**18. Plant cell remodelling proteins**

In addition to lignocellulose-degrading enzymes, there are also enzymes involved in remod‐ elling the cell wall, which could facilitate its later degradation.

#### **18.1. Expansins**

Expansins are pH-dependent wall-loosening proteins required for cell enlargement and ex‐ pansion in many developmental processes. Although to date their precise mechanism of ac‐ tion remains unclear, evidence point toward a role in dissociating the cell wall polysaccharide complex that links together wall components, thus promoting slippage be‐ tween wall polymers and, eventually, expansion in cell wall [148-149].

These proteins are coded by large multigene families present from bryophytes to angio‐ sperms and also present in monocotyledonous plants (rice, maize), dicotyledonous plants (*Arabidopsis*), ferns and mosses.

Expansins have no hydrolytic activity (glucosidase) and therefore, it has been suggested to work by breaking hydrogen bonds between cellulose fibres or between cellulose and other polysaccharides (xyloglucans), using a non-enzymatic mechanism (Figure 8) [150-153].

Expansins have molecular weights ranging from 25 to 28 kDa and, like cellulases, have a two-domain modular structure and an approximately 20 amino acids-long amino-terminal signal peptide [149].

*Domain I* occupies the N-terminal part of the protein, and it has a DPBB (Double Psi Beta Barrel) structure. It is homologous to the catalytic domain of members of glycoside hydro‐ lase family 45 (GH45), which includes mainly β-1,4-endoglucanases of fungal origin. The DPBB domain of members of this family adopts a six-stranded beta barrel structure forming a substrate-binding groove. Despite the presence of the GH45 catalytic domain in expansins, no hydrolytic activity has been detected for the latter [5].

*Domain II*, at the C-terminal end, is homologous to group II pollen allergens from grasses. Some authors have speculated that this might be a polysaccharide-binding domain, due to the presence of aromatic and polar amino acids on the protein surface, where two trypto‐ phan and one tyrosine would form a planar platform of aromatic residues favouring this binding (149, 154). Domain II folds as a β-sandwich formed by two sheets of four antiparal‐ lel β strands each (Figure 8). In fact, a β-sandwich formed by 3 to 6 β strands per sheet is the most common fold in carbohydrate-binding modules of proteins binding substrates such as crystalline cellulose or chitin [155].

lose is still a slow process because the substrate is insoluble and poorly accessible to

138 Sustainable Degradation of Lignocellulosic Biomass - Techniques, Applications and Commercialization

To overcome this situation scientists have optimized ratio of cellulolytic enzymes, and it was found that the best saccharification of crystalline cellulose is achieved with the enzyme blend: 60:20:20 (CBHI:CBHII:EGI) wherein a saturated level of BG was included to eliminate cellobiose inhibition [146]. In a different report, the impact of the cellulase mixture composi‐ tion on cellulose conversion was modelled, and the findings suggested different optimum ratios for substrates with different characteristics, specifically degrees of polymerization and

Also, researchers have pointed out the use of proteins that relax plant cell wall structure as a complementary activity before action of cellulases in order to improve saccharification.

In addition to lignocellulose-degrading enzymes, there are also enzymes involved in remod‐

Expansins are pH-dependent wall-loosening proteins required for cell enlargement and ex‐ pansion in many developmental processes. Although to date their precise mechanism of ac‐ tion remains unclear, evidence point toward a role in dissociating the cell wall polysaccharide complex that links together wall components, thus promoting slippage be‐

These proteins are coded by large multigene families present from bryophytes to angio‐ sperms and also present in monocotyledonous plants (rice, maize), dicotyledonous plants

Expansins have no hydrolytic activity (glucosidase) and therefore, it has been suggested to work by breaking hydrogen bonds between cellulose fibres or between cellulose and other polysaccharides (xyloglucans), using a non-enzymatic mechanism (Figure 8) [150-153].

Expansins have molecular weights ranging from 25 to 28 kDa and, like cellulases, have a two-domain modular structure and an approximately 20 amino acids-long amino-terminal

*Domain I* occupies the N-terminal part of the protein, and it has a DPBB (Double Psi Beta Barrel) structure. It is homologous to the catalytic domain of members of glycoside hydro‐ lase family 45 (GH45), which includes mainly β-1,4-endoglucanases of fungal origin. The DPBB domain of members of this family adopts a six-stranded beta barrel structure forming a substrate-binding groove. Despite the presence of the GH45 catalytic domain in expansins,

enzymes.

surface area [147].

**18.1. Expansins**

(*Arabidopsis*), ferns and mosses.

signal peptide [149].

