Cellulase fractionation

The E-3 peak was high in Avicelase activity and in protein content as compared with CMCase activity. This peak was further fractionated on a Bio-gel P-100 column five protein peaks (E-3-1 to E-3-5) were obtained, of which E-3-2 peak was highest among them in Avicelase activity and protein content. The elution patterns are shown in Figure 3, and the time course of hydrolysis of CMC by these cellulase fractions measured by a decrease in the viscosity is shown in Figure 4. Randomness of them is in the order of E-3-5 < E-3-2 < E-3-1 E-3-4 E-3-3. The E-3-2 fraction was subjected to further purification on a CM-Sephadex C-50 column because E-3-5 was very low in the Avicelase activity. 

Specific Adsorption on Different Cellulose Columns. Purification of cellulolytic enzymes, including the Ci enzyme, has been carried out by Li et al. (32) by specific adsorption. By passing the crude enzyme through a column of Avicel the Ci fraction was retained by the column while 95% of the cellulase activity could be eluted. The cellulase fraction was then further purified by passing a column of alkali-swollen cellulose where the cellulase was retained. The enzyme could, however, be eluted by buffer in a high yield from the column. The adsorption on an alkali-swollen cellulose column was then repeated. The yield of enzyme obtained with the specific adsorption method is surprisingly high if compared with the yield on Sephadex and on polyacrylamide gel columns. 

Figure 4. Content of galacturonic acid in the soiuble fraction after hydrolysis of AIS with polygalacturonases [a], with polygalacturonases and pectin methylesterase [b], with pectinases and cellulases [c].

Figure 2. Enzyme activities in analytical QM anion exchange column fractions detected with substrates selected for cellulases. Major peaks are numbered. (Reproduced with permission from ref. 9. Copyright 1990 Society of Fermentation Technology, Japan.)...

Three laccase preparations (I, II and III) were isolated from the racellular culture medium of Coriolus versicolor by consecutive fractionation through Sephadex G50 and DEAE Sephadex A25 (84). The laccase III preparation at pH 4.0 reduced the apparent molecular weight of a lignin-derived fraction that had been obtained by eluting the water-soluble extract of a cellulase treated ezomatsu wood residue through... 

Elimination of Cellulases from Xylanases. Classical methods of protein fractionation can be used for to separate cellulases and xylanases on a large scale only when they differ considerably in molecular weight or isoelectric point. The Tricho-derma harzianum enzymes were separated by ultrafiltration because the xylanase was smaller and passed through the membrane into the ultrafiltrate 18). Fractional precipitation with organic solvents is another possibility (7). 

Figure 2. Zymogram of gCenA (A-I) and ngCenA (J-Q) after incubation with C. fimi protease. Cellulases, bound to Avicel, were incubated with protease or control buffer for 72 hr at 30° C, then centrifuged to give cellulose-bound (A-E, J-N) and supernatant (F-I, O-Q) fractions. Products were separated on a SDS gel, replicated onto CMC-agarose and developed with Congo red. A,J. buffer control (4°C incubation) B,F,K,0, protease C,G,L,P, protease + PMSF control D,H,M,Q, buffer control E,I,N, buffer + PMSF control.

One of the favored organisms for study of cellulolysis by Trichoderma is T. reesei. Consequently, many mutant strains which hyperproduce cellulase have been obtained by treatment with ultraviolet light, gamma irradiation, the linear accelerator, diethyl sulphate and N-methyl-N -nitro-N-nitroso-guanidine (7). Whereas much of the study of T. reesei has been with cellulose as substrate, it is relevant to consider the other fractions of natural lignocelluloses hemicellulose and holocellulose (the combined cellulose and hemicellulose fraction). 

The following experiments were conducted using fraction C-l-A, which was thought to be representative of the LCC s (17). The acidic LCC fraction, C-l-A, was partially hydrolyzed with a cellulase preparation, Cellu-losin AC, and changes in the lignin and sugar distributions were analyzed... 

Fractionation Data and Distribution Analysis of the HEC Before Hydrolysis. The results of the fractionation of HEC not subjected to cellulase attack are given in Table I. It appeared that the distribution of these fractionation data could be described by the Lansing-Kraemer distribution (39), also known as the logarithmic normal distribution, i.e. ... 

Fractionation Data and Distribution Analysis of HEC After One Hour of Cellulase Attack. The results of the gel chromatographic separation of HEC after one hour of enzymic hydrolysis are given in Table II. These fractionation data did not correspond to any of the distribution functions mentioned by Peebles (41) and by Tung (42). In the middle of the distribution it corresponded to the Lansing-Kraemer distribution functions, but deviations occurred at the low- and high-molecular-weight ends. 

Fractionation Data and Distribution Analysis of HEC After One Day of Cellulase Attack. In Table III, the fractionation data of HEC are given after one day of enzymic hydrolysis. As after one hour of enzymic hydrolysis, no theoretical distribution function accorded well with the fractionation data, but we evaluated the parameters by numerical analysis, using the Gauss-Laguerre method (43,44). This method has one advantage over other numerical methods, e.g., (45)—all the calculations involved can be done manually without the need of high-speed computers. 

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. 

The most intensive fractionation studies have been performed on the cellulase elaborated by T. koningii (5,6,9,16,26,27) and T. viride (8,10, 28-32). In a typical separation, normally by chromatography on DEAE-Sephadex, three protein peaks are obtained (Figure 1). The first and second contain Cx Ci is in the third. Cx and / -glucosidase are separated by chromatography on SE-Sephadex and Ci is purified by repeated chromatography on DEAE-Sephadex and by isoelectric focusing. 

Five endoglucanases have been isolated from S. pulverulentum cellu-lase (23), four from T. viride Onozuka cellulase (37), and three from T. viride Meicelase (31), but there are many other reports of the fractionation of multiple Cx components. P. funiculosum cellulase contains three and possibly four endoglucanases (Figure 7), while we have reported T. koningii cellulase to contain five (17) and recently six (46). 

Fractionation and Purification of Ex-1 Cellulase Component from Driselase. Driselase powder (50g) was extracted with several aliquots of water and the precipitate formed upon salting out with ammonium sulfate (on a saturation between 20% and 80%) was fractionated on a DEAE-Sephadex A-50 column. Each fraction was tested for -glucosi-dase, xylanase, CMCase, Avicelase activities, and protein content. The elution patterns are shown in Figures 1 and 2. 

Previously, both in our laboratory and elsewhere, cellulases subjected to purification procedures were obtained from commercial sources (5,6, 8,9,10,13,39,46). Three cellobiases and several endoglucanases and cellobiohydrolases from commercial preparations were purified in our laboratory. While use of protease inhibitors in the fractionation procedures minimized proteolysis during enzyme purification, the existence of enzymes proteolytically modified, presumedly during prolonged fermentation (required for obtaining high titres for commercial production), was a source of confusion, as previously explained. Therefore, we prepared T. reesei cellulase harvested from young culture broth. This was used to carry out the enzyme purification procedures described below. 

The development of the sequential elution methods makes it possible not only to cleanly fractionate the three cellulase components, but to do the fractionation with very little loss of enzyme. The total recovery of major enzyme components, summarized in Table III, is considerably higher than those reported previously by other researchers (8,9). Table III also gives the molecular weights of the three enzyme components. 

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... 

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