Endocellulase activity

Figure 1. Induction of xylanases in Trichoderma harzianum. The ratios of xylanase to endocellulase activities were determined in the culture filtrates of T. harzianum grown on steam treated aspenwood. Aspen chips were steam treated from 20 to 240 s at 240° C to produce a series of samples with a variable content of xylan and cellulose. The specific content of these carbohydrates were expressed as the ratio of xylan to glucan. Enzyme activities were determined on culture filtrates of 300 mL batch cultures of T. harzianum grown on these wood samples at a loading of 1% (w/v) as described by Saddler and Mes-Hartree (66). Enzyme activities were also determined in culture filtrates of T. harzianum grown on Avicel, Solka Floe and oat spelts xylan.

Figure 2. Heat inactivation profile of the xylanase in the culture filtrate from Thermoascus aurantiacus. Xylanase enzyme was prepared as described by Tan et al. (74). Xylanase and endocellulase activities were determined after the enzyme was incubated for the specified time at 70°C. Aliquots were removed and assayed at 50°C by the method described by Yu et al. (75). Activities were expressed as a percentage of the control stored at 4°C.

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. 

Different water-soluble cellulose derivatives have been used for the determination of endocellulase activities. Carboxymethylcellulose (CMC) has been proposed as substrate and recently Almin et al. (3) have improved their method for the calculation of endocellulase activities, using a medium-molecular-weight CMC (Mv = 299,000 and Mw = 142,000) with well-defined physicochemical properties. No corrections... 

Absolute Viscosimetric Method for the Determination of Endocellulase Activities. Experimental Setup of the Viscosimetric Measurements. The substrate, buffer, and enzyme solutions are prepared in the same manner as described in the section on light scattering. However, no clarification of the substrate solution by pressure filtration is needed. 

The final concentration of the buffered HEC was about 2.5 X 10"3 g/mL and the enzyme concentration about 3 X 10 4 FIP units/mL. The FIP (F6d6xation Internationale Pharmaceutique) unit is a unit of endocellulase activity (49) calculated from capillary viscosimetric measurements. 

Calculation of the Endocellulase Activity from the Intrinsic Viscosity Values. The enzymic degradation of polymeric substrates can occur at different bonds in the same substrate molecule, and the enzymic activity has to be defined here as the initial number of moles of glyco-sidic bonds split per second (53). This definition corresponds to the definition of the katal, symbolyzed kat. This unit is defined as the catalytic amount of any catalyst (including any enzyme) that catalyzes a reaction rate of one mole per second in an assay system (54), and it is recommended by the International Union of Pure and Applied Chemistry (55) for the quantitative evaluation of catalytic activities. 

This study describes an absolute method for the evaluation of endo-/ -glucanase (Cx) or endocellulase activities in the cellulase complex without the need of a time-consuming isolation of the endocellulase fractions. In the method proposed by Almin et al. (3), an assumption was needed in the theoretical derivation of endocellulase activities, namely, that Mv/Mn is constant in the initial stages of the enzymic reactions. We found, however, a linear relationship between Mv and STn. They used CMC as substrate, and since the mode of action of endocellu-lases on CMC can be regarded as similar to that on HEC, it is possible that their assumption is not fulfilled. 

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