Quantitative Analysis of Free D-threonines: A Novel Exploitation of L-threionine Aldolase

In biological systems the majority of amino acids consist of the stereoisomer L-amino acids, but there is also the presence of D-amino acids which have a different stereochemistry. The main purpose of this experiment is to isolate the threonine D-amino acids by using L-threonine aldolase (LTA) enzyme to catalyze the reaction of L-threonine and leave D-threonine and D-allo threonine to be isolated by Mass Spectrophotometry (MS/MS). To isolate D-threonine and D-allo threonine the LTA enzyme from E. coli must be isolated. To do this the E.coli were transformed and had a plasmid inserted into its genome to produce enzyme expression. Once enzyme was expressed purification of the enzyme LTA took place along with its refolding from solution and kinetics to determine the protein\u27s concentration. The protein expression proved successful from PCR results that showed LTA enzyme gene expression, SDS-PAGE analysis showed that positive LTA expression as well. The activity of the enzyme was measured using Beer-Lambert\u27s law which showed tho the reaction occurred in a 5mM L-threonine substrate solution, and a mouse plasma solution, which both showed activity