(567bz) Lysis Techniques and Reaction Media for a Cell-Free Enzyme System Producing Succinic Acid and Acetoin

The enzyme system belonging to Bacillus licheniformis involved in the conversion from pyruvate to products was studied and its medium for activity optimized. Various lysis techniques including cell disruption by Misonix sonication and lysis by use of a Parr Cell Disruption Vessel were utilized and optimized for extraction of the enzymes from the microbial cell. Production of intermediates and byproducts such as 16mM acetoin and 13mM succinic acid from 87mM pyruvate by the cell-free enzyme system aided in determining the activity of the enzymes extracted, as did optical density measurements. Analysis of products was determined using HPLC with dilute sulfuric acid as the mobile phase. The results of the body of work conclude that the Misonix Sonicator was better suited for sonication of cells and retention of enzyme activity than was the Parr Cell Disruption Vessel. The retention of ADH activity obtained from lysis by the Misonix Sonicator (62%) was twice that of the retention of ADH activity obtained from the Parr Cell Disruption Vessel (30%). Other results include the observation that the cell-free enzymes reacted to form product. This answers the question as to whether or not the enzymes would complete fermentation outside of the cells, as they do inside the cells. The cell-free enzymes formed succinic acid and acetoin and no 2,3-butanediol. Interestingly, the whole cell fermentation produced 2,3-butanediol and no succinic acid.