Irpex lacteus の2種セルラーゼの純化とその性質

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Two cellulase components, tentatively named CMCase and Avicelase, were obtained from a culture filtrate of Irpex lacteus (Polyporus tulipiferae) grown on a synthetic medium by precipitation with ammonium sufate and by subsequent column chromatography. A cellulase preparation was fractionated from a concentrated culture filtrate of this fungus with ammonium sulfate between 20 and 80% saturation. This cellulase preparation was subjected to DEAE-Sephadex A-50 column chromatography. Of the four peaks of cellulase resolved, one eluted with a 0.3〜0.4M acetate buffer at pH 4.0 showed appreciably high CMC-and Avicelsaccharifying activitiesm but it was contaminated with high α-amylase and β-glucosidase activities. Therefore, this fraction was rechromatographed under the same conditions, and two cellulase peaks were obtained, One of them was still contaminated with β-glucosidase and α-amylase, and this fraction was further subjected to the same column chromatography.Of the two cellulase peaks obtained, one showed appreciably high CMC-and Avicel-saccharifying activities and it was practically free from α-amylase but contaminated with a reduced quantity of β-glucosidase. This cellulase fraction was further purified by a gel filtration on Sephadex G-100,and two peaks of cellulase free from β-glucosidase were obtained. The cellulase component first eluted showed a higher saccharifying action on CMC than on Avicel, namely 26.28 and 3.57 on CMC and Avicel respectively. In contract, the one secondly eluted showed the cellulase activities in reverse relation, its specific actions on CMC and Avicel being 2.30 and 13.15,respectively. Moreover, the two cellulase components hydrolyzed cellooligosaccharides from cellotriose to cellohexose, but the rate of their hydrolysis by CMCase was much greater than that by Avicelase. Thus, they showed both so-called C_1-and C_x-cellulase activities, although the proportion of these activities varied from each other.These two cellulase components showed similarly a simgle peak on electrophoresis in a cellulose acetate film and ultracentrifuge analysis. Their sedimentation coefficientw were 2.14 and 0.47 S. The molecular weights calculated therefrom were approximately 35,600 and 7,700,respectively. The amino acid composition of the two cellulases were equally rich in aspartic acid, threonine, serine, glutamic acid. However, they were poor in basic and sulphur-containing amino acid. They also contained approximately 10% carbohydrate which was composed of glucose, mannose, xylose, galactosamine and glucosamine in order of their amounts.It is particularly noteworthy that a light molecular weight of Avicelase seems to be very important for this enzyme to attach native cellulose and other highly crystalline cellulosic substrates in contrast to CMCase, which has a larger molecular size; Irpex lacteus seems to attack cellulosic substrates more efficiently through the synergistic action of these two cellulases having different specificities.
社団法人日本生物工学会の論文
1970-10-25

Irpex lacteus の2種セルラーゼの純化とその性質

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