Cellulase stimulation

In addition, a new class of proteins (a-expansins) has been discovered in plants that appear to disrupt the interactions between cellulose chains, without hydrolytic activity, to allow cell wall expansion [18]. A different, but related, set of molecules (P-expansins) has been found in pollen [19]. These molecules appear to help the growing pollen tube penetrate through the cell walls in the ovule allowing fertihzation. There is prehminary evidence that the addition of expansin to cellulases stimulates crystalhne cellulose hydrolysis [20]. 

The results of this analysis are obviously preliminary. We present them in the hope that we can stimulate both discussion and research in the area of cellulase domain analysis. It is our intention to fully develop the sequence analysis of these domains, and to develop logical criteria for family membership within each of the domain families. 

Langsford et al. reported that Cellulomonas fimi culture supernatants contained cellulase and proteinase activities, for which there appeared to be a relationship. Glucose repressed the synthesis of both activities and cellulose induced both 60), Adding cellulose to Cellulomonas sp. (NRCC 2406) cultures stimulated growth and improved production of cellulases 61). Optimum conditions for growth and cellulase production were pH 6.5 and 30 C. The addition of glucose in the presence of cellulose inhibited growth. Several species of Cellulomonas have cellobiose phosphorylase. 

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. 

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... 

The two compounds which stimulate cellulase production, sophorose and lactose, obviously do not promote amylase production. This is especially evident in the case of sophorose, since the reduction in amylase... 

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. 

Pseudomonas fluorescens produced two extracellular (A and B) and one cell-bound (C) cellulase components, the latter being released by treatment with EDTA-lysozyme in isotonic sucrose. Culture with 0.5% glucose formed little cellulase. Cellobiose stimulated only the synthesis of C. The formation of A and B was strikingly enhanced in cultures with cellulose, sophorose, or continuous low concentration of cellobiose. The absence of extracellular cellulase synthesis in 0.5% cellobiose culture may be caused by catabolite repression. The three cellulases were purified and characterized. None of them split cellobiose, but all hydrolyzed various cellodextrins and celluloses. C easily attacked cellotriose and cellotriosyl sorbitol, but A and B had no effect. When pure B was incubated with broken spheroplasts of sophorose-grown cells, a cellulase component indistinguishable from A was formed. 

Influences of glucose, cellobiose, sophorose, and cellulose, when they were each used as a C-source, upon the formation of both cell-bound and extracellular cellulases during the growth of this pseudomonad are shown in Figure 1. Glucose supported the bacterium for an excellent growth, but only slightly stimulated the formation of cellulases, and the enzymes produced were distributed almost equally to the cell and the culture medium. In the cellulose and sophorose cultures, the formation of cellulases, particularly that of extracellular component, was enhanced prominently (exo-type synthesis), whereas cellobiose which was a main end-product of enzymatic cellulolysis stimulated the formation of cell-bound component (endo-type synthesis). Thus, an apparent difference in the distribution of extracellular and cell-bound cellulases was noticed between the cultures with cellobiose and sophorose or cellulose. 

The simliar C-supply controlling culture was applied to Trichoderma viride, but no stimulated formation of cellulase was found in both cell-bound and extracellular fractions (unpublished data in our laboratory). 

E. T. Reese We have found that nearly all cellulolytic organisms, where cellulase is induced, do produce small amounts of constitutive enzyme, very very low levels. Your measurements here indicate similar very low levels of enzymatic activity. Under the conditions that you use I notice that the glucose did not disappear very rapidly. In our work on cellulase induction, we actually try to reduce the rate at which the substrate is consumed. I am wondering whether your conditions, where it is obvious that the organism wasn t very happy with its environment, were actually limiting growth and so stimulating induction ... 

Product Inhibition. In most microbiological and biochemical systems accumulation of end products exercises an inhibitory effect on the rate of the forward reaction. Stimulation by end product is thermodynamically improbable. One of the major products of hydrolysis of cellulose is cellobiose. There is a stimulation by cellobiose of Cx activity of Streptomyces spp. filtrates only when the substrate is solubilized by the introduction of various substituents—e.g., CMC, hydroxycellulose, cellulose acetate, etc. Stimulation is absent when unsubstituted cellulose is used (14). On the other hand, product inhibition is common. Cellobiose inhibits the hydrolysis of cellulose by filtrates of most of the 36 organisms tested. This action of cellobiose is believed to be that of an end product inhibiting an enzymatic hydrolysis in much the same manner that maltose inhibits hydrolysis of starch by a-amylase. The inhibitory effect of products varies with the organism from which the cellulase is derived. Thus, lactose is a very good inhibitor of the enzyme from... 

A 10% wheat bran suspension in water was autoclaved for 30 min at 115 °C and the resulting soluble (liquor) and insoluble components (residue) were separated by filtration. The liquor and residue fiactions were then used alone or mixed with 1 % MCC as carbon sources for P. decumbens growth. The biomass in cultures grown on wheat-bran liquor plus MCC was less than that on wheat-bran residues plus MCC, but the cellulase activity released was higher (Fig. 4). The liquor apparently contained a factor that stimulated cellulase synthesis and/or secretion. In contrast, the xylanase activity in the supernatants of cultures supplemented with wheat-bran liquor was lower than that in cultures supplemented with the wheat-bian residues, which were in consistent with the residues having higher hemicellulose content. 

Stimulation of Growth and Production of Cellulase and Xylanase by the Soluble Components of Wheat Bran... 

Taken together, the results of this research demonstrate the importance of knowing and regulating the starch and soluble protein contents of wheat bran supplements from different sources when adding wheat bran to stimulate cellulase and xylanase production by P. decumbens. Our results also predict that adding cello-oligosaccharides directly to P. decumhens fermentation could significantly improve industrial-scale biomass hydrolysis by P. decumbens. 

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