Avicelase activity

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.

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. 

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-... 

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 ( ).

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... 

Figure 4. Preparative isoelectric focusing in a granulated gel of a desalted lyophilized culture filtrate (50 mg protein) from the late exponential growth phase of Thermoactinomyces sp. Separation carried out for 48 hr at a constant power of 8 W (29). (C>) CM-cellulase activity (A) Avicelase activity (6).

The Avicelase activity is less stable than the CM-cellulase activity (Figure 9), and, similar to the CM-cellulase activity, the stability is lowest at pH 7.3. The stability of the activity at 55°C over 24 hr is only marginally affected. At 60°C, 50% of the activity is left after 24 hr, and at 65°C, the half-life of the enzyme activity is 3-4 hr. 

Figure 9. Stability of Avicelase activity in culture filtrate from Thermoactinomyces sp. Same symbols as Figure 8.

The enzyme system is compartmentalized into extracellular CM-cellulase and Avicelase activities and cell-associated / -glucosidase activity, which permits optimal proportions of these activities to be used in saccharifications. 

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