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

An M29 strain that can grow under highly alkaline conditions from 40 degrees C to 65 degrees 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 degrees C. The purified enzyme was stable from pH 9.5 to 10.5 and had a 1 h half-life at 80 degrees C. Kinetic experiments at 80 degrees C with polygalacturonic acid as substrate revealed K-m and V-max 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. (c) 2013 The Authors. Published by Elsevier B.V. All rights reserved.