Cellulases components

Chromophoric substrates were also used as tools in the study of the binding of several cellulase components to their natural substrates (such as Avicel). This is illustrated here in the investigation of the synergy in binding of CBH I and CBH II from Trichoderma reesei onto Avicel. The enzymes were differentiated with CNPL (see above), which was a substrate only for CBH I (core I). Thus, the amount of CBH II adsorbed when a mixture of both enzymes was added, either simultaneously or sequentionally, to Avicel was calculated from the amount of CBH I bound (activity measurements with CNPL) subtracted from the values for total protein binding (280 nm absorbance reading). The results obtained from these experiments are summarized as follows ... 

The Mode of Enzymatic Degradation of Cellulose Based on the Properties of Cellulase Components... 

In previous work, we obtained several cellulase components from culture filtrates of Irpex lacteus (Polyporus tulipiferae) or from Driselase, a commercial enzyme preparation of this fungus they behaved practically as a single protein (1,2,3). They were different in randomness of the hydrolysis of carboxymethyl cellulose (CMC), expressed as the ratio... 

With this background, we then tried to obtain the possible exocellulase component from I. lacteus using Driselase as the starting material, and we succeeded in isolating it. Its enzymatic properties were also precisely investigated and the possible role of this cellulase component in vivo was discussed with reference to the properties of other cellulase components already published. This report deals with these results, but most of the data for F-l and S-l were cited from previous work (2,3). 

Fractionation and Purification of Ex-1 Cellulase Component from Driselase. Driselase powder (50g) was extracted with several aliquots of water and the precipitate formed upon salting out with ammonium sulfate (on a saturation between 20% and 80%) was fractionated on a DEAE-Sephadex A-50 column. Each fraction was tested for -glucosi-dase, xylanase, CMCase, Avicelase activities, and protein content. The elution patterns are shown in Figures 1 and 2. 

Table IV. Amino Acid Composition of Various Cellulase Components from I. lacteus°...

Comparison of Randomness of Ex-1 and Endocellulases on the Hydrolysis of CMC and Cotton. That Ex-1 is least random (as compared with S-l and F-l) on the hydrolysis of CMC and cotton was verified by the observations of the relationships between fluidity of CMC or the decrease in degree of polymerization (DP) of cotton and the simultaneous production of reducing power. These results are shown in Figures 16 and 17. Further, as shown in Figures 18 and 19, the difference in the hydrolysis patterns of both types of cellulase becomes more clear with the comparison between time-course patterns of changes in the viscosity of CMC by both Ex-1 and En-1. The latter is a typical endo-cellulase component as described relative to Figure 12. 

The synergistic effect caused by a mixture of a typical endocellulase, F-l (CMCase), and an endocellulase of lower randomness (Avicelase) is slightly smaller than that caused by a mixture of F-l and Ex-1 (an exocellulase of Avicelase type) in the hydrolysis of both CMC and Avicel. This may be explained by the postulation that this kind of synergistic effect should be caused by the cooperation between cellulase components of extremely different types of hydrolysis. Consequently, the... 

Mutarotation of Hydrolysis Products by Ex-1. The mutarotation of hydrolysis products from cellopentaitol by Ex-1 was investigated. For comparison those by S-l and F-l were observed. As shown in Figure 20, the mutarotation of hydrolysis products by Ex-1 (exclusively G2) increases, indicating that products are released in the / -cellobiose configuration. Entirely similar results were observed for the hydrolysis products by S-l and F-l (a mixture of Gi, G2, and reduced G3 in different proportion for each reaction mixture). Therefore, these cellulase components belong to the same group, as far as the mutarotation pattern is concerned. 

We thus elucidated that three of the four cellulase components are endo- or random-type and the other is exo-type. However, it is difficult to distinguish between the components of least or lowest random-type and those of exo-type. It is rather easy to identify an endo-type cellulase component. In contrast, it is very difficult to determine a cellulase to be exo-type because if the enzyme has a glycosyl-transferring activity the hydrolysis product is not a single sort, which is one of the necessary conditions to be an exo-type. Based on our experiments, measurement of the time course of CMC using a sample of medium substitution degree seems to be the best method of diagnosis to determine a cellulase component to be endo- or exo-type. With some enzymes, direction of mutarotation of reaction products is useful to resolve this problem, as is illustrated by the classic example of the starch hydrolysis by a- and /3-amylases. If this is true for our cellulases, the mutarotation of reaction products would be a... 

