**8. Cellulose degradation mediated by non-cellulosomal enzymes**

Aerobic cellulolytic bacteria and fungi use a system for cellulose degradation consisting of sets of soluble cellulases. Cellulases are inducible enzymes by cellulosic substrates, which are syn‐ thesized by a large diversity of microorganisms including both fungi and bacteria during their growth on cellulosic materials.These microorganisms can be aerobic, anaerobic, mesophilic or thermophilic. Among them, the genera of *Clostridium, Cellulomonas, Thermomonospora, Tricho‐ derma,* and *Aspergillus* are the most extensively studied cellulases producers [91].

Extremely thermophilic cellulose-degrading microorganisms are of particular and biotech‐ nological interest owing to the presence of highly thermostable enzymes. A deeper analysis

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The group of thermophilic cellulolytic prokaryotes includes two aerobic species, *Rhodother‐ mus marinus* and *Acidothermus cellulolyticus*, and numerous anaerobes of the genera *Caldicel‐ lulosiruptor*, *Clostridium*, *Spirochaeta*, *Fervidobacterium* and *Thermotoga* (reviewed by (100)).

All members of the genus *Caldicellulosiruptor* are extremely thermophilic, cellulolytic, and non-spore-forming anaerobes with Gram-positive type cell wall, capable of fermenting dif‐ ferent types of carbohydrates and have been isolated mostly from neutral or slightly alkaline

Recently, thermostable cellulases have also been reported in the thermophilic *Geobacillus* sp. R7 that produces a cellulase with a high hydrolytic potential when grown on pretreated ag‐ ricultural residues (corn stover and prairie cord grass). In fact, it was demonstrated that *Geo‐ bacillus* sp. R7 can ferment the lignocellulosic substrates to ethanol in a single step, improving bioethanol production with important potential for cost reductions. Cellulases genes were also identified in several *Sulfolobales* strains, however, their physiological func‐

Another thermophlic bacterium *Anaerocellum thermophilum* degrade lignocellulosic biomass

While cellulases are widespread in Fungi and Bacteria, only one archaeal cellulase, an endo‐ glucanase from *Pyrococcus furiosus*, has been reported. This enzyme exhibits a significant hy‐ drolyzing activity toward crystalline cellulose even tough it lacks a CBD, the role of this intracellular enzyme in Archaea is unclear, given that Archaea are apparently unable to

In the alkali tolerant fungus *Penicillium citrinum* an alkali tolerant and thermostable cellulases were found which may have potential effectiveness as additives to laundry detergents [105].

In this search to improve cellulases activity, hybrids of hyperthermostable glycoside hydro‐ lases have been constructed as reported by [106], for example, using the structural compati‐ bility of two hyperthermostable family 1 glycoside hydrolases, *P. furiosus* CelB and *Sulfolobus solfataricus* LacS a library of hybrids using DNA family shuffling was created.

This study demonstrates that extremely thermostable enzymes with limited homology and different mechanisms of stabilization can be efficiently shuffled to form stable hybrids with

Alkaliphilic, thermophylic and halophilic microbial species have the potential to yield val‐ uable new products for biotechnological industry. Alkaliphilic polymer-degrading enzymes such as proteases, lipases and cellulases are most frequently isolated from *Bacillus* or related species. Cellulases and lipases are important not only as components of washing detergents, but they are also applied in the paper and pulp, pharmaceutical, food, leather, chemical or

geothermal springs in New Zealand, Iceland and California [100].

of these organisms is reported in [89, 98-99].

tion is not well understood [101-102].

grow on cellulose [103-104].

improved catalytic features.

waste treatment industries [107-108].

untreated as well as crystalline cellulose and xylan [86].

The aerobic cellulase mechanism evolved in terrestrial microorganisms that colonise solid substrates and therefore secrete cellulases to enable degradation of the substrate. Because of the recalcitrance of plant cell walls some cellulolytic microorganisms secrete up to 50% of their total protein during growth on biomass or cellulose [53, 90].

Cellulases are composed of independently folding and structurally and functionally discrete units called domains, making cellulases modular enzymes. Structurally fungal cellulases are simpler as compared to bacterial cellulosomes [32, 88, 92].

Fungal cellulases have two independent domains: a catalytic domain (CD) and a cellulosebinding domain (CBD), which is joined by a short poly linker region to the catalytic domain at the N-terminal. The CBD is comprised of approximately 35 amino acids, and the linker region is rich in serine and threonine [93].

It is clear that the role of the CBD is to bind the enzyme to the cellulose so that the CD keep closer to the substrate and it also gives the CD time to move the chain into its active site be‐ fore the enzyme diffuses away from the cellulose. It is still not clear whether the CBD also can modify cellulose or otherwise assist cellulose hydrolysis by the catalytic domain [94].

The mixture of free cellulases act synergistically to degrade crystalline cellulose increasing the specific activity up to fifteen fold higher than that of any individual cellulase [95].
