Purification and characterization of an enoyl-coA hydratase encoded by the yhaR gene from Bacillus subtilis strain 168
- UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
- Michael A. Maybin (Creator)
- Institution
- The University of North Carolina at Greensboro (UNCG )
- Web Site: http://library.uncg.edu/
- Advisor
- Jason Reddick
Abstract: 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 mmg 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 high performance liquid chromatography and liquid chromatography coupled to mass spectrometry 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 for the reaction products 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.
Purification and characterization of an enoyl-coA hydratase encoded by the yhaR gene from Bacillus subtilis strain 168
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Created on 5/1/2015
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Additional Information
- Publication
- Thesis
- Language: English
- Date: 2015
- Keywords
- Bacillus Subtilis, Crotonase, Mmg operon, YhaR
- Subjects
- Bacillus subtilis $x Genetics
- Bacillus subtilis $x Metabolism