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Title: The physiology, biochemistry and genetics of propane metabolism in Rhodococcus rhodochrous PNKb1
Author: Mihdhir, Alaa
ISNI:       0000 0001 3397 9647
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1993
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The metabolism of propane by R.rhodochrous PNKbl has been investigated using various techniques. SDS-PAGE of cell-free extracts of R.rhodochrous PNKbl grown on propane and some of the potential oxidation intermediates in the metabolism of propane showed the synthesis of three specific polypeptides of approximately 69, 59 and 57 kDa. A putative propane oxygenase activity was detected in cell-free extracts only by the formation of 1,2-epoxypropane from propene. Propane was shown to be a competitive inhibitor of propene oxidation activity. Activity of the putative propane oxygenase was only observed in propane and acetol-grown cells. The effect of various inhibitors on the formation of 1,2-epoxypropane from propene in whole cells of propane and acetol-grown R.rhodochrous PNKbl was investigated. This showed that the epoxide formation was inhibited by the same inhibitors under both conditions, suggesting a relationship between the metabolism of propane and acetol. Acetol monooxygenase (AMO) activity was also detected in acetol-grown cells. This enzyme was partially purified using gel filtration and ion-exchange chromatography. However, neither NAD+ nor NADP+ -dependent acetol dehydrogenase activity was measured in cell-free extracts of acetol-grown cells, indicating that acetol is not metabolized via a pyruvate pathway. NAD+- dependent secondary alcohol dehydrogenase activity in cell-free extracts of propane and propan-2-ol-grown cells was shown to be higher than the primary alcohol dehydrogenase. 1,2-Propanediol dehydrogenase activity in 1,2- propanediol-grown cells was six-fold higher than the corresponding activity obtained in propane-grown cells, indicating that there is more than one alcohol dehydrogenase involved in the metabolism of propane. NAD+-dependent secondary alcohol dehydrogenase was synthesized in cell-free extracts of propane grown-cells. This enzyme was also synthesized after growth on some of the potential intermediates in the subterminal oxidation pathway including 1,2-propanediol. NTG-generated mutants blocked in certain steps of the propane oxidation pathway were used to study the terminal and subterminal oxidation of propane metabolism. Mutants defective in propane metabolism (alkˉ) were also unable to grow on acetol, but still grew on terminal and subterminal intermediates of propane oxidation. SDS-PAGE analysis of alkˉ mutants grown under propane-inducing condition showed the synthesis of at least one of the propane-specific polypeptides. This indicated that those mutants are defective in a regulatory gene(s) (alkR) of propane/acetol oxygenase. Culture supernatants during growth on propane and potential intermediates of propane metabolism were analysed. This showed that methyl acetate is not an intermediate in the metabolism of propane. Ultra-thin sections of propane-grown R.rhodochrous PNKbl showed the involvement of unusual intracellular structures at the poles of the cells. These structures could be associated with propane assimilation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QD Chemistry ; QR Microbiology