**18. Plant cell remodelling proteins**

elling the cell wall, which could facilitate its later degradation.

tween wall polymers and, eventually, expansion in cell wall [148-149].

no hydrolytic activity has been detected for the latter [5].

(a G2A protein from *Phleum pratense*; PDB 1WHO). In (a), the domain I forms a barrel; amino-acid residues that are conserved in expansins are indicated in the single-letter amino-acid code. Images reproduced with publisher BioMed permission [5].

**Figure 8.** a) Expansin proposed activity; b) Expansin domain I (the catalytic domain of a GH45 endoglucanase from *Humicolainsolens*; PBD, 2ENG); c) Expansin domain II

Expansins are classified in four families: α-expansins (EXPA), β-expansins (EXPB), α-expan‐ sin like-proteins (EXLA) and β-expansin like-proteins (EXLB) [5].

The *EXPA family* includes proteins participating in the relaxation and extension of plant cell walls through a pH-dependent mechanism; these proteins would participate in develop‐ mental processes such as organogenesis, the degradation of cell walls during the ripening of fruits and other processes where relaxation of the cell wall is crucial [156-159].

The *EXPB family* includes group I pollen allergens from grasses. These proteins are secreted by pollen and have been suggested to soften the tissues of the stigma and style to facilitate the penetration of the pollen tube [154].

EXPB proteins, unlike EXPA members, relax specifically the cell walls of grass cells, proba‐ bly reflecting differences regarding the organization of cell walls between grasses and dicot‐ yledonous plants. Although an HFD motif, that is known to form part of the active site of endoglucanases, has been found in domain I of EXPA and EXPB family members, they do not have hydrolytic activity [5, 160].

The *EXLA and EXLB families* do not have this sequence motif, which suggests that their mode of action differs to that of the other expansins. The EXLA and EXLB families are com‐ prised of proteins identified by sequence analysis which, despite possessing the two- organi‐ zation typical of expansins, have a number of divergent sequence features that separate them from the EXPA and EXPB families [161].

rate of release of reducing sugars from agave fibre. Something similar was observed when a cucumber expansin was incubated with a compound of cellulose and xyloglucans of bacteri‐ al origin and occurred a rapid relaxation of the structure of this compound, suggesting that expansins modulate the binding between cellulose fibres and xyloglucans, relaxing or break‐

Hydrolysis of Biomass Mediated by Cellulases for the Production of Sugars

http://dx.doi.org/10.5772/53719

141

Given the optimum pH of LOOS1 (pH 5) and other expansin like proteins, they could be ap‐ plied to processes of saccharification of natural substrates, facilitating the release of reducing sugars together with cellulases. For example, it might be used as an additive to obtain fer‐

In [173], used swollenin as a pretreatment of cellulosic substrates and observed that even in non-saturating concentrations, a significant accelerated hydrolysis occurred. They also cor‐ related particle size and crystallinity of the cellulosic substrates with initial hydrolysis rates, and it could be shown that the swollenin induced-reduction in particle size and crystallinity

It is not surprising that the idea of using plant expansins in saccharification processes has

The efficient enzymatic saccharification of cellulose has been a challenge over the past 50 years, mainly due to its crystallinity, which make it a recalcitrance substrate with a high po‐

The bioconversion of cellulose to ethanol is the process where most interest has been fo‐ cused. Fortunately, increasing of the loosened cellulose surface area by the use of non-hy‐ drolytic proteins, a process known amorphogenesis, would allow access to hydrolytic

Cellulose biodegradation represents the major carbon flow from fixed carbon sinks to at‐ mospheric CO2, this process is very important in several agricultural and waste treatment processes. Also, cellulose contained in plant wastes could be used as a raw material to pro‐ duce sustainable products and bioenergy to replace depleting fossil fuels. However, one of the most important and difficult technological challenges is to overcome the recalcitrance of natural cellulosic materials, which must be enzymatically hydrolysed to produce fermenta‐ ble sugars. In order to achieve this goal, new enzymes with cellulolytic activities are being improved and organisms with novel properties have been found. Although the efforts are being directed to improve cellulolytic activity, proteins capable to relax plant cell structure (expansins, swollenins and loosenin) could be used as a biological pretreatment since they would be disrupting crystalline structure of cellulose making it more accessible to the en‐

mentable sugars from pretreated yellow poplar as reported in [172].

enzymes making the saccharification process more efficient [177].

ing the bonds keeping them together [171].

resulted in high cellulose hydrolysis rates.

tential to be used as a carbon source.

zymes and enhancing sugar releasing.

been patented [174-176].

