Starch-zone電気永動ならびにカラムクロマトグラフィによるTrichoderma virideよりの各種cellulase成分の分離

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The present work was carried out for the purpose of finding out any cellulase components with a possible different specificity in Meicelase, a crude cellulase preparation from Trichoderma viride manufactured by Meiji Seika Co., using soluble and insoluble celluloses as substrates.The lyophilized powder of precipitate from a Meicelase solution with ammonium sulfate was subjected to starch-zone electrophoresis in a sodium phosphate buffer of 0.05 ionic strength at pH 6.8 and a veronal buffer of the same ionic strength at pH 8.5. The pattern of CMC-saccharifying activity at pH 6.8 consisted of less resolved three peaks, but that of filter paper-saccharifying activity showed two main peaks. The activity of filter paper-disintegration was resolved into four main peaks, some of which overlap the peaks of filter paper-saccharifying activity to resemble more closely the pattern of the latter than that of CMC-saccharifying activity. The activities of β-glucosidase measured for aryl β-glucosides and cellobiose were separated into two components under these electrophoretic conditions and each component showed a similar substrate specificity toward these substrates.Chromatography of the cellulase preparation on hydroxyapatite column using a gradient elution with phosphate buffers of 0.001 M to 0.4 M at pH 6.0 gave two completely sepatated fraction. THe fraction eluted with a low concentration of the buffer showed much stronger CMCase activity than the other eluted later, whereas reverse relation was found with the filter paper-saccharifying and -disintegrating activities. The latter fraction, therefore, possesses a higher activity toward filter paper but little for CMC.DEAE-Sephadex column chromatography using a gradient elution with a acetate buffers of 0.02 M to 1 M at pH 4.0 gave several peaks for any of the three activities. The β-glucosidase activity, however, was resolved into two fractions. They merged with each of the first two out of three major peaks of CMCase activity, but the last peak of CMCase was completely separated from the β-glucosidase activity. The first eluting fraction of CMCase, on the contrary, contained practically no filter paper-disintegrating activity. Thus, some peaks of an activity measured by a single method merged with some peaks of the other while the other peaks of them were mutually more or less overlapped or completely separated. However, the pattern of filter paper-disintegrating activity as a whole resembled, to some extent, that of saccharifying one, whereas that of CMCase activity was very different from them. From this result and the fact that decrease in the degree of polymerization of filter paper after complete disintegration by a crude cellulase preparation was approximately fifteen per cent of the ouginal and loss in weight of the substrate also was of the same order as this percentage, the disintegration of filter paper seemed to be accomplished by the collaboration of hydrolytic action of more than one component belonging to a less random type.A considerable low recovery of filter paper-saccharifying as well as -disintegrating activity was found upon starch-zone electrophoresis and column chromatography, whreas the recovery of protein and the CMCase activity was very high upon the same resolution procedures. The fact suggests the existence of a synergestic effect of each component for the hydrolysis of filter paper by crude enzyme materials.
社団法人日本生物工学会の論文
1964-02-15

Starch-zone電気永動ならびにカラムクロマトグラフィによるTrichoderma virideよりの各種cellulase成分の分離

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