Avicel cellulases

Native or processed cellulose (e.g., cotton, Avicel, filter paper) and its soluble derivatives (e.g., CMC, HEC3) are substrates most often used in the study of cellulases. The classification based on the use of these substrates (l,4-/ -D-glucan cellobiohydrolases (CBH), exo-cellulases, Avicelases and... 

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 ... 

Figure 2. Zymogram of gCenA (A-I) and ngCenA (J-Q) after incubation with C. fimi protease. Cellulases, bound to Avicel, were incubated with protease or control buffer for 72 hr at 30° C, then centrifuged to give cellulose-bound (A-E, J-N) and supernatant (F-I, O-Q) fractions. Products were separated on a SDS gel, replicated onto CMC-agarose and developed with Congo red. A,J. buffer control (4°C incubation) B,F,K,0, protease C,G,L,P, protease + PMSF control D,H,M,Q, buffer control E,I,N, buffer + PMSF control.

Figure 6. Isoelectric focusing of the Ci component of F. solani cellulase in an ampholyte solution covering the pH range 4.6-5.1 in an LKB 110-mL column. The C. component used was that which was eluted from a DEAE-Sephadex column as a single component (12). Activity to HsP04-swollen cellulose (- A activity to Avicel with CL and /3-glucosidase added to show synergistic activity (- O -) pH gradient (- -). From...

As further evidence, we demonstrated by paper chromatography that hydrolysis products from cellooligosaccharides by Ex-1 are Gi and G2 from G3, and Gi, G2, and G3 from G5, but only G2 from G4, Ge, CMC, cellodextrine, and insoluble cellulose such as Avicel, swollen cellulose, absorbent cotton, and filter paper (Figures 13 and 14). However, G3 was formed from G6 when Ex-1 was incubated with a mixture of G6 and Gi. There is no indication that G6 was split by this cellulase into G3 plus G3, but rather that G2 produced from G6 was transferred immediately to Gi to form G3. The results are shown in Figure 15. 

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... 

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

Figure 2. Development of CM-cellulase activity in a culture of Thermo-actinomyces sp., grown on 1% Avicel (PH 102) at 55°C in a 14 L batch fermentor. Uncontrolled pH 7% shake flask inoculum. (O) Whole culture broth (O) culture filtrate (0) culture solids (6).

Zheng, Y. Tsao, G. T. Avicel Hydrolysis by Cellulase Enzyme in Supercritical C02. Biotechnol. Lett. 1996, 18, 451 -54. 

Because of its ability to produce and secrete the complete set of cellulolytic enzymes, thus making it particularly potent in hydrolyzing the cellulose polymer to glucose monomers, the soft-rot fungus Trichoderma, in particular T. reesei has been the focus of cellulase research for decades (8). The preferred substrates used by most researchers for cellulase production are pure celluloses such as Avicel, Solka-floc, and cotton (9). Cellulase production by Trichoderma is controlled by a complex metabolic regulation (10-12). Cellulose acts (indirectly) as an inducer for the production of cellulases. Expression of cellulases is furthermore subject to repres-... 

Fig. 6. Saccharification of Avicel cellulose by T. reesei cellulase without (panel 0) and with (panel 1) Orpinomyces BglA. Concentrations of sugars were measured using an HPLC system (see Materials and Methods) after 16 h of reaction.

In 1972 Ogawa and Toyama (56) purified three components— A-I-a, A-I-b, and A-II-1—which were adsorbed on a gauze column during purification from Cellulase Onozuka P1500, a commercial preparation of T. viride cellulase. These three components had molecular weights of 32,000, 48,000, and 48,000 as determined by gel filtration and contained 7-16% carbohydrate. Each is reported to carry out the random hydrolysis of CM-cellulose and to degrade hydrocellulose (Avicel) and cellooligosaccharides except for cellobiose. The order of reactivity toward either cotton or Avicel was A-II-1 > A-I-b > A-I-a. The proteins adsorbed on cellulose comprised 38% of the total cellulase protein. 

None of the six enzymes cleaved cellobiose, but in comparing the products released from swollen Avicel by hydrocellulase C with those from cellulodextrin by the Cellulases, further evidence of contamination of cellulases II and III was found. For the latter cases both glucose and cellobiose are produced whereas both hydrocellulase C and Cellulase IV yield predominantly cellobiose. Cellulase IV produced much more cellobiose than glucose from cellotetraose whereas the other Cellulases yielded only slightly more cellobiose than glucose. 

In addition to cleaving individual cellulose chains into small oligosaccharides, cellulases can also cleave cellulose particles into smaller particles. This activity is called fragmentation or short fiber formation. Quantitative studies of fragmentation utilized uniform sieved Avicel particles as the substrate and a particle counter to measure the number and size of the particles produced [111, 112]. Fragmentation activity could only be shown with particles larger than... 

Zheng, Y., and Tsao, G.T. (1996) Avicel hydrolysis by cellulase enzyme in supercritical C02. Biotechnol. Lett.,... 

As currently understood, the cellulase complex contains the following components (listed in the order in which their action on cellulose occurs) 1. Ci is an enzyme whose action is unspecified. It is required for the hydrolysis of highly oriented solid cellulose (Cotton, Avicel, etc.) by 0-1 — 4 glucanases. 

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