Review article

Cellulose-binding domains

Biotechnological applications

Ilan Levy, Oded Shoseyov*

The Institute of Plant Science and Genetics in Agriculture and The Otto Warburg Centre for Agricultural

Biotechnology, The Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of

Jerusalem, PO Box 12, Rehovot 76100, Israel

Accepted 6 January 2002

Abstract

Many researchers have acknowledged the fact that there exists an immense potential for the

application of the cellulose-binding domains (CBDs) in the field of biotechnology. This becomes

apparent when the phrase ‘‘cellulose-binding domain’’ is used as the key word for a computerized

patent search; more then 150 hits are retrieved. Cellulose is an ideal matrix for large-scale affinity

purification procedures. This chemically inert matrix has excellent physical properties as well as low

affinity for nonspecific protein binding. It is available in a diverse range of forms and sizes, is

pharmaceutically safe, and relatively inexpensive. Present studies into the application of CBDs in

industry have established that they can be applied in the modification of physical and chemical

properties of composite materials and the development of modified materials with improved

properties. In agro-biotechnology, CBDs can be used to modify polysaccharide materials both in vivo

and in vitro. The CBDs exert nonhydrolytic fiber disruption on cellulose-containing materials. The

potential applications of ‘‘CBD technology’’ range from modulating the architecture of individual cells

to the modification of an entire organism. Expressing these genes under specific promoters and using

appropriate trafficking signals, can be used to alter the nutritional value and texture of agricultural

crops and their final products.

D 2002 Elsevier Science Inc. All rights reserved.

Keywords: Cellulose; Cell wall; Cellulose-binding domain (CBD); Pulp; Paper; Biotechnology

0734-9750/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved.

PII: S0734 -9750 (02 )00006 -X

* Corresponding author. The Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew

University of Jerusalem, PO Box 12, Rehovot 76100, Israel. Tel.: +972-8-9481084; fax: +972-8-9462283.

E-mail address: shoseyov@agri.huji.ac.il (O. Shoseyov).

www.elsevier.com/locate/biotechadv

Biotechnology Advances 20 (2002) 191–213

1. Introduction

It was proposed in late 1940s, that the initial stage in the enzymatic degradation of

crystalline cellulose involves the action of an unknown nonhydrolytic component termed C1.

This component was thought to be responsible for destabilization (nonhydrolytic disruption)

of the cellulose structure, making the substrate accessible to the enzyme, Cx component

(Reese et al., 1950). The cellulose-binding domain (CBD) was first demonstrated in the

fungus Trichoderma reesei and the bacterium Cellulomonas fimi (Van Tilbeurgh et al., 1986;

Gilkes et al., 1988). The connecting linker between the CBD moiety and the enzyme proved

to be susceptible to proteolysis, thus allowing for isolation of the individual domain by

limited proteolysis. Forty years after the C1–CX model was proposed, the first C1 component

was cloned from Clostridium cellulovorans and C. fimi (Shoseyov et al., 1990; Shoseyov and

Doi, 1990; Din et al., 1991; Goldstein et al., 1993). This achievement gave researchers an

opportunity to study the C1–CX hypothesis. To date, domain structures and biochemical

functions of many CBDs have been deciphered (for review, see Gilkes et al., 1991; Davis,

1998; Tomme et al., 1995b, 1998).

In earlier studies of CBD–cellulose interactions, the presence of a CBD was shown to

increase the effective concentration of enzyme on insoluble cellulose substrates, thereby

assisting the enzyme through the phase transfer from soluble fraction (the enzyme) to

insoluble fraction (the substrate) (Shoseyov and Doi, 1990; Beguin and Aubert, 1994; Din et

al., 1994; Linder et al., 1995; Tomme et al., 1995a; Bolam et al., 1998; Suurnakki et al.,

2000).

CBDs have been found in hydrolytic and nonhydrolytic proteins. In proteins that possess

hydrolytic activity (cellulases, xylenases), the CBD is a discrete domain that concentrates the

catalytic domains on the surface of the insoluble cellulose substrate (Gilkes et al., 1991;

Tomme et al., 1995a,b, 1998; Linder et al., 1997; Teeri et al., 1998). The CBDs present in

proteins that do not have hydrolytic activity compose part of a scaffolding subunit that

organizes the catalytic subunits into a cohesive multienzyme complex known as a cellulo-

some. The enzymatic complex was found to function more efficiently in the degradation of

cellulosic substrates (Woodward et al., 1988; Shoseyov and Doi, 1990; Doi et al., 1994;

Beguin and Alzari, 1998; Bayer et al., 1998a,b). Removal of the CBD from the cellulase

molecule or from the scaffolding in cellulosomes dramatically decreased enzymatic activity

(Van Tilbeurgh et al., 1986; Tomme et al., 1988; Hefford et al., 1992; Goldstein et al., 1993;

Coutinho et al., 1993; Carrard and Linder, 1999).

CBDs have also been found in several polysaccharide-degrading enzymes. In T. reesei,

CBD has been identified in hemicellulase, endo-mannanase and acetyl-xylanesterase (Stal-

brand et al., 1995; Margolles-Clark et al., 1996). CBDs have been recognized in xylanase

originating from Clostridium thermocellum (Kulkarni et al., 1999; Kim et al., 2000), esterase

from Penicillium funiculosum (Kroon et al., 2000), and pectate lyase in Pseudomonas

cellulosa (Brown et al., 2001). In addition, there exists the intriguing presence of such a

domain in b-glucosidase located in Phanerochaete chrysosporium (Lymar et al., 1995). The

presence of putative CBDs in plant endoglucanases has also been reported (Catala and

Bennett, 1998; Trainotti et al., 1999). Expansins, that are believed to play a role in

I. Levy, O. Shoseyov / Biotechnology Advances 20 (2002) 191–213192

nonhydrolytic cell wall expansion, are homologues to CBDs and possess cellulose binding

capabilities in vitro (Cosgrove, 2000).

