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Michael Maybin

Posted on April 1, 2015


Date - April 1, 2015
12:00 am - 2:00 pm


MS Thesis – 602
Title: “Purification and characterization of an enoyl-CoA hydratase encoded by the yhaR gene from Bacillus subtilis strain 168”
Advisor Dr. Reddick


Bacillus subtilis is widely used as a model organism for the study of sporulation. While its genome has been sequenced the function of many encoded proteins have not been investigated. The mother cell metabolic gene (mmg) operon is an example that contains mmgA, mmgB, and mmgC which all encode for proteins in a fatty acid degradation pathway, however the pathway is incomplete. YhaR is a candidate gene which encodes for a putative hydratase that may help complete the fatty acid degradation pathway. The yhaR gene was cloned in Escherichia coli and the protein was overproduced and purified to a yield of 8 mg per L of culture. The activity of the purified enzyme was tested by HPLC and LC-MS analysis, using butenoyl-CoA as the substrate. These experiments revealed that the enzyme was active in producing 3- hydroxybutyryl-CoA, with a pH optimum of 6.5. LC-MS data from the product peak displayed the expected 852 m/z for the 3-hydroxybutyryl-CoA product. Kinetics experiments revealed that at 37 oC and pH 6.5 the enzyme has a kcat of 6.1 X 10-3 ± 9.2 X 10-4 s -1 . Butenoyl-CoA substrate was found to have a Km of 0.14 ± 0.03 mM.