Difference between revisions of "Part:BBa K1398002"

Latest revision as of 18:38, 2 November 2014

NemA (N-ethylmaleimide reductase)

N-ethylmaleimide reductase, created for use as a Trinitroltoluene and Nitroglycerine degrading protein. NemA is a flavoprotein that primarily catalyses the reduction of N-ethylmaleimide (NEM), which is toxic to cell growth. However, it is also involved in the degradation of other toxic compounds for their reuse in nitrogen metabolism. Some of these compounds include PETN, quinones and chromate. It increases the resistance of organisms to the toxic effects of these compounds. This sequence only encodes for the protein, an attached His (x6) Tag to allow for purification and a double-STOP codon. It therefore requires regulatory sequences (promoter/RBS/terminator) to be added for expression. The protein has been codon-optimised for expression in E. coli.

Exeter iGEM 2014

The Exeter 2014 team used Raman spectroscopy to show the breakdown of Nitroglycerin by NemA. The image below shows negative controls proving that NemA cannot perform this catalytic function without cofactors: FMN, NADH and NADPH. The conditions for this activity were room temperature and pressure and a physiologically relevant pH of 7.

The minus protein control is greater than 100% of the starting concentration. This is likely a measurement error whereby the solvent has evaporated whilst focusing the Raman laser increasing the concentration of the compound of interest.

The graph below shows how initial reaction velocity varies with initial concentration of the nitroglycerin in each reaction mixture. The concentrations of substrates and NemA are kept constant.

From this data a Hanes-Woolf plot was produced and allowed the Vmax and Km values to be determined: 6 mM and 21 umol per mg per min respectively.

Full article at: http://2014.igem.org/Team:Exeter/EnzymeValidation

Sequence and Features