Cellulase detection

Total pectinase, cellulase and lipase activities secreted by colonies were detected on BSM plates containing respectively 1% of citrus pectin, 2% Walseth cellulose and 1% olive oil + rhodamine. After few days at 30°C, pectin plates were covered by 1% CTAB for Ihour, positive colonies became surrounded by a clear halo walseth plates are not stained the halo is visible directly on positive clones lipase activity is revealed under UV on oil-rhodamine plates. 

Both digester systems exhibit extremely low levels of detectable cellulase activities (exoglucanase, endoglucanase, and -glucosidase) when compared to industrial saccharifying processes (See Table III) in which the hydrolysis of cellulose in the feedstock is optimized with respect to enzyme loading. Therefore, the data indicate the level of improvement that may be made to attain maximum rates for cellulose hydrolysis in the anaerobic reactor system. 

Figure 1. Enzyme activities in crude extracts made from cultures of different ages detected with substrates selected for cellulases, hemicellulases, amylases, and pectinases. Cultures were grown at 22 C. Black bars indicate activity for 105-day-old cultures given a 12-day-long cold shock at 5 C starting on day 90. (Reproduced with permission from ref. 8. Copyright 1985 American Society for Microbiology.)...

Figure 1. Analytical isoelectric focusing of cellulases from Trichodtrma ree-sei. Detection of CBH I and EG I activities using MeUmbLac, in the absence (A) and presence (B) of 10 mM cellobiose. Lane 1, EG I lane 2, EG I (iso-components) lane 3, CBH I (pi 3.9 component) lane 4, EG I-CBH I mixture). Gels were flooded with the fluorogenic substrate (pH 5.0) and after 5-10 min (room temperature) photographed (Polaroid 57, green filter) on a long wavelength UV-transilluminator (8).

Remove both the Suba-Seal and polythene cap and place the filter tube in the rubber cone adapter (42 x 27 mm) used with the adapter funnel attached to the Buchner flask, or other suitable device. Wash any particles from the polythene cap into the filter tube, apply just sufficient suction to remove the gamanase-cellulase solution, then wash the residual undigested fibre with hot distilled water (approximately 80°C). Finally wash well with acetone, leave to air dry in a fume cupboard, and when no smell of acetone can be detected, dry in an oven overnight at 100°C ( 2°C). Cool in a desiccator and weigh the filter tube plus residue. 

Loewenberg and Chapman (40), working with a different strain of the same organism, found that there was a 6-hr lag before any CM-cellulase (measured viscosimetrically) could be detected in the extracellular medium, whereas Nisizawa et al. (27) reported a lag period of... 

The cellobiase activity in culture filtrates of T. reesei was small relative to that of cellobiohydrolase and endoglucanase. The possibility that cellobiase of T. reesei is either an intracellular or membrane-bound enzyme was indicated by experiments in which cellobiose or other carbon sources were used as the substrate for culture growth. While cellobiose can be taken up rapidly by the fungus, very little cellulase activity could be detected in the filtrate see Table IV—cellobiose as carbon source). Furthermore, the appearance of cellobiase did not parallel the appearance of cellulase activity. Increasing culture incubation time did, however, result in increasing cellobiase activity in the filtrate. This data suggested that at least some of the cellobiase present in the filtrate might have been an intracellular cellobiase which was, perhaps, released when some of... 

All naturally occurring fungal strains of Trichoderma require an inducer for cellulase synthesis. In the absence of an inducer such as cellulose, cellobiose (21,22), or sophorose (12,13,14,23), Trichoderma does not make any detectable cellulase complex enzymes. The true physiological inducer of cellulase is currently unknown. Insoluble cellulose is presumably not such an inducer since there is no way for the internal cell machinery to sense the presence of this insoluble material. However, a small transglycosylation product such as sophorose, 2-0-/ -glucopyranosyl-D-glucose, may well be the natural inducer. 

Only minor amounts of additional reducing sugars from the xylan decomposition were detected in the reaction solutions. Acid hydrolysis showed, however, that higher-molecular xylan fragments were liberated by the combined cellulase-mannanase treatment. At the end of the experi-... 

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... 

Antibodies to BI cellulase failed to detect any of this protein among the translation products of pea mRNA in experiments where BS cellulase was clearly synthesized (II). There are several possible reasons for this failure, including the alternative that no message for BI cellulase exists as such. Structural modifications often occur during processing of extracellular proteins, and these may be so extensive that antibodies to the final form of the protein do not recognize the precursor. Such modifica-... 

In the case of pH strategies III and IV, no remarkable differences in the growth and production rate of cellulases were seen up to h 52 of fermentation (data not shown), after which deactivation of cellulases was detected. 

Kinetics studies were conducted at 55°C in a jacketed batch reactor. Shredded wastepaper (10 g / L) was added to 500 mL or 1L of citrate buffer, pH 4.8, and heated to the assay temperature. A specified quantity of either soluble or immobilized cellulase was added to the reactor to initiate hydrolysis. Samples were collected at regular intervals over 30-60 min, and centrifuged to separate solids. The DNS assay (4) was used to detect sugars formed during hydrolysis experiments. The supernatant from the centrifuge tube and the DNS solution were mixed and cooked for exactly 5 min in boiling water. Finally, the sample was transferred to a methacrylate cuvet, and its absorbance was measured at 540 nm. 

---