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Title: Activity and thermostability of L-phenylalanine ammonia-lyase in water and organic solvents
Author: Rees, D. G.
Awarding Body: University College of Swansea
Current Institution: Swansea University
Date of Award: 1994
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L-phenylalanine ammonia-lyase (EC:4,3,1,5), (PAL), catalyses the non-oxidative deamination of L-phenylalanine to trans-cinnamic acid and ammonia. PAL from the red basidiomycetous yeast Rhodosporidium toruloides NCYC 819 was studied with the ultimate aim of examining enzyme properties in water immiscible organic solvents. This is the first demonstration of stability and activity of an ammonia-lyase in organic solvents. Reversal of this reaction has received attention as a possible enzymatic route to the commercially important amino acid L-phenylalanine (which is itself a precursor to the artificial sweetener Aspartame). The growth and induction of PAL by R.toruloides in media containing various nutrient combinations was examined especially in relation to the influence of nitrogen sources on PAL induction. PAL is not appreciably induced except under conditions where L-phenylalanine provides the only or best available source of carbon and/or nitrogen. It was found, in contrast to some previous reports that if L-phenylalanine is present in addition to D-glucose and ammonium ions then the enzyme is induced but at a lower level. The enzyme was partially purified from R.toruloides cell extracts using protamine sulphate, ammonium sulphate fractionation, and DEAE-Sephacel column chromatography steps. This resulted in a PAL preparation 10-fold purified over the crude extract with over 35% yield and an estimated purity of at least 60%. This preparation had a pH optimum between 7.9 and 8.5 (typical for this enzyme) and exhibited biphasic kinetics (i.e. a nonlinear Eadie-Hofstee plot) with the resulting Michaelis constants determined as Kmhigh = 1121 μM and K_m^low = 248 μM. The stability to heating of the partially purified PAL preparations in water was examined and half lives of 95 hours at 30oC, 21 hours at 50oC and 1.4 min at 70oC were obtained. The rate of inactivation at 60oC was found to depend markedly on the pH of the PAL solution with stability falling sharply either side of the range 5.5 to 7.0.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available