Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.382931
Title: The separation and immobilisation of a yeast intracellular enzyme
Author: Sadler, Andrew Michael
ISNI:       0000 0001 3544 5999
Awarding Body: University of Surrey
Current Institution: University of Surrey
Date of Award: 1988
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Abstract:
The aim of this work was to investigate the problems involved in production and separation of a yeast microsomal enzyme, cytochrome P450. Immobilisation of the microsomal preparation, and utilisation of the immobilised system for the removal of polycyclic aromatic hydrocarbons, particularly the carcinogen benzo(a)pyrene [B(a)P], from aqueous systems was also investigated. High recoveries of the microsomal enzyme were obtained using rapid, low-speed centrifugation (5 minutes, 3000xg) by prior precipitation with a non-ionic polymer, polyethylene glycol (PEG). PEG was found to effectively replace centrifugal acceleration. A semi-empirical mathematical model of the process based on the size distribution of the agglomerates formed and the proportion of the agglomerates sedimented by centrfugation was developed. The effect of PEG was consistent with its increasing the mean effective diameter of protein agglomerates in proportion to PEG concentration to an exponent of 0.619 and with its increasing the spread of the size distribution in proportion to the mean effective agglomerate diameter. Binding spectra studies established that B(a)P binds to the active site of yeast cytochrome P450 and is unaffected by PEG. The B(a)P assay by fluorescence following hexane extraction was also unaffected by PEG. The stability of the microsomal cytochrome P450 preparation at different temperatures was investigated. The enzyme half-life was 66 minutes at 37° and was also unaffected by PEG. The enzyme preparation was satisfactorily immoblilsed by encapsulation in calcium alginate gel and enzyme distribution profiles were determined in sections of alginate stained with coomasie blue by a novel technique using a scanning optical densitometer, Diffusivity coefficients of B(a)P and NADP in alginate gel beads were determined as 7.5 and 2.5 x 10[-10] m[2]/S, respectively by the Tanaka method, fitting solute depletion profiles to Crank's theoretical model. The microsomal enzyme preparation immobilised in alginate beads was used to remove B(a)P from aqueous solution. B(a)P removal was shown to be by a non-specific affinity absorption mechanism and the removal profile was found to correspond well to a theoretical model of a diffusion-reaction system, for an affinity ligand system. The activaiton energy for deactivation of microsomal P450 was measured as 33.56kJ/mole.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.382931  DOI: Not available
Keywords: Microsomal enzyme recovery
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