Characterization of cyanobacterial fatty aldehyde deformylase and biodegradation of hydrocarbons by S. oneidensis MR-1
2012-01
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Characterization of cyanobacterial fatty aldehyde deformylase and biodegradation of hydrocarbons by S. oneidensis MR-1
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2012-01
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Petroleum-like high-energy compounds are biosynthesized by microorganisms. With increasing interest in biofuels, the mechanisms of hydrocarbon biosynthesis are under intense study. One mechanism of hydrocarbon biosynthesis involves the deformylation of fatty aldehyde to produce alkanes/alkenes. The gene encoding the deformylase enzyme from Nostoc punctiforme PCC 73102 was cloned into pET28b+ vector and transformed into E. coli BL21. The protein was expressed and purified using a Ni-NTA column. The purification process was optimized for the production of stable and active enzyme. The enzyme was characterized by ultraviolet-visible (UV-Vis) spectroscopy, electron paramagnetic spectroscopy (EPR) and inductively coupled plasma-mass spectrometry (ICP-MS). ICP-MS showed that 2 iron atoms were present per subunit of protein. In vitro activity assay was demonstrated using NADPH, spinach ferredoxin reductase, spinach ferredoxin and octadecanal (substrate). The activity was low, 4 nmoles of heptadecane (product) formed per 2 mg/ml protein in 2 hours. Octadecanoic acid, octadecanol and butanal were not the substrates. In an effort to increase activity, a crude lysate from Nostoc punctiforme PCC 73102 was added to reaction mixture, but no increase in activity was observed. Further, studies were carried out to study biodegradation of long chain ketone and alkene by S. oneidensis MR-1. This study was done in order to maximize production of fuel-like molecules produced by S. oneidensis MR-1. Resting cell assay was carried out to study biodegradation. There was no evidence for the biodegradation of 14-heptacosanone and cis-9-tricosene, compounds mimicking those produced by recombinant S. oneidensis MR-1.
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University of Minnesota M.S. thesis. January 2012. Major: Microbial engineering. Advisor: Lawrence P Wackett. 1 computer file (PDF); ix, 64 pages.
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Golwala, Deep. (2012). Characterization of cyanobacterial fatty aldehyde deformylase and biodegradation of hydrocarbons by S. oneidensis MR-1. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/121093.
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