Browsing by Author "Burton, Stephanie G"

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    Improving the production of a thermostable amidase through optimising IPTG induction in a highly dense culture of recombinant Escherichia coli.
    (Elsevier, 2010) Olaofe, Oluwafemi A; Burton, Stephanie G; Cowan, Don A; Harrison, Susan T L
    The production of a novel thermostable amidase (EC 3.5.1.4) from Geobacillus pallidus RAPc8 using recombinant Escherichia coli BL21 (DE3) was investigated. Volumetric and specific enzyme activities were investigated in relation to inducer concentration in a batch process using a defined medium with glucose as the carbon source. While IPTG is routinely used to induce expression of genes under the control of lac promoter, the impact of high biomass concentration on IPTG induction has not been reported rigorously. In this study, biomass production was unaffected by IPTG concentration across the range 0–1000 M. Induction of recombinant protein expression by 400 M IPTG at late lag phase of growth (3rd hour) inhibited cell growth while induction at early exponential phase of growth (5th hour) gave a 3 fold increase in volumetric amidase activity compared to induction at mid exponential phase (8th hour). Protein production increased by a factor of two with IPTG addition, independent of IPTG concentration in the range of 40–1000 M. Amidase activity, measured on a volumetric basis and relative to protein and biomass concentrations, increased with increasing IPTG concentration up to 400 M. While inducer concentrations are typically reported on a volumetric basis, their mode of action is consistent with a biomass dependence. Analysis of the data across a range of biomass concentration confirmed that induction was a function of inducer concentration per unit biomass. The amidase enzyme was predominantly soluble and cytoplasmic with less than 3% retained within the cell debris.
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    Laccase-mediated oxidation of totarol
    (2007) Ncanana, Sandile; Baratto, Lara; Roncaglia, Lucia; Riva, Sergio; Burton, Stephanie G
    In this study, novel dimers of the biologically active phenolic compound totarol were synthesized using the phenol oxidase enzyme laccase, obtained from Trametes pusbescens, in organic solvent medium. Two dimeric products, linked either by carbon-carbon or by carbon-oxygen bonds, were isolated and characterized. The effect of changes in various parameters such as solvent, temperature, pH and buffer concentration on the conversion of totarol by laccase was investigated. The nature of the organic solvent, in particular, was found to affect the nature and the ratio of the products obtained.