Avicelase

Native or processed cellulose (e.g., cotton, Avicel, filter paper) and its soluble derivatives (e.g., CMC, HEC3) are substrates most often used in the study of cellulases. The classification based on the use of these substrates (l,4-/ -D-glucan cellobiohydrolases (CBH), exo-cellulases, Avicelases and... 

Figure 7. Residual activities of papain-digested CBH I (open symbols) and CBH II (closed symbols). The enzymes ( 180 /jM) were incubated (pH 5.0, 25°C) with 0.6 fiM papain (300 1). Aliquots (50 /d) were removed at times indicated to measure the Avicelase activities (A, A) or to measure their activities against a small, chromophoric substrate as described in the text (CNPL in the case of CBH I, -o- MeUmbGs in the case of CBH II, - -). Residual activities are given as percent of the original.

Another method which may become a useful technique for selective inactivation of cellulases in enzyme mixtures is the use of selective heat inactivation. While establishing the thermostability properties of crude xylanases from a fungal strain Y-94, Mitsuishi et al. (80) observed differential heat labilities of the cellulase and xylanase activities in the culture filtrate. After an incubation period of 20 minutes at 65°C, the xylanase activity was reduced by 5-10% whereas the Avicelase and /3-glucosidase activities were reduced by 100% and 60%, respectively. We have observed a similar temperature dependency of xylanase and cellulase activities in T. auranti-acus. As indicated in Figure 2, treatment of the culture filtrate at 70°C for 20 minutes resulted in less than a 5% loss in xylanase activity whereas cellulase activities were reduced by 40-50%. A similar effect has also been observed for the xylanases and cellulase enzymes produced in culture filtrates from T. harzianum (93). Further work in the area of heat treatments may improve the effectiveness of cellulase inactivation. Since the cellulase activities of some enzyme preparations can be more rapidly inactivated on... 

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. 

Table III. Molecular Weight, Carbohydrate Content, and the Ratio of Avicelase to CMCase Activity...

Component Molecular Weight Carbohydrate Content as Glucose (%) Avicelase CMCase... 

Avicelase and CMCase activities were measured for 1-hr and 30-min incubations, respectively. 

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... 

The categories of substrates which are used for assays of cellulase enzymes are shown in Table I. The use of crystalline, insoluble forms of cellulose as substrates makes assays difficult and has led to such trivial names as Avicelase activity. These assays are useful as indications of the capacity of an enzyme system to degrade native cellulose and indicate the presence of CBH enzyme which cannot be assayed in the presence of endoglucanases or / -glucosidase. The susceptibility to enzymatic attack generally increases with the hydration of the polymer chains that accom-... 

Additions° glucosidase canase Avicelase milliunits/mL supemate Protein mg/mL supemate... 

Figure 9. Enzyme production by T. reesei QM 9414 incubated in the presence of ImM sophorose. The incubation medium (27) included 17mM potassium phosphate buffer, pH 6.0, at 28°C. The appearance of aryl-p-D-glucosidase activity (A) in the extracellular medium is delayed in comparison to endoglucanase activity (O) and Avicelase activity ( ).

Sprucewood holocellulose was treated with an endo-p-1,4-mannanase isolated from Aspergillus niger and an endo-/3-1,4-xylanase, two avicelases, and a cellobiohydrolase C isolated from Trichoderma viride. The mannanase hydrolyzed about a quarter of the mannan in 2-3 days without xylan or cellulose degradation. The xylanase hydrolyzed about half the xylan with 10% mannan solubilization. The three cellulases hydrolyzed up to 45% of the cellulose and 20% of the xylan, accompanied by 40-70% solubilization of the mannan. Combined xylanase-mannanase treatment hydrolyzed about half the xylan and mannan. Addition of mannanase to to cellulose-treated samples increased the degradation of the cellulose and mannan. Micromorphological studies of the variously treated specimens revealed a loss of substances in P/Slf T, and adjacent zones of S2 of the tracheid wall. 

In the present work, one of the endo-/M,4-xylanase (E.C. 3.2.1.8), the endo-/ -l,4-mannanase (E.C. 3.2.1.78), and the avicelases used in the former experiments with beechwood holocellulose (10,11) were applied to sprucewood holocellulose in order to obtain a better understanding of the individual and combined actions of these enzymes on the complex carbohydrate skeleton of wood. The experiments could be conducted only with a limited number of samples therefore, the figures given in this chapter have to be considered as preliminary results. 

Enzymes. The mannanase was isolated from Aspergillus niger (11) fraction 4 b was used throughout the experiments (cf. Table 1 in (11)). The xylanase 2, the avicelase 1, and the avicelase 2 were isolated from Trichoderma viride (10). The properties of these enzymes have also been described in the papers cited above. The cellobiohydrolase C was kindly supplied by Dr. R. D. Brown, Jr., and Dr. E. K. Gum, Jr. (Virginia Polytechnic Institute, Blacksburg). The isolation (from Trichoderma viride) and the properties of the cellobiohydrolase C are described in their 1974 paper (12). 

