Cellulolytic Microorganisms

Selective Production of Xylanases by Cellulolytic Microorganisms. Until recently there was little information on common or separate genetic control of cellulase and xylanase synthesis in microorganisms (60). Studies on this subject were complicated by the fact that numerous microbial ceUulases and xylanases are non-specific with respect to cellulose and xylan as substrates. As could be expected from a comparison of both polysaccharide structures, non-specificity is more frequently observed with cel-lulases, because their substrate binding sites can easily accommodate substrate using an unsubstituted p-(l 4)-linked chain of D-xylopyranosyl units. 

Xylan fragments induced xylanase also in non-cellulolytic microorganisms like Streptomyces sp. (71) and yeasts of the genus Cryptococcus and Trichosporon (72-74). In these strains xylanase could be efficiently induced also by methyl P-D-xylopyranoside, which is extremely slowly metabolized in the cells (72-74). TTie glycoside accelerated xylanase production in hyperproducing color variants of A. pullu-lans (75) and in several strains of Aspergillus (70). Low cost and easy preparation of methyl p-D-xylopyranoside favors its use for large-scale xylanase production. 

No doubt the cost of xylanolytic enzymes will be one of the factors determining their application in the pulp and paper industry as well as in other areas. Economically feasible xylanase production can be achieved in paper mills employing xylanase-positive transformants of common industrially used microorganisms that are capable of utilizing inexpensive carbon sources originating there. A substantial improvement in the production of xylanolytic systems can be expeaed from mutants of non-cellulolytic microorganisms that are resistant to catabolic repression. Such mutants usually exhibit hyperproduction of extracellular enzymes. 

A given microorganism may produce one or more enzymes of each type. An understanding of the role of each enzyme in cellulose biodegradation requires their purification and characterization, and an analysis of the ways in which they interact with the substrate and with each other. However, it is often quite difficult to determine the number and type of truly different enzymes produced by an organism. Many cellulolytic microorganisms secrete proteases, which may degrade some or all of the cellulases to smaller,... 

High levels of Ci are found in culture filtrates of only a few cellulolytic microorganisms high levels of Cx are found in many. Many culture filtrates rich in Cx contain no Ci, but the converse is not true. Good examples of culture filtrates rich in C i are those from the fungi Fusarium solani (12, 20, 21), Penicillium funiculosum (13, 21), Sporotrichum pul-verulentum (23,24), and Trichoderma koningii (25,26,27), but culture filtrates of T. viride (28-32), particularly T. xeesei (33) (formerly T. viride QM 6a) and derived mutants (34), appear to be the best. 

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). 

An indirect use of cellulase enzymes is the growth of cellulolytic microorganisms on waste cellulose to obtain single cell protein (64). The use of a combined culture of the bacteria Cellulomonas, which breaks down cellulose to cellobiose, and Alcaligenes, which can utilize this inhibitory product, resulted in rapid growth. The increasing need for protein and the availability of waste cellulose have focused much attention on this area. 

Oince cellulose is insoluble high polymers under physiological condi- tions, cellulase which is destined to attack it has been expected to be an extracellular enzyme. In fact, most of cellulolytic microorganisms secrete some cellulase components into the culture medium, and almost all work on the cellulase have been performed using these extracellular components. In the cultures of cellulolytic bacteria, cellulases are not only found in their culture filtrates, but also are generally obtainable from the cells by treatment with autolytic agents—e.g., toluene (9, 19, 30). These facts indicate that at least certain components are existent within the bacterial cells and that their physiological function may be distinct from that of the extracellular components. 

It has been frequently reported that some cellulolytic microorganisms can degrade cellulosic materials only after some kind of modification of the cellulose has occurred. Other organisms degrade cellulose in the native form, such as cotton fibers. Only the latter organisms have been considered as truly cellulolytic. 

In 1950 Reese and his co-workers (43) introduced their Ci, Cx hypothesis. It was postulated that truly cellulolytic microorganisms are equipped with both Ci and Cx enzymes, while microorganisms able to hydrolyze only modified cellulose lack the Ci enzyme. 

However, cell-free enzyme preparations from many, according to the above definition, truly cellulolytic microorganisms fail to solubilize native cotton completely unless this is first converted into a reactive form by swelling in acidic or basic milieu (14,41). Thus, these organisms do not seem to produce a Ci enzyme. The most well-known example is culture filtrate from Myrothecium verrucaria (26, 55). 

The susceptibility of cellulose to enzymatic hydrolysis is determined largely by its accessibility to extracellular enzymes secreted by or bound on the surface of cellulolytic microorganisms. Direct physical contact between these enzymes and the cellulosic substrate molecules is an essential prerequisite to hydrolysis. Since cellulose is an insoluble and structurally complex substrate, this contact can be achieved only by diffusion of the enzymes from the organism into the complex structural matrix of the cellulose. Any structural feature that limits the accessibility of the cellulose to enzymes by diffusion within the fiber will diminish the susceptibility of the cellulose of that fiber to enzymatic degradation. In this review, the influence of eight such structural features have been discussed in detail. 

The accessibility of cellulose to the extracellular enzymes of cellulolytic microorganisms is determined in part by its distribution within the cell wall and the nature of the structural relationships among the various cell-wall constituents. These relationships are reviewed briefly. 

To appreciate fully the influence of the structural features of natural fibers on their susceptibility and resistance to enzymatic degradation, it is necessary to understand the relationship between cellulolytic microorganisms, their extracellular enzymes, and the fiber substrate itself. 

Mineral Constituents. Cellulose fibers usually contain about 1% ash. The mineral elements contained in the ash include all those essential for the growth and development of cellulolytic microorganisms. This fact has led to speculations and certain experimental treatments by Baechler (4) and others showing that chelating agents applied to cotton or wood can so effectively bind these essential mineral elements that the growth of cellulolytic microorganisms is prevented. 

A recent review by Scheffer and Cowling (60) summarizes extensive evidence that the natural durability of many wood species is because of the toxicity of certain phenolic substances that are deposited in the process of heartwood formation. These substances act as poisons to the cellulolytic microorganism rather than by direct action on the enzymatic process of deterioration. 

Ruminant nutritionists are primarily concerned with the microbial degradation and utilization of cellulose and other carbohydrates and the factors which will stimulate the same. This is in contrast to the interests of research workers in textile and wood industries whose main object is to prevent the decomposition of valuable products by cellulolytic microorganisms. Since a proper understanding of the mechanisms of breakdown of cellulose is essential to develop methods to prevent or stimulate the same, there is a convergence of interests at the enzymic level. 

OOD CELLULOSE AND HEMICELLULOSES represent a rich nutrient source that can easily be exploited by numerous insects and wood-degrading fungi and bacteria. The third main wood component, lignin, affords some protection to carbohydrates, particularly against purely cellulolytic microorganisms. 

L. R. (2013) Development and evaluation of methods to infer biosynthesis and substrate consumption in cultures of cellulolytic microorganisms. Biotechnol. Bioeng., 110 (9), 2380-2388. 

There has been widespread use of antibiotics, specifically the ionophore monensin (brand name Rumensin), to increase feed efficiency in beef and dairy production (147,148) in both the U.S. and Canada, but not in Europe. The use of monensin has recently been extended in the U.S. for lactating and dry cow rations to improve feed efficiency (149) a use that was previously permitted in Canada. Monensin is very sensitive to cellulolytic microorganisms that include Butyrivibrio fihrisolvens (147), and there is concern that this will reduce the microbial population of the... 

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