Hydrolysis of CMC

The E-3 peak was high in Avicelase activity and in protein content as compared with CMCase activity. This peak was further fractionated on a Bio-gel P-100 column five protein peaks (E-3-1 to E-3-5) were obtained, of which E-3-2 peak was highest among them in Avicelase activity and protein content. The elution patterns are shown in Figure 3, and the time course of hydrolysis of CMC by these cellulase fractions measured by a decrease in the viscosity is shown in Figure 4. Randomness of them is in the order of E-3-5 < E-3-2 < E-3-1 E-3-4 E-3-3. The E-3-2 fraction was subjected to further purification on a CM-Sephadex C-50 column because E-3-5 was very low in the Avicelase activity. 

Figure 4. Decrease in specific viscosity during the hydrolysis of CMC solution by E-3-1, E-3-2, E-3-3, E-3-4, and E-3-5. Reaction mixture consists of 3.0 mL 1% CMC, 7.0 mL 0.1M sodium acetate buffer, pH 4.0, and 2.0 mL enzyme solution containing an amount of protein equivalent to an optical density of 0.05 at 280 nm. The reaction mixture was carried out at 30°C in an Ostwald viscometer.

Similar hydrolysis patterns were observed for the hydrolysis of CMC of different degrees of substitution, and they were entirely different from the patterns obtained by En-1, which was fractionated and purified from the E-4 peak of Figure 1. The purification procedure is not given in this chapter. These time-course patterns are shown in Figures 11 and 12. 

Comparison of Randomness of Ex-1 and Endocellulases on the Hydrolysis of CMC and Cotton. That Ex-1 is least random (as compared with S-l and F-l) on the hydrolysis of CMC and cotton was verified by the observations of the relationships between fluidity of CMC or the decrease in degree of polymerization (DP) of cotton and the simultaneous production of reducing power. These results are shown in Figures 16 and 17. Further, as shown in Figures 18 and 19, the difference in the hydrolysis patterns of both types of cellulase becomes more clear with the comparison between time-course patterns of changes in the viscosity of CMC by both Ex-1 and En-1. The latter is a typical endo-cellulase component as described relative to Figure 12. 

Figure 16. Relationship between increase in fluidity and reducing power during the hydrolysis of CMC by Ex-1, S-l, and F-l. Reaction conditions for viscosity measurement 3.0 mh 1% CMC, 7.0 mL 0.1 M sodium acetate buffer, pH 4.0, and 2.0 mL enzyme solution. The reaction mixture for reducing power measurement was made under standard conditions. Final enzyme concentrations 3.0 X 10 3%, 8.0 X 10 3%, and 6.3 X 10 3% for Ex-1, S-l, and F-l, respectively.

Synergistic Action of Ex-1 with other Endocellulases on the Hydrolysis of CMC and Avicel. The synergistic action of Ex-1 with F-l was investigated in the hydrolysis of both CMC and Avicel. For comparison, we investigated a similar action between S-l and F-l. The results are shown in Table V. 

Figure 19. Decrease in specific viscosity during the hydrolysis of CMC solution by En-1. Reaction conditions are the same as in Figure 18 final enzyme concentration 2.2 X 10 4%.

The possibility that BS and BI cellulase could act synergistically, as has been recorded for many components of fungal cellulolytic complexes (1,2), was tested in several assay systems by adding the enzymes separately or together at the same total activity levels (CMCase units). The assays included the hydrolysis of CMC, cellohexaose, and cellulose powder. The results (not shown here) indicated that the pea cellulases were no more or less effective when added together than when added singly, i.e., there is no indication of any interaction between the enzymes, or any preference by one for the products generated by the other. 

Figure 9. Relation between fluidity and reducing power during the hydrolysis of CMC by Pseudomonas CeUutases A, B, and C...

Rabinovich, M. L, Klesov, A. A, Berezin, 1. V. (1977). The Kinetics of Enzyme Action Tsellyuhticheskih Geotrilium Candidum, Viscometric Analysis of the Kinetics of Hydrolysis of CMC. Bioorganic Chemistry 3(3), 8405 14. 

The above examples show that different approaches of analysis are required for different types of poly(saccharides). Whereas enzymic hydrolysis of CMC was not appropriate for many CMC formulations and a chemical method had to be developed, the enzymic method for the determination of starch purity in high purity starches could be significantly improved. 

Although its two domains could function independently, removal of the substrate-binding domain of ngCenA reduced enzymatic activity against microcrystalline cellulose but not against CMC or amorphous cellulose (12). This suggested that the substrate-binding domain played a critical role in the hydrolysis of crystalline cellulose. 

The effects of dilution of the micellar surface charge on the rate of alkaline hydrolysis of a betaine ester surfactant have been investigated for a mixture of decyl betainate and a nonionic surfactant with a similar CMC. It was shown that the relation between micellar composition and the hydrolysis rate essentially parallels the relation between micellar composition and counterion binding to mixed micelles made up of ionic and nonionic surfactants [20]. 

In previous work, we obtained several cellulase components from culture filtrates of Irpex lacteus (Polyporus tulipiferae) or from Driselase, a commercial enzyme preparation of this fungus they behaved practically as a single protein (1,2,3). They were different in randomness of the hydrolysis of carboxymethyl cellulose (CMC), expressed as the ratio... 

