamarind seed polysaccharide); and galactomammans, particularly low galactose galactomannans, such as locust bean gum (LBG) (which as a mannan backbone of ss 1-4 linked mannose residues, with single unit galactose side chains linked a 1-6 to the backbone), enzyme modified guar (EMG) (guar gum has the same structural units as LBG but has a much higher level of galactose substitution, to the extent that there is not enough accessible mannan backbone through which to bind cellulose. EMG is produced by enzymic removal from guar gum of a controllable percentage of the galactose residues to produce a range of materials that are capable of binding to cellulose, but are cheaper and more consistently available than LBG. See Bulpin et al. Carbohydrate Polymers 12 (1990) 155-168 for a discussion of EMG), tara glactomannan and cassia galactomannan. These materials are commercially available and thus provide potentially useful sources of suitable polysaccharides. These materials have the advantage of being relatively cheap.

The polysaccharide desirably has side chain galactose residues susceptible to oxidation by galactose oxidase, for production of an aldehyde group for coupling of CBDs as will be described below. TXG, LBG and EMG have such galactose residues.

The polysaccharide, however, need not be capable of binding to cellulose. Polysaccharides such as guar gum fall into this category.

The conjugate preferably comprises a plurality of CBDs attached to the polysaccharide, and desirably multiple (e. g. 3 to 6) CBDs are attached to the polysaccharide at spaced intervals along the length of the polysaccharide backbone. The CBDs attached to a polysaccharide may be the same or different to each other.

The CBD may be attached to the polysaccharide in any convenient way, generally by chemical linking e. g. using either the amine or carboxyl end group of the CBD or by disulphide bridging. One convenient method of attachment to polysaccharides having galactose side chains involves enzymically oxidising the galactose, e. g. using galactose oxidase, to produce an aldehyde group to which an amino group of a protein can be chemically linked. As noted above, TXG, LBG and EMG have suitable galactose side chains. For polysaccharides not having suitable galactose side chains, different methods of chemical linking of proteins can be used. Alternative techniques include limited periodate oxidation, which required the polysaccharide to have two adjacent hydroxyl groups in cis orientation, and results in the production of aldehyde groups which can be reductively aminated. A further possibility is reaction with cyanogen bromide (CNBr) which inserts into sugar rings at vicinal diols, both in the backbone and side chains, to provide an isourea linkable to the amino groups of proteins. It is preferred to use chemical techniques that do not affect the polysaccharide backbone length.

It is found that the CBD conjugates in accordance with the invention can cause aggregation of cellulose via crosslinking, which has possible practical and commercial implications in industrial processes using cellulosic material. Where the polysaccharide is capable of binding to cellulose, only one attached CBD is required (although more are preferred) to produce a conjugate having 2 separate sites capable of binding to cellulose. Where the polysaccharide is not capable of binding to cellulose at least 2 (and preferably more) attached

CBDs are required to produce a conjugate having 2 separate sites capable of binding to cellulose.

In a further aspect the invention provides a CBD conjugate comprising a polysaccharide capable of binding to cellulose, attached to at least two CBDs.

In another aspect the invention provides a CBD conjugate comprising a polysaccharide not capable of binding to cellulose, attached to at least two CBDs.

In another aspect the invention provides a CBD conjugate, whereby at least three CBDs are attached to a polypeptide or protein.

The polypeptide or protein entity performs the function of supporting the CBDs, or serves as a"scaffold"to which the CBDs are attached, and for convenience will be referred to herein as a scaffold.

In a further aspect the invention provides a CBD conjugate comprising a scaffold to which at least three CBDs are attached.

The scaffold may comprise a polysaccharide, as discussed above.

Alternatively the polysaccharide may comprise a protein. It is known to produce so-called CBD dimers. These comprise two CBDs linked by a short flexible peptide, about 5 or 6 amino acids long, produced as a fusion protein by recombinant DNA technology. It is also an aspect of this invention that it is possible to produce genetic fusion proteins of multiple copies of CBD whereby more than 2 CBD domains have been expressed as a single entity. Furthermore, it is also possible to produce hybrid CBD biopolymer on a polypeptide chain or protein chain characterised in that different CBD molecules from different genetic origins (represented by different sequences) are present on the same fusion protein.

