Cellobiose inhibition

The hydrolytic activities of the intact enzymes were comparable, but CBH I was much more sensitive to product (cellobiose) inhibition. Both core enzymes exhibited a strongly reduced activity (50-90%) which was correlated with the absence of the binding domain and their consequent lower binding capacity on Avicel. The activities of CBH I and Core I on amorphous cellulose were, however, comparable. 

K — reaction rate constant Eads = grams of absorbed enzyme/gram of cellulose 12 — cellobiose inhibition concentration, g/L... 

The products (glucose and cellobiose) inhibit the cellulase enzyme competitively. 

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... 

Fig. 6. Glucose and cellobiose inhibition of enzymatic saccharification of cellulose. <25p cellulose (Solka Floe) in 10 ml tubes, agitated, O-O-O-O-control, A-A-A-A-control + 5mg/ml of cellobiose, - - -control +10 mg/ml of d-glucose. Reaction time —10 min. Temp. — 50° C, Cellulase —1.86 FP...

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. 

Fig. 7. Lineweaver-Burk plots for no inhibition, glucose and cellobiose inhibition in <25 p cellulose, enzyme activity —2.0FP, temp. 50 C...

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 induction of this operon responds to the intracellular concentration of cAMP, which is determined by the carbon source available to the cell. When cells are grown on cellobiose or cellulose that do not inhibit adenylate cyclase, cAMP is made in sufficient quantities for induction of cellulase. On the contrary, when cells are grown on glucose or other readily metabolized carbohydrates that do inhibit adenylate cyclase. 

S. Vonhoff, K. Piens, M. Pipelier, C. Braet, M. Claeyssens, and A. Vasella, Inhibition of cellobiohy-drolases from Trichoderma reesei. Synthesis and evaluation of some glucose-, cellobiose-, and cellotriose-derived hydroximolactams and imidazoles, Helv. Chim. Acta, 82 (1999) 963-980. 

The active center of CBH I (or Core I) did not seem to be implicated in the adsorption on Avicel since no influence of small, soluble ligands (e.g., cellobiose) was observed (16). The importance of an adjacent site for the binding was, however, suspected because of inhibition of adsorption by large molecular weight ligands such as xylan (DP =... 

When suitable strains to exploit natural substrates are available, their exploitation of cellulolytic substrates in pure culture could be restricted by the end-products of cellulolysis (cellobiose and glucose) repressing enzyme synthesis or inhibition/inactivation of further enzymic activity (Fig. 1). One of the simplest routes to relieve this difficulty was to use non-celluloly tic... 

Colvin155 was the first to postulate a lipid-bound D-glucose as an intermediate in the biosynthesis of bacterial cellulose. Lipid-sugar derivatives, tentatively identified as lipid-diphosphate-D-glucose, lipid-diphosphate-cellobiose, and, perhaps, higher polymers, were detected in this system.128 These lipid-sugar compounds, which were acid- and alkali-labile, seemed to be formed prior to cellulose, and their formation was inhibited by adding... 

There are two reasons for the measurable cellobiose concentration in the T. viride cellulase hydrolyzed syrups. The most likely is that T. viride has rather poor / -glucosidase activity so that cellobiose accumulates. Evidence of this is that additions of / -glucosidase to the T. viride cellulase improves its activity. A second reason is that the / -glucosidase enzyme is strongly glucose inhibited. Hence the rate of cellobiose hydrolysis slows down as the glucose concentration rises, allowing cellobiose to accumulate. 

Table VI presents a kinetic model for preferred growth of a microorganism on cellobiose (C2) but having an extracellular / -glucosidase (E2) that is glucose inhibited and in which the glucose uptake is repressed. This model readily fits the cellobiose data shown by the solid lines in Figure 9 (20).

Cellobiose, a dimer of /3-1,4-linked glucose, is reported to be a cellulase inducer in T. reesei as well as in several other fungi (20,28,33, 34). But whether cellobiose is a true inducer is questionable since Reese et al. (35) reported that cellobiose could induce as well as inhibit cellulase biosynthesis. The same is also true for glucose. Whether glucose or cellobiose is an inducer or inhibitor depends on the concentration of sugars in the environment. 

In addition to catabolite repression, the cellulase enzymes themselves are subject to end-product inhibition. For example, as glucose accumulates during saccharification, it interacts noncompetively with cellobiase to inhibit further activity of this enzyme (6). Similar inhibition of endoglucanases occurs when cellobiose accumulates in a saccharification reactor (18,19,20). 

Saponins appear to lower plasma LDL cholesterol concentration by interfering with cholesterol absorption. Studies in rats and monkeys fed naturally occurring saponins exhibited significant reductions in cholesterol absorption efficiency and an increase in fecal cholesterol excretion (Malinow et al., 1981 Nakamura et al., 1999 Sidhu et al., 1987). Decreased bile acid absorption and increased excretion has also been reported in animals fed saponins (Malinow et al., 1981 Nakamura et al., 1999 Stark and Madar, 1993). One possible mechanism of action for decreased cholesterol absorption is the ability of saponins to form insoluble complexes with cholesterol (Gestetner et al., 1972 Malinow et al., 1977). In an effort to isolate the specific properties of saponins, Malinow (1985) prepared a variety of synthetic saponins in which the complex carbohydrate moieties of native plant saponins were replaced with simplified carbohydrates such as glucose or cellobiose. One of these synthetic saponins, tiqueside (Pfizer, Inc.), can effectively precipitate cholesterol from micelle solutions in vitro and inhibit cholesterol absorption in a variety of animals (Harwood et al., 1993) and in humans (Harris et al., 1997). But despite ample data showing the formation of a saponin/cholesterol complex in vitro, there is essentially no definitive evidence that complexation occurs in the intestinal lumen (Morehouse et al., 1999). 

Physical or chemical modification of a substrate may additionally selectively affect transformation or uptake Keil and Kirchman (1992) compared the degradation of Rubisco uniformly labeled with 3H amino acids produced via in vitro translation to Rubisco that was reductively methylated with 3H-methane. Although both Rubisco preparations were hydrolyzed to lower molecular weights at approximately the same rate, little of the methylated protein was assimilated or respired. The presence of one substrate may also inhibit uptake of another, as has been demonstrated for anaerobic rumen bacteria. Transport and metabolism of the monosaccharides xylose and arabinose were strongly reduced in Ruminococcus albus in the presence of cellobiose (a disaccharide of glucose), likely because of repression of pentose utilization in the presence of the disaccharide. Glucose, in contrast, competitively inhibited xylose transport and showed noncompetitive inhibition of arabinose transport, likely because of inactivation of arabinose permease (Thurston et al., 1994). 

To measure kinetic parameters, hydrolysis rates were determined by varying the concentrations of pNPG (0.05-10 mM) and cellobiose (0.04-16 mM). The inhibition by glucose was evaluated with only pNPG (10 mM) as substrate, whereas the inhibitory effect of glucono-1,5-lactone was verified with both pNPG (10 mM) and cellobiose (1.0 mM) as substrates. Km, Vmax, and fC, values were calculated from Lineweaver-Burk plots. 

---