Tuffnell, Janet M. (1983) Studies on amino acid assays using Escherichia coli. Doctoral thesis, Durham University.
The methods most widely used for the assay of amino acids, peptides and proteins are surveyed and the advantages of microbiological assays are discussed.Comparisons are-made between the use of 'natural' auxotrophs commonly used for assay and 'artificial' mutant strains of E.coli as assay organisms. A novel assay is described in which an E.coli Lys (^-) auxotroph is used to measure Lys-dependent protein synthesis. The response to a given quantity of Lys is constant irrespective of whether it is free, peptide-bound or present in a complex mixture. Since the measured response is enzyme synthesis rather than growth the technique has the advantage of sensitivity and speed. The enzyme measured is the inducible enzyme β-galactosidase, which liberates ơ-nitrophenol fron the chromogenic substrate a ơ-nitrophenol-β-D-galactopyranoside. The number of active enzyme molecules that can be synthesised by the auxotroph depend solely on the Lys present. Thus the amount of available Lys in a digest can be determined by reference to a calibration curve of enzyme activity v. Lys concentration.Optimisation of the method and the isolation, selection and characterisation of the mutants to assay for available Lys. Met and Trp is described, and the-sensitivity of the assay is similar for all three amino acids. Application of the technique to measure Lys, Met and Trp present in digests of a variety of pure proteins, feed meals and rice varieties is described and results are compared with published data. The assay response to Met(O) led to the study of the effects of physiological status of E. coli on oxidation and reduction of Met(O) and on cleavage of MetO(_2)) residues. Finally, since the auxotrophic requirement for the Lys" Met" strain could be replaced by B(_12) a preliminary investigation of the use of the technique to assay for this vitamin is reported.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||15 May 2013 14:15|