Cellobiose enzymic

The jS-D-glucosidase of Mucor racemosus was shown to be biosynthesized when the organism was grown in the presence of such diverse carbon sources as glycerol, lactate, o-xylose, o-ribose, methyl and phenyl a-o-glucopyrano-sides, maltose, and cellobiose. Enzyme biosynthesis was strongly repressed in the presence of hexoses and of adenosine 3, 5 -monophosphate. The role of adenosine 3, 5 -monophosphate in the control of jS-D-glucosidase synthesis in M. racemosus was discussed. 

The glycosidic bond to an anomeric carbon can be either a or (3. Maltose, the disaccharide obtained by enzyme-catalyzed hydrolysis of starch, consists of two cv-D-glucopyranose units joined by a 1->4-o-glycoside bond. Cellobiose, the disaccharide obtained by partial hydrolysis of cellulose, consists of two /3-o-glucopyranose units joined by a 1—>4-/3-glycoside bond. 

The Enzymes II (E-IIs) of the phosphoenolpyruvate (P-enolpyruvate)-dependent phosphotransferase system (PTS) are carbohydrate transporters found only in prokaryotes. They not only transport hexoses and hexitols, but also pentitols and disaccharides. The PTS substrates are listed in Table I. The abbreviations used (as superscripts) throughout the text for these substrates are as follows Bgl, jS-gluco-side Cel, cellobiose Fru, fructose Glc, glucose Gut, glucitol Lac, lactose Man, mannose Mtl, mannitol Nag, iV-acetylglucosamine Scr, sucrose Sor, sorbose Xtl, xylitol. 

Kinetics of Bound and Free Enzyme. The kinetics of the IME were obtained with the recirculating differential reactor system as described above. The appropriate flow rate, the temperature optimum, and pH optimum as described above were used to most accurately establish the kinetic parameters for this IME emgmie. Substrate solutions from 3 to 150 mM cellobiose in 10 mM sodium acetate were appropriate for this portion of the study. Results were analyzed with the ENZFTT software package (Elsevier Publishers) that permits precise Lineweaver-Burk regressions. 

Cellobiohydrolase I (CBH I, 1,4-jS-D-glucan-cellobiohydrolase, E.C. 3.2.1.91) is the main protein (ca. 60%) of the cellulase complex produced by T. reesei strains. CBH I hydrolyses crystalline cellulose, acid swollen cellulose and 4-methylumbelliferyl-cellodex-trins by cleaving off the terminal cellobiose unit from the non reducing end of the chain. It operates with retention of configuration in the reaction products 19,20. The abundance of this enzyme and its stability has facihtated its purification to homogeneity... 

Figure 6. Specific enthalpy of denaturation for native CBH I, plotted as a function of the overall observed as the enzyme molecule is progressively destabilized by increasing the pH. Dot-centered circles represent the specific enthalpy in the absence of cellobiose the straight line is a linear least-squares best fit to these data points, plus the empirically derived intersection point (reference 2, see Discussion) represented by the crossed circle at upper right. The squares represent enthalpies measured at pH 4.80 and pH 8.34 in the presence of the indicated concentrations of cellobiose.

What we observe, both at the pH value (4.80) chosen to be near the activity optimum for the enzyme and at the value (8.34) chosen to produce substantial pH-stress, is that in the presence of cellobiose the enzyme has marked higher T values, but the overall shape of the denaturation envelope is very similar to diat observed in the absence of the inhibitor. In addition, the overall AH° values in the presence of even quite high concentrations of inhibitor are very close to those observed at lower temperatures in the absence of inhibitor, rather than resembling the values that the linear regression of Figure 6 would seem to imply for denaturation processes at these elevated temperatures. 

CelMbrio gUvus. This organism grows well on cellobiose, cellodextrins, and cellulose, but less well on glucose conditions for optimized growth or enzyme production were not... 

The first group is comprised that of compounds which potentially could be degraded to form G-l-P by phosphorolysis by beta-linkage specific enzymes. They include IPTGlu, cellobiose, sophorose, salicin, and sucrose. Addition of these compounds, or exogenous G-l-P, to Solka Floe fermentations improved maximum cellulase yields from 171 to 309%, and the time period for enzyme synthesis was reduced from 95 to 59% compared with using Solka Floe only. 

This enzyme [EC 5.1.3.11] catalyzes the interconversion of cellobiose to o-glucosyl-o-mannose. 

Coupling of an aryl 1-S-cellobioside to an affinity carrier was therefore expected to be useful in the chromatographic fractionation of endo and exo enzymes, e.g., from Tr. r. Preliminary tests indicated that CBH I and CBH II (prepurified by ion-exchange chromatography) were completely retained by the affinity support (4 -aminobenzyl 1-S-cellobioside coupled to Affigel-10 from Biorad). Desorption was achieved differentially by 0.1M lactose (elutes CBH I) and 0.01M cellobiose (elutes CBH I and CBH II). Attempts to elute the enzymes with 1M KC1, ethyleneglycol or glucose solutions were unsuccessful (11). 

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. 

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