2 research outputs found
Extraction and partial purification of Aspergillus flavus cell wall associated saponin hydrolase
In spite of the importance of saponin hydrolase (SH) enzyme, in the production
of biologically active compounds from natural saponins, it is surprising that many aspects of its
nature are unknown. The results of the present work revealed that Aspergillus flavus was capable
of expressing three SH forms; extracellular, intracellular and cell wall-bound forms. SH cell
bound enzyme constituted to more than 75% of the total enzymatic activity in the production
medium. The sequential extraction process of SH cell bound enzyme revealed that 47.5% of SH
was cytosolic and the rest (52.5%) was associated with the cell wall. The highest SH extraction
yield was achieved when 0.25 M Tris-HCl lysis buffer supplemented with 1% Triton X-100 for
24 h at 4-25 °C and pH 8 were applied. Under these optimized conditions, A. flavus SH yield
increased from 23.6 to 85.83%. The partial purification was achieved by applying successively
acetone precipitation, lyophilization, dialysis, and anion exchange chromatography on Fractogel
EMD DEAE-650S to the extract. The specific activity of the enzyme extract was 0.27 U/mg after
75% acetone fractionation, while that after anion exchange chromatography was 0.65 U/mg
protein. The final enzyme preparation was 7.3-fold purer than the crude extract