Mutagenic studies into the catalytic versatility of soluble methane monoxygenase

• Main Author: Nichol, Tim
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2011
• Subjects: Geochemistry; Soil sciences
• ISBN: 0355431114; 9780355431117

Soil samples from the surface and subsurface of the floodplain of the River Rother were analysed for selected heavy metal concentrations at 12 locations along the river in order to discover whether the high concentrations, reported in previous studies from 1982 to 2002, had changed. The aim was to review the levels of heavy metal contamination along the floodplain of the Rother. Thirty-seven soil samples were taken along the River floodplain, at different sites and from different depths, collected in two phases. Samples collected, were dried, ground, digested and analysed by Atomic Absorption (AA) and Inductively Coupled Plasma (ICP) techniques.While the results from up-stream sites were mainly satisfactory, according to the New Dutch List Standard and Kelly Indices, the results obtained from downstream sites were variable probably due to a flood event (November 2002) and/or earlier tipping of wastes. Previous research identified a major heavy metal contamination plume within the sediments along the Rother. By 1995 the plume appears to have divided with one part migrating faster and therefore separating from the second (Duty, 1995). This is further supported by the evidence presented for lead and zinc (Figure 6.1). The present study confirms the two plumes at Renishaw and at Catcliffe indicating a further migration even over the short timescale.Natural hydrological cleansing processes will continue to operate but may accelerate with increases in periods of heavy rainfall causing more flood episodes to occur. Even though there is metal contamination it is not having a deleterious effect on the biodiversity present along the river corridor.Recommendations for reclamation are presented for the purpose of cleaning up the contamination and reusing the derelict land in some meaningful way. The areas of heavy contamination have been subject to major clean-up processes involving not only the removal or burial of the metals but re-landscaping the sites to make them suitable for active recreation. Examples of such reclamation have been completed at Poolsbrook and Rother Valley Country Park and are still being carried out at the present time at the Avenue site and at Orgreave.The implications for the future use of the river corridor as part of the leisure and tourism scene look very good as the worst sites are reclaimed and the others are given over to passive recreation and conservation.If such actions are taken then all areas of the floodplain have the potential for re-use at some future date when the river corridor, at surface level, would be clean and restored to a tranquil fluvial environment with floristically rich riverside meadows interspersed with sites of active water-based recreation.Soluble methane monooxygenase (sMMO) is a multicomponent bacterial enzyme that catalyzes the oxidation of methane to methanol, as well as oxidizing many other adventitious substrates. A number of mutagenic studies were carried out on the sMMO enzyme of Methylosinus trichosporium OB3b in order to gain insight into sMMO and probe how structural aspects relate to function of the enzyme. Leu110 within the hydroxylase a-subunit of sMMO has been proposed as a possible gating residue, controlling access of substrate to the active site (Rosenzweig et al. 1997). A range of site directed mutants were created at the 110 position and screened for activity with a number of aromatic substrates. All mutants showed relaxed regioselectivity with all substrates assayed. However no evidence of a gating residue was found, indicating that Leu110 is more important in determining regioselectivity than substrate access to the active site.Comparison to the highly similar butane monooxygenase led to the creation of three site directed mutants: M184V F282L and C151T. M184V and C151T showed small changes in regioselectivity and reduced activity with most substrates. The M184V mutant showed relaxed regioselectivity and a novel oxidation product with the substrate mesitylene which may have implications for substrate trafficking. The F282L mutant produced a stable enzyme which had no activity with any of the substrates tested, showing Phe282 is important for the enzyme function.A random mutagenesis experiment was devised and a colorimetric screen for the oxidation of triaromatic compounds was used to screen mutant libraries for activity towards anthracene and phenanthrene. However no activity towards triaromatic compounds was detected. In order to improve the cloning strategies and to make creation of mutant libraries easier, a novel expression vector pT2ML was created. The pT2ML vector reduces the number of cloning steps required to make soluble methane monooxygenase mutants. This expression system was used to make a site directed mutants F188Aand N116G in order to complement previous site directed mutant studies, as well as a recombinant wild type mutant in order to asses the activity of the new expression system which is comparible to the wild type enzyme.