Cellulase system production

Webb, 1963), but, rather, is a more stable species. However, the ES complex does pass through a truly activated state in the course of coupling, and, in the case of a solid system, through a chain of such activated states. In the case of cellulose-cellulase system products (cellobiose or glucose) seem to be unbound to the enzyme but in others the products form complexes which are comparable to the ES complexes. Because cellulose is an insoluble substrate it has not been proven that there exists a product enzyme complex that is comparable to the enzyme substrate complex. The mechanism of the cellulose breakdown is an extremely complex system. 

Production of Trichoderma reesei Cellulase System with High Hydrofytic Potential by Solid-State Fermentation... 

A literature survey indicated that very little work has been done to produce an optimal cellulase system as described above. Here, we used solid-state fermentation (SSF) to achieve this objective. SSF processes, such as the "koji" process, have been used extensively for amylase production on wheat bran in Japan its application was extended to cellulase production on wheat bran and Ugnocellulosic materials by Toyama (13), Since then, wheat bran has become an important substrate for producing various products by SSF (14-20), In this study, we tested various lignocellulosic substrates for the production of cellulase and )3-glucosidase from T, reesei QMY-1 by SSF. 

Pretreatment of Substrate. Several different lignocelluloses were pretreated with NaOH. This pretreatment partially solubilizes the hemicelluloses and lignin and swells the cellulose so that the organism can utilize it for its growth and for production of a cellulase system in SSF. The treated lignocelluloses were not washed. The NaOH treatment is done with a minimum amount of water so that, after the addition of nutrient solution and inoculum, the moisture content is less than 80% wt/wt and there is no free water in the medium. More water was added to make suspensions of different lignocellulosic substrates of the desired concentration (1% or 5%) for liquid-state (submerged) fermentation (LSF). 

R. flavefaciens cells, during growth in pure culture, released cellulase, endoglucanase, and xylanase into the culture fluid. This microorganism hydrolyzed cellulose to yield only cellobiose as a product 49). It had been reported that a ceUobiose phosphorylase and glucokinase were present in R flavefaciens (8). The Ruminococcus cellulase system was repressed by disaccharides such as cellobiose, sucrose, and lactose 50). 

The stimulation of the synthesis of the cellulase system of T. reesei QM 9414 by sophorose was established as shown by the results of experiments summarized in Table IV. Other than sophorose, of the glycosides and oligosaccharides tested, only lactose caused even a limited production of the enzymes of the cellulase system. Lactose is not as closely related structurally to sophorose as is, for example, the disaccharide laminaribiose it is more closely related structurally to cellobiose, which, despite being the major product of cellulose breakdown, does not promote enzyme production under the conditions of this experiment. It was noted that both intra- and extracellular constitutive enzyme levels produced by cells growing on glucose (or by resting cells without inducer, Table IV) are less than 0.5% of the fully induced levels and thus are negligible. 

Table IV. Effect of Several Carbohydrates on the Production of the Enzymes of the Cellulase System by T. reesei...

The fact that transglycosylation products of one of the enzymic components of the cellulase system of T. reesei can stimulate the production of the system may be of great importance in elucidating the mechanisms of the processes involved in the biosynthesis of these enzymes. Sophorose may be an early transglycosylation product, whose... 

By using both enzymes in combination, the yield of crystalline D-glucose is increased.100 The use of cellulosic wastes for the production of glucose is of commercial interest, and in consequence much research work has been done on the characterization of cellulases. In particular, the cellulase system of the fungus T. ressei has the full complement of enzymes required to degrade crystalline cellulose. This and other fungal extracellular cellulases are commercially available.101... 

Fortunately, at this time, there were developments in the techniques for the fractionation of macromolecules at a 1967 ACS Symposium, a review of the existing fractionation procedures for cellulases (4) revealed that, although notable successes had been achieved, better methods for the purification and characterization of the enzyme components were needed. Towards the end of 1961, developments in the production of gel-filtration media and of ion-exchange forms of the same materials made possible the application of these techniques to the separation of the components of the cellulase system. 

The nature of this inhibition has not been explained. Product inhibition in the case of dehydrogenation of dienedol and of hydrocortisone by cells of Septomyxa is also reported (Chen et al., 1962). For product inhibition of cellulose-cellulase system, no linearity is noticeable. Data (Ghose, 1969d) on high rate saccharification of cellulose at lower substrate concentrations are presented in Table 2 and Fig. 3. 

If inhibition is caused by product, then I may be replaced by P and Ki by Kp, The inhibition mechanism may be described as formation of a complex between the enzyme and the inhibitor. This results in the partial loss of the compatibility to form the product In such a situation, therefore, the amount of product expected in the uninhibited reaction is always eater than that in the inhibited regardless of the nature of inhibition. The Eq. (18) should thus represent the model for competitive substrate or product inhibition. The rate of reaction should thus vary with either substrate concentration (in absence of inhibitor or product) or inhibitor (or product) concentration. It is not possible to evaluate the interdependence of the inhibition process between the three independent components the substrates, the products and known inhibitors. The Cellulose-cellulase system is one of competitive inhibition by two products of the process. This follows from the similarity of values of equilibrium constants, Kp for both cellobiose and glucose. The reciprocal plots for no inhibition, glucose and cellobiose inhibition based on the data of Table 4 are presented in Fig. 7. 

The presence of strong product inhibition and interference of the non-cellulosic components in the overall reaction results in the kinetics more complicated than in a soluble one phase enzyme-substrate system. The magnitude of activation energy (14,870cal/gm mole Das, 1969) estimated for rice hull-cellulase system falls in the range such that the reactions could be either a diffusion in aqueous phase or a chemical reaction. The involvement of large insoluble molecules suggests that both diffusional and chemical reaction processes are active. 

The second step in the direct ethanol process is that of enzyme production. The Gulf process utilizes a mutant strain of Trichoderma reesei, grown continuously to produce a complete cellulase system. The residence time is 48 hours. Enzyme production begins on a spore plate with subsequent scale-up to the enzyme production vessel size to be used. Our pilot plant facility has 300-gal enzyme reactors. 

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