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. 2009 Jun;37(2):121-7.
doi: 10.4489/MYCO.2009.37.2.121. Epub 2009 Jun 30.

Purification and Characterization of Intracellular Cellulase from Aspergillus oryzae ITCC-4857.01

Most Ferdousi Begum  1 , Nurul Absar
Affiliations

Purification and Characterization of Intracellular Cellulase from Aspergillus oryzae ITCC-4857.01

Most Ferdousi Begum et al. Mycobiology. 2009 Jun.

Abstract

Purification and characterization of intracellular cellulase produced by A. oryzae ITCC-4857.01 are reported. The enzyme was purified by ion-exchange chromatography using DEAE-cellulose followed by Gel filtration. The purification achieved was 41 fold from the crude extract with yield of 27%. The purified enzyme showed single band on poly acrylamide gel. The molecular weight as determined by SDS-PAGE and gel filtration was 38 KDa and 38.6 KDa respectively and contained only one subunit. The enzyme is glycoprotien as nature and contained 0.67% neutral sugar. The apparent Km value of the enzyme against cellulose was 0.83%. The enzyme showed the highest relative ativities on CMC followed by avicel, salicin and filter paper. The optimum pH of activity was 5.5 and very slight activity was observed at or above pH 7.5 as well as bellow pH 3.5. The optimum tempreture of the activity was 45℃ and the highest activity was exhibited in 35 to 45℃. The enzyme lost their activities almost completely (95~100%) at 80 ℃ or above and as well as bellow 25℃.

Keywords: Aspergillus; DEAE-cellulose chromatography; Gel filtration; Intracellular cellulase; SDS-PAGE.

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Figures

Fig. 1
Fig. 1
Ion-exchange chromatography of intracellular crude enzyme solution on DEAE cellulose. The crude enzyme solution (35 mg) was applied to the column (2.1 × 35 cm) preequilibrated with 0.2 M sodium acetate buffer pH 5.2 at 4℃ and eluted by stepwise increases of NaCl concentration in the same buffer. Flow rate 36 ml/hour.
Fig. 2
Fig. 2
Gel filtration of F-1 fraction on Sephadex G-75. Fractions F-1 obtained by DEAE-cellulose chromatography was applied to the column (3 × 120 cm) preequilibrated with 0.2M Sodium acetate buffer, pH 5.2 at 4℃ and developed with the same buffer.
Fig. 3
Fig. 3
Disc electrophoretic pattens of different fractions on 7.5% polyacrylamide gel. A, Crude enzyme; B, F-1 (obtained after DEAE-cellulose); C, F-1a (obtained after gel filtration).
Fig. 4
Fig. 4
SDS-polyacrylamide gel electrophoretic pattern of F-1a fraction on 7.5% polyacrylamide gel. A, Abscence of beta-mercaptaethanol; B, Presence of beta-mercaptaethanol.
Fig. 5
Fig. 5
Ultraviolate absorption spectra of intracellular cellulase.
Fig. 6
Fig. 6
Lineweaver-Burk double reciprocal plot for the determination of Km value of purified intracellular cellulase form A. oryzae.
Fig. 7
Fig. 7
Effect of pH on the activities of intracellular cellulases of A. oryzae ITCC-4857.01.
Fig. 8
Fig. 8
Effect of temperature on the activities of intracellular cellulases of A. oryzae ITCC-4857.01.
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