Today, more than 200 putative sequences, in over 40 different species, have been

identified. The binding domains are classified into 14 different families based on amino

acid sequence, binding specificity, and structure (Gilkes et al., 1991; Tomme et al., 1995a,b,

1998; Bayer et al., 1998a). Families V and VIII contain only one member each, while

Families I, II, and III consist of 40 or more members (Tomme et al., 1998). The CBDs can

contain 30–180 amino acids, and exist as a single, double, or triple domain in one protein.

Their location within the parental protein can be either C- or N-terminal and occasionally

centrally positioned in the polypeptide chain. The affinity and specificity towards different

cellulose allomorphs can vary (for an extended review on CBDs, see Gilkes et al., 1991;

Henrissat, 1994; Tomme et al., 1995b, 1998; Bayer et al., 1998a,b. Extensive data and

classification can de found in the Carbohydrate-Binding Module Family Server at http://

afmb.cnrs-mrs.fr/~pedro/CAZY/cbm.html). Three-dimensional structures of representative

members of CBD Families I, IIa, III, IV, V, VI, IX, and XV have been resolved by

crystallography and NMR (Xu et al., 1995; Tormo et al., 1996; Johnson et al., 1996; Brun

et al., 1997; Sakon et al., 1997; Mattinen et al., 1998; Notenboom et al., 2001; Szabo et al.,

2001; Czjzek et al., 2001). Data from these structures indicate that CBDs from different

families are structurally similar and that their cellulose binding capacity can be attributed, at

least in part, to several aromatic amino acids that compose their hydrophobic surface. The

positions and angles between these aromatic amino acids differ between various CBD

members. CBDCex, from Family IIa, contains a b-barrel-type backbone that displays

aromatic amino acids on a relatively flat surface (Din et al., 1994; Tormo et al., 1996;

Nagy et al., 1998). On the other hand, CBDN1 from Family V, displays its aromatic amino

acids in a narrow groove (Johnson et al., 1996; Tomme et al., 1996). CBDs from the same

organism can differ in their binding specificity (Carrard and Linder, 1999) and, occasion-

ally, two CBDs located on the same enzyme can also exhibit this distinction (Brun et al.,

2000).

Biochemical studies have shown that the course of events leading to the binding of CBD to

cellulose is directed by several driving forces. In the case of CBDCex, which binds to

crystalline cellulose, the process is entropically driven. The decrease in entropy can be

attributed to a net loss in conformational freedom of the polysaccharide and protein side

chains. Water hydration upon binding may be another factor leading to lower entropy (Creagh

et al., 1996). On the other hand, binding of CBDN1 to amorphous cellulose is driven by

enthalpy. This force can be attributed to heat release, which occurs upon complex formation

that transpires through hydrogen and van der Waals bonding between the equatorial hydroxyl

of the glucopyranosyl ring and the polar amino acids (Brun et al., 2000). Although the

interaction of the CBD with the cellulose is occasionally irreversible, contact with the

cellulose surface is dynamic. Jervis et al. (1997) demonstrated by using fluorescence recovery

techniques, that CBDCex is mobile on the surface of crystalline cellulose when it appears in

isolated form or as a module in xylanase. Furthermore, it was hypothesized that the binding of

Family IIa CBD from C. fimi to cellulose occurs either along or across the chain (McLean et

al., 2000).

I. Levy, O. Shoseyov / Biotechnology Advances 20 (2002) 191–213 193

CBDs, constituting isolated modules, are utilized in many different applications. This

article will review the potential applications of CBDs in diverse fields of biotechnology.

2. Bioprocessing

Large-scale recovery and purification of biologically active molecules continues to be a

challenge for many biotechnology companies. Various purification procedures have been

developed, of which biospecific affinity purification (affinity chromatography) has become

one of the most rapidly developing divisions of immobilized affinity ligand technology. To

date, several affinity tags have been developed that vary in size from several amino acids to a

complete protein. Each individual affinity-based purification system embodies specific

advantages. Cellulose, when compared with most immobilization systems, is an economical

support-matrix for large-scale protein purification (for review, see Harakas, 1994; Wilchek

and Chaiken, 2000; Lowe, 2001). The wide use of CBD as an affinity tag in expression and

purification is illustrated in Fig. 1. This subject has been extensively reviewed (Ong et al.,

1989; Greenwood et al., 1992; Bayer et al., 1994; Tomme et al., 1998; Saleemuddin, 1999)

and described in several patents (Shoseyov et al., 1997, 1998a,b; Kilburn et al., 1999a,b;

Fig. 1. CBD-based expression and purification of recombinant proteins. Protein expression and purification via

CBD involves several steps. (1) Gene fusion between cbd and a gene of interest. (2) Transformation of ligated

plasmid vector into a prokaryotic or eukaryotic expression system. (3) Overexpression of the recombinant protein.

(4) Purification by immobilization of CBD-tagged protein on cellulose. (5) Reconstitution of the target protein by

(A) gentle elution of target protein from cellulosic matrix, (B) addition of a ligand or a substrate, or (C) proteolytic

cleavage of the engineered sequence located between the CBD and the target protein.