Avicelases or Cellobiohydrolase C. Treatment of sprucewood holocellulose with the three different cellulose-splitting enzymes gave very similar results. The cellulose was hydrolyzed to about 25-45% in 48 hr (Table I). Cellobiose was the predominant reaction product, but the amount of glucose increased considerably with incubation time (Table II). Acid hydrolysis of the reaction solutions showed that higher-... 

Avicelases or Cellobiohydrolase C - - Mannanase. The action of mannanase on delignified sprucewood holocelluloses that were treated with avicelase 1, avicelase 2, or cellobiohydrolase C for 48 hr gave similar degradation patterns. The rate of total degradation increased rapidly when the mannanase was added the rate was most pronounced in the case of avicelase 1 (Figure 4). 

Addition of mannanase resulted in high amounts of mannobiose and mannose. The amount of mannobiose relative to mannose was somewhat higher than in the case of the combined xylanase-mannanase treatment (Table II). The ratios of mannose. -galactose in the acid hydrolysates of the reaction solutions at the end of the experiment were 1 0.05 for the two avicelases and 1 0.03 for the cellobiohydrolase C. 

Avicelases or Cellobiohydrolase C. In holocellulose treated with an avicelase or with cellobiohydrolase C, the degradation of the tracheid wall is very intense. This is mostly concentrated in the Si and adjacent zones of S2 as well as in the tertiary wall region (Figure 9). In the middle part... 

Figure 9. Avicelase-1-treated early-wood tracheid with degradation especially in Si and partially in T. Scale = 1 pm.

Avicelase or Cellobiohydrolase + Mannanase. The treatment of spruce holocellulose first with avicelase or cellobiohydrolase and subsequently with mannanase effected an overall intensive degradation of... 

Figure 10. Avicelase-treated early -wood tracheid wall evincing partial dissolution of wall substance in S2 near a pit chamber. Scale = 1 fim.

Figure 13. Avicelase 1 + mannan-ase treatment. Degradation of S2 in an earlywood tracheid snowing lamellar aggregation pattern. Scale = 1 fim.

The properties of the enzymes used in this study have been described in former publications (10,11,15). Important for the following interpretation are their hydrolytic specificities. The xylanase did not hydrolyze either isolated mannans or celluloses—or only to a very small extent (10). The same is true for the mannanase with respect to xylans and celluloses (11,15). The avicelases, which were not purified to the same extent as the xylanase and mannanase, did not hydrolyze mannans, but they degraded xylans besides crystalline cellulose (10). Also, the highly purified cellobiohydrolase C (12) degraded xylan to some extent (Dr. E. K. Gum, Jr., personal communication). 

The catalytic action of the two avicelases and the cellobiohydrolase C seems at least to be different from those of the xylanases isolated from Trichoderma viride i.e., the degradation products have a higher degree of polymerization even after prolonged incubation and, in the case of sprucewood holocellulose, no arabinose is liberated. 

This chapter deals with three aspects of the cellulolytic enzyme system of Thermoactinomyces sp. the location of the CM-cellulase, Avicelase, and / -glucosidase (cellobiase) activities in the culture, the multiplicity of the extracellular enzyme system, and the stability of the different activities as a function of pH, temperature, and time. The results are discussed with reference to saccharification of cellulosic materials. 

Extracellular Cellulolytic Activities. The appearance of the CM-cellulase activity in a culture of Thermoactinomyces grown on 1% microcrystalline cellulose is shown in Figure 2. The extracellular CM-cellulase activity approached a maximum of 14-16 mg reducing sugar (RS) mL-1 min"1 within 18-24 hr. The Avicelase activity of the culture filtrate developed simultaneously with the CM-cellulase activity and amounted to 3 mg RS mL"1 hr"1. The extracellular protein concentration reached 1.7 mg/mL in the stationary phase (6). 

The CM-cellulase activity of the solids fraction shows a skewed curve over the period of 4-24 hr with a maximum of 3 mg RS mL"1 min"1 around 8 hr, at which point it makes up about 50% of the activity in the whole culture broth (Figure 2). No activity could be detected in the solids fraction in the late stationary growth phase. Within experimental error, the CMC activity of the culture filtrate plus that of the culture solids equals the activity of the whole broth. Similarly, it was found for Thermoactinomyces, strain MJ0r, grown on 0.5% microcrystalline cellulose, that there was a lag before an appearance of extracellular cellulolytic activity, as compared with the activity in the whole culture broth (4). In a culture of Thermoactinomyces, strain YX, the CM-cellulase activity can be desorbed readily by washing the solids fraction with water. These wash fractions also show Avicelase activity (6). This result, and the fact... 

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