WO-A-94/07998 discloses a CBD linked to a catalytically active domain (CAD), with an optional second CBD of the opposed end of the CAD, resulting in the structure CBD-CAD-CBD. There is no specific disclosure of such a structure having been produced.

All specific examples are based on a 43 kDa cellulase with one CBD.

In yet another aspect, the invention provides a CBD conjugate comprising a scaffold to which are attached at least two CBDs spaced apart by a distance in the range ( > CBD dimer, < CBACAD-CBD). Suitable spacing of the CBDs is at least 200 Angstroms.

Conjugates in accordance with the invention can cause aggregation of cellulose, as noted.

The invention therefore provides a method of aggregating cellulose, comprising contacting cellulose with a CBD conjugate comprising a scaffold attached to at least one CBD, the conjugate having at least two sites capable of binding cellulose.

The conjugate preferably has at least two or three CBDs, more preferably more than three CBDs. The CBDs are preferably spaced apart along the length of the scaffold at a spacing of at least 200 Angstroms. The CBDs may be the same or different.

The scaffold may comprise a protein, with the conjugate conveniently being in the form of a fusion protein, or a polysaccharide, e. g. as discussed above.

The conjugates and method of the invention find potential in a number of applications, particularly in the laundry business for creating multiple binding events between cellulosic based surfaces/materials. The CBD conjugates can be used to increase the strength and wear of the fabric by crosslinking fibres. The CBD conjugates may also be used as delivery vehicles to deposit materials in any stage of the laundering

process. This latter application can be achieved by coating the benefit agent (either directly by chemical means or indirectly via a compound associated with the benefit agent e. g. capsule or particle). Examples of such benefit agents are softening agents, finishing agents/protective agents, fragrances (perfumes), bleaching agents.

Examples of softening agents are clays, cationic surfactants or silicon compounds. Examples of finishing agents/protective agents are polymeric lubricants, soil repelling agents, soil release agents, photo-protective agents (sunscreens), antistatic agents, dye-fixing agents, anti-bacterial agents and anti-fungal agents. The fragrances or perfumes may be encapsulated, e. g. in latex microcapsules or gelatin based coacervates.

The invention will be further described by way of illustration, in the following examples and with reference to the accompanying Figures in which: Figure 1 is a schematic illustration of a hypothetical model for the aggregation of cellulose particles by a CBD-LBG conjugate Figure 2 illustrates the results of 14% SDS-PAGE to demonstrate the efficiency of cross linking CBD to LBG ; Figure 3 are photographs illustrating the effect of CBD-LBG conjugate in aggregating acid swollen cellulose particles, for LBG, CBDcc uncoupled and CBDcc conjugated to LBG.

Figure 4 are photographs similar to Figure 3 illustrating the effect of CBDcc2-LBG conjugates in aggregating Sigmacell; and Figure 5 illustrates a series of photographs whereby CBD fusion proteins are capable of crosslinking and aggregating chitin polysaccharides.

Examples 1.0 The following examples concern experiments with the following CBDs (Clostridium cellulovorans recombinant, single (CBDcc) and dimer (CBDcc2) ) and a fungal CBD (Thermomonospora fusca E3 (CBDE3). The CBD fusion proteins containing one, two or three CBD's (using the sequence from T. Reesei cellobiohydrolase I) were constructed according to standard molecular biology techniques.

In general terms, the experiments involved making and evaluating novel CBD-LBG conjugates with an ability physically to aggregate cellulosic particles (as a model of cellulosic fibres). This was achieved by oxidising the galactose side chains of LBG and cross linking the cellulose binding domain (by reductive amination). The resulting product was a soluble polysaccharide with multiple cellulose binding groups displayed on the surface. The ability of the new conjugate to bind to cellulosic materials was examined. It was anticipated that cellulose binding would be via the CBD and the LBG, thus exploiting the dual binding modes.

In order to assess the conjugation efficiency of the CBD-LBG molecule, a biotinylation step was initially introduced, whereby the biotin could be used as a reporter molecule. Once the conjugation parameters were established, then this biotinylation step was omitted, and the influence of the multivalent-cellulose binding domain species in aggregating cellulosic particles followed by light microscopy.