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Meade, 2000; Meade et al., 2001); therefore, this section will review only current develop-

ments.

Recent reports relating to multifunctional studies have further affirmed the feasibility of

employing CBD as an affinity tag. Atrazin dechlorinating enzyme (Kauffmann et al., 2000),

a-amylase (Bjornvad et al., 1998), lipase B (Rotticci-Mulder et al., 2001), glucoamylase

(Jiang and Radford, 2000), and organophosphorus hydrolase (Richins et al., 2000) have been

expressed as CBD-fused enzymes while retaining their high specific activity. In addition,

human T cell connective tissue-activator peptide-III (CTAP-III) (Rechter et al., 1999), human

hsp60 epitope (Shpigel et al., 1998a,b), protein A (Shpigel et al., 2000), and murine stem-cell

factor (SCF) (Doheny et al., 1999; Boraston et al., 2001) were also expressed as CBD-fused

proteins. All these studies established the fact, that CBDs can be employed as high-capacity

purification tags for the isolation of biologically active target peptides, at relatively low cost.

Matrix-assisted refolding of recombinant proteins is one of the approaches taken in order

to prevent the aggregation of protein during the course of renaturation. At the present, only

histidine and arginine tags have been found to be suitable for this process as they maintain

their matrix binding ability under denaturing conditions (Stempfer et al., 1996; Glansbeek et

al., 1998) Recently, a CBD derived from Clostridium thermocellum was used as a tag for

matrix-assisted refolding of a single-chain antibody expressed in Escherichia coli. This CBD

binds to cellulose in the presence of 6 M urea. The method was shown to provide a threefold

increase in protein yields when compared to standard refolding procedures (Berdichevsky et

al., 1999a).

Phage display technology is a proven tool for isolating biologically active molecules

(Cortese et al., 1996; Forrer et al., 1999; Johnsson and Ge, 1999; Rodi and Makowski, 1999;

Gaskin et al., 2001). One of the limitations preventing extensive implementation of this

technology is the relatively high proportion of clones that lack insertions within the library. In

a recent study, CBD from Clostridium thermocellum was fused to a single-chain antibody

(scFv) and expressed as scFv–CBD phage display library. The CBD tag allowed for rapid

recovery of phages that displayed functional inserts, thus increasing the efficiency of the

screening process for recombinant antibodies (Berdichevsky et al., 1999b).

Direct passive coating of proteins to plastic can result in partial or total denaturation of the

adsorbed molecule. This can be attributed to hydrophobic interaction between the protein and

the solid phase. (Suter and Butler, 1986; Schwab and Bosshard, 1992). Recently, Levy and

Shoseyov (2002) used phage display of a random peptide library to screen for peptides that

enable indirect immobilization of proteins to a solid surface via CBD. It was demonstrated

that the affinity between the four amino acids tag and a CBD could be employed to

immobilize horseradish peroxidase (HRP) on CBD-precoated cellulosic surfaces. This

technique enables researches to tailor-make fusion tags that will mediate indirect noncovalent

immobilization of proteins to solid matrixes.

The binding of CBD to cellulose can be classified as reversible (Family I) or irreversible

(Families II and III). When Family I CBD is used as an immobilizing tag, a low-rate column

leakage is often observed (Linder et al., 1996). In order to overcome this problem, Linder et

al. (1998) constructed a chimerical protein that was composed of CBDHII and CBHI from T.

reesei, and a single-chain antibody. A significant decrease in protein leakage was observed

I. Levy, O. Shoseyov / Biotechnology Advances 20 (2002) 191–213 195

with this unique structure, indicating that CBD–antibody fused proteins are suitable tools for

affinity chromatography.

Haynes et al. (2000) proposed a novel two-phase separation system to purify proteins from

aqueous solutions by utilizing Family IV CBD that binds to water-soluble cellulosic materials

such as hydroxyethylcellulose. The system was composed of a phase-forming polysaccharide

polymer to which CBD can bind and a phase-inducing agent such as polyethylene glycol. The

solution that contains a CBD-fused peptide or protein was mixed with the phase-forming

oligosaccharide followed by the addition of the phase-inducing agent. The two phases were

then separated and the target protein purified. This system can be very effective for the

separation of proteins from fermentation broths as well as from other aqueous solutions.

Production of recombinant proteins in plants has been recently recognized as one of the

most cost-effective production systems. However, a major drawback of this system lies in the

fact that plants contain high levels of polysaccharides and phenolic components which

interferes with the purification process (Herbers and Sonnewald, 1999; Hood and Jilka, 1999;

Doran, 2000; Giddings, 2001). The utilization of CBDs in the production of CBD fusion

proteins in plants enables efficient production, taking advantage of the fact that the plant cell

wall is made of cellulose; thus, the plant manufactures both the protein and its purification

matrix (Shani and Shoseyov, 2001).

