Abstract
An -amylase was purified from the American cockroach Periplaneta americana (Linnaeus) to homogeneity by four steps purification via ammonium sulphate crude extract precipitation, Sephacryl S-100 HR gel permeation chromatography, anion exchange chromatography on DEAE-Sepharose CL-6B and hydrophobic interaction chromatography on phenyl Sepharose CL-4B. The purification was approximatively 38.42 fold with a 24.31% yield. Optimums pH and temperature of the purified -amylase were found to be 5.6 and 55°C, respectively. The enzyme was stable up to 55°C and its pH stability was in range of 5.6 - 6.6. The KM and Vmax of the enzyme with soluble starch as substrate were 5 mg/ml and 100 imol/min/mg, respectively, and the energy of activation (Ea), was 50.32 Kj/mol. The -amylase was inhibited by Tris, Fe3+, Ba2+, Mo+ and EDTA. While Ca2+, K+, Cu2+, Mg2+ and para-hydroxymercuribenzoate (pHMB) activated the enzyme. Analysis of the amylolytic reaction products by HPLC showed the presence of maltose and maltodextrin but not glucose in the starch hydrolysate (2 h of reaction). This result indicated that the amylolytic enzyme of P. americana is an -amylase (an endoamylase). The purified -amylase hydrolysed maltopentose, maltohexose and maltoheptose. Maltose, maltotriose and maltotetrose were not hydrolysed by this enzyme. Therefore, the purified -amylase is active only on substrates with more than four residues of glucose.
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