Comparative evaluation of different cell disruption methods for the release of hydrogenase from H_2-producing bacterium E.harbinenase YUAN-3~T

A comparative evaluation of three different cell-disruption methods for the release of hydrogenase from H 2-producing bacterium E.harbinenase YUAN-3 T was investigated.The cell disruption techniques evaluated in this study were ultrasonication,high-speed homogenization and bead milling.Ultrasonication process was found to be the most effective method in terms of cell disruption.As for the specific activity of hydrogenase,there is no significant difference among the three kinds of methods.An orthogonal experiment L 9 (3 4) was designed to optimize the procedures of ultrasonication for cell disruption.The optimized ultrasonication disruption conditions were the treatment at 250 W,20 kHz,30 s /15 s and 0.30 g bacteria cell (dry weight) in 15 mL suspension buffer.As a result,the optimized conditions allow the hydrogenase to maintain the active form with the yield of 93.95 mg protein /g cell and the final activity of 0.252 μmol /min /mg protein.In this work,we have developed and optimized an ultrasonication protocol for YUAN-3 T cells,which is adapted to laboratoryscale release of hydrogenase proteins.