Cloning, purification and biochemical properties of a thermostable pectinase from Bacillus halodurans M29

Abstract

An M29 strain that can grow under highly alkaline conditions from 40 °C to 65 °C was isolated and identified as Bacillus halodurans. The isolate was a Gram-positive, spore-forming, aerobic, and alkaliphilic bacterium. A pectinase was cloned from M29 and expressed in Escherichia coli JM109 (DE3). A 39 kDa protein with pectinase activity was purified by heat treatment and with DEAE-Sepharose Fast Flow from culture supernatant to gel electrophoretic homogeneity. Optimal activity was achieved at pH 10 and 80 °C. The purified enzyme was stable from pH 9.5 to 10.5 and had a 1 h half-life at 80 °C. Kinetic experiments at 80 °C with polygalacturonic acid as substrate revealed Km and Vmax values of 4.1 g L−1 and 351 U mg−1 protein, respectively. The pectinase from B. halodurans showed high thermostability and may be a valuable candidate enzyme in bioscouring.