Analysis of the Enzyme System Responsible for 2, 3-Butanediol Production by Bacillus Licheniformis

Bacillus licheniformis is one of the top microbial producers of 2, 3–butanediol. This alcohol has been used extensively as antifreeze, converted to diacetyl for use as a food additive, among other uses of which consideration as a fuel additive has become increasingly popular. The enzyme system belonging to B. licheniformis involved in the conversion from pyruvate to 2, 3-butanediol was studied and its minimal medium for activity optimized. Various lysis techniques including cell disruption by sonication and lysis by use of a pressure bomb were utilized and optimized for extraction of the enzymes from the microbial cell. The microorganism was harvested after growth of varying time periods in a complex medium and lysis was completed by one of the aforementioned procedures. Production of intermediates such as acetoin and diacetyl, in addition to 2, 3-butanediol by the cell-free enzyme system reacting in a minimal buffer medium 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. Additional studies including enzyme purification and enzyme activity stabilization were performed as well as an analysis of the enzyme kinetics.