Abstract
The purification of a thermostable Caldocellum saccharolyticum β-glucosidase expressed in Escherichia coli was investigated using heat precipitation of unclarified cell homogenates. Heat treatment at 70 °C was capable of purification with respect to cell debris, small particulates and the majority of cell protein, although E. coli proteins were even more efficiently removed at 80 °C and above. For thermostable proteins expressed in E. coli, a precipitation temperature of 80 °C or greater is recommended for optimal removal of contaminant proteins. In small-scale heating trials, heating rate was found to influence enzyme yield significantly. Losses were minimised when ‘flash-heating’ was employed. The successful single-step removal of particulates, labile protein and nucleic acids was achieved by simultaneous heat-treatment and polyethyleneimine addition, although the purification achieved was additive rather than synergistic.
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