Extraction, Purification and Characterization of Linamarase from Cassava Root Parenchyma of the High-cyanogen Cultivar KU-50

Abstract

Linamarin from cassava peel KU-50 cultivar was extracted and purified by 0.25 M chilled H2SO4and two steps in high-performance liquid chromatography (HPLC) using NH2 Lichrospher 100 column(size 4 x 250 mm) and 70% (v/v) and 80% (v/v) acetonitrile as the mobile phase, respectively. Then, it wasdetected the purity by thin layer chromatography (TLC). Crude linamarase was extracted from cassava rootparenchyma with buffer A [0.1 M phosphate buffer pH 6.0 containing 1% polyvinylpyrrolidone K30 (PVP)],buffer B [0.1 M phosphate buffer pH 6.0 containing 1% PVP and 1% Triton X-100], buffer C [0.1 Mphosphate buffer pH 6.0 containing 1% PVP and 1% Tween 20] and buffer D [0.1 M phosphate buffer pH6.0 containing 1% PVP and 1% Tween 80]. The results showed that buffer C is the highest ability forextraction of linamarase from cassava parenchyma tissue. The linamarase was then precipitated and purifiedby ammonium sulfate precipitation and DEAE-Toyopearl column, respectively. The molecular weightof the purified linamarase was estimated to be about 65 kDa. The optimum pH and temperature for linamarasewere pH 7.0 and 50 °C, respectively. The purified linamarase was stable in the broad pH range of 4.0 to 7.0and 30 to 60 °C. Ethylenediaminetetraacetic acid (EDTA) was not affect slightly on the activity of thepurified enzyme. However, linamarase activity was increased with Na+, Mn2+, Ca2+, Zn2+ and Mg2+ while itwas strongly inhibited by Fe3+ and Cd2+.

Keywords : Cassava Root KU-50 cultivar / Extraction of Linamarase / Hydrophobicity /Purification of Linamarase / Linamarin