Cellulose-binding domains

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.

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, C_x 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–C_X 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–C_X 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, Tomme et al., 1995b, Tomme et al., 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 cellulosome. 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, Bayer et al., 1998b. 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 Stalbrand 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 β-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 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, Tomme et al., 1995b, Tomme et al., 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, Tomme et al., 1998, Bayer et al., 1998a, Bayer et al., 1998b. 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. CBD_Cex, from Family IIa, contains a β-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, CBD_N1 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, occasionally, 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 CBD_Cex, 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 CBD_N1 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 CBD_Cex 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).

CBDs, constituting isolated modules, are utilized in many different applications. This article will review the potential applications of CBDs in diverse fields of biotechnology.