Purification and physicochemical properties of - amylase from cockroach, Periplaneta americana (LINNAEUS), for starches saccharification

  • EA Dué
  • JPEN Kouadio
  • HT Kouakou
  • S Dabonné
  • SL Niamké
  • LP Kouamé
Keywords: -Amylase, Periplaneta americana, cockroach, enzyme purification, saccharification, maltopentaose.

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 ìmol/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.
Section
Articles

eISSN: 1684-5315