Cellulase substrate specificity

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... 

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. 

The Tricboderma viride Cellulase Complex. Many cellulolytic organisms secrete cellulase as a multiple component enzyme system. Separation of physically distinguishable components by electrophoretic or chromatographic techniques has been repeatedly demonstrated. It is more difficult to prove that these components are distinct enzymes with different functions in terms of substrate specificity and mode—or mecha-... 

Electrophoretic properties of typical cellulase preparations, an extracellular cellulase from a culture on 0.5% cellulose and a cell-bound cellulase from that on 0.5% cellobiose, were compared in respect to their behavior in zone electrophoresis on cellulose acetate film. As shown in Figure 2, the former was separated into two components, A (fast moving to the cathode) and B (almost no moving). With the latter, a single component was detected under the same conditions. This fast moving component was in approximate agreement with component A in regard to its mobility, but as will be mentioned later, there was considerable difference in substrate specificity and other properties. Therefore, it seems to be a different component, and is referred to as component C. 

The multiplicity of cellulases from various organisms has been studied mainly on the basis of fractionation procedure, and their presence has often been assumed to imply the physiological significance for a complete degradation of native cellulose by utilizing each different substrate specificity. Consequently, any difference in their enzymatic prop-... 

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. 

Figure 3. Illustrations based on AZCL substrates. Three yeast colonies known to contain different cellulase genes were grown for three days on agar plates containing 0.1 % AZCL HE-cellulose (upper) and 0.1 % AZCL xyloglucan (lower). A clear difference in substrate specificity can be observed.

Besides chitinases, several other commercial enzymes possess chitinolytic activity. For instance, lysozymes, chitosanases, cellulase, and Af-acetylhexosaminidase also have the ability to hydrolyze chitin polymers, even though their substrate specificities are different from that of chitinases. Furthermore, chitinases from different sources may exhibit different chitin modification modes, facilitating its potential use in industrial applications. 

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. ... 

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

Chromogenlc substrates Ostazln Brilliant Red hydroxyethylcellulose (OBR-HEC), a soluble covalently dyed cellulose derivative, a substrate specific for endo-l,4-3-glucanases (cellulases) [4] Remazol Brilliant Blue xylan (RBB-X), a soluble covalently dyed beechwood 4-0-methyl-D-glucurono-D-xylan, a specific substrate for endo-l,4-B-xylanases [4]. Both substrates are available on request from Chemapol Ltd., Praha, Czechoslovakia. 

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... 

The degradation of hemicelluloses also proceeds via endo- and exohydrolases. The substrate specificity depends on the monosaccharide building blocks and on the type of binding, e. g., endo-1,4-P-D-xylanase, endo-l,5-a-L-arabinase. These enzymes occur in plants and microorganisms, frequently together with cellulases. 

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. 

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-... 

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