Cellulolytic action

As can be seen, the cellulolytic action of the fungus has indeed brought about a two-fold increase in the yield of extractable lignin. The native and enzymatically liberated lignins were then chemically compared and found to be identical in all respects examined (Table 4). 

Thiooligosaccharides can be inhibitors of cellulases and are useful for elucidation of the molecular mechanism of the cellulolytic actions. Hemithiocello-oligosaccharides from tetraose to tetradecaose were synthesized by a cellulase mixture using 4-thio-/ -cellobiosyl fluoride as an activated donor in a buffer/ acetonitrile solvent system.139... 

Thus a biospecific adsorbant was obtained selective desorption should therefore permit successive elution of cellulolytic enzymes. It was thus found that the method was very useful in the purification of the cellobio-hydrolases from Tr. r., starting from very crude culture filtrates. Addition of glucose (0.1M) or gluconolactone suppressed the action of glucosidases present, preventing deterioration of the columns. The capacities exceeded 10 mg CBH I per ml gel (prepared with CNBr activated Sepharose). 

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. 

Tt is a widely recognized fact that true cellulolytic microorganisms A produce three basic cellulase components IS), and that these enzyme components act in concert to hydrolyze crystalline cellulose to glucose (6). Many research laboratories have undertaken the task to purify cellulose components from various cellulolytic microorganisms and to study the mechanisms of cellulose hydrolysis. Much information has accumulated concerning the mode of action of cellulose hydrolysis since Reese et al. first proposed the Ci-C concept (7). In spite of this, however, conflicting reports still flourish concerning the composition of the "cellulase complex, the multiplicity of cellulase components, the biosynthesis of cellulose, and the mechanisms of cellulose hydrolysis. 

To hydrolyze crystalline cellulose efficiently by enzymatic means, the inaccessibility of crystalline structures must be overcome. T. reesei and some other true cellulolytic microorganisms produce a cellulase complex that is capable of efficiently hydrolyzing crystalline cellulose. One explanation of this capability was first proposed by Mandels and Reese (7). In this model, two factors, Ci and C worked together to disrupt and hydrolyze cellulose. Ci first disrupted the crystalline structure of the cellulose while Cx attacked the available sites formed by Ci. In other words, Ci and C exhibit synergism in hydrolyzing cellulose. Since then, the combined action of cellobiohydrolase ( Ci ) and endoglucanase ( C ) has been identified as the source of the apparent synergism (6,26,55). 

Work in several laboratories (22, 27, 55, 56) has shown a pattern of cellulase action in cellulolytic organisms which requires at least one of a set of three closely related enzymes in order to hydrolyze crystalline cellulose effectively. These enzymes often possess little ability to degrade either CM-cellulose (as measured viscosimetrically) or crystalline cellulose. Nevertheless they are characterized by the capacity to cleave swollen cellulose or cellooligosaccharides almost entirely to cellobiose by virtue of their / -( - 4)glucan cellobiohydrolase activity. Recognition of this pattern has been difficult because prior to this report the three enzymes had not been purified and characterized apart from contaminating enzyme activity. 

Vegetable matters of wool are normally removed by a process known as carbonising. Carbonisation of wool with inorganic acid may cause some degradation of the fibre. The replacement of carbonisation by the use of enzymes, such as cellulases, ligninases, hydrolases, lyases and oxidoreductases are reported [93]. A biochemical alternative using complex combination of enzymes to the chemical process of carbonising with sulphuric acid is also reported [94]. The amount ol sulphuric acid required for carbonisation can be reduced by the action of cellulolytic and pectinolytic enzymes [95]. 

Reaction Kinetics, Molecular Action, and Mechanisms of Cellulolytic Proteins... 

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]. 

Work needs to be done to find the specific activity of purified enzymes on the cellulose structure, especially the synergism between different cellulolytic enzymes. With this understood, one can then undertake the task of studying the endoglucanase-cellobiohydrolase system. This requires the identification of binding sites, reaction sites, and changes in cellulose structure as a result of the enzyme action. 

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

No attempt was made at this symposium to include all aspects of cellulose decomposition. Surveys of active organisms and interrelationships of organisms growing on complex substrates are certainly important problems. So are the properties of enzymes and the means of enhancing and preventing their action. All impinge on the aim of this symposium which is to stress the practical application of cellulolytic systems to worldwide problems. 

The terms cellulolytic enzymes and cellulases now enjoy a workable definition. This has been made possible through the separation of the components of the cellulolytic complex, as well as some clarification of their mechanism of action. New viscometric, isoelectric focusing, gel filtration, and other techniques for the determination of the enzymic components have been devised. Detailed observations on the molecular sites of hydrolytic reactions, the susceptibility of the enzymic reactions to metallic ions, proteolytic enzymes, and other chemicals also provided considerable insight into the nature of cellulase. 

T he first part of this paper will be concerned with an experimental approach to the cellulase complex which involves (1) the effect of supramolecular structure on activity of cellulolytic systems, (2) the effect of chemical modification of cellulose on the activity of cellulase, and (3) the use of oligosaccharides in determination of the mode of action of cellulolytic components. 

Use of Oligosaccharides in Determination of the Mode of Action of Cellulolytic Components. Broadly the oligosaccharides invite three approaches to determination of the site of action isotopic labelling, chemical labelling, and use of unlabelled substrates. 

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