Thesis (M. Phil.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 145-174).

An L-haloacid dehalohenase from the psychrophilic marine bacteria Psychromonas ingrahamii has been cloned, over-expressed in a bacterial expression system and biochemically characterised. The enzyme is stable at temperatures of up to 60ºC for 90 min and shows highest activity towards substrates with short carbon chains (≤C3). The enzyme is stable in up to 30% ethanol, methanol and DMSO when incubated for 1 h. The Km for the enzyme is 1.36 mM. The genome of AQP5750 from the Aquapharm Biodiscovery Ltd Microbial library was sequenced. An L-haloacid dehalohenase and a haloalkane dehalogenase gene were identified within the genome. The AQP5750 L-haloacid dehalohenase has been cloned, over-expressed in a bacterial expression system, biochemically characterised, crystallized and the native and crystal complex structure with chloropropionic acid (MCP) determined. The L-haloacid dehalohenase from AQP5750 shows highest activity at 55ºC towards brominated substrates with short carbon chains (≤C3). The enzyme shows increased activity of 150% in 40% DMSO and 123% in 30% methanol. The L-haloacid dehalohenase crystal complex structure with covalently bound MCP confirmed Asp 18 as the main catalytic residue. Residues His 183, Asp 186 and Glu 21 in the active site are proposed to be involved in activation of the catalytic water which attacks the ester intermediate in the second part of the SN2 dehalogenase mechanism. The AQP5750 haloalkane dehalogenase has been cloned, over-expressed in a bacterial expression system, crystallized and the native and complex structure with 1-hexanol has been determined. Substrate specificity experiments showed that the haloalkane dehalogenase from AQP5750 does not show high activity towards substrates used by other haloalkane dehalogenases with high amino acid sequence identity. The large active site cavity and the presence of Ser 176 and Arg 136 in the hydrophobic binding pocket may alter the binding of substrates tested which could account for the low activity observed.

published_or_final_version / Botany / Master / Master of Philosophy

Thesis (M. Phil.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 119-135).

published_or_final_version / abstract / toc / Botany / Master / Master of Philosophy

published_or_final_version / abstract / toc / Botany / Master / Master of Philosophy

published_or_final_version / Botany / Master / Master of Philosophy

The first project is focused on kinetic analysis of two enzymes: Rsc1362 (Ralstonia solanacearum GMI1000) and PA0810 (Pseudomonas aeruginosa PA01). Rsc1362 had a kcat of 504±66 min-1 and a KM of 0.06±0.02 mM, PA0810 had a kcat of 2.6±0.6 min-1 and a KM of 0.44±0.2 mM. A lack of environmental context for a chloroacetate dehalogenase was noted in Pseudomonas aeruginosa PA01. The second project focuses on kinetic and stable isotope fractionation of 1,2- dichloroethane by DhlA (Xanthobacter autotrophicus GJ10), and Jann2620 (Jannaschia CCS1). Although both enzymes had different kinetics (DhlA: KM = 4.8±0.6 mM and kcat = 133±8 min-1, Jann2620: KM = 25.9±2.3 mM and kcat = ~1.7 min-1), they fractionated similarly (ε values of -33.9‰ and -32.9‰ for DhlA and Jann2620, respectively). As calculated AKIE values were similar to the expected values of an abiotic reaction, it was determined that neither enzyme masks the intrinsic fractionation.

The first project is focused on kinetic analysis of two enzymes: Rsc1362 (Ralstonia solanacearum GMI1000) and PA0810 (Pseudomonas aeruginosa PA01). Rsc1362 had a kcat of 504±66 min-1 and a KM of 0.06±0.02 mM, PA0810 had a kcat of 2.6±0.6 min-1 and a KM of 0.44±0.2 mM. A lack of environmental context for a chloroacetate dehalogenase was noted in Pseudomonas aeruginosa PA01. The second project focuses on kinetic and stable isotope fractionation of 1,2- dichloroethane by DhlA (Xanthobacter autotrophicus GJ10), and Jann2620 (Jannaschia CCS1). Although both enzymes had different kinetics (DhlA: KM = 4.8±0.6 mM and kcat = 133±8 min-1, Jann2620: KM = 25.9±2.3 mM and kcat = ~1.7 min-1), they fractionated similarly (ε values of -33.9‰ and -32.9‰ for DhlA and Jann2620, respectively). As calculated AKIE values were similar to the expected values of an abiotic reaction, it was determined that neither enzyme masks the intrinsic fractionation.

Thesis (M. Phil.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.