3. Targeting

Cellulose is a major constituent of many commercial products; therefore, targeting of

functional molecules to cellulose-containing materials can be mediated by CBDs. The

commercial potential of CBD in this context was first realized for denim stonewashing. In

the late 1980s, cellulases were employed as an alternative to the original abrasive stones. The

presence of CBD allowed for the targeting of the enzyme onto the garment. The final product

was fabric or a garment with a ‘‘stone-washed’’ or ‘‘worn’’ look exhibiting localized variation

in color density. The first cellulases were crude enzymes produced from Trichoderma and

Humicola species that included cellulases other than endoglucanases. The use of enzyme

mixtures was problematic since some of the enzymes present in the mixtures contained

cellulolytic activity towards insoluble cellulose and this occasionally caused a decrease in

fiber strength (Cavaco-Paulo et al., 1996, 1998a,b; Eriksen, 1996). It was found that

subjecting dyed denim to enzymes with pectolytic activity, such as pectate lyases, pectin

lyases, or polygalacturonases, resulted in a ‘‘stone-washed’’ appearance. With the introduc-

tion of recombinant enzyme technology, the strong affinity between cellulose and CBD was

utilized for enzyme targeting to garments (Andersen et al., 2001). This development

eventually evolved into an alternative process that completely replaced the traditional stones

(Kalum and Andersen, 2000; Andersen et al., 2001; Miettinen-Oinonen et al., 2001).

The strong affinity that exists between cellulose and CBD is used in many applications

associated with the textile industry. Numerous laundry powders contain recombinant enzymes

that do not possess a native affinity to the cellulosic fabric (amylases, proteases, lipases, and

oxidoreductases). The performance of these enzymes, under conventional washing condi-

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tions, can be improved by increasing their affinity to the textile substrate. This can be

achieved by fusion to CBDs (von der Osten et al., 2000b). Additional substances can also be

targeted to cellulosic fabrics. Fragrance-bearing particles, conjugated to CBD, can be added

to laundry powder hence reducing the amount of fragrance needed in the product (Berry et al.,

2001).

Threads are exposed to considerable mechanical strain during the weaving process and in

order to prevent tearing, they are reinforced by gelatinous substances by a process termed

‘‘sizing.’’ The most popular material used for this procedure is starch, but substances such as

PVA, PVP, PAA or other cellulose derivatives such as CMC, hydroxyethyl–cellulose,

hydroxypropyl–cellulose, and methylcellulose are also employed. A contradictory effect of

the sizing agents is that fabrics are not able to absorb finishing agents, such as dyes, that are

frequently dissolved in water. In order to improve the enzymatic ‘‘desizing’’ process, target

enzymes can be fused to CBD, in this manner increasing the affinity of enzymes to the

cellulosic fabric (von der Osten et al., 2000a).

Antimicrobial agents can be targeted to polysaccharide materials. Emerson et al. (1998)

proposed the targeting of aromatic aldehydes or alcohols to cellulose-containing materials.

Aromatic aldehydes and alcohols, including benzaldehyde, acetaldehyde cinnamaldehyde,

piperonal, and vanillin, are known to be effective disinfectants for bacteria, fungi, and viruses

and are nontoxic to humans or animals. Targeting can be attained with the assistance of CBD

and may be useful for directly impregnating surfaces such as paper or wood (Emerson et al.,

1998).

Another interesting application is oral care products. Fuglsang and Tsuchiya (2001)

applied CBD to orally present polysaccharides (fructan and glucan) that are known to be

involved in dental plaque. They found that CBD disperses oral polysaccharides thereby

removing and preventing plaque formation. In addition, they established that CBD could be

fused to enzymes that are capable of dental plaque polysaccharide degradation and that they

could be employed safely in improved plaque removal. Research carried out by Fuglsang and

Tsuchiya (2001) concluded that CBD, on its own or combined with other ingredients when

used in conventional oral hygiene, will remove existing plaque or prevent its formation.

Cellulases are employed in the degradation of gums that are part of the dough structure in

breads. The process must be mild, since excess activity can damage dough structure and

lower the quality of the final product. Enzymatic activity of Trichoderma cellulase is too

aggressive; consequently, Aspergillus cellulase is used in its place (Godfrey, 1996). Fuglsang

and Jorgensen (1998) demonstrated another use for CBDs in the baking industry. An

antistaling enzyme such as amylolytic enzymes was fused to CBD and used to retard staling

and aging of baked bread.

4. Cell immobilization

Cell immobilization technology has many applications in biotechnology. The applications

range from ethanol production and phenol degradation (Mordoccoa et al., 1999; Nigam,

2000) to mammalian cell attachment (Yamada, 1983; Kleinman et al., 1987), and whole-cell

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diagnostics (Gunneriusson et al., 1996; Stahl and Uhlen, 1997; Samuelson et al., 2000).

Several industrial technologies have been developed to immobilize cells; however, they have

serious drawbacks. Hollow fibers are expensive and undergo a steady decline in filtration rate

(Kang et al., 1990). Covalent immobilization results in loss of cell viability (Jirku, 1999)

while cell entrapment is affected by a high degree of mass transfer resistance between the cell

and its surroundings (Pilkington et al., 1998).

Whole-cell immobilization to cellulosic material was first demonstrated when E. coli

surface-anchored CBD, derived from C. fimi, was attached to cellulose (Francisco et al.,

1993). In this study, recombinant E. coli cells expressed surface-exposed CBD that enabled

high affinity and specific immobilization onto the cellulose surface. Subsequently, it was

shown that immobilization via CBDCex derived from C. fimi provided a monolayer of cells on

different cellulosic supports. The cells bound tightly to cellulose at a wide range of pHs and

the extent of immobilization was dependent on the amount of surface-exposed CBD (Wang et

al., 2001). In a different study, Staphylococcus carnosus was chosen to display CBDCel6A

from T. reesei on its cell surface and the addition of the CBD predisposed the anchoring of

bacterial cells to cotton fibers (Lehtio et al., 2001).