2.0 Materials and Methods 2.1 Oxidation of LBG by galactose oxidase LBG and galactose oxidase were obtained from Sigma Chemical Company (G0793 and G7907, respectively). 100 mg LBG was dissolved in 100 ml sodium phosphate, pH 7.0. The resulting solution was heated to 80-90 C with constant stirring for 30

minutes and then cooled to room temperature. Galactose oxidase was dissolved in O. 1M sodium phosphate, pH 7.0, to a final concentration of 50pg/ml. Aliquots from 200-400pl of this solution were added to 1.2 ml LBG solution and the reaction mixtures were incubated at 370C for 5.5 hours.

2.2 Conjugation of CBD to oxidised LBG 0.5 mg CBD was equilibrated into 0.1 M sodium phosphate, pH 6.5, using a 10 kDa Amicon Microcentricon. This CBD preparation (total volume of 0.4ml) was added to 0.6 ml oxidised LBG/galactose oxidase, and incubated for 2 hours at room temperature. Sodium cyanoborohydride (NaBH3CN, 10 pl at 20 mg/ml) was added and the tubes were left at ambient temperature overnight. Excess NaBH3CN and uncoupled CBD were removed by concentration with a 50 kDa Amicon Microcentricon.

2.3 Binding analysis of H3PO4 Avicel and Sigmacell by CBD-LBG conjugates Particle aggregation by the conjugates was investigated with the assumption that complexes, as shown in Figure 1, would form. Using mesh filters, a solution of 1-30 pM H3PO4, Avicel particles and 1-50 pl Sigmacell (Sigmacell is another grade of crystalline cellulose) particles were prepared. 50 p1 of each cellulosic material was pipetted into a 0.45 pM filter plate and was and filtered 3 times with 50 mM sodium acetate, pH 5.0.

Particles were resuspended in 100 pl CBD-LBG conjugate and incubated on a plate shaker for 1 hour at room temperature. The

final volume in wells was reduced to 20 pi by filtering and 2. 5 pi aliquots were removed for analysis by phase contrast microscopy (magnification used was x10x12. 5 for each sample).

2.4 Determination of conjugation by gel electrophoresis A 14% SDS-PAGE gel showed that conjugation efficiency was high (performed using a Novex XCell II system with loading of 10 pg

CBD protein, Figure 2). Equivalent CBD protein levels had been loaded as comparable standards with the presumption that the CBD-LBG conjugate (high molecular weight) would not enter the gel matrix. The CBDcc conjugate had a minor proportion of the CBD remaining uncoupled, whereas with the dimer, CBDcc2, all protein had coupled. The favourable coupling results obtained with CBDcc and CBDcc2 are not surprising, for 7 lysines per domain are available for chemical binding. The CBDE3 protein was visible in the conjugate mix and confirms the poor conjugation chemistry obtained with this protein.

2.5 Binding CBD-LBG conjugates to cellulosic particles The proposed scheme for binding and crosslinking using CBD conjugates is shown in Figure 1. The binding of the different CBD-LBG conjugates and their derivatives was tested by the following method, and results are shown in Figure 3 and Figure 4, for H3P04 Avicel and Sigmacell, respectively. These figures include various CBD-LBG molecules coupled or uncoupled in the

aggregation of two cellulosic materials, H3P04, Avicel and Sigmacell. 50 pl 1% (w/v) cellulosic material was pipetted into a filter plate well (Millipore). This cellulosic material was washed 3 times with 50 mM sodium acetate, pH 5.0. 100 pl CBDLBG conjugate was added (containing at 70 pg CBD) and incubated with the cellulosic material for 1 hour at room temperature on a shaking platform. The volume was then reduced to 20 pl by filtering, and 2.5 pl of this solution was pipetted onto a glass slide and observed by phase contrast microscopy. Each original picture has a magnification factor of x125. In Figure 3, with the CBDcc-LBG conjugate present, the size of the aggregated cellulose is 0. 8mm in diameter.

