Extracellular cellulolytic activities

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... 

The extracellular cellulolytic activities, CM-cellulase and Avicelase, are stable over 24 hr at 55°C in the pH range of 6.0-7.3, which suggests that no enzymatic activity would be lost in a saccharification under these conditions and that the enzymes would be recoverable. 

The strains were cultured on Mandels medium + 1% citrus pectin for 5 days and the enzymatic activities of culture filtrates were determined on three substrates citrus pectin, polygalacturonic acid and filter paper, (a) extracellular proteins are in p.g/ml. (b) p>ectinolytic activities on pectin (PC) and on polygalacturonic acid (TO) and Pectin esterase (PE) are in units/ml. (c) total cellulolytic activity (filter paper, fp) are in mg of liberated reducing sugars/ml. 

Fig. 1. Cellulolytic and xylanolytic activities and extracellular protein during time course of the cultivation of P. brasilianum IBT 20888. (A) Cellulolytic activities on cellulose (B) cellulolytic activities on xylan (C) xylanase activities on cellulose and (D) xylanolytic activities on xylan. ( ) BG ( ) CBH ( T ) EG ( O ) BX ( O ) AF ( V ) EX (+) extracellular protein ( —) cultivation on cellulose and (----) cultivation on xylan.

When cultivated in a liquid medium with cellulose, Chaetomium thermophile produced extracellular cellulolytic enzymes. Ion-exchange chromatography and gel filtration gave three electrophoretically pure components a typical Cx endo-enzyme (pi 4.6, mol. wt. 4.1 x 10 ) active on carboxymethylcellulose in solution but of low effect on cellulose a Ci exo-enzyme (p7 4.6, mol. wt. 6.7 X 10 ) active on cellulose but hardly on carboxymethylcellulose and a... 

H. Murata, J. L. McEvoy, A. Chatterjee, A. Collmer, A. K. Chatterjee. Molecular cloning of an aepA gene that activates production of extracellular pectolytic, cellulolytic, and proteolytic enzymes in Erwinia carotovora subsp. carotovora. Mol. Plant Micr. Interact. 4 239 (1991). 

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. 

This is a preliminary report of our work. Our results to date indicate that cellulolytic enzymes may be found extracellularly, both in the culture medium and attached to the surface of hyphae, and, perhaps, intracellu-larly. In addition, they may be found in active and inactive forms in all places. The binding of the enzymes to the mycelium and the natural release mechanism is unknown. In order to investigate induction and repression mechanisms and the influence of chemical and environmental factors on the synthesis of cellulolytic enzymes, total enzyme activity must be measured. Further work along these lines is in progress. 

All the strains were found to utilize cellulose and produce extracellular cellulase and xylanase enzymes. F. oxysporum 841 was found to be a potential mould for direct conversion of cellulose to ethanol/acetic acid. Though other species, e.g. F. oxysporum 2018, 62287 and 62291 also produced comparable amounts of cellulase and xylanase enzymes, only traces of ethanol and acetic acid were detected in non-aerated cultures. Maximiun cellulase activity was observed after 96 h of bioconversion process. F. oxysporum VTT-D-80134, however, was not found to produce sufficient cellulolytic and xylanolytic activities to convert cellulose or hemicellulose directly into ethanol [81]. 

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