Purification and characterization of a new thermoalkaliphilic pectate lyase from Actinomadura keratinilytica Cpt20

Abstract

This study was carried out to investigate the purification and biochemical characterization of a new extracellular alkaliphilic and thermostable pectate lyase (Pel-20) isolated from Actinomadura keratinilytica strain Cpt20. Pure protein was obtained after sequential chromatographies on a fast performance liquid chromatography (FPLC) and high performance liquid chromatography (HPLC) columns. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme was a monomer with a molecular mass of 34125.11-Da. The enzyme had an NH2-terminal sequence of GFATNQGGTTGGAGGTLS, thus, sharing high homology with actinomycetes pectate lyase family. The results showed that this enzyme was completely inhibited by EDTA, which supports its belonging to the pectate lyase superfamily. It showed optimum activity at pH 10.5 and 70°C. The thermoactivity and thermostability of Pel-20 were enhanced in the presence of 1mM Ca2+. Its half-life times at 70, 80, 90, and 100°C were 18, 12, 7, and 2h, respectively. Its kinetic parameters, Km and Vmax values were 0.45mM and 21,700U/mg, respectively. Low-esterified pectin was the optimum substrate for the Pel-20. However, higher-esterified pectin was also weakly cleaved. Overall, the alkaliphilicity and thermostability properties of Pel-20 make it a potential candidate for future application in industrial bioprocesses.