Molecular weight of cellulase

An important parameter influencing the mode of action of cellulases is the accessibility of the cellulose to the enzymes. The molecular weights of cellulases range between 30 and 80 kDa. A comparison of the size of cellulase (3-8 nm) and the pore size of cotton swollen in water (1-7 nm) shows very clearly that cellulases can penetrate the cellulose to a limited extent only. In addition, the enzyme reaction takes place preferentially on amorphous cellulose because the more compact, crystalline cellulose structures do not offer any space for such macromolecules. Thus - provided of enzyme and process parameters have been selected correctly -cellulases act mainly on the textile surface. In this way interesting effects on cellu-losic fibers can be achieved. 

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... 

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. 

Physical Properties. All of the cellulase (CMCase) activity which develops in auxin-treated pea apices dissolves in salt solutions (e.g., phosphate buffer, 20mM, pH 6.2, containing 1M NaCl). Gel chromatography of such extracts indicates the presence of two cellulase components with similar levels of activity and elution volumes corresponding to molecular weights of about 20,000 and 70,000 (Figure 1). If the tissue is extracted with buffer alone, only the smaller cellulase dissolves (referred to as buffer-soluble or BS cellulase). The larger buffer-insoluble (BI) cellulase can then be extracted from the residue by salt solutions. This simple extraction procedure effectively separates the two cellulases, and can be used as an initial step for their estimation or purification. 

Figure 1. Elution profiles of cellulase activity from Sephadex G-100 gel chromatographs of crude extracts of auxin-treated pea apices. BS cellulose activity has an elution volume corresponding to a molecular weight of 20,000. BI cellulase activity dissolves in 1M NaCl and elutes with a molecular weight of 70,000. These values correspond to those observed for purified cellulases (3), indicating that the enzymes were not altered in molecular weight during purification, and could be effectively separated by differential extraction.

According to the International Union of Biochemistry, one enzyme international unit (IU) was defined as the enzyme strength which can catalyze 1 jumole of substrate per minute. In describing the activity of the cellulase, one IU is equivalent to the strength to release 1 /rmoles of glucose per minute, because the molecular weight of the substrate, cellulose polymer, is not well defined. 

In 1972 Ogawa and Toyama (56) purified three components— A-I-a, A-I-b, and A-II-1—which were adsorbed on a gauze column during purification from Cellulase Onozuka P1500, a commercial preparation of T. viride cellulase. These three components had molecular weights of 32,000, 48,000, and 48,000 as determined by gel filtration and contained 7-16% carbohydrate. Each is reported to carry out the random hydrolysis of CM-cellulose and to degrade hydrocellulose (Avicel) and cellooligosaccharides except for cellobiose. The order of reactivity toward either cotton or Avicel was A-II-1 > A-I-b > A-I-a. The proteins adsorbed on cellulose comprised 38% of the total cellulase protein. 

At the end of the fermentation, protein is separated from cell mass by filtration, typically with a rotary vacuum filter. The crude enzyme concentration is often lower than suitable for commercial applications, so the concentration of enzyme is increased by ultrafiltration. Most cellulase enzymes have a molecular weight of 25,000 to 75,000 and are retained by ultrafiltration membranes of 5000 molecular weight cutoff. The membranes permit the passage of low molecular weight salts, sugars and other impurities, and are sometimes operated in a diafiltration mode to increase the purity of the enzymes. The crude broth at this point is dark brown. 

From Sephadex G-10 and G-15 gel filtration, the molecular weight of CF was calculated as about 700 daltons (16). To obtain further information about the chemical nature of CF, the active CF fraction, isolated after Sephadex G-10 gel filtration, was treated with different enzymes. Enzymes proteinase K, cellulase, a and / amylase did not reduce the CF activity (data not shown). 

Porcine pancreas lipase (PPL) pol5mierized 3-hydroxybutyric and 12-hydroxydodecanoic acids in anhydrons hydrophobic solvents (79). The molecular weight of the polymer from the former was low (ca 500), whereas the polymerization of the latter at 75°C prodnced the polymer with molecnlar weight of 3x10 . A cellulase-assisted pol5mierization of chiral flnorinated compound having carboxylic acid and phenolic groups produced the aromatic polyester (72). 

Since cellulases are commercially available, we looked at this reaction further. A number of several commercial cellulases were screened for their ability to reduce the Brookfield viscosity of xanthan solution. Mannosidase and glucosidase were also used in combination with cellulase with the hope of exposing the cellulose backbone by removing the side chains. All of the cellulases tested were found to be somewhat active toward r ucing the molecular weight of xanthan gum (Table 3). 

Table 3. Determination of Molecular Weight and Subunit Molecular Weight of Purified 3-glucosidases, Cellulases and the Sugar Content. 

The metabolite 5 was incubated with cellulase and a new peak corresponding to 4 appeared via concomitant decrease of the peak due to 5. LC-APCI-MS analysis showed that the molecular weight of 5 was 521. With the methylated 5, introduction of the two methyl groups were shown by a MS peak at mtz 548 [M-H]". When 5 was acetylated, a MS peak at mtz 730 [M-H] was observed. In order to estimate the functional groups of 5 in detail, the successive derivatization (methylation followed by acetylation) was conducted. LC-APCI-... 

Molecular weight Most of the cellulases that show chitosanolytic activity have the apparent molecular mass in the range of 23-55kDa, which is consistent with that of specific chitosanases. But there are also few exceptions such as the molecular weights of those from T. viride [29, 56] and T. reesei PC-3-7[57] are 66kDa and 97kDa, respectively. 

Figure 14. Change (solid line) in apparent molecular weight distribution of water-soluble extract from a cellulase-treated ezomatsu wood residue (dotted line) at pH 4.0 brought about by the laccase HI preparation from C. versicolor, Sephadex GIO/H2O elution profiles adapted and redrawn from reference 85.

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). 

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