The cellulase components that are synthesized in the presence of sophorose were investigated by the basic procedures previously described (1,2,4) for the isolation of cellulolytic components from commercial cellulase preparations. The purification to homogeneity of the proteins that yield the three predominant bands when the crude preparation is subjected to disc gel electrophoresis was accomplished by ion exchange chromatography. 

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. 

The development of the sequential elution methods makes it possible not only to cleanly fractionate the three cellulase components, but to do the fractionation with very little loss of enzyme. The total recovery of major enzyme components, summarized in Table III, is considerably higher than those reported previously by other researchers (8,9). Table III also gives the molecular weights of the three enzyme components. 

Table VII. Hydrolysis of Avicel by Purified Cellulase Components, Alone and in Combination...

Physical Properties. All of the cellulase (CMCase) activity which develops in auxin-treated pea apices dissolves in salt solutions (e.g., phosphate buffer, 20mM, pH 6.2, containing 1M NaCl). Gel chromatography of such extracts indicates the presence of two cellulase components with similar levels of activity and elution volumes corresponding to molecular weights of about 20,000 and 70,000 (Figure 1). If the tissue is extracted with buffer alone, only the smaller cellulase dissolves (referred to as buffer-soluble or BS cellulase). The larger buffer-insoluble (BI) cellulase can then be extracted from the residue by salt solutions. This simple extraction procedure effectively separates the two cellulases, and can be used as an initial step for their estimation or purification. 

Foam fractionation is a promising technique for concentrating proteins because of its simplicity and low operating cost. One such protein that can be foamed is the enzyme cellulase. The use of inexpensively purified cellulase may be a key step in the economical production of ethanol from biomass. We conducted foam fractionation experiments at total reflux using the cellulase component P-glucosidase to study how continuous shear affects P-glu-cosidase in a foam such as a fermentation or foam fractionation process. The experiments were conducted at pH 2.4, 5.4, and 11.6 and airflow rates of 3,... 

The effect of reflux time at pH 5.4 (holding the airflow rate at 10 cc/min) is shown in Fig. 3. It is observed that the activity held at about 95% of the initial activity until about 6 min of refluxing, at which time the activity dropped to about 80% of the initial activity. This means that with comparable aeration shear, experienced in a fermentor or a foam fractionation column, the activity of this cellulase component remains relatively intact for up to 6 min. At pH 3.5 and 4.0, when the activities were measured, following foaming for 6-8 min, the activities decreased only slightly (to about 95%). This low airflow rate usually led to foamate (recovered solution at the top of the foam) that was highly enriched. 

Research to improve ethanol fermentation has also focused on the development of a high solids, continuous feeding process as well as improved yeast strains. Other related developments include processes for the use of biomass feedstocks, such as cellulosic waste material.40"44 The Genencor-NREL collaboration funded by the U.S. DOE has (a) put in place the tools required for continual improvement of biomass-derived cellulases, (b) built a suite of enzymes with enhanced thermostability and improved specific performance at elevated temperatures, (c) developed an improved production strain for the enhanced cellulase components and demonstrated an enhanced production process. Therefore, plans are in place to provide a developmental product(s) in support of continued industry development. 

Bartley, T. D., Murphy-Holland, K., and Eveleigh, D. E., A method for the detection and differentiation of cellulase components in polyacrylamide gels. Anal Biochem 1984, 140 (1), 157-61. 

Recent developments in enzyme manufacturing have led to commercial products that contain a preponderance of one cellulase component. These mono-component enzymes are produced from modified Humicola strains and are primarily endo-glucanases active at pH 7-7.5 and are referred to as alkaline cellulases . 

The relationship between catabolite repression by glucose and induction of cellulase by sophorose has been studied in T. viride by Nisizawa and co-workers (36, 37). The induction by sophorose (10 M) was competitively repressed by glucose and other metabolites such as pyruvate. Since glucose was an effective repressor when added one hour after the previous addition of actinomycin D, it was concluded that the repression takes place at the translational level. Previous work indicated (26) that the sophorose induction led to the formation of a cellulase component designated FII, which is the source of cellulase II discussed below. In higher plants indoleacetic acid (38) and abscisic acid (39) have been shown to stimulate cellulase production. 

In 1968 Nisizawas laboratory (55) purified three cellulase components from Meicelase, which is a commercial cellulase from Trichoderma viride. These three components—Cellulases II, III, and IV—contained 16.8, 15.6, and 10.4% carbohydrate, respectively, and were active in hydrolyzing cellooligosaccharides, CM-cellulose, and cotton. They were inactive toward cellobiose and p-nitrophenyl-yS-D-glucoside. 

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