A different strategy for cell immobilization was demonstrated by fusing the cell attachment

peptide, RGD, to CBDCenA from C. fimi. This novel approach enabled cell immobilization

without the need for expensive attachment factors (Wierzba et al., 1995). Recently, it has been

demonstrated that stem cell factor immobilized onto cellulose via CBDCex from C. fimi is

more potent in stimulating the proliferation of factor-dependent cell lines when compared to

the soluble unbound growth factor (Doheny et al., 1999).

Surface-exposed CBD is an efficient means of whole-cell immobilization. The process is

uncomplicated, mild, and inexpensive. Furthermore, this CBD technology provides an

enhanced method for growth factor and cytokine presentation in primary cell cultures.

5. Protein engineering with CBD

Protein engineering, using CBDs, is an emerging field. High-level expression vectors have

been designed for the production of CBD-fused proteins. Graham et al. (1995) and

Hasenwinkle et al. (1997) constructed an expression vector for C- or N-terminal CBD-fused

proteins (pTugA and pTugK) based on CBDCex from C. fimi. Other studies have shown that

expressing foreign proteins fused to CBD, for the most part, resulted in high expression levels

(Shpigel et al., 1998b, 1999, 2000; Doheny et al., 1999; Rechter et al., 1999; Richins et al.,

2000; Kauffmann et al., 2000; Levy and Shoseyov, 2001; Rotticci-Mulder et al., 2001;

Boraston et al., 2001). Based on these developments, Novagen has utilized this technology to

add to their pET expression vector panel, a group of expression vectors (pET34–38) that

incorporate CBDs as their fusion tags (Novy et al., 1997).

The beneficial effect that CBD has on the expression of proteins was demonstrated in

several studies. Replacing the CBD of endo-1,4-b-glucanase from Bacillus subtilis (Ben) with

the CBD of exoglucanase I (Texl) from T. viride resulted in high expression levels in E. coli

(Kim et al., 1998). Similar results were reported by Otomo et al. (1999). Segmental isotope

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labeling is a new technique that enables observation, by NMR, of signals emitted by selected

N- or C-terminal regions along a peptide chain. Low levels of protein expression will result in

low levels of segmentally labeled protein and an ineffective signal (Yamazaki et al., 1998).

Maximum isotope labeling was achieved with increased levels of expression when the N-

terminal fragment of a specific protein was fused to a CBD (Otomo et al., 1999).

Several studies have demonstrated the potential for employing CBD for modification of

the activity characteristics of an enzyme. Addition of CBD, derived from cellobiohydrolase II

of T. reesei, to the T. harzianum chitinase resulted in increased hydrolytic activity of insoluble

substrates (Limon et al., 2001). Replacing the CBD of endo-1,4-b-glucanase from B. subtilis

(Ben) with the CBD of exoglucanase I (Texl) from T. viride affected a higher binding

capability and enhanced hydrolytic activity on microcrystalline cellulose. In addition, the

hybrid enzyme was more resistant to tryptic digestion (Kim et al., 1998). CBDs can be

designed to conform to diverse reaction conditions. Linder et al. (1999) rationally modified

the small CBD from Cel7A cellobiohydrolase from T reesei, to be sensitive to pH. By

replacing the tyrosine residues in two different positions with histidine, a definite pH

dependency was obtained. As a result of this manipulation, the binding efficiency of the

mutant CBD, at optimal pH, was inferior to that of the wild type.

CBD technologies are a valuable tool in the emerging field of designed protein scaffolds.

Engineered protein scaffolds are novel types of ligand receptors designed for use in various

applications relating to research and medicine (reviewed in Skerra, 2000). Within this group,

one can find the so-called ‘‘knottins,’’ which are a family of rather small proteins that binds to

a wide range of molecular targets such as proteins, sugars, and lipids (Le Nguyen et al.,

1990). The natural knottins vary both in length and sequence; consequently, the core of the

knottins can be a suitable scaffold for novel binding activities. Smith et al. (1998) utilized the

flat hydrophobic face of the wedge-shaped CBD from T. reesei for the introduction of random

mutations in seven side chains. The mutated CBD was then displayed on phage and screened

on various targets (cellulose, a-amylase, alkaline phosphatase, and b-glucuronidase). While

selection experiments for a-amylase and b-glucuronidase failed, CBD variants with affinity

to alkaline phosphatase were successfully isolated with the highest KD being 10 mm. A similar

approach was taken by Lehtio et al. (2000) when screening for a-amylase inhibition in a

combinatorial library of CBD scaffold that was displayed on phage. The library in use was

comprised of variants of the CBD (cellobiohydrolase Cel7A from T. reesei) that were

randomized in 11 positions located in the surface domain. Using this library, two CBD

variants were found to selectively inhibit a-amylase and that were capable of competing with

the binding of the amylase inhibitor, acarbose (Lehtio et al., 2000). Using the same CBD

displayed library, Wernerus et al. (2001) generated a metal-binding protein. This engineered

CBD protein (that was deficient in cellulose binding capacity) was displayed on the surface of

S. carnosus and conferred nickel binding properties to the bacteria. The study demonstrated,

for the first time, that it is feasible to engineer a metal binding protein and to display it on the

surface of gram-positive bacterium (Wernerus et al., 2001). Fierobe et al. (2001) used a

different strategy when they designed and produced active cellulosome. To construct the

desired complex, a series of chimerical scaffolds was prepared. The molecular building

blocks were obtained from the two clostridia cellulosomes, C. thermocellum and C.

