Substrate specificity of cellulase

In the presence of high concentrations of auxin, etiolated pea Pisum sativum) epicotyls synthesized a buffer-soluble cellulase (pH optimum 5.5) and a salt-soluble cellulase (pH optimum 7.0). The buffer-soluble enzyme gave two bands, and the salt-soluble enzyme several bands, on polyacrylamide gel electrophoresis. It was concluded that ethylene- and auxin-treated pea epicotyls synthesize different forms of cellulase. The kinetics and substrate specificities of cellulases from auxin-treated pea epicotyls have been investigated. ... 

In relation to the substrate specificity of cellulase catalysis, a-cellobiosyl fluoride (of-CF, the anomer of j0-CF) was not reacted under reaction conditions similar to those of -CF polymerization, of-CF being recovered unchanged (Scheme 8). This suggests that o -CF cannot be recognized by cel-... 

Discrimination of Cellulase Components A, B, and C. The physical and chemical properties as well as substrate specificities of the highly purified cellulases of Ps. fluorescens have been characterized and are summarized in Table IV. Cellulase A is different from Cellulase B in the mobility on zone electrophoresis and in the pattern of Sephadex G-25 chromatography, but similar in substrate specificity toward several reduced and nonreduced cello-oligosaccharides. On the other hand, Cellulase C is different from A in the pattern of DEAE-Sephadex chromatography and the substrate specificity, and from B in all respects. These characteristics of each cellulase component are therefore different enough to be used as criteria to discriminate one from the other. 

Table 2. Substrate Specificity of Purified 3-glucosidases and Cellulases.

The postulation of a model on cellulose degradation obviously depends on firm knowledge of the kinetics and substrate specificities of individual cellulases. Investigations in this area have been crowned by success, especially because of... 

A derivatized form of pneumococcal type III polysaccharide has been used to distinguish between three jS-D-glucan hydrolases (including cellulase) from a Streptomyces species. Oligosaccharides also released from carboxymethyl-cellulose, lichenin, and oat glucan by the enzymes were identified, allowing the substrate specificities of the enzymes to be deduced. 

Cellulase enzyme complexes consist of three major types of proteins that synergistically catalyze the breakdown of a cellulosic substrate. Because the enzymes are strictly substrate-specific in their action, any change in the structure or accessibility of the substrate can have a considerable influence on the course of the hydrolysis reaction. A pretreatment method based on exposing cellulosic substrate to phosphoric acid solution [9] and addition of the nonionic... 

In the treatment of cellulose pulps one essential criterion for a suitable enzyme preparation is that its cellulase activity should be as low as possible, or preferably absent completely. As even extremely low cellulase activities may ruin pulp quality, Trichoderma enzyme preparations are unlikely to be suitable for these applications. Many bacterial and fungal enzymes with low cellulase activity have been shown to be suitable for treatment of pulps 14, 15, 16,17), Regulation of the often synchronous production of cellulolytic and hemicellulolytic enzymes in micro-organisms is not well understood, and is further complicated by substrate cross-specificity of these enzymes. Enzymes with both endoglucanase and xylanase activity have been reported for bacteria 18, 19) and fungi 20, 21, 22), In addition to selection of strain and... 

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. 

A third water-soluble substrate for endocellulase is hydroxyethyl-cellulose (HEC). As early as 1931, Ziese (5) used HEC and Sandegren et al. (6) defined the activity as being proportional to the change of the inverse of the specific viscosity per time unit. Child et al. (7) have also used HEC and based their calculation -on a linearization of the viscosity measurements according to Eriksson et al. (8). They defined an enzymic activity unit as the amount of enzyme causing a viscosity change of 0.001 rjre 126 min"1. The exponential factor was used in order to linearize the data. This unit is useful for comparison of cellulases of different origin, but it is based on an empirical relationship. The authors made an evaluation of their method in comparison with CMC hydrolysis. 

Hormone-treated pea seedlings generate two physically distinct cellulases (EC 3.2.1.4), with similar substrate specificities, Km values, and inhibitor sensitivities. They may be effectively separated by sequential extraction with buffer and salt and they appear to possess identical active sites but different apoprotein structures. The question arises of why this tissue should elaborate two hydrolases which catalyze the same reactions. The cellulase that forms first is synthesized by and accumulates in vesicles, where it would never encounter cellulose, while the other is concentrated on the inner wall microfibrils. It is suggested that only the latter cellulase functions to hydrolyze cellulose. A precursor/ product relationship between them could explain their distribution and developmental kinetics, but physical and chemical differences mitigate against this interpretation. 

The classification of pectic enzymes in general, their occurrence in higher plants and micro-organisms and the properties of pectic enzymes from some plants and food grade micro-organisms are described with special emphasis on their substrate specificity. Their technological roles and applications, also in combination with (hemi-)cellulases, in a variety of processes are discussed. Evidence is presented for the existence of a new type of pectic enzyme which acts specifically in the hairy regions of pectic substances. 

Both the quantity and properties of cellulases produced by microorganisms depend on the culture conditions. Commonly, cellulases are produced by culture of the organism either (a) in a liquid medium, which may be stationary, shaken, or submerged with aeration, or (b) by a Koji process on a solid substrate such as wheat bran (7). The complexity of the crude cellulosic carbon source usually leads to the production of a mixture of hydrolytic enzymes which may include amylases, proteases, chitinases, etc., in addition to the cellulases. Separation of proteins from culture filtrates by high resolution techniques such as chromatography, electrophoresis, or electrofocusing often reveals a number of enzyme species which may differ in specificity toward cellulosic substrates. These forms may represent ... 

As understanding of the components of the cellulase complex has increased, we can better define the mode of action by which native cellulose is depolymerized. The Ci-Cx model has directed attention to the specific steps in the crystalline cellulose breakdown. The use of nomenclature describing the catalytic function of each specific enzyme should encourage higher standards of purity. The ubiquity of the substrate invites imaginative applications of cellulases. 

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