Thermoactinomyces activities

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).

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.

Fig. 10. Sequence alignment of subtilisins S41, SSII, S39, BPN, E, Carlsberg, and thermitase. Thermitase is an homologous subtilisin-like protease from the thermophilic bacterium Thermoactinomyces vulgaris. Residues conserved in four or more of the sequences are shaded. The positions of mutations discovered during the directed evolution of the various subtilisins are indicated above the alignment. E-subtilisin E, F-subtilisin S41, S-subtilisin SSII, B-subtilisin BPN. Active site residues are indicated (A).

Thermitase, a thermostable extracellular serine protease from the thermophrhc microorganism Thermoactinomyces vulgaris, with an esterase/protease activity ratio >1000 1 shows a broad amino acid side-chain tolerance and cleaves methyl, ethyl, benzyl, ethox-ybenzyl, and ferf-butyl esters from a variety of Nps-, Boc-, Bpoc-, and Z-protected di- and oligopeptides in high yields at pH 8 and 33-55 °C (Scheme 15 ).[28,29,60-62] jjj addition, it is specific for the a-carboxy groups of Asp and Glu. 

A new cytotoxic substance named mechercharmycin A 164, and its linear congener mechercharmycin B 165 were isolated from marine-derived Thermoactinomyces sp. YM3-25l/ 164 exhibited relatively strong antitumor activity, whereas 165 exhibited almost no such activity. 

PheDH from Thermoactinomyces intermedius ATCC 33 205 was utilized recently to synthesize allysine ethylene acetal [(S)-2-amino-5-(l,3-dioxolan-2-yl)-pentanoic acid (2)] from the corresponding keto acid with regeneration of NAD+ cofactor by FDH/ formate[58 (Fig. 15.3-4) the specific activity towards the keto acid was 16% compared to the standard substrate phenylpyruvate. 

An a-amylase preparation from Thermoactinomyces vulgaris has been used to hydrolyse pullulan to panose. The digest was applied to a carbon-Celite column and eluted with a linear gradient of propan-l-ol from 0 to 5%. From the trisaccharide fractions eluted, panose was prepared in about 70% yield. The production of amylase activity by Thermomonospora curvata has been investigated. ... 

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