Purification and characterization of amylase from roots of Paederia foetida Linn

The root extract of Paederia foetida Linn. has been traditionally utilized for the improvement of taste and texture of various foods in Thailand. To identify a factor for the improvement, we performed biochemical analyses. Enzyme assay and zymographic method revealed that there is amylolytic activity in the root extract. This and SDS-polyacrylamide gel electrophoresis (PAGE) analyses suggest that an enzyme exhibiting amylolytic activity is a dominant protein in the extracts. By using DEAE-column chromatography, the amylase was purified to homogeneity, having a molecular mass on SDS-PAGE of 60 kDa. The purified enzyme showed pH- and temperature-optimum activities at 7.0 and at 50 °C, respectively. The enzyme activity was found to be stable in the pH and temperature ranges of 6.0-7.5 and of 30-60 °C, respectively, and was inhibited completely by the addition of Hg2+ and Cu2+ and partially by Fe3+. The amylase was active on starch>dextrin>amylopectin>glycogen>β-limit dextrin, but was inactive on pullulan and starch azure. HPLC analysis of starch hydrolysate by the enzyme showed maltose as a main product with no detectable glucose. The Km value for starch of the purified enzyme was determined to be 2.7±0.24 mg ml−1. Taken together, it is suggested that an enzyme responsible for amylolytic activity in the root extracts of Paederia foetida Linn. is β-amylase.