I. Levy, O. Shoseyov / Biotechnology Advances 20 (2002) 191–213 199

cellulolyticum. The designed chimerical cellulosomes exhibited enhanced synergistic action

on crystalline cellulose (Fierobe et al., 2001). It seems evident that CBD has the potential of

being used as a molecular scaffold (Shoseyov and Doi, 1990; Doi et al., 1994; Beguin and

Alzari, 1998; Bayer et al., 1998a,b).

The characteristics of motifs that bind to GroEL were analyzed using affinity panning of

immobilized GroEL chaperonin for a phage display library of randomized fungal CBD. This

study revealed that GroEL could bind a wide range of structures with exposed side chains; a

finding that further substantiates the unfolding activity of GroEL by binding extended

conformation of the substrate (Chatellier et al., 1999).

6. Diagnostics

Biosensors have enormous potential in the analysis of complex systems due to the high

specificity and sensitivity of biomolecules (Hill and Davis, 1999; Turner, 2000; Scheller et

al., 2001). In bioprocesses such as fermentation, optimization can only be achieved if the

different components in the bioreactor are monitored and controlled. In order to address this

problem, Phelps et al. (1994) harnessed CBDs as a tool for glucose biosensing. This novel

approach is based on the reversible immobilization of chemically conjugated CBD–glucose

oxidase (CBDCex from C. fimi) that can be repeatedly loaded onto a cellulose probe. The

binding of CBDCex is reversible and consequently when the enzyme activity deteriorates, the

sensor can be regenerated by eluting the original bound enzyme and substituting it with a

fresh source (Phelps et al., 1994, 1995; Turner et al., 1997).

Staphylococcal Protein A is a cell wall protein consisting of five specific Fc IgG-binding

domains which, when bound to the antibody, do not interfere with antigen binding ability

(Moks et al., 1986). Protein Awas fused to the Clostridium cellulovorans CBD and was used

for antibody purification on a cellulose column. It was further demonstrated that this

bifunctional protein could be employed in combination with cellulosic microtiter plates as

an attractive diagnostic matrix for antigen immobilization. Prot A–CBD complex could also

be used as a signal-amplification reagent based on the ability of Prot A–CBD to link

prestained cellulose particles to primary antibodies in a Western blot technique (Shoseyov et

al., 1999; Shpigel et al., 2000).

In recent years, the demand for rapid microbial testing has steadily increased. Various

detection methods have been developed that are based on nucleic acid hybridization and

immunological assays. An important factor that guides the development of these systems, is

the shortening of the time required for pathogen detection. These methods require a high

pathogen to cell concentration and therefore, an enrichment step is needed prior to the

detection stage (Swaminathan and Feng, 1994; Blackburn et al., 1994). PCR-based detection

assays have also been developed that increase detection sensitivity owing to high-specific

pathogen detection competence in the presence of large bacterial background. Even so, when

pathogen levels are less then 103 CFU/g, an additional 6–8 h enrichment step is require

(Swaminathan and Feng, 1994). Recently Shoseyov (1998) and Siegel and Shoseyov (2001)

developed a system based on CBD, which enables rapid detection of pathogenic microbes in

I. Levy, O. Shoseyov / Biotechnology Advances 20 (2002) 191–213200

food samples (illustrated in Fig. 2). In this method, CBD is conjugated to a bacteria-binding

protein such as an epitope specific monoclonal antibody and is loaded on to a cellulosic

matrix (e.g., cotton gauze) that acts as a bacterial cell concentrator (Fig. 2A). The structure of

the cotton gauze enables passage of relatively large volumes of liquids so sufficient bacteria

can be isolated, even from dilute samples. The bacteria can then be further enriched with a

short growing period (Fig. 2B) or eluted from the loaded matrix, all the while maintaining a

very low bacterial background (Fig. 2C) (Shoseyov, 1998; Siegel and Shoseyov, 2001). The

eluted bacteria can be utilized for enumeration and/or classification. The advantage of CBDs

in diagnostics can be attributed to the wealth of different cellulosic matrices that possess very

low nonspecific binding to proteins.

7. Fiber modification

Din et al. (1991) reported that CBDCenA from C. fimi endoglucanase A is capable of

nonhydrolytic disruption activity of cellulose fibers that results in small particle release. In

addition, it was shown that CBDCenA could prevent the flocculation of microcrystalline

Fig. 2. CBD-based pathogen detection system. The method involves conjugation of CBD to a bacteria-binding

protein that is subsequently loaded onto a cellulosic matrix column (e.g., cotton gauze). This column acts as a

bacterial cell concentrator (A). If the anticipated concentration is insufficient, brief growth period prior to elution

from the loaded matrix can be applied to increase bacterial count (B). The resultant isolated bacterial flora contains

a very low undesirable background (C). The eluted bacteria can be further analyzed quantitatively or classified

into types by means of ELISA, lateral flow detection, or plating onto selective or differential media (D).

I. Levy, O. Shoseyov / Biotechnology Advances 20 (2002) 191–213 201

bacterial cellulose (Gilkes et al., 1993). Similar phenomena were observed for other CBDs

(Krull et al., 1988; Boraston et al., 1998; Banka et al., 1998; Gao et al., 2001; Levy et al.,

2002a).

