Cellulose-binding domains

Cellulose-binding domains

(Parte 2 de 6)

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).

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 introduction 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- 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 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 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 I 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 m. 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 1 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.

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).

(Parte 2 de 6)

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