**19. Conclusions**

Another group included in the expansin superfamily is the *expansin-like X family* (EXLX), comprising proteins that exhibit weak sequence homology with the domains of EXPA and EXPB members, and identified in organisms other than plants, such as the mucilaginous fungus *Dictyostelium* and the bacteria *Bacillus subtilis,* and *Hahella chejuensis* [161-164].

The denomination of expansin or expansin-like is reserved for proteins exhibiting both do‐ main I and domain II. Proteins with only one of these domains are not classified as expan‐ sins [161]. However other proteins with similar disrupting activity of the cell wall have been reported.

Expansins and expansin-like proteins have been detected in angiosperms such as *Arabidopsis thaliana, Oryza sativa, Zea mays* and *Triticum aestivum,* gymnosperms such as pine and pop‐ lar, ferns such as *Regnellidium diphyllum* and *Marsilea quadrifolia* and the moss *Physcomitrella patens.* Some members of the expansin superfamily have been found even in a potato-infect‐ ing nematode, *Globodera rostochiensis*, where they are hypothesized to promote the infection process [165-169].

#### **18.2. Novel proteins with expansin-like activity**

Proteins with expansin-like activity called swollenins and loosenins have been identified in ascomycete and basidiomycete fungi such as *T. reesei, A. fumigatus* and *B. adusta* [6-8, 170].

In [7], a swollenin gene from *T. reesei* denominated *swo1,* was cloned and expressed in *Sac‐ charomyces cerevisiae,* coding for a protein that modifies the structure of cellulose in swollen regions of cotton fibres (hence the name) without releasing reducing sugars. Swo1 is a fun‐ gal expansin-like protein, containing a pollen allergen domain and a cellulose-binding do‐ main.

Proteins with expansin activity could be used to improve the efficiency of cellulose biocon‐ version processes. For example, a swollenin purified from *A. fumigatus* has been used in combination with cellulases to facilitate the saccharification of microcrystalline cellulose (Avicel) [8]. In [163] also is described the synergism of an EXLX from *B. subtilis* in the enzy‐ matic hydrolysis of cellulose and recently, and a new protein with expansin activity from the basidiomycete fungus *B. adusta,* denominated loosenin (LOOS1] was cloned and charac‐ terized [6].

Not only expansins, but also swollenins and loosenin represent good candidate as pretreat‐ ment to enhance sugar production from plant biomass. For example, loosenin activity was efficient to release reducing sugars (after cellulase treatment) from *Agave tequilana,* a crop ex‐ tensively grown in some areas of Mexico, which shredded fibrous waste is usually burnt or left to decompose. Indeed, *A. tequilana* fiber became a susceptible substrate for a cocktail of commercial cellulases and xylanases in the presence of LOOS1. Loosenin shows optimum activity at the same pH as most cellulolytic enzymes, opening the possibility to use them as a mixture. This protein is able to relax the structure of cotton, enhancing up to 7.5-fold the rate of release of reducing sugars from agave fibre. Something similar was observed when a cucumber expansin was incubated with a compound of cellulose and xyloglucans of bacteri‐ al origin and occurred a rapid relaxation of the structure of this compound, suggesting that expansins modulate the binding between cellulose fibres and xyloglucans, relaxing or break‐ ing the bonds keeping them together [171].

Given the optimum pH of LOOS1 (pH 5) and other expansin like proteins, they could be ap‐ plied to processes of saccharification of natural substrates, facilitating the release of reducing sugars together with cellulases. For example, it might be used as an additive to obtain fer‐ mentable sugars from pretreated yellow poplar as reported in [172].

In [173], used swollenin as a pretreatment of cellulosic substrates and observed that even in non-saturating concentrations, a significant accelerated hydrolysis occurred. They also cor‐ related particle size and crystallinity of the cellulosic substrates with initial hydrolysis rates, and it could be shown that the swollenin induced-reduction in particle size and crystallinity resulted in high cellulose hydrolysis rates.

It is not surprising that the idea of using plant expansins in saccharification processes has been patented [174-176].

The efficient enzymatic saccharification of cellulose has been a challenge over the past 50 years, mainly due to its crystallinity, which make it a recalcitrance substrate with a high po‐ tential to be used as a carbon source.

The bioconversion of cellulose to ethanol is the process where most interest has been fo‐ cused. Fortunately, increasing of the loosened cellulose surface area by the use of non-hy‐ drolytic proteins, a process known amorphogenesis, would allow access to hydrolytic enzymes making the saccharification process more efficient [177].