Lee et al. (2000) provided physical evidence for the involvement of CBD in fiber surface

alteration following cellulase treatment. In their study, two cellulases from T. reesei,

exoglucanase CBH I and EGase EG II, were applied separately and in combination onto

cotton fibers. Treatment with CBH I resulted in the appearance of distinct tracks along the

longitudinal axis of the fiber, when visualized by atomic force microscopy, whereas EG II

treatment appeared to cause peeling and smoothing of the fiber surface. When cellulase from

Thermotoga maritime, which lacked a CBD, was used, no effect on the surface of the cotton

fiber was detected (Lee et al., 2000). Additional information to support this observation came

from the study carried out by Suurnakki et al. (2000). In this application, the actions of

endoglucanases, cellobiohydrolases, and the catalytic domains from T. reesei on bleached

chemical pulp were compared. The presence of CBD in the endoglucanases enhanced

enzymatic hydrolysis of cellulose (primarily crystalline cellulose). According to this research,

the presence of CBD in the intact enzyme had a beneficial effect on pulp properties such as

viscosity and strength after PFI refining (Suurnakki et al., 2000).

The tensile strength of paper is determined by its intrinsic fiber strength as well as by the

amount and strength of the fiber-to-fiber bonds (Roberts, 1996). Today, it is known that part

of the strength is owing to the strength of the fibers themselves, but most of the dry strength is

a product of the bonds that exists between the fibers (Spence, 1987; Xu and Yang, 1999).

Intrafiber bonding improves stress transfer between the fibers and is considered to be one of

the most important factors affecting overall stress development in the fiber web when under

tensile deformation (Askling et al., 1998; Gassan and Bledzki, 1998). Earlier studies have

shown that the low strength of dry-formed structures can be improved by adding binder

materials or bicomponent fibers (Villalobos, 1981). Small cross-linking agents can easily

penetrate into the pore structures of cellulose and form intrafiber cross-links. However, such

molecules have only a small effect on the dry tensile strength of the paper. Conversely, large

cross-linking molecules reinforce the fiber-to-fiber bonds, resulting in a marked increase in

dry strength (Xu and Yang, 1999). Recently, we demonstrated that CBDs could modify paper

properties. Two CBDs belonging to Family III (from C. cellulovorans) that had been fused

together to form a cellulose cross-linking protein (CCP) were applied onto filter paper.

Treatment of the filter paper with CBD or CCP significantly improved its tensile strength

(Levy et al., 2001, 2002b). We propose that the increase in stress to failure caused by CBD

and CCP is related to the nature of the CBD’s binding site, which is a large hydrophobic

planar surface with several attachment sites (Gilkes et al., 1992; Din et al., 1994; White et al.,

1994; Xu et al., 1995; Tormo et al., 1996). Current opinion states that paper dry strength is

improved by any factor that facilitates the formation of hydrogen bonds between fibers

(Spence, 1987). CCP is an efficient cross-linker due to its larger size and to the number of

attachment sites it contains that enable it to cross-link cellulosic materials. Applying a single

CBD molecule to the paper also improved its mechanical properties, but to a lesser extent

when compared to CCP. In addition, papers treated with the CCP became more hydrophobic

and demonstrated water-repellent properties (Fig. 3). At optimum CCP concentration, all of

I. Levy, O. Shoseyov / Biotechnology Advances 20 (2002) 191–213202

the binding sites in CCP are attached to the cellulosic surface and this results in improved

mechanical properties (Fig. 4A). At high CCP concentrations, most of the binding sites on the

cellulose are occupied by single CBD moieties; consequently, the second CBD moiety (the

nonbound moiety) of CCP exposes a hydrophobic surface and in this manner increases

surface hydrophobicity (Fig. 4B). Applying CBD to cellulose fibers has a potential for use in

paper recycling. It has been demonstrated that the application of CBD on secondary fibers,

such as old paperboard containers, results in increased tensile and burst indexes as well as

improvement in pulp drainage (Pala et al., 2001).

Another study demonstrated that polysaccharide structure modification could be achieved

using isolated CBDs. In this study, the surface area of a polysaccharide (ramie cotton fibers)

was roughened after treatment with CBD (CBDCenA from C. fimi). It was proposed that these

treatments could be used in order to alter dyeing characteristics of cellulose fibers (Gilkes et

al., 1998). Cavaco-Paulo et al. (1999) demonstrated the effect of CBD on the dye affinity to

cotton fibers. The treated fibers demonstrated increased levels of dye affinity following

treatments with Family II CBD from C. fimi. This was especially notable with acid dyes.

Bjorkquist et al. (2001) employed a different approach. They demonstrated that an amino acid

sequence of less then 30 amino acids can mimic the high affinity of CBD for cellulose

(‘‘mimic CBD’’). They proposed that a hybrid protein, composed of a ‘‘mimic CBD’’ and

‘‘benefit agents’’ could be used in fiber care. The benefit agents could be enzymes, perfumes,

Fig. 3. Interfacial contact angle between CCP-treated filter paper and water. Water droplets (20 ml) were onto CCP-treated paper or on nontreated filter papers and pictures were taken in time laps of 25 ms. The nontreated paper

frame was taken 25 ms after the water came in contact with the paper, whereas the CCP-treated frame was taken 2

min after the water came in contact with the paper. In order to prevent paper wetting, the paper surface must

present low energy on which the initial contact angle of a drop of water is higher than 90�. When the contact angle

is less then 90�, wetting, spreading, and penetration occur (Spence, 1987). From the picture, it is clear that the

CCP-treated paper wetting angle is higher then 90�; therefore, this paper possesses a hydrophobic surface (Levy etal., 2002b).

I. Levy, O. Shoseyov / Biotechnology Advances 20 (2002) 191–213 203

I. Levy, O. Shoseyov / Biotechnology Advances 20 (2002) 191–213204

antiseptics, insecticides, bleaching agents, softeners, dye fixatives, soil release agents, and

brightness.

