Cellulase, preparation

MSW-fed digesters are operating under less than optimal enzyme titers. Table IV shows the activity optima for selected cellulase related enzymes found in actual laboratory digesters 51) and the optima for a typical commercially available cellulase preparation. 

The commercial cellulase preparation used in this study was Genencor GC 123 (lot D7246), which demonstrates 90 IFPU/mL at 45 C. 

Similar results were obtained with crude cellulase preparations from Penicillium pinophilum (12). The general applicability of this biospecific chromatography is illustrated by the isolation of the EGD from C. t., cloned in E. c. (Fig. 6). 

The following experiments were conducted using fraction C-l-A, which was thought to be representative of the LCC s (17). The acidic LCC fraction, C-l-A, was partially hydrolyzed with a cellulase preparation, Cellu-losin AC, and changes in the lignin and sugar distributions were analyzed... 

The action of various cellulase preparations on solutions of commercial xanthans, and those from laboratory strains containing both pyruvate and acetate, pyruvate only, or acetate only, in the unordered state has been... 

Table IV. Degradation of Cotton Cellulose by Cellulase Preparations from Three Cellulolytic Fungi in the Presence of Either Air, Nitrogen, or Oxygen ...

Reese (18) attaches a great deal of significance to the increase in the fragment of cotton fiber which was soluble in alkali after treatment with a cellulase preparation (Ci + Cx + / -glucosidase) that contained methocel to inhibit Cx activity. He interprets this data to indicate that Ci acts first. 

If the mechanism of cellulase action can be explained simply in terms of sequential action of endo- and exoglucanases, it is logical to expect that Ci from one cellulase preparation should act synergistically with Cx from another, at least in those enzyme systems from which both Cx and Ci have been isolated. Synergism between Ci of P. funiculosum and Cx of T. viride has already been demonstrated (22), and the results in Table V show that "cross-synergism of this type is shown by many different mixtures of the Ci and Cx components of F. solani, T, koningii, and P. funiculosum cellulases. In each case, a marked potentiation in activity is observed. 

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. 

There is one sharp, distinct peak of endoglucanase activity from young fermentation broth. In comparison, the ten-day-old culture exhibited additional endoglucanase peaks, while in the fourteen-day-old culture four different endoglucanase activity peaks were observed. The activity peaks of enzyme obtained from older cultures are similar to those obtained from commercial cellulase preparations as shown in Figure 1. Similar results have been observed by Nakayama (16). 

This data, together with the observation of prolific protease activity in crude commercial cullulase preparations that are probably obtained from older cultures, has led us to speculate that the multiple enzyme peaks in the older cultures could have resulted from protease modification of one parent endoglucanase. This prompted us to discontinue the use of commercial cellulase preparations. 

T ine structural studies on woody cell walls attacked by ectoenzymes of fungi in situ are numerous (cf. 1,2). In contrast, investigations on the selective degradation of cell walls by enzymes isolated from fungi are few. Jutte and Wardrop (3) attempted the use of crude commercial cellu-lase preparations to determine the degradation pattern of Valonia cellulose and beechwood fibers. Similar use of commercial preparations of enzymes was made by Reis and Roland (4) to evaluate the nature of diverse cell walls and to show the distribution of polysaccharides. An endo-/ -l,4-xylanase with specific xylanolytic activities was isolated from a commercial cellulase preparation using chromatographic methods and... 

The degradation rates of cellulose obtained with the individual cellulases were, in each case, higher than the corresponding values reached under the same conditions with beechwood holocellulose (10). The same tendency had already been found in preliminary tests with crude cellulase preparations (13 cf.23). 

Cellulase activities of T. reesei broths are normally reported to lie between 400 and 600 FPU/g total protein.188 Our work to assess the specific activities of T. reesei cellulase preparations has led us to the direct comparison of commercial cellulase products, typically highly selected T. reesei mutants, and reconstituted, purified cellulase enzymes (Table 33.4). Although the range of specific activities found from this internally consistent study generally agrees with the literature, our estimation of the... 

TABLE 33.4 Specific Activities of Various Trichoderma reesei Cellulase Preparations... 

A mathematical model has also been proposed for evaluating cellulase preparations. Sattler et al.209 describe a relationship between hydrolysis extent, reaction time, and enzyme concentration. This procedure permits the effectiveness of different enzymes and of different pretreatment methods to be ranked. This method examines cellulose hydrolysis data collected from hyperbolic functions of substrate concentration versus cellulase enzyme concentration at various timed incubations. The model is based on a double reciprocal plot of the relationship... 

Adney, W. S., Mohagheghi, A., Thomas, S. R., and Himmel, M. E., Comparison of protein contents of cellulase preparations in a worldwide round-robin assay. In Enzymatic Degradation of Insoluble Carbohydrates, 1995 Vol. 618, pp. 256-271. 

Although several properties of each of the three proteins have been investigated, most of our work has been done with the C form because (a) it has the highest affinity for crystalline cellulose (b) it is the predominant form in most, but not aU, T. viride cellulase preparations (c) it contains more carbohydrate than the A and B forms, suggesting that they may be degradation products from or intermediates in the biosynthesis of hydrocellulase C and (d) it forms a slightly more active hydrocellulase system when recombined with the enzymes of fraction I. 

Cellulases have been limited to a few specific applications, but economic and ecological factors have increased interest in their potential value. They have been used mainly as a component in digestive aids with other hydrolytic enzymes (Table VII). Toyama reported (62) in 1968 that commercial cellulase preparations were exported from Japan at a rate of 500 kg per month. 

Fig. 3. Process flowsheet for cellulase production in wheat bran culture [25]. A Submerged seed culture of T. viride, B oil-free compressed air C air filter D inoculum E exhaust air F sample collection G centrifugal pump H automatic wheat bran culture of T. viride I water spray I ammonium sulfate, alcohol and water J conveyor belt J screw conveyor K hopper L extraction column M storage tank centrifuge 0 precipitation P mixing tank Q ion-exchange column R membrane concentrator S spray dryer T filter press U rotary dryer V mixer W cellulase preparation and salt stabilizer... 

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