Team:NYMU-Taipei/ymim2

Results

In the figure above, it showed that most nitrates were converted into ammonia. And the productivity of nitrogen was quite low.

After 1440 min(1 day), only 10-12 mM of nitrogen were produced by an E.coli. But if we combined the cell growth of E.coli with the equation, the result would be different.

This figure showed us the relationship between different enzyme concentration and time.

Even though the concentration of nitrogen we produced was low, but the toxicity of ammonia was much lower than the NOx produced by the industrial factories. It might still be a good way to apply our pathway to eliminate the toxic NOx compounds.

Enzyme	Substrate	Kcat (min-1)	Km(mM)	Organism	Reference
Nitrate Reductase (Nap)	Nitrate	3480	0.112	Paracoccus pantotrophus	(1)
Nitrite Reductase (Nir)	Nitrite	480	0.053	Pseudomonas aeruginosa	(2)
Nitric Oxide Reductase (Nor)	Nitric Oxide	12660	0.06	Fusarium oxysporum	(3)
Nitrous Oxide Reductase (Nos)	Nitrous Oxide	16800	0.0056	Paracoccus denitrificans	(4)

Enzyme	Substrate	Vmax (mM /min-1)	Km(mM)	Organism	Reference
Nitrate Reductase (Nap)	Nitrate	27.885	0.47	Escherichia coli	(5)
Nitrite Reductase (Nfr)	Nitrite	46.14	0.022	Escherichia coli	(6)
Nitrite Reductase (Nir)	Nitrite	0.465	0.4	Escherichia coli	(7)

Parameter	Description	Value	Units	Reference
	Copy Number in E.coli	300	Copy Number per Cell	Part Registry
	Ptrc Promoter Strength	0.00003156	Polymerase per min	(8)(9)(10)
	Degradation Rate of mRNA in E.coli	0.102	min-1	(11)
	Degradation Rate of Enzyme in E.coli	0.0348	min-1	(11)
	Translation rate in E.coli	720~1260	Amino Acids/min	(12)
	Avogadaros' Number	6.02*	mmole-1	N/A
	Cell Volume of E.coli	6.5*	liter	(13)
	Translation rate of Nap	0.763	Number of mRNA/min	Estimated
	Translation rate of Nir	0.96	Number of mRNA/min	Estimated
	Translation rate of Nor	1.668	Number of mRNA/min	Estimated
	Translation rate of Nos	1.602	Number of mRNA/min	Estimated

[1]Andrew J. GATES, David J. RICHARDSON, Julea N. BUTT(2008) Voltammetric characterization of the aerobic energy-dissipating nitrate reductase of Paracoccus pantotrophus : exploring the activity of a redox-balancing enzyme as a f unction of electrochemical potential. Biochem. J. (2008) 409, 159–168

[2]Serena Rinaldo, Francesca Cutruzzolà(2007) Nitrite Reductases in Denitrification. Biology of the Nitrogen Cycle 37-55

[3]Naoki Toritsuka, Hirofumi Shoun, Udai P. Singh, Sam-Yong Park , Tetsutaro Iizuka, Yoshitsugu Shiro(1997) Functional and structural comparison of nitric oxide reductases from denitrifying fungi Cylindrocarpon tonkinense and Fusarium oxysporum. Biochimica et Biophysica Acta 1338 1997 93–99

[4]Stuart W. Snyder, Thomas C. Hollocher(1987) Purification and some characteristics of  nitrous oxide reductase from Paracoccus denitrificans. The Journal of Biology Chemistry VOl. 262, No.14. Issue of May 15. , pp. 6515-6525

[5] Robert A. LAZZARINI, Daniel E. Atkinson(1961) A Triphosphopyridine Nucleotide-specific Nitrite Reductase from Escherichia coli. The Journal of Biology Chemistry VOl. 236, No.12. December

[6] Michael W. W. AdamsS, Leonard E. Mortenson(1982) The Effect of Cyanide and Ferricyanide on the Activity of the Dissimilatory Nitrate Reductase of Escherichia coli. The Journal of Biology Chemistry VOl. 257, No.4. Issue of February 25, pp. 1791-1799

[7] Lewis M. Siegel, David C. Rueger, Michael J. Barber, Rick J. Krueger(1982) Escherichia coli Sulfite Reductase Hemoprotein Subuni. The Journal of Biology Chemistry VOl. 257, No.11. Issue of June 10, pp. 6343-6350

[8] Hsin-Ho Huang, Daniel Camsund, Peter Lindblad and Thorsten Heidorn(2010) Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology. Nucleic Acids Research, Vol. 38, No. 8 2577–2593

[9] Michael Brunner, Hermann Bujard(1987) Promoter recognition and promoter strength in the Escherichia coli system. The EMBO Journal vol.6 no.10 pp.3139-3144

[10]Thomas A . Clarke , Paul C. Mills, Susie R. Poock,bJulea N. Butt, Myles R. Cheesman, Jeffrey A. Cole, Jay C. D. Hinton, Andrew M. Hemmings, Gemma Kemp, Christopher A . G . So¨derberg, Stephen Spiro, Jessica Van Wonderen, David J. Richardson(2008)Escherichia coli Cytochrome c Nitrite Reductase NrfA. Methods in Enzymology, Vol. 437 pp.64-77

[11] Kmalendu Nath, Arthur L. Koch(1971) Protein Degradation in Escherichia coli. The Journal of Biology Chemistry VOl. 246, No.22. Issue of November 25, pp. 6956-6967

[12]Thomas A . Clarke , Paul C. Mills, Susie R. Poock,bJulea N. Butt, Myles R. Cheesman, Jeffrey A. Cole, Jay C. D. Hinton, Andrew M. Hemmings, Gemma Kemp, Christopher A . G . So¨derberg, Stephen Spiro, Jessica Van Wonderen, David J. Richardson(2008)Escherichia coli Cytochrome c Nitrite Reductase NrfA. Methods in Enzymology, Vol. 437 pp.64-77

[13] Cell Volume Increase in Escherichia coli after Shifts to Richer Media, H. E. KUBITSCHEK ,Biological and Medical Research Division, Argonne National Laboratory,9700 South Cass Avenue, Argonne, Illinois 60439