Cellulase activity requirement

Assays for endo-l,4- -glucanase [EC 3.2.1.4] (i.e., CMCase) and saccharifying cellulase (i.e., international filter paper U, IFPU) activities partially followed the methods recommended in the 1987 lUPAC report (65). When even undiluted enzyme samples fail to give the required glucose yield under prescribed assay conditions, the lUPAC committee recommends a less precise method. In the current study, cellulase activities in digester extracts were so low that the CMCU could only be defined as follows one CMC unit of activity was that amount of enzyme required to liberate one Hg glucose from CMC in 60 min. 

Wheat straw. Wheat straw ground to 20 mesh was treated with 2% NaOH solution (wt/vol) in 1 2 (solidiliquid) ratio at 121 C for 0.5 h (i.e., 4 g NaOH/100 g wheat straw). Trichoderma reesei QMY-1 was grown on pretreated wheat straw in SSF as well as in LSF under otherwise identical culture conditions. The SSF was carried out with full nutrient concentrations in one set and with one-half nutrient concentrations in the other set to evaluate the possible deleterious effects of elevated osmotic pressure. T reesei QMY-1 produced FP cellulase of 8.6 lU/ml (430 lU/g cellulose or 172 lU/g substrate) in 22 days. This showed that the organism was able to tolerate the high salt concentrations required in the SSF. In contrast, when the nutrients were supplied in one-half concentration, FP cellulase activity dropped to 6.7 lU/ml (335 lU/g cellulose or 134 lU/g substrate). However, the maximum enzyme activity was obtained one week earlier (14 days) than that obtained with full salt concentrations (Table I). 

Assay of Endocellulase Activity. Cellulase is an enzyme complex a synergistic action between the components is required for a complete hydrolysis of the insoluble cellulose. There is no consensus about the substrate to be used for the cellulase activity measurements. 

Regardless of the assay used, the non-hnearity of cellulase kinetics requires that the enzyme activity be measured based on a fixed level of conversion. 

The determination of glucose is one of the most frequently perfomed routine analyses in clinical chemistry as well as in the microbiological and food industries. Here, the application of glucose electrodes appears to be the method of choice. Moreover, in combination with other enzymes, glucose oxidase sensors are applicable to the measurement of di- and polysaccharides and amylase and cellulase activity, which is required in many biotechnological processes. This versatility explains, why numerous researchers worldwide are concerned with the development and optimization of glucose sensors. 

For the concentration of enzyme solutions it is important to use methods giving the highest possible yield of enzymic activity—i.e., loss of activity should be brought to a minimum. None of the above methods will in all cases fulfill these requirements since denaturation and consequently low yield of enzymic activity are reported for all these methods. To avoid denaturation owing to precipitation as well as owing to freezedrying and evaporation, Petterson et al. (39) introduced the dextran gel concentration method of Flodin et al. (12) into cellulase research. This method was found to give essentially quantitative recovery of cellulase activity from culture solutions of Polyporus versicolor. 

Acceleration of airspace formation is attributed to production of ethylene and increased cellu-lase activity in the tissue (Kawase, 1981). The sequential processes in aerenchyma development are presented by McLeod et al. (1987). They suggest that flooding first results in soil oxygen depletion, followed by depletion of root oxygen. This results in ACC (1-aminocyclopropane-l-carboxylic acid) production that requires ATP. Ethylene is produced from ACC, and this process requires oxygen and is sensitive to temperature. Ethylene produced accelerates cellulase activity that softens tissue, resulting in the formation of aerenchyma tissue. 

Another thermophilic bacterium, Clostridium thermocellum, possesses a distinct cellulase activity that requires the presence of Ca and a reducing agent, and is more active against crystalline cellulose than amorphous cellulose (12). This particular cellulase exists as a cellulosome, an enormous complex that contains at least 15 different proteins (13), all of which have been cloned and sequenced... 

Since protein adsorption to an anion exchange resin is reversible and does not constitute a classical immobilization, the ability of the resins to retain activity under various conditions must be determined. Macrosorb KAX DEAE bound -D-glucosidase was tested with solutions of primary interest for their final application. Several batch washes of a 1% w/v slurry were required to ensure complete equilibrium elution for a given concentration, as determined from the absence of pNPG units in subsequent washes. Several salt solutions of typical fermentation media components were tested. These included 3 mM to 50 mM solutions of MgSO, KHgPO, NaQ, and sodium acetate. Also, incubations with cellulase solutions were tested to determine if the proteins present in a cellulose hydrolysis would displace the -D-glucosidase. Both of these displacement studies were carried out at 22°C and 40 C. 

The scheme proposed above requires microbial colonization of the material and excludes degradation by amylases and cellulases that are present in soils (28), but are not newly synthesized or associated with microbial cells. Active polysaccharide hydrolases are found in nearly all soils, but these enzymes are primarily bound to soil organic matter or mineral components attachment is firm enough to severely limit migration of the enzymes from surrounding soil to the film. 

New materials need to be investigated as possible specific inhibitors to the cellulase enzymes. This research approach will require the screening of chemicals against pure enzyme solutions of known activity. Specificity must be determined using a... 

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