Cellulolytic enzymes hydrolysis

Cellulose is a heterogeneous substrate that makes modeling cellulolytic enzyme hydrolysis difficult. Cellulose is composed of chains of glucose connected by P 1-4 glycosidic bonds. One chain end is termed the reducing end because the hemiacetal is able to open to expose the reducing aldehyde. The other chain end is called the non-reducing end because the 1 carbon in the hemiacetal is involv-... 

The xylanolytic enzyme system of Trichoderma reesei, a well-known producer of cellulolytic enzymes, is versatile and well suited for the total hydrolysis of different xylans. It consists of two major, specific and several non-specific xylanases, at least one / -xylosidase, a-arabinosidase and a-glucuronidase and at least two acetyl esterases. The hydrolysis of polymeric xylans starts by the action of endoxylanases. The side-groupcleaving enzymes have their highest activities towards soluble, short xylo-oligosaccharides, and make the substituted oligosaccharides again accessible for xylanases and / -xylosidase. 

While Trichoderma reesei is best known as an efficient producer of cellulolytic enzymes, it has also been reported to produce xylanase and / -xylosidase (18-20). Two xylanases and a / -xylosidase have been purified from T. reesei (10), and two xylanases (21,22) and a / -xylosidase (5) from T. viride. We have previously shown that T. reesei produces all the enzymes needed for complete hydrolysis of native substituted xylans (23). One xylanase (24), a / -xylosidase (25), an a-arabinosidase (26), and an acetyl esterase (27) of T. reesei have so far been purified. In this chapter, the mode of action of these enzymes in the hydrolysis of different xylans is discussed. 

The economic potential of enzyme hydrolysis is therefore dependent upon the evolution of (1) cheap, readily available, large biomass sources, (2) cheap and efficient pretreatment methods, and (3) cheap and potent sources of cellulolytic enzymes. 

The production of fuel ethanol from renewable lignocellulosic material ("bioethanol") has the potential to reduce world dependence on petroleum and to decrease net emissions of carbon dioxide. The lignin-hemicellulose network of biomass retards cellulose biodegradationby cellulolytic enzymes. To remove the protecting shield of lignin-hemicellulose and make the cellulose more readily available for enzymatic hydrolysis, biomass must be pretreated (1). 

Index Entries Cellulolytic enzymes hemicellulolytic enzymes enzymatic hydrolysis coinduction. 

The polysaccharides in the raw materials need to be hydrolyzed before the sugar monomers canbe fermented to ethanol. Today, enzymatic hydrolysis is regarded as a method with great potential. One major obstacle to overcome is the high cost of cellulolytic enzymes. In 2001, the United States Department of Energy formed a contract with two commercial producers of cellulolytic enzymes in an attempt to achieve a 10-fold decrease in the cost of the cellulolytic enzymes (www.ott.doe.gov/ biofuels/research partnerships.html). 

The filamentous fungi investigated showed coinduction of cellulolytic and xylanolytic enzymes. During growth on cellulose, products from the hydrolysis of cellulose also induced production of xylanolytic enzymes, and during growth on xylan, products from the hydrolysis of xylan also induced the production of cellulolytic enzymes. 

Enzymatic action can be defined on three levels operational kinetics, molecular architecture, and chemical mechanism. Operational kinetic data have given indirect information about cellulolytic enzyme mode of action along with important information useful for modeling cellulose hydrolysis by specific cellulolytic enzyme systems. These data are based on measurement of initial rates of enzyme hydrolysis with respect to purified celluloses and their water soluble derivatives over a range of concentrations of both substrate and products. The resulting kinetic patterns facilitate definition of the enzyme s mode of action, kinetic equations, and concentration based binding constants. Since these enable the enzymes action to be defined with little direct knowledge of its mechanistic basis, the rate equations obtained are referred to as operational kinetics. The rate patterns have enabled mechanisms to be inferred, and these have often coincided with more direct observations of the enzyme s action on a molecular level [2-4]. 

Improved protein separation techniques utilizing hquid chromatography and electrophoresis coupled with X-ray diffraction and NMR studies have given insights into the three-dimensional structures of cellulolytic enzymes. This molecular architecture data coupled with DNA sequence information has given clues to the chemical mechanisms of enzymatic hydrolysis and molecular interaction between cellulose and the enzymes. 

Fundamental studies of the molecular basis of cellulolytic enzymes show surface interactions are a key determinant of enzyme efficiency. Retaining and inverting mechanisms have recently been shown to have similar operations in cellulose hydrolysis as in the general-acid catalytic mechanisms of other glycolsyl hydrolases. Specific physical and chemical characteristics of the solid substrate have also been shown to influence greatly the hydrolysis rate of cellulolytic enzymes. 

Cooperative action, often designated synergy, of the three cellulolytic enzyme classes is essential for an efficient enzymatic hydrolysis process [181]. 

Size and Diffusibility of Cellulolytic Enzymes in Relation to the Capillary Structure of Cellulose. As discussed earlier, enzymatic degradation of cellulose requires that the cellulolytic and other extracellular enzymes of the organisms diffuse from the organism producing them to accessible surfaces on or in the walls of the fiber. This accessible surface is defined by the size, shape, and surface properties of the microscopic and submicroscopic capillaries within the fiber in relation to the size, shape, and diffusibility of the enzyme molecules themselves. The influence of these relationships on the susceptibility and resistance of cellulose to enzymatic hydrolysis has not been verified experimentally in natural fibers but the validity of the concepts that follow is demonstrated by the work of Stone, Scallan, Donefer, and Ahlgren (69). 

In addition to considerations of size and shape, the surface properties of the fiber capillaries and the diffusibility of the cellulolytic enzyme molecules within them can profoundly influence the susceptibility of cellulose to enzymatic hydrolysis. In contrast to inorganic catalysts, enzymes have a very strong and specific affinity for their specific substrate molecules. This affinity accounts for their susceptibility to competitive inhibitors. When the substrate exists as an insoluble polymer in a complex structural matrix, this specific affinity drastically reduces the rate of diffusion of the enzyme in the presence of the substrate. This retarded... 

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