Thermoactinomyces

Cellulolytic bacteria can be found which produce only cell-bound cellulase such as Cytophaga (12), only cell-free cellulase, such as CelMbrio vulgaris (21), Bacillus sp. (22), Clostridium sp. (23), Acetivibrio cellulofyticus (24), and Thermoactinomyces (25,26), and both cell-bound and cell-free cellulase such as Pseudomonas (27), Bacteroides succinogenes (28), and CelMbrio futvus (29). However, the location of cellulase in bacteria is also dependent upon the environments in which the bacteria are grown and the age of the culture (29,27). 

Zabriski and colleagues 145, 46] first used culture fluorescence as an on-line estimate of viable biomass during the batch cultivation of Saccharomyces cere-visiae, a species of Streptomyces, and a species of Thermoactinomyces. They simply linearized the fluorescence to biomass data in order to find a direct function between NADH-dependent culture fluorescence and biomass concentration in the bioreactor. In the following years several other authors reported - on the basis of these results - on the estimation of biomass concentration from culture fluorescence data as shown in Table 1. 

Figure 9. Growth of Thermoactinomyces on 1% cellohiose (55°C, 500-mL shake flask)...

Figure 1. Cellulose degradation, cell growth and extracellular filter-paper activity in a culture of Thermoactinomyces sp., strain YX. 40 L batch fermentor, 55°C, pH 7.2 (5).

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. 

Location and Development of Cellulolytic Activities in a Culture of Thermoactinomyces sp. 

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).

Figure 5. Analytical isoelectric focusing in the pH range 3.5-9.5 (Am-pholine PAG Plate, LKB-Produkter AB, Bromma, Sweden) of filtrate samples from a culture of Thermoactinomyces sp. Separation carried out for 1.5 hr at a constant power of 1 W/cm (30). Protein concentration 0.5 mg mL 1 50 pJL applied with glass filter paper (Whatman GF/A) (6).

Figure 5 also shows two 10-hr samples, 10a and 10b. Sample 10a was stored in solution at 4°C for one week, while sample 10b was stored frozen and then thawed immediately before application to the polyacrylamide gel. Both samples show the same protein band pattern. If proteolytic enzymes in the culture filtrate had acted on and partially degraded the extracellular proteins, a different band pattern would have been expected. Thus no product-precursor relationship appeared to exist between the various extracellular proteins in a culture filtrate of Thermoactinomyces. Moreover, it seems as if this organism produces at least three different extracellular cellulolytic enzymes simultaneously. 

Stability of Cellulolytic Activities of Thermoactinomyces with Respect to pH and Temperature... 

Figure 6. pH-activity profile of cellulolytic enzyme activities from Thermoactinomyces sp. under assay conditions. (Q) CM-cellulase, incubation time 10 min (A) Avicelase, incubation time 20 min (O) /3-glu-cosidase, incubation time 30 min. 

Figure 7. Temperature-activity profile for cellulolytic activities from Thermoactinomyces sp. under assay conditions. Same symbols and conditions as Figure 6.

The CM-cellulase activity in a culture filtrate of Thermoactinomyces is stable over a wide pH range at elevated temperatures (Figure 8). At 55°C and 60°C, the pH range studied does not influence the stability, and only about 15% of the activity is lost over 24 hr at 60°C. At 65°C, a pH of 7.3 destabilizes the activity more than a pH in the range of 6.0-6.6. In this latter pH range, 60% of the CM-cellulase activity is retained over 24 hr. 

The stability of the -glucosidase activity in the whole culture broth of Thermoactinomyces was studied at 55°C and 60°C (Figure 10). The destabilizing effect of a pH of 7.3 is even more pronounced for this... 

Figure 8. Stability of CM-cellulase activity in culture filtrate from Thermoactinomyces sp. ( ) pH 6.0 (O) pH 6.6 (A) pH 7.3 filled symbols 55°C half open symbols 60°C open symbols 65°C.

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

Despite the fact that the / -glucosidase is sensitive to high temperatures, the cellulolytic enzyme system of Thermoactinomyces has several advantages that have to be considered in the choice of an enzyme system for saccharifications. 

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