Accessory protein CBP21 [1]


In 2005, Vaaje-Kolstad et al. showed that CBP21, a non-hydrolytic 19 kDa chitin-binding protein produced by one of the most efficient bacterial chitin-degraders, Serratia marcescens, strongly increased the efficiency of chitinases in degrading certain forms of crystalline chitin (Figure 2). This was the first clear example of an accessory protein produced to improve the efficiency of enzymatic hydrolysis of an insoluble recalcitrant polysaccharide substrate. Interestingly, CBP21-like proteins can be found in many chitinolytic microorganisms, as well as in insect viruses that need to perforate the chitinous heteropolymeric peritrophicmatrix of the insect gut before infection. It could be shown that insect virus infectivity was dramatically increased when insect larvae were fed a CBP21-like protein. The functionality of CBP21 was tested in combination with several individual pure chitinases. The magnitude of the synergistic effect of CBP21 depended on the enzyme used because different enzymes exhibit different intrinsic abilities to degrade the substrate. CBP21 binds only to b-chitin, and hence can only contribute to the degradation of his substrate. There are CBP21-like proteins that are known to bind a-chitin, but their potential effect on chitin degradation has not yet been explored. Clues to the molecular function of CBP21 come from its crystal structure and site-directed mutagenesis studies, which showed that the binding surface of CBP21 contains conserved polar residues that are crucial for its synergistic effect (Figure 2c). 

enz_figura2

Interestingly, single point mutations that abolished the synergistic effect of the protein had only moderate effects on the chitin binding affinity. This demonstrates that the action of CBP21-like proteins is driven by other factors in addition to mere binding activity. An interesting possibility is that the assembly of polar amino acid side chains on the binding surface is needed to form a specific set of multiple hydrogen bonds with the substrate, thus disrupting the hydrogen-binding network between individual polymer chains. This could also explain why CBP-like proteins only bind to certain types of substrates, which have specific packing arrangements and hydrogen-bonding networks. The example of CBP21 clearly shows that in nature accessory proteins have evolved to improve substrate accessibility and to act synergistically with hydrolytic enzymes. So far, a corresponding example is lacking from cellulase research, but there are a few indications that proteins exist that are capable of disrupting cellulose and cellulose-containing heteropolymeric complexes. For example, plants produce proteins, so-called expansins, which contribute to a loosening of the cell wall necessary for plant growth. Expansins have also been detected in nematodes that are able to degrade plant cell walls. It is known that expansins mechanically weaken plant cell walls and pure cellulose paper, and the use of expansions to improve cellulase efficiency has been suggested. However, application of expansins has been hampered by the fact that these proteins are difficult to produce in vitro. Interestingly, two very recent studies showed that expansin-like grass pollen allergens, which are easy to obtain, and a non-characterized protein from corn stover  could be used to increase cellulose efficiency. Another potential CBP21 analogue that could act on cellulose is swollenin from the cellulose-degrading fungus Trichoderma reesei. Swollenin contains both a cellulosebinding domain and an expansin-like domain and, furthermore, its expression is co-regulated with cellulose expression. Swollenin showed clear disruptive effects on cellulose, but its application as an accessory protein to cellulases has so far not been described. Available information for fungal genomes suggests that expansin-like domains occur regularly in fungal proteins that act on plant polysaccharides, suggesting that these domains are indeed used for substrate disruption, either as functional parts of swollenin-like proteins or as accessory domains in cellulases and related enzymes. Based on sequence comparisons, carbohydrate-binding domains and proteins have been classified in _50 different families of carbohydrate-binding modules (CBMs). CBP21 belongs to CBM family 33. Most CBMs do not exist as free proteins but rather as substrate-binding domains in multi-domain hydrolytic enzymes, such as chitinases and cellulases (Figures 1 and 2). The roles of these domains in cellulases and chitinases have received much attention, and it is well established that they contribute to enzyme efficiency by increasing affinity for the substrate. Importantly, it has been shown that some cellulose-binding domains can change the structure of the crystalline substrate and that this ‘destructuring’ effect alone might have a positive effect on cellulase efficiency. Thus, cellulose-binding domains in cellulases might have similar effects on cellulolytic activity as CBP21-like proteins have on chitinolytic activity.

[1] Vincent G.H. Eijsink, Gustav Vaaje-Kolstad, Kjell M. Varum and Svein J. Horn Towards new enzymes for biofuels: lessons from chitinase research, Trends in Biotechnology (2008) 26 (5) 228-235.