Cellulose-binding domains

Cellulose-binding domains

(Parte 3 de 6)

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

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).

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 I, 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 I 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 I (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

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 I 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 et al., 2002b).

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).I n 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 I 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 mechanism 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 I 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 I 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).

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 I 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 cbdClos gene, 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).

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).

(Parte 3 de 6)