Heat treatment purification of thermostable cellulase and hemicellulase enzymes expressed in E. coli

Four enzymes implicated in the breakdown of cellulose and hemicellulose by the extremely thermophilic anaerobe Caldocellum saccharolyticum have been purified 7–19-fold in a single heat-treatment step. In earlier studies, C. saccharolyticum genes expressing either carboxymethyl cellulase (CMCase), β-glucosidase (E.C.3.2.1.21), xylanase (E.C.3.2.1.8), or β-xylosidase (E.C.3.2.1.37) activity had been cloned into Escherichia coli, and these clones served as enzyme sources for purification experiments. After cell lysis by a nonionic detergent-osmotic shock-lysozyme method and/or sonication, the lysate was heated to 70°C for 30–65 min, resulting in the denaturation and precipitation of most E. coli proteins. Centrifugation of the heat-treated lysates produced supernatants containing at least 50% of the original enzyme activity, with a 7–23-fold decrease in protein concentration. The method has general application as a means of obtaining thermostable enzymes free of contaminating activities, and also achieves a rapid and simple partial purification.