Biochemical Characterization of Alpha-amylase from the Digestive Fluid of Larvae of the Rhino Beetle, Oryctes owariensis
Keywords:Oryctes owariensis, alpha-amylase, Enzyme, monomeric protein, Energy production, Starch saccharification
Alpha-amylase was purified from the digestive fluid of larvae of the Rhino Beetle (Oryctes owariensis) by chromatography on ion-exchanges and hydrophobic interaction columns. The preparation was shown to be homogeneous on polyacrylamide gel. The enzyme was purified 260.20-fold to a specific activity of 128.50 (U/ mg of protein) and an overall yield of 26.22 %. SDS-PAGE revealed a single polypeptide of 53.8 Â± 1.2 kDa, thus indicating apparent homogeneity of the final enzyme preparation. Gel filtration chromatography showed that the enzyme was a 50.4 Â± 2.2 kDa monomeric protein. The purified enzyme exhibited pH and temperature optima at 7.0 and 55Â°C, respectively. The alpha-amylase was stable at 37 Â°C and its pH stability was in the range of 6.6-7.6. This enzyme exhibited a high affinity towards soluble starch with Km values of 0.6 Â±0.04. The alpha-amylase activities were stimulated by Ca2+, K+, Mg2+ and Na+ and were inhibited by EDTA, Fe2+ and Cu2+. The analysis of hydrolytic products after soluble starch hydrolysis by alpha-amylase revealed that maltose, maltotriose and maltotetraose were the major products. This indicated that this enzyme can be classified as the alpha-amylase (the endoamylase). From a physiological point of view, alpha-amylase play a fundamental role in energy production for this insect larvae. Consequently, the digestive tract of this larva could be a good source of alpha-amylase for starch saccharification.
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