The most striking and important result of this study was that conjugates of CBD and LBG could cause highly efficient physical aggregation of both types of cellulose particle. The aggregation of H3P04 Avicel by CBDcc-LBG conjugate is shown in Figure 3. Because LBG is known to bind to H3P04 Avicel, (section 3.1) the LBG alone or uncoupled (with CBD present)

was also investigated, but neither resulted in any comparable aggregation. These observations were also confirmed in the aggregation of Sigmacell (Figure 4). The divalent CBDcc2 and the CBDE3 LBG conjugates also caused aggregation of H3P04 Avicel but were not as effective as the CBDcc-LBG molecule. The LBG could participate as a molecular scaffold or as a co-binder.

An option is to use guar gum (which is readily available) which would enable an increased attachment of more CBD molecules.

Using this gum would also prevent non-specific binding of the polysaccharide to cellulosic surfaces because the open P 1-4 galactomannan regions are absent.

The crystalline cellulose, Sigmacell, was only aggregated in those samples where the CBD was coupled to the LBG. There was a dramatic effect of aggregation by the CBDcc-LBG and CBDcc2-LBG conjugates. Simple mixtures of CBD and LBG did not cause aggregation. The CBDcc2-LBG conjugate caused a high degree of aggregation, which would agree with its increased ability to bind to crystalline cellulose.

2.6 Construction of CBD fusion proteins Single, double and triple CBD domain repeats were constructed as single fusion proteins. The CBD used was the CBD sequence obtained from T. Reesei cellobiohydrolase I. Construction of such molecules was performed according to established molecular biology techniques.

The ability of the CBD proteins to aggregate chitin and microcrystalline cellulose was investigated as follows: A sub 20pm fraction of dispersed chitin fragments was placed into a lml solution of Phosphate Buffered Saline containing either: water, 100pg CBD (CBDE) or 100pg CBD-CBD-CBD (CBD3).

Samples were incubated for 60 minutes at room temperature and the presence of flocculation was observed. Figure 5 illustrates the levels of flocculation observed. This was repeated using

cellulose beads (Figure 5). In all cases evidence of aggregation of polysaccharide based particulates was observed in the samples containing the fusion protein with three CBD repeats. This highlights the need for multiple binding sites on the same molecule.

Claims

CLAIMS 1. A cellulose binding domain (CBD) conjugate, whereby at least two CBDs are attached to a polysaccharide.
2. A CBD conjugate according to claim 1, wherein the polysaccharide is capable of binding to cellulose.
3. A CBD conjugate according to any one of the preceding claims, wherein the polysaccharide has a 1-4 ss linked glycan backbone structure.
4. A CBD conjugate according to any one of the preceding claims, wherein the polysaccharide has a glucan backbone, a mannan backbone or a xylan backbone.
5. A CBD conjugate according to any one of the preceding claims, wherein the polysaccharide is selected from xyloglucans, glucomannans, mannans, galactomannans, ss (I-3), (1- 4) glucan and the xylan family incorporating glucurono-, arabino-and glucuronoarabinoxylan.
6. A CBD conjugate according to any one of the preceding claims, wherein the polysaccharide is selected from xyloglucans such as tamarind seed xyloglucan (TXG) and pea xyloglucan; and galactomannans, particularly low galactose glactomannans, such as locust bean gum (LBG), enzyme modified guar (EMG), tara galactomannan and cassia galactomannan.
7. A CBD conjugate according to any one of the preceding claims, wherein the polysaccharide has side chain galactose residues susceptible to oxidation by galactose oxidase.
8. A CBD conjugate according to any one of the preceding claims, comprising a plurality of CBDs attached to the polysaccharide.

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9. A CBD conjugate according to any one of the preceding claims, wherein the or each CBD is chemically linked to a respective aldehyde group formed on the polysaccharide.
10. A CBD conjugate according to any one of the preceding claims, wherein each CBD is chemically linked to TXG, LBG or EMG via a respective aldehyde group produced by enzymic oxidation of galactose side chains.
11. A CBD conjugate according to any one of the preceding claims, comprising a polysaccharide not capable of binding to cellulose.
12. A cellulose binding domain (CBD) conjugate, whereby at least three CBDs are attached to a polypeptide or protein.
13. A CBD conjugate according to claim 12, whereby the protein backbone is greater than 5 kDa.
14. A CBD conjugate according to claims 12-13, whereby the CBD's are linked genetically to the peptide or protein sequence.
15. A CBD conjugate according to any one of the preceding claims, whereby at least two different CBD's of different origin are attached to the backbone.