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Hydroxylation of naphthalene by Pseudomonas putida in the presence of organic solvents.

Harrop, Anthony John; (1990) Hydroxylation of naphthalene by Pseudomonas putida in the presence of organic solvents. Doctoral thesis (Ph.D.), University College London. Green open access

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Abstract

The operation of two-liquid phase bioreactors is a relatively new technology by which compounds only sparingly soluble in water can be dissolved to high concentrations in water-immiscible organic solvents so as to bring about bioconversion in an efficient manner. There are many potential advantages of operating in the presence of a second phase, however, there are also drawbacks, including the possible inactivation of the biocatalyst by the organic solvent. With the development of this new technology there comes an increasing need to understand how the catalyst interacts with its environment during the biotransformation and in particular the effect of the potentially toxic organic phase on catalytic activity and stability. This thesis describes the design of a model system involving the microbial hydroxylation of naphthalene by the Gram-negative bacterium Pseudomonas putida in the presence of a selection of organic solvents to determine biocatalyst-solvent interactions and their effects upon biocatalyst catalytic activity. Optimisation studies for the production of toluene dioxygenase, the enzyme responsible for the hydroxylation of many aromatic compounds including naphthalene, were performed for the constitutive mutant P. putida UV4 in order to define the enzyme production process. A correlation between solvent polarity and biocatalyst activity has been demonstrated, polar solvents appearing more toxic. These findings when compared to the steroid Δ1-dehydrogenase system of Arthrobacter simplex a Gram-positive bacterium, for the same solvents, show a striking quantitative difference in product formation rates; P. putida being able to bring about biotransformation in a wider range of polar organic solvents. This thesis details the biocatalyst-solvent interactions of P. putida UV4 including the effects of solvent exposure upon membrane permeability, cell viability and motility. The role of the organic-aqueous liquid-liquid interface in biocatalyst inactivation is also considered. This Gram-positive - Gram-negative difference is discussed in relation to the differences in cell wall make up. It is proposed that the presence of the outer membrane in Gram-negative bacteria shields the organism from some of the potentially toxic effects of the organic solvents employed. This Gram-positive - Gram-negative difference reactor productivity is likely to be a key consideration in the selection of biocatalysts for biotransformations in the presence of organic solvents. It is likely that the method of biocatalyst growth will alter operational stability, this along with strain development and design are also discussed including outlines for future work.

Type: Thesis (Doctoral)
Qualification: Ph.D.
Title: Hydroxylation of naphthalene by Pseudomonas putida in the presence of organic solvents.
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by Proquest
URI: https://discovery.ucl.ac.uk/id/eprint/10109354
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