Evidence for Ex-1 to be an Exo-type Component. The time course of CMC hydrolysis by Ex-1 is shown in Figure 10. The hydrolysis proceeded rapidly at first, but it reached a plateau and seemed to stop after 3 hr. This is characteristic of the hydrolysis by exo-type cellulase, as has been reported for exocellulase of glucosidase type from T. viride (7) and for another Trichoderma exocellulase of Avicelase type (10). 

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... 

We thus elucidated that three of the four cellulase components are endo- or random-type and the other is exo-type. However, it is difficult to distinguish between the components of least or lowest random-type and those of exo-type. It is rather easy to identify an endo-type cellulase component. In contrast, it is very difficult to determine a cellulase to be exo-type because if the enzyme has a glycosyl-transferring activity the hydrolysis product is not a single sort, which is one of the necessary conditions to be an exo-type. Based on our experiments, measurement of the time course of CMC using a sample of medium substitution degree seems to be the best method of diagnosis to determine a cellulase component to be endo- or exo-type. With some enzymes, direction of mutarotation of reaction products is useful to resolve this problem, as is illustrated by the classic example of the starch hydrolysis by a- and /3-amylases. If this is true for our cellulases, the mutarotation of reaction products would be a... 

Luisi has shown that membrane material itself can be formed autocatalytically in an experiment to investigate the base catalysed ester hydrolysis of hydrophobic ethylcaprylate [27], Hydrolysis occured initially at the aqueous-organic interface where the products were micelle-forming sodium caprylate and ethanol. Once the critical micelle concentration, or cmc, was reached an exponential increase in hydrolysis was observed. The rate of hydrolysis in this second phase was almost 1000 times greater than in the initial phase suggesting that a catalytic mechanism had been activated. Luisi and co-workers hypothesized that once the cmc had been reached hydrolysis occurred within the micelles and, as the reactants were then constrained within a more hydrophobic environment, the increased rate was due to autocatalysis. Below pH 7 the micelles reorganized into unstable vesicles, in the order of 150 nm across as verified by freeze-fracture electron microscopy. 

The values of the observed rate constants and, where specified, the activation energy, for the hydrolysis of micellar solutions of the surfactants are given in the cited reference, but no data are available for solutions below the CMC. 

Since phosphates and sulfates with long chain alkyl substituents form micelles at concentrations above their CMC, the hydrolysis of these esters can be subject to micellar catalysis thereby providing a simplified system in which micelle formation and structure are not alfected by the presence of a foreign solubilizate. The hydrolysis of such surfactants must be considered, however, in investigations of their effects on reaction rates. Fortunately, the rate constants for the neutral hydrolysis of esters such as sodium dodecyl sulfate are extremely slow at 90° = 296 days at pH = 8-63), and the acid-catalyzed hydrolysis of the same ester is some three orders of magnitude faster and thus is still negligible in most cases (Kurz, 1962). 

The effects of surfactants on the hydrolysis of 2,4-dinitrophenyl sulfate are, however, smaller in magnitude (Fendler et al., 1970a). Above the critical micelle concentration both CTAB and polyoxyethylene(24) dinonylphenol, Igepal DM-730, increase the rate of the neutral hydrolysis of 2,4-dinitrophenyl sulfate by factors of 3-15 and 2-58, respectively, but NaLS has no effect (Fig. 8). A good linear relationship was obtained between — 10 /(i — o) a fid lj Gj — CMC) (equation 10a) from which the binding constant between 2,4-dinitrophenyl sulfate and CTAB was calculated to be 1-9 x lO M . This value was found to agree well with... 

Catalysis arising solely from hydrophobic interactions between the reactants in model systems has been investigated recently by Knowles and Parsons (1967, 1969). The effects of hydrophobic interactions on the rate of hydrolysis, aminolysis, and imidazole-catalyzed hydrolysis of p-nitrophenyl esters were elucidated by varying the hydrocarbon chain length of the -nitrophenyl ester, the primary amine, and the N-substituted imidazole and determining the second order rate constants at concentrations well below the CMCs of the reactants, conditions under which cationic (amine) and neutral (ester) micellar catalysis is... 

Kinetic investigations of the effects of urea and similar denaturing agents on rates and thermodynamic parameters of micelle catalyzed reactions have been suggested to be more sensitive probes for the nature and extent of hydrophobic interactions than CMC determinations. Thus, Monger and Portnoy (1968) have taken advantage of the base catalyzed hydrolysis of micellar -nitrophenyl dodecanoate. The rate constant for the hydrolysis of -nitrophenyl dodecanoate decreases rapidly with increasing initial concentration of the ester (10 to 10 m), and the second-order rate constant for the base-catalyzed hydrolysis of this ester... 

Another interesting application of a structurally ill-defined assembly is the first efficient hydrolysis of an amide at room temperature and pH 8. When the cationic amide 15 (2 x 10 M) is mixed with anionic palmitate (2 x 10 M)at pH 8, an undefined molecular cluster is formed in which the water-stable amide (t./, > 1 yr) rapidly hydrolyzes = 3.1 min). The long-time contact of reactants, a state thought to be essential for enzyme-like reactions, can obviously not only be enforced by neighbouring group effects in rigid molecules or in complexes between enzyme clefts and substrates. It may also occur in very simple micellar-like clusters of extremely low cmc. 

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