8. In vivo cell wall modification

The gram-negative bacterium Acetobacter xylinum has long been regarded as a model of

cellulose biosynthesis primarily because cellulose microfibril synthesis is set apart from cell

wall formation (Ross et al., 1991). In A. xylinum, cellulose is produced as separate ribbons

composed of microfibrils and interactions with other polysaccharides do not exist as in plant

cell walls. Since polymerization and crystallization is a coupled process in A. xylinum

cellulose biosynthesis, interference with crystallization results in accelerated polymerization

(Benziman et al., 1980). Some cellulose-binding organic substances can also alter cell growth

and cellulose–microfibril assembly in vivo. Carboxymethyl cellulose (CMC) and fluorescent

brightening agents (FBAs, e.g., calcofluor white ST) prevent microfibril crystallization,

thereby enhancing polymerization. These molecules bind to the polysaccharide chains

immediately after their extrusion from the cell surface, thus preventing normal assembly of

microfibrils and cell walls (Haigler, 1991). Shpigel et al. (1998a) demonstrated that, like other

organic cellulose-binding substances, Family III CBD derived from C. cellulovorans could

modulate cellulose biosynthesis. CBD increased the rate of cellulose synthesis activity in A.

xylinum up to fivefold compared to a control. Electron microscopy of cellulose synthesized in

the presence of CBD revealed that the newly formed fibrils are spread out into a splayed

ribbon instead of the uniform, thin, packed ribbon in the control fibers. The mechanism by

which CBD affects cell wall metabolism remains unknown. A physico-mechanical mech-

anism was proposed whereby CBD slides between the cellulose fibers and separates them in a

wedge-like action (Levy et al., 2002a). This hypothesis is supported by in vitro experiments.

Petunia cell suspensions treated with increasing concentrations of CBD displayed abnormal

shedding of cell wall layers, indicating that CBD can cause nonhydrolytic cell wall disruption

in vivo (Levy et al., 2002a).

Several protocols were tested to analyze the effect of CBD on living plant cells. In these

studies, it was found that Family III CBD from C. cellulovorans could modulate cell

elongation. At low concentrations, this CBD enhanced elongation of Prunus persica L. pollen

tubes and A. thaliana root seedlings, whereas at high concentrations, CBD inhibited root

elongation in a dose-dependent manner. It was demonstrated that cellulose–xyloglucan

networks, similar to plant cell walls, could be formed when employing the A. xylinum model

system in a medium containing xyloglucan (Atalla et al., 1993; Hayashi and Ohsumi, 1994;

Hackney et al., 1994; Whitney et al., 1995). NMR analysis indicated that 80–85% of the

Fig. 4. The interaction of CCP with cellulose fibers in filter paper. Two family III CBDs were fused together to

form a CCP and applied to filter paper. The treated papers became hydrophobic and demonstrated water-repellent

properties. It is assumed that at optimum CCP concentration, all of the binding sites in CCP are attached to the

cellulosic surface, resulting in improved mechanical properties (A). At high CCP concentrations, most of the

binding sites on the cellulose are occupied by single CBD moieties. Consequently, the second CBD moiety (the

nonbound moiety) of CCP exposes a hydrophobic surface thus effecting increased surface hydrophobicity (B).

I. Levy, O. Shoseyov / Biotechnology Advances 20 (2002) 191–213 205

xyloglucan adopts a rigid conformation in all probability aligned with the cellulose chain,

whereas, the remainder is more mobile. The xyloglucan, when present during cellulose

synthesis in the A. xylinum model system, causes the cellulose to become more amorphous

and increases its tensile strength (Brett, 2000). When CBD was present, it was shown that

CBD could compete with xyloglucan for binding to cellulose (Shpigel et al., 1998a). These

findings support the hypothesis that, at least part of the effect of CBD on the plant cell wall is

via cellulose–xyloglucan interactions.

Shoseyov et al. (2001) have shown that CBD can modulate plant growth of transgenic

plants. Introduction of the Family III CBD gene from C. cellulovorans under the control of

the elongation-specific cel1 promoter into transgenic poplar plants led to a significant

increase in biomass production in selected clones when compared with wild-type control

plants (Fig. 5). Analysis of wood characteristics from transgenic poplar trees showed a

significant increase in fiber cell length as well as an increase in the average degree of

Fig. 5. Transgenic poplar (Populus tremula) plants expressing CBD. Poplar trees were transformed with cbdClosgene, fused to cel1 signal peptide under the control of Arabidopsis thaliana elongation specific promotor (cel1

promoter, Shani, 2000; Shani et al., 1997, 2000). Transgenic plants displayed faster growth rates, thicker stems,

and significant increase in wood volume (Shani, 2000).

I. Levy, O. Shoseyov / Biotechnology Advances 20 (2002) 191–213206

polymerization of cellulose. In addition, a significant decrease in microfibril angle (MFA)

was observed. All these new properties resulted in increased burst, tear, and tensile indices of

paper prepared from these fibers (Shoseyov et al., 2001; Levy et al., 2001, 2002b).

9. Concluding remarks

Cellulose is by far the most abundant biopolymer on earth. Its excellent chemical and

physical properties made it a practical product for infinite applications since the origin of

civilization. Today, we are surrounded by numerous products that are composed of cellulose.

The capability to govern the binding of biomolecules and cellulose-containing biopolymers to

cellulose during the biosynthesis products is an actuality owing to the abundance of CBDs. It

is only our imagination that limits their applications.

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