Cellulase patterns

Tuerker and Mavituna immobilized Trichoderma reesei within the open porous networks of reticulated polyurethane foam matrices. Growth pattern, glucose consumption, and cellulase production were compared with those of freely suspended cells. The method of immobilization was simple and had no detrimental effect on cell activity. Hundreds of similar projects could be cited. Not all rated the use of polymethane as the preferred technique. If a statistical analysis were conducted on aU the immobilization literature, we are sure that no single technique would be dominant. However, the combination of ease of immobilization, cost of materials, flow-through properties, control of flux rate through the immobilizing membrane, high surface-to-volume ratio, and other factors make polymethane a viable substratum for the continuous production of proteins. 

In the reported structures of products of cellulase digestion of xyloglucans from different sources, there has been considerable variation that may reflect different action-patterns of enzyme preparations as much as actual differences in the structures of the polysaccharides. On reaction of xyloglucan from the walls and culture medium of suspension-cultured, sycamore cells,26,27 four major oligosaccharide products were isolated. Stuctures were proposed for the heptamer (Z) and the nonamer (3). 

Fractionation and Purification of Ex-1 Cellulase Component from Driselase. Driselase powder (50g) was extracted with several aliquots of water and the precipitate formed upon salting out with ammonium sulfate (on a saturation between 20% and 80%) was fractionated on a DEAE-Sephadex A-50 column. Each fraction was tested for -glucosi-dase, xylanase, CMCase, Avicelase activities, and protein content. The elution patterns are shown in Figures 1 and 2. 

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. 

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. 

Mutarotation of Hydrolysis Products by Ex-1. The mutarotation of hydrolysis products from cellopentaitol by Ex-1 was investigated. For comparison those by S-l and F-l were observed. As shown in Figure 20, the mutarotation of hydrolysis products by Ex-1 (exclusively G2) increases, indicating that products are released in the / -cellobiose configuration. Entirely similar results were observed for the hydrolysis products by S-l and F-l (a mixture of Gi, G2, and reduced G3 in different proportion for each reaction mixture). Therefore, these cellulase components belong to the same group, as far as the mutarotation pattern is concerned. 

T ine structural studies on woody cell walls attacked by ectoenzymes of fungi in situ are numerous (cf. 1,2). In contrast, investigations on the selective degradation of cell walls by enzymes isolated from fungi are few. Jutte and Wardrop (3) attempted the use of crude commercial cellu-lase preparations to determine the degradation pattern of Valonia cellulose and beechwood fibers. Similar use of commercial preparations of enzymes was made by Reis and Roland (4) to evaluate the nature of diverse cell walls and to show the distribution of polysaccharides. An endo-/ -l,4-xylanase with specific xylanolytic activities was isolated from a commercial cellulase preparation using chromatographic methods and... 

Work in several laboratories (22, 27, 55, 56) has shown a pattern of cellulase action in cellulolytic organisms which requires at least one of a set of three closely related enzymes in order to hydrolyze crystalline cellulose effectively. These enzymes often possess little ability to degrade either CM-cellulose (as measured viscosimetrically) or crystalline cellulose. Nevertheless they are characterized by the capacity to cleave swollen cellulose or cellooligosaccharides almost entirely to cellobiose by virtue of their / -( - 4)glucan cellobiohydrolase activity. Recognition of this pattern has been difficult because prior to this report the three enzymes had not been purified and characterized apart from contaminating enzyme activity. 

Figure 5. Patterns of synthesis of cellulase (C), amylase (A), and fS-glucosidase (G p-nitrophenyl-f3-D-glucoside as substrate) of Ps. fluorescens in 0.5% and C-supply controlling cultures on some selected...

Figure 8. Ultracentrifugal patterns of Pseudomonas cellulases Pictures were taken at 45 minutes after rotation had reached a maximum speed of 55,430 r.p.m. at 17°C. Protein concentration was 0.45% (Cellulase A) and 0.7% (Cellulases B and C) in 1/15M phosphate buffer of pH 7.0. The synthetic boundary cell was employed for Cellulases B and C...

Discrimination of Cellulase Components A, B, and C. The physical and chemical properties as well as substrate specificities of the highly purified cellulases of Ps. fluorescens have been characterized and are summarized in Table IV. Cellulase A is different from Cellulase B in the mobility on zone electrophoresis and in the pattern of Sephadex G-25 chromatography, but similar in substrate specificity toward several reduced and nonreduced cello-oligosaccharides. On the other hand, Cellulase C is different from A in the pattern of DEAE-Sephadex chromatography and the substrate specificity, and from B in all respects. These characteristics of each cellulase component are therefore different enough to be used as criteria to discriminate one from the other. 

Figure 10. Zone electrophoretic patterns showing the conversion of Cellulase B into another cellulose component moving rapidly toward cathode by treatment with SI or S3 tor 48 hours. The patterns of highly purified Celluloses A and B are shown for comparison, but the size of peak in one run cannot be directly compared with that in another. Arrow indicates the position of start. Electrophoresis and enzyme assay as described in Figure 2...

---